CCR8 antibodies for therapeutic applications (2025)

U.S. patent number 11,427,640 [Application Number 17/358,841] was granted by the patent office on 2022-08-30 for ccr8 antibodies for therapeutic applications. This patent grant is currently assigned to BAYER AKTIENGESELLSCHAFT. The grantee listed for this patent is Bayer Aktiengesellschaft, Bayer HealthCare LLC, Bayer Intellectual Property GmbH. Invention is credited to Sandra Berndt, Katharina Filarsky, Uwe Gritzan, Sabine Hoff, Wiebke Maria Nadler, Helge Roider, Su-Yi Tseng, Christian Votsmeier.

CCR8 antibodies for therapeutic applications

Abstract

The present invention relates to tools and methods for thegeneration of antibodies which specifically bind chemokinereceptors, such as CC or CXC chemokine receptors. Provided areisolated sulfated polypeptides and conjugates thereof, which can beused for example as antigens or for off target panning tofacilitate the generation of anti-human, anti-cynomolgus, and/oranti-mouse chemokine receptor antibodies, e.g. for the generationof antibodies with fully human CDRs and/or other favorableproperties for therapeutic use. The present invention furthermorerelates to antibodies and conjugates thereof which can be obtainedby applying the aforementioned tools and methods. Provided areantibodies specifically binding to human, cynomolgus and/or murineCCR8 with favorable properties for therapeutic use, such ascross-reactive antibodies, fully human antibodies, lowinternalizing (including non-internalizing) antibodies, andantibodies efficiently inducing ADCC and/or ADCP in Treg cells.

Related U.S. Patent Documents

Foreign Application Priority Data

References Cited [Referenced By]

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Other References

Primary Examiner: Kaufman; Claire
Attorney, Agent or Firm: Quarles & Brady LLP

Claims

We claim:

1. An isolated anti-CCR8 antibody or antigen-binding fragmentthereof comprising six CDR sequences wherein each CDR has an aminoacid sequence set forth in one of (a) to (r): a. SEQ ID NO:420, SEQID NO:421, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:425 and SEQ IDNO:426, b. SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ IDNO:604, SEQ ID NO:605 and SEQ ID NO:606, c. SEQ ID NO:618, SEQ IDNO:619, SEQ ID NO:620, SEQ ID NO:622, SEQ ID NO:623 and SEQ IDNO:624, d. SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ IDNO:665, SEQ ID NO:666 and SEQ ID NO:667, e. SEQ ID NO:681, SEQ IDNO:682, SEQ ID NO:683, SEQ ID NO:685, SEQ ID NO:686 and SEQ IDNO:687, f SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ IDNO:707, SEQ ID NO:708 and SEQ ID NO:709, g. SEQ ID NO:723, SEQ IDNO:724, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:728 and SEQ IDNO:729, h. SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:747, SEQ ID NO:748 and SEQ ID NO:749, i. SEQ ID NO:763, SEQ IDNO:764, SEQ ID NO:765, SEQ ID NO:767, SEQ ID NO:768 and SEQ IDNO:769, j. SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ IDNO:787, SEQ ID NO:788 and SEQ ID NO:789, k. SEQ ID NO:803, SEQ IDNO:804, SEQ ID NO:805, SEQ ID NO:807, SEQ ID NO:808 and SEQ IDNO:809, l. SEQ ID NO:827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ IDNO: 831, SEQ ID NO:832 and SEQ ID NO:833, m. SEQ ID NO:847, SEQ IDNO: 848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO:852 and SEQ IDNO:853, n. SEQ ID NO:867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ IDNO: 871, SEQ ID NO:872 and SEQ ID NO:873, o. SEQ ID NO:887, SEQ IDNO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO:892 and SEQ IDNO:893, p. SEQ ID NO:907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ IDNO: 911, SEQ ID NO:912 and SEQ ID NO:913, q. SEQ ID NO:927, SEQ IDNO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO:932 and SEQ IDNO:933, and r. SEQ ID NO:947, SEQ ID NO: 948, SEQ ID NO: 949, SEQID NO: 951, SEQ ID NO:952 and SEQ ID NO:953.

2. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1 further comprising a variable heavy chainsequence and/or a variable light chain sequence having an aminoacid sequence set forth in one of (a) to (r) a. a variable heavychain sequence according to SEQ ID NO:419 and/or a variable lightchain sequence according to SEQ ID NO:423, b. a variable heavychain sequence according to SEQ ID NO:599 and/or a variable lightchain sequence according to SEQ ID NO:603, c. a variable heavychain sequence according to SEQ ID NO:617 and/or a variable lightchain sequence according to SEQ ID NO:621, d. a variable heavychain sequence according to SEQ ID NO:660 and/or a variable lightchain sequence according to SEQ ID NO:664, e. a variable heavychain sequence according to SEQ ID NO:680 and/or a variable lightchain sequence according to SEQ ID NO:684, f. a variable heavychain sequence according to SEQ ID NO:702 and/or a variable lightchain sequence according to SEQ ID NO:706, g. a variable heavychain sequence according to SEQ ID NO:722 and/or a variable lightchain sequence according to SEQ ID NO:726, h. a variable heavychain sequence according to SEQ ID NO:742 and/or a variable lightchain sequence according to SEQ ID NO:746, i. a variable heavychain sequence according to SEQ ID NO:762 and/or a variable lightchain sequence according to SEQ ID NO:766, j. a variable heavychain sequence according to SEQ ID NO:782 and/or a variable lightchain sequence according to SEQ ID NO:786, k. a variable heavychain sequence according to SEQ ID NO:802 and/or a variable lightchain sequence according to SEQ ID NO:806, l. a variable heavychain sequence according to SEQ ID NO:826 and/or a variable lightchain sequence according to SEQ ID NO:830, m. a variable heavychain sequence according to SEQ ID NO:846 and/or a variable lightchain sequence according to SEQ ID NO:850, n. a variable heavychain sequence according to SEQ ID NO:866 and/or a variable lightchain sequence according to SEQ ID NO:870, o. a variable heavychain sequence according to SEQ ID NO:886 and/or a variable lightchain sequence according to SEQ ID NO:890, p. a variable heavychain sequence according to SEQ ID NO:906 and/or a variable lightchain sequence according to SEQ ID NO:910, q. a variable heavychain sequence according to SEQ ID NO:926 and/or a variable lightchain sequence according to SEQ ID NO:930, or r. a variable heavychain sequence according to SEQ ID NO:946 and/or a variable lightchain sequence according to SEQ ID NO:950.

3. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1 further comprising a heavy chain sequence and/ora light chain sequence having an amino acid sequence set forth inone of (a) to (r): a. a heavy chain according to SEQ ID NO:435 anda light chain according to SEQ ID NO:436, b. a heavy chainaccording to SEQ ID NO:615 and a light chain according to SEQ IDNO:616, c. a heavy chain according to SEQ ID NO:633 and a lightchain according to SEQ ID NO:634, d. a heavy chain according to SEQID NO:676 and a light chain according to SEQ ID NO:677, e. a heavychain according to SEQ ID NO:696 and a light chain according to SEQID NO:697, f. a heavy chain according to SEQ ID NO:718 and a lightchain according to SEQ ID NO:719, g. a heavy chain according to SEQID NO:738 and a light chain according to SEQ ID NO:739, h. a heavychain according to SEQ ID NO:758 and a light chain according to SEQID NO:759, i. a heavy chain according to SEQ ID NO:778 and a lightchain according to SEQ ID NO:779, j. a heavy chain according to SEQID NO:798 and a light chain according to SEQ ID NO:799, k. a heavychain according to SEQ ID NO:818 and a light chain according to SEQID NO:819, l. a heavy chain according to SEQ ID NO:842 and a lightchain according to SEQ ID NO:843, m. a heavy chain according to SEQID NO:862 and a light chain according to SEQ ID NO:863, n. a heavychain according to SEQ ID NO:882 and a light chain according to SEQID NO:883, o. a heavy chain according to SEQ ID NO:902 and a lightchain according to SEQ ID NO:903, p. a heavy chain according to SEQID NO:922 and a light chain according to SEQ ID NO:923, q. a heavychain according to SEQ ID NO:942 and a light chain according to SEQID NO:943, and r. a heavy chain according to SEQ ID NO:962 and alight chain according to SEQ ID NO:963.

4. A polynucleotide encoding an antibody or antigen-bindingfragment according to claim 1; wherein the antibody orantigen-binding fragment comprises six CDR sequences wherein eachCDR has an amino acid sequence set forth in one of (a) to (r): a.SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ ID NO:424, SEQ IDNO:425 and SEQ ID NO:426, b. SEQ ID NO:600, SEQ ID NO:601, SEQ IDNO:602, SEQ ID NO:604, SEQ ID NO:605 and SEQ ID NO:606, c. SEQ IDNO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:622, SEQ ID NO:623and SEQ ID NO:624, d. SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663,SEQ ID NO:665, SEQ ID NO:666 and SEQ ID NO:667, e. SEQ ID NO:681,SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:685, SEQ ID NO:686 and SEQID NO:687, f. SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ IDNO:707, SEQ ID NO:708 and SEQ ID NO:709, g. SEQ ID NO:723, SEQ IDNO:724, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:728 and SEQ IDNO:729, h. SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:747, SEQ ID NO:748 and SEQ ID NO:749, i. SEQ ID NO:763, SEQ IDNO:764, SEQ ID NO:765, SEQ ID NO:767, SEQ ID NO:768 and SEQ IDNO:769, j. SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ IDNO:787, SEQ ID NO:788 and SEQ ID NO:789, k. SEQ ID NO:803, SEQ IDNO:804, SEQ ID NO:805, SEQ ID NO:807, SEQ ID NO:808 and SEQ IDNO:809, l. SEQ ID NO:827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ IDNO: 831, SEQ ID NO:832 and SEQ ID NO:833, m. SEQ ID NO:847, SEQ IDNO: 848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO:852 and SEQ IDNO:853, n. SEQ ID NO:867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ IDNO: 871, SEQ ID NO:872 and SEQ ID NO:873, o. SEQ ID NO:887, SEQ IDNO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO:892 and SEQ IDNO:893, p. SEQ ID NO:907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ IDNO: 911, SEQ ID NO:912 and SEQ ID NO:913, q. SEQ ID NO:927, SEQ IDNO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO:932 and SEQ IDNO:933, and r. SEQ ID NO:947, SEQ ID NO: 948, SEQ ID NO: 949, SEQID NO: 951, SEQ ID NO:952 and SEQ ID NO:953.

5. A vector comprising the polynucleotide according to claim 4.

6. An isolated cell comprising the polynucleotide of claim 4.

7. A pharmaceutical composition comprising the isolated anti-CCR8antibody or antigen-binding fragment of claim 2 and apharmaceutically acceptable excipient.

8. A pharmaceutical composition comprising the isolated anti-CCR8antibody or antigen-binding fragment of claim 1 and apharmaceutically acceptable excipient.

9. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1, wherein the heavy chain variable region (VH)comprises CDRs 1, 2 and 3 with the amino acid sequences set forthin SEQ ID NOs: 618, 619, and 620, respectively, and the light chainvariable region (VL) comprises CDRs 1, 2, 3 with the amino acidsequences set forth in SEQ ID NOs: 622, 623, and 624,respectively.

10. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1, wherein the heavy chain variable region (VH)comprises an amino acid sequence at least 98% identical to theamino acid sequence set forth in SEQ ID NO: 617 and the light chainvariable region (VL) comprises an amino acid sequence at least 98%identical to the amino acid sequence set forth in SEQ ID NO:621.

11. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 10, wherein the VH comprises an amino acidsequence set forth in SEQ ID NO: 617 and the VL comprises an aminoacid sequence set forth in SEQ ID NO: 621.

12. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1, wherein the heavy chain comprises an amino acidsequence at least 98% identical to the amino acid sequence setforth in SEQ ID NO: 633 and the light chain comprises an amino acidsequence at least 98% identical to the amino acid sequence setforth in SEQ ID NO: 634.

13. The isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 12, wherein the heavy chain comprises an aminoacid sequence set forth in SEQ ID NO: 633 and the light chaincomprises an amino acid sequence set forth in SEQ ID NO: 634.

14. A method for inducing antibody-dependent cellular phagocytosis(ADCP) in a target cell expressing CCR8 comprising administering toa human patient having a tumor a therapeutically effective amountof the isolated anti-CCR8 antibody or antigen-binding fragmentthereof of claim 1.

15. The method of claim 14, wherein the isolated anti-CCR8 antibodyor antigen-binding fragment thereof comprises six CDR sequencesthat have the amino acid sequences set forth in SEQ ID NO:618, SEQID NO:619, SEQ ID NO:620, SEQ ID NO:622, SEQ ID NO:623 and SEQ IDNO:624.

Description

SEQUENCE LISTING SUBMISSION

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 172086_00185_ST25. The size ofthe text file is 763,277 bytes, and the text file was created onFeb. 4, 2022.

TECHNICAL FIELD

The present invention relates to tools and methods for thegeneration of antibodies which specifically bind chemokinereceptors, such as CC or CXC chemokine receptors. Provided areisolated sulfated polypeptides and conjugates thereof, which can beused for example as antigens or for off target panning tofacilitate the generation of anti-human, anti-cynomolgus, and/oranti-mouse chemokine receptor antibodies, e.g. for the generationof antibodies with fully human CDRs and/or other favorableproperties for therapeutic use.

The present invention furthermore relates to antibodies andconjugates thereof which can be obtained by applying theaforementioned tools and methods. Provided are antibodiesspecifically binding to human, cynomolgus and/or murine CCR8 withfavorable properties for therapeutic use, such as cross-reactiveantibodies, fully human antibodies, low internalizing (includingnon-internalizing) antibodies, and antibodies efficiently inducingADCC and/or ADCP in Treg cells.

Also provided are medical uses of the inventive antibodies orconjugates and/or treatment methods comprising the administrationof these antibodies to a patient or subject, either alone or incombination. Biomarkers, stratification methods and diagnosticmethods are finally provided to predict or evaluate responsivenessto anti-CCR8 antibody monotherapy or combination therapy.

The invention furthermore provides tools and methods for producingthe foregoing antibodies, pharmaceutical compositions, diagnosticuses of the antibodies, and kits with instructions for use.

Technical Problem

Antibody Generation for CC and CXC Chemokine Receptors

CXC and CC chemokine-receptors are specific seven-transmembraneG-protein-coupled receptors that mediate cell migration inchemotactic gradients. Chemokine receptors are a challenging targetclass for antibody generation due to their inherent structural,biophysical and biological properties. There are multiple reasonsfor the difficulties to obtain optimal antibodies against chemokinereceptors which shall be discussed in an exemplary fashion, with anemphasis on human CCR8.

Chemokine receptors are characterized by seven domains embedded inthe cell membrane and therefore cannot be easily purified in theirnative confirmation. A purified, native chemokine receptor willhave been removed from the membrane environment and is thereforelikely to be conformationally compromised. These corruptedstructures are typically not suitable for antibody generation,because antibodies are required to recognize the intact antigen asit is presented at the cell surface. Although some chemokinereceptors, such as CXCR4 and CCR5, possess some inherent stabilitythat allows for purification in mild detergents, this does notapply to most chemokine receptors (Hutchings, Catherine J., et al."Opportunities for therapeutic antibodies directed atG-protein-coupled receptors." Nature reviews Drug discovery 16.11(2017): 787.). This is emphasized by the fact that until today, noX-ray crystal structure is available for CCR8 in the Protein DataBank PDB (rcsb.org).

In consequence, solubilization for obtaining the required amountsof protein in the native conformation as well as correctorientation and folding for use as immunogen is difficult forchemokine receptors (cf. Klarenbeek, Alex, et al. "Targetingchemokines and chemokine receptors with antibodies." Drug DiscoveryToday: Technologies 9.4 (2012): e237-e244.).

A schematic representation of the overall structure of human CCR8is shown in FIG. 2B. Of the 355 amino acids, residues 1-35 (Nterm), 94-107 (ECL1), 172-202 (ECL2) and 264-280 (ECL3) have beenpredicted as extracellular domains (uniprot.org). Theseextracellular domains are assumed to adopt a tertiary structure,stabilized by disulfide bonds. On average, less than 30% of achemokine receptor is thus exposed at the cell surface. Inconsequence, chemokine receptors and particularly CCR8 are noteasily accessible for antibody binding, as described also inWO200744756.

Furthermore, antibodies generated against peptides corresponding toextracellular domains of chemokine receptors often fail torecognize the intact receptor on the cell, possibly due todifferences in secondary structure. In consequence, researchers inthis field have had a low success rate in developing antibodies(Tschammer, Nuska, ed. Chemokines: chemokines and their receptorsin drug discovery. Vol. 14. Springer, 2015, Chapter by J. E. Pease& R. Horuk, section 6, page 12, 2nd para).

Antibody generation to obtain anti-murine CCR8 antibodies seemsslightly less difficult and conventional approaches such as the onedescribed by Kremer and Marquez could be reproduced by theinventors (D'Ambrosio, Daniele, and Francesco Sinigaglia. CellMigration in Inflammation and Immunity. Springer, 2004, Chapter byKremer and Marquez p. 243-260). Murine and human CCR8 have asequence identity of .about.70%, which is even lower for theextracellular domains, as described in more detail in example 2.Nevertheless, Kremer and Marquez discuss, that production ofanti-mouse chemokine-receptor monoclonal antibodies is a demandingchallenge and that antibodies against murine chemokine receptorsare comparably scarce, despite the time that has passed since theiramino acid sequences were first reported.

Therapeutic Antibodies for Ccr8 and Medical Uses Thereof

Despite the difficulties associated with their generation,antibodies targeting CC chemokine receptors have been suggested aspromising therapeutic tools in various diseases, e.g. based onmechanistic insights in diseases with involvement of immune cellsor in various cancer indications characterized by aberrantexpression of chemokine receptors.

In 2004, Curiel et al. could show for CD4+CD25+FOXP3+ regulatory Tcells (Treg cells or Tregs) in 104 individuals affected withovarian carcinoma, that human tumor Treg cells suppresstumor-specific T cell immunity and contribute to growth of humantumors in vivo (Curiel, Tyler J., et al. "Specific recruitment ofregulatory T cells in ovarian carcinoma fosters immune privilegeand predicts reduced survival." Nature medicine 10.9 (2004):942-949.).

Tumor cells and microenvironmental macrophages produce chemokinessuch as CCL22, which mediates trafficking of Treg cells to thetumor. High level of Tregs in the tumor microenvironment are notonly associated with poor prognosis in many cancers, such asovarian, breast, renal, and pancreatic cancer, but cause thesuppression of the immune response against these cancers, e.g. bysuppressing the action of the effector cells of the immune system.This specific recruitment of Treg cells thus represents a mechanismby which tumors may foster immune privilege. It has therefore beensuggested to block Treg cell migration or function to defeat humancancer.

Being aware of the finding of Curiel, two independent teams aroundPlitas/Rudensky and De Simone/Abrignani/Pagani found that tumorinfiltrating Treg cells are characterized by selective expressionof CCR8. Indeed, selectivity of Treg cell depletion for the tumorinfiltrating Tregs is important because systemic depletion of Tregcells may cause severe autoimmunity (Nishikawa, Hiroyoshi, andShimon Sakaguchi. "Regulatory T cells in tumor immunity."International journal of cancer 127.4 (2010): 759-767.). The searchfor a specific Treg marker is complicated by the fact, that Tregcells show a molecular pattern which resembles effectorlymphocytes, cf. example 11.2. Both peripheral Tregs andtumor-specific effector cells should not suffer from friendly fireduring depletion of intra-tumoral Tregs, because peripheral Tregsare important to avoid autoimmunity while tumor-specific effectorcells assist in keeping the tumor under control.

Based on these findings various teams have suggested to use CCR8antibodies for selective depletion of tumor-infiltrating regulatoryT cells. While some have presented data confirming tumor reductionin tumor models using anti-human CCR4 or anti-murine CCR8antibodies, thereby confirming the mechanistic concept of Tregdepletion, there is a need for therapeutic anti-human CCR8antibodies, e.g. with superior properties in therapeuticapplications.

PRIOR ART

1.1 Antigens, Methods & Antibodies

The selection of an antigen in antibody generation is crucial forthe properties of the resulting antibodies and may pose seriousproblems as discussed elsewhere herein for chemokine receptors suchas CCR8. In some cases, antibodies have been obtained using wholecells as antigens, which were engineered for overexpression ofchemokine receptors. At least for some chemokine receptors, theseapproaches seem to suffer from low numbers of resulting binders.Furthermore, antibodies obtained with these antigens are oftencharacterized by off-target binding and low specificity for thechemokine receptor. If whole cells are used as antigens,immunodominant epitopes can mask other less antigenic ones, wherethe less antigenic would otherwise have the potential of generatingthe desired selective and specific antibodies. For CCR8, asuccessful "whole cell" approach has been described in WO2007044756(ICOS). In brief, anti-CCR8 monoclonal antibodies were developed byimmunizing Balb/c mice with irradiated cells transfected with CCR8which had high levels of CCR8 expressed on the cell surface. Spleencells from these mice were fused by standard methods to createhybridomas producing the antibodies. Positive pools were identifiedby FACS and were cloned by limiting dilution. Two of theantibodies, 433H and 459M, showed a specific reactivity to humanCCR8 in immunohistochemistry. Antibody 433H is still available andcan be purchased from BD.

Biolegend distributes clone L263G8, a purified mouse IgG2aanti-human CCR8 antibody which has been generated using human CCR8transfectants as immunogen.

Kremer and Marquez have described the generation of monoclonalantibodies against murine CCR8 (D'Ambrosio, Daniele, and FrancescoSinigaglia. Cell Migration in Inflammation and Immunity. Springer,2004, Chapter by Kremer and Marquez p. 243-260). In brief, Kremerand Marquez describe the use of peptides derived from theextracellular domains of murine CCR8 as immunogens, but do notsuggest sulfation of the tyrosines. Indeed, the inventors foundthat the approach described by Kremer and Marquez can besuccessfully applied for antibodies recognizing murine CCR8 but haslow success rates for antibodies recognizing human CCR8.

Schaerli et al. have generated anti-human CCR8 antibodies byimmunizing rabbits with a human CCR8 peptide conjugate,corresponding to positions 1-34 of the N-terminal region of CCR8coupled to either KLH or BSA, but again, do not suggest sulfationof the tyrosines (Schaerli, Patrick, et al. "A skin-selectivehoming mechanism for human immune surveillance T cells." TheJournal of experimental medicine 199.9 (2004): 1265-1275.).

A murine monoclonal antibody binding to a 26-amino acid-containingpeptide from the extracellular N-terminal portion of CCR8 has beendescribed by Hague et al. (Hague, Nasreen S., et al. "The chemokinereceptor CCR8 mediates human endothelial cell chemotaxis induced by1-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and bylipoprotein (a)-stimulated endothelial cell conditioned medium."Blood, The Journal of the American Society of Hematology 97.1(2001): 39-45.).

None of these methods for antibody generation uses an isolatedsulfated polypeptide comprising the tyrosine rich domain (TRD) of achemokine receptor or transmembrane protein as antigen. Use of anisolated sulfated polypeptide comprising the TRD of a chemokinereceptor or transmembrane protein as antigen influences both,structural and functional features of the set of obtainedantibodies, as discussed elsewhere herein.

In consequence, the antibodies according to the current inventionare assumed to deviate in structure and function from theaforementioned prior art antibodies, e.g. at least in theiraffinity for sulfated CCR8, in their affinity for unsulfated CCR8,in their way and degree of modulating receptor signaling, in theirinternalization behavior, in cross reactivity, in clearance andpharmacokinetic behavior and finally in Treg depletion and/orefficacy for therapeutic applications.

Furthermore, the use of the antigens disclosed herein enabled thegeneration of fully human antibodies. In contrast, the discussedprior art antibodies for CCR8 are of non-human origin and deviatefrom some of the inventive antibodies at least because they do notcomprise human CDRs.

1.2 Medical Uses and Mode of Action

Cancer immunotherapy involves the use of a subject's immune systemto treat or prevent cancer. Immunotherapies typically exploit thefact that cancer cells often have subtly different molecules ontheir surface that can be detected by the immune system, the cancerantigens. Immunotherapy thus involves provocation of the immunesystem into attacking tumor cells via these cancer antigens.However, some cancers, such as solid tumors or hematologicalcancers can escape immune surveillance. For example, tumorinfiltration by regulatory T cells (Treg cells or Tregs) and, morespecifically, low ratio of effector T cells (Teff) to Tregs havebeen proposed as critical factors for hiding the tumor from theimmune system (Smyth, Mark J, Shin Foong Ngiow, and Michele W LTeng. "Targeting regulatory T cells in tumor immunotherapy."Immunology and cell biology 92.6 (2014): 473-474.).

Foxp3-expressing regulatory T cells, which are indispensable forpreventing autoimmunity, are known to effectively suppress tumorimmunity. Treg cells abundantly infiltrate into tumor tissues,which is often associated with poor prognosis in cancer patients.Removal of Treg cells enhances anti-tumor immune responses but mayalso elicit autoimmunity. A key issue for tailoring Treg-targetingcancer immunotherapy resides in specific depletion of Treg cellsinfiltrating into tumor tissues without affecting tumor-reactiveeffector T cells, while suppressing autoimmunity.

Back in 2010 various groups had already investigated that Tregremoval, e.g. by depletion with anti-CD25 antibodies, could improveantitumor immunity in mice. It had been shown that depletion ofTregs before the inoculation of tumor cells led to their efficientrejection while Treg depletion occurring simultaneously with orafter tumor inoculation resulted in no tumor regression. It hadfurthermore been suggested, that this was due to the fact, that theadministered depleting antibodies also removed CD25-expressingeffector T cells, while CD25-Foxp3+ Tregs persisted (Klages,Katjana, et al. "Selective depletion of Foxp3+ regulatory T cellsimproves effective therapeutic vaccination against establishedmelanoma." Cancer research 70.20 (2010): 7788-7799.).

In 2015, the afucosylated humanized anti-human CCR4 monoclonalantibody mogamulizumab (KW-0761) was evaluated in a clinical studyfor patients with CCR4-positive cancers (Kurose, Koji, et al."Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of ahumanized anti-CCR4 antibody, KW-0761, in cancer patients."Clinical Cancer Research 21.19 (2015): 4327-4336.). CCR4 isexpressed on regulatory T cells. Indeed, mogamulizumab efficientlydepleted Treg cells and an augmentation or induction of specificimmune responses to cancer antigens was observed. However,mogamulizumab targets both, peripheral as well as intra-tumoralTregs and further effector cell polulations, thereby leading toimmunological side effects such as skin rashes or Stevens Johnsonsyndrome.

Thus, although the great potential for Treg depletion in cancertherapies was obvious, a concept to selectively target the Tregsand to avoid overt autoimmunity was missing. This was changed withthe work of two teams around Plitas/Rudensky and DeSimone/Abrignani/Pagani in 2015 and 2016.

Plitas et al. demonstrated that CCR8 is selectively expressed byhuman breast cancer infiltrating Treg cells and immediatelyconcluded that targeting CCR8 represents a promisingimmunotherapeutic approach for the treatment of patients withbreast cancer and further tumors (Plitas, G., et al. "AbstractP4-04-11: Preferential expression of the chemokine receptor 8(CCR8) on regulatory T cells (Treg) infiltrating human breastcancers represents a novel immunotherapeutic target." (2016):P4-04; Plitas, George, et al. "Regulatory T cells exhibit distinctfeatures in human breast cancer." Immunity 45.5 (2016): 1122-1134.;U.S. Ser. No. 10/087,259).

Only shortly after publication of the initial work of Plitas etal., De Simone et al. published an analysis on the transcriptionallandscape of tumor infiltrating T regulatory cells and found thattumor-infiltrating Treg cells were highly immune suppressive andexpressed CCR8 on their cell surface as a specific signaturemolecule (De Simone, Marco, et al. "Transcriptional landscape ofhuman tissue lymphocytes unveils uniqueness of tumor-infiltrating Tregulatory cells." Immunity 45.5 (2016): 1135-1147, WO2017198631).The authors described that CCR8 correlated with poor prognosis andconcluded that CCR8 could be an interesting therapeutic target toinhibit Treg cells trafficking to tumor sites, without disturbingrecruitment of other effector T cells that do not express CCR8.

WO2018112032 and WO2018112033 describe methods of decreasing thenumber or activity of tumor infiltrating T regulatory cells (TITR)in a tumor comprising administering an agent that inducescytotoxicity in cells that express a product of a gene, e.g.CCR8.

WO2018112033 and EP3431105 describe a molecule which can modulatethe expression and/or function of at least one marker that isselectively deregulated in tumor-infiltrating regulatory T cellsfor use in the prevention and/or treatment of this tumor.

WO2018181425 relates to an antibody against CCR8 having ADCCactivity, for use in a method of treating a cancer, wherein theantibody against CCR8 is a CCR8-neutralizing antibody. WO2018181425does not disclose specific antibody sequences but refers to ratanti-mouse CCR8 clone SA214G2 distributed by BioLegend under Cat.No. 150302. This antibody was used as a tool antibody torecapitulate the previously described anti-tumorigenic effects ofTreg depletion in mice.

According to the current invention, a method is provided whichfacilitates the generation of anti-CCR8 antibodies and yieldsantibodies having superior properties for therapeutic uses. Theantibodies according to the current invention were compared withthe prior art antibodies and were found to deviate in structure,function and therapeutic efficacy, as discussed elsewhereherein.

Solution to Problem

1.1 Antibody Generation for CC and CXC Chemokine Receptors

Because prior art approaches to generate antibodies for CCchemokine receptors suffer from low success rates, and resultingantibodies often show a poor performance (cf. e.g. example 3), theinventors developed a novel method to generate antibodies forchemokine receptors in general and, in particular, to generateanti-CCR8 antibodies.

The use of a specifically modified isolated polypeptide as antigenfor the selection of human anti-human CCR8 antibodies solved theproblem to provide an improved method for antibody generationagainst CC and CXC chemokine receptors, such that e.g. fully humananti-CCR8 antibodies could be obtained for the first time, cf.example 6. In more detail, a polypeptide comprising the tyrosinerich domain of human CCR8, optionally including the LID domain, wasmodified by introduction of sulfate modifications at specificpositions in order to form the antigen, cf. example 4, Table 4.1. Aphage display approach was optionally used together with thisspecifically modified antigen to select fully human anti-human CCR8antibodies. In the alternative, the sulfated antigens can be usedin various further methods, e.g. in conventional immunizationapproaches.

The obtained antibodies showed an excellent binding profile for thesulfated antigen and the biological target expressed underphysiological conditions, cf. examples 6, 10.1.1, but a comparablylow or no affinity for the unmodified antigen, cf example 10.1.2,example 10.1.3, demonstrating that the sulfated residues wereindeed crucial for antigen antibody binding. The inventiveantibodies furthermore showed an excellent and also specificbinding on cell lines engineered to express human, cynomolgus ormurine CCR8, and also on activated human Tregs, cf. example10.1.1.

Out of the six CDR loops of an antibody, the H3 loop shows thegreatest structural diversity and is located in the center of thebinding site. It also gains the most mutations through affinitymaturation and has on average the largest number of contacts withthe antigen. It therefore plays a crucial role in antigen binding.Upon analysis of the specific structure of the antibodies obtainedwith the method according to the current invention, it wassurprisingly found that the composition of the HCDR3 wasstructurally different from usual human HCDR3 domains, cf. example9. In particular, the HCDR3 domains of the antibodies with thetherapeutically most beneficial properties and specific binding toCCR8 were characterized by an average frequency of .about.21%tyrosine residues and an average histidine content of .about.10%,emphasizing a beneficial impact of these residues on therecognition of the specific sulfated antigen (cf. Table 9.2).Without being bound by theory, the inventors believe that thesestructural features translate into certain functionalcharacteristics which make the obtained antibodies more suitablefor therapeutic applications. For example, and in contrast to thetested prior art antibodies, several antibodies according to theinvention block G protein dependent signaling, cf. example 10.4.3,but do not impact G protein independent pathways, cf. examples10.4, 10.4.1, 10.4.2, and do not substantially internalize intocells with endogenous expression of the target chemokine receptor,cf. example 10.5. In addition, particularly preferred antibodiesaccording to the current invention were specific for the targetreceptor and target cells, cf. example 10.2, 11, and are especiallysuitable/showed superior properties in methods of treatment forcancer, cf. examples 12 ff. Furthermore, use of these syntheticpolypeptides either as antigens or for off target panningfacilitated or enabled the generation of cross-reactive antibodies,such as antibodies specifically binding to human CCR8 and/orcynomolgus CCR8, cf. example 10.1.1.

1.2 Provision of Therapeutic Antibodies Specifically BindingChemokine Receptors

1.2.1 Obtaining Chemokine Receptor Antibodies with Human CDRs

While humanization of antibodies with murine CDRs may improve theimmunogenicity of an antibody, residual immunogenicity resides inthe CDR regions (Harding, Fiona A., et al. "The immunogenicity ofhumanized and fully human antibodies: residual immunogenicityresides in the CDR regions." MAbs. Vol. 2. No. 3. Taylor &Francis, 2010.). Antibodies comprising human CDRs are thus assumedto have a superior suitability as therapeutic agents for humanscompared with antibodies comprising CDRs from other species.

For example, antibodies with human CDRs can be produced via phagedisplay technologies or using transgenic animals capable ofgenerating fully human antibodies. However, it is not alwaystrivial to obtain fully human antibodies. While, in theory, thediversity of HCDR3 regions is almost unlimited, in practice thegeneration of the antibody repertoire diversity appears to becarefully regulated by multiple mechanisms, which yield an in vivorepertoire that is restricted in its diversity and constrained inits range of antigen-binding sites. This is also the case for humanphage display libraries, which are often designed to reflect the invivo repertoire. Thus, in some cases, the structural requirementsof a "complicated" antigen may be fulfilled by rodent CDRs, whilethe same structure cannot be readily recognized by human CDRs.Based on the observation that tyrosine representation is on averageapproximately 50% lower in human HCDR3 than in murine HCDR3, it canbe assumed that it is clearly more challenging to find a humananti-human antibody in cases, where the unique binding abilities oftyrosine and histidine are required to enable the target binding.Indeed, the inventors are not aware of fully human anti-human CCR8prior art antibodies. According to the current invention there areprovided CCR8 antibodies comprising human derived CDRs, and alsofully human antibodies, cf. examples 6, 7, 8.

1.2.2 Obtaining Cross Reactive Chemokine Receptor Antibodies

Cross reactive anti-chemokine receptor antibodies such as crossreactive anti-CCR8 antibodies are advantageous for the developmentof a therapeutic antibody, because they can be used in non-humananimal models to characterize the therapeutic agents with regardsto pharmacological data and safety, before the antibodies areadministered to humans. However, cross reactive antibodies e.g.with similar binding behavior in two species are difficult togenerate and cannot be easily affinity maturated, e.g. because theextracellular parts of chemokine receptors such as CCR8 have a lowhomology between species (see example 2). According to the currentinvention, cross reactive antibodies for CCR8 can be generated inparticular by using small sulfated tyrosine comprising motifs whichhave a higher conservation between species, such that crossreactive antibodies binding a chemokine receptor such as CCR8 fromtwo or more species, e.g. with affinities in the same order ofmagnitude can be obtained in an easy and convenient way, cf.examples 6, 7, 10.1.1.

1.2.3 Obtaining Chemokine Receptor Antibodies for Therapy

To induce the killing of cells expressing a target chemokinereceptor or CCR8, multiple modes of action can be envisioned. Onemode of action is the conjugation of an antibody targeting thechemokine receptor or CCR8 to a drug in the form of an antibodydrug conjugate (ADC). Other possible modes of action are inductionof antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC). For ADCC, CDC and ADCP, atwo-step mechanism is involved: On the one hand, the antibody orfragment is required to effectively bind the target cell, e.g. theTreg via CCR8, on the other hand, the FC part of the antibody (oran alternative binding moiety which can be conjugated to theantibody or fragment as described elsewhere herein) has to bind toan effector cell, which will then mediate the killing of the targetcell. For ADCP, binding to macrophages as effector cells typicallyoccurs via the interaction of the antibodies FC part withFc.gamma.RIIa (CD32a) expressed by macrophages. In contrast, ADCCis mediated via interaction of the antibody or fragment withFc.gamma.RIIIa. In humans, Fc.gamma.RIII exists in two differentforms: Fc.gamma.RIIIa (CD16a) and Fc.gamma.RIIIb (CD16b). WhileFc.gamma.RIIIa is expressed on mast cells, macrophages, and naturalkiller cells as a transmembrane receptor, Fc.gamma.RIIIb is onlyexpressed on neutrophils. These receptors bind to the Fc portion ofIgG antibodies, which then activates antibody-dependentcell-mediated cytotoxicity (ADCC) mediated by the human effectorcells.

1.2.4 Obtaining Low Internalizing (Including Non-Internalizing)Chemokine Receptor Antibodies

When the inventors analyzed known anti-human CCR8 prior artantibodies, they found that these antibodies readily internalizedinto a cell with endogenous target expression but did not reside onthe cell surface for extended periods of time. The internalizationbehavior of an antibody does not only influence its clearance andpharmacological behavior but also its suitability for a specificmode of action for therapeutic uses.

While high internalization is desirable for the generation ofcertain antibody drug conjugates, it is undesirable for ADCCinduced depletion of tumor cells or Treg cells. In more detail, anantibody drug conjugate is required to transport the drug into thecell to achieve an efficient and specific depletion of the targetcell. In contrast, the ADCC mode of action requires the exposure ofthe antibody and its Fc domain at the outside of the target cell,where the immune effector cells can bind the FC domain for thelysis of the target cell. A low or even absent internalization ratethereby increases the time of effect for an ADCC/ADCP inducingantibody.

Upon characterization of the antibodies obtained with the novelmethod, the inventors surprisingly found, that several of theantibodies according to the current invention showed a particularlylow or even absent internalization profile in cells with endogenousexpression levels of CCR8, cf. example 10.5, while the prior artantibodies recognizing the same target had a higher internalizationrate. For example, prior art antibodies 433H and L263G8 readilyinternalized into the targeted cell and did not reside on the cellsurface for extended periods of time. Due to their lowinternalization properties, some of the antibodies according to thecurrent invention are thus particularly useful in an ADCC, ADCP,CDC or a mixed approach, such as a combined ADCC/ADCP approach.

Typically, the selection of a target is an important factor whichstrongly influences the internalization of an antibody into a cell.According to Islam S A et al, CCL1 ligand binding induces Ca2+ fluxand rapid receptor internalization of CCR8 (Islam, Sabina A., etal. "Identification of human CCR8 as a CCL18 receptor CCR8 is aCCL18 receptor." The Journal of experimental medicine 210.10(2013): 1889-1898.). The inventors found it surprising that theantibody itself was able to influence the internalizationproperties in such a substantial degree. However, when theycharacterized the ability of the inventive antibodies to modulate Gprotein independent signaling pathways of their target chemokinereceptor, they found that all tested prior art antibodies not onlyblocked G protein dependent signaling, but also modulated G proteinindependent signaling, cf. examples 10.4 ff. G protein independentsignaling has been previously linked to internalization behavior,cf. Fox, James M., et al. "Structure/function relationships of CCR8agonists and antagonists: Amino-terminal extension of CCL1 by asingle amino acid generates a partial agonist." Journal ofBiological Chemistry 281.48 (2006): 36652-36661.

1.2.5 Obtaining Chemokine Receptor Antibodies Modulating TargetReceptor Signaling

There are multiple different ways how an antibody can modulate achemokine receptor. For example, an antibody can

a) block G-protein independent signaling,

b) block G-protein dependent signaling,

c) block G-protein dependent and G-protein independentsignaling,

d) increase G-protein independent signaling,

e) increase G-protein dependent signaling,

f) increase G-protein dependent and G-protein independentsignaling.

Without being bound by theory, the inventors hypothesize that thesulfated TRD of the respective chemokine receptor may be relevantfor the ligand induced signaling of the chemokine receptor.Interestingly, most of the inventive antibodies efficiently blockedligand induced G-protein dependent signaling but did not influenceG protein independent signaling, cf. examples 10.4 ff. In contrast,all prior art antibodies likewise blocked ligand induced G-proteindependent signaling but agonized G-protein independent signaling.Without being bound by theory, these differences may contribute tothe differences in internalization behavior.

Avoiding induction of G protein independent signaling by atherapeutic antibody might also be advantageous, because unspecificsignaling may result in uncontrollable downstream effects such asan increased cell proliferation, cf. Hutchings, Catherine J., etal. (2017); Webb, David R., et al. "Opportunities for functionalselectivity in GPCR antibodies." Biochemical pharmacology 85.2(2013): 147-152.; Fox, James M., et al. (2006).

1.2.6 Obtaining ADCC/ADCP Inducing Chemokine ReceptorAntibodies

The antibodies according to the current invention are characterizedby a superior induction of ADCC and ADCP, as shown e.g. in example10.3.3, 10.3.4. In some preferred embodiments, the antibodies wereengineered by elimination of the fucose at N297 (afucosylation) toobtain the high ADCC rates, cf. examples 10.3.1. Interestingly,many of the inventive anti-CCR8 antibodies seemed to induce targetcell depletion via a combined mode of action using both mechanisms,i.e. ADCC and ADCP.

1.3 Methods for Treatment

While it has been suggested by several research groups to useantibodies binding CCR8 in methods of treatment based onmechanistic insights, the way to the provision of antibodies withoptimal therapeutic properties has been tedious. By providing amethod for antibody generation according to the current invention,antibodies for chemokine receptors with superior therapeuticproperties can now be easily obtained. According to the currentinvention, various sequence defined antibodies with favorableproperties are disclosed, which can be used in a method oftreatment, e.g. as part of a conjugate, in an ADCC based approach,in an ADCP based approach, in a CDC based approach, or in a mixedADCC/ADCP based approach. Examples 12 ff. show remarkableefficacies in Treg depletion, Overall Response Rate andTumor-to-Control ratio for surrogate antibodies generated with amethod according to the current invention.

1.4 Combination Treatment

While remarkable response rates were obtained in monotherapy withthe inventive anti-CCR8 antibodies, it was surprisingly found thatthe therapeutic benefit could be further increased by combining theantibodies with immune checkpoint inhibitors, with further targetedtherapies, and even with unspecific chemotherapeutics or radiationtherapy. These combination treatments were particularly beneficialin challenging tumor models. In particular, a specific combinationtreatment scheme was found effective, where the second therapeuticagent or therapy was administered only after the anti-CCR8 antibodyhad caused substantial Treg depletion (see examples 12.6 ff.).Furthermore, it was observed that even a single anti-CCR8 antibodytreatment showed a substantial therapeutic effect (see example12.4.2).

1.5 Stratification Schemes and Diagnostic Methods

When evaluating the responsiveness of different syngeneic mousemodels to anti-CCR8 antibody treatment, the inventors couldidentify several mechanisms, biomarkers and biomarker combinationsthat were predictive for treatment efficacy and overall response.For example, it was surprisingly found that the degree ofresponsiveness to immune checkpoint inhibitors such as PD-1, PD-L1or CTLA4 antibodies was also predictive for the response toanti-CCR8 antibody treatment. Because PD-L1 expression is sometimesused as a surrogate marker to predict the responsiveness for immunecheckpoint inhibitors, the inventors evaluated if PD-L1 could alsobe used to predict the responsiveness for anti-CCR8 antibodytreatment. This hypothesis could finally be confirmed bycorrelation data, see example 12.7.1. In a further attempt toidentify those subjects that would most likely benefit fromanti-CCR8 treatment, further marker genes, such as immune cell andTreg markers were evaluated as biomarkers (see examples 12.1.2,12.7.2, 12.8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of human CC chemokine receptors and CXC chemokinereceptors. The following sequences are shown, ordered from top tobottom: hCCR1 (SEQ ID NO:109), hCCR2 (SEQ ID NO:112), hCCR3 (SEQ IDNO:115), hCCR4 (SEQ ID NO:118), hCCR5 (SEQ ID NO:121), hCCR6 (SEQID NO:124), hCCR7 (SEQ ID NO:127), hCCR8 (SEQ ID NO:130), hCCR9(SEQ ID NO:133), hCCR10 (SEQ ID NO: 136), hCXCR1 (SEQ ID NO:139),hCXCR2 (SEQ ID NO:142), hCXCR3 (SEQ ID NO:145), hCXCR4 (SEQ IDNO:148), hCXCR5 (SEQ ID NO:151), and hCXCR6 (SEQ ID NO:154).

FIG. 2A: Human CCR8 (SEQ ID NO:130), cynomolgus CCR8 (SEQ IDNO:131), and murine CCR8 (SEQ ID NO:132) sequences were retrievedfrom Uniprot and aligned with Clustal Omega.

FIG. 2B: Schematic representation of CCR8 receptor structure whichcontains 7 transmembrane domains (black boxes), three extracellularloops (ECL), several disulfide bridges (SS), and an intracellularC-terminal part as well as the extracellular N-terminal domaincomposed of the "LID" domain and the tyrosine rich domain(TRD).

FIG. 3: Evaluation of three prior art antibodies by FACS stainingin CCR8 positive target cells. None of the shown prior artantibodies showed a shift in FACS staining compared with theisotype control. hAP-0068 and MAB1429-100 were tested on 293T cellsstably transfected with human CCR8 while MAB8324 was tested onBW5147.3 cells expressing murine CCR8.

FIG. 4: Panning strategy for lead finding: Two major strategies forselections on sulfated peptides are depicted. In all strategies,prior to each round of selection a depletion step on a relevant oran irrelevant biotinylated protein was included.

FIG. 5: Panning strategy to obtain anti-murine CCR8 antibodies: Themain strategy for selection based on a mix of sulfated/non-sulfatedpeptides is depicted. Prior to each round of selection, a depletionstep on a relevant biotinylated protein was performed.

FIG. 6: FACS data of inventive antibodies binding to CHO cellsexpressing human CCR8.

FIG. 7: FACS data of inventive antibodies binding to CHO cellsexpressing cynomolgus CCR8.

FIG. 8: FACS data of candidates binding to activated human Tregs(donor 1). Right side: Percent of CCR8 expression determined usingBioLegend Clone L263G8.

FIG. 9: FACS data of inventive antibodies TPP-21181 and TPP-23411binding to CHO cells expressing human CCR8.

FIG. 10: FACS data of inventive antibodies TPP-21181 and TPP-23411binding to CHO cells expressing cynomolgus CCR8.

FIG. 11: Binding of TPP-21360, L263G8, and 433H to CHO cellsexpressing human CCR8.

FIG. 12: Binding of TPP-21360, L263G8, and 433H to CHO cellsexpressing cynomolgus CCR8. Prior art antibodies showed only verylow overall binding to cynomolgus CCR8 (low saturation).Nevertheless, EC50 values could be determined for eachantibody.

FIG. 13: Activated human Treg staining with inventive antibodyTPP-23411, gating strategy. CD4+ and CD25+ sorted cells fromperipheral blood mononuclear cells (PBMC) were activated withanti-CD3 and anti-CD28 beads (Treg expansion kit, Miltenyi). Middlepane: Cells that are CD4+CD25+CD127low and Foxp3+. Lower panel:CCR8 expression on Tregs after staining with TPP-23411 orL263G8.

FIG. 14: Binding of TPP-21360 to CHO cells with mock transfectants.No unspecific binding was observed.

FIG. 15: Off target binding in HEK cells transiently transfectedwith human CCR1. Most inventive candidates showed only low offtarget binding for CCR1.

FIG. 16: Off target binding in HEK cells transiently transfectedwith human CCR4. Most inventive candidates showed only low offtarget binding for CCR4.

FIG. 17: Staining for CCR8 expression on different immune cellpopulations from healthy donor PBMCs (CD4+ T cells, CD8+ T cells, Bcells as marked by CD19+ expression, Myeloid cells as marked byCD11b+ expression, CD4 T cells activated with anti-CD3 andanti-CD28 beads for 4 days, CD8+ T cells activated with anti-CD3and anti-CD28 beads for 4 days).

FIG. 18: CCR8 is upregulated on human Tregs after activation. Afteractivation, the percentage of CCR8+ cells increased significantlyin CD4+CD25+ purified cells with increased MFI while % of CCR8+cells increased slightly in PBMC population. Anti-CCR8 antibodyfrom BioLegend was used for these experiments. N=4 donors.

FIG. 19: ADCC induced by wild type or afucosylated inventiveantibodies TPP-19546, TPP-21360 or TPP-23411 in HEK cellsexpressing human CCR8 as target cells. Isotype control:TPP-9808.

FIG. 20: Left: Percent CCR8+ expression on day 3 for activatedhuman Tregs as target cells. Right: ADCC induced by wild type orafucosylated inventive antibody TPP-21360, TPP-23411 in activatedhuman Tregs as target cells. Isotype control: TPP-9808. Resultswere reproducible in different donors (data not shown).

FIG. 21: Protocol for ADCP assay.

FIG. 22: ADCP assay for wild type and afucosylated versions ofinventive antibodies shows induction of phagocytosis in HEK cellsexpressing human CCR8 as target cells with in vitro differentiatedM2c macrophages as effector cells. Wild type and afucosylatedversions of inventive antibodies TPP-19546, TPP-21360 and TPP-23411induced ADCP. TPP-9808 is the isotype control. Mogamulizumab is amarketed anti-CCR4 antibody.

FIG. 23: ADCP assay for wild type and afucosylated versions ofinventive antibodies TPP-21360, and TPP-23411 shows induction ofphagocytosis in activated human Tregs from two different donors astarget cells (upper or lower panel) and M2c macrophages as effectorcells. TPP-9808 is the isotype control.

FIG. 24: ADCP assay for wild type and afucosylated versions ofinventive antibodies TPP-21360, and TPP-23411 shows induction ofphagocytosis in activated human Tregs as target cells and M1macrophages as effector cells.

FIG. 25: Activation of .beta.-arrestin signaling measured with theDiscoverX assay for CCL1, TPP-23411, L263G8, or 433H on CHO cellsco-expressing human CCR8 tagged with ProLink and EA. As expected,CCR8 ligand CCL1 (human) induces .beta.-arrestin signaling. Anextra timepoint was analyzed for TPP-23411 to ensure that no.beta.-arrestin activation happened at earlier time points. None ofthe analyzed antibodies induced .beta.-arrestin signalingactivation.

FIG. 26: Evaluation of blocking of CCL1 induced .beta.-arrestinsignaling by TP-23411, L263G8, or 433H, measured with the DiscoverXassay. CCL1 was used at its EC80 (20 ng/ml). Prior art antibodiesL263G8, and 433H both blocked .beta.-arrestin signaling induced byCCL1 at low antibody concentration, while inventive antibodyTPP-23411 showed no effect at these concentrations. .beta.-arrestinsignaling has been suggested to play a role in internalization.

FIG. 27: Phospho Erk 1/2 ELISA assay. CHO cells expressing humanCCR8 were treated with CCL1, TPP-23411, Biolegend L263G8 or BDantibody 433H and cell lysates were collected at the respectivetime point.

FIG. 28: Phospho Erk 1/2 ELISA assay. Activated human Treg cellsexpressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8or BD antibody 433H, and cell lysates were collected at therespective time point. Prior art antibodies Biolegend L263G8 and BDantibody 433H both induced a significant increase of phosphorylatedErk1/2 levels, e.g. after 15 minutes in activated human Tregs,while this was not the case for inventive antibody.

FIG. 29: Phospho AKT ELISA assay. CHO cells expressing human CCR8were treated with CCL1, TPP-23411, Biolegend L263G8 or BD antibody433H and cell lysates were collected at the respective timepoint.

FIG. 30: Phospho AKT ELISA assay. Activated human Treg cellsexpressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8or BD antibody 433H, and cell lysates were collected at therespective time point. Prior art antibodies Biolegend L263G8 and BD433H both induced a significant increase of phospho AKT levels inactivated human Tregs, e.g. after 15 minutes, while this was notthe case for inventive antibodies.

FIG. 31A: Internalization studies. FACS analysis of commercialanti-human CCR8 antibodies Biolegend L263G8 or BD antibody 433Hwith endogenously CCR8-expressing cell line HuT78.

FIG. 31B: Internalization studies of commercial anti-human CCR8antibodies with endogenously CCR8-expressing cell line HuT78 basedon cytoplasmic-intensity spots. TPP-5657: Isotype control.

FIG. 32A: Internalization studies. FACS analysis of commercialanti-murine CCR8 antibody SA214G2 with murine endogenouslyCCR8-expressing cell line BW5147.3.

FIG. 32B: Internalization study of commercial anti-murine CCR8antibody SA214G2 with murine endogenously CCR8-expressing cell lineBW5147.3. The anti-murine CCR8 prior art antibody SA214G2 elicitsinternalization.

FIG. 33A: Internalization study of inventive anti-human CCR8antibodies TPP-21360 and TPP-23411 with endogenouslyCCR8-expressing cell line TALL-1. Both antibodies reveal identicalinternalization behavior, comparable with the isotype controlTPP-5657.

FIG. 33B: Internalization study of inventive anti-human CCR8antibodies TPP-21360 and TPP-23411 with endogenouslyCCR8-expressing cell line HuT78. Both antibodies reveal identicalinternalization behavior, comparable with the isotype controlTPP-5657.

FIG. 34A: FACS analysis of inventive anti-human CCR8 antibodiesTPP-21360 and TPP-23411 with endogenously CCR8-expressing cell lineTALL-1. Both antibodies reveal same binding potency to the cellline.

FIG. 34B: FACS analysis of inventive anti-human CCR8 antibodiesTPP-21360 and TPP-23411 with endogenously CCR8-expressing cell lineHuT78. Both antibodies reveal same binding potency to the cellline.

FIG. 35: CCR8 mRNA expression across 11642 samples representing acomprehensive set of human tissues and cell types as measured byaffymetrix probe 208059_at. All samples were co-normalized usingthe refRMA method (Katz, Simon, et al. "A summarization approachfor Affymetrix GeneChip data using a reference training set from alarge, biologically diverse database." BMC bioinformatics 7.1(2006): 1-11.). Dark grey box coloring indicates that the mediansample in the corresponding group has a probe signal intensitysignificantly above background noise (as estimated by affymetrix'MASS algorithm, cf. Pepper, Stuart D., et al. "The utility of MASSexpression summary and detection call algorithms." BMCbioinformatics 8.1 (2007): 1-12.). Median CCR8 expressionsignificantly above background noise is only observed in activatedregulatory T cells as well as tumor infiltrating lymphocytes.Groups are sorted based on decreasing mean expression.

FIG. 36: CCR8 mRNA expression, as measured by RNA-seq, across 50different TCGA (www.cancer.gov/tcga) tumor indications (dark greyboxes), corresponding normal tissues (white boxes), as well asacross the CCLE tumor cell line panel (Barretina, Jordi, et al."The Cancer Cell Line Encyclopedia enables predictive modelling ofanticancer drug sensitivity." Nature 483.7391 (2012): 603-607.)also grouped by tumor indications (light grey boxes). Groups aresorted based on mean expression. Indications with highest CCR8expression are breast cancer, lung adenocarcinoma (ADC) andsquamous cell carcinoma (SCC), head and neck malignancies, as wellas esophageal tumors. In all indications except pancreaticadenocarcinoma and melanoma, expression appears higher in the tumorcompared to the corresponding normal tissue. Little to noexpression is observed in corresponding tumor cell lines ofepithelial origin indicating that CCR8 is not expressed by tumorcells but exclusively by tumor infiltrating T cells. In principle,each tumor indication with CCR8+ Treg cell infiltration is supposedto be eligible for anti-CCR8 antibody treatment.

FIG. 37: Immunohistochemistry with staining of Treg markers FOXP3and CCR8 (clone 433H) in human Non-small-cell lung cancer tissue(NSCLC) or human melanoma tissue.

FIG. 38: Staining of FACS sorted T cell populations based on CCR4,CCR8, OX40, GITR, and CD25. Only CCR8 is specific for activatedTregs. OX40, GITR, and CD25 are significantly expressed onstimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). Darkgrey: isotype control, light grey: target staining.

FIG. 39: Expression of CCR8 across different T cell subsetsextracted from different tumor entities as measured by single cellRNA-seq. For these tumors, CCR8 mRNA expression is largelyrestricted to regulatory T cells (light grey boxes) residing intumor tissue while largely being absent in regulatory T cells fromnormal tissues as well as from CD4 helper T and CD8 cytotoxic Tcells (medium and dark gray boxes, respectively). Upper panel:Colorectal tumor tissue (Tumor) or adjacent normal tissue (Normal).Middle panel: Liver cancer tissue (Tumor) or adjacent normal tissue(Normal). Lower panel: Lung cancer tissue (Tumor) or adjacentnormal lung tissue and peripheral blood (Normal). Sample sizes, N,indicate the number of cells in each category. Cells weredesignated as regulatory T cells, T helper cells, or cytotoxic Tcells based on expression of marker genes FOXP3, CD4 (but noFOXP3), and CD8A/B, respectively.

FIG. 40: Treg, macrophage, T cell or tumor cell populations fromNSCLC, CRC (colorectal cancer) or RCC (renal carcinoma) werestained with anti-CCR8 antibody or isotype control and wereanalyzed by flow cytometry. X axis: Log shift up to 10.sup.5; Yaxis: Intensity normalized to mode. Lighter grey: isotype control.Darker grey: CCR8. Specific CCR8 expression on intra-tumoral humanTregs is indicated by arrows.

FIG. 41: CT26 tumor growth after treatment with different doses ofanti-CCR8 antibodies or anti-PDL1 antibody.

FIG. 42: Spider plots of CT26 tumor bearing mice after treatmentwith different doses of anti-CCR8 antibodies, anti-PDL1 antibody orisotype controls.

FIG. 43: FACS analysis of intra-tumoral immune cells 24 hours aftersecond antibody treatment. Analysis of intra-tumoral Tregs.Anti-CCR8 antibody TPP15285 leads to an average decrease ofintra-tumoral Tregs to 13%, 33%, or 47% of the Tregs in the isotypecontrol, for 10 mg/kg, 1 mg/kg or 0.1 mg/kg, respectively.Anti-CCR8 antibody TPP15286 leads to an average decrease ofintra-tumoral Tregs to 10%, 9% or 14% of the Tregs in the isotypecontrol, for 10 mg/kg, 1 mg/kg or 0.1 mg/kg, respectively, cf.Table 12.2.1. Analysis of intra-tumoral CD8+ T cells. Mean increasein % of isotype control is shown in Table 12.2.1. Analysis of ratioof CD8+ T cells to Treg cells. Anti-CCR8 antibody TPP15286 leads toan average increase of the CD8+ cell to Treg cell ratio of 44 (10mg/kg), 87 (1 mg/kg) or 68 (0.1 mg/kg). Anti-CCR8 antibody TPP15285leads to an average increase of the CD8+ cell to Treg cell ratio of64 (10 mg/kg), 16 (1 mg/kg) or 10 (0.1 mg/kg). Analysis of ratio ofCD4+ conv T cells to Treg cells.

FIG. 44: CT26 tumor growth after treatment with glycosylated(TPP-14095, TPP-14099, TPP-15285, TPP-15286) or aglycosylated(TPP-18208, TPP-18209) anti-CCR8 antibodies. Aglycosylation largelyabolished the anti-tumor effect.

FIG. 45: Spider plots of CT26 tumor bearing mice after treatmentwith glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) oraglycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies.Aglycosylation largely abolished the anti-tumor effect, suggestingan ADCC/ADCP dependent mechanism of anti-tumor efficacy.

FIG. 46: FACS analysis of immune cells 24 hours after secondantibody treatment. Absolute numbers of intra-tumoral CD45+ cells.Absolute numbers of intra-tumoral CD45+CD8+ T cells. Absolutenumbers of intra-tumoral CD8+ T cells. Absolute numbers ofintra-tumoral CD4+ conv cells. Analysis of intra-tumoral Tregdepletion by flow cytometry showing absolute cell numbers. Averagepercentage of residual Tregs relative to the respective isotypecontrol after antibody administration was 15.6% for TPP-14095,28.2% for TPP-14099, 8.7% for TPP-15285 and 36.5% for TPP-15286.For the aglycosylated antibodies no reduction in Tregs wasobserved. % CD8+ T cells of intra-tumoral CD45+ cells. CD8+ Tcells:Treg ratio. CD4+ conv:Treg ratio. Percentage Treg cells ofCD4+ T cells. Absolute numbers of intra-tumoral 4-1BB+ Treg cells.Absolute numbers of intra-tumoral 4-1BB+CD4+ conv cells.

FIG. 47: EMT6 tumor growth after treatment with different doses ofanti-CCR8 antibody TPP-15285. Significance was determined by 1-WayANOVA plus Sidak's post-test after log transform.

FIG. 48: EMT6 tumor growth at day 19 after treatment with differentdoses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody.

FIG. 49: Spider plots of EMT6-tumor bearing mice after treatmentwith different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4antibody.

FIG. 50: FACS analysis of immune cells of EMT6-tumor bearing miceafter treatment with different doses of CCR8-antibody TPP-15285 oranti-CTLA4 antibody, 24 hours after second treatment or at the endof the study. Absolute numbers of intra-tumoral Treg cells.Significant differences were observed e.g. for 10 mg/kg ofanti-CCR8 antibody at both timepoints. CD8 positive cells to Tregratio. CD4+ conv cells to Treg ratio. Percentage Treg cells of CD4+T cells.

FIG. 51: FACS analysis of immune cells of EMT6-tumor bearing miceafter treatment with different doses of anti-CCR8 antibodyTPP-15285 or anti-CTLA4 antibody, 24 hours after second treatmentor at the end of the study. Absolute numbers of intra-tumoral CD45+cells. Absolute numbers of intra-tumoral CD4+ conv cells. Absolutenumbers of intra-tumoral CD4+ T cells. Absolute numbers ofintra-tumoral activated CD8+ T cells. Absolute numbers ofintra-tumoral NK cells. Absolute numbers of intra-tumoral CD8+ Tcells. Differences were observed e.g. for 10 mg/kg of anti-CCR8antibody at the end of the study.

FIG. 52: F9 tumor growth after treatment with different doses ofanti-CCR8 antibody TPP-15285 or anti-PDL1 antibody.

FIG. 53: F9 tumor volume at day 16 after treatment start withdifferent doses of anti-CCR8 antibody TPP-15285 or anti-PDL1antibody. Significant improvement was observed at least for anantibody dose of 10 mg/kg.

FIG. 54: Spider plots of F9 tumor bearing mice after treatment withdifferent doses of anti-CCR8 antibodies or anti-PDL1 antibody.

FIG. 55: Effects of anti-CCR8 antibodies or anti-PDL1 antibody onimmune cell populations and their ratios in F9 tumors analyzed byFACS 24 hours after second antibody treatment. Anti-CCR8 antibodyincreased absolute numbers of intra-tumoral CD45+ T cells,intra-tumoral CD4+ T cells, intra-tumoral CD8+ T cells,intra-tumoral CD4+ conv T cells and intra-tumoral NK cells comparedwith the matched isotype control. Anti-CCR8 antibody at 10 mg/kgdecreased absolute numbers of intra-tumoral Treg cells (CD4+,CD25+, FoxP3+).

FIG. 56: Effects of anti-CCR8 antibodies or anti-PDL1 antibody onimmune cell populations and their ratios in F9 tumors analyzed byFACS 24 hours after second treatment plotted as CD8+ T cell to Tregratio, frequency of Treg to CD4+ T cells, frequency of 4-1BB+ cellsof CD8+ T cells, frequency of 4-1BB+ cells of Tregs and frequencyof 4-1BB+ cells of conventional CD4+ T cells. At 10 mg/kg anti-CCR8antibody increased the CD8+ cell to Treg cell ratio toapproximately 54 or higher, cf. Table 12.5.1.

FIG. 57: Efficacy of combination treatments in C38 tumor bearingmice. a. C38 tumor growth after treatment with anti-CCR8 surrogateantibodies TPP-14099 or TPP-15285 or anti-PD-L1 antibody or aftercombination treatment with 3 mg/kg anti-PDL1 antibody, 10 mg/kgTPP-15285. Significance as determined by 1-Way ANOVA plus Sidak'spost-test after log transform. b. Survival plot of C38-tumorbearing mice after treatment with anti-murine CCR8 antibodyTPP-15285 (10 mg/kg), anti-murine PD-L1 antibody (PDL1, 3 mg/kg) ora combination of TPP-15285 (10 mg/kg) and anti-murine PD-1 antibody(3 mg/kg).

FIG. 58: Analysis of tumoral Treg depletion in C38 tumors by flowcytometry (sampling 24 hours after second antibody treatment). C38tumor bearing mice were treated with anti-CCR8 surrogate antibodiesTPP-14099 or TPP-15285 or anti-PD-L1 antibody in monotherapy, orwith TPP-15285 in combination with anti-PD-L1 antibody. AbsoluteTreg depletion. CD8+ T cell/Treg cell ratios.

FIG. 59: CD11+F4/80+ macrophages in C38 tumors as analyzed by flowcytometry (sampling at study end).

FIG. 60: B16F10-OVA tumor growth after treatment with TPP-15285,anti-CTLA4 antibody or both.

FIG. 61: EMT-6 tumor growth after treatment with anti-murine CCR8antibody TPP-15285 (1 mg/kg), anti-murine PD-1 antibody (CDRs:atezolizumab, 10 mg/kg) or a combination of TPP-15285 (1 mg/kg) andanti-murine PD-1 antibody (10 mg/kg), (twice weekly i.p.). Meanwith standard deviation.

FIG. 62: FACS analysis of immune cell populations in EMT-6-tumorsanalyzed by flow cytometry, sampling 24 hours after second antibodytreatment. CD4+ T cells, Tregs, CD8+ T cells, NK cells, Ratios ofCD8+ T cells to Tregs. Box and whiskers, min to max with median(GraphPad).

FIG. 63: a. Analysis of C38 tumor bearing mice after treatment withanti-murine CCR8 antibody TPP-15285 (5 mg/kg), anti-murine PD-1antibody (CDRs: atezolizumab, 5 mg/kg) or a combination ofTPP-15285 (5 mg/kg) and anti-murine PD-1 antibody (5 mg/kg), (eachtwice weekly i.p.). a. Tumor volume. Mean with standard deviation.b. Survival plot.

FIG. 64: Analysis of tumoral CD4+ T cells, Tregs, CD8+ T cells, NKcells or ratios of CD8+ T cells to Tregs in C38-tumors by flowcytometry (sampling 24 hours after second antibody treatment).

FIG. 65: MB49 tumor growth after treatment with anti-CCR8 antibodyTPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs:atezolizumab, 10 mg/kg) alone or sequential combination treatmentof TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) (bothtwice weekly i.p.).

FIG. 66: Analysis of tumoral CD45+ cells, Tregs, CD8+ T cells, NKcells and ratio of CD8 to Tregs in MB49-tumors by flow cytometry(sampling at study end).

FIG. 67: EMT-6 tumor growth after treatment with anti-CCR8 antibodyTPP-15285 (5 mg/kg, q3/4d i.p.), Oxaliplatin (5 mg/kg, q4d i.p.),Doxorubicin (6 mg/kg, i.v. SD), Docetaxel (10 mg/kg, q2dx5, i.v.)or a combination of TPP-15285 with either Oxaliplatin, Doxorubicinor Docetaxel.

FIG. 68: MB49 tumor growth after treatment with anti-CCR8 antibodyTPP-15285, gemcitabine or a combination of TPP-15285 withgemcitabine.

FIG. 69: Lewis lung tumor growth after treatment with anti-CCR8antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1,CDRs: atezolizumab, 10 mg/kg) alone, anti-PD-L1 antibody (10 mg/kg)alone, anti-CTLA4 antibody alone, TPP-15285 (10 mg/kg) andanti-murine PD-1 antibody (10 mg/kg) in combination, or TPP-15285(10 mg/kg) and anti-PD-L1 antibody (10 mg/kg) in combination.

FIG. 70: EMT-6 tumor growth after treatment with anti-CCR8 antibodyTPP-15285 (3 mg/kg) or radiotherapy (RT, 3.times.2 Gy) alone or incombination. Significance was determined by 1-Way ANOVA plusSidak's post-test after log transform.

FIG. 71: mRNA expression levels of different immune cell markers indifferent syngeneic tumor models treated with either isotypecontrol (TPP-9809) or anti-CCR8 antibody (TPP-14099). Medianexpression in the isotype treated control groups is set to zero.Gene expression is measured in transcripts per million (TPM) asestimated by the RSEM algorithm. Treg marker Foxp3. Significantlyhigher Foxp3 levels are observed in the CT26, H22, Hepa1-6, and RM1models treated with TPP-14099, indicating increased Treginfiltration, after administration of at least three doses ofTPP-14099.

FIG. 72: Inflammation marker 1 fng. Significantly higher 1 fnglevels are observed in the H22, CT26, and RM1 models treated withTPP-14099 pointing towards a strong pro inflammatory activityinduced by TPP-14099.

FIG. 73: Macrophage marker Ms4a7. Significantly higher Ms4a7 levelsare observed in the CT26, H22, and Hepa1-6 models treated withTPP-14099, indicating increased infiltration of macrophages causedby TPP-14099.

FIG. 74: Cytotoxic T cell markers Cd8a and Cd8b1. Significantlyhigher cytotoxic T cell levels are observed in the CT26, H22, RM1,and Hepa1-6 models treated with TPP-14099 indicating the inductionof cytotoxic T cell infiltration and/or proliferation byTPP-14099.

FIG. 75: Natural killer (NK) cell marker Ncr1. Significantly higherNK cell levels are observed in the CT26, H22, and Hepa1-6 modelstreated with TPP-14099.

FIG. 76: Pan T cell markers Cd3e/d/g. Significantly higher T celllevels are observed in the CT26, H22, and Hepa1-6 models treatedwith TPP-14099.

FIG. 77: Activated Treg marker and antibody target Ccr8.Significantly higher Ccr8 levels are observed in the CT26, H22, andHepa1-6 models treated with TPP-14099.

FIG. 78: Highly specific pro-inflammatory M1 macrophage markerAcod1 (a) and highly specific anti-inflammatory M2 macrophagemarker Mrc1 (b). Markedly higher M1 macrophage levels are observedin the CT26, 4T1, H22, Hepa1-6, and RM1 models treated withTPP-14099, while no markedly higher M2 macrophage levels areobserved across all models treated with TPP-14099.

FIG. 79: Ratio of highly specific pro-inflammatory M1 macrophagemarker Acod1 and highly specific anti-inflammatory M2 macrophagemarker Mrc1. Taken together, multiple doses of anti-CCR8 antibodyTPP-14099 increased the M1/M2 macrophage ratio in these tumormodels.

FIG. 80: B cell markers Cd19 and Cd22. Markedly higher B celllevels are observed in the CT26, H22, RM1, and Hepa1-6 modelstreated with TPP-14099. Anti-CCR8 antibody seems to recruit B cellsto the tumor. Without being bound by theory, recruitment of B cellsmay affect the anti-tumor response elicited by TPP-14099.

FIG. 81: Expression levels for LTta/b as well as Cxcr5 and itsligand Cxcl13. These markers are crucial for the development oflymph nodes as well as tertiary lymphoid structures (cf. Cyster,Jason G. "Blown away: the unexpected role of lymphotoxin inlymphoid organ development." The Journal of Immunology 192.5(2014): 2007-2009, and Cupedo, Tom, et al. "Induction of secondaryand tertiary lymphoid structures in the skin." Immunity 21.5(2004): 655-667.) which are key drivers of an anti-tumor effect inhumans (cf. Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoidstructures, drivers of the anti-tumor responses in human cancers."Immunological reviews 271.1 (2016): 260-275.). All threepublications are incorporated herein by reference in theirentirety.

FIG. 82: Spider plots of tumor growth (measured in mm.sup.3) overtime in the TPP-14099 treatment group (shown as triangles) andisotype control treated group (shown as circles) for differenttumor models. Levels of T cell infiltration at the end of thestudy, as judged by Cd8a mRNA levels in corresponding bulk tumorsamples, are indicated by grey shades (black and light greycorrespond to highest and lowest Cd8a levels, respectively). In H22and CT26, tumor size showed an excellent anti-correlation with Cd8alevels.

FIG. 83: Correlation between infiltration of the tumor by differentimmune cell populations as judged by mRNA levels of differentimmune cell markers and tumor size. TPP-14099 treated tumors areshown as triangles and isotype treated tumors are shown as circles.Tumor size is measured in mm.sup.3. Strong negative correlationbetween cytotoxic T cell infiltration (as judged by Cd8a+Cd8b1 mRNAlevels) and CT26 tumor sizes. TPP-14099 treated tumors are smallerin size and show higher Cd8a+Cd8b1 T cell infiltration levels thancontrols, indicating that increased T cell infiltration uponTPP-14099 treatment causes reduced tumor growth. Strong negativecorrelation between T cell infiltration (as measured by Cd3 mRNAlevels) and CT26 tumor sizes in the TPP-14099 treated tumors.TPP-14099 treated tumors are smaller in size and show higher T cellinfiltration levels, i.e. the higher the level of Cd3 mRNA thesmaller the tumors. Negative correlation between NK cellinfiltration (as judged by NK marker Ncr1 expression) and CT26tumor sizes. TPP-14099 treated tumors are smaller in size and showhigher NK cell infiltration. Tumor size is strongly negativelycorrelated with NK infiltration, the higher the level of NK, thesmaller the tumors. Correlation between postulated induction oftertiary lymphoid structures as judged by Lta/Ltb/Cxcr5 and Cxcl13mRNA levels and CT26 tumor sizes in the TPP-14099 treated tumors.For each of the four markers, the level is strongly increased afterTPP-14099 treatment, and tumor size is strongly negativelycorrelated with the expression level, indicating that increasedformation of tertiary lymphoid structures upon TPP-14099 treatmentcauses reduced tumor growth.

FIG. 84: Correlation between Cd8a infiltration levels, as judged byCd8a mRNA expression, and tumor sizes in the TPP-14099 treatedtumors (shown as triangles) and in the isotype treated controlgroup (shown as circles) for different tumor models. TPP-14099treated tumors are smaller in size and show higher Cd8ainfiltration levels. Tumor size is strongly negatively correlatedwith Cd8a infiltration, the higher the level of Cd8a, the smallerthe tumors.

FIG. 85: Efficacy of CCR8-antibody TPP-15285 in EMT6-tumor bearingmice treated with four different treatment schemes: singletreatment, two times BIW treatment, three times BIW treatment, orfour times BIW treatment. Each group was treated with vehiclecontrol or 0.1 mg/kg, 1 mg/kg, or 5 mg/kg of TPP-15285.

FIG. 86: Therapeutic efficacies of inventive anti-CCR8 antibody,anti-PD-1 antibody, anti-PD-L1 antibody, or Paclitaxel tested aloneor in combination in MBT2 syngeneic tumor bearing mice as shown inTable 12.6.9.1.

FIG. 87: Therapeutic efficacy of inventive anti-CCR8 antibody afterquantitative depletion of CD8a+ expression in diphtheria toxintreated DTR mice. Depleting Cd8+ T cells abrogates anti-tumorgrowth effect of TPP15825 (cf. group 02b vs group 01b).

FIG. 88: Therapeutic efficacy of inventive anti-CCR8 antibody afterquantitative depletion of CD19+ expression in diphtheria toxintreated DTR mice. Depleting Cd19+ B cells enhances the anti-tumorgrowth effect of TPP15825 (cf. group 02b vs group 01b).

FIG. 89: Internalization curves for various inventive anti-CCR8antibodies in HUVEC cells. An anti-CD71 antibody with knowninternalization profile was used as positive control. Correspondingdata are shown in Table 10.5.2. None of the antibodies obtainedwith a method according to the current invention showed substantialinternalization, making them particularly useful for ADCC/ADCPbased approaches.

BRIEF DESCRIPTION OF THE SEQUENCE IDS

The sequence listing provided with the application via electronicfiling is included herein in its entirety. SEQ ID NO:1 to SEQ IDNO:108 and SEQ ID NO:157 to SEQ ID NO:168 relate to isolatedpolypeptides which can be used as antigens or for off targetpanning. SEQ ID NO:109 to SEQ ID NO:156 relate to chemokinereceptor proteins from different species. SEQ ID NO:201 to SEQ IDNO:965 relate to inventive antibodies. The column on sulfations isprovided for convenience only and is not intended to restrict therespective sequence in any way.

TABLE-US-00001 Sequence Sequence Name Region Type SEQ ID SulfatedP32246_CCR1_HUMAN_TRD ANTIGEN PRT SEQ ID NO:1 Y10, Y18Q2Y2P0_CCR1_MACFA_TRD ANTIGEN PRT SEQ ID NO:2 Y10, Y18P51675_CCR1_MOUSE_TRD ANTIGEN PRT SEQ ID NO:3 Y10, Y18P32246_CCR1_HUMAN_N term ANTIGEN PRT SEQ ID NO:4 Y10, Y18Q2Y2P0_CCR1_MACFA_N term ANTIGEN PRT SEQ ID NO:5 Y10, Y18P51675_CCR1_MOUSE_N term ANTIGEN PRT SEQ ID NO:6 Y10, Y18P41597_CCR2_HUMAN_TRD ANTIGEN PRT SEQ ID NO:7 Y26, Y28O18793_CCR2_MACMU_TRD ANTIGEN PRT SEQ ID NO:8 Y26, Y28P51683_CCR2_MOUSE_TRD ANTIGEN PRT SEQ ID NO:9 Y37, Y39P41597_CCR2_HUMAN_N term ANTIGEN PRT SEQ ID NO:10 Y26, Y28O18793_CCR2_MACMU_N term ANTIGEN PRT SEQ ID NO:11 Y26, Y28P51683_CCR2_MOUSE_N term ANTIGEN PRT SEQ ID NO:12 Y37, Y39P51677_CCR3_HUMAN_TRD ANTIGEN PRT SEQ ID NO:13 Y16, Y17Q9BDS8_CCR3_MACFA_TRD ANTIGEN PRT SEQ ID NO:14 Y16P51678_CCR3_MOUSE_TRD ANTIGEN PRT SEQ ID NO:15 Y20, Y22P51677_CCR3_HUMAN_N term ANTIGEN PRT SEQ ID NO:16 Y16, Y17Q9BDS8_CCR3_MACFA_N term ANTIGEN PRT SEQ ID NO:17 Y16P51678_CCR3_MOUSE_N term ANTIGEN PRT SEQ ID NO:18 Y20, Y22P51679_CCR4_HUMAN_TRD ANTIGEN PRT SEQ ID NO:19 (Y16, Y19, Y20), Y22G7NYB7_CCR4_MACFA_TRD ANTIGEN PRT SEQ ID NO:20 (Y16, Y19, Y20), Y22P51680_CCR4_MOUSE_TRD ANTIGEN PRT SEQ ID NO:21 (Y16, Y19, Y20), Y22P51679_CCR4_HUMAN_N term ANTIGEN PRT SEQ ID NO:22 (Y16, Y19, Y20),Y22 G7NYB7_CCR4_MACFA_N term ANTIGEN PRT SEQ ID NO:23 (Y16, Y19,Y20), Y22 P51680_CCR4_MOUSE_N term ANTIGEN PRT SEQ ID NO:24 (Y16,Y19, Y20), Y22 P51681_CCR5_HUMAN_TRD ANTIGEN PRT SEQ ID NO:25 Y3,Y10, Y14, Y15 P61814_CCR5_MACFA_TRD ANTIGEN PRT SEQ ID NO:26 Y3,Y10, Y14, Y15 P51682_CCR5_MOUSE_TRD ANTIGEN PRT SEQ ID NO:27 Y10,Y12, Y16 P51681_CCR5_HUMAN_N term ANTIGEN PRT SEQ ID NO:28 Y3, Y10,Y14, Y15 P61814_CCR5_MACFA_N term ANTIGEN PRT SEQ ID NO:29 Y3, Y10,Y14, Y15 P51682_CCR5_MOUSE_N term ANTIGEN PRT SEQ ID NO:30 Y10,Y12, Y16 P51684_CCR6_HUMAN_TRD ANTIGEN PRT SEQ ID NO:31 Y18, Y26,Y27 A0A2K5WY45_CCR6_MACFA_TRD ANTIGEN PRT SEQ ID NO:32 Y23, Y31,Y32 O54689_CCR6_MOUSE_TRD ANTIGEN PRT SEQ ID NO:33 (Y7), Y13, Y18,Y19 P51684_CCR6_HUMAN_N term ANTIGEN PRT SEQ ID NO:34 Y18, Y26, Y27A0A2K5WY45_CCR6_MACFA_N term ANTIGEN PRT SEQ ID NO:35 Y23, Y31, Y32O54689_CCR6_MOUSE_N term ANTIGEN PRT SEQ ID NO:36 (Y7), Y13, Y18,Y19 P32248_CCR7_HUMAN_TRD ANTIGEN PRT SEQ ID NO:37 Y8, Y17C0ISS3_CCR7_MACFA_TRD ANTIGEN PRT SEQ ID NO:38 Y8, Y17P47774_CCR7_MOUSE_TRD ANTIGEN PRT SEQ ID NO:39 Y8, Y17, Y20P32248_CCR7_HUMAN_N term ANTIGEN PRT SEQ ID NO:40 Y8, Y17 (w/oSignal Peptide) C0ISS3_CCR7_MACFA_N term ANTIGEN PRT SEQ ID NO:41Y8, Y17 (w/o Signal Peptide) P47774_CCR7_MOUSE_N term ANTIGEN PRTSEQ ID NO:42 Y8, Y17, Y20 (w/o Signal Peptide)P51685_CCR8_HUMAN_TRD ANTIGEN PRT SEQ ID NO:43 Y3, Y15, (16), Y17G7NYJ2_CCR8_MACFA_TRD ANTIGEN PRT SEQ ID NO:44 Y3, Y15, (16), Y17P56484_CCR8_MOUSE_TRD ANTIGEN PRT SEQ ID NO:45 Y3, Y14, Y15P51685_CCR8_HUMAN_N term ANTIGEN PRT SEQ ID NO:46 Y3, Y15, (16),Y17 G7NYJ2_CCR8_MACFA_N term ANTIGEN PRT SEQ ID NO:47 Y3, Y15,(16), Y17 P56484_CCR8_MOUSE_N term ANTIGEN PRT SEQ ID NO:48 Y3,Y14, Y15 P51685_CCR8_HUMAN_LID ANTIGEN PRT SEQ ID NO:49G7NYJ2_CCR8_MACFA_LID ANTIGEN PRT SEQ ID NO:50P56484_CCR8_MOUSE_LID ANTIGEN PRT SEQ ID NO:51P51685_CCR8_HUMAN_ECL1 ANTIGEN PRT SEQ ID NO:52G7NYJ2_CCR8_MACFA_ECL1 ANTIGEN PRT SEQ ID NO:53P56484_CCR8_MOUSE_ECL1 ANTIGEN PRT SEQ ID NO:54P51685_CCR8_HUMAN_ECL2 ANTIGEN PRT SEQ ID NO:55G7NYJ2_CCR8_MACFA_ECL2 ANTIGEN PRT SEQ ID NO:56P56484_CCR8_MOUSE_ECL2 ANTIGEN PRT SEQ ID NO:57P51685_CCR8_HUMAN_ECL3 ANTIGEN PRT SEQ ID NO:58G7NYJ2_CCR8_MACFA_ECL3 ANTIGEN PRT SEQ ID NO:59P56484_CCR8_MOUSE_ECL3 ANTIGEN PRT SEQ ID NO:60P51686_CCR9_HUMAN_TRD ANTIGEN PRT SEQ ID NO:61 Y17, Y28, Y37Q0H741_CCR9_MACFA_TRD ANTIGEN PRT SEQ ID NO:62 Y17, Y28, Y37Q9WUT7_CCR9_MOUSE_TRD ANTIGEN PRT SEQ ID NO:63 Y19, Y28P51686_CCR9_HUMAN_N term ANTIGEN PRT SEQ ID NO:64 Y17, Y28, Y37Q0H741_CCR9_MACFA_N term ANTIGEN PRT SEQ ID NO:65 Y17, Y28, Y37Q9WUT7_CCR9_MOUSE_N term ANTIGEN PRT SEQ ID NO:66 Y19, Y28P46092_CCR10_HUMAN_TRD ANTIGEN PRT SEQ ID NO:67 Y14, Y22A0A2K5UAP1_CCR10_MACFA_TRD ANTIGEN PRT SEQ ID NO:68 Y14, Y22Q9JL21_CCR10_MOUSE_TRD ANTIGEN PRT SEQ ID NO:69 Y14, Y17, Y22P46092_CCR10_HUMAN_N term ANTIGEN PRT SEQ ID NO:70 Y14, Y22A0A2K5UAP1_CCR10_MACFA_N term ANTIGEN PRT SEQ ID NO:71 Y14, Y22Q9JL21_CCR10_MOUSE_N term ANTIGEN PRT SEQ ID NO:72 Y14, Y17, Y22P25024_CXCR1_HUMAN_TRD ANTIGEN PRT SEQ ID NO:73 Y27A0A2K5X9E4_CXCR1_MACFA_TRD ANTIGEN PRT SEQ ID NO:74 Y14, Y28Q810W6_CXCR1_MOUSE_TRD ANTIGEN PRT SEQ ID NO:75 Y6, Y32P25024_CXCR1_HUMAN_N term ANTIGEN PRT SEQ ID NO:76 Y27A0A2K5X9E4_CXCR1_MACFA_N term ANTIGEN PRT SEQ ID NO:77 Y14, Y28Q810W6_CXCR1_MOUSE_N term ANTIGEN PRT SEQ ID NO:78 Y6, Y32P25025_CXCR2_HUMAN_TRD ANTIGEN PRT SEQ ID NO:79 Y23, Y25Q28519_CXCR2_MACMU_TRD ANTIGEN PRT SEQ ID NO:80 Y20, Y22P35343_CXCR2_MOUSE_TRD ANTIGEN PRT SEQ ID NO:81 Y24P25025_CXCR2_HUMAN_N term ANTIGEN PRT SEQ ID NO:82 Y23, Y25Q28519_CXCR2_MACMU_N term ANTIGEN PRT SEQ ID NO:83 Y20, Y22P35343_CXCR2_MOUSE_N term ANTIGEN PRT SEQ ID NO:84 Y24P49682_CXCR3_HUMAN_TRD ANTIGEN PRT SEQ ID NO:85 Y27, Y29A0A2K5UV19_CXCR3_MACFA_TRD ANTIGEN PRT SEQ ID NO:86 Y27, Y29O88410_CXCR3_MOUSE_TRD ANTIGEN PRT SEQ ID NO:87 Y2, Y27, Y29P49682_CXCR3_HUMAN_N term ANTIGEN PRT SEQ ID NO:88 Y27, Y29A0A2K5UV19_CXCR3_MACFA_N term ANTIGEN PRT SEQ ID NO:89 Y27, Y29O88410_CXCR3_MOUSE_N term ANTIGEN PRT SEQ ID NO:90 Y2, Y27, Y29P61073_CXCR4_HUMAN_TRD ANTIGEN PRT SEQ ID NO:91 (Y7), Y12, Y21Q28474_CXCR4_MACFA_TRD ANTIGEN PRT SEQ ID NO:92 (Y7), Y12, Y21P70658_CXCR4_MOUSE_TRD ANTIGEN PRT SEQ ID NO:93 Y9, Y14, Y23P61073_CXCR4_HUMAN_N term ANTIGEN PRT SEQ ID NO:94 (Y7), Y12, Y21Q28474_CXCR4_MACFA_N term ANTIGEN PRT SEQ ID NO:95 (Y7), Y12, Y21P70658 CXCR4_MOUSE_N term ANTIGEN PRT SEQ ID NO:96 Y9, Y14, Y23P32302 CXCR5_HUMAN_TRD ANTIGEN PRT SEQ ID NO:97 Y3, Y27G8F5K4_CXCR5_MACFA_TRD ANTIGEN PRT SEQ ID NO:98 Y3, Y27Q04683_CXCR5_MOUSE_TRD ANTIGEN PRT SEQ ID NO:99 Y3, Y14, Y20, Y26P32302_CXCR5_HUMAN_N term ANTIGEN PRT SEQ ID NO:100 Y3, Y27G8F5K4_CXCR5_MACFA_N term ANTIGEN PRT SEQ ID NO:101 Y3, Y27Q04683_CXCR5_MOUSE_N term ANTIGEN PRT SEQ ID NO:102 Y3, Y14, Y20,Y26 O00574_CXCR6_HUMAN_ TRD ANTIGEN PRT SEQ ID NO:103 Y6, Y10Q9BDS6_CXCR6_MACFA_TRD ANTIGEN PRT SEQ ID NO:104 Y4, Y7, Y39Q9EQ16_CXCR6_MOUSE_TRD ANTIGEN PRT SEQ ID NO:105 Y11, (Y15)O00574_CXCR6_HUMAN_N term ANTIGEN PRT SEQ ID NO:106 Y6, Y10Q9BDS6_CXCR6_MACFA_N term ANTIGEN PRT SEQ ID NO:107 Y4, Y7, Y39Q9EQ16_CXCR6_MOUSE_N term ANTIGEN PRT SEQ ID NO:108 Y11, (Y15)P32246_CCR1_HUMAN ANTIGEN PRT SEQ ID NO:109 Q2Y2P0_CCR1_MACFAANTIGEN PRT SEQ ID NO:110 P51675_CCR1_MOUSE ANTIGEN PRT SEQ IDNO:111 P41597_CCR2_HUMAN ANTIGEN PRT SEQ ID NO:112O18793_CCR2_MACMU ANTIGEN PRT SEQ ID NO:113 P51683_CCR2_MOUSEANTIGEN PRT SEQ ID NO:114 P51677_CCR3_HUMAN ANTIGEN PRT SEQ IDNO:115 Q9BDS8_CCR3_MACFA ANTIGEN PRT SEQ ID NO:116P51678_CCR3_MOUSE ANTIGEN PRT SEQ ID NO:117 P51679_CCR4_HUMANANTIGEN PRT SEQ ID NO:118 G7NYB7_CCR4_MACFA ANTIGEN PRT SEQ IDNO:119 P51680_CCR4_MOUSE ANTIGEN PRT SEQ ID NO:120P51681_CCR5_HUMAN ANTIGEN PRT SEQ ID NO:121 P61814_CCR5_MACFAANTIGEN PRT SEQ ID NO:122 P51682_CCR5_MOUSE ANTIGEN PRT SEQ IDNO:123 P51684_CCR6_HUMAN ANTIGEN PRT SEQ ID NO:124A0A2K5WY45_CCR6_MACFA ANTIGEN PRT SEQ ID NO:125 O54689_CCR6_MOUSEANTIGEN PRT SEQ ID NO:126 P32248_CCR7_HUMAN ANTIGEN PRT SEQ IDNO:127 C0ISS3_CCR7_MACFA ANTIGEN PRT SEQ ID NO:128P47774_CCR7_MOUSE ANTIGEN PRT SEQ ID NO:129 P51685_CCR8_HUMANANTIGEN PRT SEQ ID NO:130 G7NYJ2_CCR8_MACFA ANTIGEN PRT SEQ IDNO:131 P56484_CCR8_MOUSE ANTIGEN PRT SEQ ID NO:132P51686_CCR9_HUMAN ANTIGEN PRT SEQ ID NO:133 Q0H741_CCR9_MACFAANTIGEN PRT SEQ ID NO:134 Q9WUT7_CCR9_MOUSE ANTIGEN PRT SEQ IDNO:135 P46092_CCR10_HUMAN ANTIGEN PRT SEQ ID NO:136A0A2K5UAP1_CCR10_MACFA ANTIGEN PRT SEQ ID NO:137 Q9JL21_CCR10_MOUSEANTIGEN PRT SEQ ID NO:138 P25024_CXCR1_HUMAN ANTIGEN PRT SEQ IDNO:139 A0A2K5X9E4_CXCR1_MACFA ANTIGEN PRT SEQ ID NO:140Q810W6_CXCR1_MOUSE ANTIGEN PRT SEQ ID NO:141 P25025_CXCR2_HUMANANTIGEN PRT SEQ ID NO:142 Q28519_CXCR2_MACMU ANTIGEN PRT SEQ IDNO:143 P35343_CXCR2_MOUSE ANTIGEN PRT SEQ ID NO:144P49682_CXCR3_HUMAN ANTIGEN PRT SEQ ID NO:145 A0A2K5UV19_CXCR3_MACFAANTIGEN PRT SEQ ID NO:146 O88410_CXCR3_MOUSE ANTIGEN PRT SEQ IDNO:147 P61073_CXCR4_HUMAN ANTIGEN PRT SEQ ID NO:148Q28474_CXCR4_MACFA ANTIGEN PRT SEQ ID NO:149 P70658_CXCR4_MOUSEANTIGEN PRT SEQ ID NO:150 P32302_CXCR5_HUMAN ANTIGEN PRT SEQ IDNO:151 G8F5K4_CXCR5_MACFA ANTIGEN PRT SEQ ID NO:152Q04683_CXCR5_MOUSE ANTIGEN PRT SEQ ID NO:153 O00574_CXCR6_HUMANANTIGEN PRT SEQ ID NO:154 Q9BDS6_CXCR6_MACFA ANTIGEN PRT SEQ IDNO:155 Q9EQ16_CXCR6_MOUSE ANTIGEN PRT SEQ ID NO:156P49238_CX3CR1_HUMAN_TRD ANTIGEN PRT SEQ ID NO:157 Y14Q6Y3J6_CX3CR1_MACFA_TRD ANTIGEN PRT SEQ ID NO:158 Y20Q9Z0D9_CX3CR1_MOUSE_TRD ANTIGEN PRT SEQ ID NO:159 Y15P49238_CX3CR1_HUMAN_N Term ANTIGEN PRT SEQ ID NO:160 Y14, Y22Q6Y3J6_CX3CR1_MACFA_N Term ANTIGEN PRT SEQ ID NO:161 Y20, Y22Q9Z0D9_CX3CR1_MOUSE_N Term ANTIGEN PRT SEQ ID NO:162 Y15, Y23P25024_CXCR1_HUMAN_TRD ANTIGEN PRT SEQ ID NO:163 Y27Q2YEG0_CXCR1_MACMU_TRD ANTIGEN PRT SEQ ID NO:164 Y14, Y28Q810W6_CXCR1_MOUSE_TRD ANTIGEN PRT SEQ ID NO:165 Y6, Y32P25024_CXCR1_HUMAN_N Term ANTIGEN PRT SEQ ID NO:166 Y27Q2YEG0_CXCR1_MACMU_N Term ANTIGEN PRT SEQ ID NO:167 Y14, Y28, Y41Q810W6_CXCR1_MOUSE_N Term ANTIGEN PRT SEQ ID NO:168 Y6, Y32

TABLE-US-00002 Sequence TPP ID Sequence Name Region Type SEQ IDTPP-14095 497A-M005-E07-hIgG1Lambda VH PRT SEQ ID NO:201 TPP-14095497A-M005-E07-hIgG1Lambda HCDR1 PRT SEQ ID NO:202 TPP-14095497A-M005-E07-hIgG1Lambda HCDR2 PRT SEQ ID NO:203 TPP-14095497A-M005-E07-hIgG1Lambda HCDR3 PRT SEQ ID NO:204 TPP-14095497A-M005-E07-hIgG1Lambda VL PRT SEQ ID NO:205 TPP-14095497A-M005-E07-hIgG1Lambda LCDR1 PRT SEQ ID NO:206 TPP-14095497A-M005-E07-hIgG1Lambda LCDR2 PRT SEQ ID NO:207 TPP-14095497A-M005-E07-hIgG1Lambda LCDR3 PRT SEQ ID NO:208 TPP-14095497A-M005-E07-hIgG1Lambda VH DNA SEQ ID NO:209 TPP-14095497A-M005-E07-hIgG1Lambda VL DNA SEQ ID NO:210 TPP-14095497A-M005-E07-hIgG1Lambda HC PRT SEQ ID NO:211 TPP-14095497A-M005-E07-hIgG1Lambda LC PRT SEQ ID NO:212 TPP-14095497A-M005-E07-hIgG1Lambda HC DNA SEQ ID NO:213 TPP-14095497A-M005-E07-hIgG1Lambda LC DNA SEQ ID NO:214 TPP-14099497A-M005-F02-hIgG1Lambda VH PRT SEQ ID NO:215 TPP-14099497A-M005-F02-hIgG1Lambda HCDR1 PRT SEQ ID NO:216 TPP-14099497A-M005-F02-hIgG1Lambda HCDR2 PRT SEQ ID NO:217 TPP-14099497A-M005-F02-hIgG1Lambda HCDR3 PRT SEQ ID NO:218 TPP-14099497A-M005-F02-hIgG1Lambda VL PRT SEQ ID NO:219 TPP-14099497A-M005-F02-hIgG1Lambda LCDR1 PRT SEQ ID NO:220 TPP-14099497A-M005-F02-hIgG1Lambda LCDR2 PRT SEQ ID NO:221 TPP-14099497A-M005-F02-hIgG1Lambda LCDR3 PRT SEQ ID NO:222 TPP-14099497A-M005-F02-hIgG1Lambda VH DNA SEQ ID NO:223 TPP-14099497A-M005-F02-hIgG1Lambda VL DNA SEQ ID NO:224 TPP-14099497A-M005-F02-hIgG1Lambda HC PRT SEQ ID NO:225 TPP-14099497A-M005-F02-hIgG1Lambda LC PRT SEQ ID NO:226 TPP-14099497A-M005-F02-hIgG1Lambda HC DNA SEQ ID NO:227 TPP-14099497A-M005-F02-hIgG1Lambda LC DNA SEQ ID NO:228 TPP-15285497A-M005-F02-mIgG2aLambda VH PRT SEQ ID NO:229 TPP-15285497A-M005-F02-mIgG2aLambda HCDR1 PRT SEQ ID NO:230 TPP-15285497A-M005-F02-mIgG2aLambda HCDR2 PRT SEQ ID NO:231 TPP-15285497A-M005-F02-mIgG2aLambda HCDR3 PRT SEQ ID NO:232 TPP-15285497A-M005-F02-mIgG2aLambda VL PRT SEQ ID NO:233 TPP-15285497A-M005-F02-mIgG2aLambda LCDR1 PRT SEQ ID NO:234 TPP-15285497A-M005-F02-mIgG2aLambda LCDR2 PRT SEQ ID NO:235 TPP-15285497A-M005-F02-mIgG2aLambda LCDR3 PRT SEQ ID NO:236 TPP-15285497A-M005-F02-mIgG2aLambda VH DNA SEQ ID NO:237 TPP-15285497A-M005-F02-mIgG2aLambda VL DNA SEQ ID NO:238 TPP-15285497A-M005-F02-mIgG2aLambda HC PRT SEQ ID NO:239 TPP-15285497A-M005-F02-mIgG2aLambda LC PRT SEQ ID NO:240 TPP-15285497A-M005-F02-mIgG2aLambda HC DNA SEQ ID NO:241 TPP-15285497A-M005-F02-mIgG2aLambda LC DNA SEQ ID NO:242 TPP-15286497A-M005-E07-mIgG2aLambda VH PRT SEQ ID NO:243 TPP-15286497A-M005-E07-mIgG2aLambda HCDR1 PRT SEQ ID NO:244 TPP-15286497A-M005-E07-mIgG2aLambda HCDR2 PRT SEQ ID NO:245 TPP-15286497A-M005-E07-mIgG2aLambda HCDR3 PRT SEQ ID NO:246 TPP-15286497A-M005-E07-mIgG2aLambda VL PRT SEQ ID NO:247 TPP-15286497A-M005-E07-mIgG2aLambda LCDR1 PRT SEQ ID NO:248 TPP-15286497A-M005-E07-mIgG2aLambda LCDR2 PRT SEQ ID NO:249 TPP-15286497A-M005-E07-mIgG2aLambda LCDR3 PRT SEQ ID NO:250 TPP-15286497A-M005-E07-mIgG2aLambda VH DNA SEQ ID NO:251 TPP-15286497A-M005-E07-mIgG2aLambda VL DNA SEQ ID NO:252 TPP-15286497A-M005-E07-mIgG2aLambda HC PRT SEQ ID NO:253 TPP-15286497A-M005-E07-mIgG2aLambda LC PRT SEQ ID NO:254 TPP-15286497A-M005-E07-mIgG2aLambda HC DNA SEQ ID NO:255 TPP-15286497A-M005-E07-mIgG2aLambda LC DNA SEQ ID NO:256 TPP-1696615291-deglyco-hIgG1Lambda VH PRT SEQ ID NO:257 TPP-1696615291-deglyco-hIgG1Lambda HCDR1 PRT SEQ ID NO:258 TPP-1696615291-deglyco-hIgG1Lambda HCDR2 PRT SEQ ID NO:259 TPP-1696615291-deglyco-hIgG1Lambda HCDR3 PRT SEQ ID NO:260 TPP-1696615291-deglyco-hIgG1Lambda VL PRT SEQ ID NO:261 TPP-1696615291-deglyco-hIgG1Lambda LCDR1 PRT SEQ ID NO:262 TPP-1696615291-deglyco-hIgG1Lambda LCDR2 PRT SEQ ID NO:263 TPP-1696615291-deglyco-hIgG1Lambda LCDR3 PRT SEQ ID NO:264 TPP-1696615291-deglyco-hIgG1Lambda VH DNA SEQ ID NO:265 TPP-1696615291-deglyco-hIgG1Lambda HCDR1 DNA SEQ ID NO:266 TPP-1696615291-deglyco-hIgG1Lambda HCDR2 DNA SEQ ID NO:267 TPP-1696615291-deglyco-hIgG1Lambda HCDR3 DNA SEQ ID NO:268 TPP-1696615291-deglyco-hIgG1Lambda VL DNA SEQ ID NO:269 TPP-1696615291-deglyco-hIgG1Lambda LCDR1 DNA SEQ ID NO:270 TPP-1696615291-deglyco-hIgG1Lambda LCDR2 DNA SEQ ID NO:271 TPP-1696615291-deglyco-hIgG1Lambda LCDR3 DNA SEQ ID NO:272 TPP-1696615291-deglyco-hIgG1Lambda HC PRT SEQ ID NO:273 TPP-1696615291-deglyco-hIgG1Lambda LC PRT SEQ ID NO:274 TPP-17575497A-M181-C17-1-hIgG1Lambda VH PRT SEQ ID NO:275 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:276 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:277 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:278 TPP-17575497A-M181-C17-1-hIgG1Lambda VL PRT SEQ ID NO:279 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:280 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:281 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:282 TPP-17575497A-M181-C17-1-hIgG1Lambda VH DNA SEQ ID NO:283 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:284 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:285 TPP-17575497A-M181-C17-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:286 TPP-17575497A-M181-C17-1-hIgG1Lambda VL DNA SEQ ID NO:287 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:288 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:289 TPP-17575497A-M181-C17-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:290 TPP-17575497A-M181-C17-1-hIgG1Lambda HC PRT SEQ ID NO:291 TPP-17575497A-M181-C17-1-hIgG1Lambda LC PRT SEQ ID NO:292 TPP-17576497A-M136-O21-1-hIgG1Lambda VH PRT SEQ ID NO:293 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:294 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:295 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:296 TPP-17576497A-M136-O21-1-hIgG1Lambda VL PRT SEQ ID NO:297 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:298 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:299 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:300 TPP-17576497A-M136-O21-1-hIgG1Lambda VH DNA SEQ ID NO:301 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:302 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:303 TPP-17576497A-M136-O21-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:304 TPP-17576497A-M136-O21-1-hIgG1Lambda VL DNA SEQ ID NO:305 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:306 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:307 TPP-17576497A-M136-O21-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:308 TPP-17576497A-M136-O21-1-hIgG1Lambda HC PRT SEQ ID NO:309 TPP-17576497A-M136-O21-1-hIgG1Lambda LC PRT SEQ ID NO:310 TPP-17577497A-M178-K09-1-hIgG1Lambda VH PRT SEQ ID NO:311 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:312 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:313 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:314 TPP-17577497A-M178-K09-1-hIgG1Lambda VL PRT SEQ ID NO:315 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:316 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:317 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:318 TPP-17577497A-M178-K09-1-hIgG1Lambda VH DNA SEQ ID NO:319 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:320 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:321 TPP-17577497A-M178-K09-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:322 TPP-17577497A-M178-K09-1-hIgG1Lambda VL DNA SEQ ID NO:323 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:324 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:325 TPP-17577497A-M178-K09-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:326 TPP-17577497A-M178-K09-1-hIgG1Lambda HC PRT SEQ ID NO:327 TPP-17577497A-M178-K09-1-hIgG1Lambda LC PRT SEQ ID NO:328 TPP-17578497A-M128-B10-1-hIgG1Lambda VH PRT SEQ ID NO:329 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:330 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:331 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:332 TPP-17578497A-M128-B10-1-hIgG1Lambda VL PRT SEQ ID NO:333 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:334 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:335 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:336 TPP-17578497A-M128-B10-1-hIgG1Lambda VH DNA SEQ ID NO:337 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:338 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:339 TPP-17578497A-M128-B10-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:340 TPP-17578497A-M128-B10-1-hIgG1Lambda VL DNA SEQ ID NO:341 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:342 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:343 TPP-17578497A-M128-B10-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:344 TPP-17578497A-M128-B10-1-hIgG1Lambda HC PRT SEQ ID NO:345 TPP-17578497A-M128-B10-1-hIgG1Lambda LC PRT SEQ ID NO:346 TPP-17579497A-M180-K04-1-hIgG1Lambda VH PRT SEQ ID NO:347 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:348 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:349 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:350 TPP-17579497A-M180-K04-1-hIgG1Lambda VL PRT SEQ ID NO:351 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:352 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:353 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:354 TPP-17579497A-M180-K04-1-hIgG1Lambda VH DNA SEQ ID NO:355 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:356 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:357 TPP-17579497A-M180-K04-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:358 TPP-17579497A-M180-K04-1-hIgG1Lambda VL DNA SEQ ID NO:359 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:360 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:361 TPP-17579497A-M180-K04-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:362 TPP-17579497A-M180-K04-1-hIgG1Lambda HC PRT SEQ ID NO:363 TPP-17579497A-M180-K04-1-hIgG1Lambda LC PRT SEQ ID NO:364 TPP-17580497A-M193-A08-1-hIgG1Lambda VH PRT SEQ ID NO:365 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:366 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:367 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:368 TPP-17580497A-M193-A08-1-hIgG1Lambda VL PRT SEQ ID NO:369 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:370 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:371 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:372 TPP-17580497A-M193-A08-1-hIgG1Lambda VH DNA SEQ ID NO:373 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:374 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:375 TPP-17580497A-M193-A08-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:376 TPP-17580497A-M193-A08-1-hIgG1Lambda VL DNA SEQ ID NO:377 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:378 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:379 TPP-17580497A-M193-A08-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:380 TPP-17580497A-M193-A08-1-hIgG1Lambda HC PRT SEQ ID NO:381 TPP-17580497A-M193-A08-1-hIgG1Lambda LC PRT SEQ ID NO:382 TPP-17581497A-M177-L03-1-hIgG1Lambda VH PRT SEQ ID NO:383 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR1 PRT SEQ ID NO:384 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR2 PRT SEQ ID NO:385 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR3 PRT SEQ ID NO:386 TPP-17581497A-M177-L03-1-hIgG1Lambda VL PRT SEQ ID NO:387 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR1 PRT SEQ ID NO:388 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR2 PRT SEQ ID NO:389 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR3 PRT SEQ ID NO:390 TPP-17581497A-M177-L03-1-hIgG1Lambda VH DNA SEQ ID NO:391 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR1 DNA SEQ ID NO:392 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR2 DNA SEQ ID NO:393 TPP-17581497A-M177-L03-1-hIgG1Lambda HCDR3 DNA SEQ ID NO:394 TPP-17581497A-M177-L03-1-hIgG1Lambda VL DNA SEQ ID NO:395 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR1 DNA SEQ ID NO:396 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR2 DNA SEQ ID NO:397 TPP-17581497A-M177-L03-1-hIgG1Lambda LCDR3 DNA SEQ ID NO:398 TPP-17581497A-M177-L03-1-hIgG1Lambda HC PRT SEQ ID NO:399 TPP-17581497A-M177-L03-1-hIgG1Lambda LC PRT SEQ ID NO:400 TPP-18205004-F10_497A-M296-L12+497A-M326- VH PRT SEQ ID NO:401O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- HCDR1PRT SEQ ID NO:402 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- HCDR2 PRT SEQ ID NO:403O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- HCDR3PRT SEQ ID NO:404 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- VL PRT SEQ ID NO:405O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- LCDR1PRT SEQ ID NO:406 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- LCDR2 PRT SEQ ID NO:407O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- LCDR3PRT SEQ ID NO:408 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- VH DNA SEQ ID NO:409O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- HCDR1DNA SEQ ID NO:410 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- HCDR2 DNA SEQ ID NO:411O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- HCDR3DNA SEQ ID NO:412 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- VL DNA SEQ ID NO:413O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- LCDR1DNA SEQ ID NO:414 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- LCDR2 DNA SEQ ID NO:415O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- LCDR3DNA SEQ ID NO:416 O09-hIgG1Lambda TPP-18205004-F10_497A-M296-L12+497A-M326- HC PRT SEQ ID NO:417O09-hIgG1Lambda TPP-18205 004-F10_497A-M296-L12+497A-M326- LC PRTSEQ ID NO:418 O09-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- VH PRT SEQ ID NO:419O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- HCDR1PRT SEQ ID NO:420 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- HCDR2 PRT SEQ ID NO:421O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- HCDR3PRT SEQ ID NO:422 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- VL PRT SEQ ID NO:423O07-hIgG1Lambda

TPP-18206 002-F01_497A-M281-P02+497A-M317- LCDR1 PRT SEQ ID NO:424O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317-O07-hIgG1Lambda LCDR2 PRT SEQ ID NO:425 TPP-18206002-F01_497A-M281-P02+497A-M317- LCDR3 PRT SEQ ID NO:426O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- VH DNASEQ ID NO:427 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- HCDR1 DNA SEQ ID NO:428O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- HCDR2DNA SEQ ID NO:429 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- HCDR3 DNA SEQ ID NO:430O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- VL DNASEQ ID NO:431 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- LCDR1 DNA SEQ ID NO:432O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- LCDR2DNA SEQ ID NO:433 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- LCDR3 DNA SEQ ID NO:434O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- HC PRTSEQ ID NO:435 O07-hIgG1Lambda TPP-18206002-F01_497A-M281-P02+497A-M317- LC PRT SEQ ID NO:436O07-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- VH PRTSEQ ID NO:437 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- HCDR1 PRT SEQ ID NO:438D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- HCDR2PRT SEQ ID NO:439 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- HCDR3 PRT SEQ ID NO:440D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- VL PRTSEQ ID NO:441 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- LCDR1 PRT SEQ ID NO:442D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- LCDR2PRT SEQ ID NO:443 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- LCDR3 PRT SEQ ID NO:444D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- VH DNASEQ ID NO:445 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- HCDR1 DNA SEQ ID NO:446D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- HCDR2DNA SEQ ID NO:447 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- HCDR3 DNA SEQ ID NO:448D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- VL DNASEQ ID NO:449 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- LCDR1 DNA SEQ ID NO:450D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- LCDR2DNA SEQ ID NO:451 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- LCDR3 DNA SEQ ID NO:452D16-hIgG1Lambda TPP-18207 001-B08_497A-M301-B18+497A-M316- HC PRTSEQ ID NO:453 D16-hIgG1Lambda TPP-18207001-B08_497A-M301-B18+497A-M316- LC PRT SEQ ID NO:454D16-hIgG1Lambda TPP-19546 17578-gl33-hIgG1Lambda VH PRT SEQ IDNO:455 TPP-19546 17578-gl33-hIgG1Lambda HCDR1 PRT SEQ ID NO:456TPP-19546 17578-gl33-hIgG1Lambda HCDR2 PRT SEQ ID NO:457 TPP-1954617578-gl33-hIgG1Lambda HCDR3 PRT SEQ ID NO:458 TPP-1954617578-gl33-hIgG1Lambda VL PRT SEQ ID NO:459 TPP-1954617578-gl33-hIgG1Lambda LCDR1 PRT SEQ ID NO:460 TPP-1954617578-gl33-hIgG1Lambda LCDR2 PRT SEQ ID NO:461 TPP-1954617578-gl33-hIgG1Lambda LCDR3 PRT SEQ ID NO:462 TPP-1954617578-gl33-hIgG1Lambda VH DNA SEQ ID NO:463 TPP-1954617578-gl33-hIgG1Lambda HCDR1 DNA SEQ ID NO:464 TPP-1954617578-gl33-hIgG1Lambda HCDR2 DNA SEQ ID NO:465 TPP-1954617578-gl33-hIgG1Lambda HCDR3 DNA SEQ ID NO:466 TPP-1954617578-gl33-hIgG1Lambda VL DNA SEQ ID NO:467 TPP-1954617578-gl33-hIgG1Lambda LCDR1 DNA SEQ ID NO:468 TPP-1954617578-gl33-hIgG1Lambda LCDR2 DNA SEQ ID NO:469 TPP-1954617578-gl33-hIgG1Lambda LCDR3 DNA SEQ ID NO:470 TPP-1954617578-gl33-hIgG1Lambda HC PRT SEQ ID NO:471 TPP-1954617578-gl33-hIgG1Lambda LC PRT SEQ ID NO:472 TPP-2095018206-gl-42-hIgG1wt-Lambda VH PRT SEQ ID NO:473 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR1 PRT SEQ ID NO:474 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR2 PRT SEQ ID NO:475 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR3 PRT SEQ ID NO:476 TPP-2095018206-gl-42-hIgG1wt-Lambda VL PRT SEQ ID NO:477 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR1 PRT SEQ ID NO:478 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR2 PRT SEQ ID NO:479 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR3 PRT SEQ ID NO:480 TPP-2095018206-gl-42-hIgG1wt-Lambda VH DNA SEQ ID NO:481 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR1 DNA SEQ ID NO:482 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR2 DNA SEQ ID NO:483 TPP-2095018206-gl-42-hIgG1wt-Lambda HCDR3 DNA SEQ ID NO:484 TPP-2095018206-gl-42-hIgG1wt-Lambda VL DNA SEQ ID NO:485 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR1 DNA SEQ ID NO:486 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR2 DNA SEQ ID NO:487 TPP-2095018206-gl-42-hIgG1wt-Lambda LCDR3 DNA SEQ ID NO:488 TPP-2095018206-gl-42-hIgG1wt-Lambda HC PRT SEQ ID NO:489 TPP-2095018206-gl-42-hIgG1wt-Lambda LC PRT SEQ ID NO:490 TPP-2095518206-gl-47-hIgG1wt-Lambda VH PRT SEQ ID NO:491 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR1 PRT SEQ ID NO:492 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR2 PRT SEQ ID NO:493 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR3 PRT SEQ ID NO:494 TPP-2095518206-gl-47-hIgG1wt-Lambda VL PRT SEQ ID NO:495 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR1 PRT SEQ ID NO:496 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR2 PRT SEQ ID NO:497 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR3 PRT SEQ ID NO:498 TPP-2095518206-gl-47-hIgG1wt-Lambda VH DNA SEQ ID NO:499 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR1 DNA SEQ ID NO:500 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR2 DNA SEQ ID NO:501 TPP-2095518206-gl-47-hIgG1wt-Lambda HCDR3 DNA SEQ ID NO:502 TPP-2095518206-gl-47-hIgG1wt-Lambda VL DNA SEQ ID NO:503 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR1 DNA SEQ ID NO:504 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR2 DNA SEQ ID NO:505 TPP-2095518206-gl-47-hIgG1wt-Lambda LCDR3 DNA SEQ ID NO:506 TPP-2095518206-gl-47-hIgG1wt-Lambda HC PRT SEQ ID NO:507 TPP-2095518206-gl-47-hIgG1wt-Lambda LC PRT SEQ ID NO:508 TPP-2096518206-gl-58-hIgG1wt-Lambda VH PRT SEQ ID NO:509 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR1 PRT SEQ ID NO:510 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR2 PRT SEQ ID NO:511 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR3 PRT SEQ ID NO:512 TPP-2096518206-gl-58-hIgG1wt-Lambda VL PRT SEQ ID NO:513 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR1 PRT SEQ ID NO:514 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR2 PRT SEQ ID NO:515 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR3 PRT SEQ ID NO:516 TPP-2096518206-gl-58-hIgG1wt-Lambda VH DNA SEQ ID NO:517 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR1 DNA SEQ ID NO:518 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR2 DNA SEQ ID NO:519 TPP-2096518206-gl-58-hIgG1wt-Lambda HCDR3 DNA SEQ ID NO:520 TPP-2096518206-gl-58-hIgG1wt-Lambda VL DNA SEQ ID NO:521 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR1 DNA SEQ ID NO:522 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR2 DNA SEQ ID NO:523 TPP-2096518206-gl-58-hIgG1wt-Lambda LCDR3 DNA SEQ ID NO:524 TPP-2096518206-gl-58-hIgG1wt-Lambda HC PRT SEQ ID NO:525 TPP-2096518206-gl-58-hIgG1wt-Lambda LC PRT SEQ ID NO:526 TPP-2104517578-gl79-hIgG1-wt-Lambda VH PRT SEQ ID NO:527 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR1 PRT SEQ ID NO:528 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR2 PRT SEQ ID NO:529 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR3 PRT SEQ ID NO:530 TPP-2104517578-gl79-hIgG1-wt-Lambda VL PRT SEQ ID NO:531 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR1 PRT SEQ ID NO:532 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR2 PRT SEQ ID NO:533 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR3 PRT SEQ ID NO:534 TPP-2104517578-gl79-hIgG1-wt-Lambda VH DNA SEQ ID NO:535 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR1 DNA SEQ ID NO:536 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR2 DNA SEQ ID NO:537 TPP-2104517578-gl79-hIgG1-wt-Lambda HCDR3 DNA SEQ ID NO:538 TPP-2104517578-gl79-hIgG1-wt-Lambda VL DNA SEQ ID NO:539 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR1 DNA SEQ ID NO:540 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR2 DNA SEQ ID NO:541 TPP-2104517578-gl79-hIgG1-wt-Lambda LCDR3 DNA SEQ ID NO:542 TPP-2104517578-gl79-hIgG1-wt-Lambda HC PRT SEQ ID NO:543 TPP-2104517578-gl79-hIgG1-wt-Lambda LC PRT SEQ ID NO:544 TPP-2104717578-gl81-hIgG1-wt-Lambda VH PRT SEQ ID NO:545 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR1 PRT SEQ ID NO:546 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR2 PRT SEQ ID NO:547 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR3 PRT SEQ ID NO:548 TPP-2104717578-gl81-hIgG1-wt-Lambda VL PRT SEQ ID NO:549 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR1 PRT SEQ ID NO:550 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR2 PRT SEQ ID NO: 551 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR3 PRT SEQ ID NO:552 TPP-2104717578-gl81-hIgG1-wt-Lambda VH DNA SEQ ID NO:553 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR1 DNA SEQ ID NO:554 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR2 DNA SEQ ID NO:555 TPP-2104717578-gl81-hIgG1-wt-Lambda HCDR3 DNA SEQ ID NO:556 TPP-2104717578-gl81-hIgG1-wt-Lambda VL DNA SEQ ID NO:557 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR1 DNA SEQ ID NO:558 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR2 DNA SEQ ID NO:559 TPP-2104717578-gl81-hIgG1-wt-Lambda LCDR3 DNA SEQ ID NO:560 TPP-2104717578-gl81-hIgG1-wt-Lambda HC PRT SEQ ID NO:561 TPP-2104717578-gl81-hIgG1-wt-Lambda LC PRT SEQ ID NO:562 TPP-2118118205-gl46-hIgG1-wt-Lambda VH PRT SEQ ID NO:563 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR1 PRT SEQ ID NO:564 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR2 PRT SEQ ID NO:565 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR3 PRT SEQ ID NO:566 TPP-2118118205-gl46-hIgG1-wt-Lambda VL PRT SEQ ID NO:567 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR1 PRT SEQ ID NO:568 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR2 PRT SEQ ID NO:569 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR3 PRT SEQ ID NO:570 TPP-2118118205-gl46-hIgG1-wt-Lambda VH DNA SEQ ID NO:571 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR1 DNA SEQ ID NO:572 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR2 DNA SEQ ID NO:573 TPP-2118118205-gl46-hIgG1-wt-Lambda HCDR3 DNA SEQ ID NO:574 TPP-2118118205-gl46-hIgG1-wt-Lambda VL DNA SEQ ID NO:575 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR1 DNA SEQ ID NO:576 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR2 DNA SEQ ID NO:577 TPP-2118118205-gl46-hIgG1-wt-Lambda LCDR3 DNA SEQ ID NO:578 TPP-2118118205-gl46-hIgG1-wt-Lambda HC PRT SEQ ID NO:579 TPP-2118118205-gl46-hIgG1-wt-Lambda LC PRT SEQ ID NO:580 TPP-2118318205-gl48-hIgG1-wt-Lambda VH PRT SEQ ID NO:581 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR1 PRT SEQ ID NO:582 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR2 PRT SEQ ID NO:583 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR3 PRT SEQ ID NO:584 TPP-2118318205-gl48-hIgG1-wt-Lambda VL PRT SEQ ID NO:585 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR1 PRT SEQ ID NO:586 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR2 PRT SEQ ID NO:587 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR3 PRT SEQ ID NO:588 TPP-2118318205-gl48-hIgG1-wt-Lambda VH DNA SEQ ID NO:589 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR1 DNA SEQ ID NO:590 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR2 DNA SEQ ID NO:591 TPP-2118318205-gl48-hIgG1-wt-Lambda HCDR3 DNA SEQ ID NO:592 TPP-2118318205-gl48-hIgG1-wt-Lambda VL DNA SEQ ID NO:593 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR1 DNA SEQ ID NO:594 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR2 DNA SEQ ID NO:595 TPP-2118318205-gl48-hIgG1-wt-Lambda LCDR3 DNA SEQ ID NO:596 TPP-2118318205-gl48-hIgG1-wt-Lambda HC PRT SEQ ID NO:597 TPP-2118318205-gl48-hIgG1-wt-Lambda LC PRT SEQ ID NO:598 TPP-2136018206-gl25-gl13-hIgG1Lambda VH PRT SEQ ID NO:599 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR1 PRT SEQ ID NO:600 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR2 PRT SEQ ID NO:601 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR3 PRT SEQ ID NO:602 TPP-2136018206-gl25-gl13-hIgG1Lambda VL PRT SEQ ID NO:603 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR1 PRT SEQ ID NO:604 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR2 PRT SEQ ID NO:605 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR3 PRT SEQ ID NO:606 TPP-2136018206-gl25-gl13-hIgG1Lambda VH DNA SEQ ID NO:607 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR1 DNA SEQ ID NO:608 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR2 DNA SEQ ID NO:609 TPP-2136018206-gl25-gl13-hIgG1Lambda HCDR3 DNA SEQ ID NO:610 TPP-2136018206-gl25-gl13-hIgG1Lambda VL DNA SEQ ID NO:611 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR1 DNA SEQ ID NO:612 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR2 DNA SEQ ID NO:613 TPP-2136018206-gl25-gl13-hIgG1Lambda LCDR3 DNA SEQ ID NO:614 TPP-2136018206-gl25-gl13-hIgG1Lambda HC PRT SEQ ID NO:615 TPP-2136018206-gl25-gl13-hIgG1Lambda LC PRT SEQ ID NO:616 TPP-2341121360-hIgG1wtlambda VH PRT SEQ ID NO:617 TPP-2341121360-hIgG1wtlambda HCDR1 PRT SEQ ID NO:618 TPP-2341121360-hIgG1wtlambda HCDR2 PRT SEQ ID NO:619 TPP-2341121360-hIgG1wtlambda HCDR3 PRT SEQ ID NO:620 TPP-2341121360-hIgG1wtlambda VL PRT SEQ ID NO:621 TPP-2341121360-hIgG1wtlambda LCDR1 PRT SEQ ID NO:622 TPP-2341121360-hIgG1wtlambda LCDR2 PRT SEQ ID NO:623 TPP-2341121360-hIgG1wtlambda LCDR3 PRT SEQ ID NO:624 TPP-2341121360-hIgG1wtlambda VH DNA SEQ ID NO:625 TPP-2341121360-hIgG1wtlambda HCDR1 DNA SEQ ID NO:626 TPP-2341121360-hIgG1wtlambda HCDR2 DNA SEQ ID NO:627 TPP-2341121360-hIgG1wtlambda HCDR3 DNA SEQ ID NO:628 TPP-2341121360-hIgG1wtlambda VL DNA SEQ ID NO:629 TPP-2341121360-hIgG1wtlambda LCDR1 DNA SEQ ID NO:630 TPP-2341121360-hIgG1wtlambda LCDR2 DNA SEQ ID NO:631 TPP-2341121360-hIgG1wtlambda LCDR3 DNA SEQ ID NO:632 TPP-2341121360-hIgG1wtlambda HC PRT SEQ ID NO:633 TPP-2341121360-hIgG1wtlambda LC PRT SEQ ID NO:634 TPP-2959618206-gl13-gl14-hIgG1wtLambda VH PRT SEQ ID NO:660 TPP-2959618206-gl13-gl14-hIgG1wtLambda HCDR1 PRT SEQ ID NO:661 TPP-2959618206-gl13-gl14-hIgG1wtLambda HCDR2 PRT SEQ ID NO:662 TPP-2959618206-gl13-gl14-hIgG1wtLambda HCDR3 PRT SEQ ID NO:663 TPP-2959618206-gl13-gl14-hIgG1wtLambda VL PRT SEQ ID NO:664 TPP-2959618206-gl13-gl14-hIgG1wtLambda LCDR1 PRT SEQ ID NO:665 TPP-2959618206-gl13-gl14-hIgG1wtLambda LCDR2 PRT SEQ ID NO:666 TPP-2959618206-gl13-gl14-hIgG1wtLambda LCDR3 PRT SEQ ID NO:667 TPP-2959618206-gl13-gl14-hIgG1wtLambda VH DNA SEQ ID NO:668

TPP-29596 18206-gl13-gl14-hIgG1wtLambda HCDR1 DNA SEQ ID NO:669TPP-29596 18206-gl13-gl14-hIgG1wtLambda HCDR2 DNA SEQ ID NO:670TPP-29596 18206-gl13-gl14-hIgG1wtLambda HCDR3 DNA SEQ ID NO:671TPP-29596 18206-gl13-gl14-hIgG1wtLambda VL DNA SEQ ID NO:672TPP-29596 18206-gl13-gl14-hIgG1wtLambda LCDR1 DNA SEQ ID NO:673TPP-29596 18206-gl13-gl14-hIgG1wtLambda LCDR2 DNA SEQ ID NO:674TPP-29596 18206-gl13-gl14-hIgG1wtLambda LCDR3 DNA SEQ ID NO:675TPP-29596 18206-gl13-gl14-hIgG1wtLambda HC PRT SEQ ID NO:676TPP-29596 18206-gl13-gl14-hIgG1wtLambda LC PRT SEQ ID NO:677TPP-29596 18206-gl13-gl14-hIgG1wtLambda HC DNA SEQ ID NO:678TPP-29596 18206-gl13-gl14-hIgG1wtLambda LC DNA SEQ ID NO:679TPP-29597 18206-gl11-gl25-hIgG1wtLambda VH PRT SEQ ID NO:680TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR1 PRT SEQ ID NO:681TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR2 PRT SEQ ID NO:682TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR3 PRT SEQ ID NO:683TPP-29597 18206-gl11-gl25-hIgG1wtLambda VL PRT SEQ ID NO:684TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR1 PRT SEQ ID NO:685TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR2 PRT SEQ ID NO:686TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR3 PRT SEQ ID NO:687TPP-29597 18206-gl11-gl25-hIgG1wtLambda VH DNA SEQ ID NO:688TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR1 DNA SEQ ID NO:689TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR2 DNA SEQ ID NO:690TPP-29597 18206-gl11-gl25-hIgG1wtLambda HCDR3 DNA SEQ ID NO:691TPP-29597 18206-gl11-gl25-hIgG1wtLambda VL DNA SEQ ID NO:692TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR1 DNA SEQ ID NO:693TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR2 DNA SEQ ID NO:694TPP-29597 18206-gl11-gl25-hIgG1wtLambda LCDR3 DNA SEQ ID NO:695TPP-29597 18206-gl11-gl25-hIgG1wtLambda HC PRT SEQ ID NO:696TPP-29597 18206-gl11-gl25-hIgG1wtLambda LC PRT SEQ ID NO:697TPP-29597 18206-gl11-gl25-hIgG1wtLambda HC DNA SEQ ID NO:698TPP-29597 18206-gl11-gl25-hIgG1wtLambda LC DNA SEQ ID NO:699TPP-18206 002-F01_497A-M281-P02+497A-M317- HC DNA SEQ ID NO:700O07-hIgG1Lambda TPP-18206 002-F01_497A-M281-P02+497A-M317- LC DNASEQ ID NO:701 O07-hIgG1Lambda TPP-18429 18206-gl10-hIgG1Lambda VHPRT SEQ ID NO:702 TPP-18429 18206-gl10-hIgG1Lambda HCDR1 PRT SEQ IDNO:703 TPP-18429 18206-gl10-hIgG1Lambda HCDR2 PRT SEQ ID NO:704TPP-18429 18206-gl10-hIgG1Lambda HCDR3 PRT SEQ ID NO:705 TPP-1842918206-gl10-hIgG1Lambda VL PRT SEQ ID NO:706 TPP-1842918206-gl10-hIgG1Lambda LCDR1 PRT SEQ ID NO:707 TPP-1842918206-gl10-hIgG1Lambda LCDR2 PRT SEQ ID NO:708 TPP-1842918206-gl10-hIgG1Lambda LCDR3 PRT SEQ ID NO:709 TPP-1842918206-gl10-hIgG1Lambda VH DNA SEQ ID NO:710 TPP-1842918206-gl10-hIgG1Lambda HCDR1 DNA SEQ ID NO:711 TPP-1842918206-gl10-hIgG1Lambda HCDR2 DNA SEQ ID NO:712 TPP-1842918206-gl10-hIgG1Lambda HCDR3 DNA SEQ ID NO:713 TPP-1842918206-gl10-hIgG1Lambda VL DNA SEQ ID NO:714 TPP-1842918206-gl10-hIgG1Lambda LCDR1 DNA SEQ ID NO:715 TPP-1842918206-gl10-hIgG1Lambda LCDR2 DNA SEQ ID NO:716 TPP-1842918206-gl10-hIgG1Lambda LCDR3 DNA SEQ ID NO:717 TPP-1842918206-gl10-hIgG1Lambda HC PRT SEQ ID NO:718 TPP-1842918206-gl10-hIgG1Lambda LC PRT SEQ ID NO:719 TPP-1842918206-gl10-hIgG1Lambda HC DNA SEQ ID NO:720 TPP-1842918206-gl10-hIgG1Lambda LC DNA SEQ ID NO:721 TPP-1843018206-gl11-hIgG1Lambda VH PRT SEQ ID NO:722 TPP-1843018206-gl11-hIgG1Lambda HCDR1 PRT SEQ ID NO:723 TPP-1843018206-gl11-hIgG1Lambda HCDR2 PRT SEQ ID NO:724 TPP-1843018206-gl11-hIgG1Lambda HCDR3 PRT SEQ ID NO:725 TPP-1843018206-gl11-hIgG1Lambda VL PRT SEQ ID NO:726 TPP-1843018206-gl11-hIgG1Lambda LCDR1 PRT SEQ ID NO:727 TPP-1843018206-gl11-hIgG1Lambda LCDR2 PRT SEQ ID NO:728 TPP-1843018206-gl11-hIgG1Lambda LCDR3 PRT SEQ ID NO:729 TPP-1843018206-gl11-hIgG1Lambda VH DNA SEQ ID NO:730 TPP-1843018206-gl11-hIgG1Lambda HCDR1 DNA SEQ ID NO:731 TPP-1843018206-gl11-hIgG1Lambda HCDR2 DNA SEQ ID NO:732 TPP-1843018206-gl11-hIgG1Lambda HCDR3 DNA SEQ ID NO:733 TPP-1843018206-gl11-hIgG1Lambda VL DNA SEQ ID NO:734 TPP-1843018206-gl11-hIgG1Lambda LCDR1 DNA SEQ ID NO:735 TPP-1843018206-gl11-hIgG1Lambda LCDR2 DNA SEQ ID NO:736 TPP-1843018206-gl11-hIgG1Lambda LCDR3 DNA SEQ ID NO:737 TPP-1843018206-gl11-hIgG1Lambda HC PRT SEQ ID NO:738 TPP-1843018206-gl11-hIgG1Lambda LC PRT SEQ ID NO:739 TPP-1843018206-gl11-hIgG1Lambda HC DNA SEQ ID NO:740 TPP-1843018206-gl11-hIgG1Lambda LC DNA SEQ ID NO:741 TPP-1843218206-gl13-hIgG1Lambda VH PRT SEQ ID NO:742 TPP-1843218206-gl13-hIgG1Lambda HCDR1 PRT SEQ ID NO:743 TPP-1843218206-gl13-hIgG1Lambda HCDR2 PRT SEQ ID NO:744 TPP-1843218206-gl13-hIgG1Lambda HCDR3 PRT SEQ ID NO:745 TPP-1843218206-gl13-hIgG1Lambda VL PRT SEQ ID NO:746 TPP-1843218206-gl13-hIgG1Lambda LCDR1 PRT SEQ ID NO:747 TPP-1843218206-gl13-hIgG1Lambda LCDR2 PRT SEQ ID NO:748 TPP-1843218206-gl13-hIgG1Lambda LCDR3 PRT SEQ ID NO:749 TPP-1843218206-gl13-hIgG1Lambda VH DNA SEQ ID NO:750 TPP-1843218206-gl13-hIgG1Lambda HCDR1 DNA SEQ ID NO:751 TPP-1843218206-gl13-hIgG1Lambda HCDR2 DNA SEQ ID NO:752 TPP-1843218206-gl13-hIgG1Lambda HCDR3 DNA SEQ ID NO:753 TPP-1843218206-gl13-hIgG1Lambda VL DNA SEQ ID NO:754 TPP-1843218206-gl13-hIgG1Lambda LCDR1 DNA SEQ ID NO:755 TPP-1843218206-gl13-hIgG1Lambda LCDR2 DNA SEQ ID NO:756 TPP-1843218206-gl13-hIgG1Lambda LCDR3 DNA SEQ ID NO:757 TPP-1843218206-gl13-hIgG1Lambda HC PRT SEQ ID NO:758 TPP-1843218206-gl13-hIgG1Lambda LC PRT SEQ ID NO:759 TPP-1843218206-gl13-hIgG1Lambda HC DNA SEQ ID NO:760 TPP-1843218206-gl13-hIgG1Lambda LC DNA SEQ ID NO:761 TPP-1843318206-gl14-hIgG1Lambda VH PRT SEQ ID NO:762 TPP-1843318206-gl14-hIgG1Lambda HCDR1 PRT SEQ ID NO:763 TPP-1843318206-gl14-hIgG1Lambda HCDR2 PRT SEQ ID NO:764 TPP-1843318206-gl14-hIgG1Lambda HCDR3 PRT SEQ ID NO:765 TPP-1843318206-gl14-hIgG1Lambda VL PRT SEQ ID NO:766 TPP-1843318206-gl14-hIgG1Lambda LCDR1 PRT SEQ ID NO:767 TPP-1843318206-gl14-hIgG1Lambda LCDR2 PRT SEQ ID NO:768 TPP-1843318206-gl14-hIgG1Lambda LCDR3 PRT SEQ ID NO:769 TPP-1843318206-gl14-hIgG1Lambda VH DNA SEQ ID NO:770 TPP-1843318206-gl14-hIgG1Lambda HCDR1 DNA SEQ ID NO:771 TPP-1843318206-gl14-hIgG1Lambda HCDR2 DNA SEQ ID NO:772 TPP-1843318206-gl14-hIgG1Lambda HCDR3 DNA SEQ ID NO:773 TPP-1843318206-gl14-hIgG1Lambda VL DNA SEQ ID NO:774 TPP-1843318206-gl14-hIgG1Lambda LCDR1 DNA SEQ ID NO:775 TPP-1843318206-gl14-hIgG1Lambda LCDR2 DNA SEQ ID NO:776 TPP-1843318206-gl14-hIgG1Lambda LCDR3 DNA SEQ ID NO:777 TPP-1843318206-gl14-hIgG1Lambda HC PRT SEQ ID NO:778 TPP-1843318206-gl14-hIgG1Lambda LC PRT SEQ ID NO:779 TPP-1843318206-gl14-hIgG1Lambda HC DNA SEQ ID NO:780 TPP-1843318206-gl14-hIgG1Lambda LC DNA SEQ ID NO:781 TPP-1843618206-gl17-hIgG1Lambda VH PRT SEQ ID NO:782 TPP-1843618206-gl17-hIgG1Lambda HCDR1 PRT SEQ ID NO:783 TPP-1843618206-gl17-hIgG1Lambda HCDR2 PRT SEQ ID NO:784 TPP-1843618206-gl17-hIgG1Lambda HCDR3 PRT SEQ ID NO:785 TPP-1843618206-gl17-hIgG1Lambda VL PRT SEQ ID NO:786 TPP-1843618206-gl17-hIgG1Lambda LCDR1 PRT SEQ ID NO:787 TPP-1843618206-gl17-hIgG1Lambda LCDR2 PRT SEQ ID NO:788 TPP-1843618206-gl17-hIgG1Lambda LCDR3 PRT SEQ ID NO:789 TPP-1843618206-gl17-hIgG1Lambda VH DNA SEQ ID NO:790 TPP-1843618206-gl17-hIgG1Lambda HCDR1 DNA SEQ ID NO:791 TPP-1843618206-gl17-hIgG1Lambda HCDR2 DNA SEQ ID NO:792 TPP-1843618206-gl17-hIgG1Lambda HCDR3 DNA SEQ ID NO:793 TPP-1843618206-gl17-hIgG1Lambda VL DNA SEQ ID NO:794 TPP-1843618206-gl17-hIgG1Lambda LCDR1 DNA SEQ ID NO:795 TPP-1843618206-gl17-hIgG1Lambda LCDR2 DNA SEQ ID NO:796 TPP-1843618206-gl17-hIgG1Lambda LCDR3 DNA SEQ ID NO:797 TPP-1843618206-gl17-hIgG1Lambda HC PRT SEQ ID NO:798 TPP-1843618206-gl17-hIgG1Lambda LC PRT SEQ ID NO:799 TPP-1843618206-gl17-hIgG1Lambda HC DNA SEQ ID NO:800 TPP-1843618206-gl17-hIgG1Lambda LC DNA SEQ ID NO:801 TPP-1957118206-gl25-hIgG1Lambda VH PRT SEQ ID NO:802 TPP-1957118206-gl25-hIgG1Lambda HCDR1 PRT SEQ ID NO:803 TPP-1957118206-gl25-hIgG1Lambda HCDR2 PRT SEQ ID NO:804 TPP-1957118206-gl25-hIgG1Lambda HCDR3 PRT SEQ ID NO:805 TPP-1957118206-gl25-hIgG1Lambda VL PRT SEQ ID NO:806 TPP-1957118206-gl25-hIgG1Lambda LCDR1 PRT SEQ ID NO:807 TPP-1957118206-gl25-hIgG1Lambda LCDR2 PRT SEQ ID NO:808 TPP-1957118206-gl25-hIgG1Lambda LCDR3 PRT SEQ ID NO:809 TPP-1957118206-gl25-hIgG1Lambda VH DNA SEQ ID NO:810 TPP-1957118206-gl25-hIgG1Lambda HCDR1 DNA SEQ ID NO:811 TPP-1957118206-gl25-hIgG1Lambda HCDR2 DNA SEQ ID NO:812 TPP-1957118206-gl25-hIgG1Lambda HCDR3 DNA SEQ ID NO:813 TPP-1957118206-gl25-hIgG1Lambda VL DNA SEQ ID NO:814 TPP-1957118206-gl25-hIgG1Lambda LCDR1 DNA SEQ ID NO:815 TPP-1957118206-gl25-hIgG1Lambda LCDR2 DNA SEQ ID NO:816 TPP-1957118206-gl25-hIgG1Lambda LCDR3 DNA SEQ ID NO:817 TPP-1957118206-gl25-hIgG1Lambda HC PRT SEQ ID NO:818 TPP-1957118206-gl25-hIgG1Lambda LC PRT SEQ ID NO:819 TPP-1957118206-gl25-hIgG1Lambda HC DNA SEQ ID NO:820 TPP-1957118206-gl25-hIgG1Lambda LC DNA SEQ ID NO:821 TPP-2136018206-gl25-gl13-hIgG1Lambda HC DNA SEQ ID NO:822 TPP-2136018206-gl25-gl13-hIgG1Lambda LC DNA SEQ ID NO:823 TPP-2341121360-hIgG1lambda HC DNA SEQ ID NO:824 TPP-23411 21360-hIgG1lambdaLC DNA SEQ ID NO:825 TPP-27477 18206-gl10-gl25-hIgG1Lambda VH PRTSEQ ID NO:826 TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR1 PRT SEQID NO:827 TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR2 PRT SEQ IDNO:828 TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR3 PRT SEQ IDNO:829 TPP-27477 18206-gl10-gl25-hIgG1Lambda VL PRT SEQ ID NO:830TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR1 PRT SEQ ID NO:831TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR2 PRT SEQ ID NO:832TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR3 PRT SEQ ID NO:833TPP-27477 18206-gl10-gl25-hIgG1Lambda VH DNA SEQ ID NO:834TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR1 DNA SEQ ID NO:835TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR2 DNA SEQ ID NO:836TPP-27477 18206-gl10-gl25-hIgG1Lambda HCDR3 DNA SEQ ID NO:837TPP-27477 18206-gl10-gl25-hIgG1Lambda VL DNA SEQ ID NO:838TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR1 DNA SEQ ID NO:839TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR2 DNA SEQ ID NO:840TPP-27477 18206-gl10-gl25-hIgG1Lambda LCDR3 DNA SEQ ID NO:841TPP-27477 18206-gl10-gl25-hIgG1Lambda HC PRT SEQ ID NO:842TPP-27477 18206-gl10-gl25-hIgG1Lambda LC PRT SEQ ID NO:843TPP-27477 18206-gl10-gl25-hIgG1Lambda HC DNA SEQ ID NO:844TPP-27477 18206-gl10-gl25-hIgG1Lambda LC DNA SEQ ID NO:845TPP-27478 18206-gl11-gl25-hIgG1Lambda VH PRT SEQ ID NO:846TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR1 PRT SEQ ID NO:847TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR2 PRT SEQ ID NO:848TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR3 PRT SEQ ID NO:849TPP-27478 18206-gl11-gl25-hIgG1Lambda VL PRT SEQ ID NO:850TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR1 PRT SEQ ID NO:851TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR2 PRT SEQ ID NO:852TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR3 PRT SEQ ID NO:853TPP-27478 18206-gl11-gl25-hIgG1Lambda VH DNA SEQ ID NO:854TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR1 DNA SEQ ID NO:855TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR2 DNA SEQ ID NO:856TPP-27478 18206-gl11-gl25-hIgG1Lambda HCDR3 DNA SEQ ID NO:857TPP-27478 18206-gl11-gl25-hIgG1Lambda VL DNA SEQ ID NO:858TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR1 DNA SEQ ID NO:859TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR2 DNA SEQ ID NO:860TPP-27478 18206-gl11-gl25-hIgG1Lambda LCDR3 DNA SEQ ID NO:861TPP-27478 18206-gl11-gl25-hIgG1Lambda HC PRT SEQ ID NO:862TPP-27478 18206-gl11-gl25-hIgG1Lambda LC PRT SEQ ID NO:863TPP-27478 18206-gl11-gl25-hIgG1Lambda HC DNA SEQ ID NO:864TPP-27478 18206-gl11-gl25-hIgG1Lambda LC DNA SEQ ID NO:865TPP-27479 18206-gl13-gl14-hIgG1Lambda VH PRT SEQ ID NO:866TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR1 PRT SEQ ID NO:867TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR2 PRT SEQ ID NO:868TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR3 PRT SEQ ID NO:869TPP-27479 18206-gl13-gl14-hIgG1Lambda VL PRT SEQ ID NO:870TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR1 PRT SEQ ID NO:871TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR2 PRT SEQ ID NO:872TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR3 PRT SEQ ID NO:873TPP-27479 18206-gl13-gl14-hIgG1Lambda VH DNA SEQ ID NO:874TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR1 DNA SEQ ID NO:875TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR2 DNA SEQ ID NO:876TPP-27479 18206-gl13-gl14-hIgG1Lambda HCDR3 DNA SEQ ID NO:877TPP-27479 18206-gl13-gl14-hIgG1Lambda VL DNA SEQ ID NO:878TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR1 DNA SEQ ID NO:879TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR2 DNA SEQ ID NO:880TPP-27479 18206-gl13-gl14-hIgG1Lambda LCDR3 DNA SEQ ID NO:881TPP-27479 18206-gl13-gl14-hIgG1Lambda HC PRT SEQ ID NO:882TPP-27479 18206-gl13-gl14-hIgG1Lambda LC PRT SEQ ID NO:883TPP-27479 18206-gl13-gl14-hIgG1Lambda HC DNA SEQ ID NO:884TPP-27479 18206-gl13-gl14-hIgG1Lambda LC DNA SEQ ID NO:885TPP-27480 18206-gl13-gl20-hIgG1Lambda VH PRT SEQ ID NO:886TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR1 PRT SEQ ID NO:887TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR2 PRT SEQ ID NO:888TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR3 PRT SEQ ID NO:889TPP-27480 18206-gl13-gl20-hIgG1Lambda VL PRT SEQ ID NO:890TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR1 PRT SEQ ID NO:891TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR2 PRT SEQ ID NO:892TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR3 PRT SEQ ID NO:893TPP-27480 18206-gl13-gl20-hIgG1Lambda VH DNA SEQ ID NO:894TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR1 DNA SEQ ID NO:895TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR2 DNA SEQ ID NO:896TPP-27480 18206-gl13-gl20-hIgG1Lambda HCDR3 DNA SEQ ID NO:897TPP-27480 18206-gl13-gl20-hIgG1Lambda VL DNA SEQ ID NO:898TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR1 DNA SEQ ID NO:899TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR2 DNA SEQ ID NO:900TPP-27480 18206-gl13-gl20-hIgG1Lambda LCDR3 DNA SEQ ID NO:901TPP-27480 18206-gl13-gl20-hIgG1Lambda HC PRT SEQ ID NO:902TPP-27480 18206-gl13-gl20-hIgG1Lambda LC PRT SEQ ID NO:903TPP-27480 18206-gl13-gl20-hIgG1Lambda HC DNA SEQ ID NO:904TPP-27480 18206-gl13-gl20-hIgG1Lambda LC DNA SEQ ID NO:905TPP-29367 18206-hIgG1wtLambda VH PRT SEQ ID NO:906 TPP-2936718206-hIgG1wtLambda HCDR1 PRT SEQ ID NO:907 TPP-2936718206-hIgG1wtLambda HCDR2 PRT SEQ ID NO:908 TPP-2936718206-hIgG1wtLambda HCDR3 PRT SEQ ID NO:909 TPP-2936718206-hIgG1wtLambda VL PRT SEQ ID NO:910 TPP-2936718206-hIgG1wtLambda LCDR1 PRT SEQ ID NO:911 TPP-2936718206-hIgG1wtLambda LCDR2 PRT SEQ ID NO:912 TPP-2936718206-hIgG1wtLambda LCDR3 PRT SEQ ID NO:913 TPP-2936718206-hIgG1wtLambda VH DNA SEQ ID NO:914 TPP-2936718206-hIgG1wtLambda HCDR1 DNA SEQ ID NO:915 TPP-2936718206-hIgG1wtLambda HCDR2 DNA SEQ ID NO:916 TPP-2936718206-hIgG1wtLambda HCDR3 DNA SEQ ID NO:917

TPP-29367 18206-hIgG1wtLambda VL DNA SEQ ID NO:918 TPP-2936718206-hIgG1wtLambda LCDR1 DNA SEQ ID NO:919 TPP-2936718206-hIgG1wtLambda LCDR2 DNA SEQ ID NO:920 TPP-2936718206-hIgG1wtLambda LCDR3 DNA SEQ ID NO:921 TPP-2936718206-hIgG1wtLambda HC PRT SEQ ID NO:922 TPP-2936718206-hIgG1wtLambda LC PRT SEQ ID NO:923 TPP-2936718206-hIgG1wtLambda HC DNA SEQ ID NO:924 TPP-2936718206-hIgG1wtLambda LC DNA SEQ ID NO:925 TPP-2936818432-hIgG1wtLambda VH PRT SEQ ID NO:926 TPP-2936818432-hIgG1wtLambda HCDR1 PRT SEQ ID NO:927 TPP-2936818432-hIgG1wtLambda HCDR2 PRT SEQ ID NO:928 TPP-2936818432-hIgG1wtLambda HCDR3 PRT SEQ ID NO:929 TPP-2936818432-hIgG1wtLambda VL PRT SEQ ID NO:930 TPP-2936818432-hIgG1wtLambda LCDR1 PRT SEQ ID NO:931 TPP-2936818432-hIgG1wtLambda LCDR2 PRT SEQ ID NO:932 TPP-2936818432-hIgG1wtLambda LCDR3 PRT SEQ ID NO:933 TPP-2936818432-hIgG1wtLambda VH DNA SEQ ID NO:934 TPP-2936818432-hIgG1wtLambda HCDR1 DNA SEQ ID NO:935 TPP-2936818432-hIgG1wtLambda HCDR2 DNA SEQ ID NO:936 TPP-2936818432-hIgG1wtLambda HCDR3 DNA SEQ ID NO:937 TPP-2936818432-hIgG1wtLambda VL DNA SEQ ID NO:938 TPP-2936818432-hIgG1wtLambda LCDR1 DNA SEQ ID NO:939 TPP-2936818432-hIgG1wtLambda LCDR2 DNA SEQ ID NO:940 TPP-2936818432-hIgG1wtLambda LCDR3 DNA SEQ ID NO:941 TPP-2936818432-hIgG1wtLambda HC PRT SEQ ID NO:942 TPP-2936818432-hIgG1wtLambda LC PRT SEQ ID NO:943 TPP-2936818432-hIgG1wtLambda HC DNA SEQ ID NO:944 TPP-2936818432-hIgG1wtLambda LC DNA SEQ ID NO:945 TPP-2936919571-hIgG1wtlambda VH PRT SEQ ID NO:946 TPP-2936919571-hIgG1wtlambda HCDR1 PRT SEQ ID NO:947 TPP-2936919571-hIgG1wtlambda HCDR2 PRT SEQ ID NO:948 TPP-2936919571-hIgG1wtlambda HCDR3 PRT SEQ ID NO:949 TPP-2936919571-hIgG1wtlambda VL PRT SEQ ID NO:950 TPP-2936919571-hIgG1wtlambda LCDR1 PRT SEQ ID NO:951 TPP-2936919571-hIgG1wtlambda LCDR2 PRT SEQ ID NO:952 TPP-2936919571-hIgG1wtlambda LCDR3 PRT SEQ ID NO:953 TPP-2936919571-hIgG1wtlambda VH DNA SEQ ID NO:954 TPP-2936919571-hIgG1wtlambda HCDR1 DNA SEQ ID NO:955 TPP-2936919571-hIgG1wtlambda HCDR2 DNA SEQ ID NO:956 TPP-2936919571-hIgG1wtlambda HCDR3 DNA SEQ ID NO:957 TPP-2936919571-hIgG1wtlambda VL DNA SEQ ID NO:958 TPP-2936919571-hIgG1wtlambda LCDR1 DNA SEQ ID NO:959 TPP-2936919571-hIgG1wtlambda LCDR2 DNA SEQ ID NO:960 TPP-2936919571-hIgG1wtlambda LCDR3 DNA SEQ ID NO:961 TPP-2936919571-hIgG1wtlambda HC PRT SEQ ID NO:962 TPP-2936919571-hIgG1wtlambda LC PRT SEQ ID NO:963 TPP-2936919571-hIgG1wtlambda HC DNA SEQ ID NO:964 TPP-2936919571-hIgG1wtlambda LC DNA SEQ ID NO:965

Definitions

Unless otherwise defined, all scientific and technical terms usedin the description, figures and claims have their ordinary meaningas commonly understood by one of ordinary skill in the art. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, includingdefinitions, will prevail. If two or more documents incorporated byreference include conflicting and/or inconsistent disclosure withrespect to each other, then the document having the later effectivedate shall control. Where reference to a database is made, theeffective data shall be the version number applicable 26 May 2021,if not indicated otherwise. The materials, methods, and examplesare illustrative only and are not intended to be limiting. Unlessstated otherwise, the following terms used in this document,including the description and claims, have the definitions givenbelow.

The expression "about" or ".about." as used herein refers to avalue being within an acceptable error range for the particularvalue as determined by one of ordinary skill in the art, which willdepend in part on how the value is measured or determined, i.e., onthe limitations of the measurement system. For example, "about" canmean within 1 or more than 1 standard deviation, per the practicein the art. The term "about" is also used to indicate that theamount or value in question may be the value designated or someother value that is approximately the same. The phrase is intendedto convey that similar values promote equivalent results or effectsas described herein. In this context "about" may refer to a rangeabove and/or below of up to 10%. Wherever the term "about" isspecified for a certain assay or embodiment, that definitionprevails for the particular context.

The terms "comprising", "including", "containing", "having" etc.shall be read expansively or open-ended and without limitation. Theterm comprising when used in the specification includes "consistingof".

Singular forms such as "a", "an" or "the" include plural referencesunless the context clearly indicates otherwise. Thus, for example,reference to "a monoclonal antibody" includes a single monoclonalantibody as well as a plurality of monoclonal antibodies, eitherthe same or different. Likewise reference to "cell" includes asingle cell as well as a plurality of cells.

Unless otherwise indicated, the term "at least" preceding a seriesof elements is to be understood to refer to every element in theseries. The terms "at least one" and "at least one of" include forexample, one, two, three, four, five or more elements.

It is furthermore understood that slight variations above and belowa stated range can be used to achieve substantially the sameresults as a value within the range. Also, unless indicatedotherwise, the disclosure of ranges is intended as a continuousrange including every value between the minimum and maximumvalues.

The term "amino acid" or "amino acid residue" as used hereintypically refers to a naturally-occurring amino acid. The oneletter code is used herein to refer to the respective amino acid.As used herein, a "charged amino acid" is an amino acid which isnegatively charged or positively charged. "Negatively charged aminoacids" are aspartic acid (D) and glutamic acid (E). "Positivelycharged amino acids" are arginine (R) lysine (K) and histidine (H)."Polar amino acids" are all amino acids that form hydrogen bonds asdonors or acceptors. These are all charged amino acids andasparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y) and cysteine (C). "Polar uncharged amino acids" are asparagine(N), glutamine (Q), serine (S), threonine (T), tyrosine (Y) andcysteine (C). "Amphiphatic amino acids" are tryptophan (W),tyrosine (Y) and methionine (M). "Aromatic amino acids" arephenylalanine (F), tyrosine (Y), and tryptophan (W). "Hydrophobicamino acids" are glycine (G), alanine (A), valine (V), leucine (L),isoleucine (I), proline (P), phenylalanine (F), methionine (M) andcysteine. "Small amino acids" are glycine (G), alanine (A), serine(S), proline (P), threonine (T), aspartic acid (D) and asparagine(N).

As used herein, the terms "peptide", "polypeptide", and "protein"are used interchangeably, and refer to a compound comprised ofamino acid residues covalently linked by peptide bonds. A proteinor peptide must contain at least two amino acids, and no limitationis placed on the maximum number of amino acids. Polypeptidesinclude any peptide or protein comprising two or more amino acidsjoined to each other by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to inthe art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. "Polypeptides" include,for example, biologically active fragments, substantiallyhomologous polypeptides, oligopeptides, homodimers, heterodimers,variants of polypeptides, modified polypeptides, derivatives,analogs, fusion proteins, among others. The polypeptides includenatural peptides, recombinant peptides, synthetic peptides, or acombination thereof.

Where generic reference is made to a gene or protein from a certainspecies such as mouse, the analogue from human shall likewise bemeant, if not stated otherwise or obviously incompatible. Thisholds in particular in the context of biomarkers.

The term "isolated" when applied to a nucleic acid, polypeptide,protein or antibody, denotes that the nucleic acid, polypeptide,protein or antibody is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state. It can be in either a dry oraqueous solution. Purity and homogeneity are typically determinedusing analytical chemistry techniques such as polyacrylamide gelelectrophoresis or high-performance liquid chromatography. Aprotein, polypeptide or antibody that is the predominant speciespresent in a preparation is substantially purified. In particular,an isolated gene is separated from open reading frames that flankthe gene and encode a protein other than the gene of interest. Anisolated polypeptide may however be immobilized, e.g. on beads orparticles, e.g. via a suitable linker.

The term "purified" denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

As used herein, the term "synthetic", with reference to, forexample, a synthetic nucleic acid molecule or a synthetic gene or asynthetic peptide refers to a nucleic acid molecule or polypeptidemolecule that is produced by recombinant methods and/or by chemicalsynthesis methods. As used herein, production by recombinant meansby using recombinant DNA methods means the use of the well-knownmethods of molecular biology for expressing proteins encoded bycloned DNA.

"Post-translational modification(s)" (PTM) refer to the covalentmodification(s) of peptides or proteins, which are introducedfollowing protein biosynthesis under natural conditions. The termincludes without limitation glycosylation, phosphorylation,acylation, adenylation, farnesylation, ubiquitination, andsulfation. Post-translational modifications may influence theactivity of peptides or proteins. In 2004, Gutierrez et al. havedescribed the sulfation and glycosylation state of the murine CCR8chemokine receptor, and the way in which these post-translationalmodifications affect CCR8 activity. They suggest that tyrosines atpositions 14 and 15 in mouse CCR8 are sulfated amino acid residues,whereas asparagine 8 and threonines 10 and 12 are glycosylated.Furthermore, they show that the sulfations are important for theactivity of CCR8 (Gutierrez, Julio, et al. "Analysis ofpost-translational CCR8 modifications and their influence onreceptor activity." Journal of Biological Chemistry 279.15 (2004):14726-14733.).

A "sulfation" is a posttranslational modification where a sulfategroup is added to an amino acid such as a tyrosine residue of apolypeptide or protein. Tyrosine sulfation occurs in allmulticellular organisms. Under physiological conditions it iscatalyzed by tyrosylprotein sulfotransferases (TPSTs) 1 and 2,Golgi-resident enzymes which transfer sulfate from the cofactorPAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependenttyrosine in a protein substrate. Synthetic sulfation of tyrosinemay be performed with a technique known in the art, e.g. asdescribed in Bunschoten, Anton, et al. "A general sequenceindependent solid phase method for the site specific synthesis ofmultiple sulfated-tyrosine containing peptides." ChemicalCommunications 21 (2009): 2999-3001. A sulfated polypeptide is apolypeptide comprising at least one sulfation. A non-sulfatedpolypeptide is a polypeptide comprising no sulfation.

A "tyrosine rich domain" (TRD) is a conserved domain whichcharacterizes seven transmembrane proteins such as CXC and CCchemokine receptors. The TRD is typically located at the N terminusof the chemokine receptor and is typically linked to a LID domainvia a cysteine, cf. FIG. 2B. Thus, as used herein, the term TRDrefers to the amino acid or protein sequence of a CXC or CCchemokine receptor which is located N terminal of the firstcysteine counted from the N terminus. The TRD may or may notcomprise a signal peptide. The TRD may or may not be modified.Besides tyrosine, a TRD often comprises negatively charged aminoacid residues such as aspartic acid. TRDs have been proposed to beimportant structures for the interaction of chemokine receptorswith their endogenous ligands. Under physiological conditions, thetyrosine residues in a TRD can be sulfated, non-sulfated orpartially sulfated. The specific sequences for the TRDs of therespective chemokine receptors are provided in Table 4.1. However,it is obvious that mutations may be introduced in the TRD sequencewithout changing the overall charge and interaction pattern.Preferably, a TRD has at least a 90%, 95% or 99% sequence identityor sequence similarity with at least one TRD sequence according toTable 4.1.

The term "N terminus" or "N term" of a chemokine receptor as usedherein refers to the N terminal amino acids of the chemokinereceptor comprising at least the TRD. Where a polypeptide orprotein comprises a signal peptide, the N terminus may also referto the N terminal sequence behind the natural cleavage site of thepolypeptide or protein. According to some preferred embodiments,the N terminus comprises the LID domain and the TRD domain of achemokine receptor but does not comprise the natural cysteinebetween these two domains. Instead, the cysteine can be removed orcan be replaced by a different amino acid.

The "LID" domain of a chemokine receptor as used herein refers toan amino acid sequence located C terminal of the TRD of thechemokine receptor. TRD and LID domain are typically separated by asingle cysteine.

"Sequence identity" or "percent identity" is a number thatdescribes how similar a query sequence is to a target sequence,more precisely how many characters in each sequence are identicalafter alignment. The most popular tool to calculate sequenceidentity is BLAST (basic local alignment search tool,blast.ncbi.nlm.nih.gov), which performs comparisons between pairsof sequences, searching for regions of local similarity. Suitablealignment methods are known in the art, e.g. Needleman-Wunschalgorithm for global-global alignment, using BLOSUM62 matrix, withgap opening penalty of 11 and a gap extension penalty of 1.Afterwards, the pairs of aligned identical residues can be countedand then divided by the total length of the alignment (includinggaps, internal as well as external) to arrive at the percentidentity value.

For "percent similarity" or "sequence similarity" values, the sameapproach as for percent identity values can be used, except thatwhat is counted, instead of pairs of identical residues, is thealigned residue pairs with BLOSUM62 values that are not negative(i.e., .gtoreq.0).

"Seven transmembrane receptors" (7-TM receptors) are integralmembrane proteins that contain seven membrane-spanning helices. Asused herein, 7-TM receptors are G protein-coupled receptors.

"Chemokine receptors" are seven transmembrane receptors. Thechemokine receptor family contains 24 members in humans and can besubdivided, based on the class of chemokines they bind, into foursubfamilies: CX3CR, CXCR, CCR, and XCR, all of them activating Gproteins, and ACKR, containing 6 atypical receptors, unable toactivate G proteins upon ligand binding

"CXC chemokine receptors" (CXCR) are integral membrane proteinsthat specifically bind and respond to cytokines of the CXCchemokine family. They represent one subfamily of chemokinereceptors, a large family of G protein-linked receptors that areknown as seven transmembrane (7-TM) proteins, since they span thecell membrane seven times. There are currently seven known CXCchemokine receptors in mammals, named CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, and CXCR6. CXCR6 is more closely related in structure to CCchemokine receptors than to other CXC chemokine receptors.

"CC chemokine receptors" (CCR, also beta chemokine receptors) areintegral membrane proteins that specifically bind and respond tocytokines of the CC chemokine family. They represent one subfamilyof chemokine receptors, a large family of G protein-linkedreceptors that are known as seven transmembrane (7-TM) proteinssince they span the cell membrane seven times. The subfamily of theCC chemokine receptors comprises CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9 and CCR10.

The term "CCR8" refers to the C-C chemokine receptor type 8. TheCCR8 protein is encoded by the gene CCR8 (NCBI gene ID 1237).Synonyms for CCR8 are inter alia CC-CKR-8, CCR-8, CDw198, CKRL1,CMKBR8, CMKBRL2, GPRCY6, CY6, TER1. The CCR8 protein compriseshuman, murine, rat, Rhesus macaque and further mammalian andnon-mammalian homologues. Sequence(s) for human CCR8 are accessiblevia UniProt Identifier P51685 (CCR8 HUMAN), for instance humanisoform P51685-1 or P51685-2 (UniProt, Nov. 29, 2019). Sequence(s)for murine CCR8 are accessible via UniProt Identifier P56484(CCR8_MOUSE). Sequence(s) for Rhesus macaque CCR8 are accessiblevia UniProt Identifier 097665 (CCR8_MACMU). Different isoforms andvariants may exist for the different species and are all comprisedby the term CCR8. Also comprised are CCR8 molecules before andafter maturation, i.e., independent of cleavage of one or morepro-domains. In addition, synthetic variants of the CCR8 proteinmay be generated and are comprised by the term CCR8. The proteinCCR8 may furthermore be subject to various modifications, e.g.,synthetic or naturally occurring modifications, such as posttranslational modifications. Recombinant human CCR8 is commerciallyavailable or can be manufactured as known in the art. CCR8 is areceptor for the chemokine CCL1/SCYA1/I-309. Barington et al. havereported the importance of conserved extracellular disulfidebridges and aromatic residues in extracellular loop 2 (ECL-2) forligand binding and activation in the chemokine receptor CCR8(Barington, Line, et al. "Role of conserved disulfide bridges andaromatic residues in extracellular loop 2 of chemokine receptorCCR8 for chemokine and small molecule binding." Journal ofBiological Chemistry 291.31 (2016): 16208-16220.). Furthermore,they found that two distinct aromatic residues in ECL-2, Tyr184(Cys+1) and Tyr187 (Cys+4), were crucial for binding of the CCchemokines CCL1 (agonist) and MC148 (antagonist), respectively, butnot for small molecule binding.

"Programmed Death-1 (PD-1)" refers to an immunoinhibitory receptorbelonging to the CD28 family. PD-1 is expressed predominantly onpreviously activated T cells in vivo and binds to two ligands,PD-L1 and PD-L2. The term "PD-1" as used herein includes withoutlimitation human PD-1 (hPD-1), variants, isoforms, and specieshomologs of hPD-1, and analogs having at least one common epitopewith hPD-1. The complete hPD-1 sequence can be found under GenBankAccession No. U64863 (Nov. 29, 2019).

"Programmed Death Ligand-1 (PD-L1)" is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding toPD-1. The term "PD-L1" as used herein includes without limitationhuman PD-L1 (hPDL1), variants, isoforms, and species homologs ofhPD-L1, and analogs having at least one common epitope with hPD-L1.The complete hPD-L1 sequence can be found under GenBank AccessionNo. Q9NZQ7 (Nov. 29, 2019).

"Tumor Proportion Score" (TPS) is the percentage of viable tumorcells showing partial or complete membrane staining at anyintensity. For example, a specimen should be considered to havePD-L1 expression if TPS.gtoreq.1% and high PDL1 expression ifTPS.gtoreq.50%. For example, PD-L1 protein expression in NSCLC isusually determined by using Tumor Proportion Score (TPS).

"Combined Positive Score" (CPS) is the number of staining cells(tumor cells, lymphocytes, macrophages) divided by the total numberof viable tumor cells, multiplied by 100. For example, a specimenshould be considered to have PD-L1 expression if CPS.gtoreq.1 andto have high PD-L1 expression if CPS.gtoreq.10. The FDA hasapproved the use of the PD-L1 IHC 22C3 pharmDx assay to determine apatient's eligibility for therapeutic antibody pembrolizumab.

"FOXP3" is a 50-55 kD transcription factor, also known as Forkheadbox protein P3, Scurfin, JM2, or IPEX. It is proposed to be amaster regulatory gene and more specific marker of T regulatorycells than most cell surface markers. Transduced expression ofFOXP3 in CD4+/CD25- cells has been shown to induce GITR, CD103, andCTLA4 and impart a T regulatory cell phenotype. Biolegend antibodyclones 206D and 259D recognize a human FOXP3 epitope in the regionof amino acids 105-235. Poly6238 recognizes both human and mouseFOXP3 and was raised against the N-terminal portion of FOXP3.

The term "modulation" refers to any alteration of an existingprocess or behavior, such as blocking (antagonism) and induction(agonism). For example, modulation of G protein independentsignaling refers to any significant alteration of G-proteinindependent signaling.

The term "internalization" of an antibody, fragment or conjugaterefers to the uptake of the antibody, fragment or conjugate into acell. Preferably, internalization is determined for a cell linewith endogenous target expression, e.g. as described elsewhereherein for human or murine CCR8. Preferably, internalization isdetermined by measuring total internalized fluorescence intensityper cell and is quantified relative to an isotype control, e.g. asdescribed in example 10.5. In brief, the antibody, fragment orconjugate and a matching isotype control are labeled with a dye andinternalized fluorescence is determined and quantified for theantibody, fragment or conjugate relative to the isotype control. A"non-internalizing antibody" is defined as an antibody showingsubstantially the same internalization as a corresponding isotypecontrol. A "low internalizing antibody" is defined as an antibodyshowing an internalization which is equal to or lower than the10-fold of the internalization of the isotype control, preferablylower than the 9-, 8-, 7-, 6-, 5-, 4-, 3-, 2-, 1.5-, 1.4-, 1.3-,1.2-, or 1.1-fold of the internalization of the isotype control. A"medium internalizing antibody" is defined as an antibody showingan internalization which is equal to or lower than the 21-fold ofthe internalization of the isotype control and higher than the10-fold of the internalization of the isotype control. A "highinternalizing antibody" is defined as an antibody showing aninternalization which is higher than the 21-fold of theinternalization of the isotype control.

In the alternative, internalization can furthermore be quantifiedbased on t(1/2), i.e. as time until half of the amount of antibody,fragment or conjugate has been internalized. Preferably, antibodiesaccording to the current invention are characterized by a timeuntil half of the amount of antibody, fragment or conjugate hasbeen internalized which is >2 hours, preferably >4, >5,>6, >7, >8, >9, >10, >11, >12, >13, >14,>15, >16, >17, >18, >19, >20, >21, >22,>23, >24, >26, >28, >30, or >48 hours. Mostpreferably, antibodies according to the current invention are notinternalized at all, i.e., no time can be defined until which halfof the amount of antibody, fragment or conjugate has beeninternalized. An "isotype control" is an antibody or fragment thatdoes not bind a target but has the same class and type as thereference antibody or fragment recognizing the target.

An antibody or fragment is termed "cross-reactive" or "crossreactive" if the antibody or fragment binds an antigen from two ormore different species, e.g. with a KD value of 10-7 M or less,more preferably of less than 10-8 M, even more preferably in therange from 10-9 M to 10-11 M.

By the term "specifically binds" as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen,but does not substantially recognize or bind other molecules in asample: An antibody characterized by substantial unspecific bindingwould lack therapeutic applicability, such that these embodimentsare excluded. However, as known in the art, specific binding of anantibody or binder does not necessarily exclude an antibody orbinder binding to further antigens/target molecules. An antibodythat specifically binds to an antigen from one species may alsobind to that antigen from one or more further species. Suchcross-species reactivity does not itself alter the classificationof an antibody as specific.

In some instances, the terms "specific binding" or "specificallybinding" can be used in reference to the interaction of anantibody, a protein, or a peptide with a second chemical species,to mean that the interaction is dependent upon the presence of aparticular structure (e.g., an antigenic determinant or epitope) onthe chemical species; for example, an antibody recognizes and bindsto a specific protein structure rather than to proteins generally.If an antibody is specific for epitope "A", the presence of amolecule containing epitope A (or free, unlabeled A), in a reactioncontaining labeled "A" and the antibody, will reduce the amount oflabeled A bound to the antibody.

In case of doubt, specific binding of an antibody or binderpreferably describes binding of an antibody, antibody fragment orbinder to its antigen/target with an affinity of at least 10-7 M(as KD value; i.e. preferably those with KD values smaller than10-7 M), with the antibody or binder having an at least two timeslower affinity for a non-specific antigen which is not thepredetermined antigen/target molecule or a closely relatedantigen/target molecule.

"Polyspecificity", also "polyreactivity" or "unspecific binding"refers to the binders' or antibodies' ability to bind a defined setof unrelated antigens. Unspecific binding is substantial, if the(therapeutic) applicability of the antibody is compromised.Polyspecificity for non-protein structures including withoutlimitation target negative cell lines or tissues, baculo virusparticle (BVP), insulin or DNA, may be evaluated as known in theart and as described herein. For example, unspecific binding totarget negative human cell lines can be determined e.g. by FACSanalysis using mock transfected CHO or HEK cells. In a secondexample, unspecific binding to different tissues can be analyzed byFACS analysis of a cell line or panel of cell lines derived fromthe respective tissue. In a third example, unspecific binding toimmune cell populations can be analyzed by FACS after sorting theimmune cell populations as known in the art. In a fourth example,unspecific binding to BVP, insulin or DNA can be analyzed usingELISA, e.g. as described in Hotzel, Isidro, et al. "A strategy forrisk mitigation of antibodies with fast clearance." MAbs. Vol. 4.No. 6. Taylor & Francis, 2012.; Avery, Lindsay B., et al."Establishing in vitro in vivo correlations to screen monoclonalantibodies for physicochemical properties related to favorablehuman pharmacokinetics." MAbs. Vol. 10. No. 2. Taylor &Francis, 2018, and Jain, Tushar, et al. "Biophysical properties ofthe clinical-stage antibody landscape." Proceedings of the NationalAcademy of Sciences 114.5 (2017): 944-949, incorporated herein intheir entirety and in particular with regards to the technicaldetails necessary to analyze and quantify unspecific binding. Anantibody without substantial unspecific binding is preferablycharacterized by an unspecific binding that is lower thanunspecific binding of reference antibody Gantenerumab (Roche) andmost preferably lower than unspecific binding of reference antibodyRemicade (Janssen Biotech).

The term "off target binding" refers to the ability of an antibodyto bind individual proteins different from the intended target, forexample proteins of the targets' protein family. Off target bindingmay be evaluated using commercial assays known in the art such asthe Retrogenix off target profiling assay. In brief, antibodies aretested on microarrays containing HEK293 cells individuallyexpressing several thousand human membrane proteins and secretedproteins. Binding of the antibody to a potential off target has tobe confirmed by FACS using cells overexpressing the potential offtarget.

The term "affinity" is a term of the art and describes the strengthof binding between a binder, antibody or antibody fragment and atarget. The "affinity" of antibodies and fragments thereof for atarget can be determined using techniques well known in the art ordescribed herein, for example by ELISA, isothermal titrationcalorimetry (ITC), surface plasmon resonance (SPR), flow cytometryor fluorescent polarization assays. Preferably the affinity isprovided as dissociation constant KD.

The "dissociation constant" (KD) has molar units (M) andcorresponds to the concentration of the binder/antibody at whichhalf of the target proteins are occupied at equilibrium. Thesmaller the dissociation constant is, the higher is the affinitybetween the binder or antibody and its target. According to thecurrent invention, the antibodies preferably have a target affinityof at least 10-7 M (as KD value), more preferably of at least 10-8M, even more preferably in the range from 10-9 M to 10-11 M. The KDvalues can be preferably determined by means of surface plasmonresonance spectroscopy, e.g. as described elsewhere herein. Whereassay conditions were found to influence the determined KD, theassay setup with the least standard deviation shall be used.

"Half maximal effective concentration" (EC50) refers to theconcentration of a drug, antibody, fragment, conjugate or moleculewhich induces a response halfway between the baseline and maximumafter a specified incubation time. In the context of antibodybinding, the EC50 thus reflects the antibody concentration neededfor half-maximal binding. An EC50 can be determined if aninflection point can be determined by mathematical modeling (e.g.,non-linear regression) of the dose-response curve describing therelationship between applied drug, antibody, fragment, conjugate ormolecule concentration and signal. For example, if thedose-response curve follows a sigmoidal curve, an EC50 can bedetermined. Where the response is an inhibition, the EC50 is termedhalf maximal inhibitory concentration (IC50). EC80 can bedetermined mutatis mutandis.

The term "antibody" (Ab) refers to an immunoglobulin molecule (e.g.without limitation human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE,IgA1, IgA2, mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE orIgM, rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE or IgM, rabbitIgA1, IgA2, IgA3, IgE, IgG, IgM, goat IgA, IgE, IgG1, IgG2, IgE,IgM or chicken IgY) that specifically binds to, or isimmunologically reactive with, a particular antigen. Antibodies orantibody fragments comprise complementarity determining regions(CDRs), also known as hypervariable regions, in both the lightchain and heavy chain variable domains. The more highly conservedportions of the variable domains are called the framework (FR). Asis known in the art, the amino acid position/boundary delineating ahypervariable region of an antibody can vary, depending on thecontext and the various definitions known in the art. As usedherein, numbering of immunoglobulin amino acid residues is doneaccording to the immunoglobulin amino acid residue numbering systemof Kabat et al. The variable domains of native heavy and lightchains each comprise four FR regions. The three CDRs in each chainare held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies, see Kabat, E. A., et al."Sequences of Proteins of Immunological Interest (Natl. Inst.Health, Bethesda, Md.), GPO Publ." No 165-462 (1987). The termantibody as used herein also refers to antibody fragments, exceptwhere explicitly stated otherwise. Depending on the respectivecontext, the term antibody may also refer to any proteinaceousbinding molecule with immunoglobulin-like function.

The term "CDR" refers to the complementary determining region ofthe antibody. As known in the art complementarity-determiningregions (CDRs) are part of the variable chains in antibodies and Tcell receptors. A set of CDRs constitutes a paratope. CDRs arecrucial to the diversity of antigen specificities. There are threeCDRs (CDR1, CDR2 and CDR3), arranged non-consecutively on the aminoacid sequence of a variable domain of an antigen receptor. Sincethe antigen receptors are typically composed of two variabledomains (on two different polypeptide chains, heavy and lightchain), there are usually six CDRs for each antigen receptor thatcan collectively come into contact with the antigen. The CDRs ofthe light chain are LCDR1, LCDR2 and LCDR3. The CDRs of the heavychain are termed HCDR1, HCDR2 and HCDR3. HCDR3 is the most variablecomplementary determining region (see, e.g., Chothia, Cyrus, andArthur M. Lesk. "Canonical structures for the hypervariable regionsof immunoglobulins." Journal of molecular biology 196.4 (1987):901-917.; Kabat, E. A., et al. "Sequences of proteins ofimmunological interest. Bethesda, Md.: US Department of Health andHuman Services." Public Health Service, National Institutes ofHealth (1991): 103-511.).

The "constant region" refers to the portion of the antibodymolecule that confers effector functions. The heavy chain constantregion can be selected from any of the five isotypes: alpha (a),delta (6), epsilon (c), gamma (g), or mu (O.

The term "Fc domain", "Fc region" or "Fc part" as used hereinrefers to a C-terminal region of an antibody heavy chain thatcontains at least a portion of the constant region. The termincludes native sequence Fc regions and variant Fc regions. Forexample, a human IgG heavy chain Fc region may extend from Cys226,or from Pro230, to the carboxyl-terminus of the heavy chain.

Antibodies or binding fragments according to the current inventionmay have been modified to alter at least one constantregion-mediated biological effector function. For example, in someembodiments, an antibody may be modified to reduce or enhance atleast one constant region-mediated biological effector functionrelative to the unmodified antibody, e.g., reduced or improvedbinding to the Fc receptor (Fc.gamma.R). Fc.gamma.R binding may bereduced, e.g. by mutating the immunoglobulin constant regionsegment of the antibody at particular regions necessary forFc.gamma.R interactions (see, e.g., Canfield, Stephen M., andSherie L. Morrison. "The binding affinity of human IgG for its highaffinity Fc receptor is determined by multiple amino acids in theCH2 domain and is modulated by the hinge region." The Journal ofexperimental medicine 173.6 (1991): 1483-1491; and Lund, John, etal. "Human Fc gamma RI and Fc gamma RII interact with distinct butoverlapping sites on human IgG." The Journal of Immunology 147.8(1991): 2657-2662.). Fc.gamma.R binding may be enhanced, e.g. byafucosylation. Reducing Fc.gamma.R binding may also reduce othereffector functions which rely on Fc.gamma.R interactions, such asopsonization, phagocytosis and antigen-dependent cellularcytotoxicity ("ADCC").

Furthermore, addressing the interaction of Fc with FcRn allows tomodulate the half-life of antibodies in vivo. Abrogating theinteraction by e.g. introduction of mutation H435A leads to anextremely short half-life, since the antibody is no longerprotected from lysosomal degradation by FcRn recycling. In somepreferred embodiments according to all aspects, the antibodyaccording to the current invention comprises mutation H435A or hasotherwise been engineered for a reduced half-life.

In contrast, antibodies comprising "YTE" mutations(M252Y/S254T/T256E) and/or equivalent mutations such as "LS"mutations (M428L/N434S) have been shown to significantly extend thehalf-life by more efficient recycling from endosomes in bothpre-clincal species as well as humans (Dall'Acqua, William F., etal. "Increasing the affinity of a human IgG1 for the neonatal Fcreceptor: biological consequences." The Journal of Immunology 169.9(2002): 5171-5180.; Zalevsky, Jonathan, et al "Enhanced antibodyhalf-life improves in vivo activity." Nature biotechnology 28.2(2010): 157-159.). In some preferred embodiments according to allaspects, the antibody according to the current invention comprisesYTE mutations (M252Y/S254T/T256E) and/or equivalent mutations suchas LS (M428L/N434S) or has otherwise been engineered for animproved half-life. Suitable Fc engineering approaches forextension of half-life can be found in Haraya, Kenta, TatsuhikoTachibana, and Tomoyuki Igawa. "Improvement of pharmacokineticproperties of therapeutic antibodies by antibody engineering." Drugmetabolism and pharmacokinetics 34.1 (2019): 25-41., and/or Lee,Chang-Han, et al. "An engineered human Fc domain that behaves likea pH-toggle switch for ultra-long circulation persistence." Naturecommunications 10.1 (2019): 1-11., both incorporated herein byreference.

"Afucosylated" antibodies are antibodies engineered such that theoligosaccharides in the Fc region of the antibody do not have anyfucose sugar units. Glycosylation of an antibody can alter itsfunction. For example, if glycosylation at N297 in the CH2 domainof an IgG is completely eliminated, binding to Fc.gamma.Rs is lost.However, modulation of the specific carbohydrate composition atN297 can have the opposite effect and enhance the ADCC activity ofthe antibody. In brief, the affinity of an antibody for theactivating Fc.gamma.Rs depends on the composition of the N297N-linked oligosaccharide. There are 32 different possiblecombinations of oligosaccharides that can occur at this site.Naturally occurring human IgG and those produced by hybridomas orother common expression systems are usually composed ofN-acetylglucosamine (GlcNAc) and three mannose residues that form acore carbohydrate. This core is attached to two additional GlcNAcgroups to form biantennary branches. The addition of galactose ateach branch can occur as well as the terminal addition of sialicacid to these galactose molecules. Fucose is often part of the coreGlcNAc. This fucose, through steric hindrance, obstructs theinteraction of the antibody with the Fc.gamma.RIIIA. Thus,elimination of this fucose molecule while maintaining other formsof glycosylation at this site increases the binding of the antibodyto the activating Fc.gamma.Rs, enhancing its ability to elicit ADCCand/or ADCP (Almagro, Juan C., et al. "Progress and challenges inthe design and clinical development of antibodies for cancertherapy." Frontiers in immunology 8 (2018): 1751.). Methods ofpreparing fucose-less antibodies include growth in rat myelomaYB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8mRNA, which encodes .alpha.-1,6-fucosyltransferase, an enzymenecessary for fucosylation of polypeptides. Afucosylated antibodiesare preferred for the current invention.

"Antibody-dependent cellular cytotoxicity" ("ADCC"), also referredto as "antibody-dependent cell-mediated cytotoxicity", is amechanism of cell-mediated immune defense whereby an immune cellactively lyses a target cell, whose membrane-surface antigens havebeen bound by specific antibodies. ADCC is mediated via interactionof the antibody or fragment with Fc.gamma.RIIIa. In humans,Fc.gamma.RIII exists in two different forms: Fc.gamma.RIIIa (CD16a)and Fc.gamma.RIIIb (CD16b). While Fc.gamma.RIIIa is expressed onmonocytes, neutrophils, mast cells, macrophages, and natural killercells as a transmembrane receptor, Fc.gamma.RIIIb is only expressedon neutrophils. These receptors bind to the Fc portion of IgGantibodies, which then activates antibody-dependent cell-mediatedcytotoxicity (ADCC) mediated by the human effector cells.

Different assay systems to determine ADCC induction in humansubjects have been described in the literature and are suitable forcharacterization of the subject matter disclosed herein. Forexample, Yao-Te Hsieh et al. have studied different ADCC assaysystems, namely assays based on (i) natural killer cells from humandonors (Fc.gamma.RIIIA+primary NK), (ii) Fc.gamma.RIIIA engineeredNK-92 cells and (iii) Fc.gamma.RIIIA/NFAT-RE/luc2 engineered JurkatT cells (Hsieh, Yao-Te, et al. "Characterization of Fc.gamma.RIIIAeffector cells used in in vitro ADCC bioassay: comparison ofprimary NK cells with engineered NK-92 and Jurkat T cells." Journalof Immunological Methods 441 (2017): 56-66, incorporated herein inentirety; in particular, reference is made to the methoddescription for these assays). In brief, all three effector cellsystems differentially express Fc.gamma.RIIIA and providedose-dependent ADCC pathway activity, yet only primary NK andengineered NK-92 cells are capable of inducing ADCC-mediated celllysis. For functional assessment of ADCC activity, primary NK orNK-92 (V-158) cells thus better reflect the physiologicallyrelevant ADCC mechanism of action. As an engineered cell line,NK-92 cells may behave more reproducibly than primary NK and istherefore the preferred assay system to determine ADCC response inhuman subjects, e.g. in case of doubt.

An antibody or antigen-binding fragment inducing ADCC is anantibody which may elicit a substantial amount of lysis of targetcells in the presence of NK effector cells. Preferably, the ADCCinduction results in the lysis of at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 99% of the target cells.

"Antibody-dependent cellular phagocytosis" ("ADCP") is themechanism by which antibody-opsonized target cells activate theFc.gamma.Rs on the surface of macrophages to induce phagocytosis,resulting in internalization and degradation of the target cell.For ADCP, binding to macrophages as effector cells typically occursvia the interaction of the antibodies FC part with Fc.gamma.RIIa(CD32a) expressed by macrophages.

An antibody or antigen-binding fragment inducing ADCP is anantibody which may elicit a substantial amount of phagocytosis oftarget cells in the presence of macrophages. Preferably, the ADCPinduction results in the phagocytosis of at least 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% of the target cells. "Complement-dependentcytotoxicity" ("CDC") is an effector function of IgG and IgMantibodies.

When they are bound to a surface antigen on a target cell (e.g.bacterial or viral infected cell), the classical complement pathwayis triggered by bonding protein C1q to these antibodies, resultingin formation of a membrane attack complex (MAC) and target celllysis. Complement system is efficiently activated by human IgG1,IgG3 and IgM antibodies, weakly by IgG2 antibodies and is notactivated by IgG4 antibodies. It is one mechanism of action bywhich therapeutic antibodies--also specific embodiments of theantibodies according to the current invention--can achieve anantitumor effect. Several laboratory methods exist for determiningthe efficacy of CDC and are known in the art.

An antibody or antigen-binding fragment inducing CDC is an antibodywhich may elicit a substantial amount of formation of a membraneattack complex and lysis of target cells. Preferably, the CDCinduction results in the lysis of at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 99% of the target cells.

Antibodies comprising an Fc region may or may not comprise amodification promoting the association of the first and the secondsubunit of the Fc domain.

A "modification promoting the association of the first and thesecond subunit of the Fc domain" is a manipulation of the peptidebackbone or the post-translational modifications of an Fc domainsubunit that reduces or prevents the association of a polypeptidecomprising the Fc domain subunit with an identical polypeptide toform a homodimer. Antibodies comprising an Fc region may or may notcomprise a modification promoting the association of the first andthe second subunit of the Fc domain. A modification promotingassociation as used herein particularly includes separatemodifications made to each of the two Fc domain subunits desired toassociate (i.e. the first and the second subunit of the Fc domain),wherein the modifications are complementary to each other so as topromote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure orcharge of one or both of the Fc domain subunits so as to make theirassociation sterically or electrostatically favorable. Thus,(hetero)dimerization occurs between a polypeptide comprising thefirst Fc domain subunit and a polypeptide comprising the second Fcdomain subunit, which might be non-identical, e.g. in the sensethat further components fused to each of the subunits (e.g. antigenbinding moieties) are not the same. In some embodiments themodification promoting association comprises an amino acid mutationin the Fc domain, specifically an amino acid substitution. In aparticular embodiment, the modification promoting associationcomprises a separate amino acid mutation, specifically an aminoacid substitution, in each of the two subunits of the Fcdomain.

A "fragment" of an antibody as used herein is required tosubstantially retain the desired affinity of the full-lengthantibody. As such, suitable fragments of an anti-human CCR8antibody will retain the ability to bind to the target chemokinereceptor, e.g. to bind to human CCR8 receptor. Fragments of anantibody comprise a portion of a full-length antibody, generallythe antigen binding or variable region thereof. Examples ofantibody fragments include, but are not limited to, Fab, Fab',F(ab')2, and Fv fragments, single-chain antibody molecules,diabodies and domain antibodies, see Holt, Lucy J., et al. "Domainantibodies: proteins for therapy." Trends in biotechnology 21.11(2003): 484-490.

A "Fab fragment" contains the constant domain of the light chainand the first constant domain (CH2) of the heavy chain.

"Fab' fragments" differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH2 domainincluding one or more cysteines from the antibody hinge region.

"F(ab) fragments" are produced by cleavage of the disulfide bond atthe hinge cysteines of the F(ab')2 pepsin digestion product.Additional chemical couplings of antibody fragments are known tothose of ordinary skill in the art. Fab and F(ab')2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation of animals, and may have less non-specific tissuebinding than an intact antibody, see, e.g., Wahl, Richard L.,Charles W. Parker, and Gordon W. Philpott. "Improved radioimagingand tumor localization with monoclonal F (ab') 2." Journal ofnuclear medicine: official publication, Society of Nuclear Medicine24.4 (1983): 316-325.

An "Fv fragment" is the minimum fragment of an antibody thatcontains a complete target recognition and binding site. Thisregion consists of a dimer of one heavy and one light chainvariable domain in a tight, non-covalent association (VH-VL dimer).It is in this configuration that the three CDRs of each variabledomain interact to define an antigen binding site on the surface ofthe VH-VL dimer. Often, the six CDRs confer antigen bindingspecificity upon the antibody. However, in some instances even asingle variable domain (or half of an Fv comprising only three CDRsspecific for a target) may have the ability to recognize and bindthe antigen, although at a lower affinity than the entire bindingsite.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH andVL domains of an antibody in a single polypeptide chain. Generally,the Fv polypeptide further comprises a polypeptide linker betweenthe VH and VL domains which enables the scFv to form the desiredstructure for antigen binding.

"Single domain antibodies" are composed of single VH or VL domainswhich exhibit sufficient affinity to the target. In a specificembodiment, the single domain antibody is a camelized antibody,see, e.g., Riechmann, Lutz, and Serge Muyldermans. "Single domainantibodies: comparison of camel VH and camelised human VH domains."Journal of immunological methods 231.1-2 (1999): 25-38.

"Bispecific antibodies" are monoclonal antibodies that have bindingspecificities for at least two different epitopes on the same ordifferent antigens. In the present disclosure, one of the bindingspecificities can be directed towards the target chemokine receptorsuch as CCR8, the other can be for any other antigen, e.g., withoutlimitation for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein. Bispecific antibody constructs according to the inventionalso encompass multispecific antibody constructs comprisingmultiple binding domains/binding sites, such as trispecificantibody constructs, where the construct comprises three bindingdomains.

"Derivatized antibodies" are typically modified by glycosylation,acetylation, pegylation, phosphorylation, sulfation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein. Any ofnumerous chemical modifications may be carried out by knowntechniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative may contain one ormore non-natural amino acids, e.g., using ambrx technology, see,e.g., Wolfson, Wendy. "Amber codon flashing ambrx augments proteinswith unnatural amino acids." Chemistry & biology 13.10 (2006):1011-1012.

Antibodies according to the current invention may be derivatized,e.g. glycosylated or sulfated. "Monoclonal antibodies" aresubstantially homogenous populations of antibodies binding aparticular antigen. Monoclonal immunoglobulins may be obtained bymethods well known to those skilled in the art (see for example,Kohler, Georges, and Cesar Milstein. "Continuous cultures of fusedcells secreting antibody of predefined specificity." nature256.5517 (1975): 495-497., and U.S. Pat. No. 4,376,110). Animmunoglobulin or immunoglobulin fragment with specific bindingaffinity can be isolated, enriched, or purified from a prokaryoticor eukaryotic organism. Routine methods known to those skilled inthe art enable production of both immunoglobulins or immunoglobulinfragments and proteinaceous binding molecules withimmunoglobulin-like functions, in both prokaryotic and eukaryoticorganisms. The antibodies according to the current invention arepreferably monoclonal.

"Humanized antibodies" contain CDR regions derived from a non-humanspecies, such as mouse, that have, for example, been engrafted,along with any necessary framework back-mutations, into humansequence-derived V regions. Thus, for the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region of the recipient are replacedby residues from a hypervariable region of a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primatehaving the desired specificity, affinity, and capacity. See, forexample, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762;5,859,205, each herein incorporated by reference. In someinstances, framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore,humanized antibodies may comprise residues that are not found inthe recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance(e.g., to obtain desired affinity). In general, the humanizedantibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially allof the hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the frameworkregions are those of a human immunoglobulin sequence. The humanizedantibody optionally comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones, Peter T., et al."Replacing the complementarity-determining regions in a humanantibody with those from a mouse." Nature 321.6069 (1986):522-525.; Riechmann, Lutz, et al. "Reshaping human antibodies fortherapy." Nature 332.6162 (1988): 323-327.; and Presta, Leonard G."Antibody engineering." Current Opinion in Structural Biology 2.4(1992): 593-596., each incorporated herein by reference.

Fully human antibodies (human antibodies) comprise human derivedCDRs, i.e. CDRs of human origin. Preferably, a fully human antibodyaccording to the current invention is an antibody having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%sequence identity with the closest human VH germline gene (e.g.sequence extracted from recommended list and analyzed inIMGT/Domain-gap-align).

As accepted by usual nomenclature systems such as the INN speciessubsystem in force until 2017, fully human antibodies may comprisea low number of germline deviations compared with the closest humangermline reference determined based on the IMGT database(www.imgt.org, Nov. 29, 2019). For example, a fully human antibodyaccording to the current invention may comprise up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 12, 13, 14 or 15 germline deviations in the CDRscompared with the closest human germline reference. Fully humanantibodies can be developed from human derived B cells by cloningtechniques in combination with a cell enrichment or immortalizationstep. The majority of fully human antibodies in clinical use,however, were isolated either from immunized mice transgenic forthe human IgG locus or from sophisticated combinatorial librariesby phage display (Bruggemann, Marianne, et al. "Human antibodyproduction in transgenic animals." Archivum immunologiae ettherapiae experimentalis 63.2 (2015): 101-108.; Carter, Paul J."Potent antibody therapeutics by design." Nature reviews immunology6.5 (2006): 343-357.; Frenzel, Andre, Thomas Schirrmann, andMichael Hust. "Phage display-derived human antibodies in clinicaldevelopment and therapy." MAbs. Vol. 8. No. 7. Taylor &Francis, 2016.; Nelson, Aaron L., Eugen Dhimolea, and Janice M.Reichert. "Development trends for human monoclonal antibodytherapeutics." Nature reviews drug discovery 9.10 (2010):767-774.).

Several techniques are available to generate fully human antibodiesor to generate antibodies comprising human derived CDRs (cf.WO2008112640). Cambridge Antibody Technologies (CAT) and Dyax haveobtained antibody cDNA sequences from peripheral B cells isolatedfrom immunized humans and devised phage display libraries for theidentification of human variable region sequences of a particularspecificity. Briefly, the antibody variable region sequences arefused either with the Gene III or Gene VIII structure of the M13bacteriophage. These antibody variable region sequences areexpressed either as Fab or single chain Fv (scFv) structures at thetip of the phage carrying the respective sequences. Through roundsof a panning process using different levels of antigen bindingconditions (stringencies), phages expressing Fab or scFv structuresthat are specific for the antigen of interest can be selected andisolated. The antibody variable region cDNA sequences of selectedphages can then be elucidated using standard sequencing procedures.These sequences may then be used for the reconstruction of a fullantibody having the desired isotype using established antibodyengineering techniques. Antibodies constructed in accordance withthis method are considered fully human antibodies (including theCDRs). In order to improve the immunoreactivity (antigen bindingaffinity and specificity) of the selected antibody, an in vitromaturation process can be introduced, including a combinatorialassociation of different heavy and light chains,deletion/addition/mutation at the CDR3 of the heavy and lightchains (to mimic V-J, and V-D-J recombination), and randommutations (to mimic somatic hypermutation). An example of a "fullyhuman" antibody generated by this method is the anti-tumor necrosisfactor .alpha. antibody, Humira (adalimumab). The term"polynucleotide" refers to a recombinantly or syntheticallyproduced polymeric desoxyribonucleotide or analog thereof, or amodified polynucleotide. The term comprises double and singlestranded DNA or RNA. The polynucleotide can be integrated e.g. intominicircles, plasmids, cosmids, minichromosomes, or artificialchromosomes. The polynucleotide can be isolated or integrated inanother nucleic acid molecule, e.g. in an expression vector orchromosome of a eukaryotic host cell. The term "vector", as usedherein, refers to a nucleic acid molecule capable of propagating anucleic acid molecule to which it is linked. The term furthercomprises plasmids (non-viral) and viral vectors. Certain vectorsare capable of directing the expression of nucleic acids orpolynucleotides to which they are operatively linked. Such vectorsare referred to herein as "expression vectors". Expression vectorsfor eukaryotic use can be constructed by inserting a polynucleotidesequence encoding at least one protein of interest (POI) into asuitable vector backbone. The vector backbone can comprise thenecessary elements to ensure maintenance of the vector and, ifdesirable, to provide amplification within the host. For viralvectors, e.g. lentiviral or retroviral vectors, further virusspecific elements such as structural elements or other elements canbe required and are well known in the art. These elements can befor instance provided in cis (on the same plasmid) or in trans (ona separate plasmid). Viral vectors may require helper viruses orpackaging lines for large-scale transfection. Vectors may containfurther elements such as e g enhancer elements (e.g. viral,eukaryotic), introns, and viral origins of plasmid replication forreplication in mammalian cells. According to the current invention,expression vectors typically have a promoter sequence that drivesexpression of the POI. Expression of the POI and/or selectivemarker protein may be constitutive or regulated (e.g. inducible byaddition or removal of small molecule inductors). Preferredregulatory sequences for mammalian host cell expression includeviral elements that direct high levels of expression of a POI inmammalian cells, such as regulatory elements, promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40(SV40), adenovirus, (e.g., the adenovirus major late promoter AdLP) or polyoma. For further description of viral regulatoryelements, and sequences thereof, see e.g., U.S. Pat. Nos.5,168,062, 4,510,245 and 4,968,615.

The term "linker" or "spacer" as used herein refers to any moleculeenabling a direct topological connection between two moieties. Amoiety may be inter alia a polypeptide, a protein, an antibody, anantibody fragment, a cytotoxic moiety, a binding moiety, a moietyfor detection such as a fluorophore, a moiety for immobilization orretrieval such as beads or magnetic beads, a reactive moiety, orany other molecule. The two moieties may be of the same type ordifferent. Linkers may be part of conjugates and may evencontribute to their function. For instance, for a conjugatecomprising a polypeptide and a biotin, the presence of a spacer ofapproximately 4 .ANG. (.about.5 atoms) between the carboxy group ofthe biotin and the 1st bulky amino acid of the peptide allows thebiotin to reach the (strept)avidin binding pocket. Various linkersare known in the art and can be selected based on the moietieswhich shall be connected. The linker length typically rangesbetween 4 atoms and more than 200 atoms. Linkers exceeding 60 atomsin length generally comprise a population of compounds having anaverage length.

"Linkers for polypeptides" may be attached through an amide linkageor any other functional residue. Linkers for polypeptides may beattached N-terminal or C-terminal of the polypeptide or may beattached via a reactive functional group or amino acid side chain.Polypeptides may be coupled for example to biotin, proteins such ashuman serum albumin (HSA), carrier proteins such as keyhole limpethemocyanin (KLH), ovalbumin (OVA) or bovine serum albumin (BSA),fluorescent dyes, short amino acid sequences such as Flag tag, HAtag, Myc tag or His tag, reactive tags such as maleimides,iodoacetamides, alkyl halides, 3-mercaptopropyl or 4-azidobutyricacid, or to various further suitable moieties. Non-limitingexamples for suitable linkers, e.g. for conjugation ofpolypeptides, include beta-alanine, 4-aminobutyric acid (GABA),(2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava),6-aminohexanoic acid (Ahx), PEG2 spacer (8-amino-3,6-dioxaoctanoicacid), PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), PEG4spacer (15-amino-4,7,10,13-tetraoxapenta-decanoic acid), and Ttds(Trioxatridecan-succinamic acid). In some cases, the linker may bederived from a reactive moiety, e.g. maleimides, iodoacetamides,alkyl halides, 3-mercaptopropyl or 4-azidobutyric acid. In somecases, the linker may comprise polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol or polypropylene glycol.

"Linkers for antibodies" are linkers establishing a covalentconnection between different antibody portions and include peptidelinker and non-proteinaceous polymers, including but not limited topolyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,or copolymers of polyethylene glycol, polypropylene glycol.

"Treating" a disease in a subject or "treating" a subject having adisease refers to subjecting the subject to a pharmaceuticaltreatment, e.g., the administration of a drug, such that at leastone symptom of the disease is decreased or prevented fromworsening.

The terms "prevent", "preventing", "prevention" and the like referto reducing the probability of developing a disease, disorder, orcondition in a subject, who does not have, but is at risk of orsusceptible to developing a disease, disorder, or condition.

The term "effective amount" or "therapeutically effective amount"are used interchangeably herein and refer to an amount sufficientto achieve a particular biological result or to modulate orameliorate a symptom in a subject, or the time of onset of asymptom, typically by at least about 10%; usually by at least about20%, preferably at least about 30%, or more preferably at leastabout 50%. Efficacy of the use of an antibody in cancer therapy canbe assessed based on the change in tumor burden. Both tumorshrinkage (objective response) and time to the development ofdisease progression are important endpoints in cancer clinicaltrials. Standardized response criteria, known as RECIST (ResponseEvaluation Criteria in Solid Tumors), were published in 2000. Anupdate (RECIST 1.1) was released in 2009. RECIST criteria aretypically used in clinical trials where objective response is theprimary study endpoint, as well as in trials where assessment ofstable disease, tumor progression or time to progression analysesare undertaken because these outcome measures are based on anassessment of anatomical tumor burden and its change over thecourse of the trial. An effective amount for a particular subjectmay vary depending on factors such as the condition being treated,the overall health of the subject, the method, route, and dose ofadministration and the severity of side effects. When incombination, an effective amount is in ratio to a combination ofcomponents and the effect is not limited to individual componentsalone.

If not defined otherwise, "Complete Response" (CR) is defined asdisappearance of all target lesions. Any pathological lymph nodes(whether target or non-target) must have reduction in short axis to<10 mm. For "Partial Response" (PR) at least a 30% decrease inthe sum of diameters of target lesions has to be reached, taking asreference the baseline sum diameters. For "Progressive Disease"(PD) at least a 20% increase in the sum of diameters of targetlesions, taking as reference the smallest sum on study (thisincludes the baseline sum if that is the smallest on study). Inaddition to the relative increase of 20%, the sum must alsodemonstrate an absolute increase of at least 5 mm. In "StableDisease" (SD) neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD is observed, taking asreference the smallest sum diameters while on study.

Secondary outcome measures that can be used to determine thetherapeutic benefit of the inventive antibodies described hereininclude the following: "Objective Response Rate" (ORR) is definedas the proportion of subjects who achieve a complete response (CR)or partial response (PR). "Progression Free Survival" (PFS) isdefined as the time from the first dose date of an antibody toeither disease progression or death, whichever occurs first."Overall Survival" (OS) is defined as the length of time fromeither the date of diagnosis or the start of treatment for adisease, that patients diagnosed with the disease are still alive."Duration of Overall Response" (DOR) is defined as the time fromthe participant's initial CR or PR to the time of diseaseprogression. "Depth of Response" (DpR) is defined as the percentageof tumor shrinkage observed at the maximal response point comparedto baseline tumor load. Clinical endpoints for both ORR and PFS canbe determined based on RECIST 1.1 criteria described above.

Where non-human subjects are analyzed, the aforementionedparameters to determine therapeutic efficacy and benefit have to beadapted as discussed elsewhere herein, cf. example 12 ff.

Typical "subjects" according to the current invention include humanand non-human subjects. Subjects can be mammals such as mice, rats,cats, dogs, primates and/or humans.

"Pharmaceutical compositions" (also "therapeutic formulations") ofthe antibody, fragment or conjugate can be prepared by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers,e.g. according to Remington's Pharmaceutical Sciences (18th ed.;Mack Pub. Co.: Eaton, Pa., 1990), e.g. in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients,or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbicacid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride,benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,or dextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as Tween.RTM., Pluronic.RTM. orpolyethylene glycol (PEG).

A "host cell" is a cell that is used to receive, maintain,reproduce and amplify a vector. A host cell also can be used toexpress the polypeptide, e.g. an antibody or fragment thereofencoded by the vector. The nucleic acid contained in the vector isreplicated when the host cell divides, thereby amplifying thenucleic acids. Preferred host cells are mammalian cells, such asCHO cells or HEK cells. Further preferred host cells are ratmyeloma YB2/0 cell.

A "cell with endogenous target expression" is a cell whichexpresses a target protein at a level which is comparable to thephysiological or diseased situation. Typically, cells which havebeen engineered for overexpression express a target protein at muchhigher levels.

The term "intra-tumoral", "intratumoral", "tumor infiltrating" or"tumoral" in the context of cells, structures, proteins,antibodies, or markers refers to their localization within thetumor tissue. Cells which are "positive" or "+" for a certainmarker or protein are cells characterized by substantial expressionof that marker or protein. Marker or protein expression can bedetermined and quantified as known in the art, e.g. to definedifferent cell populations. For the characterization of (immune)cell populations, the marker expression can be determined by FACSor using any other technique described herein.

"Leukocytes" are immune cells expressing CD45. "CD45+ cells", asused herein, refer to all leukocytes. CD45 can be used as a markerto distinguish immune cells and non-immune cells.

The term "lymphocyte" refers to all immature, mature,undifferentiated, and differentiated white lymphocyte populations,including tissue specific and specialized varieties. Itencompasses, by way of non-limiting example, B cells, T cells, NKTcells, and NK cells. In some embodiments, lymphocytes include all Bcell lineages including pre-B cells, progenitor B cells, earlypro-B cells, late pro-B cells, large pre-B cells, small pre-Bcells, immature B cells, mature B cells, plasma B cells, memory Bcells, B-1 cells, B-2 cells, and anergic AN1/T3 cellpopulations.

"T cells" are immune cells expressing TCR.alpha..beta., CD3, andCD8 or CD4. As used herein, the term includes naive T cells, CD4+ Tcells, CD8+ T cells, regulatory T cells, memory T cells, activatedT cells, anergic T cells, tolerant T cells, chimeric B cells, andantigen-specific T cells and further T cell populations known inthe art. In some embodiments, the presence of a T cell receptor(TCR) on the cell surface distinguishes T cells from otherlymphocytes.

"CD8+ T cells" (also "cytotoxic T cell", "TC", "cytotoxic Tlymphocyte", "CTL", "T-killer cell", "cytolytic T cell", "CD8+T-cell" or "killer T cell") are T cells expressing CD3, CD45 andCD8. CD8+ T cells can kill cancer cells, cells that are infected(particularly with viruses), or otherwise damaged cells. "CD4+ Tcells" (also "T helper cells", "Th cells") are immune cellsexpressing CD3, CD4 and CD45. There are several subsets of T helpercells, such as, without limitation, Th1, Th2, and Th17. CD4+ Tcells help suppress or regulate immune responses. They areessential in B cell antibody class switching, in the activation andgrowth of cytotoxic T cells, and in maximizing bactericidalactivity of phagocytes such as macrophages.

As used herein, the term "Treg cells" (also "Tregs", "regulatory Tcells", "T regulatory cells", "suppressor T cells") refers toimmune cells expressing CD3, CD4, CD45, and FoxP3, and furthermoreexpressing high levels of CD25 and low levels of CD127.Identification of Treg cells may be performed as describedelsewhere herein. Treg cells typically also express high levels ofCTLA-4, GITR, and LAG-3. In the literature, Tregs have furthermorebeen classified based on memory marker CD45RO.

Under physiological conditions, Treg cells maintain immunologicaltolerance. During an immune response, Treg cells stop Tcell-mediated immunity and suppress auto-reactive T cells that haveescaped negative selection within the thymus. Treg cells can alsosuppress other types of immune cells such as NK cells and B cells.Adaptive Treg cells (called Th3 or Tr1 cells) are thought to begenerated during an immune response.

Treg cells furthermore play an important role in immune escape bysuppressing antitumor immunity, thereby providing an environment ofimmune tolerance. T cells that recognize cancer cells are oftenpresent in large numbers in tumors, but their cytotoxic function issuppressed by nearby immune-suppressor cells. Tregs are abundant inmany different cancers, are highly enriched in the tumormicroenvironment, and are well known for their role in tumorprogression.

"Activated Treg cells" express CD4, CD45, FoxP3, CD69 and CCR8, andfurthermore have a high expression of CD25, and have a lowexpression of CD127. CD69 is a T cell activation marker.

"CCR8 positive regulatory T cells" or "CCR8+ regulatory T cells"are Tregs expressing CCR8. "CD4conv cells" are conventional CD4+,CD25- T cells.

"Gamma delta T cells" are T cells that express a distinctive T-cellreceptor, TCR.gamma..delta., on their surface. Gamma delta T cellsalso express CD3.

"B cells" are immune cells expressing CD19, and mature B cellsexpress CD20 and CD22. B cells upon activation via CD40 undergodifferentiation where somatic hypermutation and enhancedimmunoglobulin class switch occur resulting in mature B cells orplasma cells (capable of secreting Abs). B cells are involved inhumoral immunity of the adaptive immune system, and are antigenpresenting cells.

"Macrophages" are immune cells expressing low CD14, high CD16,CD11b, CD68, CD163, and CD206. Macrophages engulf and digestcellular debris, foreign substances, microbes or cancer cells byphagocytosis. Besides phagocytosis, macrophages play a criticalrole in innate immunity and also help initiate adaptive immunity byrecruiting other immune cells. For example, macrophages areimportant as antigen presenters to T cells. Macrophages thatencourage inflammation are called M1 macrophages, whereas thosethat decrease inflammation and encourage tissue repair are calledM2 macrophages.

As used herein, "M1 macrophages" are a subset of macrophagesexpressing ACOD1. M1 macrophages have pro-inflammatory,bactericidal, and phagocytic functions.

As used herein, "M2 macrophages" are a subset of macrophagesexpressing MRC1 (CD206). M2 macrophages secrete anti-inflammatoryinterleukins, play a role in wound healing and are needed forrevascularization and reepithelialization. Tumor-associatedmacrophages are mainly of the M2 phenotype and seem to activelypromote tumor growth.

"Dendritic Cells" (DCs) are bone marrow derived leukocytes and arethe most potent type of antigen-presenting cells. DCs arespecialized to capture and process antigens, converting proteins topeptides that are presented on major histocompatibility complex(MHC) molecules recognized by T cells. As defined herein, DCs arecharacterized by expression of CD1c, CD14, CD16, CD141, CD11c andCD123. Different subpopulations of Dendritic cells exist. In human,DC1 are immunogenic while DC2 cells are tolerogenic.

Mature DC express CD83, while plasmacytoid DC express CD123. "NKcells" (also natural killer cells) are immune cells which expressCD45, CD16, CD56, NKG2D, but are CD3 negative. NK cells do notrequire activation to kill cells that are missing "self" markers ofMHC class 1. NCR1 (also referred to as CD335 or NKp46) is expressedon NK cells and on a subset of NKT cells.

"Natural killer T (NKT) cells" are a heterogeneous group of T cellsthat share properties of both T cells and natural killer cells.

"iNKT cells" (also "invariant natural killer T cells") expressinvariant .alpha..beta. TCR (V.alpha.24-J.alpha.18, CD24lo),CD44hi, NK1.1 (mouse), and NKG2D. The invariant TCR recognizesglycoplipid antigen presented by non-polymorphic MHC class I-likemolecule, CD1d. These cells can influence an immune response byrapidly producing large amounts of cytokines, i.e. IFNg.

As known in the art, "effector cells" are immune cells thatactively support immune response after stimulation. As used herein,effector cells refer to immune cells expressing Fc.gamma. receptorsand are therefore able to mediate ADCC or ADCP. Non-limitingexamples of effector cells are monocytes, neutrophils, mast cells,and, preferably, macrophages, and natural killer cells.

"Tertiary lymphoid structures" are intra-tumoral structurescharacterized by increased expression of LTta, LTtb, Cxcr5 andCxcl13.

The term "chimeric antigen receptor" or "CAR" as used herein,refers to an artificial T cell surface receptor that is engineeredto be expressed on an immune effector cell and specifically bind anantigen. CARs may be used as a therapy with adoptive cell transfer.Monocytes are removed from a patient (blood, tumor or ascitesfluid) and modified so that they express the receptors specific toa particular form of antigen. In some embodiments, the CARs havebeen expressed with specificity to a tumor associated antigen. CARsmay also comprise an intracellular activation domain, atransmembrane domain and an extracellular domain comprising a tumorassociated antigen binding region. In some aspects, CARs comprisefusions of single-chain variable fragments (scFv) derivedmonoclonal antibodies, fused to CD3-zeta transmembrane andintracellular domain. The specificity of CAR designs may be derivedfrom ligands of receptors (e.g., peptides). In some embodiments, aCAR can target cancers by redirecting a monocyte/macrophageexpressing the CAR specific for tumor associated antigens.

Dosing schemes are abbreviated as known in the art, e.g. every day(QD), every 2 days (Q2D), or every 3 days (Q3D).

EMBODIMENTS

Antigens and Antibodies Binding Chemokine Receptors

Aspect 1--Antigen

According to a first aspect there is provided an isolated sulfatedpolypeptide comprising the tyrosine rich domain (TRD) of a seventransmembrane receptor. A tyrosine rich domain is a conserved, Nterminal domain, which characterizes seven transmembrane receptorssuch as CXC and CC chemokine receptors, cf. example 1. As usedherein, the term TRD refers to the amino acid or protein sequenceof a CXC or CC chemokine receptor which is located N terminal ofthe first cysteine counted from the N terminus. Besides tyrosine, aTRD typically comprises negatively charged amino acid residues suchas aspartic acid. The TRDs for all CC and CXC chemokine receptorsare listed in example 4, Table 4.1 for mouse, monkey and human.

Tyrosine sulfation is a ubiquitous posttranslational proteinmodification which occurs in all multicellular organisms. It iscatalyzed by tyrosylprotein sulfotransferases (TPSTs) 1 and 2,Golgi-resident enzymes which transfer sulfate from the cofactorPAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependenttyrosine in a protein substrate. Currently, only a small fractionof sulfated proteins is known and the understanding of thebiological sulfation mechanisms and specific modification positionsis still in progress, cf. example 4. While post-translationalmodifications, such as glycosylation, phosphorylation, acylation,adenylation, farnesylation, ubiquitination, and sulfation arefrequent in proteins of living systems, antibody generation istypically performed based on the unmodified target sequence.

The inventors developed an unusual approach for antibodygeneration, and surprisingly found that the synthetically sulfatedpolypeptides according to the first aspect could be used toincrease both, the success rates for antibody generation forchemokine receptors per se, and also the success rates forchemokine receptor antibodies with superior functional propertiesfor therapeutic uses, as discussed elsewhere herein. The increasedsuccess rate for highly specific chemokine receptor antibodies wasparticularly surprising, because research antibodies which aredesigned for the sequence independent detection of sulfatedtyrosine as a post-translational modification are rare.

The seven transmembrane receptor may be from any species expressingchemokine receptors characterized by a TRD, e.g. human, monkey,Macaca fascicularis (cynomolgus monkey), Macaca mulatta (Rhesusmacaque), rodent, mouse, rat, horse, bovine, pig, dog, cat andcamel.

According to some first embodiments of the first aspect, there isprovided an isolated polypeptide, wherein the isolated polypeptidecomprises the tyrosine rich domain (TRD) of a seven transmembranereceptor, further characterized in that at least 25%, at least 50%,or at least 75% of the tyrosine residues of the TRD aresulfated.

For example, in a highly successful antibody campaign, the TRD ofhuman or cynomolgus CCR8 was sulfated at positions Y3, Y15 and Y17,while Y16 was omitted, i.e. 75% of tyrosines in the TRD weresulfated (Table 6.1). In another example, the TRD of human CCR4 wassulfated at positions 19 and 22 and was used for off-targetbinding, i.e. 50% of the tyrosines were sulfated (Table 8.1). Inyet another approach, the TRD of murine CCR4 was sulfated atposition 22 and used for off target binding, i.e. 25% of tyrosinesin the TRD were sulfated (Table 6.1).

Table 4.1 shows a list of sulfated peptides including the preferredpositions for tyrosine sulfations. Without being bound by theory,the inventors believe that the introduction of additional chargesin the form of tyrosine sulfations tailors the antibody torecognize a specific pattern of negative charges. This recognitionseems to require an increased percentage of tyrosines andpositively charged amino acids, at least in the HCDR3 of theantibody, i.e., the use of the isolated sulfated polypeptidesaccording to the current invention also influenced the structuralcomposition of the antibodies and in particular the amino acidcomposition of the HCDR3, cf. example 9.

According to some second embodiments of the first aspect, which maybe or may not be the same as the first embodiments according to thefirst aspect, there is provided an isolated polypeptide, whereinthe seven transmembrane receptor is human, cynomolgus or mouse.

In some of these second embodiments the seven transmembranereceptor is murine. In some preferred of these second embodimentsthe seven transmembrane receptor is human and/or cynomolgus. Insome preferred of these second embodiments the seven transmembranereceptor is human. In some of these second embodiments the seventransmembrane receptor is cynomolgus.

According to some third embodiments of the first aspect, which maybe or may not be the same as the first and/or second embodimentsaccording to the first aspect, there is provided an isolatedpolypeptide, wherein the seven transmembrane receptor is achemokine receptor, preferably a) a CC chemokine receptor such asCCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b) aCXC chemokine receptor such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,or CXCR6, or c) CX3CR1 or CXCR1.

In some of these third embodiments, the seven transmembranereceptor is a CC chemokine receptor or a CXC chemokine receptor. Insome of these third embodiments, the seven transmembrane receptoris a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9 or CCR10.

In some preferred of these third embodiments, the seventransmembrane receptor is CCR8 or CCR4. In some highly preferred ofthese third embodiments, the seven transmembrane receptor is CCR8.In some of these third embodiments, the seven transmembranereceptor is a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3,CXCR4, CXCR5, or CXCR6. In some of these third embodiments, theseven transmembrane receptor is CX3CR1 or CXCR1.

In some preferred embodiments of the first aspect, there isprovided an isolated polypeptide which comprises the TRD of a humanor cynomolgus seven transmembrane receptor, further characterizedin that at least 25%, at least 50%, or at least 75% of the tyrosineresidues of the TRD are sulfated, wherein the seven transmembranereceptor is a CC chemokine receptor or a CXC chemokine receptor,and preferably, wherein the seven transmembrane receptor is CCR8 orCCR4.

According to some embodiments A of the third embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:1 (CCR1_HUMAN_TRD), preferably whereinat least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:7(CCR2_HUMAN_TRD), preferably wherein at least Y26 has beensulfated, or c) SEQ ID NO:13 (CCR3_HUMAN_TRD), preferably whereinY16 and/or Y17 have been sulfated, or d) SEQ ID NO:19(CCR4_HUMAN_TRD), preferably wherein at least Y22 has been sulfatedand preferably furthermore Y16, Y19 and/or Y20 have been sulfated,or e) SEQ ID NO:25 (CCR5_HUMAN_TRD), preferably wherein two, threeor all of Y3, Y10, Y14 and Y15 have been sulfated, or f) SEQ IDNO:31 (CCR6_HUMAN_TRD), preferably wherein at least two or three ofY18, Y26 and Y27 have been sulfated, or g) SEQ ID NO:37(CCR7_HUMAN_TRD), preferably wherein one or both of Y8 and Y17 havebeen sulfated, or h) SEQ ID NO:43 (CCR8_HUMAN_TRD), preferablywherein at least two or all of Y3, Y15 and Y17 have been sulfated,or i) SEQ ID NO:61 (CCR9_HUMAN_TRD), wherein at least Y28 andpreferably also Y17 and/or Y37 have been sulfated, or j) SEQ IDNO:67 (CCR10_HUMAN_TRD), preferably wherein at least one or both ofY14 and Y22 have been sulfated, or k) SEQ ID NO:73(CXCR1_HUMAN_TRD), preferably wherein Y27 has been sulfated, or l)SEQ ID NO:79 (CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25have been sulfated, or m) SEQ ID NO:85 (CXCR3_HUMAN_TRD),preferably wherein at least one or both of Y27 and Y29 have beensulfated, or n) SEQ ID NO:91 (CXCR4_HUMAN_TRD), preferably whereinat least Y12 and/or Y21 have been sulfated, or o) SEQ ID NO:97(CXCR5_HUMAN_TRD), preferably wherein at least one of Y3 and Y27have been sulfated, or p) SEQ ID NO:103 (CXCR6_HUMAN_TRD),preferably wherein at least one or both of Y6 and Y10 has beensulfated, or q) SEQ ID NO:157 (CX3CR1_HUMAN_TRD), preferablywherein at least Y14 has been sulfated, or r) SEQ ID NO:163(CXCR1_HUMAN_TRD), preferably wherein at least Y27 has beensulfated.

According to some embodiments B of the third embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:3 (CCR1_MOUSE_TRD), preferably whereinat least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:9(CCR2_MOUSE_TRD), preferably wherein at least Y37 and/or Y39 hasbeen sulfated, or c) SEQ ID NO:15 (CCR3_MOUSE_TRD), preferablywherein Y20 and/or Y22 has been sulfated, or d) SEQ ID NO:21(CCR4_MOUSE_TRD), preferably wherein at least Y22 has been sulfatedand preferably furthermore Y16, Y19 and/or Y20 have been sulfated,or e) SEQ ID NO:27 (CCR5_MOUSE_TRD), preferably wherein two orthree of Y10, Y12 and Y16 have been sulfated, or f) SEQ ID NO:33(CCR6_MOUSE_TRD), preferably wherein at least two or three of Y13,Y18 and Y19 have been sulfated, g) SEQ ID NO:39 (CCR7_MOUSE_TRD),preferably wherein one or both of Y8 and Y17 and optionally Y20have been sulfated have been sulfated, or h) SEQ ID NO:45(CCR8MOUSE_TRD), preferably wherein at least two or all of Y3, Y14and Y15 have been sulfated, or i) SEQ ID NO:63 (CCR9_MOUSE_TRD),preferably wherein at least Y28 has been sulfated, and preferablyalso Y19 has been sulfated, or j) SEQ ID NO:69 (CCR10_MOUSE_TRD),preferably wherein at least one, two or all of Y14, Y17 and Y22 hasbeen sulfated, or k) SEQ ID NO:75 (CXCR1_MOUSE_TRD), preferablywherein at least Y6 has been sulfated, or l) SEQ ID NO:81(CXCR2_MOUSE_TRD), preferably wherein Y24 has been sulfated, or m)SEQ ID NO:87 (CXCR3_MOUSE_TRD), preferably wherein at least one orboth of Y27 and Y29 have been sulfated, or n) SEQ ID NO:93(CXCR4_MOUSE_TRD), preferably wherein at least Y23, Y13 and/or Y14have been sulfated, or o) SEQ ID NO:99 (CXCR5_MOUSE_TRD),preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26have been sulfated, or p) SEQ ID NO:105 (CXCR6_MOUSE_TRD),preferably wherein at least one or both of Y11 and Y15 have beensulfated, or q) SEQ ID NO:159 (CX3CR1_MOUSE_TRD), preferablywherein at least Y15 has been sulfated, or r) SEQ ID NO:165(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has beensulfated.

According to some embodiments C of the third embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:2 (CCR1_MACFA_TRD), preferably whereinat least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:8(CCR2_MACMU_TRD), preferably wherein at least Y26 has beensulfated, or c) SEQ ID NO:14 (CCR3_MACFA_TRD), preferably whereinY16 has been sulfated, or d) SEQ ID NO:20 (CCR4MACFA_TRD),preferably wherein at least Y22 has been sulfated and preferablyfurthermore Y16, Y19 and/or Y20 have been sulfated, or e) SEQ IDNO:26 (CCR5_MACMU_TRD), preferably wherein two, three or all of Y3,Y10, Y14 and Y15 have been sulfated, or f) SEQ ID NO:32(CCR6_MACFA_TRD), preferably wherein at least two or three of Y23,Y31 and Y32 have been sulfated, or g) SEQ ID NO:38(CCR7_MACFA_TRD), preferably wherein one or both of Y8 and Y17 havebeen sulfated, or h) SEQ ID NO:44 (CCR8_MACFA_TRD), preferablywherein at least two or all of Y3, Y15 and Y17 have been sulfated,or i) SEQ ID NO:62 (CCR9_MACFA_TRD), preferably wherein at leastY28, and preferably also Y17 and/or Y37 has been sulfated, or j)SEQ ID NO:68 (CCR10_MACFA_TRD), preferably wherein at least one orboth of Y14 and Y22 has been sulfated, or k) SEQ ID NO:74(CXCR1_MACFA_TRD), preferably wherein at least one of Y14 and Y28has been sulfated, or l) SEQ ID NO:80 (CXCR2_MACFA_TRD), preferablywherein Y20 and/or Y22 have been sulfated, or m) SEQ ID NO:86(CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27and Y29 have been sulfated, or n) SEQ ID NO:92 (CXCR4_MACFA_TRD),preferably wherein at least Y12 and/or Y21 have been sulfated, oro) SEQ ID NO:98 (CXCR5_MACFA_TRD), preferably wherein at least oneof Y3 and Y27 have been sulfated, or p) SEQ ID NO:104(CXCR6_MACFA_TRD), preferably wherein at least two or all of Y4, Y7and Y39 have been sulfated, or q) SEQ ID NO:158 (CX3CR1_MACFA_TRD),preferably wherein at least Y20 has been sulfated, or r) SEQ IDNO:164 (CXCR1 MACMU TRD), preferably wherein at least Y14 has beensulfated.

In some fourth embodiments of the first aspect, which may be or maynot be the same as the first, second and/or third embodimentsaccording to the first aspect, the isolated polypeptide comprisesthe N terminus of the seven transmembrane receptor including TRDand LID domain, preferably wherein at least the cysteine betweenthe TRD and the LID domain has been removed or has been alteredinto a different amino acid.

The extracellular domains of chemokine receptors can be structuredinto four regions: (i) an N-terminal domain which can be subdividedinto (a) the membrane-distal tyrosine-rich domain (TRD), (b) acysteine, and (c) a LID domain, (iii) an extracellular domain 1(ECL1), (iii) an extracellular domain 2 (ECL2), and (iv) anextracellular domain 3 (ECL3).

Some of the polypeptides according to the current invention aredifficult to handle, e.g. due to a high tendency for aggregation.Without being bound by theory, the inventors believe, that the high"stickiness" results from the increased numbers of charged aminoacids and charged sulfate residues. For the polypeptides comprisingthe N terminus of a chemokine receptor, the aggregation propertiescould be ameliorated by removal or amino acid exchange of thecysteine between TRD and LID domain. Optimal results were obtainedby altering the cysteine into a serine (example 5, Table 4.1).

In some of the fourth embodiments according to the first aspect,the cysteine may be omitted and TRD and LID are directly connected.In some different of the fourth embodiments according to the firstaspect, the cysteine may be replaced by a different polar unchargedamino acid. In some preferred of the fourth embodiments accordingto the first aspect, the cysteine is replaced by a serine (cf.Table 4.1). In some of the fourth embodiments according to thefirst aspect, the cysteine may be replaced by at least onedifferent amino acid.

According to some embodiments A of the fourth embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:4 (CCR1_HUMAN_N term), wherein at leastY10 and/or Y18 have been sulfated, b) SEQ ID NO:10 (CCR2_HUMAN_Nterm), wherein at least Y26 has been sulfated, c) SEQ ID NO:16(CCR3_HUMAN_N term), wherein Y16 and/or Y17 have been sulfated, d)SEQ ID NO:22 (CCR4_HUMAN_N term), wherein at least Y22 has beensulfated and preferably furthermore Y16, Y19 and/or Y20 have beensulfated, e) SEQ ID NO:28 (CCR5_HUMAN_N term), wherein two, threeor all of Y3, Y10, Y14 and Y15 have been sulfated, f) SEQ ID NO:34(CCR6_HUMAN_N term), wherein at least two or three of Y18, Y26 andY27 have been sulfated, g) SEQ ID NO:40 (CCR7_HUMAN_N term),wherein one or both of Y8 and Y17 have been sulfated, h) SEQ IDNO:46 (CCR8_HUMAN_N term), wherein at least two or all of Y3, Y15and Y17 have been sulfated, i) SEQ ID NO:64 (CCR9_HUMAN_N term),wherein at least Y28, and preferably also Y17 and/or Y37 have beensulfated, j) SEQ ID NO:70 (CCR10_HUMAN_N term), wherein at leastone or both of Y14 and Y22 have been sulfated, k) SEQ ID NO:76(CXCR1_HUMAN_N term), wherein Y27 has been sulfated, l) SEQ IDNO:82 (CXCR2_HUMAN_N term), wherein Y23 and/or Y25 have beensulfated, m) SEQ ID NO:88 (CXCR3_HUMAN_N term), wherein at leastone or both of Y27 and Y29 have been sulfated, n) SEQ ID NO:94(CXCR4_HUMAN_N term), wherein at least Y12 and/or Y21 have beensulfated, o) SEQ ID NO:100 (CXCR5_HUMAN_N term), wherein at leastone of Y3 and Y27 have been sulfated, or p) SEQ ID NO:106(CXCR6_HUMAN_N term), wherein at least one or both of Y6 and Y10have been sulfated, or q) SEQ ID NO:160 (CX3CR1_HUMAN_N term),preferably wherein at least Y14 has been sulfated, or r) SEQ IDNO:166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 hasbeen sulfated.

According to some embodiments B of the fourth embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:6 (CCR1_MOUSE_N term), preferablywherein at least Y10 and/or Y18 have been sulfated, or b) SEQ IDNO:12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/orY39 has been sulfated, or c) SEQ ID NO:18 (CCR3_MOUSE_N term),preferably wherein Y20 and/or Y22 has been sulfated, or d) SEQ IDNO:24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has beensulfated and preferably furthermore Y16, Y19 and/or Y20 have beensulfated, or e) SEQ ID NO:30 (CCR5_MOUSE_N term), preferablywherein two or three of Y10, Y12 and Y16 have been sulfated, or f)SEQ ID NO:36 (CCR6_MOUSE_N term), preferably wherein at least twoor three of Y13, Y18 and Y19 have been sulfated, g) SEQ ID NO:42(CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17and optionally Y20 have been sulfated, or h) SEQ ID NO:48(CCR8_MOUSE_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y14 and Y15 have been sulfated, or i) SEQ IDNO:66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has beensulfated, and preferably also Y19 has been sulfated, or j) SEQ IDNO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, twoor all of Y14, Y17 and Y22 has been sulfated, or k) SEQ ID NO:78(CXCR1_MOUSE_N term), preferably wherein at least Y6 has beensulfated, or l) SEQ ID NO:84 (CXCR2_MOUSE_N term), preferablywherein Y24 has been sulfated, or m) SEQ ID NO:90 (CXCR3_MOUSE_Nterm), preferably wherein at least one or both of Y27 and Y29 havebeen sulfated, or n) SEQ ID NO:96 (CXCR4_MOUSE_N term), preferablywherein at least Y23 and/or Y14 have been sulfated, or o) SEQ IDNO:102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/orY14 and/or Y20 and/or Y26 have been sulfated, or p) SEQ ID NO:108(CXCR6_MOUSE_N term), preferably wherein at least one or both ofY11 and Y15 have been sulfated, or q) SEQ ID NO:162 (CX3CR1_MOUSE_Nterm), preferably wherein at least Y15 has been sulfated, or r) SEQID NO:168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 hasbeen sulfated.

According to some embodiments C of the fourth embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90%, 95% or 98% sequenceidentity with a) SEQ ID NO:5 (CCR1_MACFA_N term), preferablywherein at least Y10 and/or Y18 have been sulfated, or b) SEQ IDNO:11 (CCR2_MACMU_N term), preferably wherein at least Y26 has beensulfated, or c) SEQ ID NO:17 (CCR3_MACFA_N term), preferablywherein Y16 has been sulfated, or d) SEQ ID NO:23 (CCR4_MACFA_Nterm), preferably wherein at least Y22 has been sulfated andpreferably furthermore Y16, Y19 and/or Y20 have been sulfated, ore) SEQ ID NO:29 (CCR5_MACMU_N term), preferably wherein two, threeor all of Y3, Y10, Y14 and Y15 have been sulfated, or f) SEQ IDNO:35 (CCR6_MACFA_N term), preferably wherein at least two or threeof Y23, Y31 and Y32 have been sulfated, or g) SEQ ID NO:41(CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17have been sulfated, or h) SEQ ID NO:47 (CCR8_MACFA_N term),preferably wherein at least two or all of Y3, Y15 and Y17 have beensulfated, or i) SEQ ID NO:65 (CCR9_MACFA_N term), preferablywherein at least Y28, and preferably also Y17 and/or Y37 have beensulfated, or j) SEQ ID NO:71 (CCR10_MACFA_N term), preferablywherein at least one or both of Y14 and Y22 has been sulfated, ork) SEQ ID NO:77 (CXCR1_MACFA_N term), preferably wherein at leastone of Y14 and Y28 has been sulfated, or l) SEQ ID NO:83(CXCR2MACFA_N term), preferably wherein Y20 and/or Y22 have beensulfated, or m) SEQ ID NO:89 (CXCR3_MACFA_N term), preferablywherein at least one or both of Y27 and Y29 have been sulfated, orn) SEQ ID NO:95 (CXCR4_MACFA_N term), preferably wherein at leastY12 and/or Y21 have been sulfated, or o) SEQ ID NO:101(CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27have been sulfated, or p) SEQ ID NO:107 (CXCR6_MACFA_N term),preferably wherein at least two or all of Y4, Y7 and Y39 have beensulfated, or q) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferablywherein at least Y20 or Y22 has been sulfated, or r) SEQ ID NO:167(CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 hasbeen sulfated.

In some of the fourth embodiments of the first aspect, the isolatedsulfated polypeptide comprises a sequence according to a) SEQ IDNO:4 (CCR1_HUMAN_N term), SEQ ID NO:5 (CCR1_MACFA_N term), or SEQID NO:6 (CCR1_MOUSE_N term), preferably wherein at least Y10 and/orY18 have been sulfated, or b) SEQ ID NO:10 (CCR2_HUMAN_N term), orSEQ ID NO:11 (CCR2_MACMU_N term), preferably wherein at least Y26has been sulfated, or c) SEQ ID NO:12 (CCR2_MOUSE_N term),preferably wherein at least Y37 and/or Y39 has been sulfated, or d)SEQ ID NO:16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17have been sulfated, or e) SEQ ID NO:17 (CCR3_MACFA_N term),preferably wherein Y16 has been sulfated, or f) SEQ ID NO:18(CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 has beensulfated, or g) SEQ ID NO:22 (CCR4_HUMAN_N term), SEQ ID NO:23(CCR4_MACFA_N term), or SEQ ID NO:24 (CCR4_MOUSE_N term),preferably wherein at least Y22 has been sulfated and preferablyfurthermore Y16, Y19 and/or Y20 have been sulfated, or h) SEQ IDNO:28 (CCR5_HUMAN_N term), or SEQ ID NO:29 (CCR5_MACMU_N term),preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, or i) SEQ ID NO:30 (CCR5_MOUSE_N term), preferablywherein two or three of Y10, Y12 and Y16 have been sulfated, or j)SEQ ID NO:34 (CCR6_HUMAN_N term), preferably wherein at least twoor three of Y18, Y26 and Y27 have been sulfated, or k) SEQ ID NO:35(CCR6_MACFA_N term), preferably wherein at least two or three ofY23, Y31 and Y32 have been sulfated, or 1) SEQ ID NO:36(CCR6_MOUSE_N term), preferably wherein at least two or three ofY13, Y18 and Y19 have been sulfated, or m) SEQ ID NO:40(CCR7_HUMAN_N term), or SEQ ID NO:41 (CCR7_MACFA_N term),preferably wherein one or both of Y8 and Y17 have been sulfated, orn) SEQ ID NO:42 (CCR7_MOUSE_N term), preferably wherein one or bothof Y8 and Y17 and optionally Y20 have been sulfated have beensulfated, or o) SEQ ID NO:46 (CCR8 HUMAN_N term with C.dbd.X or S),or SEQ ID NO:47 (CCR8_MACFA_N term with C.dbd.X or S), preferablywherein at least two or all of Y3, Y15 and Y17 have been sulfated,or p) SEQ ID NO:48 (CCR8_MOUSE_N term with C.dbd.X or S),preferably wherein at least two or all of Y3, Y14 and Y15 have beensulfated, or q) SEQ ID NO:64 (CCR9_HUMAN_N term), or SEQ ID NO:65(CCR9_MACFA_N term), preferably wherein at least Y28, andpreferably also Y17 and/or Y37 has been sulfated, or r) SEQ IDNO:66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has beensulfated, and preferably also Y19 has been sulfated, or s) SEQ IDNO:70 (CCR10_HUMAN_N term), or SEQ ID NO:71 (CCR10_MACFA_N term),preferably wherein at least one or both of Y14 and Y22 has beensulfated, or t) SEQ ID NO:72 (CCR10_MOUSE_N term), preferablywherein at least one, two or all of Y14, Y17 and Y22 has beensulfated, or u) SEQ ID NO:76 (CXCR1_HUMAN_N term), preferablywherein Y27 has been sulfated, or v) SEQ ID NO:77 (CXCR1_MACFA_Nterm), preferably wherein at least one of Y14 and Y28 has beensulfated, or w) SEQ ID NO:78 (CXCR1_MOUSE_N term), preferablywherein at least Y6 has been sulfated, or x) SEQ ID NO:82 (CXCR2HUMAN_N term), preferably wherein Y23 and/or Y25 have beensulfated, or y) SEQ ID NO:83 (CXCR2MACFA_N term), preferablywherein Y20 and/or Y22 have been sulfated, or z) SEQ ID NO:84(CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, oraa) SEQ ID NO:88 (CXCR3_HUMAN_N term), SEQ ID NO:89 (CXCR3_MACFA_Nterm), or SEQ ID NO:90 (CXCR3_MOUSE_N term), preferably wherein atleast one or both of Y27 and Y29 have been sulfated, or bb) SEQ IDNO:94 (CXCR4_HUMAN_N term), or SEQ ID NO:95 (CXCR4_MACFA_N term),preferably wherein at least Y12 and/or Y21 have been sulfated, orcc) SEQ ID NO:96 (CXCR4_MOUSE_N term), preferably wherein at leastY23 and/or Y14 have been sulfated, or dd) SEQ ID NO:100(CXCR5_HUMAN_N term), or SEQ ID NO:101 (CXCR5_MACFA_N term),preferably wherein at least one of Y3 and Y27 have been sulfated,or ee) SEQ ID NO:102 (CXCR5_MOUSE_N term), preferably wherein atleast Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, orff) SEQ ID NO:106 (CXCR6_HUMAN_N term), preferably wherein at leastone or both of Y6 and Y10 has been sulfated, or gg) SEQ ID NO:107(CXCR6_MACFA_N term), preferably wherein at least two or all of Y4,Y7 and Y39 have been sulfated, or hh) SEQ ID NO:108 (CXCR6_MOUSE_Nterm), preferably wherein at least one or both of Y11 and Y15 havebeen sulfated, or ii) SEQ ID NO:160 (CX3CR1_HUMAN_N term),preferably wherein at least Y14 has been sulfated, or jj) SEQ IDNO:161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 orY22 has been sulfated, or kk) SEQ ID NO:162 (CX3CR1_MOUSE_N term),preferably wherein at least Y15 has been sulfated, or ll) SEQ IDNO:166 (CXCR1 HUMAN_N term), preferably wherein at least Y27 hasbeen sulfated, or mm) SEQ ID NO:167 (CXCR1_MACMU_N term),preferably wherein at least Y14 or Y28 has been sulfated, or nn)SEQ ID NO:168 (CXCR1_MOUSE_N term), preferably wherein at least Y6has been sulfated.

In some of the first, second, third or fourth embodiments of thefirst aspect, the isolated sulfated polypeptide comprises asequence according to or having at least 90% sequence identity witha) SEQ ID NO:1 (CCR1_HUMAN_TRD), SEQ ID NO:4 (CCR1_HUMAN_N term),SEQ ID NO:2 (CCR1_MACFA_TRD), SEQ ID NO:5 (CCR1_MACFA_N term), SEQID NO:3 (CCR1_MOUSE_TRD) or SEQ ID NO:6 (CCR1_MOUSE_N term),preferably wherein at least Y10 and/or Y18 have been sulfated, orb) SEQ ID NO:7 (CCR2_HUMAN_TRD), SEQ ID NO:10 (CCR2_HUMAN_N term),SEQ ID NO:8 (CCR2_MACMU_TRD) or SEQ ID NO:11 (CCR2_MACMU_N term),preferably wherein at least Y26 has been sulfated, or c) SEQ IDNO:9 (CCR2_MOUSE_TRD) or SEQ ID NO:12 (CCR2_MOUSE_N term),preferably wherein at least Y37 and/or Y39 has been sulfated, or d)SEQ ID NO:13 (CCR3_HUMAN_TRD) or SEQ ID NO:16 (CCR3_HUMAN_N term),preferably wherein Y16 and/or Y17 have been sulfated, or e) SEQ IDNO:14 (CCR3_MACFA_TRD) or SEQ ID NO:17 (CCR3_MACFA_N term),preferably wherein Y16 has been sulfated, or f) SEQ ID NO:15(CCR3_MOUSE_TRD) or SEQ ID NO:18 (CCR3_MOUSE_N term), preferablywherein Y20 and/or Y22 have been sulfated, or g) SEQ ID NO:19(CCR4_HUMAN_TRD), SEQ ID NO:22 (CCR4_HUMAN_N term), SEQ ID NO:20(CCR4_MACFA_TRD), SEQ ID NO:23 (CCR4_MACFA_N term), SEQ ID NO:21(CCR4_MOUSE_TRD) or SEQ ID NO:24 (CCR4_MOUSE_N term), preferablywherein at least Y22 has been sulfated and preferably furthermoreY16, Y19 and/or Y20 have been sulfated, or h) SEQ ID NO:25(CCR5_HUMAN_TRD), SEQ ID NO:28 (CCR5_HUMAN_N term), SEQ ID NO:26(CCR5_MACMU_TRD) or SEQ ID NO:29 (CCR5_MACMU_N term), preferablywherein two, three or all of Y3, Y10, Y14 and Y15 have beensulfated, or i) SEQ ID NO:27 (CCR5_MOUSE_TRD) or SEQ ID NO:30(CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12and Y16 have been sulfated, or j) SEQ ID NO:31 (CCR6_HUMAN_TRD) orSEQ ID NO:34 (CCR6_HUMAN_N term), preferably wherein at least twoor three of Y18, Y26 and Y27 have been sulfated, or k) SEQ ID NO:32(CCR6_MACFA_TRD) or SEQ ID NO:35 (CCR6_MACFA_N term), preferablywherein at least two or three of Y23, Y31 and Y32 have beensulfated, or l) SEQ ID NO:33 (CCR6_MOUSE_TRD) or SEQ ID NO:36(CCR6_MOUSE_N term), preferably wherein at least two or three ofY13, Y18 and Y19 have been sulfated, or m) SEQ ID NO:37(CCR7_HUMAN_TRD), SEQ ID NO:40 (CCR7_HUMAN_N term), SEQ ID NO:38(CCR7_MACFA_TRD) or SEQ ID NO:41 (CCR7_MACFA_N term), preferablywherein one or both of Y8 and Y17 have been sulfated, or n) SEQ IDNO:39 (CCR7_MOUSE_TRD) or SEQ ID NO:42 (CCR7_MOUSE_N term),preferably wherein one or both of Y8 and Y17 and optionally Y20have been sulfated have been sulfated, or o) SEQ ID NO:43(CCR8_HUMAN_TRD), SEQ ID NO:44 (CCR8_MACFA_TRD), SEQ ID NO:46(CCR8_HUMAN_N term with C.dbd.X or S), or SEQ ID NO:47(CCR8_MACFA_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, or p) SEQ IDNO:45 (CCR8_MOUSE_TRD) or SEQ ID NO:48 (CCR8_MOUSE_N term withC.dbd.X or S), preferably wherein at least two or all of Y3, Y14and Y15 have been sulfated, or q) SEQ ID NO:61 (CCR9_HUMAN_TRD),SEQ ID NO:64 (CCR9_HUMAN_N term), SEQ ID NO:62 (CCR9_MACFA_TRD) orSEQ ID NO:65 (CCR9_MACFA_N term), preferably wherein at least Y28,and preferably also Y17 and/or Y37 has been sulfated, or r) SEQ IDNO:63 (CCR9_MOUSE_TRD) or SEQ ID NO:66 (CCR9_MOUSE_N term),preferably wherein at least Y28 has been sulfated, and preferablyalso Y19 has been sulfated, or s) SEQ ID NO:67 (CCR10_HUMAN_TRD),SEQ ID NO:70 (CCR10_HUMAN_N term), SEQ ID NO:68 (CCR10_MACFA_TRD)or SEQ ID NO:71 (CCR10_MACFA_N term), preferably wherein at leastone or both of Y14 and Y22 has been sulfated, or t) SEQ ID NO:69(CCR10_MOUSE_TRD) or SEQ ID NO:72 (CCR10_MOUSE_N term), preferablywherein at least one, two or all of Y14, Y17 and Y22 has beensulfated, or u) SEQ ID NO:73 (CXCR1_HUMAN_TRD) or SEQ ID NO:76(CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, orv) SEQ ID NO:74 (CXCR1_MACFA_TRD) or SEQ ID NO:77 (CXCR1_MACFA_Nterm), preferably wherein at least one of Y14 and Y28 has beensulfated, or w) SEQ ID NO:75 (CXCR1_MOUSE_TRD) or SEQ ID NO:78(CXCR1_MOUSE_N term), preferably wherein at least Y6 has beensulfated, or x) SEQ ID NO:79 (CXCR2_HUMAN_TRD) or SEQ ID NO:82(CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have beensulfated, or y) SEQ ID NO:80 (CXCR2_MACFA_TRD) or SEQ ID NO:83(CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have beensulfated, or z) SEQ ID NO:81 (CXCR2_MOUSE_TRD) or SEQ ID NO:84(CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, oraa) SEQ ID NO:85 (CXCR3_HUMAN_TRD), SEQ ID NO:88 (CXCR3_HUMAN_Nterm), SEQ ID NO:86 (CXCR3_MACFA_TRD), SEQ ID NO:89 (CXCR3_MACFA_Nterm), SEQ ID NO:87 (CXCR3_MOUSE_TRD) or SEQ ID NO:90(CXCR3_MOUSE_N term), preferably wherein at least one or both ofY27 and Y29 have been sulfated, or bb) SEQ ID NO:91(CXCR4_HUMAN_TRD), SEQ ID NO:94 (CXCR4_HUMAN_N term), SEQ ID NO:92(CXCR4_MACFA_TRD) or SEQ ID NO:95 (CXCR4_MACFA_N term), preferablywherein at least Y12 and/or Y21 have been sulfated, or cc) SEQ IDNO:93 (CXCR4_MOUSE_TRD) or SEQ ID NO:96 (CXCR4_MOUSE_N term),preferably wherein at least Y23 and/or Y14 have been sulfated, ordd) SEQ ID NO:97 (CXCR5_HUMAN_TRD), SEQ ID NO:100 (CXCR5_HUMAN_Nterm), SEQ ID NO:98 (CXCR5_MACFA_TRD) or SEQ ID NO:101(CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27have been sulfated, or ee) SEQ ID NO:99 (CXCR5_MOUSE_TRD) or SEQ IDNO:102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/orY14 and/or Y20 and/or Y26 have been sulfated, or ff) SEQ ID NO:103(CXCR6_HUMAN_TRD) or SEQ ID NO:106 (CXCR6_HUMAN_N term), preferablywherein at least one or both of Y6 and Y10 has been sulfated, orgg) SEQ ID NO:104 (CXCR6_MACFA_TRD) or SEQ ID NO:107 (CXCR6_MACFA_Nterm), preferably wherein at least two or all of Y4, Y7 and Y39have been sulfated, or hh) SEQ ID NO:105 (CXCR6_MOUSE_TRD) or SEQID NO:108 (CXCR6_MOUSE_N term), preferably wherein at least one orboth of Y11 and Y15 have been sulfated, or ii) SEQ ID NO:157(CX3CR1_HUMAN_TRD) or SEQ ID NO:160 (CX3CR1_HUMAN_N term),preferably wherein at least Y14 has been sulfated, or jj) SEQ IDNO:158 (CX3CR1_MACFA_TRD), preferably wherein at least Y20 has beensulfated, or kk) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferablywherein at least Y20 or Y22 has been sulfated, or ll) SEQ ID NO:159(CX3CR1_MOUSE_TRD) or SEQ ID NO:162 (CX3CR1_MOUSE_N term),preferably wherein at least Y15 has been sulfated, or mm) SEQ IDNO:163 (CXCR1_HUMAN_TRD) or SEQ ID NO:166 (CXCR1_HUMAN_N term),preferably wherein at least Y27 has been sulfated, or nn) SEQ IDNO:164 (CXCR1_MACMU_TRD), preferably wherein at least Y14 has beensulfated or oo) SEQ ID NO:167 (CXCR1_MACMU_N term), preferablywherein at least Y14 or Y28 has been sulfated, or pp) SEQ ID NO:165(CXCR1_MOUSE_TRD) or SEQ ID NO:168 (CXCR1_MOUSE_N term), preferablywherein at least Y6 has been sulfated.

Preferably, the isolated polypeptide according to the currentaspect is immobilized, e.g. via a linker. Immobilization may occurwithout limitation on suitable beads, particles, proteins, or solidsupports.

Aspect 2--Conjugate Comprising Isolated Polypeptide

According to a second aspect of the current invention, there isprovided a conjugate comprising an isolated sulfated polypeptideaccording to the first aspect.

For example, the conjugate may comprise a polypeptide according tothe first embodiments of the first aspect. For example, theconjugate may comprise a polypeptide according to the secondembodiments of the first aspect. For example, the conjugate maycomprise a polypeptide according to the third embodiments of thefirst aspect. For example, the conjugate may comprise a polypeptideaccording to some embodiments A, B, C of the third embodiments ofthe first aspect. For example, the conjugate may comprise apolypeptide according to the fourth embodiments of the firstaspect. For example, the conjugate may comprise a polypeptideaccording to some embodiments A, B, C of the fourth embodiments ofthe first aspect.

For example, the isolated sulfated polypeptide may be attached to atag or linker for immobilization or retrieval. Suitable tags areselected from the tags known in the art, for example small organicmolecules like biotin (which binds strongly and non-covalently tostreptavidin), derivatives thereof, or short peptide sequence suchas Flag tag, HA tag, Myc tag or His tag (cf. Table 4.1 or example10.1.2). For some applications, the tag can be a protein, such ashuman serum albumin (cf. Table 4.1 or example 10.1.2), or a carrierprotein, such as KLH, OVA or BSA, or a larger structure such as abead or magnetic particle. Preferably, the tag can be attached viathe C term or at the N term of the TRD or the N terminus of thechemokine receptor but may also be attached via a reactive residueor amino acid side chain, such as a lysine within the TRD. In someembodiments, the tag is attached via a linker, which can be anylinker known in the art. Suitable linker includetrioxatridecan-succinamic acid (Ttds) linker (cf. Table 4.1),beta-alanine, GABA, AEA, Ava, Ahx, PEG2 spacer, PEG3 spacer, PEG4spacer, O1Pen, O2Oc or O1Pen-O1.

Aspect 3--Antigen Production Method

Sulfated peptides may be difficult to synthesize e.g. because thesulfate is unstable under acidic conditions (Houben-Weyl, Methodsof Organic Chemistry Vol. E 22b, Synthesis of Peptides andPeptidomimetics, 4th Edition, section 6.6.1.2 Synthesis of SulfatedTyrosine Peptides with Tyrosine 0-Sulfate Synthons, p. 440 ff. in:Felix, Arthur et al.: 2004).

According to a 3rd aspect there is provided a method for productionof the isolated sulfated polypeptide according to the first aspector conjugate according to the second aspect, wherein the methodcomprises synthesis of the isolated polypeptide and sulfation ofthe respective tyrosine residues. Synthesis of the isolatedpolypeptide according to the first aspect and sulfation of therespective tyrosine residues may occur as described in example 5,or may occur according to any other method known in the art. Inchapter 6.6.1 "Methods of Organic Chemistry Vol. E 22b, Synthesisof Peptides and Peptidomimetics", Houben-Weyl describe variouschemical approaches for the synthesis of sulfated tyrosinepeptides. The chapter, and in particular these methods, areincorporated herein by reference in their entirety.

For example, synthesis may be performed using the sequenceindependent solid phase method previously described by Bunschotenet al. (Bunschoten, Anton, et al. "A general sequence independentsolid phase method for the site specific synthesis of multiplesulfated-tyrosine containing peptides." Chemical communications 21(2009): 2999-3001.), incorporated herein in its entirety. In brief,a peptide is synthesized according to the Fmoc-tBu-strategy,followed by selective deprotection of the tyrosine residues to besulfated and introduction of a protected sulfate group. Uponcompletion of the synthesis of the sulfated peptide, it is cleavedfrom the resin by acidolysis and protection groups are removed withthe exception of the sulfate protection group, thereby preventingundesired acid induced removal of the sulfate group(s) during thisstep. Finally, the sulfate protecting groups can be removed in aslightly acidic reductive step, leaving the sulfate groupsuntouched.

Chen et al. have reported a short and efficient one-step route tosY-containing peptides, wherein Fmoc-protected fluorosulfatedtyrosine (Y(OSO2F)) is incorporated into the peptide of interestthrough an Fmoc-based solid-phase synthetic strategy (cf. Chen,Wentao, et al. "Synthesis of Sulfotyrosine-Containing Peptides byIncorporating Fluorosulfated Tyrosine Using an Fmoc-BasedSolid-Phase Strategy." Angewandte Chemie 128.5 (2016): 1867-1870.),incorporated herein in its entirety. Standard simultaneouspeptide-resin cleavage and removal of the acid-labile side-chainprotecting groups yield the crude peptides containingfluorosulfated tyrosine. Basic ethylene glycol, serving as solventand reactant, transforms the fluorosulfated tyrosine peptides intosulfotyrosine peptides in high yield.

Global sulfation of polypeptides is likewise possible, for exampleusing sulfur trioxide-pyridine. According to the current invention,this route can be used, if all tyrosines have to be sulfated or if"crude" mixtures of partially sulfated polypeptides shall be usedfor further steps. Furthermore, sulfation may occur enzymatically,e.g. in a biotransformation reaction using the natural sulfationenzymes or engineered versions thereof. Preferably, the sulfationof the respective tyrosine may occur chemically or enzymatically.Preferably, the synthesis of the isolated polypeptide occurs usingFmoc-tBu-strategy. According to some embodiments of the 3rd aspect,the method comprises purifying the obtained polypeptides.Purification may occur for example by HPLC, e.g. using a C18column. According to some embodiments of the 3rd aspect, the methodcomprises analytical characterization of the polypeptide. Withoutlimitation, analytical characterization may be performed usingspectroscopic methods or mass spectrometry.

Aspect 4--Uses of the Antigen/Methods Compr. Use of the Antigen

According to a 4.sup.th aspect there is provided the use of theisolated sulfated polypeptide according to the first aspect orconjugate according to the second aspect for antibody generation,as antigen or for off-target panning, and/or for characterizationof an antibody. For example, the isolated sulfated polypeptideaccording to the first aspect or conjugate according to the secondaspect can be used for the generation of a fully human antibody ora fragment thereof. For example, the isolated sulfated polypeptideaccording to the first aspect or conjugate according to the secondaspect can be used for the generation of a cross reactiveantibody.

According to some first embodiments according to the 4.sup.thaspect, there is provided the use of the isolated sulfatedpolypeptide according to the first aspect or conjugate according tothe second aspect for antibody generation. In particular, theisolated sulfated polypeptide according to the first aspect may beused to facilitate the generation of antibodies specificallyrecognizing chemokine receptors, as described elsewhere herein.

According to some second embodiments according to the 4.sup.thaspect, which may be the same as or different from the firstembodiments according to the 4.sup.th aspect, the isolated sulfatedpolypeptide according to the first aspect or conjugate according tothe second aspect is used as antigen, e.g. to select antibodies,antibody fragments or molecules specifically binding to chemokinereceptors, cf examples 6 and 8.

According to some third embodiments according to the 4.sup.thaspect, which may be the same as or different from the first and/orsecond embodiments according to the 4.sup.th aspect, the isolatedsulfated polypeptide according to the first aspect or conjugateaccording to the second aspect is used for off-target panning toselect antibodies which do not bind a certain seven transmembranereceptor, e.g. a chemokine receptor (off-target receptor), cfexamples 6 and 8.

According to some fourth embodiments, the method according to the4.sup.th aspect comprises the use of the isolated sulfatedpolypeptide according to the first aspect or conjugate according tothe second aspect for characterization of an antibody. Preferably,the antibody is an antibody according to the current invention. Forexample, the characterization of the antibody may comprise the useof ELISA, surface plasmon resonance, mass spectrometry, competitionassays, staining, IHC, FACS, or various further assays known in theart.

Aspect 5--Antibody Production Method

According to a 5.sup.th aspect, there is provided a method forobtaining an antibody or binder, the method comprising the use ofthe isolated sulfated polypeptide according to the first aspect orconjugate according to the second aspect.

According to some first embodiments, the method according to the5th aspect comprises the use of the isolated sulfated polypeptideaccording to the first aspect or conjugate according to the secondaspect as antigen.

According to some preferred of the first embodiments of the5.sup.th aspect, the method comprises the use of at least onefurther isolated polypeptide or conjugate thereof, wherein the atleast one further isolated polypeptide comprises a TRD a) of aseven transmembrane receptor different from the first seventransmembrane receptor, or b) of the first seven transmembranereceptor derived from a different species, preferably wherein theat least one further isolated polypeptide is an isolatedpolypeptide according to the first aspect.

According to some embodiments A of the first embodiments of the5.sup.th aspect, the method comprises the use of at least onefurther isolated polypeptide or conjugate, preferably according tothe first or second aspect, preferably either as antigen or foroff-target selection. For these embodiments, the first isolatedsulfated polypeptide comprises a TRD of a first seven transmembranereceptor (e.g. a chemokine receptor) and the further isolatedpolypeptide or conjugate comprises a TRD of a seven transmembranereceptor different from the first seven transmembrane receptor.Where the further isolated polypeptide or conjugate is used asantigen, the method is a method for the production of antibodies orbinders recognizing at least two different seven transmembranereceptors, e.g. two different chemokine receptor familymembers.

Where the further isolated polypeptide or conjugate is used for offtarget panning, the method is a method for the production ofantibodies or binders recognizing only a specific seventransmembrane receptor, e.g. to avoid off target binding to thefurther chemokine receptor family members.

According to some embodiments B of the first embodiments of the5.sup.th aspect, the method comprises the use of at least onefurther isolated polypeptide or conjugate, preferably according tothe first or second aspect, either as antigen or for off-targetselection. For these embodiments, the first isolated polypeptidecomprises a TRD of a first seven transmembrane receptor of a firstspecies (e.g. a human chemokine receptor) and the further isolatedpolypeptide comprises a TRD of the same seven transmembranereceptor derived from a different species (e.g. a cynomolguschemokine receptor). For example, the first polypeptide maycomprise the TRD of a human chemokine receptor, and the secondpolypeptide may comprise the TRD of a cynomolgus chemokinereceptor.

These embodiments B are of particular advantage, because thegeneration of chemokine receptor antibodies or binders which arecross reactive for both human and a suitable model species isdifficult for chemokine receptors, in particular for CCR8. Whilethe overall consensus between the transmembrane domains iscomparably high, the extracellular domains of chemokine receptorshave a low consensus between the different chemokine receptorfamily members, and also between the species for a given chemokinereceptor (FIG. 1, FIG. 2A). However, according to the currentinvention it was now found, that small sulfated tyrosine comprisingmotifs within the TRD are sufficiently conserved for a givenchemokine receptor to be used to obtain high numbers ofcross-reactive antibodies. Example 6 describes the generation ofcross-reactive antibodies for human and cynomolgus, and example10.1.1 shows excellent affinities in both species for variousantibodies according to the current invention. Compared withrodents and mice, cynomolgus is a preferred model system, becausemouse models have failed to predict immunological side effects.

The method according to the 5.sup.th aspect may be any method forantibody generation known in the art. For example, the method maybe a conventional immunization method, e.g. wherein the polypeptideaccording to the first aspect is conjugated to KLH and isadministered to a suitable animal for immunization.

For example, after immunization, the splenocytes of the immunizedanimals can be used for the generation of hybridoma cells, whichproduce the antibodies of the animals, and in a second step can bescreened for antibodies specifically binding to the antigen bymethods known in the art like e.g. ELISA as well as binding to anoff-target, if applicable. Alternatively, the splenocytes can bedirectly screened for the production of antibodies binding to theantigen in droplet based microfluidic systems or in cell cultureassays. Afterwards only selected splenocytes are directly appliedto sequencing to obtain the sequence of the antibody or hybridomageneration. Another method can comprise using the antigen forpanning with an antibody library which for example can be a phagedisplay library, e.g. without limitation as described elsewhereherein, or alternatively a mammalian library. After panning of thelibrary on the antigen the enriched antibodies can be screened forspecific binding to the antigen by methods known in the art likee.g. ELISA or SPR, as described in more detail herein.

According to some second embodiments of the 5.sup.th aspect, whichmay be the same as or different from the first embodiments of the5.sup.th aspect, the method for obtaining an antibody according tothe 5.sup.th aspect, is a method comprising the use of a phagedisplay library, a transgenic animal, or any other techniqueavailable in the art for the generation of antibodies comprisinghuman CDRs.

In some embodiments A of the second embodiments of the 5.sup.thaspect, the method comprises the use of a human phage displaylibrary, cf. example 6 and example 8. For example, the phagedisplay library can be a fully human antibody phage displaylibrary, such as the Biolnvent n-CoDeR Fab lambda library. In somepreferred embodiments, the Phage display library is enriched fortyrosine and/or histidine content. In a first step, the phagedisplay library comprising bacteriophages displaying antibodies orantibody fragments on their outside may be combined with theimmobilized isolated polypeptide according to the first aspect orconjugate according to the second aspect to allow binding to theisolated polypeptide or conjugate.

In an optional second depletion step, which may occur before orafter the first step, the phage display library comprisingbacteriophages displaying antibodies or antibody fragments on theiroutside may be combined with an immobilized isolated polypeptide orconjugate according to the first aspect, being different from theisolated polypeptide of the first step, to deplete off-targetbinders.

In an optional third step, which may occur before or after thefirst step, or may occur before or after the second step, the phagedisplay library comprising bacteriophages displaying antibodies orantibody fragments on their outside may be combined with animmobilized isolated polypeptide according to the first aspect orconjugate according to the second aspect being different from theisolated polypeptide of the first step and from the isolatedpolypeptide of the optional second step, to obtain cross reactivebinders. Each of the steps may be repeated multiple times, e.g.one, two, three, four, five, six, seven eight, nine, ten or moretimes. Binding antibodies or fragments can be retrieved forexpansion by infection of suitable bacterial hosts and the DNA canbe sequenced as known in the art to obtain the sequence of theseven transmembrane receptor antibody or fragment.

In some embodiments B of the second embodiments of the 5.sup.thaspect, the method for obtaining an antibody according to the 5thaspect, is a method comprising the use of transgenic animals asdescribed by Lonberg (Lonberg, Nils. "Human antibodies fromtransgenic animals." Nature biotechnology 23.9 (2005): 1117-1125.),incorporated herein in its entirety. For example, the transgenicanimal may be a XenoMouse (Abgenix Inc., Fremont, Calif., e.g. U.S.Pat. No. 5,939,598), the HuMAb Mouse (GenPharm-Medarex, San Jose,Calif.), the RenMab Mouse (Biocytogen), or any other animal knownin the art for the generation of fully human antibodies.

In some embodiments C of the second embodiments of the 5.sup.thaspect, the method for obtaining an antibody according to the 5thaspect, is a method comprising the use of in vitro activated Bcells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of whichis incorporated herein by reference in its entirety).

According to some third embodiments of the 5.sup.th aspect there isprovided a method for obtaining an antibody or antibody fragment ora binder, which specifically binds to a human and/or cynomolgusand/or murine CC or CXC chemokine receptor, the method comprisinga) (synthetically) sulfating a polypeptide comprising a tyrosinerich domain (TRD) and b) selection of an antibody, antibodyfragment or binder recognizing the sulfated polypeptide, and c)optionally producing the antibody, antibody fragment or binder.

In some embodiments, the use according to the 4.sup.th aspect, orthe method according to the 5.sup.th aspect is a use/method toobtain an antibody with favorable properties as described elsewhereherein, preferably wherein the antibody a) comprises human derivedCDRs, and/or b) is a human, rat or murine IgG antibody, preferablya human IgG1 antibody or a murine IgG2a antibody, and/or c) iscross reactive for two different seven transmembrane receptors,and/or d) is cross reactive for a human and a cynomolgus seventransmembrane receptor, and/or e) is characterized by a HCDR3region comprising between 10 and 34% of tyrosine and/or between 2and 20% of histidine, preferably between 7 and 20% of histidineand/or f) does not modulate G protein independent signaling of thechemokine receptor, and/or g) is a non-internalizing antibody or ischaracterized by an internalization into a cell with endogenoustarget expression which is lower than the 1.5, 2, 3, 4, 5, 6, 7, or10-fold of the internalization of the isotype control.

The method according to the current aspect can be used to obtainhigh numbers of antibodies binding chemokine receptors.Interestingly, a comparably high number of these antibodies werecharacterized by properties differentiating them from the knownantibodies obtained with conventional methods as describedelsewhere herein.

Aspect 6--Antibody Defined by Antigen

According to the current invention, there is provided an isolatedantibody or antigen-binding fragment thereof or a binder obtainedwith a method or use according to a previous aspect.

As will be appreciated by skilled artisans, antibodies and/orbinding fragments are "modular" in nature. Throughout thedisclosure, various specific aspects and embodiments of the various"modules" composing the antibodies and/or binding fragments aredescribed. As specific non-limiting examples, various specificembodiments of VH CDRs, VH chains, VL CDRs and VL chains orfunctional features are described. It is intended that all of thespecific embodiments may be combined with each other as though eachspecific combination were explicitly described individually. Asspecific non-limiting examples, various specific functionalembodiments are described. It is intended that all of the specificembodiments may be combined with each other as though each specificcombination were explicitly described individually.

According to a 6.sup.th aspect, there is provided an isolatedantibody or antigen-binding fragment thereof, (specifically)binding to a first isolated sulfated polypeptide which comprisesthe tyrosine rich domain (TRD) of a seven transmembrane receptor,wherein at least 25%, at least 50% or at least 75% of the tyrosineresidues of the TRD are sulfated. Preferably, the first isolatedsulfated polypeptide furthermore comprises the LID domain of theseven transmembrane receptor. Preferably, the cysteine between theTRD and the LID domain has been removed or has been exchanged intoa different amino acid. Preferably, the first isolated sulfatedpolypeptide comprises the N terminus of a seven transmembranereceptor including its tyrosine rich domain (TRD) and optionallyincluding its LID domain, and even more preferably, at least 25%,at least 50% or at least 75% of the tyrosine residues of the TRDare sulfated.

In preferred embodiments, the isolated antibody or antigen-bindingfragment thereof binds its target(s) with a KD value of <5E-8 M,<4E-8 M, <3E-8 M, <2E-8 M, <1E-8 M, <9E-9 M,<8E-9 M, <7E-9 M, <6E-9 M, <5E-9 M, <4E-9 M,<3E-9 M, <2.5E-9 M, <2E-9 M, <1.5E-9 M, <1E-9 M,<9E-10 M, <8E-10 M, <7E-10 M, <6E-10 M, <5E-10 M,<4E-10 M, <3E-10 M, <2.5E-10 M, <2E-10 M, <1.5E-10M, <1E-10 M, or <9E-11 M. For example, the inventive antibodymay bind its target(s) with a KD value between <8E-9 M and>4E-10 M. Where the isolated antibody or antigen-bindingfragment thereof binds more than one target, most preferably itbinds its' targets with an affinity in the same order ofmagnitude.

According to some preferred embodiments, the isolated antibody orantigen-binding fragment thereof a) comprises human derived CDRs,and/or b) is cross reactive for human and cynomolgus, and/or c) ischaracterized by a HCDR3 region comprising between 10 and 34% oftyrosine and/or between 7 and 20% of histidine, and/or d) does notmodulate G protein independent signaling of the seven transmembranereceptor, and/or e) is a non-internalizing antibody or ischaracterized by an internalization into a cell with endogenoustarget expression which is lower than the 1.5, 2, 3, 4, 5, 6, 7, or10-fold of the internalization of the isotype control, and/or f)induces ADCC and/or ADCP and/or g) is a human, rat or murine IgGantibody, preferably a human IgG1 antibody or a murine IgG2aantibody, and/or h) is an scFv, Fab, Fab' or a F(ab')2fragment.

According to some first embodiments of the 6.sup.th aspect, theseven transmembrane receptor is a chemokine receptor. In some ofthese first embodiments, the seven transmembrane receptor is a CCchemokine receptor or a CXC chemokine receptor. In some of thesefirst embodiments, the seven transmembrane receptor is a CCchemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9 or CCR10. In some preferred of these firstembodiments, the seven transmembrane receptor is CCR8 or CCR4. Insome most preferred of these first embodiments, the seventransmembrane receptor is CCR8. In some of the first embodiments,the seven transmembrane receptor is a CXC chemokine receptor, suchas CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of thefirst embodiments, the seven transmembrane receptor is CX3CR1 orCXCR1.

According to some second embodiments of the 6.sup.th aspect, whichmay or may not be the same as the first embodiments of the 6.sup.thaspect, the seven transmembrane receptor may be from any speciesexpressing chemokine receptors characterized by a TRD, e.g. human,monkey, Macaca fascicularis (cynomolgus monkey), Macaca mulatta(Rhesus macaque), rodent, mouse, rat, horse, bovine, pig, dog, catand camel. In some of these second embodiments of the 6.sup.thaspect, the seven transmembrane receptor is murine. In some mostpreferred of these second embodiments of the sixth aspect, theseven transmembrane receptor is human. In some of these secondembodiments of the sixth aspect, the seven transmembrane receptoris cynomolgus. In some preferred of these embodiments, the seventransmembrane receptor is human, cynomolgus or mouse. In some ofthese preferred second embodiments of the sixth aspect, the seventransmembrane receptor is human or cynomolgus.

In some preferred embodiments, the seven transmembrane receptor isa human, cynomolgus or mouse seven transmembrane receptor and theseven transmembrane receptor is a) a CC chemokine receptor,preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 orCCR10, b) a CXC chemokine receptor, preferably CXCR1, CXCR2, CXCR3,CXCR4, CXCR5, or CXCR6, or c) CX3CR1 or CXCR1.

According to some third embodiments of the 6.sup.th aspect, whichmay be or may not be the same as the first and/or secondembodiments according to the 6.sup.th aspect, the (first) isolatedsulfated polypeptide comprises or consists of a sequence accordingto a) SEQ ID NO:1 (CCR1_HUMAN_TRD), SEQ ID NO:4 (CCR1_HUMAN_Nterm), SEQ ID NO:2 (CCR1_MACFA_TRD), SEQ ID NO:5 (CCR1_MACFA_Nterm), SEQ ID NO:3 (CCR1_MOUSE_TRD) or SEQ ID NO:6 (CCR1_MOUSE_Nterm), preferably wherein at least Y10 and/or Y18 have beensulfated, or b) SEQ ID NO:7 (CCR2_HUMAN_TRD), SEQ ID NO:10(CCR2_HUMAN_N term), SEQ ID NO:8 (CCR2_MACMU_TRD) or SEQ ID NO:11(CCR2_MACMU_N term), preferably wherein at least Y26 has beensulfated, or c) SEQ ID NO:9 (CCR2_MOUSE_TRD) or SEQ ID NO:12(CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 hasbeen sulfated, or d) SEQ ID NO:13 (CCR3_HUMAN_TRD) or SEQ ID NO:16(CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have beensulfated, or e) SEQ ID NO:14 (CCR3_MACFA_TRD) or SEQ ID NO:17(CCR3_MACFA_N term), preferably wherein Y16 has been sulfated, orf) SEQ ID NO:15 (CCR3_MOUSE_TRD) or SEQ ID NO:18 (CCR3_MOUSE_Nterm), preferably wherein Y20 and/or Y22 has been sulfated, or g)SEQ ID NO:19 (CCR4_HUMAN_TRD), SEQ ID NO:22 (CCR4_HUMAN_N term),SEQ ID NO:20 (CCR4_MACFA_TRD), SEQ ID NO:23 (CCR4_MACFA_N term),SEQ ID NO:21 (CCR4_MOUSE_TRD) or SEQ ID NO:24 (CCR4_MOUSE_N term),preferably wherein at least Y22 has been sulfated and preferablyfurthermore Y16, Y19 and/or Y20 have been sulfated, or h) SEQ IDNO:25 (CCR5_HUMAN_TRD), SEQ ID NO:28 (CCR5_HUMAN_N term), SEQ IDNO:26 (CCR5_MACMU_TRD) or SEQ ID NO:29 (CCR5_MACMU_N term),preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, or i) SEQ ID NO:27 (CCR5_MOUSE_TRD) or SEQ ID NO:30(CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12and Y16 have been sulfated, or j) SEQ ID NO:31 (CCR6_HUMAN_TRD) orSEQ ID NO:34 (CCR6_HUMAN_N term), preferably wherein at least twoor three of Y18, Y26 and Y27 have been sulfated, or k) SEQ ID NO:32(CCR6_MACFA_TRD) or SEQ ID NO:35 (CCR6_MACFA_N term), preferablywherein at least two or three of Y23, Y31 and Y32 have beensulfated, or l) SEQ ID NO:33 (CCR6_MOUSE_TRD) or SEQ ID NO:36(CCR6_MOUSE_N term), preferably wherein at least two or three ofY13, Y18 and Y19 have been sulfated, or m) SEQ ID NO:37(CCR7_HUMAN_TRD), SEQ ID NO:40 (CCR7_HUMAN_N term), SEQ ID NO:38(CCR7_MACFA_TRD) or SEQ ID NO:41 (CCR7_MACFA_N term), preferablywherein one or both of Y8 and Y17 have been sulfated, or n) SEQ IDNO:39 (CCR7_MOUSE_TRD) or SEQ ID NO:42 (CCR7_MOUSE_N term),preferably wherein one or both of Y8 and Y17 and optionally Y20have been sulfated have been sulfated, or o) SEQ ID NO:43(CCR8_HUMAN_TRD), SEQ ID NO:44 (CCR8_MACFA_TRD), SEQ ID NO:46(CCR8_HUMAN_N term with C.dbd.X or S), or SEQ ID NO:47(CCR8_MACFA_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, or p) SEQ IDNO:45 (CCR8_MOUSE_TRD) or SEQ ID NO:48 (CCR8_MOUSE_N term withC.dbd.X or S), preferably wherein at least two or all of Y3, Y14and Y15 have been sulfated, or q) SEQ ID NO:61 (CCR9_HUMAN_TRD),SEQ ID NO:64 (CCR9_HUMAN_N term), SEQ ID NO:62 (CCR9_MACFA_TRD) orSEQ ID NO:65 (CCR9_MACFA_N term), preferably wherein at least Y28,and preferably also Y17 and/or Y37 has been sulfated, or r) SEQ IDNO:63 (CCR9_MOUSE_TRD) or SEQ ID NO:66 (CCR9_MOUSE_N term),preferably wherein at least Y28 has been sulfated, and preferablyalso Y19 has been sulfated, or s) SEQ ID NO:67 (CCR10_HUMAN_TRD),SEQ ID NO:70 (CCR10_HUMAN_N term), SEQ ID NO:68 (CCR10_MACFA_TRD)or SEQ ID NO:71 (CCR10_MACFA_N term), preferably wherein at leastone or both of Y14 and Y22 has been sulfated, or t) SEQ ID NO:69(CCR10_MOUSE_TRD) or SEQ ID NO:72 (CCR10_MOUSE_N term), preferablywherein at least one, two or all of Y14, Y17 and Y22 has beensulfated, or u) SEQ ID NO:73 (CXCR1_HUMAN_TRD) or SEQ ID NO:76(CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, orv) SEQ ID NO:74 (CXCR1_MACFA_TRD) or SEQ ID NO:77 (CXCR1_MACFA_Nterm), preferably wherein at least one of Y14 and Y28 has beensulfated, or w) SEQ ID NO:75 (CXCR1_MOUSE_TRD) or SEQ ID NO:78(CXCR1_MOUSE_N term), preferably wherein at least Y6 has beensulfated, or x) SEQ ID NO:79 (CXCR2_HUMAN_TRD) or SEQ ID NO:82(CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have beensulfated, or y) SEQ ID NO:80 (CXCR2_MACFA_TRD) or SEQ ID NO:83(CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have beensulfated, or z) SEQ ID NO:81 (CXCR2_MOUSE_TRD) or SEQ ID NO:84(CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, oraa) SEQ ID NO:85 (CXCR3_HUMAN_TRD), SEQ ID NO:88 (CXCR3_HUMAN_Nterm), SEQ ID NO:86 (CXCR3_MACFA_TRD), SEQ ID NO:89 (CXCR3_MACFA_Nterm), SEQ ID NO:87 (CXCR3_MOUSE_TRD) or SEQ ID NO:90(CXCR3_MOUSE_N term), preferably wherein at least one or both ofY27 and Y29 have been sulfated, or bb) SEQ ID NO:91(CXCR4_HUMAN_TRD), SEQ ID NO:94 (CXCR4_HUMAN_N term), SEQ ID NO:92(CXCR4_MACFA_TRD) or SEQ ID NO:95 (CXCR4_MACFA_N term), preferablywherein at least Y12 and/or Y21 have been sulfated, or cc) SEQ IDNO:93 (CXCR4_MOUSE_TRD) or SEQ ID NO:96 (CXCR4_MOUSE_N term),preferably wherein at least Y23 and/or Y14 have been sulfated, ordd) SEQ ID NO:97 (CXCR5_HUMAN_TRD), SEQ ID NO:100 (CXCR5_HUMAN_Nterm), SEQ ID NO:98 (CXCR5_MACFA_TRD) or SEQ ID NO:101(CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27have been sulfated, or ee) SEQ ID NO:99 (CXCR5_MOUSE_TRD) or SEQ IDNO:102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/orY14 and/or Y20 and/or Y26 have been sulfated, or ff) SEQ ID NO:103(CXCR6_HUMAN_TRD) or SEQ ID NO:106 (CXCR6_HUMAN_N term), preferablywherein at least one or both of Y6 and Y10 has been sulfated, orgg) SEQ ID NO:104 (CXCR6_MACFA_TRD) or SEQ ID NO:107 (CXCR6_MACFA_Nterm), preferably wherein at least two or all of Y4, Y7 and Y39have been sulfated, or hh) SEQ ID NO:105 (CXCR6_MOUSE_TRD) or SEQID NO:108 (CXCR6_MOUSE_N term), preferably wherein at least one orboth of Y11 and Y15 have been sulfated, or ii) SEQ ID NO:157(CX3CR1_HUMAN_TRD) or SEQ ID NO:160 (CX3CR1_HUMAN_N term),preferably wherein at least Y14 has been sulfated, or jj) SEQ IDNO:158 (CX3CR1_MACFA_TRD), preferably wherein at least Y20 has beensulfated, or kk) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferablywherein at least Y20 or Y22 has been sulfated, or ll) SEQ ID NO:159(CX3CR1_MOUSE_TRD) or SEQ ID NO:162 (CX3CR1_MOUSE_N term),preferably wherein at least Y15 has been sulfated, or mm) SEQ IDNO:163 (CXCR1_HUMAN_TRD) or SEQ ID NO:166 (CXCR1_HUMAN_N term),preferably wherein at least Y27 has been sulfated, or nn) SEQ IDNO:164 (CXCR1_MACMU_TRD), preferably wherein at least Y14 has beensulfated or oo) SEQ ID NO:167 (CXCR1_MACMU_N term), preferablywherein at least Y14 or Y28 has been sulfated, or pp) SEQ ID NO:165(CXCR1_MOUSE_TRD) or SEQ ID NO:168 (CXCR1_MOUSE_N term), preferablywherein at least Y6 has been sulfated.

According to some embodiments A of the third embodiments of the6.sup.th aspect, the (first) isolated sulfated polypeptidecomprises or consists of a sequence according to a. SEQ ID NO:1(CCR1_HUMAN_TRD), preferably wherein at least Y10 and/or Y18 havebeen sulfated, or b. SEQ ID NO:7 (CCR2_HUMAN_TRD), preferablywherein at least Y26 has been sulfated, or c. SEQ ID NO:13(CCR3_HUMAN_TRD), preferably wherein Y16 and/or Y17 have beensulfated, or d. SEQ ID NO:19 (CCR4_HUMAN_TRD), preferably whereinat least Y22 has been sulfated and preferably furthermore Y16, Y19and/or Y20 have been sulfated, or e. SEQ ID NO:25 (CCR5_HUMAN_TRD),preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, or f. SEQ ID NO:31 (CCR6_HUMAN_TRD), preferablywherein at least two or three of Y18, Y26 and Y27 have beensulfated, or g. SEQ ID NO:37 (CCR7_HUMAN_TRD), preferably whereinone or both of Y8 and Y17 have been sulfated, or h. SEQ ID NO:43(CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15and Y17 have been sulfated, or i. SEQ ID NO:61 (CCR9_HUMAN_TRD),preferably also Y17 and/or Y37 has been sulfated, or j. SEQ IDNO:67 (CCR10_HUMAN_TRD), preferably wherein at least one or both ofY14 and Y22 has been sulfated, or k. SEQ ID NO:73(CXCR1_HUMAN_TRD), preferably wherein Y27 has been sulfated, or l.SEQ ID NO:79 (CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25have been sulfated, or m. SEQ ID NO:85 (CXCR3_HUMAN_TRD),preferably wherein at least one or both of Y27 and Y29 have beensulfated, or n. SEQ ID NO:91 (CXCR4_HUMAN_TRD), preferably whereinat least Y12 and/or Y21 have been sulfated, or o. SEQ ID NO:97(CXCR5_HUMAN_TRD), preferably wherein at least one of Y3 and Y27have been sulfated, or p. SEQ ID NO:103 (CXCR6_HUMAN_TRD),preferably wherein at least one or both of Y6 and Y10 has beensulfated, or q. SEQ ID NO:157 (CX3CR1_HUMAN_TRD), preferablywherein at least Y14 has been sulfated, or r. SEQ ID NO:163(CXCR1_HUMAN_TRD), preferably wherein at least Y27 has beensulfated.

According to some embodiments B of the third embodiments of theaspect, the (first) isolated sulfated polypeptide comprises orconsists of a sequence according to a) SEQ ID NO:3(CCR1_MOUSE_TRD), preferably wherein at least Y10 and/or Y18 havebeen sulfated, or b) SEQ ID NO:9 (CCR2_MOUSE_TRD), preferablywherein at least Y37 and/or Y39 has been sulfated, or c) SEQ IDNO:15 (CCR3_MOUSE_TRD), preferably wherein Y20 and/or Y22 has beensulfated, or d) SEQ ID NO:21 (CCR4_MOUSE_TRD), preferably whereinat least Y22 has been sulfated and preferably furthermore Y16, Y19and/or Y20 have been sulfated, or e) SEQ ID NO:27 (CCR5_MOUSE_TRD),preferably wherein two or three of Y10, Y12 and Y16 have beensulfated, or f) SEQ ID NO:33 (CCR6_MOUSE_TRD), preferably whereinat least two or three of Y13, Y18 and Y19 have been sulfated, g)SEQ ID NO:39 (CCR7_MOUSE_TRD), preferably wherein one or both of Y8and Y17 and optionally Y20 have been sulfated have been sulfated,or h) SEQ ID NO:45 (CCR8_MOUSE_TRD), preferably wherein at leasttwo or all of Y3, Y14 and Y15 have been sulfated, or i) SEQ IDNO:63 (CCR9_MOUSE_TRD), preferably wherein at least Y28 has beensulfated, and preferably also Y19 has been sulfated, or j) SEQ IDNO:69 (CCR10_MOUSE_TRD), preferably wherein at least one, two orall of Y14, Y17 and Y22 has been sulfated, or k) SEQ ID NO:75(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has beensulfated, or l) SEQ ID NO:81 (CXCR2_MOUSE_TRD), preferably whereinY24 has been sulfated, or m) SEQ ID NO:87 (CXCR3_MOUSE_TRD),preferably wherein at least one or both of Y27 and Y29 have beensulfated, or n) SEQ ID NO:93 (CXCR4_MOUSE_TRD), preferably whereinat least Y23 and/or Y14 have been sulfated, or o) SEQ ID NO:99(CXCR5_MOUSE_TRD), preferably wherein at least Y3 and/or Y14 and/orY20 and/or Y26 have been sulfated, or p) SEQ ID NO:105(CXCR6_MOUSE_TRD), preferably wherein at least one or both of Y11and Y15 have been sulfated, or q) SEQ ID NO:159 (CX3CR1_MOUSE_TRD),preferably wherein at least Y15 has been sulfated, or r) SEQ IDNO:165 (CXCR1_MOUSE_TRD), preferably wherein at least Y6 has beensulfated.

According to some embodiments C of the third embodiments of the6.sup.th aspect, the (first) isolated sulfated polypeptidecomprises or consists of a sequence according to a) SEQ ID NO:2(CCR1_MACFA_TRD), preferably wherein at least Y10 and/or Y18 havebeen sulfated, or b) SEQ ID NO:8 (CCR2_MACMU_TRD), preferablywherein at least Y26 has been sulfated, or c) SEQ ID NO:14(CCR3_MACFA_TRD), preferably wherein Y16 has been sulfated, or d)SEQ ID NO:20 (CCR4MACFA_TRD), preferably wherein at least Y22 hasbeen sulfated and preferably furthermore Y16, Y19 and/or Y20 havebeen sulfated, or e) SEQ ID NO:26 (CCR5_MACMU_TRD), preferablywherein two, three or all of Y3, Y10, Y14 and Y15 have beensulfated, or f) SEQ ID NO:32 (CCR6_MACFA_TRD), preferably whereinat least two or three of Y23, Y31 and Y32 have been sulfated, or g)SEQ ID NO:38 (CCR7_MACFA_TRD), preferably wherein one or both of Y8and Y17 have been sulfated, or h) SEQ ID NO:44 (CCR8_MACFA_TRD),preferably wherein at least two or all of Y3, Y15 and Y17 have beensulfated, or i) SEQ ID NO:62 (CCR9_MACFA_TRD), preferably whereinat least Y28, and preferably also Y17 and/or Y37 has been sulfated,or j) SEQ ID NO:68 (CCR10_MACFA_TRD), preferably wherein at leastone or both of Y14 and Y22 has been sulfated, or k) SEQ ID NO:74(CXCR1_MACFA_TRD), preferably wherein at least one of Y14 and Y28has been sulfated, or l) SEQ ID NO:80 (CXCR2_MACFA_TRD), preferablywherein Y20 and/or Y22 have been sulfated, or m) SEQ ID NO:86(CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27and Y29 have been sulfated, or n) SEQ ID NO:92 (CXCR4_MACFA_TRD),preferably wherein at least Y12 and/or Y21 have been sulfated, oro) SEQ ID NO:98 (CXCR5_MACFA_TRD), preferably wherein at least oneof Y3 and Y27 have been sulfated, or p) SEQ ID NO:104(CXCR6_MACFA_TRD), preferably wherein at least two or all of Y4, Y7and Y39 have been sulfated, or q) SEQ ID NO:158 (CX3CR1_MACFA_TRD),preferably wherein at least Y20 has been sulfated, or r) SEQ IDNO:164 (CXCR1_MACMU_TRD), preferably wherein at least Y14 has beensulfated.

According to some fourth embodiments of the 6.sup.th aspect, whichmay or may not be the same as the first, second and/or thirdembodiments of the 6.sup.th aspect, the first isolated sulfatedpolypeptide comprises the N terminus of a seven transmembranereceptor including tyrosine rich domain (TRD) and preferably LIDdomain, and at least 25%, at least 50% or at least 75% of thetyrosine residues of the TRD are sulfated, preferably wherein atleast one/the cysteine between the TRD and the LID domain has beenremoved or has been exchanged into a different amino acid.

According to some embodiments A of the fourth embodiments of the6.sup.th aspect, the (first) isolated sulfated polypeptidecomprises or consists of a sequence according to a) SEQ ID NO:4(CCR1_HUMAN_N term), preferably wherein at least Y10 and/or Y18have been sulfated, b) SEQ ID NO:10 (CCR2_HUMAN_N term), preferablywherein at least Y26 has been sulfated, c) SEQ ID NO:16(CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have beensulfated, d) SEQ ID NO:22 (CCR4_HUMAN_N term), preferably whereinat least Y22 has been sulfated and preferably furthermore Y16, Y19and/or Y20 have been sulfated, e) SEQ ID NO:28 (CCR5_HUMAN_N term),preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, f) SEQ ID NO:34 (CCR6_HUMAN_N term), preferablywherein at least two or three of Y18, Y26 and Y27 have beensulfated, g) SEQ ID NO:40 (CCR7_HUMAN_N term), preferably whereinone or both of Y8 and Y17 have been sulfated, h) SEQ ID NO:46(CCR8_HUMAN_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, i) SEQ ID NO:64(CCR9_HUMAN_N term), preferably wherein at least Y28, andpreferably also Y17 and/or Y37 have been sulfated, j) SEQ ID NO:70(CCR10_HUMAN_N term), preferably wherein at least one or both ofY14 and Y22 have been sulfated, k) SEQ ID NO:76 (CXCR1_HUMAN_Nterm), preferably wherein Y27 has been sulfated, l) SEQ ID NO:82(CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have beensulfated, m) SEQ ID NO:88 (CXCR3_HUMAN_N term), preferably whereinat least one or both of Y27 and Y29 have been sulfated, n) SEQ IDNO:94 (CXCR4_HUMAN_N term), preferably wherein at least Y12 and/orY21 have been sulfated, o) SEQ ID NO:100 (CXCR5_HUMAN_N term),preferably wherein at least one of Y3 and Y27 have been sulfated,or p) SEQ ID NO:106 (CXCR6_HUMAN_N term), preferably wherein atleast one or both of Y6 and Y10 have been sulfated, q) SEQ IDNO:160 (CX3CR1_HUMAN_N term), preferably wherein at least Y14 hasbeen sulfated, or r) SEQ ID NO:166 (CXCR1_HUMAN_N term), preferablywherein at least Y27 has been sulfated.

According to some embodiments B of the fourth embodiments of the6.sup.th aspect, the (first) isolated sulfated polypeptidecomprises or consists of a sequence according to a) SEQ ID NO:6(CCR1_MOUSE_N term), preferably wherein at least Y10 and/or Y18have been sulfated, or b) SEQ ID NO:12 (CCR2_MOUSE_N term),preferably wherein at least Y37 and/or Y39 has been sulfated, or c)SEQ ID NO:18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22has been sulfated, or d) SEQ ID NO:24 (CCR4_MOUSE_N term),preferably wherein at least Y22 has been sulfated and preferablyfurthermore Y16, Y19 and/or Y20 have been sulfated, or e) SEQ IDNO:30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10,Y12 and Y16 have been sulfated, or f) SEQ ID NO:36 (CCR6_MOUSE_Nterm), preferably wherein at least two or three of Y13, Y18 and Y19have been sulfated, g) SEQ ID NO:42 (CCR7_MOUSE_N term), preferablywherein one or both of Y8 and Y17 and optionally Y20 have beensulfated have been sulfated, or h) SEQ ID NO:48 (CCR8_MOUSE_N termwith C.dbd.X or S), preferably wherein at least two or all of Y3,Y14 and Y15 have been sulfated, or i) SEQ ID NO:66 (CCR9_MOUSE_Nterm), preferably wherein at least Y28 has been sulfated, andpreferably also Y19 has been sulfated, or j) SEQ ID NO:72(CCR10_MOUSE_N term), preferably wherein at least one, two or allof Y14, Y17 and Y22 has been sulfated, or k) SEQ ID NO:78(CXCR1_MOUSE_N term), preferably wherein at least Y6 has beensulfated, or l) SEQ ID NO:84 (CXCR2_MOUSE_N term), preferablywherein Y24 has been sulfated, or m) SEQ ID NO:90 (CXCR3_MOUSE_Nterm), preferably wherein at least one or both of Y27 and Y29 havebeen sulfated, or n) SEQ ID NO:96 (CXCR4_MOUSE_N term), preferablywherein at least Y13 and/or Y14 have been sulfated, or o) SEQ IDNO:102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/orY14 and/or Y20 and/or Y26 have been sulfated, or p) SEQ ID NO:108(CXCR6_MOUSE_N term), preferably wherein at least one or both ofY11 and Y15 have been sulfated, or q) SEQ ID NO:162 (CX3CR1_MOUSE_Nterm), preferably wherein at least Y15 has been sulfated, or r) SEQID NO:168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 hasbeen sulfated.

According to some embodiments C of the fourth embodiments of the6.sup.th aspect, the (first) isolated sulfated polypeptidecomprises or consists of a sequence according to a) SEQ ID NO:5(CCR1_MACFA_N term), preferably wherein at least Y10 and/or Y18have been sulfated, or b) SEQ ID NO:11 (CCR2_MACMU_N term),preferably wherein at least Y26 has been sulfated, or c) SEQ IDNO:17 (CCR3_MACFA_N term), preferably wherein Y16 has beensulfated, or d) SEQ ID NO:23 (CCR4_MACFA_N term), preferablywherein at least Y22 has been sulfated and preferably furthermoreY16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:29(CCR5_MACMU_N term), preferably wherein two, three or all of Y3,Y10, Y14 and Y15 have been sulfated, or f) SEQ ID NO:35(CCR6_MACFA_N term), preferably wherein at least two or three ofY23, Y31 and Y32 have been sulfated, or g) SEQ ID NO:41(CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17have been sulfated, or h) SEQ ID NO:47 (CCR8_MACFA_N term withC.dbd.X or S), preferably wherein at least two or all of Y3, Y15and Y17 have been sulfated, or i) SEQ ID NO:65 (CCR9_MACFA_N term),preferably wherein at least Y28, and preferably also Y17 and/or Y37has been sulfated, or j) SEQ ID NO:71 (CCR10_MACFA_N term),preferably wherein at least one or both of Y14 and Y22 has beensulfated, or k) SEQ ID NO:77 (CXCR1_MACFA_N term), preferablywherein at least one of Y14 and Y28 has been sulfated, or l) SEQ IDNO:83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 havebeen sulfated, or m) SEQ ID NO:89 (CXCR3_MACFA_N term), preferablywherein at least one or both of Y27 and Y29 have been sulfated, orn) SEQ ID NO:95 (CXCR4_MACFA_N term), preferably wherein at leastY12 and/or Y21 have been sulfated, or o) SEQ ID NO:101(CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27have been sulfated, or p) SEQ ID NO:107 (CXCR6_MACFA_N term),preferably wherein at least two or all of Y4, Y7 and Y39 have beensulfated, or q) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferablywherein at least Y20 or Y22 has been sulfated, or r) SEQ ID NO:167(CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 hasbeen sulfated.

According to some fifth embodiments of the aspect, which may be ormay not be the same as the first, second, third and/or fourthembodiments according to the 6.sup.th aspect, the isolated antibodyor antigen-binding fragment thereof, (specifically) binds to asecond isolated sulfated polypeptide which comprises the tyrosinerich domain (TRD) of a seven transmembrane receptor.

Preferably the seven transmembrane receptor of the TRD comprised bythe second isolated sulfated polypeptide a) is different from theseven transmembrane receptor of the TRD comprised by the firstisolated sulfated polypeptide, or is b) the corresponding seventransmembrane receptor of the TRD comprised by the first isolatedsulfated polypeptide but from a different species.

According to some preferred embodiments, the second isolatedsulfated polypeptide which comprises the tyrosine rich domain (TRD)of a seven transmembrane receptor is different from the firstisolated sulfated polypeptide which comprises the tyrosine richdomain (TRD) of a seven transmembrane receptor and is an isolatedpolypeptide with a sequence according to any of embodiments A, B,or C according to the third and/or fourth embodiments of the6.sup.th aspect. For example, the first isolated sulfatedpolypeptide may comprise the TRD of a seven transmembrane receptorof a first species and the second isolated sulfated polypeptide maycomprise the TRD of a seven transmembrane receptor of a secondspecies, preferably wherein the species are human and cynomolgus,or human and mouse, or human and rat.

According to some embodiments A of the fifth embodiments of the6.sup.th aspect, the antibody or fragment specifically binds to a)a first isolated sulfated polypeptide comprising SEQ ID NO:1(CCR1_HUMAN_TRD), preferably wherein at least Y10 and/or Y18 havebeen sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:2 (CCR1_MACFA_TRD), preferably wherein atleast Y10 and/or Y18 have been sulfated, or b) a first isolatedsulfated polypeptide comprising SEQ ID NO:7 (CCR2_HUMAN_TRD),preferably wherein at least Y26 has been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:8(CCR2_MACMU_TRD), preferably wherein at least Y26 has beensulfated, or c) a first isolated sulfated polypeptide comprisingSEQ ID NO:13 (CCR3_HUMAN_TRD), preferably wherein Y16 and/or Y17have been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:14 (CCR3_MACFA_TRD), preferably wherein Y16has been sulfated, or d) a first isolated sulfated polypeptidecomprising SEQ ID NO:19 (CCR4_HUMAN_TRD), preferably wherein atleast Y22 has been sulfated and preferably furthermore Y16, Y19and/or Y20 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:20 (CCR4_MACFA_TRD), preferablywherein at least Y22 has been sulfated and preferably furthermoreY16, Y19 and/or Y20 have been sulfated, or e) a first isolatedsulfated polypeptide comprising SEQ ID NO:25 (CCR5_HUMAN_TRD),preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:26 (CCR5_MACMU_TRD), preferably wherein two,three or all of Y3, Y10, Y14 and Y15 have been sulfated, or f) afirst isolated sulfated polypeptide comprising SEQ ID NO:31(CCR6_HUMAN_TRD), preferably wherein at least two or three of Y18,Y26 and Y27 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:32 (CCR6_MACFA_TRD), preferablywherein at least two or three of Y23, Y31 and Y32 have beensulfated, or g) a first isolated sulfated polypeptide comprisingSEQ ID NO:37 (CCR7_HUMAN_TRD), preferably wherein one or both of Y8and Y17 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:38 (CCR7_MACFA_TRD), preferablywherein one or both of Y8 and Y17 have been sulfated, or h) a firstisolated sulfated polypeptide comprising SEQ ID NO:43(CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15and Y17 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:44 (CCR8_MACFA_TRD), preferablywherein at least two or all of Y3, Y15 and Y17 have been sulfated,or i) a first isolated sulfated polypeptide comprising SEQ ID NO:61(CCR9_HUMAN_TRD), preferably also Y17 and/or Y37 has been sulfated,and a second isolated sulfated polypeptide comprising SEQ ID NO:62(CCR9_MACFA_TRD), preferably wherein at least Y28, and preferablyalso Y17 and/or Y37 has been sulfated, or j) a first isolatedsulfated polypeptide comprising SEQ ID NO:67 (CCR10_HUMAN_TRD),preferably wherein at least one or both of Y14 and Y22 has beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:68 (CCR10_MACFA_TRD), preferably wherein at least one or bothof Y14 and Y22 has been sulfated, or k) a first isolated sulfatedpolypeptide comprising SEQ ID NO:73 (CXCR1_HUMAN_TRD), preferablywherein Y27 has been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:74 (CXCR1_MACFA_TRD), preferablywherein at least one of Y14 and Y28 has been sulfated, or l) afirst isolated sulfated polypeptide comprising SEQ ID NO:79(CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25 have beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:80 (CXCR2_MACFA_TRD), preferably wherein Y20 and/or Y22 havebeen sulfated, or m) a first isolated sulfated polypeptidecomprising SEQ ID NO:85 (CXCR3_HUMAN_TRD), preferably wherein atleast one or both of Y27 and Y29 have been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:86(CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27and Y29 have been sulfated, or n) a first isolated sulfatedpolypeptide comprising SEQ ID NO:91 (CXCR4_HUMAN_TRD), preferablywherein at least Y12 and/or Y21 have been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:92(CXCR4_MACFA_TRD), preferably wherein at least Y12 and/or Y21 havebeen sulfated, or o) a first isolated sulfated polypeptidecomprising SEQ ID NO:97 (CXCR5_HUMAN_TRD), preferably wherein atleast one of Y3 and Y27 have been sulfated, and a second isolatedsulfated polypeptide comprising SEQ ID NO:98 (CXCR5_MACFA_TRD),preferably wherein at least one of Y3 and Y27 have been sulfated,or p) a first isolated sulfated polypeptide comprising SEQ IDNO:103 (CXCR6_HUMAN_TRD), preferably wherein at least one or bothof Y6 and Y10 has been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:104 (CXCR6_MACFA_TRD), preferablywherein at least two or all of Y4, Y7 and Y39 have been sulfated q)a first isolated sulfated polypeptide comprising SEQ ID NO:157(CX3CR1_HUMAN_TRD), preferably wherein at least Y14 has beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:158 (CX3CR1_MACFA_TRD), preferably wherein at least Y20 hasbeen sulfated, or r) a first isolated sulfated polypeptidecomprising SEQ ID NO:163 (CXCR1_HUMAN_TRD), preferably wherein atleast Y27 has been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:164 (CXCR1_MACMU_TRD), preferablywherein at least Y14 has been sulfated.

According to some embodiments B of the fifth embodiments of the6.sup.th aspect, the antibody or fragment specifically binds to a)a first isolated sulfated polypeptide comprising SEQ ID NO:4(CCR1_HUMAN_N term), preferably wherein at least Y10 and/or Y18have been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:5 (CCR1_MACFA_N term), preferably wherein atleast Y10 and/or Y18 have been sulfated, b) a first isolatedsulfated polypeptide comprising SEQ ID NO:10 (CCR2_HUMAN_N term),preferably wherein at least Y26 has been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:11 (CCR2_MACMU_Nterm), preferably wherein at least Y26 has been sulfated, c) afirst isolated sulfated polypeptide comprising SEQ ID NO:16(CCR3HUMAN_N term), preferably wherein Y16 and/or Y17 have beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:17 (CCR3_MACFA_N term), preferably wherein Y16 has beensulfated, d) a first isolated sulfated polypeptide comprising SEQID NO:22 (CCR4_HUMAN_N term), preferably wherein at least Y22 hasbeen sulfated and preferably furthermore Y16, Y19 and/or Y20 havebeen sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:23 (CCR4_MACFA_N term), preferably wherein atleast Y22 has been sulfated and preferably furthermore Y16, Y19and/or Y20 have been sulfated, e) a first isolated sulfatedpolypeptide comprising SEQ ID NO:28 (CCR5_HUMAN_N term), preferablywherein two, three or all of Y3, Y10, Y14 and Y15 have beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:29 (CCR5_MACMU_N term), preferably wherein two, three or allof Y3, Y10, Y14 and Y15 have been sulfated, f) a first isolatedsulfated polypeptide comprising SEQ ID NO:34 (CCR6_HUMAN_N term),preferably wherein at least two or three of Y18, Y26 and Y27 havebeen sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:35 (CCR6_MACFA_N term), preferably wherein atleast two or three of Y23, Y31 and Y32 have been sulfated, g) afirst isolated sulfated polypeptide comprising SEQ ID NO:40(CCR7_HUMAN_N term), preferably wherein one or both of Y8 and Y17have been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:41 (CCR7_MACFA_N term), preferably wherein oneor both of Y8 and Y17 have been sulfated, h) a first isolatedsulfated polypeptide comprising SEQ ID NO:46 (CCR8_HUMAN_N termwith C.dbd.X or S), preferably wherein at least two or all of Y3,Y15 and Y17 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:47 (CCR8_MACFA_N term with C.dbd.Xor S), preferably wherein at least two or all of Y3, Y15 and Y17have been sulfated, i) a first isolated sulfated polypeptidecomprising SEQ ID NO:64 (CCR9_HUMAN_N term), preferably wherein atleast Y28, and preferably also Y17 and/or Y37 have been sulfated,and a second isolated sulfated polypeptide comprising SEQ ID NO:65(CCR9_MACFA_N term), preferably wherein at least Y28, andpreferably also Y17 and/or Y37 has been sulfated, j) a firstisolated sulfated polypeptide comprising SEQ ID NO:70(CCR10_HUMAN_N term), preferably wherein at least one or both ofY14 and Y22 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:71 (CCR10_MACFA_N term),preferably wherein at least one or both of Y14 and Y22 has beensulfated, k) a first isolated sulfated polypeptide comprising SEQID NO:76 (CXCR1_HUMAN_N term), preferably wherein Y27 has beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:77 (CXCR1_MACFA_N term), preferably wherein at least one ofY14 and Y28 has been sulfated, l) a first isolated sulfatedpolypeptide comprising SEQ ID NO:82 (CXCR2_HUMAN_N term),preferably wherein Y23 and/or Y25 have been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:83(CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have beensulfated, m) a first isolated sulfated polypeptide comprising SEQID NO:88 (CXCR3_HUMAN_N term), preferably wherein at least one orboth of Y27 and Y29 have been sulfated, and a second isolatedsulfated polypeptide comprising SEQ ID NO:89 (CXCR3_MACFA_N term),preferably wherein at least one or both of Y27 and Y29 have beensulfated, n) a first isolated sulfated polypeptide comprising SEQID NO:94 (CXCR4_HUMAN_N term), preferably wherein at least Y12and/or Y21 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:95 (CXCR4_MACFA_N term),preferably wherein at least Y12 and/or Y21 have been sulfated, o) afirst isolated sulfated polypeptide comprising SEQ ID NO:100(CXCR5_HUMAN_N term), preferably wherein at least one of Y3 and Y27have been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:101 (CXCR5_MACFA_N term), preferably whereinat least one of Y3 and Y27 have been sulfated, or p) a firstisolated sulfated polypeptide comprising SEQ ID NO:106(CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6and Y10 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:107 (CXCR6_MACFA_N term),preferably wherein at least two or all of Y4, Y7 and Y39 have beensulfated, or q) a first isolated sulfated polypeptide comprisingSEQ ID NO:160 (CX3CR1_HUMAN_N term), preferably wherein at leastY14 has been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably whereinat least Y20 or Y22 has been sulfated, or r) a first isolatedsulfated polypeptide comprising SEQ ID NO:166 (CXCR1_HUMAN_N term),preferably wherein at least Y27 has been sulfated, and a secondisolated sulfated polypeptide comprising SEQ ID NO:167(CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 hasbeen sulfated.

According to some embodiments C1 of the fifth embodiments of the6.sup.th aspect, the antibody or fragment specifically binds to afirst isolated sulfated polypeptide comprising a sequence accordingto some embodiments A of the third embodiments of the 6.sup.thaspect and binds to a second isolated sulfated polypeptidecomprising a sequence according to some embodiments B or C of thethird embodiments of the 6.sup.th aspect, wherein preferably thefirst and the second polypeptide comprise the TRD of the samereceptor but from different species.

According to some embodiments C2 of the fifth embodiments of the6.sup.th aspect, the antibody or fragment specifically binds to afirst isolated sulfated polypeptide comprising a sequence accordingto some embodiments A of the fourth embodiments of the 6.sup.thaspect and binds to a second isolated sulfated polypeptidecomprising a sequence according to some embodiments B or C of thefourth embodiments of the 6.sup.th aspect, wherein preferably thefirst and the second polypeptide comprise the TRD of the samereceptor but from different species.

According to some sixth embodiments of the 6.sup.th aspect, whichmay be or may not be the same as the first, second, third, fourthand/or fifth embodiments according to the 6.sup.th aspect, thedissociation constant or the EC50 of the antibody orantigen-binding fragment for binding the first isolated sulfatedpolypeptide and/or for binding said seven transmembrane receptor isbelow 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or0.25 nM.

According to some embodiments A of the sixth embodiments of the6.sup.th aspect, the dissociation constant or the EC50 of theantibody or antigen-binding fragment for binding the first isolatedsulfated polypeptide and/or for binding said seven transmembranereceptor is below 200 nM, 199 nM, 198 nM, 197 nM, 196 nM, 195 nM,194 nM, 193 nM, 192 nM, 191 nM, 190 nM, 189 nM, 188 nM, 187 nM, 186nM, 185 nM, 184 nM, 183 nM, 182 nM, 181 nM, 180 nM, 179 nM, 178 nM,177 nM, 176 nM, 175 nM, 174 nM, 173 nM, 172 nM, 171 nM, 170 nM, 169nM, 168 nM, 167 nM, 166 nM, 165 nM, 164 nM, 163 nM, 162 nM, 161 nM,160 nM, 159 nM, 158 nM, 157 nM, 156 nM, 155 nM, 154 nM, 153 nM, 152nM, 151 nM, 150 nM, 149 nM, 148 nM, 147 nM, 146 nM, 145 nM, 144 nM,143 nM, 142 nM, 141 nM, 140 nM, 139 nM, 138 nM, 137 nM, 136 nM, 135nM, 134 nM, 133 nM, 132 nM, 131 nM, 130 nM, 129 nM, 128 nM, 127 nM,126 nM, 125 nM, 124 nM, 123 nM, 122 nM, 121 nM, 120 nM, 119 nM, 118nM, 117 nM, 116 nM, 115 nM, 114 nM, 113 nM, 112 nM, 111 nM, 110 nM,109 nM, 108 nM, 107 nM, 106 nM, 105 nM, 104 nM, 103 nM, 102 nM, 101nM, 100 nM, 99 nM, 98 nM, 97 nM, 96 nM, 95 nM, 94 nM, 93 nM, 92 nM,91 nM, 90 nM, 89 nM, 88 nM, 87 nM, 86 nM, 85 nM, 84 nM, 83 nM, 82nM, 81 nM, 80 nM, 79 nM, 78 nM, 77 nM, 76 nM, 75 nM, 74 nM, 73 nM,72 nM, 71 nM, 70 nM, 69 nM, 68 nM, 67 nM, 66 nM, 65 nM, 64 nM, 63nM, 62 nM, 61 nM, 60 nM, 59 nM, 58 nM, 57 nM, 56 nM, 55 nM, 54 nM,53 nM, 52 nM, 51 nM, 50 nM, 49 nM, 48 nM, 47 nM, 46 nM, 45 nM, 44nM, 43 nM, 42 nM, 41 nM, 40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM,34 nM, 33 nM, 32 nM, 31 nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25nM, 24 nM, 23 nM, 22 nM, 21 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM,15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM,0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, or 0.1 nM.

Preferably, the dissociation constant or the EC50 of the antibodyor antigen-binding fragment for binding the first isolated sulfatedpolypeptide and/or for said seven transmembrane receptor is below10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 0.2 nM, 0.15 nM, or 0.1nM.

Preferably, EC50 can be determined in CHO cells overexpressing thetarget. As disclosed for instance in example 10.1.1, the antibodiesobtained with a method disclosed herein have excellent affinitiesfor their respective target. For example, TPP-21181, TPP-17578,TPP-19546, TPP-18206, TPP-21360 and TPP-23411 bound human CCR8 withan EC50 of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, .about.0.9 nM or 1.7 nMin CHO cells engineered to overexpress CCR8. Also, TPP-21181,TPP-17578, TPP-19546, TPP-18206, TPP-21360 and TPP-23411 boundcynomolgus CCR8 with an EC50 of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM,.about.0.55 nM or 0.9 nM. In addition, TPP-17578, TPP-19546,TPP-18206, and TPP-21360 bound to human regulatory T cells with anEC50 of 25 nM, 15 nM, 23 nM or 10 nM. In addition, anti-murine CCR8antibody TPP-14099 binds CHO cells expressing murine CCR8 with anEC50 of 3 nM and murine iTregs with an EC50 of 13.2 nM, cf. Table10.1.1.5.

According to some embodiments B of the sixth embodiments of theaspect, which may or may not be the same as the embodiments A ofthe sixth embodiments of the 6.sup.th aspect, the dissociationconstant or the EC50 of the antibody or antigen-binding fragmentfor binding the second isolated sulfated polypeptide and/or forbinding the second seven transmembrane receptor is below 200 nM,199 nM, 198 nM, 197 nM, 196 nM, 195 nM, 194 nM, 193 nM, 192 nM, 191nM, 190 nM, 189 nM, 188 nM, 187 nM, 186 nM, 185 nM, 184 nM, 183 nM,182 nM, 181 nM, 180 nM, 179 nM, 178 nM, 177 nM, 176 nM, 175 nM, 174nM, 173 nM, 172 nM, 171 nM, 170 nM, 169 nM, 168 nM, 167 nM, 166 nM,165 nM, 164 nM, 163 nM, 162 nM, 161 nM, 160 nM, 159 nM, 158 nM, 157nM, 156 nM, 155 nM, 154 nM, 153 nM, 152 nM, 151 nM, 150 nM, 149 nM,148 nM, 147 nM, 146 nM, 145 nM, 144 nM, 143 nM, 142 nM, 141 nM, 140nM, 139 nM, 138 nM, 137 nM, 136 nM, 135 nM, 134 nM, 133 nM, 132 nM,131 nM, 130 nM, 129 nM, 128 nM, 127 nM, 126 nM, 125 nM, 124 nM, 123nM, 122 nM, 121 nM, 120 nM, 119 nM, 118 nM, 117 nM, 116 nM, 115 nM,114 nM, 113 nM, 112 nM, 111 nM, 110 nM, 109 nM, 108 nM, 107 nM, 106nM, 105 nM, 104 nM, 103 nM, 102 nM, 101 nM, 100 nM, 99 nM, 98 nM,97 nM, 96 nM, 95 nM, 94 nM, 93 nM, 92 nM, 91 nM, 90 nM, 89 nM, 88nM, 87 nM, 86 nM, 85 nM, 84 nM, 83 nM, 82 nM, 81 nM, 80 nM, 79 nM,78 nM, 77 nM, 76 nM, 75 nM, 74 nM, 73 nM, 72 nM, 71 nM, 70 nM, 69nM, 68 nM, 67 nM, 66 nM, 65 nM, 64 nM, 63 nM, 62 nM, 61 nM, 60 nM,59 nM, 58 nM, 57 nM, 56 nM, 55 nM, 54 nM, 53 nM, 52 nM, 51 nM, 50nM, 49 nM, 48 nM, 47 nM, 46 nM, 45 nM, 44 nM, 43 nM, 42 nM, 41 nM,40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM, 34 nM, 33 nM, 32 nM, 31nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25 nM, 24 nM, 23 nM, 22 nM,21 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25nM, 0.2 nM, 0.15 nM, or 0.1 nM.

Preferably, the dissociation constant (KD) or the EC50 of theantibody or antigen-binding fragment for binding the secondisolated sulfated polypeptide and/or for binding the second seventransmembrane receptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or0.25 nM.

According to some embodiments AB of the sixth embodiments of the6.sup.th aspect, the dissociation constant (KD) or the EC50 of theantibody or antigen-binding fragment for binding the first isolatedsulfated polypeptide and/or the first seven transmembrane receptoris below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and thedissociation constant (KD) or the EC50 of the antibody orantigen-binding fragment for binding the second isolated sulfatedpolypeptide and/or the second seven transmembrane receptor is below10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

According to some seventh embodiments of the 6.sup.th aspect, whichmay be or may not be the same as the first, second, third, fourth,fifth and/or sixth embodiments according to the 6.sup.th aspect,the dissociation constant (KD) of the antibody for binding thefirst isolated sulfated polypeptide is lower than the dissociationconstant (KD) of the antibody for binding a first isolatednon-sulfated polypeptide having the same sequence as the firstisolated sulfated polypeptide. Preferably, the antibody does notsubstantially bind a first isolated non-sulfated polypeptide havingthe same sequence as the first isolated sulfated polypeptide.

According to some embodiments A of the seventh embodiments of the6.sup.th aspect, the dissociation constant or the EC50 of theantibody for binding the first isolated non-sulfated polypeptide ishigher than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM,450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 .mu.M, 1.25.mu.M, 1.5 .mu.M, 1.75 .mu.M, 2 .mu.M, 2.25 .mu.M, 2.5 .mu.M, 2.75.mu.M, or 3 .mu.M, or is not detectable. Preferably, thedissociation constant or the EC50 of the antibody for binding thefirst isolated non-sulfated polypeptide is higher than 100 nM, 250nM, 500 nM, 1 .mu.M, 2 .mu.M or 3 .mu.M, or is not detectable.

According to some embodiments B of the seventh embodiments of the6.sup.th aspect, which may be the same as the embodiments A of theseventh embodiments of the 6.sup.th aspect, the dissociationconstant or the EC50 of the antibody or fragment for binding thefirst isolated sulfated polypeptide is below 10 nM, 5 nM, 2.5 nM, 1nM, 0.5 nM or 0.25 nM, and the dissociation constant or the EC50 ofthe antibody or fragment for binding the first isolatednon-sulfated polypeptide is higher than 10 nM, 25 nM, 50 nM, 100nM, 250 nM or 500 nM, or is not detectable.

The antibody according to the 6.sup.th aspect may comprise CDRsderived from human, monkey, Macaca fascicularis (cynomolgusmonkey), Macaca mulatta (Rhesus macaque), rodent, mouse, rat,horse, bovine, pig, dog, cat and camel. Preferably, the antibodyaccording to the 6.sup.th aspect comprises human, rat or mousederived CDRs.

According to some 8.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth and/or seventh embodiments ofthe sixth aspect, the isolated antibody or antigen-binding fragmentcomprises human derived CDRs. According to some highly preferredembodiments, the human derived CDRs comprise no deviations or lessthan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 deviationsfrom the closest human germline. The closest human germline may bedetermined as known in the art, e.g. in silico using IgBLAST (Ye,Jian, et al. "IgBLAST: an immunoglobulin variable domain sequenceanalysis tool." Nucleic acids research 41.W1 (2013): W34-W40.) withdata retrieved from the IMGT human germline database. Theantibodies according to these 8.sup.th embodiments can be obtainedas described e.g. in example 6 or 8, or as described elsewhereherein.

According to some 9.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, or 8.sup.thembodiments of the 6.sup.th aspect, the isolated antibody orantigen-binding fragment is cross reactive for human andcynomolgus, cf. example 10.1.1. Preferably, the dissociationconstant (KD) or the EC50 of the antibody or antigen-bindingfragment for binding a human chemokine receptor is below 200 nM,150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM, such asbelow 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Preferably, thedissociation constant (KD) or the EC50 of the antibody orantigen-binding fragment for binding a cynomolgus chemokinereceptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM.

According to some 10.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, 8.sup.th, and/or9.sup.th embodiments of the 6.sup.th aspect, the isolated antibodyor antigen-binding fragment is characterized by a HCDR3 regiondeviating in composition from average HCDR3 regions. Preferably theisolated antibody or antigen-binding fragment is characterized by aHCDR3 region comprising between 10 and 34% of tyrosine and/or atleast one histidine, preferably between 2 and 20% of histidine,most preferably between 7 and 20% of histidine.

In some embodiments A of the 10.sup.th embodiments of the 6.sup.thaspect, the isolated antibody or antigen-binding fragment comprisesa HCDR3 having a) >0 and <35%, >8 and <34%, .gtoreq.10and <34%, or >15 and <31% tyrosine (Y) residues, and/or b)>0 and .ltoreq.16%, .gtoreq.2 and .ltoreq.20% or .gtoreq.7 and.ltoreq.20% histidine (H) residues, and c) preferably >0 and.ltoreq.18%, .gtoreq.7 and .ltoreq.10% or .gtoreq.0 and .ltoreq.7%arginine (R) residues, and/or d) preferably >0 and .ltoreq.25%,.gtoreq.7 and .ltoreq.16% or .gtoreq.7 and .ltoreq.13% asparticacid (D) residues, and/or e) preferably no lysine (K) residues,and/or f) preferably no glutamic acid (E) residues.

In some embodiments B of the 10.sup.th embodiments of the 6.sup.thaspect, which may be the same as or different from the embodimentsA, the isolated antibody or antigen-binding fragment comprises aHCDR3 having a) >0 and <47%, >22 and <50%, >10 and<34%, or >15 and <47% charged amino acids, and/or b) >0and <32%, >8 and <30%, or >10 and <37% positivelycharged amino acids, and/or c) >0 and <26%, .gtoreq.7 and<16%, or >7 and <14% negatively charged amino acids.

In some embodiments C of the 10.sup.th embodiments of the 6.sup.thaspect, which may be the same as or different from embodiments Aand/or B, the isolated antibody or antigen-binding fragmentcomprises a HCDR3 having >0 and .ltoreq.42%, >10 and.ltoreq.42% or .gtoreq.36 and .ltoreq.43% histidine and tyrosineresidues in total.

Each of the embodiments and descriptions disclosed for CCR8according to aspect 10 is disclosed herein for chemokine receptorantibodies in general, e.g. with the necessary changes. Theinventors believe that the sulfated TRD motif rather than thespecific CCR8 sequence promotes the increased frequencies fortyrosine and/or histidine.

According to some 11.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, and/or10.sup.th embodiments of the 6.sup.th aspect, the isolated antibodyor antigen-binding fragment at least partially modulates CCR8signaling. For example, an antibody according to the currentinvention can a) block G-protein independent signaling, and/or b)block G-protein dependent signaling, and/or c) block G-proteindependent and G-protein independent signaling, and/or d) increaseG-protein independent signaling, and/or e) increase G-proteindependent signaling, and/or f) increase G-protein dependent andG-protein independent signaling.

In the particular context, modulation refers to blocking of ligandinduced G-protein independent signaling and induction of G-proteinindependent signaling.

According to some preferred embodiments, the antibody or fragmentdoes not modulate G protein independent signaling of the seventransmembrane receptor. According to some preferred embodiments,the antibody or fragment does not block ligand induced G-proteinindependent signaling of the target protein. According to somepreferred embodiments, the antibody or fragment does not induceG-protein independent signaling

According to some preferred embodiments, the antibody or fragmentblocks G protein dependent signaling. G protein dependent signalingcan be analyzed by chemotaxis assay or preferably by calcium fluxassay, as described elsewhere herein.

According to some preferred embodiments, the antibody or fragmentblocks G protein dependent signaling of the chemokine receptor butdoes not block G protein independent signaling of the chemokinereceptor, cf. example 10.4. G protein independent signaling of thechemokine receptor is any signaling activity which is not G proteindependent signaling.

According to some 12.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, 8.sup.th, 9.sup.th,10.sup.th and 11.sup.th embodiments of the 6.sup.th aspect, theisolated antibody or antigen-binding fragment is a lowinternalizing or non-internalizing antibody or antigen bindingfragment.

Because overexpression may impact the internalization behavior andis less suited to model internalization in a therapeutic setting,internalization is preferably determined using a model cell linewith endogenous expression of the target chemokine receptor, cf.example 10.5. For example, internalization can be determined over atime frame or for specific time points. Preferably internalizationcan be determined after 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h,24 h or 48 h in a cell endogenously expressing the target.

According to some embodiments A of the 12th embodiments of the 6thaspect, the antibody or antigen-binding fragment has aninternalization rate in the same order of magnitude as theinternalization rate of the isotype control.

According to some embodiments B of the 12th embodiments of the 6thaspect, the antibody or antigen-binding fragment is characterizedby an internalization into a cell with endogenous target expressionwhich is lower than the 1.5, 2, 3, 4, 5, 6, 7, or 10-fold of theinternalization of the isotype control. The isotype control for anantibody can be selected as known in the art to match the isotypeof the antibody as closely as possible, but without binding to thetarget.

According to some embodiments C of the 12th embodiments of the 6thaspect, the antibody or antigen-binding fragment is characterizedby an internalization into a cell with endogenous target expressionwhich is lower than 150%, 175%, 200%, 300%, 400% or 500% of theinternalization of the isotype control e.g. after 15 min, 30 min, 1h, 2 h, 3 h, 6 h, 12 h, 24 h or 48 h.

According to some 13.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th and/or 12.sup.th embodiments of the 6.sup.thaspect, the isolated antibody or antigen-binding fragment inducesADCC and/or ADCP of cells expressing the target receptor of theantibody.

In some preferred embodiments of the 13th embodiments of the 6thaspect, the antibody or antigen-binding fragment isafucosylated.

In some embodiments A1 of the 13.sup.th embodiments of the 6thaspect, the antibody or antigen-binding fragment binds to human Fcgamma receptor IIIA variant V176 (CD16a) with a dissociationconstant (KD) lower than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or200 nM. Preferably, the KD can be determined using surface plasmonresonance.

In some embodiments B1 of the 13.sup.th embodiments of the 6thaspect, which may be the same as or different from the embodimentsA1, the antibody or antigen-binding fragment inducesantibody-dependent cell-mediated cytotoxicity (ADCC) in targetcells expressing the target receptor via human effector cells, suchas human NK cells. ADCC induction can be analyzed with an assayknown in the art, for example as described according to examples10.3.3 ff. or elsewhere herein. Preferably the assay is performedwith Treg cells wherein at least 80% or 85% of the Treg cellsexpress CCR8.

In some embodiments C1 of the 13.sup.th embodiments of the 6thaspect, which may be the same as or different from the embodimentsA1 or B1, the ADCC-induced maximal depletion of cells expressingthe target receptor is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,70%, 80%, 90%, 95%, 98% or 99%.

In some embodiments D1 of the 13.sup.th embodiments of the 6thaspect, which may be the same as or different from the embodimentsA1, B1 or C1, the EC50 for ADCC-induced depletion of targetexpressing cells is below 500 .mu.M, 400 .mu.M, 300 .mu.M, 200.mu.M, 100 .mu.M, 50 .mu.M, 25 .mu.M, 20 .mu.M, 12.5 .mu.M, 10.mu.M, 5 .mu.M or 2.5 .mu.M.

In some embodiments A2 of the 13.sup.th embodiments of the 6thaspect, which may or may not be the same as the embodiments A1, B1,C1 and/or D1, the antibody or antigen-binding fragment binds tohuman Fc gamma RITA (CD32a) with a dissociation constant (KD) lowerthan 30 .mu.M, 20 .mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M.

In some embodiments B2 of the 13.sup.th embodiments of the 6thaspect, which may be the same as the embodiments A1, B1, C1 and/orD1, and which may be the same as the embodiments A2, the antibodyor antigen-binding fragment induces antibody-dependentcell-mediated phagocytosis (ADCP) in cells expressing the targetreceptor via human effector cells, such as human macrophages. Forexample, the human macrophages can be M2 or M1 macrophages.

In some embodiments C2 of the 13.sup.th embodiments of the 6thaspect, which may be the same as the embodiments A1, B1, C1 and/orD1, and which may be the same as the embodiments A2 and/or B2, theADCP-induced maximal depletion of cells expressing the targetreceptor is at least 5, 10, 15, 20, 25, 30, 40 or 50%.

In some embodiments D2 of the 13.sup.th embodiments of the 6thaspect, which may be the same as the embodiments A1, B1, C1 and/orD1, and which may be the same as the embodiments A2, B2, and/or C2,the EC50 for ADCP-induced depletion of activated human regulatory Tcells is below 1500 .mu.M, 1000 .mu.M, 500 .mu.M, 250 .mu.M, 200.mu.M, 150 .mu.M, 100 .mu.M, 75 .mu.M, 50 .mu.M, 25 .mu.M or 10.mu.M.

In some preferred of the 13.sup.th embodiments of the 6th aspect,there is provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to a chemokine receptor, wherein theantibody or antigen-binding fragment a) binds to human Fc gammareceptor IIIA variant V176 (CD16a) with a dissociation constant(KD) lower than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM,and/or b) binds to human Fc gamma RITA (CD32a) with a dissociationconstant (KD) lower than 30 .mu.M, 20 .mu.M, 10 .mu.M, 5 .mu.M or 1.mu.M.

In some preferred of the 13.sup.th embodiments of the 6th aspect,there is provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to a chemokine receptor, wherein theantibody or antigen-binding fragment a) induces antibody-dependentcell-mediated cytotoxicity (ADCC) in target cells expressing thehuman chemokine receptor via human effector cells, such as human NKcells, and/or b) induces antibody-dependent cell-mediatedphagocytosis (ADCP) in target cells expressing the human chemokinereceptor via human effector cells, such as human macrophages.

In some preferred of the 13.sup.th embodiments of the 6th aspect,there is provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to a chemokine receptor, wherein a)the ADCC-induced maximal depletion in target cells expressing thehuman chemokine receptor is at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or b) theADCP-induced maximal depletion in target cells expressing the humanchemokine receptor is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or50%, and/or c) the maximal depletion of target cells expressing thehuman chemokine receptor, is at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 70%, 80%, 90%, 95% or 99%.

In some preferred of the 13.sup.th embodiments of the 6th aspect,there is provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to a chemokine receptor, wherein a)the EC50 for ADCC-induced depletion of target cells expressing thehuman chemokine receptor is below 200 .mu.M, 100 .mu.M, 50 .mu.M,25 .mu.M, 12.5 .mu.M, 10 .mu.M or 5 .mu.M and/or b) the EC50 forADCP-induced depletion of target cells expressing the humanchemokine receptor is below 500 .mu.M, 250 .mu.M, 200 .mu.M, 150.mu.M, 100 .mu.M, 75 .mu.M, 50 .mu.M or 25 .mu.M.

According to some 14.sup.th embodiments of the 6.sup.th aspect,which may be and are suggested to be combined with the first,second, third, fourth, fifth, sixth, seventh, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th and/or 13.sup.th embodiments of the6.sup.th aspect, the isolated antibody or antigen-binding fragmentcomprises a human, rat or murine IgG antibody, preferably a humanIgG1 antibody or a murine IgG2a antibody, cf. example 6 and 8.According to some 15th embodiments of the 6.sup.th aspect, whichmay be and are suggested to be combined with the first, second,third, fourth, fifth, sixth, seventh, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th and/or 14.sup.thembodiments of the 6.sup.th aspect, the isolated antibody orantigen-binding fragment is an scFv, Fab, Fab' or a F(ab')2fragment, cf. example 6.

The antibodies according to the current aspect can be conjugated,e.g. as discussed elsewhere herein. The antibodies according to thecurrent aspect can be used in the treatment of a tumor or a diseasecharacterized by the involvement of cells expressing the seventransmembrane receptor, e.g. as discussed elsewhere herein. Theantibodies according to the current aspect can be used as adiagnostic agent in vivo or in vitro, e.g. as discussed elsewhereherein. Furthermore, there is provided a kit comprising an antibodyaccording to the current aspect with instructions for use.

Preferred Combinations According to the 6.sup.th Aspect

The following embodiments disclose preferred combinations of thesixth aspect, thereby emphasizing the modular nature of theinvention. Provided according to a preferred embodiment I is anisolated antibody or antigen-binding fragment thereof, specificallybinding to a first isolated sulfated polypeptide which comprisesthe tyrosine rich domain (TRD) of a seven transmembrane receptor,and optionally its LID domain, wherein at least 25%, at least 50%or at least 75% of the tyrosine residues of the TRD are sulfated.Provided according to a preferred embodiment II is the isolatedantibody or antigen-binding fragment according to preferredembodiment I, wherein the cysteine between the TRD and the LIDdomain has been removed or has been exchanged into a differentamino acid. Provided according to a preferred embodiment III is theisolated antibody or antigen-binding fragment according topreferred embodiment I or II, wherein the seven transmembranereceptor is a human, cynomolgus or mouse seven transmembranereceptor. Provided according to a preferred embodiment IV is theisolated antibody or antigen-binding fragment according to any ofpreferred embodiments I, II or III, wherein the seven transmembranereceptor is a) a CC chemokine receptor, preferably CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b) a CXCchemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,or CXCR6, or c) CX3CR1 or CXCR1. Provided according to a preferredembodiment V is an isolated antibody or antigen-binding fragmentaccording to any of preferred embodiments I, II, III or IV, saidfirst isolated sulfated polypeptide comprising or consisting of asequence according to a. SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:2, SEQID NO:5, SEQ ID NO:3 or SEQ ID NO:6, preferably wherein at leastY10 and/or Y18 have been sulfated, or b. SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:8 or SEQ ID NO:11, preferably wherein at least Y26 hasbeen sulfated, or c. SEQ ID NO:9 or SEQ ID NO:12, preferablywherein at least Y37 and/or Y39 has been sulfated, or d. SEQ IDNO:13 or SEQ ID NO:16, preferably wherein Y16 and/or Y17 have beensulfated, or e. SEQ ID NO:14 or SEQ ID NO:17, preferably whereinY16 has been sulfated, or f. SEQ ID NO:15 or SEQ ID NO:18preferably wherein Y20 and/or Y22 have been sulfated, or g. SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:21 orSEQ ID NO:24, preferably wherein at least Y22 has been sulfated andpreferably furthermore Y16, Y19 and/or Y20 have been sulfated, orh. SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:26 or SEQ ID NO:29,preferably wherein two, three or all of Y3, Y10, Y14 and Y15 havebeen sulfated, or i. SEQ ID NO:27 or SEQ ID NO:30, preferablywherein two or three of Y10, Y12 and Y16 have been sulfated, or j.SEQ ID NO:31 or SEQ ID NO:34, preferably wherein at least two orthree of Y18, Y26 and Y27 have been sulfated, or k. SEQ ID NO:32 orSEQ ID NO:35, preferably wherein at least two or three of Y23, Y31and Y32 have been sulfated, or l. SEQ ID NO:33 or SEQ ID NO:36,preferably wherein at least two or three of Y13, Y18 and Y19 havebeen sulfated, or m. SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:38 orSEQ ID NO:41, preferably wherein one or both of Y8 and Y17 havebeen sulfated, or n. SEQ ID NO:39 or SEQ ID NO:42, preferablywherein one or both of Y8 and Y17 and optionally Y20 have beensulfated, or o. SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46 or SEQ IDNO:47, preferably wherein at least two or all of Y3, Y15 and Y17have been sulfated, or p. SEQ ID NO:45 or SEQ ID NO:48, preferablywherein at least two or all of Y3, Y14 and Y15 have been sulfated,or q. SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:62 or SEQ ID NO:65,preferably wherein at least Y28, and preferably also Y17 and/or Y37has been sulfated, or r. SEQ ID NO:63 or SEQ ID NO:66, preferablywherein at least Y28 has been sulfated, and preferably also Y19 hasbeen sulfated, or s. SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:68 orSEQ ID NO:71, preferably wherein at least one or both of Y14 andY22 has been sulfated, or t. SEQ ID NO:69 or SEQ ID NO:72,preferably wherein at least one, two or all of Y14, Y17 and Y22 hasbeen sulfated, or u. SEQ ID NO:73 or SEQ ID NO:76, preferablywherein Y27 has been sulfated, or v. SEQ ID NO:74 or SEQ ID NO:77,preferably wherein at least one of Y14 and Y28 has been sulfated,or w. SEQ ID NO:75 or SEQ ID NO:78, preferably wherein at least Y6has been sulfated, or x. SEQ ID NO:79 or SEQ ID NO:82, preferablywherein Y23 and/or Y25 have been sulfated, or y. SEQ ID NO:80 orSEQ ID NO:83, preferably wherein Y20 and/or Y22 have been sulfated,or z. SEQ ID NO:81 or SEQ ID NO:84, preferably wherein Y24 has beensulfated, or aa. SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:86, SEQ IDNO:89, SEQ ID NO:87 or SEQ ID NO:90, preferably wherein at leastone or both of Y27 and Y29 have been sulfated, or bb. SEQ ID NO:91,SEQ ID NO:94, SEQ ID NO:92 or SEQ ID NO:95, preferably wherein atleast Y12 and/or Y21 have been sulfated, or cc. SEQ ID NO:93 or SEQID NO:96, preferably wherein at least Y23 and/or Y14 have beensulfated, or dd. SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:98 or SEQID NO:101, preferably wherein at least one of Y3 and Y27 have beensulfated, or ee. SEQ ID NO:99 or SEQ ID NO:102, preferably whereinat least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, orff. SEQ ID NO:103 or SEQ ID NO:106, preferably wherein at least oneor both of Y6 and Y10 has been sulfated, or gg. SEQ ID NO:104 orSEQ ID NO:107, preferably wherein at least two or all of Y4, Y7 andY39 have been sulfated, or hh. SEQ ID NO:105 or SEQ ID NO:108,preferably wherein at least one or both of Y11 and Y15 have beensulfated, or ii. SEQ ID NO:157 or SEQ ID NO:160, preferably whereinat least Y14 has been sulfated, or jj. SEQ ID NO:158, preferablywherein at least Y20 has been sulfated, or kk. SEQ ID NO:161,preferably wherein at least Y20 or Y22 has been sulfated, or ll.SEQ ID NO:159 or SEQ ID NO:162, preferably wherein at least Y15 hasbeen sulfated, or mm. SEQ ID NO:163 or SEQ ID NO:166, preferablywherein at least Y27 has been sulfated, or nn. SEQ ID NO:164,preferably wherein at least Y14 has been sulfated or oo. SEQ IDNO:167, preferably wherein at least Y14 or Y28 has been sulfated,or pp. SEQ ID NO:165 or SEQ ID NO:168, preferably wherein at leastY6 has been sulfated.

Provided according to a preferred embodiment VI is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I, II, III, IV or V, wherein the dissociation constantor the EC50 of the antibody for binding the first isolated sulfatedpolypeptide and/or for said seven transmembrane receptor is below150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Providedaccording to a preferred embodiment VII is an isolated antibody orantigen-binding fragment according to any of preferred embodimentsI to VI, wherein the isolated antibody or antigen-binding fragmentspecifically binds to a second isolated sulfated polypeptide whichcomprises the TRD of a seven transmembrane receptor, preferablywherein the seven transmembrane receptor of the TRD comprised bythe second isolated sulfated polypeptide a. is different from theseven transmembrane receptor of the TRD comprised by the firstisolated sulfated polypeptide, or b. is the corresponding seventransmembrane receptor of the TRD comprised by the first isolatedsulfated polypeptide but from a different species.

Provided according to a preferred embodiment VIII is the isolatedantibody or antigen-binding fragment according to preferredembodiment VII, wherein the dissociation constant or the EC50 ofthe antibody for binding the second isolated sulfated polypeptideand/or for binding the second seven transmembrane receptor is below10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Provided according to apreferred embodiment IX is an isolated antibody or antigen-bindingfragment according to any of preferred embodiments I to VIII,wherein the dissociation constant (KD) of the antibody for bindingthe first isolated sulfated polypeptide is lower than thedissociation constant (KD) of the antibody for binding a firstisolated non-sulfated polypeptide having the same sequence as thefirst isolated sulfated polypeptide. Provided according to apreferred embodiment X is the isolated antibody or antigen-bindingfragment according to preferred embodiment IX, wherein thedissociation constant and/or EC50 of the antibody for binding thefirst isolated non-sulfated polypeptide is higher than 150 nM, 250nM, 500 nM, 1 .mu.M, 2 .mu.M or 3 .mu.M, or is not detectable.Provided according to a preferred embodiment XI is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments IX or X, wherein the dissociation constant or the EC50of the antibody or fragment for binding the first isolated sulfatedpolypeptide is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM,and wherein the dissociation constant of the antibody or fragmentfor binding the first isolated non-sulfated polypeptide is higherthan 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or is notdetectable. Provided according to a preferred embodiment XII is theisolated antibody or antigen-binding fragment according to any ofpreferred embodiments I to XI, wherein the antibody compriseshuman, rat or mouse derived CDRs. Provided according to a preferredembodiment XIII is the isolated antibody or antigen-bindingfragment according to any of preferred embodiments I to XII,wherein the antibody a. comprises human derived CDRs, and/or b. iscross reactive for human and cynomolgus, and/or c. is characterizedby a HCDR3 region comprising between 10 and 34% of tyrosine and/orbetween 2 and 20% of histidine, and/or d. does not modulate Gprotein independent signaling of the seven transmembrane receptor,and/or e. is a non-internalizing antibody or is characterized by aninternalization into a cell with endogenous target expression whichis lower than the 1.5, 2, 3, 4, 5, 6, 7, or 10-fold of theinternalization of the isotype control, and/or f. induces ADCCand/or ADCP g. is a human, rat or murine IgG antibody, preferably ahuman IgG1 antibody or a murine IgG2a antibody, and/or h. is anscFv, Fab, Fab' or a F(ab')2 fragment.

Provided according to a preferred embodiment XIV is a conjugatecomprising an antibody or antigen-binding fragment according to anyof preferred embodiments I to XIII, preferably wherein theconjugate comprises a. a radioactive element, b. a cytotoxic agent,such as an auristatin, a maytansinoid, a kinesin-spindle proteininhibitor, a nicotinamide phosphoribosyltransferase inhibitor or apyrrolobenzodiazepine derivative, c. a further antibody orantigen-binding fragment, or d. a chimeric antigen receptor.

Provided according to a preferred embodiment XV is an antibody orantigen-binding fragment according to any of preferred embodimentsI to XIII or a conjugate according to preferred embodiment XIV foruse in the treatment of a tumor or a disease characterized by theinvolvement of cells expressing the seven transmembrane receptor,optionally in combination with an antibody targeting a checkpointinhibitor. Provided according to a preferred embodiment XVI is theantibody or antigen-binding fragment according to any of preferredembodiments I to XIII or a conjugate according to preferredembodiment XIV for use as a diagnostic agent in vivo or in vitro.Provided according to a preferred embodiment XVII is a kitcomprising an antibody or antigen-binding fragment according to anyof preferred embodiments I to XIII or a conjugate according topreferred embodiment XIV with instructions for use.

Antibodies Binding CCR8

The following aspects 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and18 relate to antibodies specifically binding to CCR8. While eachaspect is described individually, the different structural orfunctional aspects may be combined and are suggested to be combinedwith each other, except where obviously incompatible. Furthermore,each embodiment in each aspect may be combined with each embodimentin the same or in a different aspect, except where obviouslyincompatible.

If not explicitly stated otherwise, the CCR8 may be from anyspecies e.g. human, monkey, Macaca fascicularis (cynomolgusmonkey), Macaca mulatta (Rhesus macaque), rodent, mouse, rat,horse, bovine, pig, dog, cat and camel CCR8. Antibodies bindingCCR8 from at least two species, wherein one species is human, arehighly preferred, and are suggested to be combined with eachembodiment disclosed for these aspects. Antibodies having humanderived CDRs are highly preferred and are suggested to be combinedwith each embodiment disclosed for these aspects. According to thecurrent aspects, antibodies having a HCDR3 domain characterized bya frequency of at least 21% tyrosine residues and/or at least 2%,7% or 10% histidine are highly preferred and are suggested to becombined with each embodiment disclosed for the current aspect.According to the current aspects, antibodies inducing both, ADCCand ADCP, such as afucosylated antibodies, are highly preferred,and are suggested to be combined with each embodiment disclosed forthese aspects. According to the current aspects, low internalizingor non-internalizing antibodies or fragments are highly preferredand are suggested to be combined with each embodiment disclosed forthese aspects. Some highly preferred characterizing features whichare applicable for each of the described aspects are describedunder section "Preferred combinations according to `allaspects`".

Aspect 7--CCR8 Antibody Binding Sulfated TRD

The extracellular domains of human CCR8 can be structured into fourregions:

(i) an N-terminal domain which can be subdivided into

a) the membrane-distal tyrosine-rich domain (TRD), formed by aminoacids 1 to 24 (SEQ ID NO:43) b) a cysteine at amino acid position25, and c) a LID domain, formed by amino acids 26 to 35 (SEQ IDNO:49) (iii) an extracellular domain 1 (ECL1) according to SEQ IDNO:52, (iii) an extracellular domain 2 (ECL2) according to SEQ IDNO:55, and (iv) an extracellular domain 3 (ECL3) according to SEQID NO:58.

According to a 7th aspect, which may be or may not be the same asthe 6.sup.th aspect, there is provided an isolated antibody orantigen-binding fragment thereof, specifically binding to thesulfated tyrosine rich domain of CCR8.

For example, the antibody or antigen-binding fragment binds to afirst isolated sulfated polypeptide which comprises a) the tyrosinerich domain (TRD) of CCR8 or b) the N term of CCR8 including theTRD, optionally wherein at least the cysteine between the TRD andthe LID domain has been removed or has been exchanged into adifferent amino acid. A sulfated polypeptide according to thecurrent aspect is a polypeptide wherein preferably at least 25%, atleast 50% or at least 75% of the tyrosine residues of the TRD aresulfated. Where the polypeptide comprises the N term of CCR8including the TRD, preferably the cysteine between the TRD and theLID domain has been exchanged into a serine or has been removed.Without being bound by theory, the specific recognition of theprovided sulfation pattern for CCR8 seems to influence if theantibody competes with CCL1, the natural ligand for CCR8, and alsoif and how the antibody agonizes or antagonizes the CCR8 signaling,e.g. as described according to the 11.sup.th aspect. In somepreferred embodiments according to the current aspect, theinventive antibody binds sulfated TRD of human and/or cynomolgusCCR8 with a KD value of <5E-8 M, <4E-8 M, <3E-8 M,<2E-8 M, <1E-8 M, <9E-9 M, <8E-9 M, <7E-9 M,<6E-9 M, <5E-9 M, <4E-9 M, <3E-9 M, <2.5E-9 M,<2E-9 M, <1.5E-9 M, <1E-9 M, <9E-10 M, <8E-10 M,<7E-10 M, <6E-10 M, <5E-10 M, <4E-10 M, <3E-10 M,<2.5E-10 M, <2E-10 M, <1.5E-10 M, <1E-10 M, or<9E-11 M. For example, the inventive antibody may bind sulfatedTRD of human and/or cynomolgus CCR8 with a KD value between<8E-9 M and >4E-10 M. For example, the inventive antibody maybind sulfated TRD of human and/or cynomolgus CCR8 with a KD valuebetween <8E-9 M and >5.5E-10 M. In some further preferred ofthese embodiments, the inventive antibody binds the sulfated N term(i.e. comprising the aforementioned sulfated TRD) of human and/orcynomolgus CCR8 with substantially the same KD value. In some mostpreferred of these embodiments, the inventive antibody doessubstantially not bind non-sulfated TRD of human and/or cynomolgusCCR8.

In some preferred embodiments according to the current aspect, theinventive antibody binds sulfated N term of human and/or cynomolgusCCR8 with a KD value of <5E-8 M, <4E-8 M, <3E-8 M,<2E-8 M, <1E-8 M, <9E-9 M, <8E-9 M, <7E-9 M,<6E-9 M, <5E-9 M, <4E-9 M, <3E-9 M, <2.5E-9 M,<2E-9 M, <1.5E-9 M, <1E-9 M, <9E-10 M, <8E-10 M,<7E-10 M, <6E-10 M, <5E-10 M, <4E-10 M, <3E-10 M,<2.5E-10 M, <2E-10 M, <1.5E-10 M, <1E-10 M, <9E-11M, <8E-11 M, <7E-11 M, <6E-11 M, or <5E-11 M. Forexample, the inventive antibody may bind sulfated N term of humanand/or cynomolgus CCR8 with a KD value between <8E-9 M and>8E-11 M. For example, the inventive antibody may bind sulfatedN term of human and/or cynomolgus CCR8 with a KD value between<8E-9 M and >7.5E-11 M. In some further preferred of theseembodiments, the inventive antibody binds sulfated TRD (i.e., asubsequence of the aforementioned sulfated N term) of human and/orcynomolgus CCR8 with substantially the same KD value or a KD valuewithin the same order of magnitude. In some most preferred of theseembodiments, the inventive antibody does substantially not bindnon-sulfated TRD of human and/or cynomolgus CCR8.

According to some first embodiments of the 7th aspect, the firstisolated sulfated polypeptide comprises a) SEQ ID NO:43 (CCR8HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 andY17 have been sulfated, b) SEQ ID NO:44 (CCR8_MACFA_TRD),preferably wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and/or c) SEQ ID NO:45 (CCR8_MOUSE_TRD), preferablywherein at least two or all of Y3, Y14 and Y15 have beensulfated.

According to some second embodiments of the 7th aspect, the firstisolated sulfated polypeptide comprises a) SEQ ID NO:46(CCR8_HUMAN_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, b) SEQ ID NO:47(CCR8_MACFA_N term with C.dbd.X or S), preferably wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, and/or c) SEQ IDNO:48 (CCR8_MOUSE_N term with C.dbd.X or S), preferably wherein atleast two or all of Y3, Y14 and Y15 have been sulfated.

According to some third embodiments of the 7th aspect, which may bethe same as or different from the first and/or second embodiments,the antibody or antigen-binding fragment specifically binds to thefirst isolated sulfated polypeptide with a dissociation constant oran EC50 of <15 nM, <10 nM, <5 nM, <1 nM or <0.6nM.

According to some preferred of these embodiments, the isolatedantibody or antigen-binding fragment thereof, specifically bindswith a dissociation constant or an EC50 of <15 nM, <10 nM,<5 nM, <1 nM or <0.6 nM a) to human CCR8 or to an isolatedpolypeptide according to SEQ ID NO:46, wherein at least two or allof Y3, Y15 and Y17 have been sulfated, b) to cynomolgus CCR8 or toan isolated polypeptide according to SEQ ID NO:47, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, and/or c) tomurine CCR8 or to an isolated polypeptide according to SEQ IDNO:48, wherein at least two or all of Y3, Y14 and Y15 have beensulfated.

According to some fourth embodiments of the 7.sup.th aspect, whichmay be and are suggested to be combined with the first, second,and/or third embodiments of the 7.sup.th aspect, the dissociationconstant (KD) of the antibody for binding the first isolatedsulfated polypeptide is lower than the dissociation constant (KD)of the antibody for binding an isolated non-sulfated polypeptidehaving the same sequence as the first isolated sulfatedpolypeptide.

According to some embodiments A of these fourth embodiments, thedissociation constant or EC50 of the antibody for binding theisolated non-sulfated polypeptide is higher than 1 pM, 10 nM, 25nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM,450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 .mu.M, 1.25.mu.M, 1.5 .mu.M, 1.75 .mu.M, 2 .mu.M, 2.25 .mu.M, 2.5 .mu.M, 2.75.mu.M, or 3 .mu.M, 5 .mu.M or is not detectable. Preferably, thedissociation constant or EC50 of the antibody for binding theisolated non-sulfated polypeptide is higher than 100 nM, 250 nM,500 nM, 1 .mu.M, 2 .mu.M or 3 .mu.M, or is not detectable.According to some embodiments B of these fourth embodiments, whichmay be the same as the embodiments A of the these secondembodiments, the dissociation constant or the EC50 of the antibodyfor binding the first isolated sulfated polypeptide is below 10 nM,5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociationconstant or the EC50 of the antibody for binding the isolatednon-sulfated polypeptide is higher than 10 nM, 25 nM, 50 nM, 100nM, 250 nM or 500 nM, or is not detectable.

Aspect 8--CCR8 Antibody Comprising Human CDRs

According to an 8th aspect, which may be or may not be the same asthe 6.sup.th or 7.sup.th aspect, there is provided an isolatedantibody or antigen-binding fragment thereof, (specifically)binding to CCR8, wherein the antibody comprises human derived CDRs.Most preferably, the antibody or antigen-binding fragment thereof,(specifically) binds to the sulfated TRD of CCR8. According to somepreferred embodiments, a single CDR comprises less than 1, 2, 3, 4,5 or 6 deviations from the closest human germline. According tosome highly preferred embodiments, the human derived CDRs compriseno deviations or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 deviations from the closest human germline. Theclosest human germline may be determined as known in the art, e.g.in silico using IgBLAST with data retrieved from the IMGT humangermline database, as discussed elsewhere herein.

While humanization of antibodies with non-human CDRs may improvethe immunogenicity, residual immunogenicity resides in the CDRregions (Harding, Fiona A., et al. "The immunogenicity of humanizedand fully human antibodies: residual immunogenicity resides in theCDR regions." MAbs. Vol. 2. No. 3. Taylor & Francis, 2010.).Antibodies according to the current aspect comprising human derivedCDRs and a low number of germline deviations are thus assumed tohave a superior suitability as therapeutic agents.

Antibodies comprising human derived CDRs can be obtained asdescribed herein, for example by using a human phage displaylibrary as described in examples 4 and 6. In the alternative,antibodies with human CDRs can also be produced using transgenicmice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes.Furthermore, antibodies comprising human CDRs may also be generatedby using in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610and 5,229,275, each of which is incorporated herein by reference inits entirety).

The provided isolated antibody or antigen-binding fragmentcomprising human derived CDRs is preferably also an antibodyaccording to any of aspects 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18or a combination thereof.

Aspect 9--Cross Reactive CCR8 Antibody

According to a 9th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th and/or 8.sup.th aspect, there is provided anisolated antibody or antigen-binding fragment thereof, specificallybinding to CCR8, wherein the antibody or antigen-binding fragmentis cross reactive for CCR8 from at least two species, preferablyselected from human, monkey, Macaca fascicularis (cynomolgusmonkey), Macaca mulatta (Rhesus macaque), rodent, mouse, rat,horse, bovine, pig, dog, cat and camel, even more preferablyselected from human, cynomolgus and mouse. According to some mostpreferred embodiments, the antibody or antigen-binding fragment iscross reactive for human and cynomolgus CCR8.

In preferred embodiments, the antibody or antigen-binding fragmentbinds the CCR8 from a first species with a first dissociationconstant KD and binds the CCR8 from a second species with a seconddissociation constant KD, wherein the first and the seconddissociation constant are in the same order of magnitude. Asunderstood by the skilled person, an order-of-magnitude differencebetween two values is a factor of 10.

Cross reactive anti-CCR8 antibodies are advantageous for thedevelopment of a therapeutic antibody because they can be used innon-human animal models to characterize the therapeutic agents withregards to pharmacological data and safety before the antibodiesare administered to humans. However, cross reactive antibodies withsimilar binding behavior in two species are difficult to generate,because the parts of CCR8 which can be used for antibodyrecognition have a low homology between species (see example 2).According to the current invention, cross reactive antibodies forCCR8 can be generated by using small sulfated tyrosine comprisingmotifs which have a higher conservation between species, such thatcross reactive antibodies binding CCR8 from two or more specieswith affinities in the same order of magnitude could be obtained inan easy and convenient way. In more detail, the antibodiesspecifically bind sulfated TRD motifs and cross-reactive antibodiescan be obtained because these sulfated TRD motifs are conservedbetween species.

In some preferred embodiments according to the current aspect, theinventive antibody binds sulfated TRD of human and cynomolgus CCR8with a KD value of <5E-8 M, <4E-8 M, <3E-8 M, <2E-8 M,<1E-8 M, <9E-9 M, <8E-9 M, <7E-9 M, <6E-9 M,<5E-9 M, <4E-9 M, <3E-9 M, <2.5E-9 M, <2E-9 M,<1.5E-9 M, <1E-9 M, <9E-10 M, <8E-10 M, <7E-10 M,<6E-10 M, <5E-10 M, <4E-10 M, <3E-10 M, <2.5E-10 M,<2E-10 M, <1.5E-10 M, <1E-10 M, or <9E-11 M. Forexample, the inventive antibody may bind sulfated TRD of human andcynomolgus CCR8 with a KD value between <8E-9 M and >4E-10 M.For example, the inventive antibody may bind sulfated TRD of humanand cynomolgus CCR8 with a KD value between <8E-9 M and>5.5E-10 M. In some further preferred of these embodiments, theinventive antibody binds the sulfated N term (i.e. comprising theaforementioned sulfated TRD) of human and cynomolgus CCR8 withsubstantially the same KD value or a KD within one order ofmagnitude. In some most preferred of these embodiments, theinventive antibody does substantially not bind non-sulfated TRD ofhuman and cynomolgus CCR8.

In some preferred embodiments according to the current aspect, theinventive antibody binds sulfated N term of human and cynomolgusCCR8 with a KD value of <5E-8 M, <4E-8 M, <3E-8 M,<2E-8 M, <1E-8 M, <9E-9 M, <8E-9 M, <7E-9 M,<6E-9 M, <5E-9 M, <4E-9 M, <3E-9 M, <2.5E-9 M,<2E-9 M, <1.5E-9 M, <1E-9 M, <9E-10 M, <8E-10 M,<7E-10 M, <6E-10 M, <5E-10 M, <4E-10 M, <3E-10 M,<2.5E-10 M, <2E-10 M, <1.5E-10 M, <1E-10 M, <9E-11M, <8E-11 M, <7E-11 M, <6E-11 M, or <5E-11 M. Forexample, the inventive antibody may bind sulfated N term of humanand cynomolgus CCR8 with a KD value between <8E-9 M and>8E-11 M. For example, the inventive antibody may bind sulfatedN term of human and cynomolgus CCR8 with a KD value between<8E-9 M and >7.5E-11 M. In some further preferred of theseembodiments, the inventive antibody binds sulfated TRD (i.e., asubsequence of the aforementioned sulfated N term) of human andcynomolgus CCR8 with substantially the same KD value or a value inthe same order of magnitude. In some most preferred of theseembodiments, the inventive antibody does substantially not bindnon-sulfated TRD of human and cynomolgus CCR8.

According to some first embodiments of the 9th aspect, the antibodyspecifically binds to a) a first isolated sulfated polypeptidecomprising SEQ ID NO:46 (CCR8_HUMAN_N term with C.dbd.X or S),preferably wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and a second isolated sulfated polypeptide comprising SEQID NO:47 (CCR8_MACFA_N term with C.dbd.X or S), preferably whereinat least two or all of Y3, Y15 and Y17 have been sulfated, or b) afirst isolated sulfated polypeptide comprising SEQ ID NO:43(CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15and Y17 have been sulfated, and a second isolated sulfatedpolypeptide comprising SEQ ID NO:44 (CCR8_MACFA_TRD), preferablywherein at least two or all of Y3, Y15 and Y17 have beensulfated.

Preferably, the EC50 of the antibody or antigen-binding fragmentfor binding human CCR8 or for binding the first isolated sulfatedpolypeptide is below 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1nM, 0.5 or 0.25 nM, such as below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or0.25 nM. Preferably, the EC50 of the antibody or antigen-bindingfragment for binding cynomolgus CCR8 or for binding the secondisolated sulfated polypeptide is below 10 nM, 5 nM, 2.5 nM, 1 nM,0.5 or 0.25 nM. In some preferred embodiments, the antibodies bindthe first isolated sulfated polypeptide and the second isolatedsulfated polypeptide with substantially the same affinity.

According to some second embodiments of the 9th aspect, theantibody specifically binds to a) a first isolated sulfatedpolypeptide comprising SEQ ID NO:46 (CCR8_HUMAN_N term with C.dbd.Xor S), preferably wherein at least two or all of Y3, Y15 and Y17have been sulfated, and a second isolated sulfated polypeptidecomprising SEQ ID NO:48 (CCR8_MOUSE_N term with C.dbd.X or S),preferably wherein at least two or all of Y3, Y14 and Y15 have beensulfated, or b) a first isolated sulfated polypeptide comprisingSEQ ID NO:43 (CCR8_HUMAN_TRD), preferably wherein at least two orall of Y3, Y15 and Y17 have been sulfated, and a second isolatedsulfated polypeptide comprising SEQ ID NO:45 (CCR8_MOUSE_TRD),preferably wherein at least two or all of Y3, Y14 and Y15 have beensulfated.

Preferably, the EC50 of the antibody or antigen-binding fragmentfor binding human CCR8 or for binding the first isolated sulfatedpolypeptide is below 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1nM, 0.5 or 0.25 nM, such as below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or0.25 nM. Preferably, the EC50 of the antibody or antigen-bindingfragment for binding murine CCR8 or for binding the second isolatedsulfated polypeptide is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or0.25 nM. In some preferred embodiments, the antibodies bind thefirst isolated sulfated polypeptide and the second isolatedsulfated polypeptide with substantially the same affinity.

The provided isolated antibody or antigen-binding fragment ispreferably also an antibody according to any of aspects 7, 8, 10,11, 12, 13, 14, 15, 16, 17 or 18 or a combination thereof.

Aspect 10--CCR8 Antibody Defined by HCDR3 Structure

According to a 10th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th and/or 9.sup.th aspect, there isprovided an isolated antibody or antigen-binding fragment thereof,specifically binding to CCR8, wherein the antibody orantigen-binding fragment is characterized by a HCDR3 region withincreased histidine and/or tyrosine frequency. As understood by theskilled person, although the frequency for each amino acid isdiscussed separately, the respective frequencies are interrelatedand are suggested to be combined, except where the skilled personimmediately recognizes their incompatibility. For determination ofCDR regions, the immunoglobulin amino acid residue numbering systemof Kabat was used herein and shall be decisive in case ofdoubt.

It was surprisingly found, that the CCR8 antibodies according tothe current invention were characterized by a HCDR3 havingsubstantially higher frequencies of tyrosine residues than would beexpected for a random fully human HCDR3 with matched length(.about.10%, cf. Zemlin, Michael, et al. "Expressed murine andhuman CDR-H3 intervals of equal length exhibit distinct repertoiresthat differ in their amino acid composition and predicted range ofstructures." Journal of molecular biology 334.4 (2003): 733-749.).On average, the initial set of inventive antibodies comprised.about.20.6% tyrosine residues, while the optimized set of specifichuman CCR8 binding antibodies comprised on average 21% tyrosineresidues in the HCDR3.

Furthermore, the number of positively charged amino acids and inparticular the number of histidine residues was higher thanexpected, in particular in the optimized set of antibodies withimproved therapeutic features, cf. example 9. On average, whensummed up, histidine residues and tyrosine residues accounted for31% of all amino acids in the HCDR3 of the optimized set of CCR8binding antibodies. Interestingly, the used antigen comprising thesulfated TRD, is characterized by tyrosine residues and aparticularly high number of negative charges, which was furtherincreased by sulfation. Without being bound by theory, theinventors believe that the preference for a high tyrosine andhistidine content in the CDRH3 of the inventive antibodies iscaused by the particular sulfated motif of the antigen and that ahigh tyrosine/histidine content in the HCDR3 promotes the specificrecognition of the antigen. Without being bound by theory, theinventors furthermore believe, that this specific recognitioninfluences the characteristics of the obtained antibodies as amodulator of CCR8, e.g. as discussed in aspect 11.

In some highly preferred embodiments of the 10.sup.th aspect, thereis provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to CCR8, wherein the antibody orfragment is characterized by a HCDR3 region comprising between 10and 34% of tyrosine and/or between 2 and 20% of histidine.

The provided isolated antibody or antigen-binding fragment ispreferably also an antibody according to any of aspects 7, 8, 9,11, 12, 13, 14, 15, 16, 17 or 18 or a combination thereof.

In some first embodiments of the 10.sup.th aspect, the isolatedantibody or antigen-binding fragment comprises a HCDR3 having a)>0 and <35%, >8 and <34%, .gtoreq.10 and .ltoreq.34%,or >15 and <31% tyrosine (Y) residues, and/or b) >0 and.ltoreq.16%, .gtoreq.2 and .ltoreq.20% or .gtoreq.7 and .ltoreq.20%histidine (H) residues, and c) preferably >0 and .ltoreq.18%,.gtoreq.7 and .ltoreq.10% or .gtoreq.0 and .ltoreq.7% arginine (R)residues, and/or d) preferably >0 and .ltoreq.25%, .gtoreq.7 and.ltoreq.16% or .gtoreq.7 and .ltoreq.13% aspartic acid (D)residues, and/or e) preferably no lysine (K) residues, and/or f)preferably no glutamic acid (E) residues.

In some second embodiments of the 10.sup.th aspect, which may bethe same as or different from the first embodiments, the isolatedantibody or antigen-binding fragment comprises a HCDR3 having a)>0 and <47%, >22 and <50%, >10 and <34%, or>15 and <47% charged amino acids, and/or b) >0 and<32%, >8 and <30%, or >10 and <37% positivelycharged amino acids, and/or c) >0 and <26%, .gtoreq.7 and<16%, or >7 and <14% negatively charged amino acids.

In some third embodiments of the 10.sup.th aspect, which may be thesame as or different from the first and/or second embodiments, theisolated antibody or antigen-binding fragment comprises a HCDR3having >0 and .ltoreq.42%, .gtoreq.10 and .ltoreq.42% or.gtoreq.36 and .ltoreq.43% histidine and tyrosine residues intotal.

Tyrosine (Y)

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 33%, 31%, 25%,23%, 21%, 20%, 18%, 15%, 10%, 9%, 8% or 0% tyrosine. In some highlypreferred embodiments, the isolated antibody or antigen-bindingfragment comprises a HCDR3 having (a) >0 and <35%, (b) >8and <34%, (c) >10 and <34%, or (d) >15 and <31%tyrosine. In some embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having >8, >9,>10, >11, >12, >13, >14, >15, >16, >17,>18, >19, >20, >21, >22, >23, >24, >25,>26, >27, >28, >29, >30, >31, >32, >33%tyrosine residues. In some embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having <35, <34,<33, <32, <31, <30, <29, <28, <27, <26,<25, <24, <23, <22, <21, <20, <19, <18,<17, <16, <15, <14 <13, <12, <11, <10,<9 or <8% tyrosine residues.

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having >0 and <34%, >1 and<34%, >2 and <34%, >3 and <34%, >4 and <34%,>5 and <34%, >6 and <34%, >7 and <34%, >8 and<34%, >9 and <34%, >10 and <34%, >11 and <34%,>12 and <34%, >13 and <34%, >14 and <34%, >15and <34%, >16 and <34%, >17 and <34%, >18 and<34%, >19 and <34%, >20 and <34%, >21 and<34%, >22 and <34%, >23 and <34%, >24 and<34%, >25 and <34%, >26 and <34%, >27 and<34%, >28 and <34%, >29 and <34% or >30 and<34% tyrosine residues.

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having >0 and <36%, >0 and<35%, >0 and <34%, >0 and <33%, >0 and <32%,>0 and <31%, >0 and <30%, >0 and <29%, >0 and<28%, >0 and <27%, >0 and <26%, >0 and <25%,>0 and <24%, >0 and <23%, >0 and <22%, >0 and<21%, >0 and <20%, >0 and <19%, >0 and <18%,>0 and <17%, >0 and <16%,or >0 and <15%, >0and <14%, >0 and <13%, >0 and <12%, >0 and<11%, >0 and <10%, >0 and <9%, >0 and <8%,>0 and <7%, >0 and <6%, >0 and <5% tyrosineresidues.

Charged aa

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 47%, 39%, 31%,28%, 27%, 25%, 23%, 20%, 18%, 17%, 16%, 15%, 9% or 0% charged aminoacids. In some highly preferred embodiments, the isolated antibodyor antigen-binding fragment comprises a HCDR3 having (a) >0 and<47%, (b) >22 and <50%, (c) >10 and <34%, or (d)>15 and <47% charged amino acids.

Positively Charged aa

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 31%, 23%, 18%,15%, 10%, 8%, 7%, or 0% positively charged amino acids. In somehighly preferred embodiments, the isolated antibody orantigen-binding fragment comprises a HCDR3 having (a) >0 and<32%, (b) >8 and <30%, or (c) >10 and <37%positively charged amino acids.

Negatively Charged aa

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 25%, 17%, 15%,11%, 10%, 9%, 8%, 7%, or 0% negatively charged amino acids. In somehighly preferred embodiments, the isolated antibody orantigen-binding fragment comprises a HCDR3 having (a) >0 and<26%, (b) >7 and <16%, or (c) >7 and <14% negativelycharged amino acids.

Histidine (H)

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 0%, 3%, 7%, 8%,10% or 15 histidine residues. Preferably, the isolated antibody orantigen-binding fragment comprises a HCDR3 having at least onehistidine residue. In some highly preferred embodiments, theisolated antibody or antigen-binding fragment comprises a HCDR3having (a) >0 and .ltoreq.16%, (b) .gtoreq.2 and .ltoreq.20% or(c) .gtoreq.7 and .ltoreq.20% histidine residues.

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having .gtoreq.0, >0, >1, >2,>3, >4, >5, >6, >7, >8, >9, >10, >11,>12, >13, >14, >15, >16, >17, >18, >19,>20% histidine residues. In some embodiments the isolatedantibody or antigen-binding fragment comprises a HCDR3 having<20, <19, <18, <17, <16, <15, <14<13,<12, <11, <10, <9, <8, <7, <6, <5, <4,<3, <2 or <1% histidine residues.

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having >0 and <24%, >1 and<24%, >2 and <24%, >3 and <24%, >4 and <24%,>5 and <24%, >6 and <24%, >7 and <24%, >8 and<24%, >9 and <24%, >10 and <24%, >11 and <24%,>12 and <24%, >13 and <24%, >14 and <24%, >15and <24%, >16 and <24%, >17 and <24%, >18 and<24%, >19 and <24%, >20 and <24%, >21 and<24%, >22 and <24% or >23 and <24% histidineresidues.

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having >0 and <24%, >0 and<23%, >0 and <22%, >0 and <21%, >0 and <20%,>0 and <19%, >0 and <18%, >0 and <17%, >0 and<16%, or >0 and <15%, >0 and <14%, >0 and<13%, >0 and <12%, >0 and <11%, >0 and <10%,>0 and <9%, >0 and <8%, >0 and <7%, >0 and<6%, >0 and <5%, >0 and <4%, >0 and <3%, >0and <2% or >0 and <1% histidine residues.

Histidine+Tyrosine

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 42%, 38%, 33%,31%, 30%, 24%, 23%, 18%, 15%, 10%, 9% or 0% histidine and tyrosineresidues in total. Preferably, the isolated antibody orantigen-binding fragment comprises a HCDR3 having at least onehistidine residue. In some highly preferred embodiments, theisolated antibody or antigen-binding fragment comprises a HCDR3having (a) >0 and .ltoreq.42%, (b) .gtoreq.10 and .ltoreq.42% or(c) .gtoreq.36 and .ltoreq.43% histidine and tyrosine residues intotal.

Arginine (R)

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 0%, 7%, 8%, 10%,15% or 18% arginine residues. In some highly preferred embodiments,the isolated antibody or antigen-binding fragment comprises a HCDR3having (a) >0 and .ltoreq.18%, (b) .gtoreq.7 and .ltoreq.10% or(c) .gtoreq.0 and .ltoreq.7% arginine residues.

Lysine (K)

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having 0% to 8% lysine residues. In someembodiments, the isolated antibody or antigen-binding fragmentcomprises a HCDR3 having no lysine residues.

Aspartic Acid (D)

In some preferred embodiments the isolated antibody orantigen-binding fragment comprises a HCDR3 having 0%, 7%, 8%, 9%,10%, 11%, 15%, 16%, 17% or 25% aspartic acid residues. In somehighly preferred embodiments, the isolated antibody orantigen-binding fragment comprises a HCDR3 having (a) >0 and.ltoreq.25%, (b) .gtoreq.7 and .ltoreq.16% or (c) .gtoreq.7 and.ltoreq.13% aspartic acid residues.

Glutamic acid (E)

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having 0%, 8%, or 9% glutamic acidresidues. In some highly preferred embodiments, the isolatedantibody or antigen-binding fragment comprises a HCDR3 having noglutamic acid residues.

Length

In some embodiments the isolated antibody or antigen-bindingfragment comprises a HCDR3 having a length of 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.Preferably, the isolated antibody or antigen-binding fragmentcomprises a HCDR3 having a length of 10, 11, 12 or 13 amino acidresidues or a length between 8 and 13 amino acid residues.

Aspect 11--Blocking/Neutral Ccr8 Antibody

There are multiple different ways how an antibody can modulate CCR8signaling. For example, an antibody can a) block G-proteinindependent signaling, b) block G-protein dependent signaling, c)block G-protein dependent and G-protein independent signaling, d)increase G-protein independent signaling, e) increase G-proteindependent signaling, f) increase G-protein dependent and G-proteinindependent signaling.

According to an 11th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th and/or 10.sup.th aspect,there is provided an isolated antibody or antigen-binding fragmentthereof, specifically binding to CCR8, wherein the antibody orantigen-binding fragment at least partially modulates CCR8signaling. For CCR8, G-protein independent signaling pathways are.beta.-arrestin signaling (example 10.4.1), phospho Erk1/2signaling (phosphorylation of Erk1/2), and phospho Akt signaling(phosphorylation of AKT) (example 10.4.2). Further subtypes ofantagonists or agonist can be defined based on the impact on thesethree G-protein independent signaling pathways.

According to some first embodiments of the 11.sup.th aspect, theantibody or antigen-binding fragment a) does not block CCL1 induced.beta.-arrestin signaling and/or b) does not induce ERK1/2phosphorylation and/or c) does not induce AKT phosphorylation.

Example 10.4.1 shows that the prior art antibodies 433H and L268G8efficiently blocked CCL1 induced .beta.-arrestin signaling, e.g.with IC50 values below 20 nM (cf. table 10.4.1.1), while no IC50value could be determined for inventive antibodies such asTPP-23411.

According to some embodiments A of the first embodiments of the11.sup.th aspect, the antibody or antigen-binding fragment does notblock CCL1 induced .beta.-arrestin signaling. In case of doubt, anantibody blocks CCL1 induced .beta.-arrestin signaling, if the IC50is below 100 nM. In case of doubt, an antibody does not block CCL1induced .beta.-arrestin signaling, if the IC50 is .gtoreq.100 nM,or if no IC50 can be determined with the assay system describedherein. CCL1 induced .beta.-arrestin signaling has been linked toreceptor internalization as discussed for the 12.sup.th aspect.

Example 10.4.2, FIG. 27, 28 show that prior art antibodies inducephosphorylation of Erk1/2 in CHO cells expressing human CCR8 orhuman activated Tregs, e.g. after 15 minutes, at least by a factorof 1.5. Instead, the inventive antibody TPP-23411 did not induce asignificant phosphorylation of Erk1/2.

According to some embodiments B of the first embodiments of the11.sup.th aspect, the antibody or antigen-binding fragment does notinduce ERK1/2 phosphorylation. In case of doubt, an antibodyinduces phosphorylation of Erk1/2, if--using an assay as describedherein--at least a 1.5-fold increase of Erk1/2 phosphorylationlevels over control can be detected. In case of doubt, an antibodydoes not induce phosphorylation of Erk1/2, if no significantincrease of Erk1/2 phosphorylation levels is detected or if theincrease is below 1.5-fold over control.

Example 10.4.2, FIG. 30, show that prior art antibodies inducephosphorylation of AKT, e.g. after 15 minutes at least by a factorof 1.5. Instead, the inventive antibody TPP-23411 did not induce asignificant phosphorylation of AKT.

According to some embodiments C of the first embodiments of the11.sup.th aspect, the antibody or antigen-binding fragment does notinduce AKT phosphorylation. In case of doubt, an antibody inducesphosphorylation of AKT, if--using an assay as described herein--atleast a 1.5-fold increase of AKT phosphorylation levels overcontrol can be detected. In case of doubt, an antibody does notinduce phosphorylation of AKT, if no significant increase of AKTphosphorylation levels is detected or if the increase is below1.5-fold over control.

Antibodies or fragments which show no induction of G-proteinindependent signaling pathways such as AKT or ERK1/2phosphorylation are assumed to have advantages in therapy becauseinduction of G-protein dependent signaling may lead to unwantedeffects and side effects.

To analyze G-protein dependent signaling, the Ca flux assay asknown in the art and as described herein can be used as a read out.The majority of tested inventive antibodies were found to be fullyefficacious antagonists of G protein dependent Ca signaling,however, differences in the IC50 could be determined in comparisonwith the prior art antibodies.

Example 10.4.3 shows that several of the tested anti-CCR8antibodies blocked CCL1 induced G protein dependent Ca signaling,e.g. with IC50 values in the low nM or even sub nM range.

According to some second embodiments of the 11.sup.th aspect, whichcan be the same as or different from the first embodiments, theantibody or antigen-binding fragment blocks CCL1 induced calciumsignaling. In some preferred of these embodiments, the antibody orantigen binding fragment blocks CCL1 induced calcium signaling,e.g. with an IC50<1 nM, <0.5 nM or <0.01 nM.

According to some third embodiments of the 11.sup.th aspect, whichcan be the same as or different from the first embodiments, theantibody or antigen-binding fragment does not block CCL1 induced Gprotein dependent calcium signaling.

Aspect 12--None and/or Low Internalizing CCR8 Antibody

An anti-CCR8 antibody can be a non-internalizing, lowinternalizing, medium internalizing or high internalizing antibodyor antigen-binding fragment.

According to a 12th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th and/or11.sup.th aspect, there is provided an isolated antibody orantigen-binding fragment thereof, specifically binding to CCR8,wherein the antibody or antigen-binding fragment is anon-internalizing or low internalizing antibody or antigen bindingfragment.

Depending on the specific mode of action of an antibody, fragmentor conjugate, internalization into a cell may be desired or has tobe avoided, as discussed elsewhere herein. The antibodies accordingto the 12th aspect are particularly suited for an ADCC/ADCPapproach, or any other mode of action relying on the antibody orantigen binding fragment for recruiting effector cells.

Because overexpression may impact the internalization behavior andis less suited to model internalization in a therapeutic setting,internalization is preferably determined using a model cell linewith endogenous expression of the target. Where the target is humanCCR8, the cell endogenously expressing the target is preferablyHuT78. Where the target is murine CCR8, the cell endogenouslyexpressing the target is preferably murine BW5147.3. HuT78 andmBW5147.3 can be obtained from ATCC.

While all prior art antibodies readily internalized into cells withendogenous target expression, as shown in example 10.5, the testedantibodies TPP-21360, TPP-21047 (data not shown) and TPP-23411 andvarious further antibodies according to the current invention didnot significantly internalize. For example, internalization can bedetermined over a time frame or for specific time points.Preferably internalization can be determined after 15 min, 30 min,1 h, 2 h, 3 h, 6 h, 12 h, 24 h or 48 h in a cell endogenouslyexpressing the target.

According to some first embodiments of the 12.sup.th aspect, theantibody or antigen-binding fragment is characterized by aninternalization into a cell with endogenous target expression whichis lower than the 1.5, 2, 3, 4, 5, 6, 7, or 10-fold of theinternalization of the isotype control. The isotype control for anantibody can be selected as known in the art to match the isotypeof the antibody as closely as possible, but without binding thetarget.

According to some of these embodiments, the antibody orantigen-binding fragment is characterized by an internalizationinto a cell with endogenous target expression which is lower than150%, 175%, 200%, 300%, 400% or 500% of the internalization of theisotype control e.g. after 15 min, 30 min, 1 h, 2h, 3 h, 6 h, 12 h,24 h or 48 h, preferably wherein the cell with endogenous targetexpression is a HuT78 lymphoma cell.

According to some preferred of these embodiments, the antibody orantigen-binding fragment has an internalization rate in the sameorder of magnitude as the internalization rate of the isotypecontrol.

According to some second embodiments of the 12.sup.th aspect, whichmay be the same as or different from the first embodiments, theantibody or antigen-binding fragment specifically binds human CCR8and is characterized by a time until half of the amount ofantibody, fragment or conjugate has been internalized which is>2 hours, preferably >4, >5, >6, >7, >8, >9,>10, >11, >12, >13, >14, >15, >16, >17,>18, >19, >20, >21, >22, >23, >24, >26,>28, >30, or >48 hours in a cell endogenously expressingthe target, or no such time can be determined at all, preferablywherein the cell endogenously expressing the target is a HuT78lymphoma cell. For example, the anti-CCR8 antibody has a lowerinternalization rate than antibodies 433H and L263G8 (BioLegendCat. No. 360602).

According to some third embodiments of the 12.sup.th aspect, whichmay be the same as or different from the first or secondembodiments, the antibody or antigen-binding fragment specificallybinds murine CCR8 and has a lower internalization rate thanantibody SA214G2 in a cell endogenously expressing the target,preferably wherein the cell endogenously expressing the target is amurine lymphoma cell line BW5147.3.

The provided isolated antibody or antigen-binding fragment ispreferably also an antibody according to any of aspects 7, 8, 9,10, 11, 13, 14, 15, 16, 17 or 18 or a combination thereof.

Aspect 13--ADCC/ADCP Inducing CCR8 Antibody

To induce the killing of CCR8 expressing cells such as activatedTregs, multiple modes of action can be envisioned. One mode ofaction is the conjugation of an antibody targeting CCR8 to a drugin the form of an antibody drug conjugate (ADC). Other possiblemodes of action are ADCC, CDC and ADCP. For ADCC, CDC and ADCP, atwo-step mechanism is involved: On the one hand, the antibody orfragment is required to effectively bind the target cell, e.g. theTreg via CCR8, on the other hand, the FC part of the antibody (oran alternative binding moiety which can be conjugated to theantibody or fragment as described elsewhere herein) has to bind toan effector cell, which will then mediate the killing of the targetcell. For ADCP, binding to macrophages as effector cells typicallyoccurs via the interaction of the antibodies FC part withFc.gamma.RIIa (CD32a) expressed by macrophages. In contrast, ADCCis mediated via interaction of the antibody or fragment withFc.gamma.RIIIa. In humans, Fc.gamma.RIII exists in two differentforms: Fc.gamma.RIIIa (CD16a) and Fc.gamma.RIIIb (CD16b). WhileFc.gamma.RIIIa is expressed on mast cells, macrophages, and naturalkiller cells as a transmembrane receptor, Fc.gamma.RIIIb is onlyexpressed on neutrophils. These receptors bind to the Fc portion ofIgG antibodies, which then activates antibody-dependentcell-mediated cytotoxicity (ADCC) mediated by the human effectorcells.

In case of doubt, where ADCC and/or ADCP induction is analyzed, atleast 80% and preferably at least 85% of the target cells arerequired to show CCR8 expression.

According to a 13th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.thand/or 12.sup.th aspect, there is provided an isolated antibody orantigen-binding fragment thereof, specifically binding to CCR8,wherein the antibody or antigen-binding fragment induces ADCCand/or ADCP.

For example, there is provided an isolated antibody orantigen-binding fragment thereof specifically binding to CCR8,wherein the antibody or antigen-binding fragment is afucosylatedand a) induces antibody-dependent cell-mediated cytotoxicity (ADCC)in target cells expressing human CCR8 via human effector cells,such as human NK cells, and b) induces antibody-dependentcell-mediated phagocytosis (ADCP) in target cells expressing humanCCR8 via human effector cells, such as human macrophages,preferably wherein the maximal ADCC and ADCP induced in vitrodepletion of target cells expressing human CCR8 is at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%. In case ofdoubt, ADCC and ADCP shall be determined using target cells whereat least 85% of the cells express CCR8.

In some preferred embodiments of the 13th aspect, the antibody orantigen-binding fragment is afucosylated. Afucosylated antibodiesare antibodies engineered such that the oligosaccharides in the Fcregion of the antibody do not have any fucose sugar units.Afucosylated antibodies can be obtained as known in the art, e.g.as described in example 10.3. Example 10.3.2 demonstrates theimproved binding of the afucosylated versions of the inventiveantibodies to Fc.gamma.RIIIa. The binding to Fc.gamma.RIIa waslikewise slightly improved. Afucosylation furthermore increased thebinding to cynomolgus Fc gamma RIII, cf. Table 10.3.2.1.

ADCC

In some first embodiments A of the 13th aspect, the antibody orantigen-binding fragment binds to human Fc gamma receptor IIIAvariant V176 (CD16a) with a dissociation constant (KD) lower than530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM.

In some first embodiments B of the 13th aspect, which may be thesame as or different from the first embodiments A, the antibody orantigen-binding fragment induces antibody-dependent cell-mediatedcytotoxicity (ADCC) in target cells expressing human CCR8 via humaneffector cells, such as human NK cells. ADCC induction can beanalyzed with an assay known in the art, for example as describedaccording to examples 10.3.3 ff.

In some first embodiments C of the 13th aspect, which may be thesame as or different from the first embodiments A or B, theADCC-induced maximal depletion of activated human regulatory Tcells is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%,90%, 95%, 98% or 99%, preferably, where at least 85% of theactivated human regulatory T cells have CCR8 expression, cf. Table10.3.3.1.2 and Table 10.3.3.1.3.

In some first embodiments D of the 13th aspect, which may be thesame as or different from the first embodiments A, B or C, the EC50for ADCC-induced depletion of activated human regulatory T cells isbelow 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM,12.5 pM, 10 pM, 5 pM or 2.5 pM. Preferably, at least 85% of theactivated human regulatory T cells have CCR8 expression.

ADCP

In some second embodiments A of the 13th aspect, which may or maynot be the same as the first embodiments A, B, C and/or D, theantibody or antigen-binding fragment binds to human Fc gamma RITA(CD32a) with a dissociation constant (KD) lower than 30 .mu.M, 20.mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M.

In some second embodiments B of the 13th aspect, which may be thesame as the first embodiments A, B, C and/or D, and which may bethe same as the second embodiments A, the antibody orantigen-binding fragment induces antibody-dependent cell-mediatedphagocytosis (ADCP) in target cells expressing human CCR8 via humaneffector cells, such as human macrophages. For example, the humanmacrophages can be M2c or M1 macrophages.

In some second embodiments C of the 13th aspect, which may the sameas the first embodiments A, B, C and/or D, and which may the sameas the second embodiments A and/or B, the ADCP-induced maximaldepletion of activated human regulatory T cells is at least 5%,10%, 15%, 20%, 25%, 30%, 40% or 50%.

In some second embodiments D of the 13th aspect, which may the sameas the first embodiments A, B, C and/or D, and which may the sameas the second embodiments A, B and/or C, the EC50 for ADCP-induceddepletion of activated human regulatory T cells is below 1500 pM,1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25pM or 10 pM.

In some preferred embodiments there is provided an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein the antibody or antigen-binding fragment a) bindsto human Fc gamma receptor IIIA variant V176 (CD16a) with adissociation constant (KD) lower than 530 nM, 500 nM, 450 nM, 400nM, 300 nM or 200 nM, and/or b) binds to human Fc gamma RITA(CD32a) with a dissociation constant (KD) lower than 30 pM, 20 pM,10 pM, 5 pM or 1 pM.

In some preferred embodiments there is provided an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein the antibody or antigen-binding fragment a)induces antibody-dependent cell-mediated cytotoxicity (ADCC) intarget cells expressing human CCR8 via human effector cells, suchas human NK cells, and/or b) induces antibody-dependentcell-mediated phagocytosis (ADCP) in target cells expressing humanCCR8 via human effector cells, such as human macrophages.

In some preferred embodiments there is provided an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein a) the ADCC-induced maximal depletion of activatedhuman regulatory T cells is at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98% or 99%, and/or b) the ADCP-induced maximal depletionof activated human regulatory T cells is at least 5%, 10%, 15%,20%, 25%, 30%, 40% or 50%, and/or c) the maximal depletion ofintra-tumoral regulatory T cells, in vitro or in a subject, is atleast 50%, 60%, 70%, 80%, 90%, 95% or 99%.

In some preferred embodiments there is provided an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein a) the EC50 for ADCC-induced depletion ofactivated human regulatory T cells is below 200 pM, 100 pM, 50 pM,25 pM, 12.5 pM, 10 pM or 5 pM and/or b) the EC50 for ADCP-induceddepletion of activated human regulatory T cells is below 500 pM,250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

The provided isolated antibody or antigen-binding fragment ispreferably also an antibody according to any of aspects 14, 15, 16,17 or 18 or a combination thereof.

Aspect 14--Treg Modulatory Ccr8 Antibody

The therapeutic in vivo effects of the antibodies according to thecurrent invention are shown in examples 12 ff. The ability of anantibody to modulate absolute and relative numbers of severalimmune cell populations is structurally realized by a combinationof target binding properties and FC receptor interaction, e.g. asdescribed according to the 7.sup.th, 10.sup.th, 12.sup.th and/or13.sup.th aspect or a combination thereof. According to a 14thaspect, which may be or may not be the same as the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,and/or 13.sup.th aspect, there is provided an isolated antibody orantigen-binding fragment thereof, specifically binding to CCR8,wherein the antibody or antigen-binding fragment depletes activatedregulatory T cells, preferably intra-tumoral Tregs. Example 12.1.1shows high levels of T reg depletion obtained with the inventiveantibodies in various mouse models, wherein the surrogateantibodies are characterized by comparable functional features asthe provided anti-human CCR8 antibodies. Interestingly, a superiortherapeutic response was observed in those cases, where the Tregdepletion was at least 50%. Treg depletion may be measured in vitroor in vivo. As understood by the skilled person, the Treg depletionmay be a temporal depletion. For example, Treg depletion may beanalyzed 24, 48 or 72 hours after treatment.

According to some first embodiments of the 14th aspect, aneffective dose of the antibody or antigen-binding fragment ischaracterized by a maximal depletion of activated or intra-tumoralregulatory T cells, in vitro or in a subject, of at least 45%, 50%,55%, 60%, 70%, 80%, 90%, 95% or 99%. Determination in vitro or invivo may occur as known in the art. Determination in vivo may occuras described in examples 12 ff. Suitable subjects include forexample human and non-human, such as mouse (e.g. CT26 model orEMT-6 model), rodent, or cynomolgus.

According to some second embodiments of the 14th aspect, which mayor may not be the same as the first embodiments, an effective doseof the antibody or antigen-binding fragment decreases the number ofactivated or intra-tumoral regulatory T cells, in vitro or in asubject, to less than 55%, 50%, 40%, 30%, 25%, 20%, 10%, 5% or 1%.The decrease may be a temporary decrease. For example, and withoutbeing bound by theory, the pool of intra-tumoral Tregs may besubsequently replenished.

According to some third embodiments of the 14th aspect, which mayor may not be the same as the first and/or second embodiments, aneffective dose of the antibody or antigen-binding fragmentincreases the ratio of intra-tumoral CD8+ T cells to intra-tumoralTregs, in vitro or in a subject, to at least 5, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, or higher. The increase maybe a temporary increase.

According to some fourth embodiments of the 14th aspect, which mayor may not be the same as the first, second and/or thirdembodiments, an effective dose of the antibody or antigen-bindingfragment decreases the percentage of regulatory T cells ofintra-tumoral CD4+ T cells, in vitro or in a subject, to <30%,<20%, <10% or <5%. The decrease may be a temporarydecrease.

In preferred embodiments, an effective dose of the antibody orantigen-binding fragment a) decreases the number of activated orintra-tumoral regulatory T cells, in vitro or in a subject, to lessthan 30%, 25%, 20%, 10%, 5% or 1% and/or b) increases the ratio ofintra-tumoral CD8+ T cells to intra-tumoral Tregs, in vitro or in asubject, to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, or higher and/or c) decreases the percentage ofregulatory T cells of intra-tumoral CD4+ T cells, in vitro or in asubject, to <30%, <20%, <10% or <5%.

Antibodies according to the current aspect were superior in termsof therapeutic efficacy, see examples 12 ff. The provided isolatedantibody or antigen-binding fragment is preferably also an antibodyaccording to any of aspects 15, 16, 17 or 18 or a combinationthereof.

Aspect 15--Immune Cell Modulatory Ccr8 Antibody

According to a 15th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th,12.sup.th, 13.sup.th and/or 14.sup.th aspect, there is provided anisolated antibody or antigen-binding fragment thereof, specificallybinding to CCR8, wherein an effective dose of the antibody orantigen-binding fragment increases the absolute numbers of immunecells in a defined tumor volume, e.g. at least by a factor of 1.5,2, 2.5, 3, 3.5 or 4.

Example 12.8 shows a massive increase in the mRNA expression levelsfor various global or specific immune cell markers in responsivetumors from different subjects at the study end. An increase wasobserved e.g. for macrophages (in particular M1 macrophages), CD8+T cells, NK cells, CD3+ T cells, B cells, and interestingly alsoactivated Tregs, suggesting an increased infiltration of theseimmune cell populations within the tumor after chronic treatment.The increase in absolute immune cell numbers was also confirmed byFACS, see example 12.3 (CT26), example 12.4 (EMT6), example 12.5(F9). Suitable subjects include for example human and non-humansubjects, such as mouse, rodent, or cynomolgus. For example, theincrease of immune cells can be determined after one, two, three orfour effective doses of the antibody, cf. example 12.3, 12.4.2 andalso after chronic treatment, e.g. after the final treatment. Forexample, the tumor may be a tumor characterized by tumorinfiltrating lymphocytes, e.g. a tumor characterized by tumorinfiltrating T cells or a tumor characterized by expression ofproinflammatory cytokines.

According to some first embodiments of the 15th aspect, the immunecells are at least one, two, three, four, five, six, seven, eightor nine selected from a) (intra-tumoral) CD45+ cells, b)(intra-tumoral) CD8+ T cells, c) (intra-tumoral) CD4+ T cells, d)(intra-tumoral) macrophages, such as M1 macrophages or M2macrophages, e) (intra-tumoral) NK cells, f) (intra-tumoral) Bcells, g) (intra-tumoral) Dendritic cells, h) (intra-tumoral) Gammadelta T cells (unconventional T cells), and i) iNKT cells.

The respective cell types and populations are defined as known inthe art, and in particular as defined elsewhere herein.

According to some highly preferred of these first embodiments,three or more effective doses of the antibody or antigen-bindingfragment, in a tumor, increase a) the number of intra-tumoral CD8+T cells to at least 150%, 200%, 250%, 300% or 350%, b) the ratio ofintra-tumoral CD8+ T cells to intra-tumoral regulatory T cells toat least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, or higher, c) the number of intra-tumoral macrophages atleast by a factor of 2, 3, 4, or 5, d) the number of intra-tumoralACOD1+ macrophages (M1 macrophages) at least by a factor of 2, 3,4, 5, 6, 7, 8, 9 or 10, e) the ratio of ACOD1+ macrophages (M1macrophages) to MRC1+ macrophages (M2 macrophages) to at least 1.5,2, 3, 4, 5, 10, or higher, f) the number of intra-tumoral NK cellsto at least 140% or 200%, g) the number of intra-tumoral CD3+ Tcells to at least 150%, 200%, 300% or 400%, h) the number ofintra-tumoral B cells at least by a factor of 2, 5, 10, 20, 30 or40, i) the number of intra-tumoral CD45+ T cells to at least 150%,200% or 300%, and/or j) the number of intra-tumoral CD4+ T cells toat least 150%, 200%, 300% or 400%.

The tumor is preferably a tumor characterized by tumor infiltratinglymphocytes. As understood by the skilled person, the tumor ischaracterized by cells expressing the target CCR8 of the inventiveantibody or fragment, e.g. intratumoral Tregs. Preferably, thetumor is a tumor in a syngeneic tumor model derived from a BALB/cstrain. For example, as shown in example 12.1.1, the tumor can bederived from the CT26 model, EMT-6 model, F9 model, C38, H22 orB16F10-OVA model, where the antibody specifically binds to murineCCR8. For example, where the antibody specifically binds to humanCCR8, the tumor can be selected from Adrenal cancer (e.g.Adrenocortical carcinoma or Pheochromocytoma), Bladder cancer (e.g.Transitional cell carcinoma, Transitional cellcarcinoma-Papillary), Brain cancer (e.g. Glioma-Astrocytoma,Glioma-Astrocytoma-Glioblastoma, Glioma-Oligoastrocytoma,Glioma-Oligodendroglioma), Breast cancer (e.g. ADC, ADC-Ductal,ADC-Ductal-TNBC, ADC-Ductal-TPBC, ADC-Lobular), Colorectal cancer(e.g. ADC), Esophageal cancer (e.g. ADC), Esophageal cancer (e.g.SCC), Gastric cancer (e.g. ADC, ADC-Diffuse, ADC-Intestinal,ADC-Intestinal-Tubular), Head and Neck cancer (e.g. Laryngealcancer-SCC, SCC, Oral cancer-SCC), Kidney cancer (e.g. ccRCC,Chromophobe, Papillary, Papillary-Type I, Papillary-Type II), Livercancer (e.g. HCC), Lung cancer (e.g. NSCLC-ADC, NSCLC-ADC-Mixed,NSCLC-SCC, SCLC), Mesothelioma (e.g. Epithelioid), Ovarian cancer(e.g. ADC-Cystadenocarcinoma-Papillary serous), Pancreatic cancer(e.g. ADC-Ductal), Prostate cancer (e.g. ADC-Acinar type), Sarcoma(e.g. Leiomyosarcoma, Liposarcoma-Dedifferentiated, Malignantfibrous histiocytoma), Skin cancer (e.g. Melanoma), Testicularcancer (e.g. Germ cell tumor-Seminoma), Thymoma, Thyroid cancer(e.g. Follicular carcinoma, Papillary carcinoma-Classical variant),Uterine cancer (e.g. Cervical-SCC, Cervical-SCC-Keratinizing,Cervical-SCC-Non-keratinizing, Endometrial-ADC-Endometrioid,Endometrial-ADC-Papillary serous,Endometrial-Carcinosarcoma-Malignant mixed mullerian tumor), cf.Table 11.1.2.

In some of these embodiments, the immune cells are a) CD8+ T cells,CD4+ T cells, and macrophages, b) CD8+ T cells, CD4+ T cells, NKcells, c) CD8+ T cells, CD4+ T cells, CD45+ cells, d) CD8+ T cells,CD4+ T cells, NK cells and macrophages, e) CD8+ T cells, CD4+ Tcells, NK cells, and macrophages, e.g. ACOD1+ macrophages, f) CD8+T cells, CD4+ T cells, NK cells, CD45+ cells, g) CD8+ T cells, CD4+T cells, NK cells, CD45+ cells, and macrophages, e.g. ACOD1+macrophages, h) CD8+ T cells, ACOD1+ macrophages (M1 macrophages),and B cells, i) CD8+ T cells, CD4+ T cells, and dendritic cells,wherein the dendritic cells are characterized by expression ofCD1c, CD14, CD16, CD141, CD11c and CD123, or j) CD8+ T cells andAcod1+ macrophages (M1 macrophages).

In some preferred of these embodiments, the immune cells are a)CD8+ T cells, CD4+ T cells, and macrophages, or b) CD8+ T cells,CD4+ T cells, NK cells and macrophages, or c) CD8+ T cells, ACOD1+macrophages (M1 macrophages), and B cells.

According to some further embodiments, the immune cells are CD45+cells and CD8+ T cells, CD45+ cells and CD4+ T cells, CD45+ cellsand macrophages (such as M1 macrophages or M2 macrophages), CD45+cells and NK cells, CD45+ cells and B cells, CD45+ cells andDendritic cells, CD45+ cells and Gamma delta T cells, CD45+ cellsand iNKT cells, CD8+ T cells and CD4+ T cells, CD8+ T cells andmacrophages (such as M1 macrophages or M2 macrophages), CD8+ Tcells and NK cells, CD8+ T cells and B cells, CD8+ T cells andDendritic cells, CD8+ T cells and Gamma delta T cells, CD8+ T cellsand iNKT cells, CD4+ T cells and macrophages (such as M1macrophages or M2 macrophages), CD4+ T cells and NK cells, CD4+ Tcells and B cells, CD4+ T cells and Dendritic cells, CD4+ T cellsand Gamma delta T cells, CD4+ T cells and iNKT cells, macrophages(such as M1 macrophages or M2 macrophages) and NK cells,macrophages (such as M1 macrophages or M2 macrophages) and B cells,macrophages (such as M1 macrophages or M2 macrophages) andDendritic cells, macrophages (such as M1 macrophages or M2macrophages) and Gamma delta T cells, macrophages (such as M1macrophages or M2 macrophages) and iNKT cells, B cells andDendritic cells, B cells and Gamma delta T cells, B cells and iNKTcells, Dendritic cells and Gamma delta T cells, Dendritic cells andiNKT cells, or Gamma delta T cells and iNKT cells.

The provided isolated antibody or antigen-binding fragment ispreferably also an antibody according to any of aspects 16, 17 or18 or a combination thereof.

Aspect 16--CCR8 Antibody Forming Tertiary Lymphoid Structures

According to a 16th aspect, which may be or may not be the same asthe 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th,12.sup.th, 13.sup.th, 14.sup.th and/or 15.sup.th aspect, there isprovided an isolated antibody or antigen-binding fragment thereof,specifically binding to CCR8, wherein the antibody orantigen-binding fragment induces the formation of tertiary lymphoidstructures. Tertiary lymphoid structures are intra-tumoralstructures characterized by increased expression of LTta, LTtb,Cxcr5 and its ligand Cxcl13. Tertiary lymphoid structures have beendescribed as key drivers of an anti-tumor effect in humans. Withoutbeing bound by theory, the inventors believe, that the formation ofthese substructures might contribute to the altered pattern inimmune cells and an anti-tumor effect of the inventive antibodies,e.g. in humans. As discussed in example 12.8, chronic treatmentwith the inventive antibodies consistently increased the expressionlevels for LTta, LTtb as well as Cxcr5 and its ligand Cxcl13.

SEQ Defined Antibodies

The antibodies according to the following two aspects 17 and 18were obtained with a method according to aspect 3 or 4. Theresulting antibodies according to aspect 17 and 18 specificallybind to a polypeptide comprising the sulfated TRD of a humanchemokine receptor, e.g. wherein at least 50% of the tyrosineresidues have been sulfated. As discussed previously herein, themethod influences the specific structural features of the HCDR3 ofthe obtained antibodies and may also influence functional features,such as the modulation of G protein independent signaling orinternalization behavior.

Where CDRs, variable heavy chains, variable light chains, heavychains or light chains are disclosed, the modular nature of theantibodies typically allows their combination, but also thecombination with the various functional features, as describedelsewhere herein.

Aspect 17--Anti-Human Ccr8 Antibody (SEQ Defined)

According to a 17.sup.th aspect, which may be or may not be thesame as the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14.sup.th,15.sup.th and/or 16.sup.th aspect, there is provided an isolatedanti-CCR8 antibody or antigen-binding fragment thereof.

Preferably the antibodies according to the current aspect bind (a)to an isolated polypeptide according to SEQ ID NO:43 and/or SEQ IDNO:46, wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and (b) to an isolated polypeptide according to SEQ IDNO:44 and/or SEQ ID NO:47, wherein at least two or all of Y3, Y15and Y17 have been sulfated, and the antibodies are thus crossreactive for human and cynomolgus CCR8. Furthermore, the antibodiesare preferably characterized by properties making them particularlysuitable for therapy, e.g. are antibodies according to at least oneor more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises at leastone, two, and preferably three, four, five or six CDR sequence(s)having at least 90%, 95%, 98% or 100% sequence identity with any ofa) SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:262, SEQID NO:263 or SEQ ID NO:264 (TPP-16966), b) SEQ ID NO:276, SEQ IDNO:277, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:281 or SEQ IDNO:282 (TPP-17575), c) SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296,SEQ ID NO:298, SEQ ID NO:299 or SEQ ID NO:300 (TPP-17576), d) SEQID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:316, SEQ IDNO:317 or SEQ ID NO:318 (TPP-17577), e) SEQ ID NO:330, SEQ IDNO:331, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:335 or SEQ IDNO:336 (TPP-17578), f) SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350,SEQ ID NO:352, SEQ ID NO:353 or SEQ ID NO:354 (TPP-17579), g) SEQID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:370, SEQ IDNO:371 or SEQ ID NO:372 (TPP-17580), h) SEQ ID NO:384, SEQ IDNO:385, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:389 or SEQ IDNO:390 (TPP-17581), i) SEQ ID NO:402, SEQ ID NO:403, SEQ ID NO:404,SEQ ID NO:406, SEQ ID NO:407 or SEQ ID NO:408 (TPP-18205), j) SEQID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ ID NO:424, SEQ IDNO:425 or SEQ ID NO:426 (TPP-18206), k) SEQ ID NO:438, SEQ IDNO:439, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:443 or SEQ IDNO:444 (TPP-18207), l) SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458,SEQ ID NO:460, SEQ ID NO:461 or SEQ ID NO:462 (TPP-19546), m) SEQID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:478, SEQ IDNO:479 or SEQ ID NO:480 (TPP-20950), n) SEQ ID NO:492, SEQ IDNO:493, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:497 or SEQ IDNO:498 (TPP-20955), o) SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512,SEQ ID NO:514, SEQ ID NO:515, or SEQ ID NO:516 (TPP-20965), p) SEQID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:532, SEQ IDNO:533 or SEQ ID NO:534 (TPP-21045), q) SEQ ID NO:546, SEQ IDNO:547, SEQ ID NO:548, SEQ ID NO:550, SEQ ID NO:551 or SEQ IDNO:552 (TPP-21047), r) SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566,SEQ ID NO:568, SEQ ID NO:569 or SEQ ID NO:570 (TPP-21181), s) SEQID NO:582, SEQ ID NO:583, SEQ ID NO:584, SEQ ID NO:586, SEQ IDNO:587 or SEQ ID NO:588 (TPP-21183), t) SEQ ID NO:600, SEQ IDNO:601, SEQ ID NO:602, SEQ ID NO:604, SEQ ID NO:605 or SEQ IDNO:606 (TPP-21360), u) SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620,SEQ ID NO:622, SEQ ID NO:623 or SEQ ID NO:624 (TPP-23411), v) SEQID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:665, SEQ IDNO:666 or SEQ ID NO:667 (TPP-29596), w) SEQ ID NO:681, SEQ IDNO:682, SEQ ID NO:683, SEQ ID NO:685, SEQ ID NO:686 or SEQ IDNO:687 (TPP-29597), x) SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705,SEQ ID NO:707, SEQ ID NO:708 or SEQ ID NO:709 (TPP-18429), y) SEQID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:727, SEQ IDNO:728 or SEQ ID NO:729 (TPP-18430), z) SEQ ID NO:743, SEQ IDNO:744, SEQ ID NO:745, SEQ ID NO:747, SEQ ID NO:748 or SEQ IDNO:749 (TPP-18432), aa) SEQ ID NO:763, SEQ ID NO:764, SEQ IDNO:765, SEQ ID NO:767, SEQ ID NO:768 or SEQ ID NO:769 (TPP-18433),bb) SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ ID NO:787, SEQID NO:788 or SEQ ID NO:789 (TPP-18436), cc) SEQ ID NO:803, SEQ IDNO:804, SEQ ID NO:805, SEQ ID NO:807, SEQ ID NO:808 or SEQ IDNO:809 (TPP-19571), dd) SEQ ID NO:827, SEQ ID NO: 828, SEQ ID NO:829, SEQ ID NO: 831, SEQ ID NO:832 or SEQ ID NO:833 (TPP-27477),ee) SEQ ID NO:847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 851,SEQ ID NO:852 or SEQ ID NO:853 (TPP-27478), ff) SEQ ID NO:867, SEQID NO: 868, SEQ ID NO: 869, SEQ ID NO: 871, SEQ ID NO:872 or SEQ IDNO:873 (TPP-27479), gg) SEQ ID NO:887, SEQ ID NO: 888, SEQ ID NO:889, SEQ ID NO: 891, SEQ ID NO:892 or SEQ ID NO:893 (TPP-27480),hh) SEQ ID NO:907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 911,SEQ ID NO:912 or SEQ ID NO:913 (TPP-29367), ii) SEQ ID NO:927, SEQID NO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO:932 or SEQ IDNO:933 (TPP-29368), and/or jj) SEQ ID NO:947, SEQ ID NO: 948, SEQID NO: 949, SEQ ID NO: 951, SEQ ID NO:952 or SEQ ID NO:953(TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a HCDR3sequence having at least 90%, 95%, 98% or 100% sequence identitywith any of SEQ ID NO:260 (TPP-16966), SEQ ID NO:278 (TPP-17575),SEQ ID NO:296 (TPP-17576), SEQ ID NO:314 (TPP-17577), SEQ ID NO:332(TPP-17578), SEQ ID NO:350 (TPP-17579), SEQ ID NO:368 (TPP-17580),SEQ ID NO:386 (TPP-17581), SEQ ID NO:404 (TPP-18205), SEQ ID NO:422(TPP-18206), SEQ ID NO:440 (TPP-18207), SEQ ID NO:458 (TPP-19546),SEQ ID NO:476 (TPP-20950), SEQ ID NO:494 (TPP-20955), SEQ ID NO:512(TPP-20965), SEQ ID NO:530 (TPP-21045), SEQ ID NO:548 (TPP-21047),SEQ ID NO:566 (TPP-21181), SEQ ID NO:584 (TPP-21183), SEQ ID NO:602(21360), or SEQ ID NO:620 (TPP-23411), SEQ ID NO:663 (TPP-29596),SEQ ID NO:683 (TPP-29597), SEQ ID NO:705 (TPP-18429), SEQ ID NO:725(TPP-18430), SEQ ID NO:745 (TPP-18432), SEQ ID NO:765 (TPP-18433),SEQ ID NO:785 (TPP-18436), SEQ ID NO:805 (TPP-19571), SEQ ID NO:829(TPP-27477), SEQ ID NO:849 (TPP-27478), SEQ ID NO:869 (TPP-27479),SEQ ID NO:889 (TPP-27480), SEQ ID NO:909 (TPP-29367), SEQ ID NO:929(TPP-29368), or SEQ ID NO:949 (TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises at leastone, two, and preferably three, four, five or six CDR sequencesaccording to a) SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ IDNO:262, SEQ ID NO:263 and SEQ ID NO:264 (TPP-16966), b) SEQ IDNO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:281and SEQ ID NO:282 (TPP-17575), c) SEQ ID NO:294, SEQ ID NO:295, SEQID NO:296, SEQ ID NO:298, SEQ ID NO:299 and SEQ ID NO:300(TPP-17576), d) SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ IDNO:316, SEQ ID NO:317 and SEQ ID NO:318 (TPP-17577), e) SEQ IDNO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:335and SEQ ID NO:336 (TPP-17578), f) SEQ ID NO:348, SEQ ID NO:349, SEQID NO:350, SEQ ID NO:352, SEQ ID NO:353 and SEQ ID NO:354(TPP-17579), g) SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ IDNO:370, SEQ ID NO:371 and SEQ ID NO:372 (TPP-17580), h) SEQ IDNO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:389and SEQ ID NO:390 (TPP-17581), i) SEQ ID NO:402, SEQ ID NO:403, SEQID NO:404, SEQ ID NO:406, SEQ ID NO:407 and SEQ ID NO:408(TPP-18205), j) SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ IDNO:424, SEQ ID NO:425 and SEQ ID NO:426 (TPP-18206), k) SEQ IDNO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:443and SEQ ID NO:444 (TPP-18207), l) SEQ ID NO:456, SEQ ID NO:457, SEQID NO:458, SEQ ID NO:460, SEQ ID NO:461 and SEQ ID NO:462(TPP-19546), m) SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ IDNO:478, SEQ ID NO:479 and SEQ ID NO:480 (TPP-20950), n) SEQ IDNO:492, SEQ ID NO:493, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:497and SEQ ID NO:498 (TPP-20955), o) SEQ ID NO:510, SEQ ID NO:511, SEQID NO:512, SEQ ID NO:514, SEQ ID NO:515, and SEQ ID NO:516(TPP-20965), p) SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ IDNO:532, SEQ ID NO:533 and SEQ ID NO:534 (TPP-21045), q) SEQ IDNO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:550, SEQ ID NO:551and SEQ ID NO:552 (TPP-21047), r) SEQ ID NO:564, SEQ ID NO:565, SEQID NO:566, SEQ ID NO:568, SEQ ID NO:569 and SEQ ID NO:570(TPP-21181), s) SEQ ID NO:582, SEQ ID NO:583, SEQ ID NO:584, SEQ IDNO:586, SEQ ID NO:587 and SEQ ID NO:588 (TPP-21183), t) SEQ IDNO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:604, SEQ ID NO:605or SEQ ID NO:606 (TPP-21360), u) SEQ ID NO:618, SEQ ID NO:619, SEQID NO:620, SEQ ID NO:622, SEQ ID NO:623 or SEQ ID NO:624(TPP-23411), v) SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ IDNO:665, SEQ ID NO:666 or SEQ ID NO:667 (TPP-29596), w) SEQ IDNO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:685, SEQ ID NO:686or SEQ ID NO:687 (TPP-29597), x) SEQ ID NO:703, SEQ ID NO:704, SEQID NO:705, SEQ ID NO:707, SEQ ID NO:708 or SEQ ID NO:709(TPP-18429), y) SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ IDNO:727, SEQ ID NO:728 or SEQ ID NO:729 (TPP-18430), z) SEQ IDNO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:747, SEQ ID NO:748or SEQ ID NO:749 (TPP-18432), aa) SEQ ID NO:763, SEQ ID NO:764, SEQID NO:765, SEQ ID NO:767, SEQ ID NO:768 or SEQ ID NO:769(TPP-18433), bb) SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQID NO:787, SEQ ID NO:788 or SEQ ID NO:789 (TPP-18436), cc) SEQ IDNO:803, SEQ ID NO:804, SEQ ID NO:805, SEQ ID NO:807, SEQ ID NO:808or SEQ ID NO:809 (TPP-19571), dd) SEQ ID NO:827, SEQ ID NO: 828,SEQ ID NO: 829, SEQ ID NO: 831, SEQ ID NO:832 or SEQ ID NO:833(TPP-27477), ee) SEQ ID NO:847, SEQ ID NO: 848, SEQ ID NO: 849, SEQID NO: 851, SEQ ID NO:852 or SEQ ID NO:853 (TPP-27478), ff) SEQ IDNO:867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 871, SEQ IDNO:872 or SEQ ID NO:873 (TPP-27479), gg) SEQ ID NO:887, SEQ ID NO:888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO:892 or SEQ ID NO:893(TPP-27480), hh) SEQ ID NO:907, SEQ ID NO: 908, SEQ ID NO: 909, SEQID NO: 911, SEQ ID NO:912 or SEQ ID NO:913 (TPP-29367), ii) SEQ IDNO:927, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ IDNO:932 or SEQ ID NO:933 (TPP-29368), and/or jj) SEQ ID NO:947, SEQID NO: 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ ID NO:952 or SEQ IDNO:953 (TPP-29369), optionally wherein up to one, two, three, fouror five mutations have been introduced into at least one CDR.Preferably, the HCDR3 comprises or has been engineered to compriseat least one or more histidine residues as described elsewhereherein.

For example, tyrosine may be exchanged with a positively chargedamino acid such as histidine and vice versa. For example, apositively charged amino acid may be exchanged with a differentpositively charged amino acid, a negatively charged amino acid maybe exchanged with a different negatively charged amino acid, apolar amino acid may be exchanged with a different polar aminoacid, a polar uncharged amino acid may be exchanged with adifferent polar uncharged amino acid, a small amino acid may beexchanged with a different small amino acid, an amphiphatic aminoacid may be exchanged with a different amphiphatic amino acid, anaromatic amino acid may be exchanged with a different aromaticamino acid. As understood by the skilled person, in particularthose amino acid exchanges are possible, which are not altering thespecific interaction between the antibody and the sulfated TRD ofCCR8.

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a variableheavy chain sequence and/or a variable light chain sequence havingat least 90%, 95%, 98% or 100% sequence identity with a) a variableheavy chain sequence according to SEQ ID NO:257 and/or a variablelight chain sequence according to SEQ ID NO:261 (TPP-16966), b) avariable heavy chain sequence according to SEQ ID NO:275 and/or avariable light chain sequence according to SEQ ID NO:279(TPP-17575), c) a variable heavy chain sequence according to SEQ IDNO:293 and/or a variable light chain sequence according to SEQ IDNO:297 (TPP-17576), d) a variable heavy chain sequence according toSEQ ID NO:311 and/or a variable light chain sequence according toSEQ ID NO:315 (TPP-17577), e) a variable heavy chain sequenceaccording to SEQ ID NO:329 and/or a variable light chain sequenceaccording to SEQ ID NO:333 (TPP-17578), f) a variable heavy chainsequence according to SEQ ID NO:347 and/or a variable light chainsequence according to SEQ ID NO:351 (TPP-17579), g) a variableheavy chain sequence according to SEQ ID NO:365 and/or a variablelight chain sequence according to SEQ ID NO:369 (TPP-17580), h) avariable heavy chain sequence according to SEQ ID NO:383 and/or avariable light chain sequence according to SEQ ID NO:387(TPP-17581), i) a variable heavy chain sequence according to SEQ IDNO:401 and/or a variable light chain sequence according to SEQ IDNO:405 (TPP-18205), j) a variable heavy chain sequence according toSEQ ID NO:419 and/or a variable light chain sequence according toSEQ ID NO:423 (TPP-18206), k) a variable heavy chain sequenceaccording to SEQ ID NO:437 and/or a variable light chain sequenceaccording to SEQ ID NO:441 (TPP-18207), l) a variable heavy chainsequence according to SEQ ID NO:455 and/or a variable light chainsequence according to SEQ ID NO:459 (TPP-19546), m) a variableheavy chain sequence according to SEQ ID NO:473 and/or a variablelight chain sequence according to SEQ ID NO:477 (TPP-20950), n) avariable heavy chain sequence according to SEQ ID NO:491 and/or avariable light chain sequence according to SEQ ID NO:495(TPP-20955), o) a variable heavy chain sequence according to SEQ IDNO:509 and/or a variable light chain sequence according to SEQ IDNO:513 (TPP-20965), p) a variable heavy chain sequence according toSEQ ID NO:527 and/or a variable light chain sequence according toSEQ ID NO:531 (TPP-21045), q) a variable heavy chain sequenceaccording to SEQ ID NO:545 and/or a variable light chain sequenceaccording to SEQ ID NO:549 (TPP-21047), r) a variable heavy chainsequence according to SEQ ID NO:563 and/or a variable light chainsequence according to SEQ ID NO:567 (TPP-21181), s) a variableheavy chain sequence according to SEQ ID NO:581 and/or a variablelight chain sequence according to SEQ ID NO:585 (TPP-21183), t) avariable heavy chain sequence according to SEQ ID NO:599 and/or avariable light chain sequence according to SEQ ID NO:603(TPP-21360), u) a variable heavy chain sequence according to SEQ IDNO:617 and/or a variable light chain sequence according to SEQ IDNO:621 (TPP-23411), v) a variable heavy chain sequence according toSEQ ID NO:660 and/or a variable light chain sequence according toSEQ ID NO:664 (TPP-29596), w) a variable heavy chain sequenceaccording to SEQ ID NO:680 and/or a variable light chain sequenceaccording to SEQ ID NO:684 (TPP-29597), x) a variable heavy chainsequence according to SEQ ID NO:702 and/or a variable light chainsequence according to SEQ ID NO:706 (TPP-18429), y) a variableheavy chain sequence according to SEQ ID NO:722 and/or a variablelight chain sequence according to SEQ ID NO:726 (TPP-18430), z) avariable heavy chain sequence according to SEQ ID NO:742 and/or avariable light chain sequence according to SEQ ID NO:746(TPP-18432), aa) a variable heavy chain sequence according to SEQID NO:762 and/or a variable light chain sequence according to SEQID NO:766 (TPP-18433), bb) a variable heavy chain sequenceaccording to SEQ ID NO:782 and/or a variable light chain sequenceaccording to SEQ ID NO:786 (TPP-18436), cc) a variable heavy chainsequence according to SEQ ID NO:802 and/or a variable light chainsequence according to SEQ ID NO:806 (TPP-19571), dd) a variableheavy chain sequence according to SEQ ID NO:826 and/or a variablelight chain sequence according to SEQ ID NO:830 (TPP-27477), ee) avariable heavy chain sequence according to SEQ ID NO:846 and/or avariable light chain sequence according to SEQ ID NO:850(TPP-27478), ff) a variable heavy chain sequence according to SEQID NO:866 and/or a variable light chain sequence according to SEQID NO:870 (TPP-27479), gg) a variable heavy chain sequenceaccording to SEQ ID NO:886 and/or a variable light chain sequenceaccording to SEQ ID NO:890 (TPP-27480), hh) a variable heavy chainsequence according to SEQ ID NO:906 and/or a variable light chainsequence according to SEQ ID NO:910 (TPP-29367), ii) a variableheavy chain sequence according to SEQ ID NO:926 and/or a variablelight chain sequence according to SEQ ID NO:930 (TPP-29368), or jj)a variable heavy chain sequence according to SEQ ID NO:946 and/or avariable light chain sequence according to SEQ ID NO:950(TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a) avariable heavy chain sequence according to SEQ ID NO:257 and/or avariable light chain sequence according to SEQ ID NO:261(TPP-16966), b) a variable heavy chain sequence according to SEQ IDNO:275 and/or a variable light chain sequence according to SEQ IDNO:279 (TPP-17575), c) a variable heavy chain sequence according toSEQ ID NO:293 and/or a variable light chain sequence according toSEQ ID NO:297 (TPP-17576), d) a variable heavy chain sequenceaccording to SEQ ID NO:311 and/or a variable light chain sequenceaccording to SEQ ID NO:315 (TPP-17577), e) a variable heavy chainsequence according to SEQ ID NO:329 and/or a variable light chainsequence according to SEQ ID NO:333 (TPP-17578), f) a variableheavy chain sequence according to SEQ ID NO:347 and/or a variablelight chain sequence according to SEQ ID NO:351 (TPP-17579), g) avariable heavy chain sequence according to SEQ ID NO:365 and/or avariable light chain sequence according to SEQ ID NO:369(TPP-17580), h) a variable heavy chain sequence according to SEQ IDNO:383 and/or a variable light chain sequence according to SEQ IDNO:387 (TPP-17581), i) a variable heavy chain sequence according toSEQ ID NO:401 and/or a variable light chain sequence according toSEQ ID NO:405 (TPP-18205), j) a variable heavy chain sequenceaccording to SEQ ID NO:419 and/or a variable light chain sequenceaccording to SEQ ID NO:423 (TPP-18206), k) a variable heavy chainsequence according to SEQ ID NO:437 and/or a variable light chainsequence according to SEQ ID NO:441 (TPP-18207), l) a variableheavy chain sequence according to SEQ ID NO:455 and/or a variablelight chain sequence according to SEQ ID NO:459 (TPP-19546), m) avariable heavy chain sequence according to SEQ ID NO:473 and/or avariable light chain sequence according to SEQ ID NO:477(TPP-20950), n) a variable heavy chain sequence according to SEQ IDNO:491 and/or a variable light chain sequence according to SEQ IDNO:495 (TPP-20955), o) a variable heavy chain sequence according toSEQ ID NO:509 and/or a variable light chain sequence according toSEQ ID NO:513 (TPP-20965), p) a variable heavy chain sequenceaccording to SEQ ID NO:527 and/or a variable light chain sequenceaccording to SEQ ID NO:531 (TPP-21045), q) a variable heavy chainsequence according to SEQ ID NO:545 and/or a variable light chainsequence according to SEQ ID NO:549 (TPP-21047), r) a variableheavy chain sequence according to SEQ ID NO:563 and/or a variablelight chain sequence according to SEQ ID NO:567 (TPP-21181), s) avariable heavy chain sequence according to SEQ ID NO:581 and/or avariable light chain sequence according to SEQ ID NO:585(TPP-21183), t) a variable heavy chain sequence according to SEQ IDNO:599 and/or a variable light chain sequence according to SEQ IDNO:603 (TPP-21360), u) a variable heavy chain sequence according toSEQ ID NO:617 and/or a variable light chain sequence according toSEQ ID NO:621 (TPP-23411), v) a variable heavy chain sequenceaccording to SEQ ID NO:660 and/or a variable light chain sequenceaccording to SEQ ID NO:664 (TPP-29596), w) a variable heavy chainsequence according to SEQ ID NO:680 and/or a variable light chainsequence according to SEQ ID NO:684 (TPP-29597), x) a variableheavy chain sequence according to SEQ ID NO:702 and/or a variablelight chain sequence according to SEQ ID NO:706 (TPP-18429), y) avariable heavy chain sequence according to SEQ ID NO:722 and/or avariable light chain sequence according to SEQ ID NO:726(TPP-18430), z) a variable heavy chain sequence according to SEQ IDNO:742 and/or a variable light chain sequence according to SEQ IDNO:746 (TPP-18432), aa) a variable heavy chain sequence accordingto SEQ ID NO:762 and/or a variable light chain sequence accordingto SEQ ID NO:766 (TPP-18433), bb) a variable heavy chain sequenceaccording to SEQ ID NO:782 and/or a variable light chain sequenceaccording to SEQ ID NO:786 (TPP-18436), cc) a variable heavy chainsequence according to SEQ ID NO:802 and/or a variable light chainsequence according to SEQ ID NO:806 (TPP-19571), dd) a variableheavy chain sequence according to SEQ ID NO:826 and/or a variablelight chain sequence according to SEQ ID NO:830 (TPP-27477), ee) avariable heavy chain sequence according to SEQ ID NO:846 and/or avariable light chain sequence according to SEQ ID NO:850(TPP-27478), ff) a variable heavy chain sequence according to SEQID NO:866 and/or a variable light chain sequence according to SEQID NO:870 (TPP-27479), gg) a variable heavy chain sequenceaccording to SEQ ID NO:886 and/or a variable light chain sequenceaccording to SEQ ID NO:890 (TPP-27480), hh) a variable heavy chainsequence according to SEQ ID NO:906 and/or a variable light chainsequence according to SEQ ID NO:910 (TPP-29367), ii) a variableheavy chain sequence according to SEQ ID NO:926 and/or a variablelight chain sequence according to SEQ ID NO:930 (TPP-29368), or jj)a variable heavy chain sequence according to SEQ ID NO:946 and/or avariable light chain sequence according to SEQ ID NO:950(TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a heavychain sequence and/or a light chain sequence having at least 90%,95%, 98% or 100% sequence identity with a) a heavy chain accordingto SEQ ID NO:273 and a light chain according to SEQ ID NO:274(TPP-16966), b) a heavy chain according to SEQ ID NO:291 and alight chain according to SEQ ID NO:292 (TPP-17575), c) a heavychain according to SEQ ID NO:309 and a light chain according to SEQID NO:310 (TPP-17576), d) a heavy chain according to SEQ ID NO:327and a light chain according to SEQ ID NO:328 (TPP-17577), e) aheavy chain according to SEQ ID NO:345 and a light chain accordingto SEQ ID NO:346 (TPP-17578), f) a heavy chain according to SEQ IDNO:363 and a light chain according to SEQ ID NO:364 (TPP-17579), g)a heavy chain according to SEQ ID NO:381 and a light chainaccording to SEQ ID NO:382 (TPP-17580), h) a heavy chain accordingto SEQ ID NO:399 and a light chain according to SEQ ID NO:400(TPP-17581), i) a heavy chain according to SEQ ID NO:417 and alight chain according to SEQ ID NO:418 (TPP-18205), j) a heavychain according to SEQ ID NO:435 and a light chain according to SEQID NO:436 (TPP-18206), k) a heavy chain according to SEQ ID NO:453and a light chain according to SEQ ID NO:454 (TPP-18207), l) aheavy chain according to SEQ ID NO:471 and a light chain accordingto SEQ ID NO:472 (TPP-19546), m) a heavy chain according to SEQ IDNO:489 and a light chain according to SEQ ID NO:490 (TPP-20950), n)a heavy chain according to SEQ ID NO:507 and a light chainaccording to SEQ ID NO:508 (TPP-20955), o) a heavy chain accordingto SEQ ID NO:525 and a light chain according to SEQ ID NO:526(TPP-20965), p) a heavy chain according to SEQ ID NO:543 and alight chain according to SEQ ID NO:544 (TPP-21045), q) a heavychain according to SEQ ID NO:561 and a light chain according to SEQID NO:562 (TPP-21047), r) a heavy chain according to SEQ ID NO:579and a light chain according to SEQ ID NO:580 (TPP-21181), s) aheavy chain according to SEQ ID NO:597 and a light chain accordingto SEQ ID NO:598 (TPP-21183), t) a heavy chain according to SEQ IDNO:615 and a light chain according to SEQ ID NO:616 (TPP-21360), u)a heavy chain according to SEQ ID NO:633 and a light chainaccording to SEQ ID NO:634 (TPP-23411), v) a heavy chain accordingto SEQ ID NO:676 and a light chain according to SEQ ID NO:677(TPP-29596), w) a heavy chain according to SEQ ID NO:696 and alight chain according to SEQ ID NO:697 (TPP-29597), x) a heavychain according to SEQ ID NO:718 and a light chain according to SEQID NO:719 (TPP-18429), y) a heavy chain according to SEQ ID NO:738and a light chain according to SEQ ID NO:739 (TPP-18430), z) aheavy chain according to SEQ ID NO:758 and a light chain accordingto SEQ ID NO:759 (TPP-18432), aa) a heavy chain according to SEQ IDNO:778 and a light chain according to SEQ ID NO:779 (TPP-18433),bb) a heavy chain according to SEQ ID NO:798 and a light chainaccording to SEQ ID NO:799 (TPP-18436), cc) a heavy chain accordingto SEQ ID NO:818 and a light chain according to SEQ ID NO:819(TPP-19571), dd) a heavy chain according to SEQ ID NO:842 and alight chain according to SEQ ID NO:843 (TPP-27477), ee) a heavychain according to SEQ ID NO:862 and a light chain according to SEQID NO:863 (TPP-27478), ff) a heavy chain according to SEQ ID NO:882and a light chain according to SEQ ID NO:883 (TPP-27479), gg) aheavy chain according to SEQ ID NO:902 and a light chain accordingto SEQ ID NO:903 (TPP-27480), hh) a heavy chain according to SEQ IDNO:922 and a light chain according to SEQ ID NO:923 (TPP-29367),ii) a heavy chain according to SEQ ID NO:942 and a light chainaccording to SEQ ID NO:943 (TPP-29368), or jj) a heavy chainaccording to SEQ ID NO:962 and a light chain according to SEQ IDNO:963 (TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a) a heavychain according to SEQ ID NO:273 and a light chain according to SEQID NO:274 (TPP-16966), b) a heavy chain according to SEQ ID NO:291and a light chain according to SEQ ID NO:292 (TPP-17575), c) aheavy chain according to SEQ ID NO:309 and a light chain accordingto SEQ ID NO:310 (TPP-17576), d) a heavy chain according to SEQ IDNO:327 and a light chain according to SEQ ID NO:328 (TPP-17577), e)a heavy chain according to SEQ ID NO:345 and a light chainaccording to SEQ ID NO:346 (TPP-17578), f) a heavy chain accordingto SEQ ID NO:363 and a light chain according to SEQ ID NO:364(TPP-17579), g) a heavy chain according to SEQ ID NO:381 and alight chain according to SEQ ID NO:382 (TPP-17580), h) a heavychain according to SEQ ID NO:399 and a light chain according to SEQID NO:400 (TPP-17581), i) a heavy chain according to SEQ ID NO:417and a light chain according to SEQ ID NO:418 (TPP-18205), j) aheavy chain according to SEQ ID NO:435 and a light chain accordingto SEQ ID NO:436 (TPP-18206), k) a heavy chain according to SEQ IDNO:453 and a light chain according to SEQ ID NO:454 (TPP-18207), l)a heavy chain according to SEQ ID NO:471 and a light chainaccording to SEQ ID NO:472 (TPP-19546), m) a heavy chain accordingto SEQ ID NO:489 and a light chain according to SEQ ID NO:490(TPP-20950), n) a heavy chain according to SEQ ID NO:507 and alight chain according to SEQ ID NO:508 (TPP-20955), o) a heavychain according to SEQ ID NO:525 and a light chain according to SEQID NO:526 (TPP-20965), p) a heavy chain according to SEQ ID NO:543and a light chain according to SEQ ID NO:544 (TPP-21045), q) aheavy chain according to SEQ ID NO:561 and a light chain accordingto SEQ ID NO:562 (TPP-21047), r) a heavy chain according to SEQ IDNO:579 and a light chain according to SEQ ID NO:580 (TPP-21181), s)a heavy chain according to SEQ ID NO:597 and a light chainaccording to SEQ ID NO:598 (TPP-21183), t) a heavy chain accordingto SEQ ID NO:615 and a light chain according to SEQ ID NO:616(TPP-21360), u) a heavy chain according to SEQ ID NO:633 and alight chain according to SEQ ID NO:634 (TPP-23411), v) a heavychain according to SEQ ID NO:676 and a light chain according to SEQID NO:677 (TPP-29596), w) a heavy chain according to SEQ ID NO:696and a light chain according to SEQ ID NO:697 (TPP-29597), x) aheavy chain according to SEQ ID NO:718 and a light chain accordingto SEQ ID NO:719 (TPP-18429), y) a heavy chain according to SEQ IDNO:738 and a light chain according to SEQ ID NO:739 (TPP-18430), z)a heavy chain according to SEQ ID NO:758 and a light chainaccording to SEQ ID NO:759 (TPP-18432), aa) a heavy chain accordingto SEQ ID NO:778 and a light chain according to SEQ ID NO:779(TPP-18433), bb) a heavy chain according to SEQ ID NO:798 and alight chain according to SEQ ID NO:799 (TPP-18436), cc) a heavychain according to SEQ ID NO:818 and a light chain according to SEQID NO:819 (TPP-19571), dd) a heavy chain according to SEQ ID NO:842and a light chain according to SEQ ID NO:843 (TPP-27477), ee) aheavy chain according to SEQ ID NO:862 and a light chain accordingto SEQ ID NO:863 (TPP-27478), ff) a heavy chain according to SEQ IDNO:882 and a light chain according to SEQ ID NO:883 (TPP-27479),gg) a heavy chain according to SEQ ID NO:902 and a light chainaccording to SEQ ID NO:903 (TPP-27480), hh) a heavy chain accordingto SEQ ID NO:922 and a light chain according to SEQ ID NO:923(TPP-29367), ii) a heavy chain according to SEQ ID NO:942 and alight chain according to SEQ ID NO:943 (TPP-29368), or jj) a heavychain according to SEQ ID NO:962 and a light chain according to SEQID NO:963 (TPP-29369).

Also provided according to the current aspect are variousantibodies comprising random permutations of the CDRs of theanti-CCR8 antibodies provided with the sequence listings.

Preferably, the antibody or antigen-binding fragment according tothe current aspect is afucosylated, as described elsewhere herein.Preferably, the antibody or antigen-binding fragment according tothe current aspect induces ADCC and/or ADCP, as described elsewhereherein. Preferably, the antibody or antigen-binding fragmentaccording to the current aspect is a non-internalizing orlow-internalizing antibody, as described elsewhere herein.

Aspect 18--Anti-Murine Ccr8 Antibody (SEQ Defined)

According to an 18.sup.th aspect, which may be or may not be thesame as the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14.sup.th,15.sup.th and/or 16.sup.th aspect, there is provided an isolatedanti-CCR8 antibody or antigen-binding fragment thereof. Preferably,the antibodies according to the current aspect bind to murine CCR8,in particular to an isolated polypeptide according to SEQ ID NO:45and/or SEQ ID NO:48, wherein at least two or all of Y3, Y14 and Y15have been sulfated. The CDRs of the antibodies according to thecurrent aspect are preferably human derived CDRs. Furthermore, theantibodies are characterized by properties making them particularlysuitable for therapy, e.g. are antibodies according to at least oneor more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Insummary, the antibodies according to the current aspect can be usedas surrogate antibodies recognizing murine CCR8 and have superiortherapeutic properties as discussed e.g. in example 12.

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises at leastone, two, three, four, five or six CDR sequence(s) having at least90%, 95%, 98% or 100% sequence identity with any of a) SEQ IDNO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:207and SEQ ID NO:208 (TPP-14095), b) SEQ ID NO:216, SEQ ID NO:217, SEQID NO:218, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:222(TPP-14099), c) SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ IDNO:234, SEQ ID NO:235 and SEQ ID NO:236 (TPP-15285), or d) SEQ IDNO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:249and SEQ ID NO:250 (TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a HCDR3sequence having at least 90%, 95%, 98% or 100% sequence identitywith any of SEQ ID NO:204, SEQ ID NO:218, SEQ ID NO:232, or SEQ IDNO:246.

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises at leastone, two, and preferably three, four, five or six CDR sequencesaccording to a) SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ IDNO:206, SEQ ID NO:207 and SEQ ID NO:208 (TPP-14095), b) SEQ IDNO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:221or SEQ ID NO:222 (TPP-14099), c) SEQ ID NO:230, SEQ ID NO:231, SEQID NO:232, SEQ ID NO:234, SEQ ID NO:235 and SEQ ID NO:236(TPP-15285), or d) SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQID NO:248, SEQ ID NO:249 and SEQ ID NO:250 (TPP-15286), optionallywherein up to one, two, three, four or five mutations have beenintroduced into at least one CDR.

For example, tyrosine may be exchanged with a positively chargedamino acid such as histidine and vice versa. For example, apositively charged amino acid may be exchanged with a differentpositively charged amino acid, a negatively charged amino acid maybe exchanged with a different negatively charged amino acid, apolar amino acid may be exchanged with a different polar aminoacid, a polar uncharged amino acid may be exchanged with adifferent polar uncharged amino acid, a small amino acid may beexchanged with a different small amino acid, an amphiphatic aminoacid may be exchanged with a different amphiphatic amino acid, anaromatic amino acid may be exchanged with a different aromaticamino acid. As understood by the skilled person, in particularthose amino acid exchanges are possible, which are not altering thespecific interaction between the antibody and the sulfated TRD ofCCR8. Amino acid exchanges introducing histidine are preferred.

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a variableheavy chain sequence and/or a variable light chain sequence havingat least 90%, 95%, 98% or 100% sequence identity with a) a variableheavy chain sequence according to SEQ ID NO:201 and a variablelight chain sequence according to SEQ ID NO:205 (TPP-14095), b) avariable heavy chain sequence according to SEQ ID NO:215 and avariable light chain sequence according to SEQ ID NO:219(TPP-14099), c) a variable heavy chain sequence according to SEQ IDNO:229 and a variable light chain sequence according to SEQ IDNO:233 (TPP-15285), or d) a variable heavy chain sequence accordingto SEQ ID NO:243 and a variable light chain sequence according toSEQ ID NO:247 (TPP-15286).

According to some preferred embodiments, the anti-CCR8 antibody orantigen-binding fragment thereof comprises e) a variable heavychain sequence according to SEQ ID NO:201 and a variable lightchain sequence according to SEQ ID NO:205 (TPP-14095), f) avariable heavy chain sequence according to SEQ ID NO:215 and avariable light chain sequence according to SEQ ID NO:219(TPP-14099), g) a variable heavy chain sequence according to SEQ IDNO:229 and a variable light chain sequence according to SEQ IDNO:233 (TPP-15285), or h) a variable heavy chain sequence accordingto SEQ ID NO:243 and a variable light chain sequence according toSEQ ID NO:247 (TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a heavychain sequence and/or a light chain sequence having at least 90%,95%, 98% or 100% sequence identity with a) a heavy chain accordingto SEQ ID NO:211 and/or a light chain according to SEQ ID NO:212(TPP-14095), b) a heavy chain according to SEQ ID NO:225 and/or alight chain according to SEQ ID NO:226 (TPP-14099), c) a heavychain according to SEQ ID NO:239 and/or a light chain according toSEQ ID NO:240 (TPP-15285), or d) a heavy chain according to SEQ IDNO:253 and/or a light chain according to SEQ ID NO:254(TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8antibody or antigen-binding fragment thereof comprises a) a heavychain according to SEQ ID NO:211 and/or a light chain according toSEQ ID NO:212 (TPP-14095), b) a heavy chain according to SEQ IDNO:225 and/or a light chain according to SEQ ID NO:226 (TPP-14099),c) a heavy chain according to SEQ ID NO:239 and/or a light chainaccording to SEQ ID NO:240 (TPP-15285), or d) a heavy chainaccording to SEQ ID NO:253 and/or a light chain according to SEQ IDNO:254 (TPP-15286).

Preferred Combinations According to "all Aspects"

The following embodiments are particularly preferred embodiments ofaspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 ("allaspects" according to this section).

In preferred embodiments of all aspects, the antibody orantigen-binding fragment the isolated antibody or antigen-bindingfragment thereof specifically binds to the sulfated tyrosine richdomain of CCR8. In preferred embodiments of all aspects, theantibody or antigen-binding fragment is characterized by a HCDR3region comprising between 10 and 34% of tyrosine and/or between 2and 20% of histidine. In preferred embodiments of all aspects, theantibody or antigen-binding fragment is non-internalizing or ischaracterized by an internalization into a cell with endogenoustarget expression which is lower than the 1.5, 2, 3, 4, 5, 6, 7, or10-fold of the internalization of the isotype control.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment comprises human (derived) CDRs. Forexample, the antibody can be a human anti-human CCR8 antibody. Inpreferred embodiments of all aspects, the antibody orantigen-binding fragment is cross reactive for CCR8 from at leasttwo species, preferably selected from human, monkey, Macacafascicularis (cynomolgus monkey), Macaca mulatta (Rhesus macaque),rodent, mouse, rat, horse, bovine, pig, dog, cat and camel, evenmore preferably selected from human, cynomolgus and mouse.According to some most preferred of these embodiments, the antibodyor antigen-binding fragment is cross reactive for human andcynomolgus CCR8.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment binds the CCR8 from a first species with afirst dissociation constant KD and binds the CCR8 from a secondspecies with a second dissociation constant KD, wherein the firstand the second dissociation constant are in the same order ofmagnitude.

In preferred embodiments of all aspects the antibody orantigen-binding fragment binds with a KD value of <5E-8 M,<4E-8 M, <3E-8 M, <2E-8 M, <1E-8 M, <9E-9 M,<8E-9 M, <7E-9 M, <6E-9 M, <5E-9 M, <4E-9 M,<3E-9 M, <2.5E-9 M, <2E-9 M, <1.5E-9 M, <1E-9 M,<9E-10 M, <8E-10 M, <7E-10 M, <6E-10 M, <5E-10 M,<4E-10 M, <3E-10 M, <2.5E-10 M, <2E-10 M, <1.5E-10M, <1E-10 M, or <9E-11 M a) to an isolated polypeptideaccording to SEQ ID NO:43 and/or SEQ ID NO:46, wherein at least twoor all of Y3, Y15 and Y17 have been sulfated, and/or b) to anisolated polypeptide according to SEQ ID NO:44 and/or SEQ ID NO:47,wherein at least two or all of Y3, Y15 and Y17 have been sulfated,and/or c) to an isolated polypeptide according to SEQ ID NO:45and/or SEQ ID NO:48, wherein at least two or all of Y3, Y14 and Y15have been sulfated.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment (specifically) binds with an EC50 of<15 nM, <10 nM, <5 nM, <1 nM or <0.6 nM a) to humanCCR8 and/or to an isolated polypeptide according to SEQ ID NO:43and/or SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17have been sulfated, and/or b) to cynomolgus CCR8 and/or to anisolated polypeptide according to SEQ ID NO:44 and/or SEQ ID NO:47,wherein at least two or all of Y3, Y15 and Y17 have been sulfated,and/or c) to murine CCR8 and/or to an isolated polypeptideaccording to SEQ ID NO:45 and/or SEQ ID NO:48, wherein at least twoor all of Y3, Y14 and Y15 have been sulfated, and optionally bindswith an EC50 of <50 nM, <25 nM, <15 nM or <10 nM toactivated human regulatory T cells.

According to some most preferred of these embodiments, the EC50 ofthe antibody or antigen-binding fragment for binding to human CCR8or to an isolated polypeptide according to SEQ ID NO:46, wherein atleast two or all of Y3, Y15 and Y17 have been sulfated, is below 10nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and the EC50 of theantibody or antigen-binding fragment for binding to cynomolgus CCR8or to an isolated polypeptide according to SEQ ID NO:47, wherein atleast two or all of Y3, Y15 and Y17 have been sulfated, is below 10nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

As disclosed for instance in example 10.1.1, the antibodiesaccording to the current invention have excellent affinities fortheir respective target. For example, cross reactive antibodiesTPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360 and TPP-23411bound human CCR8 with an EC50 of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM,.about.0.9 nM or 1.7 nM. Also, TPP-21181, TPP-17578, TPP-19546,TPP-18206, TPP-21360 and TPP-23411 bound cynomolgus CCR8 with anEC50 of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, .about.0.55 nM or 0.9 nM. Inaddition, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bound tohuman regulatory T cells with an EC50 of 25 nM, 15 nM, 23 nM or 10nM. In addition, anti-murine CCR8 antibody TPP-14099 binds CHOcells expressing murine CCR8 with an EC50 of 3 nM and murine iTregswith an EC50 of 13.2 nM, cf. Table 10.1.1.5.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment, preferably at a concentration of 10 nM,a) does not or not substantially or to a lower degree thanantibodies L263G8 and 433H block CCL1 induced .beta.-arrestinsignaling, and/or b) does not or not substantially or to a lowerdegree than antibodies L263G8 and 433H induce ERK1/2phosphorylation and/or c) does not or not substantially or to alower degree than antibodies L263G8 and 433H induce AKTphosphorylation.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment blocks G protein dependent signaling ofthe chemokine receptor.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment is afucosylated.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment a) binds to human Fc gamma receptor IIIAvariant V176 (CD16a) with a dissociation constant (KD) lower than530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM, and/or b) bindsto human Fc gamma RITA (CD32a) with a dissociation constant (KD)lower than 30 .mu.M, 20 .mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment a) induces antibody-dependentcell-mediated cytotoxicity (ADCC) in target cells expressinghuman

CCR8 via human effector cells, such as human NK cells, and/or b)induces antibody-dependent cell-mediated phagocytosis (ADCP) intarget cells expressing human CCR8 via human effector cells, suchas human macrophages.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment induces ADCC and/or ADCP and a) theADCC-induced maximal depletion of activated human regulatory Tcells is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or99%, and/or b) the ADCP-induced maximal depletion of activatedhuman regulatory T cells is at least at least 5%, 10%, 15%, 20%,25%, 30%, 40% or 50%, and/or c) the maximal depletion ofintra-tumoral regulatory T cells, in vitro or in a subject, is atleast 50%, 60%, 70%, 80%, 90%, 95% or 99%.

In preferred embodiments of all aspects, the EC50 of the antibodyor antigen-binding fragment a) for ADCC-induced depletion ofactivated human regulatory T cells is below 100 pM, 50 pM, 25 pM,12.5 pM, 10 pM or 5 pM and/or b) for ADCP-induced depletion ofactivated human regulatory T cells is below 500 pM, 250 pM, 200 pM,150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

In preferred embodiments of all aspects, an effective dose of theantibody or antigen-binding fragment a) decreases the number ofactivated or intra-tumoral regulatory T cells, in vitro or in asubject, to less than 30%, 25%, 20%, 10%, 5% or 1% and/or b)increases the ratio of intra-tumoral CD8+ T cells to intra-tumoralTregs, in vitro or in a subject, to at least 5, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, or higher and/or c)decreases the percentage of regulatory T cells of intra-tumoralCD4+ T cells, in vitro or in a subject, to <30%, <20%,<10% or <5%.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment increases the absolute numbers of immunecells in a defined tumor volume, at least by a factor of 1.5, 2,2.5, 3, 3.5 or 4, preferably wherein the immune cells are selectedfrom a) (intra-tumoral) CD45+ cells, b) (intra-tumoral) CD8+ Tcells, c) (intra-tumoral) CD4+ T cells, d) (intra-tumoral)macrophages, such as M1 macrophages or M2 macrophages, e)(intra-tumoral) NK cells, f) (intra-tumoral) B cells, g)(intra-tumoral) Dendritic cells, h) (intra-tumoral) Gamma delta Tcells, i) (intra-tumoral) iNKT cells, or any combinationthereof.

For example, the immune cells are a) CD8+ T cells, CD4+ T cells,and macrophages, or b) CD8+ T cells, CD4+ T cells, NK cells andmacrophages, or c) CD8+ T cells, ACOD1+ macrophages (M1macrophages), and B cells.

In preferred embodiments of all aspects, three or more effectivedoses of the antibody or antigen-binding fragment, in a tumor,increase a) the number of intra-tumoral CD8+ T cells to at least150%, 200%, 250%, 300% or 350%, b) the ratio of intra-tumoral CD8+T cells to intra-tumoral regulatory T cells to at least 4, 5, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or higher,c) the number of intra-tumoral macrophages at least by a factor of2, 3, 4, or 5, d) the number of intra-tumoral ACOD1+ macrophages(M1 macrophages) at least by a factor of 2, 3, 4, 5, 6, 7, 8, 9 or10, e) the ratio of ACOD1+ macrophages (M1 macrophages) to MRC1+macrophages (M2 macrophages) to at least 1.5, 2, 3, 4, 5, 10, orhigher, f) the number of intra-tumoral NK cells to at least 140% or200%, g) the number of intra-tumoral CD3+ T cells to at least 150%,200%, 300% or 400%, h) the number of intra-tumoral B cells at leastby a factor of 2, 5, 10, 20, 30 or 40, i) the number ofintra-tumoral CD45+ T cells to at least 150%, 200% or 300%, and/orj) the number of intra-tumoral CD4+ T cells to at least 150%, 200%,300% or 400%, preferably wherein the tumor is characterized bytumor infiltrating lymphocytes, such as tumor infiltrating Tcells.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment induces formation of tertiary lymphoidstructures.

In preferred embodiments of all aspects, the antibody is an IgGantibody, preferably a human IgG1 or a murine IgG2a.

In preferred embodiments of all aspects, the antibody orantigen-binding fragment binds with an EC50 of <15 nM, <10nM, <5 nM, <1 nM or <0.6 nM a) to human CCR8 or to anisolated polypeptide according to SEQ ID NO:46, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, and/or b) tocynomolgus CCR8 or to an isolated polypeptide according to SEQ IDNO:47, wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and/or c) to murine CCR8 or to an isolated polypeptideaccording to SEQ ID NO:48, wherein at least two or all of Y3, Y14and Y15 have been sulfated.

In preferred embodiments of all aspects, the EC50 of the antibodyor antigen-binding fragment for binding to human CCR8 or to anisolated polypeptide according to SEQ ID NO:46, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and the EC50 of the antibody orantigen-binding fragment for binding to cynomolgus CCR8 or to anisolated polypeptide according to SEQ ID NO:47, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

In preferred embodiments of all aspects, the isolated antibody orantigen-binding fragment binds with an EC50 of <50 nM, <25nM, <15 nM or <10 nM to activated human regulatory Tcells.

In preferred embodiments of all aspects, the dissociation constantof the antibody for binding the first isolated non-sulfatedpolypeptide is higher than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM,350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1.mu.M, 1.25 .mu.M, 1.5 .mu.M, 1.75 .mu.M, 2 .mu.M, 2.25 .mu.M, 2.5.mu.M, 2.75 .mu.M, or 3 .mu.M, or is not detectable. Preferably,the dissociation constant of the antibody for binding the firstisolated non-sulfated polypeptide is higher than 100 nM, 250 nM,500 nM, 1 .mu.M, 2 .mu.M or 3 .mu.M, or is not detectable.

Most Preferred Combinations of "all Aspects"

Provided according to a preferred embodiment I is an isolatedantibody or antigen-binding fragment thereof specifically bindingto CCR8, wherein the antibody or antigen-binding fragment isnon-internalizing or is characterized by an internalization into acell with endogenous target expression which is lower than the 1.5,2, 3, 4, 5, 6, 7, or 10-fold of the internalization of the isotypecontrol.

Provided according to a preferred embodiment II is an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein the antibody or fragment is characterized by aHCDR3 region comprising between 10 and 34% of tyrosine and/orbetween 2 and 20% of histidine.

Provided according to a preferred embodiment III is an isolatedantibody or antigen-binding fragment thereof specifically bindingto CCR8, wherein the antibody comprises human derived CDRs.

Provided according to a preferred embodiment IV is an isolatedantibody or antigen-binding fragment thereof specifically bindingto CCR8, wherein the antibody or antigen-binding fragment is crossreactive for CCR8 from at least two species, preferably selectedfrom human, cynomolgus and mouse, most preferably wherein theantibody or antigen-binding fragment is cross reactive for humanand cynomolgus CCR8.

Provided according to a preferred embodiment V is an isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein the antibody or antigen-binding fragment, a. doesnot block CCL1 induced .beta.-arrestin signaling and/or b. does notinduce ERK1/2 phosphorylation and/or c. does not induce AKTphosphorylation.

Provided according to a preferred embodiment VI is an isolatedantibody or antigen-binding fragment thereof specifically bindingto CCR8, wherein the antibody or antigen-binding fragment isafucosylated and a. induces antibody-dependent cell-mediatedcytotoxicity (ADCC) in target cells expressing human CCR8 via humaneffector cells, such as human NK cells, and b. inducesantibody-dependent cell-mediated phagocytosis (ADCP) in targetcells expressing human CCR8 via human effector cells, such as humanmacrophages, c. wherein the maximal ADCC and ADCP induced in vitrodepletion of target cells expressing human CCR8 is at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%.

Provided according to a preferred embodiment VII is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments II to VI, wherein the antibody or antigen-bindingfragment is non-internalizing or is characterized by aninternalization into a cell with endogenous target expression whichis lower than the 1.5, 2, 3, 4, 5, 6, 7, or 10-fold of theinternalization of the isotype control.

Provided according to a preferred embodiment VIII is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I, or III to VII, wherein the antibody orantigen-binding fragment is characterized by a HCDR3 regioncomprising between 10 and 34% of tyrosine and/or between 2 and 20%of histidine.

Provided according to a preferred embodiment IX is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to II, or IV to VIII, wherein the antibody compriseshuman derived CDRs.

Provided according to a preferred embodiment X is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to III, or V to IX, wherein the antibody orantigen-binding fragment is cross reactive for CCR8 from at leasttwo species, preferably selected from human, cynomolgus and mouse,most preferably wherein the antibody or antigen-binding fragment iscross reactive for human and cynomolgus CCR8.

Provided according to a preferred embodiment XI is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to X, wherein the antibody or antigen-bindingfragment binds the CCR8 from a first species with a firstdissociation constant KD and binds the CCR8 from a second specieswith a second dissociation constant KD, wherein the first and thesecond dissociation constant are in the same order ofmagnitude.

Provided according to a preferred embodiment XII is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to IV, or VI to XI, wherein the antibody orantigen-binding fragment, a. does not block CCL1 induced.beta.-arrestin signaling and/or b. does not induce ERK1/2phosphorylation and/or c. does not induce AKT phosphorylation.

Provided according to a preferred embodiment XIII is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to V, or VII to XII, wherein the antibody orantigen-binding fragment a. induces antibody-dependentcell-mediated cytotoxicity (ADCC) in target cells expressing humanCCR8 via human effector cells, such as human NK cells, and b.induces antibody-dependent cell-mediated phagocytosis (ADCP) intarget cells expressing human CCR8 via human effector cells, suchas human macrophages, and optionally c. wherein the maximal ADCCand ADCP induced in vitro depletion of target cells expressinghuman CCR8 is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%,90%, 95% or 99%.

Provided according to a preferred embodiment XIV is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to XIII, wherein the antibody or antigen-bindingfragment specifically binds to the sulfated tyrosine rich domain ofCCR8.

Provided according to a preferred embodiment XV is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to XIV, wherein the antibody or antigen-bindingfragment specifically binds (i) with an EC50 of <15 nM, <10nM, <5 nM, <1 nM or <0.6 nM a. to human CCR8 and/or to anisolated polypeptide according to SEQ ID NO:46, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, and/or b. tocynomolgus CCR8 and/or to an isolated polypeptide according to SEQID NO:47, wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and/or c. to murine CCR8 and/or to an isolatedpolypeptide according to SEQ ID NO:48, wherein at least two or allof Y3, Y14 and Y15 have been sulfated, and/or (ii) with an EC50 of<50 nM, <25 nM, <15 nM or <10 nM to activated humanregulatory T cells.

Provided according to a preferred embodiment XVI is the isolatedantibody or antigen-binding fragment thereof according to any ofpreferred embodiments I to XV, a. wherein the antibody orantigen-binding fragment binds to human Fc gamma receptor IIIAvariant V176 (CD16a) with a dissociation constant (KD) lower than530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM, and/or b. whereinthe antibody or antigen-binding fragment binds to human Fc gammaRITA (CD32a) with a dissociation constant (KD) lower than 30 .mu.M,20 .mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M.

Provided according to a preferred embodiment XVII is the isolatedantibody or antigen-binding fragment thereof according to any ofpreferred embodiments I to XVI, wherein the antibody orantigen-binding fragment is afucosylated.

Provided according to a preferred embodiment XVIII is the isolatedantibody or antigen-binding fragment thereof according to any ofpreferred embodiments I to XVII, wherein the antibody orantigen-binding fragment a. induces antibody-dependentcell-mediated cytotoxicity (ADCC) in target cells expressing humanCCR8 via human effector cells, such as human NK cells, and/or b.induces antibody-dependent cell-mediated phagocytosis (ADCP) intarget cells expressing human CCR8 via human effector cells, suchas human macrophages, preferably, wherein c. the ADCC-inducedmaximal depletion of activated human regulatory T cells is at least25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98% or99%, and/or d. the ADCP-induced maximal depletion of activatedhuman regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%,40% or 50%, and/or e. the maximal ADCC and ADCP induced in vitrodepletion of activated human regulatory T cells is at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%.

Provided according to a preferred embodiment XIX is the isolatedantibody or antigen-binding fragment according to any of preferredembodiments I to XVIII, wherein a. the EC50 of the antibody orantigen-binding fragment for ADCC-induced depletion of activatedhuman regulatory T cells is below 100 pM, 50 pM, 25 pM, 12.5 pM, 10M or 5 pM and/or b. the EC50 of the antibody or antigen-bindingfragment for ADCP-induced depletion of activated human regulatory Tcells is below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pMor 25 pM.

Provided according to a preferred embodiment XX is the isolatedantibody or antigen-binding fragment according to any previouspreferred embodiment, wherein an effective dose of the antibody orantigen-binding fragment a. decreases the number of activated orintra-tumoral regulatory T cells, in vitro or in a subject, to lessthan 50%, 40%, 30%, 25%, 20%, 10%, 5% or 1% and/or b. increases theratio of intra-tumoral CD8+ T cells to intra-tumoral Tregs, invitro or in a subject, to at least 5, 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, or higher and/or c. decreases thepercentage of regulatory T cells of intra-tumoral CD4+ T cells, invitro or in a subject, to <30%, <20%, <10% or <5%.

Provided according to a preferred embodiment XXI is the isolatedantibody or antigen-binding fragment according to any previouspreferred embodiment, wherein an effective dose of the antibody orantigen-binding fragment induces formation of tertiary lymphoidstructures in a tumor.

Provided according to a preferred embodiment XXII is the isolatedantibody or antigen-binding fragment thereof according to anyprevious preferred embodiment, wherein the antibody is an IgGantibody, preferably a human IgG1 or a murine IgG2a, and or whereinthe antigen-binding fragment is an scFv, Fab, Fab' or a F(ab')2fragment.

Provided according to a preferred embodiment XXIII is a conjugatecomprising an antibody or antigen-binding fragment according to anyof preferred embodiments I to XXII, preferably wherein theconjugate comprises a. a radioactive element, b. a cytotoxic agent,such as an auristatin, a maytansinoid, a kinesin-spindle proteininhibitor, a nicotinamide phosphoribosyltransferase inhibitor or apyrrolobenzodiazepine derivative, c. a further antibody orantigen-binding fragment, or d. a chimeric antigen receptor.

Provided according to a preferred embodiment XXIV is apharmaceutical composition comprising an antibody orantigen-binding fragment according to any of preferred embodimentsI to XXII or a conjugate according to preferred embodimentXXIII.

Provided according to a preferred embodiment XXV is thepharmaceutical composition according to preferred embodiment XXIV,comprising one or more further therapeutically active compounds,preferably selected from a. an antibody or compound targeting acheckpoint protein, such as PD1, PD-L1 or CTLA-4, preferablywherein the antibody or compound targeting the checkpoint proteinis Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab,Cemiplimab, Dostarlimab or Ipilimumab, b. an antibody targeting afurther chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CX3CR1 or CXCR1, c. an antibody or a small molecule targeting HER2and/or EGFR, d. the standard of care for any of head and neckcancer, breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma and esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, e. a chemotherapeutic agent,preferably a taxane, paclitaxel, doxorubicin, cis-platin,carboplatin, oxaliplatin, or gemcitabine, e. a B cell depletingagent, such as an anti-CD19 antibody or an anti-CD20 antibodyand/or f. a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1.

Provided according to a preferred embodiment XXVI is an antibody orantigen-binding fragment according to any of preferred embodimentsI to XXII or a conjugate according to preferred embodiment XXIII ora pharmaceutical composition according to preferred embodiment XXIVor XXV for use as a medicament.

Provided according to a preferred embodiment XXVII is the antibodyor antigen-binding fragment according to any of preferredembodiments I to XXII or a conjugate according to preferredembodiment XXIII or a pharmaceutical composition according topreferred embodiment XXIV or XXV for use in the treatment of atumor or a disease characterized by CCR8 positive cells, such asCCR8 positive regulatory T cells.

Provided according to a preferred embodiment XXVIII is an antibodyor antigen-binding fragment according to any of preferredembodiments I to XXII or a conjugate according to preferredembodiment XXIII or a pharmaceutical composition according topreferred embodiment XXIV or XXV for use in simultaneous, separate,or sequential combination (i) with one or more furthertherapeutically active compounds, preferably selected from a. anantibody or a small molecule targeting a checkpoint protein, suchas PD1, PD-L1 or CTLA-4, preferably wherein the antibody or smallmolecule targeting a checkpoint protein is Nivolumab,Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab,Dostarlimab or Ipilimumab, b. an antibody targeting a furtherchemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CX3CR1 or CXCR1, c. an antibody targeting a protein which isspecifically expressed by the tumor cells, d. an antibody or asmall molecule targeting HER2 and/or EGFR, e. the standard of carefor any of head and neck cancer, breast cancer, gastric cancer,lung cancer, squamous cell carcinoma, esophageal tumor, melanoma,bladder cancer, liver cancer, and/or prostate cancer, f. achemotherapeutic agent, preferably a taxane, paclitaxel,doxorubicin, cis-platin, carboplatin, oxaliplatin, or gemcitabineand/or g. a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1, and/or (ii) with radiation therapy, and/or(iii) with depletion of intra-tumoral B cells, in the treatment ofa tumor or a disease characterized by CCR8 positive cells, such asCCR8 positive regulatory T cells.

Provided according to a preferred embodiment XXIX is the antibody,fragment, conjugate or pharmaceutical composition for use accordingto preferred embodiment XXVII or XXVIII, wherein the tumor isselected from the group of T-cell acute lymphoblastic leukemia,breast cancer, triple-negative breast cancer, triple positivebreast cancer, non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), testicular cancer, gastric cancer, head and necksquamous cell carcinoma, thymoma, esophageal adenocarcinoma,colorectal cancer, pancreatic adenocarcinoma, ovarian cancer orcervical cancer, acute myeloid leukemia, kidney cancer, bladdercancer, skin cancer, melanoma, thyroid cancer, mesothelioma,sarcoma and prostate cancer, B cell lymphoma, T cell lymphoma, orany other cancer involving CCR8 expressing cells, preferablywherein the tumor is selected from head and neck cancer, breastcancer, gastric cancer, lung cancer, squamous cell carcinoma,esophageal tumor, melanoma, bladder cancer, liver cancer, prostatecancer, B cell lymphoma and T cell lymphoma. Provided according toa preferred embodiment XXX is the antibody, fragment, conjugate orpharmaceutical composition for use according to preferredembodiment XXVI to XXIX, wherein the use comprises determining a.presence or quantity of tumor infiltrating lymphocytes, b. presenceor quantity of macrophages and/or NK cells, c. presence or quantityof CCR8 positive or FOXP3 positive regulatory T cells, d. tumormutational burden, e. cancer staging, f. presence, level oractivation of interferon-stimulated genes or proteins, g. CCR8expression, h. presence or quantity of complement factor proteins,serpins, and/or MHC components, i. presence or quantity ofcytokines, such as inflammatory or suppressive cytokines, j.activation of immune gene expression, and/or k. immune checkpoint(protein) expression, such as PD-(L)1 or CTLA4 expression, l.presence or quantity of tumor infiltrating CD19+ B cells, m.presence or quantity of tumor infiltrating CD8+ T cells; to predictor monitor treatment success.

Provided according to a preferred embodiment XXXI is apolynucleotide encoding an antibody or antigen-binding fragmentaccording to any of preferred embodiments I to XXII.

Provided according to a preferred embodiment XXXII is a vectorcomprising a polynucleotide according to preferred embodimentXXXI.

Provided according to a preferred embodiment XXXIII is an isolatedcell arranged for production of an antibody or antigen-bindingfragment according to any of preferred embodiments I to XXII.

Provided according to a preferred embodiment XXXIV is a method ofproducing an antibody or antigen-binding fragment according to anyof preferred embodiments I to XXII or a conjugate according topreferred embodiment XXIII, comprising culturing a cell accordingto preferred embodiment XXXIII and optionally purification of theantibody or antigen-binding fragment.

Provided according to a preferred embodiment XXXV is the antibodyor antigen-binding fragment according to any of preferredembodiments I to XXII or a conjugate according to preferredembodiment XXIII for use as a diagnostic agent in vivo or invitro.

Provided according to a preferred embodiment XXXVI is kitcomprising an antibody or antigen-binding fragment according to anyof preferred embodiments I to XXII or a conjugate according topreferred embodiment XXIII or a pharmaceutical compositionaccording to preferred embodiment XXIV or XXV with instructions foruse.

Aspect 19--Antibody Conjugates

In addition to naked antibodies, various further antibody- orantibody fragment-based conjugates can be designed using theantibodies or antigen binding fragments disclosed herein. Theseconjugates may be conjugates for diagnosis, therapy, researchapplications, and various other purposes. Provided are for exampleantibodies or antigen binding fragments thereof conjugated toradionuclides, cytotoxic agents, organic compounds, protein toxins,immunomodulators such as cytokines, fluorescent moieties, cells,further antibodies or antigen binding fragments thereof.

The conjugates disclosed herein, e.g. antibody drug conjugates(ADCs), targeted thorium conjugates (TTCs), bispecific antibodiesetc., are "modular" in nature. Throughout the disclosure, variousspecific embodiments of the various "modules" composing theconjugates are described. As specific non-limiting examples,specific embodiments of antibodies or fragments thereof, linkers,and cytotoxic and/or cytostatic agents that may compose the ADCsare described. It is intended that all of the specific embodimentsdescribed may be combined with each other as though each specificcombination was explicitly described individually.

According to a 19.sup.th aspect, there is provided a conjugatecomprising an antibody or antigen-binding fragment according to the6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th,12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.thand/or 18.sup.th aspect or a combination thereof. For example, theconjugate comprises an antibody or antigen-binding fragmentaccording to the 6.sup.th aspect. For example, the conjugatecomprises an antibody or antigen-binding fragment according to the7.sup.th aspect. For example, the conjugate comprises an antibodyor antigen-binding fragment according to the 8.sup.th aspect. Forexample, the conjugate comprises an antibody or antigen-bindingfragment according to the 9.sup.th aspect. For example, theconjugate comprises an antibody or antigen-binding fragmentaccording to the 10.sup.th aspect. For example, the conjugatecomprises an antibody or antigen-binding fragment according to the11.sup.th aspect. For example, the conjugate comprises an antibodyor antigen-binding fragment according to the 12.sup.th aspect. Forexample, the conjugate comprises an antibody or antigen-bindingfragment according to the 13.sup.th aspect. For example, theconjugate comprises an antibody or antigen-binding fragmentaccording to the 14.sup.th aspect. For example, the conjugatecomprises an antibody or antigen-binding fragment according to the15.sup.th aspect. For example, the conjugate comprises an antibodyor antigen-binding fragment according to the 16.sup.th aspect. Forexample, the conjugate comprises an antibody or antigen-bindingfragment according to the 17.sup.th aspect. For example, theconjugate comprises an antibody or antigen-binding fragmentaccording to the 18.sup.th aspect.

According to some preferred embodiments the conjugate comprises (a)a radioactive element, (b) a cytotoxic agent, such as anauristatin, a maytansinoid, a kinesin-spindle protein (KSP)inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor or a pyrrolobenzodiazepine derivative, (c) a furtherantibody or antigen-binding fragment, (d) a chimeric antigenreceptor.

Radioconjugates

According to some first embodiments of the 19th aspect, theconjugate comprises or is arranged to comprise a radionuclide, suchas a beta particle, an alpha particle, or an Auger electronemitter. Suitable beta emitters according to the current inventionare for example yttrium-90, iodine-131, strontium-89-chloride,lutetium-177, holmium-166, rhenium-186, rhenium-188, copper-67,promethium-149, gold-199, and rhodium-105. Suitable Auger electronemitters, according to the current invention are for examplebromine-77, indium-111, iodine-123, and iodine-125. Suitable alphaemitters, according to the current invention are for examplethorium-227, bismuth-213, radium-223, actinium-225 andastatine-211.

For example, thorium-227 (227Th) can be efficiently complexed withoctadentate 3,2-hydroxypyridinone (3,2-HOPO) chelators that areconjugated to antibodies according to the current invention,resulting in highly stable targeted thorium-227 conjugates (TTCs),as described in example 13. Targeted thorium conjugates (TTCs)comprise three main building blocks. Following the .beta.-particledecay of actinium-227, the first building block,.alpha.-particle-emitting radionuclide 227Th is purified by ionexchange chromatography. The second building block is a chelator,such as a siderophore-derived chelator containing HOPO groupsbearing four 3-hydroxy-N-methyl-2-pyridinone moieties on asymmetrical polyamine scaffold functionalized with a carboxylicacid linker for bioconjugation. Conjugation to a targeting moietycan be achieved through the amide bond formation with the.epsilon.-amino groups of lysine residues. These octadentate3,2-HOPO chelators can be efficiently labeled with 227Th, with highyield, purity, and stability at ambient conditions. Compared withthe tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA)chelator, which often requires heating, the HOPO chelators aresuperior due to efficient radiolabeling at ambient temperatures andhigh stability of formed complexes. The third building block is thetargeting moiety, that is the chemokine receptor antibody orantigen binding fragment thereof according to any of the aspects 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

ADCs

According to some second embodiments of the 19th aspect, theconjugate comprises a cytotoxic agent, e.g. to form an antibodydrug conjugate (ADC).

In some preferred embodiments, the cytotoxic agent is anauristatin, a maytansinoid, a kinesin-spindle protein (KSP)inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor or a pyrrolobenzodiazepine derivative. Generation ofconjugates comprising maytansinoid may occur as described in Chari,Ravi V J, et al. "Immunoconjugates containing novel maytansinoids:promising anticancer drugs." Cancer research 52.1 (1992): 127-131,or EP2424569 B1, both incorporated herein in their entirety.Generation of conjugates comprising kinesin-spindle protein (KSP)inhibitors may occur as described in WO2019243159 A1, incorporatedherein in its entirety. Generation of conjugates comprising anicotinamide phosphoribosyltransferase (NAMPT) inhibitor may occuras described in WO2019149637 A1, incorporated herein in itsentirety. Generation of conjugates comprising apyrrolobenzodiazepine may be obtained as described in EP3355935 A1,incorporated herein in its entirety.

The cytotoxic and/or cytostatic agent of the anti-chemokinereceptor or anti-CCR8 ADC may be any agent known to inhibit thegrowth and/or replication of, and/or kill cells. Numerous agentshaving cytotoxic and/or cytostatic properties are known in theliterature. Non-limiting examples of classes of cytotoxic and/orcytostatic agents include, by way of example and not limitation,cell cycle modulators, apoptosis regulators, kinase inhibitors,protein synthesis inhibitors, alkylating agents, DNA cross-linkingagents, intercalating agents, mitochondria inhibitors, nuclearexport inhibitors, topoisomerase I inhibitors, topoisomerase IIinhibitors, RNA/DNA antimetabolites and antimitotic agents.

The linkers linking the cytotoxic and/or cytostatic agent(s) to theantigen binding moiety of an anti-chemokine receptor or anti-CCR8ADC may be long, short, flexible, rigid, hydrophilic or hydrophobicin nature, or may comprise segments that have differentcharacteristics, such as segments of flexibility, segments ofrigidity, etc. The linker may be chemically stable to extracellularenvironments, for example, chemically stable in the blood stream,or may include linkages that are not stable and release thecytotoxic and/or cytostatic agents in the extracellular milieu. Insome embodiments, the linkers include linkages that are designed torelease the cytotoxic and/or cytostatic agents upon internalizationof the anti-chemokine receptor or anti-CCR8 ADC, within the cell.In some specific embodiments, the linkers include linkages designedto cleave and/or immolate or otherwise breakdown specifically ornon-specifically inside cells. A wide variety of linkers useful forlinking drugs to antigen binding moieties such as antibodies in thecontext of ADCs are known in the art. Any of these linkers, as wellas other linkers, may be used to link the cytotoxic and/orcytostatic agents to the antigen binding moiety of theanti-chemokine receptor or anti-CCR8 ADCs, described herein.

The number of cytotoxic and/or cytostatic agents linked to theantigen binding moiety of an anti-chemokine receptor or anti-CCR8ADC (drug-to-antibody ratio: DAR) can vary and will be limited onlyby the number of available attachments sites on the antigen bindingmoiety and the number of agents linked to a single linker.Typically, a linker will link a single cytotoxic and/or cytostaticagent to the antigen binding moiety of an anti-chemokine receptoror anti-CCR8 ADC. In embodiments of anti-chemokine receptor oranti-CCR8 ADCs, which include more than a single cytotoxic and/orcytostatic agent, each agent may be the same or different. As longas the anti-chemokine receptor or anti-CCR8 ADC, does not exhibitunacceptable levels of aggregation under the conditions of useand/or storage, anti-chemokine receptor or anti-CCR8 ADCs, withDARs of twenty, or even higher, are contemplated. In someembodiments, the anti-chemokine receptor or anti-CCR8 ADCs,described herein may have a DAR in the range of about 1-10, 1-8,1-6, or 1-4. In certain specific embodiments, the anti-chemokinereceptor or anti-CCR8 ADC may have a DAR of 2, 3 or 4. In someembodiments, the anti-chemokine receptor or anti-CCR8 ADCs, arecompounds according to structural formula (I): [D-L-XY]n-Ab (I) orsalts thereof, where each "D" represents, independently of theothers, a cytotoxic and/or cytostatic agent; each "L" represents,independently of the others, a linker; "Ab" represents ananti-chemokine receptor binding moiety, e.g. an anti-CCR8 antibodyaccording to the current invention; each "XY" represents a linkageformed between a functional group Rx on the linker and a"complementary" functional group Ry on the anti-chemokine receptorbinding moiety; and n represents the DAR of the anti-chemokinereceptor ADC.

In a specific exemplary embodiment, the anti-chemokine receptorADCs are compounds according to structural formula (I) in whicheach "D" is the same and is either a cell-permeating auristatin(for example, dolastatin-10 or MMAE) or a cell-permeating minorgroove-binding DNA cross-linking agent; each "L" is the same and isa linker cleavable by a lysosomal enzyme; each "XY" is a linkageformed between a maleimide and a sulfhydryl group; "Ab" is anantibody or fragment thereof comprising six CDRs corresponding tothe six CDRs of an anti-chemokine receptor or CCR8 antibodyaccording to the current invention; and n is 2, 3 or 4. In aspecific embodiment "Ab" is a fully human antibody comprising humanderived CDRs.

Cytotoxic and cytostatic agents are agents known to inhibit thegrowth and/or replication of and/or kill cells and in particulartumor cells or intra-tumoral Treg cells. These compounds may beused in a combination therapy with an anti-chemokine receptorantibody such as a CCR8 antibody, or as part of an anti-chemokinereceptor ADC as described herein: In some embodiments, the drugmoiety of the anti-chemokine receptor or anti-CCR8 ADC is acytostatic agent selected from radionuclides, alkylating agents,DNA cross-linking agents, DNA intercalating agents (e.g., groovebinding agents such as minor groove binders), cell cyclemodulators, apoptosis regulators, kinase inhibitors, proteinsynthesis inhibitors, mitochondria inhibitors, nuclear exportinhibitors, topoisomerase I inhibitors, topoisomerase IIinhibitors, RNA/DNA antimetabolites and antimitotic agents. In someembodiments, the drug moiety of the anti-chemokine receptor oranti-CCR8 ADC is an alkylating agent selected from asaley(L-Leucine,N--[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-,ethylester); AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU(N,N'-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanedioldimethanesulfonate); (carboxyphthalato)platinum; CBDCA(cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II))); CCNU(N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (iproplatin;NSC 256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum(cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodisone;dianhydrogalactitol (5,6-diepoxydulcitol); fluorodopan((5-[(2-chloroethyl)-(2-fluoroethy)amino]-6-methyl-uracil);hepsulfam; hycanthone; indolinobenzodiazepine dimer DGN462;melphalan; methyl CCNU((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea);mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea));piperazine alkylator((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazinedihydrochloride)); piperazinedione; pipobroman(N,N'-bis(3-bromopropionyl) piperazine); porfiromycin(N-methylmitomycin C); spirohydantoin mustard; teroxirone(triglycidylisocyanurate); tetraplatin; thio-tepa(N,N',N''-tri-1,2-ethanediylthio phosphoramide);triethylenemelamine; uracil nitrogen mustard (desmethyldopan);Yoshi-864 ((bis(3-mesyloxy propyl)amine hydrochloride).

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a DNA alkylating-like agent selected fromCisplatin; Carboplatin; Nedaplatin; Oxaliplatin; Satraplatin;Triplatin tetranitrate; Procarbazine; altretamine; dacarbazine;mitozolomide; temozolomide.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is an alkylating antineoplastic agents selectedfrom Carboquone; Carmustine; Chlornaphazine; Chlorozotocin;Duocarmycin; Evofosfamide; Fotemustine; Glufosfamide; Lomustine;Mannosulfan; Nimustine; Phenanthriplatin; Pipobroman; Ranimustine;Semustine; Streptozotocin; ThioTEPA; Treosulfan; Triaziquone;Triethylenemelamine; Triplatin tetranitrate.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a DNA replication and repair inhibitor selectedfrom Altretamine; Bleomycin; Dacarbazine; Dactinomycin;Mitobronitol; Mitomycin; Pingyangmycin; Plicamycin; Procarbazine;Temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281;AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a cell cycle modulator, such as Paclitaxel;Nab-Paclitaxel; Docetaxel; Vincristine; Vinblastine; ABT-348;AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitors;Aurora B-specific kinase inhibitors and pan-Aurora kinaseinhibitors; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584;flavopyridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib(CYC-202, R-roscovitine); ZK-304709; AZD4877, ARRY-520:GSK923295A.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is an apoptosis regulator such as AT-101((-)gossypol); G3139 or oblimersen (Bcl-2-targeting antisenseoligonucleotide); IPI-194; IPI-565;N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-ylben-zoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3--nitrobenzenesulfonamide);N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide;GX-070 (Obatoclax.RTM.; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; venetoclax;agents that target TRAIL or death receptors (e.g., DR4 and DR5)such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs thattarget caspases, caspase-regulators, BCL-2 family members, deathdomain proteins, TNF family members, Toll family members, and/orNF-kappa-B proteins.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is an angiogenesis inhibitor such as ABT-869;AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632; IM-862;pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib(PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib;ABT-165; ZD-6474; DLL4 inhibitors.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a proteasome inhibitor such as Bortezomib;Carfilzomib; Epoxomicin; Ixazomib; Salinosporamide A.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a kinase inhibitor such as Afatinib; Axitinib;Bosutinib; Crizotinib; Dasatinib; Erlotinib; Fostamatinib;Gefitinib; Ibrutinib; Imatinib; Lapatinib; Lenvatinib; Mubritinib;Nilotinib; Pazopanib; Pegaptanib; Sorafenib; Sunitinib; SU6656;Vandetanib; Vemurafenib; CEP-701 (lesaurtinib); XL019; INCB018424(ruxolitinib); ARRY-142886 (selemetinib); ARRY-438162(binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus;RAD-001; rapamycin; temsirolimus; ATP-competitive TORC1/TORC2inhibitors including PI-103, PP242, PP30, Torin 1; LY294002;XL-147; CAL-120; ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941;BGT226; BEZ235; XL765.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a protein synthesis inhibitor such asStreptomycin; Dihydrostreptomycin; Neomycin; Framycetin;Paromomycin; Ribostamycin; Kanamycin; Amikacin; Arbekacin;Bekanamycin; Dibekacin; Tobramycin; Spectinomycin; Hygromycin B;Paromomycin; Gentamicin; Netilmicin; Sisomicin; Isepamicin;Verdamicin; Astromicin; Tetracycline; Doxycycline;Chlortetracycline; Clomocycline; Demeclocycline; Lymecycline;Meclocycline; Metacycline; Minocycline; Oxytetracycline;Penimepicycline; Rolitetracycline; Tetracycline; Glycylcyclines;Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid;Radezolid; Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferaseinhibitors; Chloramphenicol; Azidamfenicol; Thiamphenicol;Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin;Azithromycin; Clarithromycin; Dirithromycin; Erythromycin;Flurithromycin; Josamycin; Midecamycin; Miocamycin; Oleandomycin;Rokitamycin; Roxithromycin; Spiramycin; Troleandomycin; Tylosin;Ketolides; Telithromycin; Cethromycin; Solithromycin; Clindamycin;Lincomycin; Pirlimycin; Streptogramins; Pristinamycin;Quinupristin/dalfopristin; Virginiamycin.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a histone deacetylase inhibitor such asVorinostat; Romidepsin; Chidamide; Panobinostat; Valproic acid;Belinostat; Mocetinostat; Abexinostat; Entinostat; SB939(pracinostat); Resminostat; Givinostat; Quisinostat;thioureidobutyronitrile (Kevetrin.TM.); CUDC-10; CHR-2845(tefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat);ME-344; sulforaphane.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a topoisomerase I inhibitor such ascamptothecin; various camptothecin derivatives and analogs (forexample, NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323,NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC 618939, NSC610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830, and NSC606497); morpholinisoxorubicin; SN-38.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a topoisomerase II inhibitor such asdoxorubicin; amonafide (benzisoquinolinedione); m-AMSA(4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide);anthrapyrazole derivative ((NSC 355644); etoposide (VP-16);pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine,9-methoxy-N, N-dimethyl-5-nitro-, monomethanesulfonate); bisantrenehydrochloride; daunorubicin; deoxydoxorubicin; mitoxantrone;menogaril; N,N-dibenzyl daunomycin; oxanthrazole; rubidazone;teniposide.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a DNA intercalating agent such as anthramycin;chicamycin A; tomaymycin; DC-81; sibiromycin; pyrrolobenzodiazepinederivative; SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1-,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi-n-5(11aH)-one); SG2000 (SJG-136;(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3--dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)).

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a RNA/DNA antimetabolite such as L-alanosine;5-azacytidine; 5-fluorouracil; acivicin; aminopterin derivativeN-[2-chloro-5[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-asparticacid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-asparticacid; aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acidmonohydrate; antifolate PT523((N.alpha.-(4-amino-4-deoxypteroyl)-N.gamma.-hemiphthaloyl-L-ornithine));Baker's soluble antifol (NSC 139105); dichlorallyl lawsone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; ftorafur((pro-drug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil);5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative(N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glut-amic acid); PALA ((N-(phosphonoacetyl)-L-aspartate); pyrazofurin;trimetrexate.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a DNA antimetabolite such as 3-HP;2'-deoxy-5-fluorouridine; 5-HP; .alpha.-TGDR(.alpha.-2'-deoxy-6-thioguanosine); aphidicolin glycinate; ara C(cytosine arabinoside); 5-aza-2'-deoxycytidine; .beta.-TGDR(.beta.-2'-deoxy-6-thioguanosine); cyclocytidine; guanazole;hydroxyurea; inosine glycodialdehyde; macbecin II;pyrazoloimidazole; thioguanine; thiopurine.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC, is a mitochondria inhibitor such aspancratistatin; phenpanstatin; rhodamine-123; edelfosine;d-alpha-tocopherol succinate; compound 11.beta.; aspirin;ellipticine; berberine; cerulenin; GX015-070 (Obatoclax.RTM.;1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-; celastrol (tripterine); metformin; Brilliant green; ME-344.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC, is an antimitotic agent such as allocolchicine;auristatins, such as MMAE (monomethyl auristatin E) and MMAF(monomethyl auristatin F); halichondrin B; cemadotin; colchicine;cholchicine derivative (N-benzoyl-deacetyl benzamide);dolastatin-10; dolastatin-15; maytansine; maytansinoids, such asDM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine);rhozoxin; paclitaxel; paclitaxel derivative((2'-N-[3-(dimethylamino)propyl]glutaramate paclitaxel); docetaxel;thiocolchicine; trityl cysteine; vinblastine sulfate; vincristinesulfate.

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a nuclear export inhibitor such as callystatinA; delactonmycin; KPT-185 (propan-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2--enoate); kazusamycin A; leptolstatin; leptofuranin A; leptomycin B;ratjadone; Verdinexor((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin--2-ylprop-2-enehydrazide).

In some embodiments, the drug moiety of the anti-chemokine receptoror anti-CCR8 ADC is a hormonal therapeutics such as anastrozole;exemestane; arzoxifene; bicalutamide; cetrorelix; degarelix;deslorelin; trilostane; dexamethasone; flutamide; raloxifene;fadrozole; toremifene; fulvestrant; letrozole; formestane;glucocorticoids; doxercalciferol; sevelamer carbonate;lasofoxifene; leuprolide acetate; megesterol; mifepristone;nilutamide; tamoxifen citrate; abarelix; prednisone; finasteride;rilostane; buserelin; luteinizing hormone releasing hormone (LHRH);Histrelin; trilostane or modrastane; fosrelin; goserelin.

Any of these agents that include, or that may be modified toinclude, a site of attachment to an antibody and/or bindingfragment can be included in an anti-chemokine receptor ADC, e.g. inan anti-CCR8 ADC.

CAR T Cells

According to some third embodiments of the 19th aspect, theconjugate is a CAR T cell conjugate engineered for chemokinereceptor or CCR8 targeting. Recently, CART cells have gainedattention from their clinical successes and expedited FDAapprovals, cf. WO2020102240, incorporated herein in its entirety.In the CAR T cell approach, T cells are collected from patientblood and are then genetically engineered to express CARs that arespecific for an antigen present on tumor cells. These engineered Tcells are then re-administered to the same patient. Upon injection,CAR T cells recognize the targeted antigen on target cells toinduce target cell death. T cells expressing chimeric antigenreceptors (CAR T cells) thus constitute a novel modality formedical uses such as tumor treatment. The chimeric antigen receptor(CAR) is a genetically engineered receptor that is designed totarget a specific antigen, for example, a tumor antigen. Thistargeting can result in cytotoxicity against the tumor, forexample, such that CAR T cells expressing CARs can target and killtumors via the specific tumor antigens. According to the presentinvention, the antibodies or antigen binding fragments provided forCCR8 or chemokine receptor recognition can be used to engineer CART cells for specific recognition of CCR8 expressing cells or cellsexpressing the respective chemokine receptor. CARS according to thecurrent invention may comprise a) a recognition region, e.g., asingle chain fragment variable (scFv) region derived from aprovided anti-CCR8 or anti-chemokine receptor antibody forrecognition and binding to the CCR8 or chemokine receptor expressedby the target cell, and b) an activation signaling domain, e.g.,the CD3 chain of T cells, which can serve as a T cell activationsignal in CARs.

Preferably, the CARs according to the current invention comprise aco-stimulation domain (e.g., CD137, CD28 or CD134) to achieveprolonged activation of T cells in vivo. Addition of aco-stimulation domain enhances the in vivo proliferation andsurvival of T cells containing CARs, and initial clinical data haveshown that such constructs are promising therapeutic agents in thetreatment of diseases, such as cancer. According to the currentinvention, the CAR T cells can be used to treat any disease withlocal or systemic aberrant presence of cells expressing the targetchemokine receptor, in particular CCR8 expressing cells, such asTreg cells.

Bispecifics

According to some fourth embodiments of the 19th aspect, theconjugate is a bispecific antibody or a multispecific antibody. Insome preferred embodiments the bispecific antibody comprises atleast one Fc domain.

In some preferred embodiments of the 19th aspect, the first bindingmoiety of the bispecific antibody is an antibody or antigen bindingfragment according to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th,16.sup.th, 17.sup.th and/or 18.sup.th aspect.

In some embodiments A of the fourth embodiments of the 19th aspect,the first binding moiety of the bispecific antibody is an antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspectand the second binding moiety of the bispecific antibody is thesame or a different antibody or antigen binding fragment accordingto the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th,11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th,17.sup.th and/or 18.sup.th aspect.

In some embodiments B of the fourth embodiments of the 19th aspect,the first binding moiety of the bispecific antibody is an antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspectbinding human CCR8 and the second binding moiety of the bispecificantibody is an antibody or antigen binding fragment binding to adifferent chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, CX3CR1 or CXCR1. For example, the second binding moiety ofthe bispecific antibody is Mogamulizumab or an antigen bindingfragment thereof.

In some embodiments C of the fourth embodiments of the 19th aspect,the first binding moiety of the bispecific antibody is an antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,preferably binding CCR8, and the second binding moiety of thebispecific antibody is an antibody or antigen binding fragmentbinding to a cell-surface protein such as cell surface proteinexpressed on immune cells or a tissue- or cell type-specificantigen. In some of these embodiments, the second binding moiety ofthe bispecific antibody is an antibody or antigen binding fragmenttargeting a checkpoint protein, such as an anti-PD1 antibody, ananti-PD-L1 antibody, or a CTLA-4 antibody. Suitable checkpointprotein targeting antibodies include Nivolumab, Pembrolizumab,Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, orIpilimumab. In some other of these embodiments, the second bindingmoiety of the bispecific antibody is a HER2 targeting antibody,such as Trastuzumab, Pertuzumab and/or Margetuximab.

In some embodiments D of the fourth embodiments of the 19th aspect,the first binding moiety of the bispecific antibody is an antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,preferably binding CCR8, and the second binding moiety of thebispecific antibody is an antibody or antigen binding fragmentbinding to a cell surface molecule associated with T-cellactivation, preferably selected from CD25, CTLA-4, PD-1, LAG3,TIGIT, ICOS, and TNF receptor super family members, 4-1BB, OX-40,and GITR.

Techniques for making bi- or multispecific antibodies include, butare not limited to, recombinant co-expression of two immunoglobulinheavy chain-light chain pairs having different specificities (seeMilstein and Cuello. "Hybrid hybridomas and their use inimmunohistochemistry." Nature 305.5934 (1983): 537-540.;WO1993008829 A1, and Traunecker, Andre, Antonio Lanzavecchia, andKlaus Karjalainen. "Bispecific single chain molecules (Janusins)target cytotoxic lymphocytes on HIV infected cells." The EMBOJournal 10.12 (1991): 3655-3659.), and chemical conjugation of twodifferent monoclonal antibodies (see Staerz, Uwe D., OsamiKanagawa, and Michael J. Bevan. "Hybrid antibodies can target sitesfor attack by T cells." Nature 314.6012 (1985): 628-631.).Multispecific antibodies may also be made by cross-linking two ormore antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,and Brennan, Maureen, Peter F. Davison, and Henry Paulus."Preparation of bispecific antibodies by chemical recombination ofmonoclonal immunoglobulin G1 fragments." Science 229.4708 (1985):81-83.), using leucine zippers to produce bi-specific antibodies(see, e.g., Kostelny, Sheri A., M. S. Cole, and J. Yun Tso."Formation of a bispecific antibody by the use of leucine zippers."The Journal of Immunology 148.5 (1992): 1547-1553.), using diabodytechnology for making bispecific antibody fragments (see, e.g.,Holliger, Philipp, Terence Prospero, and Greg Winter. ""Diabodies":small bivalent and bispecific antibody fragments." Proceedings ofthe National Academy of Sciences 90.14 (1993): 6444-6448.), usingsingle-chain Fv (sFv) dimers (see, e.g. Gruber, Meegan, et al."Efficient tumor cell lysis mediated by a bispecific single chainantibody expressed in Escherichia coli." The Journal of immunology152.11 (1994): 5368-5374.), by preparing trispecific antibodies asdescribed (e.g., in Tutt, Alison, G. T. Stevenson, and M. J.Glennie. "Trispecific F (ab') 3 derivatives that use cooperativesignaling via the TCR/CD3 complex and CD2 to activate and redirectresting cytotoxic T cells." The Journal of Immunology 147.1 (1991):60-69.), and by controlled Fab arm exchange (cFAE) according toLabrijn, Aran F., et al. "Efficient generation of stable bispecificIgG1 by controlled Fab-arm exchange." Proceedings of the NationalAcademy of Sciences 110.13 (2013): 5145-5150.

Conjugates for Diagnosis and Research Applications

According to some fifth embodiments of the 19th aspect, theconjugate comprises a detectable moiety. Examples of detectablemoieties include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals, nonradioactiveparamagnetic metal ions and reactive moieties. The detectablesubstance can be coupled or conjugated either directly to theantibody or fragment thereof or indirectly, e.g. through a linkerknown in the art or another moiety, using techniques known in theart. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase(HRPO), alkaline phosphatase, .beta.-galactosidase,acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases(e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like.

Examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examplesof suitable radioactive material include 125I, 131I, 111In or99mTc.

Detection of expression of a chemokine receptor or CCR8 generallyinvolves contacting a biological sample (tumor, cells, tissue, orbody fluid of an individual) with one or more antibodies orfragments according to the current invention (optionally conjugatedto a detectable moiety), and detecting whether or not the sample ispositive for the chemokine receptor or CCR8, or whether the samplehas altered (e.g., reduced or increased) expression as compared toa control sample.

Aspect 20--Pharmaceutical Composition

According to a 20.sup.th aspect, there is provided a pharmaceuticalcomposition comprising an antibody or antigen-binding fragmentaccording to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th,11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th,17.sup.th and/or 18.sup.th aspect, or a conjugate according to the19.sup.th aspect. Preferably, the pharmaceutical compositioncomprises an antibody or fragment thereof or a conjugate accordingto any one of the embodiments described herein, or a combinationthereof, in a therapeutically effective amount together with apharmaceutically acceptable carrier, excipient, or stabilizer.

Pharmaceutical compositions can be prepared by mixing the antibody,fragment, or conjugate having the desired degree of purity withoptional physiologically acceptable carriers, excipients orstabilizers (Remington, Joseph Price. Remington: The science andpractice of pharmacy. Vol. 1. Lippincott Williams & Wilkins,2006.). Pharmaceutical compositions may be for example in the formof lyophilized formulations or aqueous solutions.

Carriers, Excipients, Stabilizers

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate buffer (e.g. PBS), citrate buffer, andother organic acid buffer; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride,benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (e.g.less than about 10 residues) polypeptide; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,or dextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as Tween.RTM., Pluronic.RTM. orpolyethylene glycol (PEG).

Multiple therapeutically active compounds According to the currentinvention a pharmaceutical composition may contain more than oneactive compound, e.g. as necessary or beneficial for the particularindication being treated.

According to some first embodiments of the 20.sup.th aspect, thepharmaceutical composition comprises one or more furthertherapeutically active compounds.

In some preferred of the first embodiments of the 20th aspect, thepharmaceutical composition comprises an antibody or antigen-bindingfragment according to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th,16.sup.th, 17.sup.th and/or 18.sup.th aspect, or a conjugateaccording to the 19.sup.th aspect and a) an antibody or a smallmolecule targeting a checkpoint protein, such as PD1, PD-L1 orCTLA-4, and/or b) an antibody targeting a further chemokinereceptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 orCXCR1, and/or c) an antibody targeting a protein which isspecifically expressed by tumor cells and/or d) an antibody or asmall molecule targeting HER2 and/or EGFR, and/or e) the standardof care for any of head and neck cancer, breast cancer, gastriccancer, lung cancer, squamous cell carcinoma, esophageal tumor,melanoma, bladder cancer, liver cancer, and/or prostate cancer,and/or f) a chemotherapeutic agent, preferably a taxane,paclitaxel, doxorubicin, cis-platin, carboplatin, oxaliplatin,paclitaxel or gemcitabine, and/or g) a targeted kinase inhibitor,such as Sorafinib, Regorafenib, or MEKi-1.

Combination with Checkpoint Inhibitors

According to some preferred embodiments A of the first embodimentsof the 20.sup.th aspect, the pharmaceutical composition furthermorecomprises an antibody or a small molecule targeting a checkpointprotein, such as PD1, PD-L1 or CTLA-4. Suitable checkpointtargeting antibodies include Nivolumab (PD1; Human IgG4),Pembrolizumab (PD1; Humanized IgG4), Atezolizumab (PD-L1; HumanizedIgG1), Avelumab (PD-L1; Human IgG1), Durvalumab (PD-L1; HumanIgG1), Cemiplimab, cemiplimab-rwlc (PD-1; Human mAb), Dostarlimab(TSR-042) (PD-1; Humanized IgG4), or Ipilimumab (CTLA-4; HumanIgG1).

In some embodiments, the antibody or a small molecule targeting acheckpoint protein targets CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4,BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049,CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof.

Combination with Chemokine Receptor Antibodies

According to some preferred embodiments B of the first embodimentsof the 20.sup.th aspect, the pharmaceutical composition furthermorecomprises an antibody or a small molecule targeting a furtherchemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CX3CR1 or CXCR1. Suitable antibodies targeting a further chemokinereceptor include the antibodies provided according to the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or18.sup.th aspect, or Mogamulizumab.

Combination with HER2 or EGFR Targeting Antibodies or Molecules

According to some preferred embodiments C of the first embodimentsof the 20.sup.th aspect, the pharmaceutical composition furthermorecomprises an antibody or a small molecule targeting HER2 and/orEGFR. Suitable antibodies targeting HER2 are Trastuzumab (HER2;Humanized IgG1), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumabemtansine (HER2; humanized IgG1; ADC), [fam-]trastuzumabderuxtecan, fam-trastuzumab deruxtecan-nxki (HER2; Humanized IgG1ADC), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2;Humanized IgG1 ADC) and/or Margetuximab (HER2; Chimeric IgG1).Suitable antibodies targeting EGFR are Cetuximab (EGFR; ChimericIgG1), Panitumumab (EGFR; Human IgG2), and Necitumumab (EGFR; HumanIgG1).

Combination with Therapeutic Antibodies

According to some embodiments D of the first embodiments of the20.sup.th aspect, the pharmaceutical composition comprises afurther therapeutic antibody selected from Muromonab-CD3 (CD3;Murine IgG2a), Efalizumab (CD11a; Humanized IgG1), Tositumomab-I131(CD20; Murine IgG2a), Nebacumab (Endotoxin; Human IgM), Edrecolomab(EpCAM; Murine IgG2a), Catumaxomab (EPCAM/CD3; Rat/mouse bispecificmAb), Daclizumab (IL-2R; Humanized IgG1), Abciximab (GPIIb/IIIa;Chimeric IgG1 Fab), Rituximab (CD20; Chimeric IgG1), Basiliximab(IL-2R; Chimeric IgG1), Palivizumab (RSV; Humanized IgG1),Infliximab (TNF; Chimeric IgG1), Trastuzumab (HER2; HumanizedIgG1), Adalimumab (TNF; Human IgG1), Ibritumomab tiuxetan (CD20;Murine IgG1), Omalizumab (IgE; Humanized IgG1), Cetuximab (EGFR;Chimeric IgG1), Bevacizumab (VEGF; Humanized IgG1), Natalizumab (a4integrin; Humanized IgG4), Panitumumab (EGFR; Human IgG2),Ranibizumab (VEGF; Humanized IgG1 Fab), Eculizumab (C5; HumanizedIgG2/4), Certolizumab pegol (TNF; Humanized Fab, pegylated),Ustekinumab (IL-12/23; Human IgG1), Canakinumab (IL-1.beta.; HumanIgG1), Golimumab (TNF; Human IgG1), Ofatumumab (CD20; Human IgG1),Tocilizumab (IL-6R; Humanized IgG1), Denosumab (RANK-L; HumanIgG2), Belimumab (BLyS; Human IgG1), Ipilimumab (CTLA-4; HumanIgG1), Brentuximab vedotin (CD30; Chimeric IgG1; ADC), Pertuzumab(HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanizedIgG1; ADC), Raxibacumab (B. anthrasis PA; Human IgG1), Obinutuzumab(CD20; Humanized IgG1 Glycoengineered), Siltuximab (IL-6; ChimericIgG1), Ramucirumab (VEGFR2; Human IgG1), Vedolizumab (a4137integrin; humanized IgG1), Nivolumab (PD1; Human IgG4),Pembrolizumab (PD1; Humanized IgG4), Blinatumomab (CD19, CD3;Murine bispecific tandem scFv), Alemtuzumab (CD52; Humanized IgG1),Evolocumab (PCSK9; Human IgG2), Idarucizumab (Dabigatran; HumanizedFab), Necitumumab (EGFR; Human IgG1), Dinutuximab (GD2; ChimericIgG1), Secukinumab (IL-17a; Human IgG1), Mepolizumab (IL-5;Humanized IgG1), Alirocumab (PCSK9; Human IgG1), Daratumumab (CD38;Human IgG1), Elotuzumab (SLAMF7; Humanized IgG1), Ixekizumab(IL-17a; Humanized IgG4), Reslizumab (IL-5; Humanized IgG4),Olaratumab (PDGFR.alpha.; Human IgG1), Bezlotoxumab (Clostridiumdifficile enterotoxin B; Human IgG1), Atezolizumab (PD-L1;Humanized IgG1), Obiltoxaximab (B. anthrasis PA; Chimeric IgG1),Brodalumab (IL-17R; Human IgG2), Dupilumab (IL-4R .alpha.; HumanIgG4), Inotuzumab ozogamicin (CD22; Humanized IgG4; ADC),Guselkumab (IL-23 p19; Human IgG1), Sarilumab (IL-6R; Human IgG1),Avelumab (PD-L1; Human IgG1), Emicizumab (Factor Ixa, X; HumanizedIgG4, bispecific), Ocrelizumab (CD20; Humanized IgG1), Benralizumab(IL-5R .alpha.; Humanized IgG1), Durvalumab (PD-L1; Human IgG1),Gemtuzumab ozogamicin (CD33; Humanized IgG4; ADC), Erenumab,erenumab-aooe (CGRP receptor; Human IgG2), Galcanezumab,galcanezumab-gnlm (CGRP; Humanized IgG4), Burosumab, burosumab-twza(FGF23; Human IgG1), Lanadelumab, lanadelumab-flyo (Plasmakallikrelin; Human IgG1), Mogamulizumab, mogamulizumab-kpkc (CCR4;Humanized IgG1), Tildrakizumab; tildrakizumab-asmn (IL-23 p19;Humanized IgG1), Fremanezumab, fremanezumab-vfrm (CGRP; HumanizedIgG2), Ravulizumab, ravulizumab-cwvz (C5; Humanized IgG2/4),Cemiplimab, cemiplimab-rwlc (PD-1; Human mAb), Ibalizumab,ibalizumab-uiyk (CD4; Humanized IgG4), Emapalumab, emapalumab-lzsg(IFNg; Human IgG1), Moxetumomab pasudotox, moxetumomabpasudotox-tdfk (CD22; Murine IgG1 dsFv immunotoxin), Caplacizumab,caplacizumab-yhdp (von Willebrand factor; Humanized Nanobody),Risankizumab, risankizumab-rzaa (IL-23 p 19; Humanized IgG1),Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; HumanizedIgG1 ADC), Romosozumab, romosozumab-aqqg (Sclerostin; HumanizedIgG2), Brolucizumab, brolucizumab-dbll (VEGF-A; Humanized scFv),Crizanlizumab; crizanlizumab-tmca (CD62 (aka P-selectin); HumanizedIgG2), Enfortumab vedotin, enfortumab vedotin-ejfv (Nectin-4; HumanIgG1 ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumabderuxtecan-nxki (HER2; Humanized IgG1 ADC), Teprotumumab,teprotumumab-trbw (IGF-1R; Human IgG1), Eptinezumab,eptinezumab-jjmr (CGRP; Humanized IgG1), Isatuximab,isatuximab-irfc (CD38; Chimeric IgG1), Sacituzumab govitecan;sacituzumab govitecan-hziy (TROP-2; Humanized IgG1 ADC),Inebilizumab (CD19; Humanized IgG1), Leronlimab (CCR5; HumanizedIgG4), Satralizumab (IL-6R; Humanized IgG2), Narsoplimab (MASP-2,Human IgG4), Tafasitamab (CD19; Humanized IgG1), REGNEB3 (Ebolavirus; mixture of 3 human IgG1), Naxitamab (GD2; Humanized IgG1),Oportuzumab monatox (EpCAM; Humanized scFv immunotoxin), Belantamabmafodotin (B-cell maturation antigen; Humanized IgG1 ADC),Margetuximab (HER2; Chimeric IgG1), Tanezumab (Nerve growth factor;Humanized IgG2), Dostarlimab (TSR-042) (PD-1; Humanized IgG4),Teplizumab (CD3; Humanized IgG1), Aducanumab (Amyloid beta; HumanIgG1), Sutimlimab (BIVV009) (C1s; Humanized IgG4), Evinacumab(Angiopoietin-like 3; Human IgG4).

Combination with Cytotoxic or Cytostatic Agents

According to some embodiments E of the first embodiments of the20.sup.th aspect, the pharmaceutical composition furthermorecomprises a cytostatic and/or cytotoxic agent selected fromradionuclides, alkylating agents, DNA cross-linking agents, DNAintercalating agents (e.g., groove binding agents such as minorgroove binders), cell cycle modulators, apoptosis regulators,kinase inhibitors, protein synthesis inhibitors, mitochondriainhibitors, nuclear export inhibitors, topoisomerase I inhibitors,topoisomerase II inhibitors, RNA/DNA antimetabolites andantimitotic agents, as described according to the secondembodiments of the 19th aspect.

In some preferred embodiments, the cytotoxic agent is anauristatin, a maytansinoid, a kinesin-spindle protein (KSP)inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor or a pyrrolobenzodiazepine derivative.

Aspect 21--MED Use/Methods of Treatment

According to a 21.sup.st aspect, there is provided the antibody orantigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,or a conjugate according to the 19.sup.th aspect or thepharmaceutical composition according to the 20.sup.th aspect foruse as a medicament. Also, there is provided the use of theantibody or antigen binding fragment according to the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or18.sup.th aspect, or a conjugate according to the 19.sup.th aspector the pharmaceutical composition according to the 20.sup.th aspectfor the manufacture of a medicament, e.g. for the treatment of atumor or a disease involving cells expressing a chemokine receptorand in particular CCR8.

Furthermore, there is provided a method of treating a disease, themethod comprising administering an effective dose of the antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,or a conjugate according to the 19.sup.th aspect or thepharmaceutical composition according to the 20.sup.th aspect to apatient in need thereof.

For therapeutic applications, the antibody or antigen bindingfragment or the conjugate or the pharmaceutical compositionaccording to the invention, can be administered to a patient orsubject, e.g. to a human or non-human subject, in apharmaceutically acceptable dosage form. For example,administration may occur intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The antibodies,fragments, conjugates and pharmaceutical compositions according tothe current invention are particularly suitable to be administeredby intra-tumoral, peri-tumoral, intra-lesional, or peri-lesionalroutes, to exert local as well as systemic therapeutic effects.

Possible administration routes include parenteral (e.g.,intramuscular, intravenous, intra-arterial, intraperitoneal, orsubcutaneous), intrapulmonary and intranasal. In addition, theantibodies, fragments, conjugates and pharmaceutical compositionsmight be administered by pulse infusion, with, e.g., decliningdoses of the antibody, fragment or conjugate. Preferably, thedosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether theadministration is brief or chronic. The amount to be administeredmay depend on a variety of factors such as the clinical symptoms,weight of the patient or subject, and whether other drugs areadministered. The skilled artisan will recognize that the route ofadministration will vary depending on the disorder or condition tobe treated.

Dosing frequency of the antibody may range from once every 3-6months to weekly, biweekly (BIW) or daily dosing. Similarly, doselevels range from a low mg fixed dose (daily, weekly, biweekly, ormonthly, depending on antibody) up to approximately 1 g doses.Dosing frequency depends on a variety of factors including theconcentration and turnover rate of the target, biodistribution ofthe target, half-life of the antibodies, fragments or conjugatesand potential pharmacodynamic effects that may enhance thebiological effects of the antibodies, fragments, conjugates andpharmaceutical compositions beyond its presence inpharmacologically relevant levels.

In some embodiments, the anti-CCR8 antibody is administered with adose of 1 mg/kg e.g. daily, weekly, q2w, q3w or q4w. In someembodiments, the anti-CCR8 antibody is administered with a dose of1 to 30 mg/kg, preferably 5 to 10 mg/kg, e.g. daily, weekly, q2w,q3w or q4w. In some embodiments, the anti-CCR8 antibody isadministered with a dose of 4 to 8 mg/kg, preferably 5 to 6 mg/kg,e.g. daily, weekly, q2w, q3w or q4w.

For the prevention or treatment of disease, the appropriate dosageof antibody will depend on the type of disease to be treated, theseverity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previoustherapy, the subject's clinical history and response to theantibody variant, and the discretion of the attending physician orhealth veterinary professional. The antibody is suitablyadministered to the subject at one time or over a series oftreatments.

As shown in example 12.4.2 it was surprisingly observed that even asingle dose might be sufficient to establish a substantialtreatment response. According to the current invention, there istherefore provided an anti-CCR8 antibody inducing ADCC and/or ADCPfor use in the treatment of a tumor, wherein only a single dose ofthe anti-CCR8 antibody is administered to a subject, such that nofurther dose of the same or a different anti-CCR8 antibody isadministered to the subject. Such a method is not only logisticallysuperior and particularly convenient for patients but also avoidsissues with patient compliance. When administered intravenously,the pharmaceutical composition comprising the antibodies, fragmentsor conjugates can be administered by infusion over a period ofabout 0.5 to about 5 hours. In some embodiments, infusion may occurover a period of about 0.5 to about 2.5 hours, over a period ofabout 0.5 to about 2.0 hours, over a period of about 0.5 to about1.5 hours, or over a period of about 1.5 hours, depending upon theantibodies, fragments, conjugates and pharmaceutical compositionsbeing administered and the amount of antibody, fragment orconjugate being administered.

Aspect 22--Second MED Use/Methods of Treatment

Treatment with an anti-CCR8 antibody according to the currentinvention showed remarkable efficacies in various syngeneic tumormodels. In some cases, administration of an effective dose of theantibody or antigen-binding fragment to a group of diseasedsubjects leads to a. a complete response in at least 15%, 20% or25% of the subjects, and/or to b. an overall response rate in atleast 40%, 50%, 60% or 70% of the subjects.

According to a 22.sup.nd aspect, there is provided the antibody orantigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,or a conjugate according to the 19.sup.th aspect or thepharmaceutical composition according to the 20.sup.th aspect foruse in the treatment of a tumor or a disease characterized bychemokine receptor positive cells, preferably CCR8 positive cells,such as CCR8 positive regulatory T cells. The treatment may occuras discussed for aspect 21.

Preferably, there is provided the antibody or antigen bindingfragment according to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th,16.sup.th, 17.sup.th and/or 18.sup.th aspect, or a conjugateaccording to the 19.sup.th aspect or the pharmaceutical compositionaccording to the 20.sup.th aspect for use in the treatment of atumor or a disease characterized by the involvement of cellsexpressing the targeted seven transmembrane receptor or chemokinereceptor.

Multiple modes of action can be envisioned for the antibodies,antigen-binding fragments or conjugates according to the currentinvention. One mode of action is the conjugation of an inventiveanti-chemokine receptor antibody to a drug in the form of anantibody drug conjugate (ADC). Another mode of action is theability of an antibody targeting a chemokine receptor to induceADCC. A third mode of action resides in the ability of the antibodytargeting chemokine receptor to induce ADCP.

The specific CCR8 antibodies or antigen binding fragments inparticular according to the 8.sup.th, 11.sup.th, 12.sup.th,13.sup.th and 14.sup.th aspect, are particularly suited for anADCC/ADCP based approach, e.g. because they are characterized by aparticularly low internalization or are non-internalizingantibodies, thereby residing on the surface of the CCR8 expressingtarget cells and causing their efficient killing, as demonstratedfor activated human Tregs in vitro (examples 10.3.3 ff, examples10.3.4 ff) or in vivo (examples 12 ff). Regulatory T cells (Tregs)promote tumor growth by suppressing the function of effector Tcells, including tumor responsive T cells in the tumormicroenvironment. In fact, Tregs are one of the key resistancemechanisms hampering the efficacy of immune checkpoint inhibitors(ICIs) in many indications.

CCR8 is a surface receptor of the GPCR family of chemokinereceptors, which is predominantly expressed on activated immunesuppressive Tregs frequently found in tumors. Unlike otherapproaches directed against these suppressive T cells, targetingCCR8 offers the opportunity to deplete intra-tumoral Tregs withoutimpacting resting Tregs or other immune cells systemically. Thus,targeting CCR8 may be superior in terms of both efficacy andsafety. This mode of action can thus be used in tumors withintra-tumoral Tregs but can also be used in other diseasescharacterized by an aberrant presence of activated, i.e. CCR8expressing Tregs.

Specific Indications

In principle, the antibodies, fragments, conjugates andpharmaceutical compositions can be used in the treatment of anycancer involving CCR8 expressing cells. For example, the cancer maycomprise tumor cells expressing CCR8, such as B cell lymphoma and Tcell lymphoma. In the alternative, the cancer may compriseintra-tumoral Tregs expressing CCR8. As shown in example 11, CCR8is upregulated in several tumor indications, such as T-cell acutelymphoblastic leukemia, breast cancer, triple-negative breastcancer, triple positive breast cancer, non-small cell lung cancer(NSCLC), small cell lung cancer (SCLC), testicular cancer, gastriccancer, head and neck squamous cell carcinoma, thymoma, esophagealadenocarcinoma, colorectal cancer, pancreatic adenocarcinoma,ovarian cancer or cervical cancer, acute myeloid leukemia, kidneycancer, bladder cancer, skin cancer, melanoma, thyroid cancer,mesothelioma, sarcoma and prostate cancer. According to somepreferred embodiments, the use as a medicament is the use in thetreatment of head and neck cancer, breast cancer, gastric cancer,lung cancer, squamous cell carcinoma, esophageal tumor, melanoma,bladder cancer, liver cancer, and/or prostate cancer.

According to some first embodiments according to the 22.sup.ndaspect the tumor is selected from Adrenal cancer (e.g.Adrenocortical carcinoma or Pheochromocytoma), Bladder cancer (e.g.Transitional cell carcinoma, Transitional cellcarcinoma-Papillary), Brain cancer (e.g. Glioma-Astrocytoma,Glioma-Astrocytoma-Glioblastoma, Glioma-Oligoastrocytoma,Glioma-Oligodendroglioma), Breast cancer (e.g. ADC, ADC-Ductal,ADC-Ductal-TNBC, ADC-Ductal-TPBC, ADC-Lobular), Colorectal cancer(e.g. ADC), Esophageal cancer (e.g. ADC), Esophageal cancer (e.g.SCC), Gastric cancer (e.g. ADC, ADC-Diffuse, ADC-Intestinal,ADC-Intestinal-Tubular), Head and Neck cancer (e.g. Laryngealcancer-SCC, SCC, Oral cancer-SCC), Kidney cancer (e.g. ccRCC,Chromophobe, Papillary, Papillary-Type I, Papillary-Type II), Livercancer (e.g. HCC), Lung cancer (e.g. NSCLC-ADC, NSCLC-ADC-Mixed,NSCLC-SCC, SCLC), Mesothelioma (e.g. Epithelioid), Ovarian cancer(e.g. ADC-Cystadenocarcinoma-Papillary serous), Pancreatic cancer(e.g. ADC-Ductal), Prostate cancer (e.g. ADC-Acinar type), Sarcoma(e.g. Leiomyosarcoma, Liposarcoma-Dedifferentiated, Malignantfibrous histiocytoma), Skin cancer (e.g. Melanoma), Testicularcancer (e.g. Germ cell tumor-Seminoma), Thymoma, Thyroid cancer(e.g. Follicular carcinoma, Papillary carcinoma-Classical variant),or Uterine cancer (e.g. Cervical-SCC, Cervical-SCC-Keratinizing,Cervical-SCC-Non-keratinizing, Endometrial-ADC-Endometrioid,Endometrial-ADC-Papillary serous,Endometrial-Carcinosarcoma-Malignant mixed mullerian tumor), B celllymphoma and T cell lymphoma (cf. Table 11.1.2).

According to some second embodiments according to the 22.sup.ndaspect the tumor is selected from T-cell acute lymphoblasticleukemia, breast cancer, triple-negative breast cancer, triplepositive breast cancer, non-small cell lung cancer (NSCLC), smallcell lung cancer (SCLC), testicular cancer, gastric cancer, headand neck squamous cell carcinoma, thymoma, esophagealadenocarcinoma, colorectal cancer, pancreatic adenocarcinoma,ovarian cancer or cervical cancer, acute myeloid leukemia, kidneycancer, bladder cancer, skin cancer, skin cancer, melanoma, thyroidcancer, mesothelioma, sarcoma and prostate cancer or any othercancer involving chemokine receptor or CCR8 expressing cells.Preferably the tumor is selected from head and neck cancer, breastcancer, gastric cancer, lung cancer, squamous cell carcinoma,esophageal tumor, melanoma, bladder cancer, liver cancer, and/orprostate cancer.

Combination Treatment

According to some third embodiments according to the 22.sup.ndaspect, which may be and are suggested to be combined with theembodiments of the first and/or second embodiments according to the22.sup.nd aspect, the use is a use in simultaneous, separate, orsequential combination with one or more further therapeuticallyactive compounds. Examples for combination treatments are providedin examples 12.6, 12.6.1, 12.6.2, 12.6.3, 12.6.4, 12.6.5, 12.6.6,12.6.7, 12.6.8 and 12.6.9, demonstrating the broad applicability ofthe anti-CCR8 antibodies according to the current invention andanti-CCR8 antibodies in general in combination treatment.

Anti-chemokine receptor antibodies, e.g. CCR8 antibodies, or ADCs,may be used adjunctive to, or with, other agents or treatmentshaving anti-cancer properties. When used adjunctively, theantibodies, fragments or conjugates and other agent(s) may beformulated together in a single, combination pharmaceuticalcomposition or formulation, as described for the 20.sup.th aspect,or may be preferably formulated and administered separately, eitheron a single coordinated dosing regimen or on different dosingregimens.

Sequential Administration: Combination Partner after Anti-CCR8Antibody Treatment

It was found according to the current invention, thatadministration of the anti-CCR8 antibody as a first agent andadministration of the one or more further therapeutically activecompounds thereafter increased the efficacy in an unforeseenway.

According to a preferred treatment scheme, treatment starts withadministration of the anti-CCR8 antibody (e.g. daily, weekly, q2w,q3w or q4w as described above), optionally followed byadministration of the one or more further therapeutically activecompounds. As described in examples 12.6 ff., starting theadministration of the second therapeutic agent or therapy after theinitial dose of anti-CCR8 antibody was associated with furtherimproved efficacy. Without being bound by theory, the time betweenthe initial doses should be chosen to allow for an intra-tumoralTreg depletion of at least 30% 40%, 50%, or 60%. The one or morefurther therapeutically active compounds or combination partner issuggested to be any of those described herein for this purpose. Forexample, the one or more further therapeutically active compoundsmay comprise a checkpoint inhibitor (e.g.anti-PD1/anti-PD-L1/anti-CTLA4 antibody), an antibody targeting aprotein which is specifically expressed by the tumor cells, or achemotherapeutic agent described herein.

In a different embodiment, treatment starts with the administrationof a chemotherapeutic agent and is followed by administration ofthe anti-CCR8 antibody (e.g. weekly or biweekly schedule).

Further Therapeutically Active Compounds for CombinationTreatment

Agents administered adjunctively with an anti-chemokine receptorantibody or ADC, e.g. with an anti-CCR8 antibody or ADC, may havecomplementary activities to the anti-chemokine receptor antibody orADC, such that the antibodies/ADCs and other agents do notadversely affect each other. Agents that may be used adjunctivelywith the anti-chemokine receptor or anti-CCR8 antibodies or ADCsaccording to the current invention can be alkylating agents,angiogenesis inhibitors, antibodies, antimetabolites, antimitotics,antiproliferatives, antivirals, aurora kinase inhibitors, apoptosispromoters (for example, Bcl-2 family inhibitors), activators ofdeath receptor pathway, Bcr-Abl kinase inhibitors, BiTE(Bi-Specific T cell Engager) antibodies, antibody drug conjugates,biologic response modifiers, cyclin-dependent kinase inhibitors,cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemiaviral oncogene homolog (ErbB2) receptor inhibitors, growth factorinhibitors, heat shock protein (HSP)-90 inhibitors, histonedeacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,inhibitors of inhibitors of apoptosis proteins (IAPs),intercalating antibiotics, kinase inhibitors, kinesin inhibitors,Jak2 inhibitors, mammalian target of rapamycin inhibitors,microRNAs, mitogen-activated extracellular signal-regulated kinaseinhibitors, multivalent binding proteins, non-steroidalanti-inflammatory drugs (NSAIDs), poly ADP (adenosinediphosphate)-ribose polymerase (PARP) inhibitors, platinumchemotherapeutics, polo-like kinase (Plk) inhibitors,phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors,purine analogs, pyrimidine analogs, receptor tyrosine kinaseinhibitors, retinoids/deltoids plant alkaloids, small inhibitoryribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitinligase inhibitors, and the like, as well as combinations of one ormore of these agents.

According to some highly preferred embodiments of the thirdembodiments of the 22.sup.nd aspect, there is provided the antibodyor antigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,or a conjugate according to the 19.sup.th aspect or thepharmaceutical composition according to the 20.sup.th aspect foruse in simultaneous, separate, or sequential combination

(i) with one or more further therapeutically active compounds,preferably selected from

a) an antibody or a small molecule targeting a checkpoint protein,such as PD1, PD-L1 or CTLA-4, b) an antibody targeting a furtherchemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CX3CR1 or CXCR1, c) an antibody targeting a protein which isspecifically expressed by the tumor cells, d) an antibody or asmall molecule targeting HER2 and/or EGFR, e) the standard of carefor any of head and neck cancer, breast cancer, gastric cancer,lung cancer, squamous cell carcinoma, esophageal tumor, melanoma,bladder cancer, liver cancer, and/or prostate cancer, and/or f) achemotherapeutic agent, preferably a taxane, paclitaxel,doxorubicin, cis-platin, carboplatin, oxaliplatin or gemcitabine,g) a targeted kinase inhibitor, such as Sorafinib, Regorafenib, orMEKi-1, and/or (ii) with radiation therapy, and or (iii) withdepletion of intra-tumoral B cells, e.g. in the treatment of atumor or a disease, preferably characterized by chemokine receptorpositive cells, such as CCR8 positive cells, such as CCR8 positiveregulatory T cells.

Suitable taxanes may be selected e.g. from paclitaxel, abraxane,cabazitaxel, or docetaxel, or derivatives thereof.

Embodiments wherein the anti-CCR8 antibody is combined withdoxorubicin, taxanes, cis-platin or carboplatin, embodimentswherein the anti-CCR8 antibody is combined with trastuzumab orpertuzumab and embodiments wherein the anti-CCR8 antibody iscombined with a PD-L1 antibody such as atezolizumab areparticularly suitable for the treatment of breast cancer, such asstage IV breast cancer. Embodiments wherein the anti-CCR8 antibodyis combined with cis-/carboplatin, paclitaxel, docetaxel orgemcitabine, embodiments wherein the anti-CCR8 antibody is combinedwith avastin, EGFR inhibitor (such as Tarceva or Iressa), ALKinhibitor, ROS inhibitor, BRAF inhibitor or NTRK inhibitor, andembodiments wherein the anti-CCR8 antibody is combined withnivolumab, pembrolizumab or atezolizumab, or a derivative of any ofthese, are particularly suitable for the treatment of NSCLC.Embodiments wherein the anti-CCR8 antibody is combined withnivolumab, pembrolizumab or ipilimumab (CTLA4), anti IL2 antibodyor a derivative of any of these, embodiments wherein the anti-CCR8antibody is combined with BRAF inhibitor or MEK inhibitor andembodiments wherein the anti-CCR8 antibody is combined withdarcabazine, paclitaxel, carboplatin, or cis-platin areparticularly suitable for the treatment of melanoma.

Combination with Checkpoint Inhibitors

Tumors were found to be particularly responsive to anti-CCR8antibody therapy, if the tumor was responsive to immune checkpointinhibition, or if the tumor showed high or medium immuneinfiltration and if substantial numbers of CD8+ T cells and/or NKcells were present or could be induced, e.g. in addition to highCCR8/FoxP3 ratios (cf. example 12.6 ff, cf. responsive syngeneicmodels).

According to some particularly preferred embodiments A of the thirdembodiments according to the 22.sup.nd aspect, the one or morefurther therapeutically active compounds comprise(s) an antibody ora small molecule targeting a checkpoint protein, such as PD1, PD-L1or CTLA-4. Suitable checkpoint targeting antibodies includeNivolumab (PD1; Human IgG4), Pembrolizumab (PD1; Humanized IgG4),Atezolizumab (PD-L1; Humanized IgG1), Avelumab (PD-L1; Human IgG1),Durvalumab (PD-L1; Human IgG1), Cemiplimab, cemiplimab-rwlc (PD-1;Human mAb), Dostarlimab (TSR-042) (PD-1; Humanized IgG4), orIpilimumab (CTLA-4; Human IgG1). In some embodiments, the antibodyor small molecule targeting a checkpoint protein targets and/orinhibits a checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1,B7-H3, B7-H4, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160,CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combinationthereof. Examples 12.6 ff. show a superior therapeutic effectobtained by combination of an antibody targeting checkpoint proteinPD-L1 and an inventive antibody according to the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or18.sup.th aspect. Examples 12.6 ff. demonstrate that combination ofan inventive antibody according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspectand an antibody targeting checkpoint protein CTLA4 showed likewiseexcellent efficacy in a tumor model.

Combination with Chemokine Receptor Antibodies

According to some preferred embodiments B of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) an antibody targetinga further chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, CX3CR1 or CXCR1. Suitable antibodies targeting a furtherchemokine receptor include the antibodies provided according to the6.sup.th aspect, e.g. a CCR5 antibody, CXCR5 antibody, CCR4antibody, or Mogamulizumab.

Combination with HER2 or EGFR Targeting Antibodies or Molecules

According to some preferred embodiments C of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a small molecule or anantibody targeting HER2 and/or EGFR. Suitable antibodies targetingHER2 are Trastuzumab (HER2; Humanized IgG1), Pertuzumab (HER2;humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1;ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki(HER2; Humanized IgG1 ADC), Sacituzumab govitecan; sacituzumabgovitecan-hziy (TROP-2; Humanized IgG1 ADC) and/or Margetuximab(HER2; Chimeric IgG1). Suitable antibodies targeting EGFR areCetuximab (EGFR; Chimeric IgG1), Panitumumab (EGFR; Human IgG2),and Necitumumab (EGFR; Human IgG1).

Combination with Therapeutic Antibodies

According to some embodiments D of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a further therapeutic antibodyselected from Muromonab-CD3 (CD3; Murine IgG2a), Efalizumab (CD11a;Humanized IgG1), Tositumomab-I131 (CD20; Murine IgG2a), Nebacumab(Endotoxin; Human IgM), Edrecolomab (EpCAM; Murine IgG2a),Catumaxomab (EPCAM/CD3; Rat/mouse bispecific mAb), Daclizumab(IL-2R; Humanized IgG1), Abciximab (GPIIb/IIIa; Chimeric IgG1 Fab),Rituximab (CD20; Chimeric IgG1), Basiliximab (IL-2R; ChimericIgG1), Palivizumab (RSV; Humanized IgG1), Infliximab (TNF; ChimericIgG1), Trastuzumab (HER2; Humanized IgG1), Adalimumab (TNF; HumanIgG1), Ibritumomab tiuxetan (CD20; Murine IgG1), Omalizumab (IgE;Humanized IgG1), Cetuximab (EGFR; Chimeric IgG1), Bevacizumab(VEGF; Humanized IgG1), Natalizumab (a4 integrin; Humanized IgG4),Panitumumab (EGFR; Human IgG2), Ranibizumab (VEGF; Humanized IgG1Fab), Eculizumab (C5; Humanized IgG2/4), Certolizumab pegol (TNF;Humanized Fab, pegylated), Ustekinumab (IL-12/23; Human IgG1),Canakinumab (IL-113; Human IgG1), Golimumab (TNF; Human IgG1),Ofatumumab (CD20; Human IgG1), Tocilizumab (IL-6R; Humanized IgG1),Denosumab (RANK-L; Human IgG2), Belimumab (BLyS; Human IgG1),Ipilimumab (CTLA-4; Human IgG1), Brentuximab vedotin (CD30;Chimeric IgG1; ADC), Pertuzumab (HER2; humanized IgG1),Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), Raxibacumab(B. anthrasis PA; Human IgG1), Obinutuzumab (CD20; Humanized IgG1Glycoengineered), Siltuximab (IL-6; Chimeric IgG1), Ramucirumab(VEGFR2; Human IgG1), Vedolizumab (a4137 integrin; humanized IgG1),Nivolumab (PD1; Human IgG4), Pembrolizumab (PD1; Humanized IgG4),Blinatumomab (CD19, CD3; Murine bispecific tandem scFv),Alemtuzumab (CD52; Humanized IgG1), Evolocumab (PCSK9; Human IgG2),Idarucizumab (Dabigatran; Humanized Fab), Necitumumab (EGFR; HumanIgG1), Dinutuximab (GD2; Chimeric IgG1), Secukinumab (IL-17a; HumanIgG1), Mepolizumab (IL-5; Humanized IgG1), Alirocumab (PCSK9; HumanIgG1), Daratumumab (CD38; Human IgG1), Elotuzumab (SLAMF7;Humanized IgG1), Ixekizumab (IL-17a; Humanized IgG4), Reslizumab(IL-5; Humanized IgG4), Olaratumab (PDGFR.alpha.; Human IgG1),Bezlotoxumab (Clostridium difficile enterotoxin B; Human IgG1),Atezolizumab (PD-L1; Humanized IgG1), Obiltoxaximab (B. anthrasisPA; Chimeric IgG1), Brodalumab (IL-17R; Human IgG2), Dupilumab(IL-4R .alpha.; Human IgG4), Inotuzumab ozogamicin (CD22; HumanizedIgG4; ADC), Guselkumab (IL-23 p19; Human IgG1), Sarilumab (IL-6R;Human IgG1), Avelumab (PD-L1; Human IgG1), Emicizumab (Factor Ixa,X; Humanized IgG4, bispecific), Ocrelizumab (CD20; Humanized IgG1),Benralizumab (IL-5R .alpha.; Humanized IgG1), Durvalumab (PD-L1;Human IgG1), Gemtuzumab ozogamicin (CD33; Humanized IgG4; ADC),Erenumab, erenumab-aooe (CGRP receptor; Human IgG2), Galcanezumab,galcanezumab-gnlm (CGRP; Humanized IgG4), Burosumab, burosumab-twza(FGF23; Human IgG1), Lanadelumab, lanadelumab-flyo (Plasmakallikrelin; Human IgG1), Mogamulizumab, mogamulizumab-kpkc (CCR4;Humanized IgG1), Tildrakizumab; tildrakizumab-asmn (IL-23 p19;Humanized IgG1), Fremanezumab, fremanezumab-vfrm (CGRP; HumanizedIgG2), Ravulizumab, ravulizumab-cwvz (C5; Humanized IgG2/4),Cemiplimab, cemiplimab-rwlc (PD-1; Human mAb), Ibalizumab,ibalizumab-uiyk (CD4; Humanized IgG4), Emapalumab, emapalumab-lzsg(IFNg; Human IgG1), Moxetumomab pasudotox, moxetumomabpasudotox-tdfk (CD22; Murine IgG1 dsFv immunotoxin), Caplacizumab,caplacizumab-yhdp (von Willebrand factor; Humanized Nanobody),Risankizumab, risankizumab-rzaa (IL-23 p19; Humanized IgG1),Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; HumanizedIgG1 ADC), Romosozumab, romosozumab-aqqg (Sclerostin; HumanizedIgG2), Brolucizumab, brolucizumab-dbll (VEGF-A; Humanized scFv),Crizanlizumab; crizanlizumab-tmca (CD62 (aka P-selectin); HumanizedIgG2), Enfortumab vedotin, enfortumab vedotin-ejfv (Nectin-4; HumanIgG1 ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumabderuxtecan-nxki (HER2; Humanized IgG1 ADC), Teprotumumab,teprotumumab-trbw (IGF-1R; Human IgG1), Eptinezumab,eptinezumab-jjmr (CGRP; Humanized IgG1), Isatuximab,isatuximab-irfc (CD38; Chimeric IgG1), Sacituzumab govitecan;sacituzumab govitecan-hziy (TROP-2; Humanized IgG1 ADC),Inebilizumab (CD19; Humanized IgG1), Leronlimab (CCR5; HumanizedIgG4), Satralizumab (IL-6R; Humanized IgG2), Narsoplimab (MASP-2,Human IgG4), Tafasitamab (CD19; Humanized IgG1), REGNEB3 (Ebolavirus; mixture of 3 human IgG1), Naxitamab (GD2; Humanized IgG1),Oportuzumab monatox (EpCAM; Humanized scFv immunotoxin), Belantamabmafodotin (B-cell maturation antigen; Humanized IgG1 ADC),Margetuximab (HER2; Chimeric IgG1), Tanezumab (Nerve growth factor;Humanized IgG2), Dostarlimab (TSR-042) (PD-1; Humanized IgG4),Teplizumab (CD3; Humanized IgG1), Aducanumab (Amyloid beta; HumanIgG1), Sutimlimab (BIVV009) (C1s; Humanized IgG4), Evinacumab(Angiopoietin-like 3; Human IgG4).

The combination treatment with any of these further therapeuticantibodies is preferred in particular for a tumor specificallyexpressing the target of the selected further therapeuticantibody.

According to some embodiments E of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a further therapeutic antibodytargeting CD16a, ILDR2, PSMA or Mesothelin.

Combination with Cytotoxic or Cytostatic Agents

Several chemotherapeutics such as doxorubicin and oxaliplatininduce immunogenic cell death (ICD). ICD however was found to berelated to the response to anti-CCR8 antibodies, such that acombination therapy can add further benefit to the application ofmonotherapy.

According to some embodiments F of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a cytostatic agent selected fromradionuclides, alkylating agents, DNA cross-linking agents, DNAintercalating agents (e.g., groove binding agents such as minorgroove binders), cell cycle modulators, apoptosis regulators,kinase inhibitors, protein synthesis inhibitors, mitochondriainhibitors, nuclear export inhibitors, topoisomerase I inhibitors,topoisomerase II inhibitors, RNA/DNA antimetabolites andantimitotic agents.

Alkylating Agent

According to some embodiments F1 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) an alkylating agent selected fromasaley (L-Leucine,N--[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-,ethylester); AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU(N,N'-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanedioldimethanesulfonate); (carboxyphthalato)platinum; CBDCA(cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II))); CCNU(N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (iproplatin;NSC 256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum(cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodisone;dianhydrogalactitol (5,6-diepoxydulcitol); fluorodopan((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil);hepsulfam; hycanthone; indolinobenzodiazepine dimer DGN462;melphalan; methyl CCNU((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea);mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea));piperazine alkylator((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazinedihydrochloride)); piperazinedione; pipobroman(N,N'-bis(3-bromopropionyl) piperazine); porfiromycin(N-methylmitomycin C); spirohydantoin mustard; teroxirone(triglycidylisocyanurate); tetraplatin; thio-tepa(N,N',N''-tri-1,2-ethanediylthio phosphoramide);triethylenemelamine; uracil nitrogen mustard (desmethyldopan);Yoshi-864 ((bis(3-mesyloxy propyl)amine hydrochloride).

DNA Alkylating-Like Agent

According to some embodiments F2 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) an DNA alkylating-like agent selectedfrom Cisplatin; Carboplatin; Nedaplatin; Oxaliplatin; Satraplatin;Triplatin tetranitrate; Procarbazine; altretamine; dacarbazine;mitozolomide; temozolomide.

Alkylating Antineoplastic Agent

According to some embodiments F3 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) an alkylating antineoplastic agentselected from Carboquone; Carmustine; Chlornaphazine;Chlorozotocin; Duocarmycin; Evofosfamide; Fotemustine;Glufosfamide; Lomustine; Mannosulfan; Nimustine; Phenanthriplatin;Pipobroman; Ranimustine; Semustine; Streptozotocin; ThioTEPA;Treosulfan; Triaziquone; Triethylenemelamine; Triplatintetranitrate.

DNA Replication and Repair Inhibitor

According to some embodiments F4 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a DNA replication and repair inhibitorselected from Altretamine; Bleomycin; Dacarbazine; Dactinomycin;Mitobronitol; Mitomycin; Pingyangmycin; Plicamycin; Procarbazine;Temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281;AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

Cell Cycle Modulator

According to some embodiments F5 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a cell cycle modulator, such asPaclitaxel; Nab-Paclitaxel; Docetaxel; Vincristine; Vinblastine;ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinaseinhibitors; Aurora B-specific kinase inhibitors and pan-Aurorakinase inhibitors; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584;flavopyridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib(CYC-202, R-roscovitine); ZK-304709; AZD4877, ARRY-520:GSK923295A.

Apoptosis Regulator

According to some embodiments F6 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) an apoptosis regulator such as AT-101((-)gossypol); G3139 or oblimersen (Bcl-2-targeting antisenseoligonucleotide); IPI-194; IPI-565;N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(-((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitroben-zenesulfonamide);N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide;GX-070 (Obatoclax.RTM.; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; venetoclax;agents that target TRAIL or death receptors (e.g., DR4 and DR5)such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs thattarget caspases, caspase-regulators, BCL-2 family members, deathdomain proteins, TNF family members, Toll family members, and/orNF-kappa-B proteins.

Angiogenesis Inhibitor

According to some embodiments F7 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) an angiogenesis inhibitor such asABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632;IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034);vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap;vandetanib; ABT-165; ZD-6474; DLL4 inhibitors.

Proteasome Inhibitor

According to some embodiments F8 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a proteasome inhibitor such asBortezomib; Carfilzomib; Epoxomicin; Ixazomib; SalinosporamideA.

Kinase Inhibitor

According to some embodiments F9 of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a kinase inhibitor such as Afatinib;Axitinib; Bosutinib; Crizotinib; Dasatinib; Erlotinib;Fostamatinib; Gefitinib; Ibrutinib; Imatinib; Lapatinib;Lenvatinib; Mubritinib; Nilotinib; Pazopanib; Pegaptanib;Sorafenib; Sunitinib; SU6656; Vandetanib; Vemurafenib; CEP-701(lesaurtinib); XL019; INCB018424 (ruxolitinib); ARRY-142886(selemetinib); ARRY-438162 (binimetinib); PD-325901; PD-98059;AP-23573; CCI-779; everolimus; RAD-001; rapamycin; temsirolimus;ATP-competitive TORC1/TORC2 inhibitors including PI-103, PP242,PP30, Torin 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127;ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765, Regorafenib,and MEKi-1.

Protein Synthesis Inhibitor

According to some embodiments F10 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a protein synthesisinhibitor such as Streptomycin; Dihydrostreptomycin; Neomycin;Framycetin; Paromomycin; Ribostamycin; Kanamycin; Amikacin;Arbekacin; Bekanamycin; Dibekacin; Tobramycin; Spectinomycin;Hygromycin B; Paromomycin; Gentamicin; Netilmicin; Sisomicin;Isepamicin; Verdamicin; Astromicin; Tetracycline; Doxycycline;Chlortetracycline; Clomocycline; Demeclocycline; Lymecycline;Meclocycline; Metacycline; Minocycline; Oxytetracycline;Penimepicycline; Rolitetracycline; Tetracycline; Glycylcyclines;Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid;Radezolid; Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferaseinhibitors; Chloramphenicol; Azidamfenicol; Thiamphenicol;Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin;Azithromycin; Clarithromycin; Dirithromycin; Erythromycin;Flurithromycin; Josamycin; Midecamycin; Miocamycin; Oleandomycin;Rokitamycin; Roxithromycin; Spiramycin; Troleandomycin; Tylosin;Ketolides; Telithromycin; Cethromycin; Solithromycin; Clindamycin;Lincomycin; Pirlimycin; Streptogramins; Pristinamycin;Quinupristin/dalfopristin; Virginiamycin.

Histone Deacetylase Inhibitor

According to some embodiments F11 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a histone deacetylaseinhibitor such as Vorinostat; Romidepsin; Chidamide; Panobinostat;Valproic acid; Belinostat; Mocetinostat; Abexinostat; Entinostat;SB939 (pracinostat); Resminostat; Givinostat; Quisinostat;thioureidobutyronitrile (Kevetrin.TM.); CUDC-10; CHR-2845(tefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat);ME-344; sulforaphane.

Topoisomerase I Inhibitor

According to some embodiments F12 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a topoisomerase Iinhibitor such as camptothecin; various camptothecin derivativesand analogs (for example, NSC 100880, NSC 603071, NSC 107124, NSC643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028,NSC 176323, NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830,and NSC 606497); morpholinisoxorubicin; SN-38.

Topoisomerase II Inhibitor

According to some embodiments F13 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a topoisomerase IIinhibitor such as doxorubicin; amonafide (benzisoquinolinedione);m-AMSA (4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide);anthrapyrazole derivative ((NSC 355644); etoposide (VP-16);pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine,9-methoxy-N, N-dimethyl-5-nitro-, monomethanesulfonate); bisantrenehydrochloride; daunorubicin; deoxydoxorubicin; mitoxantrone;menogaril; N,N-dibenzyl daunomycin; oxanthrazole; rubidazone;teniposide.

DNA Intercalating Agent

According to some embodiments F14 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a DNA intercalatingagent such as anthramycin; chicamycin A; tomaymycin; DC-81;sibiromycin; pyrrolobenzodiazepine derivative; SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1-,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepi-n-5(11aH)-one); SG2000 (SJG-136;(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3--dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)).

RNA/DNA Antimetabolite

According to some embodiments F15 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a RNA/DNAantimetabolite such as gemcitabine, L-alanosine; 5-azacytidine;5-fluorouracil; acivicin; aminopterin derivative N-[2-chloro-5[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-asparticacid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-asparticacid; aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acidmonohydrate; antifolate PT523((Na-(4-amino-4-deoxypteroyl)-N.gamma.-hemiphthaloyl-L-ornithine));Baker's soluble antifol (NSC 139105); dichlorallyl lawsone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; ftorafur((pro-drug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil);5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative(N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glut-amic acid); PALA ((N-(phosphonoacetyl)-L-aspartate); pyrazofurin;trimetrexate.

DNA Antimetabolite

According to some embodiments F16 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a DNA antimetabolitesuch as 3-HP; 2'-deoxy-5-fluorouridine; 5-HP; .alpha.-TGDR(.alpha.-2'-deoxy-6-thioguanosine); aphidicolin glycinate; ara C(cytosine arabinoside); 5-aza-2'-deoxycytidine; .beta.-TGDR(.beta.-2'-deoxy-6-thioguanosine); cyclocytidine; guanazole;hydroxyurea; inosine glycodialdehyde; macbecin II;pyrazoloimidazole; thioguanine; thiopurine.

Mitochondria Inhibitor

According to some embodiments F17 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a mitochondriainhibitor such as pancratistatin; phenpanstatin; rhodamine-123;edelfosine; d-alpha-tocopherol succinate; compound 11.beta.;aspirin; ellipticine; berberine; cerulenin; GX015-070(Obatoclax.RTM.; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-; celastrol (tripterine); metformin; Brilliant green; ME-344.

Antimitotic Agent

According to some embodiments F18 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) an antimitotic agentsuch as allocolchicine; auristatins, such as MMAE (monomethylauristatin E) and MMAF (monomethyl auristatin F); halichondrin B;cemadotin; colchicine; cholchicine derivative (N-benzoyl-deacetylbenzamide); dolastatin-10; dolastatin-15; maytansine;maytansinoids, such as DM1(N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine); rhozoxin;paclitaxel; paclitaxel derivative((2'-N-[3-(dimethylamino)propyl]glutaramate paclitaxel); docetaxel;thiocolchicine; trityl cysteine; vinblastine sulfate; vincristinesulfate.

Nuclear Export Inhibitor

According to some embodiments F19 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a nuclear exportinhibitor such as callystatin A; delactonmycin; KPT-185(propan-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2--enoate); kazusamycin A; leptolstatin; leptofuranin A; leptomycin B;ratjadone; Verdinexor((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin--2-ylprop-2-enehydrazide).

Hormonal Therapeutics

According to some embodiments F20 of the third embodimentsaccording to the 22.sup.nd aspect, the one or more furthertherapeutically active compounds comprise(s) a hormonaltherapeutics such as anastrozole; exemestane; arzoxifene;bicalutamide; cetrorelix; degarelix; deslorelin; trilostane;dexamethasone; flutamide; raloxifene; fadrozole; toremifene;fulvestrant; letrozole; formestane; glucocorticoids;doxercalciferol; sevelamer carbonate; lasofoxifene; leuprolideacetate; megesterol; mifepristone; nilutamide; tamoxifen citrate;abarelix; prednisone; finasteride; rilostane; buserelin;luteinizing hormone releasing hormone (LHRH); Histrelin; trilostaneor modrastane; fosrelin; goserelin. According to some embodiments Gof the third embodiments according to the 22.sup.nd aspect, the oneor more further therapeutically active compounds comprise(s) anantibody targeting myeloid suppressive cells.

According to some embodiments H of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) agents that prime antigen presentingcells (APC), which in turn can activate T cells.

According to some embodiments I of the third embodiments accordingto the 22.sup.nd aspect, the one or more further therapeuticallyactive compounds comprise(s) a BiTE antibody. BiTE antibodies arebispecific antibodies that direct T-cells to attack cancer cells bysimultaneously binding the two cells. The T-cell then attacks thetarget cell, i.e. the Treg. For example, a BiTE approach iscomplementary to the ADCC based approach of a naked CCR8antibody.

Standard Therapy

According to some embodiments J of the third embodiments accordingto the 22.sup.nd aspect, the combination is a combination with thestandard of care for the tumor or the disease characterized bychemokine receptor positive, e.g. CCR8 positive cells. The standardof care can be the standard of care for any of T-cell acutelymphoblastic leukemia, breast cancer, triple-negative breastcancer, triple positive breast cancer, non-small cell lung cancer(NSCLC), testicular cancer, gastric cancer, head and neck squamouscell carcinoma, thymoma, esophageal adenocarcinoma, colorectalcancer, pancreatic adenocarcinoma, ovarian cancer or cervicalcancer, acute myeloid leukemia, kidney cancer, bladder cancer,melanoma, thyroid cancer, mesothelioma, sarcoma and prostatecancer.

According to some preferred embodiments, the standard of care isthe standard of care for the treatment of head and neck cancer,breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer. In case of doubt, the standard ofcare is defined as in the "S3 Leitlinie" for the respective tumor,e.g. Leitlinie Analkarzinom, Aktinische Keratose andPlattenepithelkarzinom der Haut, Chronische Lymphatische Leukamie(CLL), Endometriumkarzinom, Follikulares Lymphom,Harnblasenkarzinom, Hautkrebs-Pravention, Hepatozellulares Karzinom(HCC), Hodentumoren, Hodgkin-Lymphom, Kolorektales Karzinom,Larynxkarzinom, Lungenkarzinom, Magenkarzinom, Mammakarzinom,Melanom, Mundhoehlenkarzinom, Nierenzellkarzinom, Ovarialkarzinom,Osophaguskarzinom, Palliativmedizin, Pankreaskarzinom,Peniskarzinom, Prostatakarzinom, Psychoonkologie, SupportiveTherapie, Zervixkarzinom, being effective on 2020 Jun. 26, cf.www.leitlinienprogramm-onkologie.de/leitlinien, incorporated hereinby reference in its entirety as available Jan. 6, 2021.

Radiation Therapy

According to some embodiments K of the third embodiments accordingto the 22.sup.nd aspect, which may be and are suggested to becombined with the embodiments of the first and/or secondembodiments according to the 22.sup.nd aspect, the use is a use insimultaneous, separate, or sequential combination with radiationtherapy.

For example, the radiation therapy may be a therapy involving aradiation scheme as known in the art, e.g. comprising external beamtherapy by x-ray with a typical total dose of 40 Gy fractionatedinto 15 daily treatments or a total dose of 70 Gy fractionated to37 daily treatments. For example, the radiation therapy may bebrachytherapy. Example 12.6.8 shows a combination treatment with ananti-CCR8 antibody and radiotherapy.

NK Cell Therapy

According to some embodiments L of the third embodiments accordingto the 22.sup.nd aspect, which may be and are suggested to becombined with the embodiments of the first and/or secondembodiments according to the 22.sup.nd aspect, the use is a use insimultaneous, separate, or sequential combination with NK celltherapy. The NK cell therapy can for example comprise the use of anengineered NK cell line as described in EP1771471 or can comprisethe use of isolated primary NK cells, potentially includingmodifications such as a chimeric receptor, cf. WO2006052534.

Targeting Cell Surface Molecules Associated with T-CellActivation

According to some embodiments M of the third embodiments of the22.sup.nd aspect, there is provided the antibody or antigen bindingfragment according to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th,10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th,16.sup.th, 17.sup.th and/or 18.sup.th aspect, or a conjugateaccording to the 19.sup.th aspect or the pharmaceutical compositionaccording to the 20.sup.th aspect for use in simultaneous,separate, or sequential combination with one or more furthertherapeutically active compounds targeting cell surface moleculesassociated with T-cell activation, selected from CD25, CTLA-4,PD-1, LAG3, TIGIT, ICOS, and TNF receptor super family members,4-1BB, OX-40, and GITR.

B Cell Depletion

According to some embodiments N of the third embodiments accordingto the 22.sup.nd aspect, which may be and are suggested to becombined with the embodiments of the first and/or secondembodiments according to the 22.sup.nd aspect, the use is a use insimultaneous, separate, or sequential combination withintra-tumoral B cell depletion. Intra-tumoral B cell depletion mayoccur by administration of an antibody or other molecule or anyfurther treatment specifically targeting intra-tumoral B cells,preferably CD19+ B cells. Example 12.3.2 shows a treatment where ananti-CCR8 antibody was administered and intra-tumoral CD19+ B cellswere depleted. While CD8+ T cell depletion abolished the beneficialeffect of the treatment with an anti-CCR8 antibody, it wassurprisingly found, that B cell depletion improved the beneficialeffect of the treatment with an anti-CCR8 antibody. Withoutlimitation, B cell depletion may occur by using an antibodytargeting CD19 or CD20 or another suitable B cell marker.

Stratification/Prediction and Monitoring Schemes

The stratification steps described according to the followingembodiments are also provided as stand-alone methods, e.g. forstratification, diagnosis, or monitoring of treatment success foran anti-CCR8 antibody treatment.

According to some fourth embodiments according to the 22.sup.ndaspect, which may be and are suggested to be combined with theembodiments of the first, second and/or third embodiments accordingto the 22.sup.nd aspect, the use comprises determining certainparameters to predict or monitor treatment success, e.g. forstratifying a group of subjects or patients to predict tumorresponse or to monitor treatment success. For example, the subjectsmay be human or non-human, such as mouse, rodent, orcynomolgus.

According to some embodiments A of the fourth embodiments accordingto the 22.sup.nd aspect, the use comprises determining a. presenceor quantity of tumor infiltrating lymphocytes, b. presence orquantity of macrophages and/or NK cells, c. presence or quantity ofCCR8 positive or FOXP3 positive regulatory T cells, d. tumormutational burden, e. cancer staging, f. presence, level oractivation of interferon-stimulated genes or proteins, g. CCR8expression, h. presence or quantity of complement factor proteins,serpins, and/or MHC components, i. presence or quantity ofcytokines, such as inflammatory or suppressive cytokines, j.activation of immune gene expression, k. immune checkpoint proteinexpression, such as PD-(L)1 or CTLA4 expression, l. presence orquantity of tumor infiltrating CD19+ B cells, and/or m. presence orquantity of tumor infiltrating CD8+ T cells, for stratifying agroup of subjects or patients to predict tumor response, or forpredicting or monitoring treatment success.

According to some embodiments A1 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningthe presence or quantity of (a) tumor infiltrating lymphocytes, (b)macrophages and/or NK cells, and/or (c) CCR8 positive or FOXP3positive regulatory T cells. Evaluation of presence or quantity ofcell types such as (a) tumor infiltrating lymphocytes, (b)macrophages and/or NK cells, and/or (c) CCR8 positive regulatory Tcells may occur using biopsy, such as skin biopsy, surgical biopsy,endoscopic biopsy or needle biopsy, such as fine-needle aspiration,core needle biopsy, vacuum-assisted biopsy, or image-guided biopsyand subsequent staining. The number or relative amount of (a) tumorinfiltrating lymphocytes, (b) macrophages and/or NK cells, and/or(c) CCR8 positive regulatory T cells may then be compared to areference, e.g. a reference sample or a reference value. Therelative presence or quantity of (a) tumor infiltratinglymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8positive regulatory T cells may be used to predict a favorableresponse to treatment with an antibody or fragment according to thecurrent invention. In the alternative, a scoring system may beused, e.g. to determine the number or relative amount of tumorinfiltrating lymphocytes, such as the scoring system described inZhang, Dachuan, et al. "Scoring system for tumor-infiltratinglymphocytes and its prognostic value for gastric cancer." Frontiersin immunology 10 (2019): 71.

According to some embodiments A2 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningtumor mutational burden for predicting and monitoring tumorresponse. Tumor mutational burden (TMB) is a biomarker thatmeasures the number of somatic mutations present in a cancerpatient's tumor and is quantified as mutations per megabase(mut/Mb). This metric can be used in order to stratify patients,e.g. to predict or monitor response to treatment with an antibodyor conjugate according to the current invention. FDA-approved testsexist and may be used e.g. with a reference value of .gtoreq.10mut/Mb. In the alternative, microsatellite instability (MSI) may beused for stratification, which is caused by failure of the DNAmismatch repair system.

According to some embodiments A3 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningcancer staging for predicting and monitoring tumor response. Cancerstaging may be performed as known in the art, e.g. using the TNMclassification system or the FIGO staging, and may be used in orderto stratify patients, e.g. to predict or monitor response totreatment with an antibody or conjugate according to the currentinvention.

According to some embodiments A4 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningpresence, level or activation of interferon orinterferon-stimulated genes or proteins for predicting andmonitoring tumor response. An interferon-stimulated gene is a genewhose expression is stimulated by interferon, in particular byIFNg. Suitable interferon-stimulated genes or proteins includewithout limitation ACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1,AQP4, ASS1, B2M, BST2, C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D,CAMK2G, CASP1, CCL1, CCL11, CCL13, CCL14, CCL15, CCL15, CCL14,CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCL26, CCL3, CCL3L1, CCL4, CCL4L1, CCLS, CCL7, CCL8,CD40, CD44, CD58, CDCl42EP2, CDCl42EP4, CIITA, CITED1, CLDN1,CX3CL1, CXCL16, CYP27B1 DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A,FCGR1B, FLNB, GAPDH, GBP1, GBP2, GBP4, GBP5, GBP6, GCH1, GSN, HCK,HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2,HLA-DQB1, HLA-DQB2, HLA-DRA HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5,HLA-E, HLA-F, HLA-G, HLA-H, ICAM1, IFI30, IFITM1, IFITM2, IFITM3,IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1 IL23R, IRF1, IRF2, IRF3, IRF4,IRF5, IRF6, IRF7, IRF8, IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU,LGALS9, MEFV, MID1, MRC1, MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1,OAS1, OAS2, OAS3, OASL, PDE12, PML, PRKCD, PTAFR, RAB12, RAB20,RAB43, RAB7B, RPL13A, RPS6KB1 RYDEN, SEC61A1 SLC11A1 SLC26A6SLC30A8 SNCA, SP100, STAR, STAT1, STX4, STX8, STXBP1, STXBP2,STXBP3, STXBP4, SYNCRIP TDGF1, TLR2, TLR3, TLR4, TRIM21, TRIM22,TRIM25, TRIM26, TRIM31, TRIM34, TRIM38, TRIMS, TRIM62, TRIM68,TRIMS, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS, WNTSA, XCL1, XCL2,ZYX.

According to some embodiments A5 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningCCR8 expression for predicting and monitoring tumor response. CCR8expression levels may be determined, e.g. based on biopsy samples,as known in the art, e.g. using IHC, PCR or ELISA based methods,for example using an antibody, fragment or conjugate as disclosedherein.

According to some embodiments A6 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningpresence, level or activation of genes or proteins selected from(a) complement factor proteins, (b) serpins, (c) MHC components or(d) Arg2 or (e) another biomarker disclosed herein (see e.g.example 12.7.1 or 12.7.2) for predicting and monitoring tumorresponse. Suitable complement factor proteins include withoutlimitation C1R, CIS, C4A, C5ar2, F12, and MASP2, or their analoguesfrom the respective species for treatment. Suitable serpins includewithout limitation Serpina1a, Serpina1b, Serpina1d, Serpina3i, ortheir analogues from other species. Suitable MHC components includewithout limitation H2-K2, H2-T24, H2-Q10, H2-B1, H2-Q1, H2-Q5, andH2-DMb2, or their analogues from other species.

As discussed in example 12.1.2, the inventors found based on genomewide RNA-seq data of early tumors from syngeneic mouse models, thatincreased levels of the following genes strongly correlated withtumor response: Eif3j2, Eno1b, Ifi441, Hist1h2a1, Ifi202b, Hmga1b,Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41,Clrb, Cyld, Ccnblip1, Masp2, Acaa1b, C4a, Snord93, Abhd1,Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2,Igsf21, Olfr373, Cls2, Crym, Arv1, Hddc3, Plppr4, Ppplr11, Rps3a2,Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2,Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d,Cacna1a, Kcnj14, Ttc13, Farsa, Olfr1217, Jam1, H2-B1, Tnpo2, Rims3,Dock9, Car5b, Atpla4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2,Nsd3, Wt1, Nap112, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i,E112, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agtpbp1, Msh5,Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2,H2-DMb2. Interestingly, this set is highly enriched for earlycomplement factors, complement regulating factors such as Serpins,and MHC components. In particular, the presence of high levels ofComplement C1/C4 might contribute to Treg lysis. Wheredepletion/consumption of complement factors reduces the efficacy ofTreg depletion, supplementing the complement system, e.g. in acombination treatment may be an option.

According to some preferred embodiments of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningthe presence, level or activation of genes or proteins selectedfrom Eif3j2, Eno1b, Ifi441, Hist1h2al, Ifi202b, Hmga1b, Amd2,Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, Clrb,Cyld, Ccnblip1, Masp2, Acaa1b, C4a, Snord93, Abhd1, Serpina3h,H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373,Cls2, Crym, Arv1, Hddc3, Plppr4, Ppplr11, Rps3a2, Zfp459, Rnd1,Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10,Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacna1a,Kcnj14, Ttc13, Farsa, Olfr1217, Jam1, H2-B1, Tnpo2, Rims3, Dock9,Car5b, Atpla4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2, Nsd3,Wt1, Nap112, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, E112,Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agtpbp1, Msh5, Cox18,Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, H2-DMb2 orany combination thereof for predicting and monitoring tumorresponse. As immediately understood by the skilled person, for ahuman subject, the stratification, prediction or monitoring methodcomprises determining the presence, level or activation of thehuman counterparts of the provided genes or proteins.

According to some embodiments A7 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises determiningpresence or quantity of inflammatory or suppressive cytokines forpredicting and monitoring tumor response. Evaluation of presence orquantity of inflammatory or suppressive cytokines may occur asknown in the art.

According to some embodiments A8 of the fourth embodimentsaccording to the 22.sup.nd aspect, the use comprises predicting ormonitoring tumor response based on the activation of immune geneexpression. Measuring the activation of immune gene expression mayoccur using a method known in the art, e.g. using gene setenrichment analysis or using a marker-based assay known in the art.Furthermore, stratifying, predicting or monitoring may occur basedon a gene signature characteristic for activated Tregs or immunecells.

According to some highly preferred embodiments A9 of the fourthembodiments according to the 22'' aspect, the use comprisesstratifying a group of subjects or patients to predict tumorresponse based on immune checkpoint protein expression, such asPD-L1 expression, PD-1 expression or CTLA4 expression, or the usecomprises determining immune checkpoint protein expression levels,such as PD-L1 expression, PD-1 expression or CTLA4 expression forpredicting and monitoring tumor response. Programmed death-ligand 1(PD-L1) is an immune-related biomarker that can be expressed on thesurface of many tissue types, including tumor cells. PD-L1 proteinexpression can be determined by using Tumor Proportion Score (TPS)or Combined Positive Score (CPS). According to the currentinvention it was surprisingly found that the response to (mono)therapy with anti-CCR8 antibody was improved for subjects having anICI-responsive tumor and in particular a tumor with (high) PD-L1expression (cf. example 12.7). Following up on this observation,the inventors found that infiltration with FoxP3+ Treg cells waslikewise associated with high PD-L1 expression. The inventorstherefore suggest PD-L1 as a surrogate marker for stratification toovercome the problem that CCR8 is difficult to analyze andtherefore cannot be easily used to select subject populations thatcould most likely benefit from anti-CCR8 antibody treatment. Inparticularly preferred embodiments, PD-L1 expression is determinedbased on CPS or TPS. A subject or patient is determined as eligiblefor treatment with an anti-CCR8 antibody, if PD-L1 expression ispresent or high, e.g. as indicated by a CPS.gtoreq.1, preferably bya CPS.gtoreq.5, most preferably by a CPS.gtoreq.10, or as indicatedby a TPS.gtoreq.1%, preferably by a TPS.gtoreq.10%, most preferablyby a TPS.gtoreq.50%.

Aspect 23--Further and Diagnostic Uses

The anti-chemokine receptor or anti-CCR8 antibodies, fragments andconjugates as described herein may be used for a variety ofpurposes, such as to assist purification or immobilization ofchemokine receptor or CCR8 expressing cells, such as activated orintra-tumoral Tregs, for in vitro, in vivo and ex vivo applicationsor diagnostics. As a specific example, the antibodies can be usedin immunoassays for qualitatively and/or quantitatively measuringlevels of chemokine receptors or chemokine receptor expressingcells in biological samples, see, e.g., Harlow et al., Antibodies:A Laboratory Manual, Second Edition (Cold Spring Harbor LaboratoryPress, 1988).

For example, anti-chemokine receptor antibodies, anti-CCR8antibodies or antigen-binding fragments thereof can be used fordetecting the presence of chemokine receptor- or CCR8-expressingtumors. The presence or level of chemokine receptor- orCCR8-expressing cells or shed chemokine receptor or CCR8 withinvarious biological samples, including serum, and tissue biopsyspecimens, may be analyzed. In addition, anti-chemokine receptor oranti-CCR8 antibodies may be used in various imaging methodologiessuch as immunoscintigraphy with a 99Tc (or a different isotope)conjugated antibody. For example, an imaging protocol similar tothe one described using a 111In conjugated anti-PSMA antibody maybe used to detect cancer (Sodee, D. Bruce, et al. "PreliminaryImaging Results Using In-111 Labeled CYT-356 (Prostascintst) in theDetection of Recurrent Prostate Cancer." Clinical nuclear medicine21.10 (1996): 759-767.). Another method of detection that can beused is positron emitting tomography, e.g. by conjugating theantibodies of the invention with a suitable isotope (see Herzog,Hans, et al. "Measurement of pharmacokinetics of yttrium-86radiopharmaceuticals with PET and radiation dose calculation ofanalogous yttrium-90 radiotherapeutics." Journal of NuclearMedicine 34.12 (1993): 2222-2226.).

According to a 23th aspect, there is provided the antibody orantigen binding fragment according to the 6.sup.th, 7.sup.th,8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th,14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect,or a conjugate according to the 19.sup.th aspect or thepharmaceutical composition according to the 20.sup.th aspect foruse in diagnostic applications or diagnosis.

In some first embodiments of the 23th aspect, there is provided anantibody or antigen binding fragment according to the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or18.sup.th aspect, or a conjugate according to the 19.sup.th aspector the pharmaceutical composition according to the 20.sup.th aspectfor use in a method of diagnosis in vivo. The use may be a use forthe diagnosis of a tumor or a disease characterized by chemokinereceptor positive cells, e.g. CCR8 positive cells, such as CCR8positive regulatory T cells. For example, the tumor or the diseasecharacterized by chemokine receptor positive cells, is selectedfrom T-cell acute lymphoblastic leukemia, breast cancer,triple-negative breast cancer, triple positive breast cancer,non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC)testicular cancer, gastric cancer, head and neck squamous cellcarcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer,pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acutemyeloid leukemia, kidney cancer, bladder cancer, skin cancer,melanoma, thyroid cancer, mesothelioma, sarcoma and prostatecancer. According to some preferred embodiments, the use in amethod of diagnosis in vivo is the use in the diagnosis of head andneck cancer, breast cancer, gastric cancer, lung cancer, squamouscell carcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, or any other tumor describedherein.

According to some preferred embodiments, the antibody or antigenbinding fragment according to the 6.sup.th, 7.sup.th, 8.sup.th,9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th,15.sup.th, 16.sup.th, 17.sup.th and/or 18.sup.th aspect, or aconjugate according to the 19.sup.th aspect or the pharmaceuticalcomposition according to the 20.sup.th aspect is used for thestratification of subjects or patients for treatment, e.g. for thetreatment of a disease described herein.

Aspect 24--DNA/RNA for CCR8 Antibody

According to a 24.sup.th aspect, there is provided a polynucleotideencoding an antibody or antigen-binding fragment according to the6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th,12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th16.sup.th, 17.sup.thand/or 18.sup.th aspect. Preferably the polynucleotide according tothis aspect is a polynucleotide as provided according to thesequence listing.

Aspect 25--Vector for Antibody

According to a 25th aspect, there is provided a vector comprising apolynucleotide according to the 24.sup.th aspect. Various suitablevector systems are known in the art or described herein.

Aspect 26--Production Cell for Antibody

According to a 26.sup.th aspect, there is provided an isolated cellarranged for production of an antibody or antigen-binding fragmentaccording to the 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th,11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th,17.sup.th and/or 18.sup.th aspect. Preferably the isolated cellaccording to the current aspect is an eukaryotic cell, such as aCHO or HEK cell comprising a vector according to the 25.sup.thaspect. In another preferred embodiment, the cell is a rat myelomaYB2/0 cell.

Aspect 27--Production Method for Antibody

According to a 27.sup.th aspect, there is provided a method ofproducing an antibody or antigen-binding fragment according to the6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th,12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.thand/or 18.sup.th aspect, or a conjugate according to the 19.sup.thaspect, comprising culturing a cell according to the 26.sup.thaspect and optionally purification of the antibody orantigen-binding fragment. In some highly preferred embodiments, themethod comprises afucosylation of the antibody as describedelsewhere herein. In some embodiments, the method comprisespurification of the antibody.

For example, the antibody according to the present invention may bepurified by the purification process described in the following.The purification process described is suitable for a wide range ofmonoclonal IgG antibodies and is optimized for bioreactor titers upto 8 g/L with an expected harvest volume of e.g. 2000 L from asingle-use or stainless-steel bioreactor. With appropriatemodifications, antibody cultures with other titer ranges can beprocessed. Cell cultures are harvested and clarified via depthfiltration and/or charged filtration. Clarified harvest is storedin single-use bags or in a stainless-steel storage tank withoptional cooling.

General Process Conditions

Closed processing and single-use systems are preferred;stainless-steel skids or systems are also possible with appropriatecleaning and change-over procedures. Single-use bags are typicallyof low-density or ultra-low density polyethylene as contact layer.Among others, Sodium Acetate/Acetic Acid or Tris/Tris-HCl buffersystems may be used, usually in a total concentration of 50 mM andincluding 50 mM NaCl, unless otherwise noted. Buffers and processintermediates of the respective unit operations (e.g. Load, Eluateor Flowthrough) are usually 0.2 .mu.m filtered inline or with afiltration assembly, using e.g. PES membrane and/or fleece filters.These solutions are usually stored in single-use bags orstainless-steel vessels, at ambient (e.g. 18-26.degree. C.) or2-8.degree. C. conditions.

Capture

Antibody capture is performed using Protein A-based chromatography,utilizing e.g. Cytiva MabSelect SuRe, MabSelect SuRe LX, Mab SelectPrismA or Purolite A50 resins. Loading is conducted followingclarification, with or without pre-concentration byultrafiltration. The loading density is typically 45-75 g/L, usinga single or a variable flow rate to apply the clarified harvestonto the column (120-400 cm/h). The chromatography can be performedin batch mode with one or more columns, or in continuous (MCC(multi-column chromatography)/SMB (simulated moving bed)) mode withup to 8 columns. Pre- or self-packed columns are used with atypical bed height of 20 cm for batch mode. For continuous mode, acontact time of the clarified harvest to the resin of 1-4 min istargeted. The capture is performed in one or multiple cyclesdepending on the antibody titer. Prior to loading, the column isequilibrated using a Tris buffer (e.g. pH 7); washing is performedwith at least two washing steps; high NaCl concentration in thefirst wash buffer, then low NaCl concentration in the second buffer(e.g. 1st wash: 1 M NaCl, pH 5.5; 2nd wash: 50 mM NaCl, pH 6.0) inacetate buffer. Elution may for instance be achieved by low pH(e.g. 3.5) acetate buffer containing 0-50 mM NaCl in typically.ltoreq.5 column volumes (CV) with UV- or volume-controlled eluatecollection. The eluates may be pooled and well mixed at this stepor after virus inactivation. The affinity column is regenerated by.gtoreq.3 CV acetic acid (e.g. 0.1 M, incl. 500 mM NaCl),sanitization (clean-in-place, CIP) occurs with 0.5-1.0 M NaOH for30 min, re-equilibration with .gtoreq.4 CV. The column is stored ine.g. 20% EtOH or 2% BnOH.

Alternatively, affinity capture from clarified harvest can beperformed with affinity ligands on a cellulose architecture (e.g.Fibro PrismA) using the same loading schemes and buffer system asoutlined above. Regeneration or CIP block may be skipped foroptimal processing time. A contact time of 5-20 seconds can beapplied to Fibro units of varying sizes (e.g. 0.4 mL to 2.4 L).Typically, a loading capacity of 25-35 g/L unit volume can berealized. A single unit can be used to process a single batch ormultiple batches, depending on the Fibro unit and bioreactor sizesas well as titer of antibody culture. Affinity loading materialscan be further clarified by charged filters at >300 L/m2throughput prior to loading to maximize lifetime use and tominimize fouling.

Low-pH Virus Inactivation

The capture eluate is adjusted with HOAc or HCl to pH 3.7-3.9 andheld for 120-240 min in the same or a separate bag at.gtoreq.18.degree. C. to inactivate potentially present viruses.Alternatively, viral inactivation is carried out at pH 3.4-3.6 for>30 minutes. The process intermediate is then adjusted tostorage or to further processing conditions, e.g. to pH 4-7 using1-2 M Tris/-HCl titrant stock.

Polishing 1

The antibody is further polished by anion exchange chromatography(AEX), typically in the form of membrane capsule or cassettes (e.g.Sartorius Sartobind STIC PA 4 mm or 3M MA ST Polisher) inflow-through mode, with flow rates .ltoreq.350 MV/h, for removal ofprocess-related and/or product-related impurities and particles.The entire amount from one batch may be processed in one or inmultiple cycles, depending on the antibody amount. Prior toloading, the intermediate is adjusted to a target conductivity ofe.g. 8-20 mS/cm, and a target pH of e.g. 7.0 for antibodies withbasic isoelectric points. For antibodies with an isoelectric pointlower than pH 7.0, the conductivity could be 5-20 mS/cm, and the pHcan be between its isoelectric point and pH 5.0 The membrane isequilibrated with Tris buffer, pH 7.0 or Acetate buffer pH 5-6, andloaded with densities between typically 0.5-10 g antibody/mLmembrane volume (MV). A chase with equilibration buffer may beperformed and combined with the collected flow-through, thecollection criteria of which is controlled by e.g. UV or volume.The membrane adsorber may be used once or multiple times, for whichit is regenerated (1 M NaCl for 25 MV) and, re-equilibrated (Trisbuffer pH 7.0).

Polishing 2

Final polishing may for instance be achieved by cation exchangechromatography (CEX). In principle, the order of AEX and CEX unitoperations may be reversed. The CEX unit operation is usuallyperformed in bind & elute mode with flow rates between 100-200cm/h. Pre- or self-packed columns with resins e.g. Cytiva Capto SImpAct or Capto SP ImpRes may be used at 20 cm bed height. Thepolishing step is performed in one or multiple cycles depending onthe antibody amount. If needed, the load may be adjusted to targetconductivities between 5-9 mS/cm and to pH 4.5-7.5 with HOAc, Tristitrants or WFI. The load is applied at typical densities between40-105 g/L onto a CEX column equilibrated with .gtoreq.5 CV acetatebuffer (pH and conductivity matched to CEX load) and washedafterwards using .gtoreq.5 CV of the same buffer. The antibody iseluted using an acetate buffer with suitable NaCl concentration(>50 mM; <500 mM), and the eluate is collected by UV-controlin typically .ltoreq.10 CV collected eluate volume. The column isregenerated using 0.5-2 M NaCl in acetate buffer, re-equilibrated(.gtoreq.5 CV with CEX equilibration buffer), sanitized (0.5-1.0 MNaOH for .gtoreq.30 min), re-equilibrated (.gtoreq.5 CV with CEXequilibration buffer), and finally stored in e.g. 20% EtOH or 2%BnOH.

Additional Polishing

When additional polishing is needed to remove residual product- orprocess-related impurities, a mixed mode chromatography (MMC) maybe used in place of, or together with, the CEX step. Example ofmixed mode chromatographic resins are Capto adhere and Capto MMCImpRes. This unit operation is typically operated in a flow-throughmode but bind-and-elute mode may also be employed. The column canbe self- or pre-packed with a bed height of 5-20 cm at variousdiameters and operated at flow rates between 100-500 cm/h in aflowthrough mode or 100-220 cm/hr in a bind-and-elute mode.

The load is typically adjusted, if needed, to target conductivitiesbetween 3-12 mS/cm and to pH 4.2-7.5 with HOAc, Tris titrants orWFI. The load is applied at typical densities between 75-300 g/Lonto a MMC column equilibrated with .gtoreq.5 CV acetate buffer (pHand conductivity matched to MMC load). A chase with equilibrationbuffer may be performed and combined with the collectedflow-through, the collection criteria of which is controlled bye.g. UV or volume. The MMC column might be used once or multipletimes depending on the amount of antibody to be polished. Thecolumn is regenerated using 0.5-2 M NaCl in acetate buffer,re-equilibrated (.gtoreq.5 CV with equilibration buffer), sanitized(0.5-1.0 M NaOH for .gtoreq.30 min), and finally stored in e.g. 20%EtOH or 2% BnOH.

Nanofiltration

Potentially remaining adventitious viruses are removed byPDVF-based nanofiltration, e.g. using Planova BioEX with or withoutprefilter. The load is applied at constant pressure (e.g. 2.0-3.4bar) with a loading density of 1500-5000 g antibody/m.sup.2 to apre-equilibrated nanofilter (.gtoreq.5 L/m.sup.2 acetate buffer,recipe e.g. matched to MA AEX or CEX elution conditions). Thefilter may be chased using the same buffer and the integrity of thefilter is typically tested post-use. Upon failure of the integritytest, this step may be repeated. Alternatively, adventitiousviruses can be removed by cellulose-base nanofiltration with orwithout a prefilter. The load is applied at a constant pressure(e.g. 0.8-1.2 bar) with a loading density of 500-2000 gantibody/m.sup.2 to a pre-equilibrated nanofilter (.gtoreq.5L/m.sup.2 acetate buffer, recipe e.g. matched to MA AEX or CEXelution conditions).

Concentration and Buffer Exchange

The intermediate is concentrated by ultrafiltration to 15-110 g/L,diafiltered with .gtoreq.6 diafilter volumes against adiafiltration buffer (DFB) (e.g. 10 mM histidine, 10 mM methionine,30 mM arginine, pH 5.3), and, if needed, further concentrated up to200 g/L. This is typically performed using a tangential-flowfiltration device and a suitable membrane with a cutoff of 30-50kDa (e.g. Millipore Pellicon, Sartorius Hydrosart, Pall Omega) witha load of typically 300-1000 g antibody/m.sup.2. The process istypically controlled by feed or retentate flow (e.g. 2-7L/min/m.sup.2), alternatively by feed pressure (2-4 bar), and byTMP (0.7-2 bar). The membrane may be chased by DFB and the pooledretentate diluted with DFB to the target concentration. The TFFsystem and membranes are sanitized with 0.5 M NaOH and equilibratedwith acetate buffer or DFB. The membrane integrity or permeabilityis tested pre-use.

Stabilization

In an optional stabilization step, an excipient concentrate isadded to produce bulk drug substance (BDS). A bioburden reductionfiltration (e.g. 0.2 .mu.m) is performed using an e.g.pre-equilibrated filter (e.g. excipient concentrate diluted inDFB), which is tested post-use for integrity. Upon failure of theintegrity test, the filtration step may be repeated.

Fill & Freeze

The BDS is filled in suitable bags with an LDPE contact layer (e.g.5 or 10 L) with individual i.d. sampling compartments that arepreferably aseptically connected and disconnected. The bags areindividually protective packaged by optional vacu-sealing (LDPEoverwrap bags) and placed in protective shells (e.g. RoSS Shells).After an optional intermediate 2-8.degree. C. storage, the shelledbags are frozen (using a plate- or a passive freezer) andsubsequently stored at .ltoreq.-30 or .ltoreq.-60.degree. C., e.g.+/-5.degree. C., alternatively at .ltoreq.-25 or.ltoreq.-60.degree. C., e.g. +/-5.degree. C.

Aspect 28--Kit of Parts with Antibody Defined by Antigen

According to a 28th aspect, there is provided a kit comprising theantibody or antigen-binding fragment according to the 6.sup.th,7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th,13.sup.th, 14.sup.th, 15.sup.th, 16.sup.th, 17.sup.th and/or18.sup.th aspect, or a conjugate according to the 19.sup.th aspect,or the pharmaceutical composition according to the 20.sup.thaspect, with instructions for use.

The antibodies, fragments, conjugates or pharmaceuticalcompositions of the present invention can be provided in a kit,i.e., a packaged combination of reagents in predetermined amountsin one or more containers with instructions. For example, where theantibody, fragment or conjugate is a therapeutic antibody, fragmentor conjugate, the instructions for use may comprise the packageinsert. For example, the package insert may comprise informationdescribing an advantageous administration mode or combinationtreatment as described herein.

For example, where the antibody is labeled with an enzyme, the kitmay include substrates and cofactors required by the enzyme (e.g.,a substrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be variedwidely to provide for concentrations in solution of the reagentswhich substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will providea reagent solution having the appropriate concentration.

Aspect 29--Combination Treatment Comprising Anti-Ccr8 Antibody

According to one aspect there is provided an anti-CCR8 antibody anda further therapeutically active compound or therapy for use in thetreatment of a tumor, wherein the further therapeutically activecompound is a) a chemotherapeutic agent, preferably a taxane,doxorubicin, cis-platin, carboplatin, oxaliplatin, or gemcitabine,b) an antibody targeting a further chemokine receptor, such asCCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1,CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c) an antibodytargeting a protein which is specifically expressed by the tumorcells, d) an antibody or a small molecule targeting HER2 and/orEGFR, e) the standard of care for any of head and neck cancer,breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, f) a targeted kinase inhibitor,such as Sorafinib, Regorafenib, or MEKi-1, and/or g) radiationtherapy.

In other words, there is provided an anti-CCR8 antibody, preferablyan anti-CCR8 antibody inducing ADCC and/or ADCP as describedelsewhere herein, for use in the treatment of a tumor, wherein theuse comprises administration of a further therapeutically activecompound or therapy which does not target a checkpoint protein andis selected from h) a chemotherapeutic agent, preferably a taxane,paclitaxel, doxorubicin, cis-platin, carboplatin, oxaliplatin, orgemcitabine, i) an antibody targeting a further chemokine receptor,such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10,CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, j) anantibody targeting a protein which is specifically expressed by thetumor cells, k) an antibody or a small molecule targeting HER2and/or EGFR, l) the standard of care for any of head and neckcancer, breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, m) a targeted kinase inhibitor,such as Sorafinib, Regorafenib, or MEKi-1, n) radiation therapy,and/or o) (a compound or antibody for) depletion of intra-tumoral Bcells, preferably CD19+ B cells.

The anti-CCR8 antibody may be an inventive anti-CCR8 antibody asdescribed herein or any further therapeutically suitable anti-CCR8antibody known in the art. The tumor may be any tumor describedherein, e.g. for the 22.sup.nd aspect. Preferably, the tumor ordisease is characterized by chemokine receptor positive cells, suchas CCR8 positive cells, such as CCR8 positive regulatory T cells orCCR8 positive tumor cells. Preferably the tumor is an ICIresponsive tumor. Based on the examples described herein (e.g.examples 12.6 ff., see responding mouse models), it is plausible,that in particular those tumors which are or were initiallyresponsive to immune checkpoint inhibition will profit fromanti-CCR8 antibody treatment both, alone and in combination. Thefurther therapeutically active compound may be a therapeuticallyactive compound as described elsewhere herein. Suitable taxanes maybe selected e.g. from paclitaxel, abraxane, cabazitaxel, ordocetaxel, or derivatives thereof. According to a preferredembodiment, the use comprises furthermore administration of acheckpoint inhibitor such as an anti-PD-1 antibody, an anti-PD-L1antibody or a CTLA4 antibody, e.g. as described elsewhereherein.

Aspect 30--Triple Combination Treatment Comprising Anti-CCR8Antibody

According to one aspect there is provided an anti-CCR8 antibody, acheckpoint inhibitor such as an anti-PD-1 antibody, an anti-PD-L1antibody or a CTLA4 antibody, and a further therapeutically activecompound or therapy for use in the treatment of a tumor, whereinthe further therapeutically active compound or therapy ispreferably a) an antibody targeting a further chemokine receptor,such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1,b) an antibody targeting a protein which is specifically expressedby the tumor cells, c) an antibody or a small molecule targetingHER2 and/or EGFR, d) the standard of care for any of head and neckcancer, breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, e) a chemotherapeutic agent,preferably a taxane, paclitaxel, doxorubicin, cis-platin,carboplatin, oxaliplatin, or gemcitabine, f) a targeted kinaseinhibitor, such as Sorafinib, Regorafenib, or MEKi-1, and/or g)radiation therapy, h) (a compound or antibody for) depletion ofintra-tumoral B cells, preferably CD19+ B cells.

In other words, there is provided an anti-CCR8 antibody inducingADCC and/or ADCP for use in the treatment of a tumor, wherein theuse comprises administration of a further therapeutically activecompound or therapy which does not target a checkpoint protein andis selected from a) a chemotherapeutic agent, preferably a taxane,paclitaxel, doxorubicin, cis-platin, carboplatin, oxaliplatin, orgemcitabine, b) an antibody targeting a further chemokine receptor,such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10,CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c) anantibody targeting a protein which is specifically expressed by thetumor cells, d) an antibody or a small molecule targeting HER2and/or EGFR, e) a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1, f) radiation therapy, and/or depletion ofintra-tumoral B cells, preferably CD19+ B cells, and the usecomprises furthermore administration of a checkpoint inhibitor suchas an anti-PD-1 antibody, an anti-PD-L1 antibody or a CTLA4antibody, e.g. as described elsewhere herein.

Aspect 31--Sequential Combination Treatment Comprising Anti-CCR8Antibody

According to one aspect there is provided an anti-CCR8 antibody anda further therapeutically active compound or therapy for use in thetreatment of a tumor, wherein a dose of the further therapeuticallyactive compound or therapy is administered after the first dose ofthe anti-CCR8 antibody, e.g. a) after the anti-CCR8 antibody haseffected an increase of the intratumoral CD8 cell to T reg cellratio at least by a factor of 2, 3, 4 or 5 or b) after theanti-CCR8 antibody has depleted at least 40, 45, 50, 55, 60, 65 or70% of the intratumoral Treg cells.

In other words, there is provided an anti-CCR8 antibody, preferablyan anti-CCR8 antibody inducing ADCC and/or ADCP as describedelsewhere herein, for use in the treatment of a tumor, wherein theuse comprises administration of a further therapeutically activecompound or therapy, wherein a dose of the further therapeuticallyactive compound or therapy is administered after the first dose ofthe anti-CCR8 antibody, preferably a) after the anti-CCR8 antibodyhas induced an increase of the intra-tumoral CD8 cell to T reg cellratio at least by a factor of 2, 3, 4 or 5 or b) after theanti-CCR8 antibody has depleted at least 40, 45, 50, 55, 60, 65 or70% of the intra-tumoral Treg cells.

According to a preferred treatment scheme, treatment starts withadministration of the first dose of anti-CCR8 antibody (e.g. daily,weekly, q2w, q3w or q4w as described above), followed byadministration of the further therapeutically active compound ortherapy. Without being bound by theory, the time between theinitial dose of anti-CCR8 antibody and the further therapeuticallyactive compound should be chosen to allow for an intra-tumoral Tregdepletion of at least 40, 50, or 60%. The anti-CCR8 antibody may bean inventive anti-CCR8 antibody as described herein but may also beany further therapeutically suitable anti-CCR8 antibody. The tumormay be any tumor described herein, e.g. for the 22.sup.nd aspect.Preferably, the tumor or disease is characterized by chemokinereceptor positive cells, such as CCR8 positive cells, such as CCR8positive regulatory T cells or CCR8 positive tumor cells.Preferably the tumor is or was initially an ICI responsive tumor.Since acquired resistance to PD(L)-1 antibodies might strongly beinfluenced by intra-tumoral activated regulatory T cells(characterized by CCR8 expression) sequential treatment with ananti-CCR8-antibody combined with a checkpoint inhibitor isbeneficial to overcome such an acquired resistance. Administeringthe further therapeutically active compound or therapy after thefirst dose of the anti-CCR8 antibody was found to substantiallyimprove the efficacy of the treatment, even for extremelychallenging tumor models. The time between the first dose of theanti-CCR8 antibody and the dose of the further therapeuticallyactive compound shall be sufficient to induce a first anti-tumorimmune response. According to the data provided herein (cf.examples 12.6 ff), this is indicated by an increase of theintra-tumoral CD8 cell to T reg cell ratio at least by a factor of2, 3, 4 or 5 or by (temporary) depletion of at least 40, 45, 50,55, 60, 65 or 70% of the intra-tumoral Treg cells.

The further therapeutically active compound or therapy may be anycompound or therapy disclosed elsewhere herein. The therapeuticallyactive compound may or may not be a checkpoint inhibitor, e.g. ananti-PD-L1 antibody, an anti-PD1 antibody or ananti-CTLA4-antibody. Although the second therapeutic agent may be anon-targeted therapeutic, it was found that targeted therapeuticsare highly preferred for this purpose. Targeted therapeutics asdefined herein are therapeutically active compounds thatspecifically recognize tumor cells rather than all fast dividingcells or immune cell populations. Although agents targeting alldividing cells or affecting immune cell populations may be used incombination and could improve efficacy (cf. example 12.6.8,12.6.9), they may interfere with immune cell populations that wererequired for a sustainable immunological tumor response. Thisproblem was solved by use of an antibody targeting a protein whichis specifically expressed by the tumor cells. Preferred targetedtherapeutics are antibodies binding a protein which is specificallyexpressed by the tumor cells.

Preferably, the further therapeutically active compound or therapyis selected from a) an antibody or a small molecule targeting acheckpoint protein, such as PD-(L)1 or CTLA-4, b) achemotherapeutic agent, preferably a taxane, paclitaxel,doxorubicin, cis-platin, carboplatin, oxaliplatin, or gemcitabine,c) an antibody targeting a further chemokine receptor, such asCCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1,CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, d) an antibodytargeting a protein which is specifically expressed by the tumorcells, e) an antibody or a small molecule targeting HER2 and/orEGFR, f) a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1, g) the standard of care for any of head andneck cancer, breast cancer, gastric cancer, lung cancer, squamouscell carcinoma, esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, h) radiation therapy, and/or i) (acompound or antibody for) depletion of intra-tumoral B cells,preferably CD19+ B cells.

Suitable taxanes may be selected e.g. from paclitaxel, abraxane,cabazitaxel, or docetaxel, or derivatives thereof.

Preferably, the dose of the further therapeutically active compoundor therapy is the first dose of the mentioned furthertherapeutically active compound or therapy.

Aspect 32--Stratification Methods/Diagnostic Methods

As discussed elsewhere herein, the stratification steps describedfor the fourth embodiments according to the 22nd aspect may be usedeither as part of a method of treatment according to the 22.sup.ndaspect, or are provided stand-alone as a) a method to selectsubjects that will most likely profit from treatment with ananti-CCR8 antibody, b) a diagnostic method for diagnosing a tumoras sensitive for treatment with an anti-CCR8 antibody, or c) amethod to monitor the treatment success for treatment with ananti-CCR8 antibody.

For the current aspect the antibody may be an antibody according tothe current invention but may also be any therapeutically suitableanti-CCR8 antibody.

Biomarker for Anti-CCR8 Antibody Treatment

So far, no biomarkers are available to predict or monitor thetreatment success for anti-CCR8 antibodies. While the demonstratedmode of action suggests that CCR8 itself may serve as a possiblebiomarker, the inventors found that CCR8 levels are somewhatdifficult to assess in a clinical setup or within clinical studies.To solve this pressing problem, the inventors generated hypothesisand could validate some of these as suitable biomarkers withexcellent predictive power, see e.g. example 12.7.1 and 12.7.2. Thelevel of marker expression is usually determined with an assay or amethod known in the art and the level is subsequently compared witha reference value as described elsewhere herein and is subsequentlyused to predict or monitor the treatment response.

Provided is a biomarker for use in a method fordiagnosing/stratifying a subject as having a tumor that issensitive for treatment with an anti-CCR8 antibody, the usecomprising a. determining the level of the biomarker in a tumor ortumor sample comprising regulatory T cells, b. comparing the levelof the biomarker with a reference sample or value, and c.diagnosing/stratifying a subject as having a tumor that issensitive for treatment with an anti-CCR8 antibody, if the level ofthe biomarker is higher than or equal to a reference sample orvalue, wherein the biomarker is d. an immune checkpoint protein,preferably PD-1, PD-L1 or CTLA4, e. a granzyme or immune cellmarker, preferably a marker for lymphocytes, effector cells, Tcells, cytotoxic T cells, macrophage cells, M1 macrophage cells, M2macrophage cells, B cells, NK cells, or a combination thereof, f. aTreg infiltration marker, preferably CCR8, FOXP3, ICOS, CCR4,TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4,CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB,SLAMF7 or CXCL9, g. a T cell marker or cytotoxic T cell markerpreferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB,OX-40, or GITR, h. an interferon or interferon-inducible proteinpreferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 andTNF alpha, i. a complement factor, j. a serpin, and/or k. amolecule derived from a gene according to Table 12.7.2.1, 12.7.2.2or 12.7.2.3.

Preferably, the biomarker is an immune checkpoint protein, mostpreferably PD-1, PD-L1 or CTLA4. Also provided is the use of abiomarker to predict or monitor the therapeutic success of atherapy comprising the administration of an anti-CCR8 antibody, theuse comprising a. determining the level of the biomarker in asample, and b. comparing the level of the biomarker with areference sample or value, wherein the biomarker is c. an immunecheckpoint protein, preferably PD-1, PD-L1 or CTLA4, d. a granzymeor immune cell marker, preferably a marker for lymphocytes,effector cells, T cells, cytotoxic T cells, macrophage cells, M1macrophage cells, M2 macrophage cells, B cells, NK cells, or acombination thereof e. a Treg infiltration marker, preferablyselected from FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9,CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3,GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, CXCL9, f. a T cell markeror cytotoxic T cell marker, preferably selected from CD3, CD4, CD8,CD25, CXCR3, CCR5, and TNF receptor super family members such as4-1BB, OX-40, or GITR, g. an interferon-inducible protein,preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 andTNF alpha, h. a complement factor, i. a serpin and/or j. a moleculederived from a gene according to Table 12.7.2.1, 12.7.2.2 or12.7.2.3.

Preferably, the biomarker is an immune checkpoint protein, mostpreferably PD-1, PD-L1 or CTLA4.

Furthermore, there is also provided a molecule binding a biomarkerfor use in a method for diagnosing/stratifying a subject as havinga tumor that is sensitive for treatment with an anti-CCR8 antibody,the use comprising determining the level of the biomarker in atumor or tumor sample using the molecule binding the biomarker,wherein the biomarker is (preferably) a. an immune checkpointprotein, preferably PD-1, PD-L1 or CTLA4, b. a granzyme or immunecell marker, preferably a marker for lymphocytes, effector cells, Tcells, cytotoxic T cells, macrophage cells, M1 macrophage cells, M2macrophage cells, B cells, NK cells, or a combination thereof c. aTreg infiltration marker, preferably CCR8, FOXP3, ICOS, CCR4,TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4,CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB,SLAMF7 or CXCL9, d. a T cell marker or cytotoxic T cell markerpreferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB,OX-40, or GITR, e. an interferon or interferon-inducible proteinpreferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 andTNF alpha, f. a complement factor, g. a serpin, and/or h. amolecule derived from a gene according to Table 12.7.2.1, 12.7.2.2or 12.7.2.3.

The molecule binding the biomarker is preferably an antibody orantigen-binding fragment, or a conjugate thereof. Most preferably,the molecule binding the biomarker is an anti-PD-1, PD-L1 or CTLA4antibody such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab,Durvalumab, Cemiplimab, Dostarlimab or Ipilimumab.

Furthermore, there is also provided the use of a molecule binding abiomarker to predict or monitor the therapeutic success of atherapy comprising the administration of an anti-CCR8 antibody, theuse comprising determining the level of the biomarker in a tumor ortumor sample using the molecule binding the biomarker, wherein thebiomarker is (preferably) a. an immune checkpoint protein,preferably PD-1, PD-L1 or CTLA4, b. a granzyme or immune cellmarker, preferably a marker for lymphocytes, effector cells, Tcells, cytotoxic T cells, macrophage cells, M1 macrophage cells, M2macrophage cells, B cells, NK cells, or a combination thereof, c. aTreg infiltration marker, preferably CCR8, FOXP3, ICOS, CCR4,TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4,CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB,SLAMF7 or CXCL9, d. a T cell marker or cytotoxic T cell markerpreferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB,OX-40, or GITR, e. an interferon or interferon-inducible proteinpreferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 andTNF alpha, f. a complement factor, g. a serpin, and/or h. amolecule derived from a gene according to Table 12.7.2.1, 12.7.2.2or 12.7.2.3, and wherein the molecule binding the biomarker ispreferably an antibody or antigen-binding fragment, or a conjugatethereof. Most preferably, the molecule binding the biomarker is ananti-checkpoint, anti-PD-1, anti-PD-L1 or anti-CTLA4 antibody suchas Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab,Cemiplimab, Dostarlimab or Ipilimumab.

In preferred embodiments of the current aspect, the biomarker is animmune cell marker, and the molecule binding a biomarker ispreferably an antibody binding a marker for lymphocytes, effectorcells, T cells, cytotoxic T cells, macrophage cells, M1 macrophagecells, M2 macrophage cells, B cells, NK cells, or a combinationthereof.

In preferred embodiments of the current aspect, the moleculebinding the biomarker is a molecule binding a Treg infiltrationmarker, preferably an antibody binding CCR8, FOXP3, ICOS, CCR4,TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4,CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB,SLAMF7 or CXCL9.

In preferred embodiments of the current aspect, the moleculebinding the biomarker is an antibody binding a T cell markerpreferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and TNFreceptor super family members including 4-1BB, OX-40, or GITR.

In preferred embodiments of the current aspect, the moleculebinding the biomarker is an antibody binding an interferon orinterferon-inducible protein preferably selected from INF gamma,IL10, IL12p70, IL1beta, IL2 and TNF alpha.

Immune Checkpoint Protein as Biomarker for Anti-CCR8 AntibodyTreatment

According to the current invention it was surprisingly found thatthe response to (mono) therapy with anti-CCR8 antibody was improvedfor subjects that were at least initially responsive to ICItreatment, and in particular to anti-PD-(L)1 or CTLA4 antibodytreatment, see e.g. Table 12.7.1. Because PD-L1 expression is apredictor for responsiveness to anti-PD-L1 antibody treatment, theinventors hypothesized that PD-L1 expression and checkpoint proteinexpression per se might also be suitable to directly predict theresponse to anti-CCR8 antibody treatment. This hypothesis could beconfirmed by correlation data between PD-L1 expression andanti-CCR8 antibody treatment response (Table 12.8.1). PD-L1expression can be determined as known in the art, e.g. withoutlimitation by using Tumor Proportion Score (TPS), Combined PositiveScore (CPS), or mRNA expression. The inventors therefore suggestimmune checkpoint marker and particularly PD-L1 as surrogate markerfor stratification to overcome the problem that CCR8 is difficultto analyze and is therefore difficult to implement in order toselect patient populations that could most likely benefit fromanti-CCR8 antibody treatment.

Accordingly, there is provided a molecule binding an immunecheckpoint protein for use in a method for diagnosing/stratifying asubject as having a tumor that is sensitive for treatment with ananti-CCR8 antibody, the method comprising a. determining the levelof the immune checkpoint protein expression in a tumor (sample), b.comparing the level of immune checkpoint protein expression with areference sample or value, and c. diagnosing/stratifying a subjectas having a tumor that is sensitive for treatment with an anti-CCR8antibody, if the level of immune checkpoint protein expression ishigher than or equal to a reference sample or value.

Furthermore, there is provided the use of a molecule binding animmune checkpoint protein for monitoring treatment success ofanti-CCR8 antibody therapy, the method comprising a. determiningthe level of the immune checkpoint protein expression in a tumor(sample)/a sample, and b. comparing the level of immune checkpointprotein expression with a reference sample or value.

The molecule binding an immune checkpoint protein is preferably anantibody. The immune checkpoint protein is preferably PD-1, PD-L1or CTLA4. The molecule binding an immune checkpoint protein ispreferably an antibody binding PD-1, PD-L1 or CTLA4. For example,the antibody can be PD-L1 28-8, PD-L1 22C3, PD-L1 SP142, PD-L1SP263 or any other suitable antibody known in the art as companiondiagnostic. In particularly preferred embodiments, the level ofcheckpoint protein expression is determined based on CombinedPositive Score (CPS) or Tumor Proportion Score (TPS). A subject orpatients tumor is diagnosed/stratified as sensitive for treatmentwith an anti-CCR8 antibody, if immune checkpoint protein expressionis present or high, e.g. as indicated by a CPS.gtoreq.1, preferablyby a CPS.gtoreq.5, most preferably by a CPS.gtoreq.10, or asindicated by a TPS.gtoreq.1%, preferably by a TPS.gtoreq.10%, mostpreferably by a TPS.gtoreq.50%. Thus, in some preferredembodiments, the reference value is CPS=1, more preferably CPS=5 ormost preferably CPS=10. In some other or the same preferredembodiments, the reference value is TPS=1%, more preferablyTPS=10%, or most preferably TPS.gtoreq.50%.

In some preferred embodiments, PD-L1 expression is determined by aCPS.gtoreq.1, preferably by a CPS.gtoreq.5, most preferably by aCPS.gtoreq.10 or is determined by a TPS.gtoreq.1%, preferably by aTPS.gtoreq.10%, most preferably by a TPS.gtoreq.50%.

Treg Infiltration Marker as Biomarker for Anti-CCR8 AntibodyTreatment

There is provided a molecule binding a Treg infiltration marker foruse in a method for diagnosing/stratifying a subject as having atumor that is sensitive for treatment with an anti-CCR8 antibody,the method comprising a. determining the level of the Treginfiltration marker expression in a tumor (sample), b. comparingthe level of the Treg infiltration marker expression with areference sample or value, and c. diagnosing/stratifying a subjectas having a tumor that is sensitive for treatment with an anti-CCR8antibody, if the level of the Treg infiltration marker is higherthan or equal to a reference sample or value.

The Treg infiltration marker is a marker that is highly expressedon activated Tregs. Preferably, the Treg infiltration marker isselected from CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80,TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1,IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, CXCL9, cf. Table11.1.1, Table 12.8.1. The Treg infiltration marker can be differentfrom CCR8. The molecule binding a Treg infiltration marker ispreferably an antibody, e.g. an antibody binding to one or more ofthe Treg infiltration markers.

The Treg infiltration marker disclosed herein may also be used asbiomarker to control the treatment success, e.g. as describedelsewhere herein. A treatment is considered successful, if a(temporary) Treg depletion of at least 40% or 50% is obtained, asindicated e.g. by a decrease of the Treg infiltration marker by afactor of 2.

Immune Cell Marker as Biomarker for Anti-CCR8 AntibodyTreatment

There is provided a molecule binding an immune cell marker for usein a method for diagnosing/stratifying a subject as having a tumorthat is sensitive for treatment with an anti-CCR8 antibody, themethod comprising a. determining the level of the immune cellmarker expression in a tumor (sample), b. comparing the level ofthe immune cell marker expression with a reference sample or value,and c. diagnosing/stratifying a subject as having a tumor that issensitive for treatment with an anti-CCR8 antibody, if the level ofthe immune cell marker is higher than or equal to a referencesample or value.

The immune cell marker may be a marker for lymphocytes, effectorcells, T cells, cytotoxic T cells, macrophage cells, M1 macrophagecells, M2 macrophage cells, B cells, NK cells, or a combinationthereof. Suitable markers for these immune cell populations aredescribed throughout this disclosure. For example, suitable T cellmarkers include CD3 (e.g. CD3E, D and/or G). For example, suitablecytotoxic T cell markers include CD8 (e.g. CD8A and/or B). Forexample, suitable macrophage cell markers include MS4A7. Forexample, suitable macrophage M1 cell markers include Acod1. Forexample, suitable M2 macrophage cell markers include Mrc1. Forexample, suitable B cell markers include CD19, CD20, CD22 and/orMSA41.

The immune cell marker disclosed herein may also be used asbiomarker to control the treatment success, e.g. as describedelsewhere herein. A treatment is considered successful, if a(temporary) immune cell increase occurs, e.g. of at least 50%, e.g.as indicated by an increase of the immune cell infiltration markerby a factor of at least 2.

T Cell Marker as Biomarker for Anti-CCR8 Antibody Treatment

There is provided a molecule binding a T cell marker for use in amethod for diagnosing/stratifying a subject as having a tumor thatis sensitive for treatment with an anti-CCR8 antibody, the methodcomprising a. determining the level of the T cell marker expressionin a tumor (sample), b. comparing the level of the T cell markerexpression with a reference sample or value, and c.diagnosing/stratifying a subject as having a tumor that issensitive for treatment with an anti-CCR8 antibody, if the level ofthe T cell marker is higher than or equal to a reference sample orvalue.

The T cell marker may be a marker that is highly expressed on Tcells, such as activated T cells. Preferably, the T cell marker isselected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and TNF receptorsuper family members including 4-1BB, OX-40, or GITR. The moleculebinding the T cell marker is preferably an antibody, e.g. anantibody binding to one or more of the T cell markers disclosedherein.

The T cell marker disclosed herein may also be used as biomarker tocontrol the treatment success, alone or in combination with a Treginfiltration marker, e.g. as described elsewhere herein. Atreatment is considered successful, if a (temporary) T cellincrease occurs, e.g. of at least 50%, e.g. as indicated by anincrease of the T cell marker by a factor of at least 2.

Interferon or Interferon-Inducible Protein as Biomarker forAnti-CCR8 Antibody Treatment

There is provided an interferon or interferon-inducible protein foruse in a method for diagnosing/stratifying a subject as having atumor that is sensitive for treatment with an anti-CCR8 antibody orfor monitoring treatment success, the method comprising a.determining the level of interferon or interferon-inducible proteinin a tumor (sample), b. comparing the level of the interferon orinterferon-inducible protein with a reference sample or value, andc. diagnosing/stratifying a subject as having a tumor that issensitive for treatment with an anti-CCR8 antibody, if the levelinterferon or interferon-inducible protein is higher than or equalto a reference sample or value.

The interferon or interferon-inducible proteins is preferablyselected from IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alpha.According to the current invention it was found that thecorrelation of CCR8 mRNA expression with inflammation marker IFNgamma was significant for 50 tumor indications, cf. Table 11.1.2.From this it can be concluded that IFN gamma levels may be suitableas biomarkers as disclosed herein. This was confirmed e.g. inexample 12.6.2, where IFN gamma and also TNF alpha levelscorrelated with treatment response. In a different experiment(example 12.6.6), increased levels of IFN gamma, IL-1b and IL-2were associated with improved efficacy. Suitableinterferon-stimulated genes or proteins include without limitationACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1, AQP4, ASS1, B2M, BST2,C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CASP1, CCL1,CCL11, CCL13, CCL14, CCL15, CCL15, CCL14, CCL16, CCL17, CCL18,CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL3,CCL3L1, CCL4, CCL4L1, CCLS, CCL7, CCL8, CD40, CD44, CD58,CDCl42EP2, CDCl42EP4, CIITA, CITED1, CLDN1, CX3CL1, CXCL16, CYP27B1DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A, FCGR1B, FLNB, GAPDH, GBP1,GBP2, GBP4, GBP5, GBP6, GCH1, GSN, HCK, HLA-A, HLA-B, HLA-C,HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRAHLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-E, HLA-F, HLA-G, HLA-H,ICAM1, IFI30, IFITM1, IFITM2, IFITM3, IFNG, IFNGR1, IFNGR2, IL12B,IL12RB1 IL23R, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8,IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU, LGALS9, MEFV, MID1, MRC1,MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1, OAS1, OAS2, OAS3, OASL, PDE12,PML, PRKCD, PTAFR, RAB12, RAB20, RAB43, RAB7B, RPL13A, RPS6KB1RYDEN, SEC61A1 SLC11A1 SLC26A6 SLC30A8 SNCA, SP100, STAR, STAT1,STX4, STX8, STXBP1, STXBP2, STXBP3, STXBP4, SYNCRIP TDGF1, TLR2,TLR3, TLR4, TRIM21, TRIM22, TRIM25, TRIM26, TRIM31, TRIM34, TRIM38,TRIMS, TRIM62, TRIM68, TRIMS, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS,WNT5A, XCL1, XCL2, ZYX.

EXAMPLES

Example 1: Alignment CC Chemokine Receptors & CXC ChemokineReceptors

Human CC and CXC chemokine receptor sequences were retrieved fromUniprot and aligned with Clustal Omega. Results were imported inJalview for visualization (FIG. 1). Negative amino acid residues Eand D (grey), as well as tyrosine residues (Y, dark grey) andcysteine residues (C, light grey) are highlighted. While the Nterminal domains deviate in sequence, they are characterized by acomparably high number of negatively charged amino acids andtyrosine residues.

Example 2: Alignment of Human, Cynomolgus and Murine CCR8

Human CCR8, cynomolgus CCR8 and murine CCR8 sequences wereretrieved from Uniprot and aligned with Clustal Omega. Results wereimported in Jalview for visualization and calculation of sequenceidentity, see FIG. 2A. While pairwise alignment of human andcynomolgus CCR8 yields a sequence identity of 94.37%, percentagesare considerably lower between mouse and human CCR8 (PercentageID=70.99), as well as between mouse and cynomolgus CCR8 (PercentageID=71.55). Substantial differences can be observed in N term (TRD)region and C term region.

The sequence homology between the cynomolgus TRD and the human TRDis 68%, while sequence homology between the murine TRD and thehuman TRD is 52%. Nevertheless, the acidic and tyrosine-sulfatedTRD shows a negatively charged cluster which was successfully usedto generate cross-reactive antibodies, see example 6. At least theMDYT and YYPD motifs were found to be conserved between thespecies.

Example 3: Evaluation of Prior Art Antibodies for Specific Bindingto CCR8

FACS experiments were performed using CCR8 expressing cell lines toevaluate multiple prior art antibodies which are commercially soldas CCR8 binding for their specificity and staining to CCR8. Mouse Tlymphoma cell line BW5147.4 expresses murine CCR8 and humancutaneous T lymphoma, HuT78 expresses low levels of human CCR8.Prior art antibodies for chemokine receptors and in particular forCCR8 often suffer from low specificity as described e.g. by Xing etal., Appl IHC Mol Morphol (2015): "We were unable to evaluate CCR8expression by immunohistochemistry due to lack of specificity ofavailable antibodies in our experience and as previously publishedby others.", Chenivesse et al., JI (2012): "We were unable toevaluate the attraction of CD4.sup.+CCR8.sup.+ cells by CCL18because of the lack of specificity of all commercially availableanti-CCR8 Abs, which did not allow cell sorting, at least in ourhands.", or Pease, Biochem J (2011): "The latter has beenproblematic due to the lack of a seemingly reliable antibodyagainst human CCR8. For example, in one study examining CCR8expression on a population of CD4+CD25+ Tregs, CCR8 surfacestaining was undetectable using a commercially available antibody,although the cells evidently expressed functional CCR8, as assessedby CCL1-driven actin polymerization." (cf. Xing, Xiaoming, et al."Expression of the chemokine receptor gene, CCR8, is associatedWith DUSP22 rearrangements in anaplastic large cell lymphoma."Applied immunohistochemistry & molecular morphology:AIMM/official publication of the Society for AppliedImmunohistochemistry 23.8 (2015): 580; Chenivesse, Cecile, et al."Pulmonary CCL18 recruits human regulatory T cells." The Journal ofImmunology 189.1 (2012): 128-137.; and Pease, James E. "Targetingchemokine receptors in allergic disease." Biochemical Journal 434.1(2011): 11-24.).

Table 3.1 lists three analyzed commercially available antibodies,which did not specifically stain CCR8 in the inventors' hands (cf.FIG. 3). Rat IgG2B Clone #191704 (MAB1429, R&D systems) did notshow specific binding of human CCR8 and this is also in agreementwith the observations described in Example 6 of WO2007044756. AbcamE77 showed very weak signal and high background (data not shown).MM0068-4G19 (Abeam), 191704 (R&D Systems), 4G19 (Biozol(Genetex)) and 11n24 and 10k39 (both antikoerper-online.de) did notshow specific binding of human CCR8 in the inventors' hands.

TABLE-US-00003 TABLE 3.1 Selected prior art antibodies, cf. FIG. 3.Vendor Angioproteomie R&D R&D Item Rat anti-human CCR8Monoclonal rat anti-human Recombinan tMonoclonal [MM0068-4G19] CCR8Ab Rabbit IgG Clone #1055C Catalog hAP-0068 MAB1429-100 MAB8324Number Immunogen Human CCR8 transfected BaF3 mouse pro-B cell lineHEK293 human embryonic 293 cells transfected with human kidney cellline transfected CCR8 with mouse CCR8 Met1-Leu355 Accession #P51685 Reactivity Human Human mouse Antigen CCR8 CCR8 CCR8Applications FACS neutralize CCL1-induced Stains by FACS mousechemotaxis ND.sub.50 0.01- CCR8 transfectants but not 0.05 .mu.g/mLirrelevant transfectants Host rat rat rabbit Isotype Rat IgG2 RatIgG.sub.2B Clone # 191704 Clone # 1055C

The inventors found only two reliable monoclonal antibodies in theprior art which specifically bound to human CCR8: Clone 433Hdeveloped by ICOS and initially disclosed in WO2007044756 andL263G8 provided and sold by Biolegend. Both antibodies weregenerated using cells overexpressing human CCR8 as immunogen.L263G8 is not cross reactive for murine CCR8 and did not bind tomurine CCR8 in BW5147.3 cells or HEK 293T cells transfected withmCCR8.

Monoclonal Rat IgG2b, .kappa. anti-mouse CCR8 clone SA214G2(Biolegend Catalog No. 150302) stained positive for murine CCR8expressing BW5147.4 cells. This antibody was generated based oncells transfected with murine CCR8.

In summary, antibodies specifically recognizing human CC chemokinereceptors are rare for some members of this target class. The lowrates are in line with results for further commercially availableantibodies sold for human chemokine receptors.

Example 4: Antigens for CC and CXC Chemokine Receptors

Because the literature on tyrosine sulfation is incomplete andpartially contradictory, e.g. between species, the inventors usedvarious available tools, literature search and own experiences topredict sulfation sites. Table 4.1 summarizes the resultingpolypeptides suitable as antigens to obtain improved antibodies forthe respective chemokine receptors. These antigens weresuccessfully used for antibody generation, e.g. as shown in thesubsequent examples, either as antigens or for off target panning.Underlined sulfation sites were confirmed with an E-cuttoff valueof 55 by applying the algorithm according to Monigatti F. et al.(Monigatti, Flavio, et al. "The Sulfinator: predicting tyrosinesulfation sites in protein sequences." Bioinformatics 18.5 (2002):769-770.). None of the sequences had previously been included inthe training set for the hidden markov models.

Sulfation of tyrosines in brackets can be omitted. For human CCR3and human CCR8, additional sulfation sites were predicted for Y172and Y353, respectively. For monkey CCR3 and monkey CCR2 additionalsulfation sites were predicted for Y172 and Y188, respectively.Sulfation sites shown in bold have been previously described inliterature (cf. Liu, Justin, et al. "Tyrosine sulfation isprevalent in human chemokine receptors important in lung disease."American journal of respiratory cell and molecular biology 38.6(2008): 738-743; Millard; Christopher J., et al. "Structural basisof receptor sulfotyrosine recognition by a CC chemokine: theN-terminal region of CCR3 bound to CCL11/eotaxin-1." Structure22.11 (2014): 1571-1581).

Peptide Design

The N term for CCR8 is formed by the amino acids 1 to 35 within thehuman and cynomolgus CCR8 full length protein and by amino acids 1to 33 for the mouse CCR8 full length protein. The N term consistsof the TRD (amino acids 1-24 for human and cynomolgus CCR8 andamino acids 1-22 for mouse CCR8), and the LID domain (amino acids26-35 for human and cynomolgus CCR8 and amino acids 24-33 for mouseCCR8), which are connected by a cysteine. For the synthesis ofpolypeptides comprising the N term, the cysteine was replaced by aserine to avoid aggregation of the peptides. Table 5.1 shows therespective IDs of the polypeptides which were used for generationof anti-CCR8 antibodies by phage display panning as described inExample 6. To test if posttranslational modifications of proteinswere causing the difficulties for the antibody generation, peptidescomprising TRD and/or N term sequences were used, which were eitherunmodified or sulfated. Sulfation on the cynomolgus and human TRDof CCR8 were introduced on at least 50% of the tyrosines, e.g. onthe positions 3, 15 and 17 of the respective peptides. For themouse TRD and N term peptides, sulfations were introduced on thetyrosines at position 3, 14 and 15 of the respective peptides (cf.Table 6.1). In addition, peptides comprising the LID whereengineered by introduction of a C-terminal biotinylation that wasattached via a TTDS (Trioxatridecan-succinamic acid) linker. Othertags and linkers can also be used. The peptides for the TRD or Nterm were modified by introduction of a N-terminal biotinylationattached via a TTDS-Lysin linker. Again, further known linkerand/or tags can likewise be used to facilitate immobilization ofthe peptides.

TABLE-US-00004 TABLE 4.1 Polypeptides for antigen or off targetpanning for generation of chemokine receptor antibodies. At leastone sulfation at one of the listed tyrosine positions of thepolypeptide is required according to the invention, but sulfationsat two three or more sites are also possible and may be beneficial.X can be any amino acid or no amino acid, and preferably, is anamino acid different from cysteine, such as serine. SEQ ID Anti-Spe- Do- Sulfation Additional NO: gen cies main sites SequenceLength modifications (optional) 1 CCR1 HUMAN TRD Y10, Y18METPNTTEDYDTTTEFDYGDATP 23 N-terminal biotinylation (e.g. viaTTDS_Lysin linker) 2 CCR1 MACFA TRD Y10, Y18METPDTTENYDMITEFDYGDATP 23 N-terminal biotinylation (e.g. viaTTDS_Lysin linker) 3 CCR1 MOUSE TRD Y10, Y18MEISDFTEAYPTTTEFDYGDSTP 23 N-terminal biotinylation (e.g. viaTTDS_Lysin linker) 4 CCR1 HUMAN N Y10, Y18METPNTTEDYDTTTEFDYGDATPXQ 34 N-terminal biotinylation termKVNERAFGA (e.g. via TTDS_Lysin linker); C24S (to avoid aggregation)5 CCR1 MACFA N Y10, Y18 METPDTTENYDMITEFDYGDATPXH 34 N-terminalbiotinylation term KVNERAILA (e.g. via TTDS_Lysin linker); C24S (toavoid aggregation) 6 CCR1 MOUSE N Y10, Y18MEISDFTEAYPTTTEFDYGDSTPXQK 34 N-terminal biotinylation termTAVRAFGA (e.g. via TTDS_Lysin linker); C24S (to avoid aggregation)7 CCR2 HUMAN TRD Y26, Y28 MLSTSRSRFIRNTNESGEEVTTFFDYD 31 N-terminalbiotinylation YGAP (e.g. via TTDS_Lysin linker) 8 CCR2 MACMU TRDY26, Y28 MLSTSRSRFIRNTNGSGEEVTTFFDYD 31 N-terminal biotinylationYGAP (e.g. via TTDS_Lysin linker) 9 CCR2 MOUSE TRD Y37, Y39MEDNNMLPQFIHGILSTSHSLFTRSIQ 44 N-terminal biotinylationELDEGATTPYDYDDGEP (e.g. via TTDS_Lysin linker) 10 CCR2 HUMAN N Y26,Y28 MLSTSRSRFIRNTNESGEEVTTFFDYD 42 N-terminal biotinylation termYGAPXHKFDVKQIGA (e.g. via TTDS_Lysin linker); C32S (to avoidaggregation) 11 CCR2 MACMU N Y26, Y28 MLSTSRSRFIRNTNGSGEEVTTFFDYD42 N-terminal biotinylation term YGAPXHKFDVKQIGA (e.g. viaTTDS_Lysin linker); C32S (to avoid aggregation) 12 CCR2 MOUSE NY37, Y39 MEDNNMLPQFIHGILSTSHSLFTRSIQ 55 N-terminal biotinylationterm ELDEGATTPYDYDDGEPXHKTSVKQI (e.g. via TTDS_Lysin linker); GAC45S (to avoid aggregation) 13 CCR3 HUMAN TRD Y16, Y17MTTSLDTVETFGTTSYYDDVGLL 23 N-terminal biotinylation (e.g. viaTTDS_Lysin linker) 14 CCR3 MACFA TRD Y16 MTTSLDTVETFGPTSYDDDMGLL 23N-terminal biotinylation (e.g. via TTDS_Lysin linker) 15 CCR3 MOUSETRD Y20, Y22 MAFNTDEIKTVVESFETTPYEYEWAP 27 N-terminal biotinylationP (e.g. via TTDS_Lysin linker) 16 CCR3 HUMAN N Y16, Y17MTTSLDTVETFGTTSYYDDVGLLXEK 34 N-terminal biotinylation termADTRALMA (e.g. via TTDS_Lysin linker); C24S (to avoid aggregation)17 CCR3 MACFA N Y16 MTTSLDTVETFGPTSYDDDMGLLXE 34 N-terminalbiotinylation term KADVGALIA (e.g. via TTDS_Lysin linker); C24S (toavoid aggregation) 18 CCR3 MOUSE N Y20, Y22MAFNTDEIKTVVESFETTPYEYEWAP 38 N-terminal biotinylation termPXEKVRIKELGS (e.g. via TTDS_Lysin linker); C28S (to avoidaggregation) 19 CCR4 HUMAN TRD (Y16, Y19,MNPTDIADTTLDESIYSNYYLYESIPK 28 N-terminal biotinylation Y20), Y22 P(e.g. via TTDS_Lysin linker) 20 CCR4 MACFA TRD (Y16, Y19,MNPTDIADTTLDESIYSNYYLYESIPK 28 N-terminal biotinylation Y20), Y22 P(e.g. via TTDS_Lysin linker) 21 CCR4 MOUSE TRD (Y16, Y19,MNATEVTDTTQDETVYNSYYFYESM 28 N-terminal biotinylation Y20), Y22 PKP(e.g. via TTDS_Lysin linker) 22 CCR4 HUMAN N (Y16, Y19,MNPTDIADTTLDESIYSNYYLYESIPK 39 N-terminal biotinylation term Y20),Y22 PXTKEGIKAFGE (e.g. via TTDS_Lysin linker); C29S (to avoidaggregation) 23 CCR4 MACFA N (Y16, Y19, MNPTDIADTTLDESIYSNYYLYESIPK39 N-terminal biotinylation term Y20), Y22 PXTKEGIKAFGE (e.g. viaTTDS_Lysin linker); C29S (to avoid aggregation) 24 CCR4 MOUSE N(Y16, Y19, MNATEVTDTTQDETVYNSYYFYESM 39 N-terminal biotinylationterm Y20), Y22 PKPXTKEGIKAFGE (e.g. via TTDS_Lysin linker); C29S(to avoid aggregation) 25 CCR5 HUMAN TRD Y3, Y10MDYQVSSPIYDINYYTSEP 19 N-terminal biotinylation (Y14, Y15 (e.g. viaTTDS_Lysin linker) 26 CCR5 MACFA TRD Y3, Y10, MDYQVSSPTYDIDYYTSEP19 N-terminal biotinylation Y14, Y15 (e.g. via TTDS_Lysin linker)27 CCR5 MOUSE TRD Y10, Yl2, MDFQGSVPTYSYDIDYGMSAP 21 N-terminalbiotinylation Y16 (e.g. via TTDS_Lysin linker) 28 CCR5 HUMAN N Y3,Y10 MDYQVSSPIYDINYYTSEPXQKINVK 30 N-terminal biotinylation termY14, Y15 QIAA (e.g. via TTDS_Lysin linker); C20S (to avoidaggregation) 29 CCR5 MACFA N Y3, Y10, MDYQVSSPTYDIDYYTSEPXQKINVK 30N-terminal biotinylation term Y14, Y15 QIAA (e.g. via TTDS_Lysinlinker); C20S (to avoid aggregation) 30 CCR5 MOUSE N Y10, Y12,MDFQGSVPTYSYDIDYGMSAPXQKIN 32 N-terminal biotinylation term Y16VKQIAA (e.g. via TTDS_Lysin linker); C22S (to avoid aggregation) 31CCR6 HUMAN TRD Y18, Y26, MSGESMNFSDVFDSSEDYFVSVNTSY 35 N-terminalbiotinylation Y27 YSVDSEMLL (e.g. via TTDS_Lysin linker) 32 CCR6MACFA TRD Y23, Y31, MFLPTMSGESMNFSDVFDSSEDYFAS 40 N-terminalbiotinylation Y32 VNTSYYTVDSEMLL (e.g. via TTDS_Lysin linker) 33CCR6 MOUSE TRD (Y7), Y13, MNSTESYFGTDDYDNTEYYSIPPDHG 27 N-terminalbiotinylation Y18, Y19 P (e.g. via TTDS_Lysin linker) 34 CCR6 HUMANN Y18, Y26, MSGESMNFSDVFDSSEDYFVSVNTSY 47 N-terminal biotinylationterm Y27 YSVDSEMLLXSLQEVRQFSRL (e.g. via TTDS_Lysin linker); C36S(to avoid aggregation) 35 CCR6 MACFA N Y23, Y31,MFLPTMSGESMNFSDVFDSSEDYFAS 52 N-terminal biotinylation term Y32VNTSYYTVDSEMLLXTLHEVRQFSRL (e.g. via TTDS_Lysin linker); C41S (toavoid aggregation) 36 CCR6 MOUSE N (Y7), Y13,MNSTESYFGTDDYDNTEYYSIPPDHG 39 N-terminal biotinylation term Y18,Y19 PXSLEEVRNFTKV (e.g. via TTDS_Lysin linker); C28S (to avoidaggregation) 37 CCR7 HUMAN TRD Y8, Y17 QDEVTDDYIGDNTTVDYTLFESL 23N-terminal biotinylation (e.g. via TTDS_Lysin linker) 38 CCR7 MACFATRD Y8, Y17 QDEVTDDYIGDNTTVDYTLFESL 23 N-terminal biotinylation(e.g. via TTDS_Lysin linker) 39 CCR7 MOUSE TRD Y8, Y17,QDEVTDDYIGENTTVDYTLYESV 23 N-terminal biotinylation Y20 (e.g. viaTTDS_Lysin linker) 40 CCR7 HUMAN N Y8, Y17QDEVTDDYIGDNTTVDYTLFESLXSK 35 N-terminal biotinylation termKDVRNFKAW (e.g. via TTDS_Lysin linker); w/o C24S (to avoidaggregation) Signal Pep- tide 41 CCR7 MACFA NQDEVTDDYIGDNTTVDYTLFESLXSK 35 N-terminal biotinylation term Y8, Y17KDVRNFKAW (e.g. via TTDS_Lysin linker); w/o C24S (to avoidaggregation) Signal Pep- tide 42 CCR7 MOUSE N Y8, Y17,QDEVTDDYIGENTTVDYTLYESVXFK 35 N-terminal biotinylation term Y20KDVRNFKAW (e.g. via TTDS_Lysin linker); w/o C24S (to avoidaggregation) Signal Pep- tide 43 CCR8 HUMAN TRD Y3, Y15,MDYTLDLSVTTVTDYYYPDIFSSP 24 N-terminal biotinylation (16), Y17(e.g. via TTDS_Lysin linker) 44 CCR8 MACFA TRD Y3, Y15,MDYTLDPSMTTMTDYYYPDSLSSP 24 N-terminal biotinylation (16), Y17(e.g. via TTDS_Lysin linker) 45 CCR8 MOUSE TRD Y3, Y14,MDYTMEPNVTMTDYYPDFFTAP 22 N-terminal biotinylation Y15 (e.g. viaTTDS_Lysin linker) 46 CCR8 HUMAN N Y3, Y15,MDYTLDLSVTTVTDYYYPDIFSSPXD 35 N-terminal biotinylation term (16),Y17 AELIQTNGK (e.g. via TTDS_Lysin linker); C25S (to avoidaggregation) 47 CCR8 MACFA N Y3, Y15, MDYTLDPSMTTMTDYYYPDSLSSPX 35N-terminal biotinylation term (16), Y17 DGELIQRNDK (e.g. viaTTDS_Lysin linker); C25S (to avoid aggregation) 48 CCR8 MOUSE N Y3,Y14, MDYTMEPNVTMTDYYPDFFTAPXDA 33 N-terminal biotinylation term Y15EFLLRGSM (e.g. via TTDS_Lysin linker); C23S (to avoid aggregation)49 CCR8 HUMAN LID DAELIQTNGK 10 NA 50 CCR8 MACFA LID DGELIQRNDK 10NA 51 CCR8 MOUSE LID DAEFLLRGSM 10 NA 52 CCR8 HUMAN ECL1YLLDQWVFGTVMCK 14 NA 53 CCR8 MACFA ECL1 YQLDQWVFGTVMCK 14 NA 54CCR8 MOUSE ECL1 NLLDQWVFGTAMCK 14 NA

55 CCR8 HUMAN ECL2 YQVASEDGVLQCYSFYNQQTLKWKI 31 NA FTNFKM 56 CCR8MACFA ECL2 YQVASEDGVLQCYSFYNQQTLKWKI 31 NA FTNFEM 57 CCR8 MOUSEECL2 YQVASEDGMLQCFQFYEEQSLRWKL 31 NA FTHFEI 58 CCR8 HUMAN ECL3HSMHILDGCSISQQLTY 17 NA 59 CCR8 MACFA ECL3 HSMHILDGCSISQQLNY 17 NA60 CCR8 MOUSE ECL3 HDLHILDGCATRQRLAL 17 NA 61 CCR9 HUMAN TRD Y17,Y28, MTPTDFTSPIPNMADDYGSESTSSME 37 N-terminal biotinylation Y37DYVNFNFTDFY (e.g. via TTDS_Lysin linker) 62 CCR9 MACFA TRD Y17,Y28, MTPTEFTSPVPNMADDYGSESTSSME 37 N-terminal biotinylation Y37DYVNFNFTDFY (e.g. via TTDS_Lysin linker) 63 CCR9 MOUSE TRD Y19, Y28MTPTEFTSPVPNMADDYGSESTSSME 37 N-terminal biotinylation DYVNFNFTDFY(e.g. via TTDS_Lysin linker) 64 CCR9 HUMAN N Y17, Y28,MTPTDFTSPIPNMADDYGSESTSSME 48 N-terminal biotinylation term Y37DYVNFNFTDFYXEKNNVRQFAS (e.g. via TTDS_Lysin linker); C38S (to avoidaggregation) 65 CCR9 MACFA N Y17, Y28, MTPTEFTSPVPNMADDYGSESTSSME48 N-terminal biotinylation term Y37 DYVNFNFTDFYXEKNNVRQFAS (e.g.via TTDS_Lysin linker); C38S (to avoid aggregation) 66 CCR9 MOUSE NY19, Y28 MTPTEFTSPVPNMADDYGSESTSSME 48 N-terminal biotinylationterm DYVNFNFTDFYXEKNNVRQFAS (e.g. via TTDS_Lysin linker); C38S (toavoid aggregation) 67 CCR10 HUMAN TRD Y14, Y22MGTEATEQVSWGHYSGDEEDAYSAE 30 N-terminal biotinylation PLPEL (e.g.via TTDS_Lysin linker) 68 CCR10 MACFA TRD Y14, Y22MGTEATEQVSWGHYSGDEEEAYSAE 30 N-terminal biotinylation PLPEL (e.g.via TTDS_Lysin linker) 69 CCR10 MOUSE TRD Y14, Y17,MGTKPTEQVSWGLYSGYDEEAYSVG 30 N-terminal biotinylation Y22 PLPEL(e.g. via TTDS Lysin linker) 70 CCR10 HUMAN N Y14, Y22MGTEATEQVSWGHYSGDEEDAYSAE 52 N-terminal biotinylation termPLPELXYKADVQAFSRAFQPSVSLTV (e.g. via TTDS_Lysin linker); A C31S (toavoid aggregation) 71 CCR10 MACFA N Y14, Y22MGTEATEQVSWGHYSGDEEEAYSAE 52 N-terminal biotinylation termPLPELXYKADVQAFSRAFQPSVSLTV (e.g. via TTDS_Lysin linker); A C31S (toavoid aggregation) 72 CCR10 MOUSE N Y14, Y17,MGTKPTEQVSWGLYSGYDEEAYSVG 48 N-terminal biotinylation term Y22PLPELXYKADVQAFSRAFQPSVS (e.g. via TTDS_Lysin linker); C31S (toavoid aggregation) 73 CXCR HUMAN TRD Y27 MSNITDPQMWDFDDLNFTGMPPADE29 N-terminal biotinylation 1 DYSP (e.g. via TTDS_Lysin linker) 74CXCR MACFA TRD Y14, Y28 MSNATDPQMGDDDYDLNFTGMPPT 30 N-terminalbiotinylation 1 DEDYSP (e.g. via TTDS_Lysin linker) 75 CXCR MOUSETRD Y6, Y32 MAEAEYFIWTNPEGDFEKEFGNITGM 35 N-terminal biotinylation1 LPTGDYFIP (e.g. via TTDS_Lysin linker) 76 CXCR HUMAN N Y27MSNITDPQMWDFDDLNFTGMPPADE 39 N-terminal biotinylation 1 termDYSPXMLETETLNK (e.g. via TTDS_Lysin linker); C305 (to avoidaggregation) 77 CXCR MACFA N Y14, Y28 MSNATDPQMGDDDYDLNFTGMPPT 40N-terminal biotinylation 1 term DEDYSPXRLETQSLNK (e.g. viaTTDS_Lysin linker); C31S (to avoid aggregation) 78 CXCR MOUSE N Y6,Y32 MAEAEYFIWTNPEGDFEKEFGNITGM 44 N-terminal biotinylation 1 termLPTGDYFIPXKRVPITNR (e.g. via TTDS_Lysin linker); C365 (to avoidaggregation) 79 CXCR HUMAN TRD Y23, Y25 MEDFNMESDSFEDFWKGEDLSNYSY38 N-terminal biotinylation 2 SSTLPPFLLDAAP (e.g. via TTDS_Lysinlinker) 80 CXCR MACMU TRD Y20, Y22 FNMESDSFEDLWKGEDFSNYSYSSDL 35N-terminal biotinylation 2 PPSLPDVAP (e.g. via TTDS_Lysin linker)81 CXCR MOUSE TRD Y24 MGEFKVDKFNIEDFFSGDLDIFNYSSG 37 N-terminalbiotinylation 2 MPSILPDAVP (e.g. via TTDS_Lysin linker) 82 CXCRHUMAN N Y23, Y25 MEDFNMESDSFEDFWKGEDLSNYSY 48 N-terminalbiotinylation 2 term SSTLPPFLLDAAPXEPESLEINK (e.g. via TTDS_Lysinlinker); C39S (to avoid aggregation) 83 CXCR MACMU N Y20, Y22FNMESDSFEDLWKGEDFSNYSYSSDL 45 N-terminal biotinylation 2 termPPSLPDVAPXRPESLEINK (e.g. via TTDS_Lysin linker); C36S (to avoidaggregation) 84 CXCR MOUSE N Y24 MGEFKVDKFNIEDFFSGDLDIFNYSSG 47N-terminal biotinylation 2 term MPSILPDAVPXHSENLEINS (e.g. viaTTDS_Lysin linker); C38S (to avoid aggregation) 85 CXCR HUMAN TRDY27, Y29 MVLEVSDHQVLNDAEVAALLENFSS 36 N-terminal biotinylation 3SYDYGENESDS (e.g. via TTDS_Lysin linker) 86 CXCR MACFA TRD Y27, Y29MVLEVSDHQVLNDAEVAALLENFSS 36 N-terminal biotinylation 3 SYDYGENESDS(e.g. via TTDS_Lysin linker) 87 CXCR MOUSE TRD Y2, Y27,MYLEVSERQVLDASDFAFLLENSTSP 41 N-terminal biotinylation 3 Y29YDYGENESDFSDSPP (e.g. via TTDS_Lysin linker) 88 CXCR HUMAN N Y27,Y29 MVLEVSDHQVLNDAEVAALLENFSS 42 N-terminal biotinylation 3 termSYDYGENESDSXXTSPP (e.g. via TTDS_Lysin linker); C37S, C38S (toavoid aggregation) 89 CXCR MACFA N Y27, Y29MVLEVSDHQVLNDAEVAALLENFSS 42 N-terminal biotinylation 3 termSYDYGENESDSXXTSPP (e.g. via TTDS_Lysin linker); C37S, C38S (toavoid aggregation) 90 CXCR MOUSE N Y2, Y27,MYLEVSERQVLDASDFAFLLENSTSP 52 N-terminal biotinylation 3 term Y29YDYGENESDFSDSPPXPQDFSLNFDR (e.g. via TTDS_Lysin linker); C42S (toavoid aggregation) 91 CXCR HUMAN TRD (Y7), Y12,MEGISIYTSDNYTEEMGSGDYDSMKE 27 N-terminal biotinylation 4 Y21 P(e.g. via TTDS_Lysin linker) 92 CXCR MACFA TRD (Y7), Y12,MEGISIYTSDNYTEEMGSGDYDSIKEP 27 N-terminal biotinylation 4 Y21 (e.g.via TTDS_Lysin linker) 93 CXCR MOUSE TRD Y9, Y14,MEPISVSIYTSDNYSEEVGSGDYDSN 29 N-terminal biotinylation 4 Y23 KEP(e.g. via TTDS_Lysin linker) 94 CXCR HUMAN N (Y7), Y12,MEGISIYTSDNYTEEMGSGDYDSMKE 38 N-terminal biotinylation 4 term Y21PXFREENANFNK (e.g. via TTDS_Lysin linker); C28S (to avoidaggregation) 95 CXCR MACFA N (Y7), Y12, MEGISIYTSDNYTEEMGSGDYDSIKEP38 N-terminal biotinylation 4 term Y21 XFREENAHFNR (e.g. viaTTDS_Lysin linker); C28S (to avoid aggregation) 96 CXCR MOUSE N Y9,Y14, MEPISVSIYTSDNYSEEVGSGDYDSN 40 N-terminal biotinylation 4 termY23 KEPXFRDENVHFNR (e.g. via TTDS_Lysin linker); C30S (to avoidaggregation) 97 CXCR HUMAN TRD Y3, Y27 MNYPLTLEMDLENLEDLFWELDRLD 37N-terminal biotinylation 5 NYNDTSLVENHL (e.g. via TTDS_Lysinlinker) 98 CXCR MACFA TRD Y3, Y27 MNYPLMLEMDLENLEDLFLEFDKFD 37N-terminal biotinylation 5 NYNDTSLVENHL (e.g. via TTDS_Lysinlinker) 99 CXCR MOUSE TRD Y3, Y14, MNYPLTLDMGSITYNMDDLYKELAF 39N-terminal biotinylation 5 Y20, Y26 YSNSTEIPLQDSNF (e.g. viaTTDS_Lysin linker) 100 CXCR HUMAN N Y3, Y27MNYPLTLEMDLENLEDLFWELDRLD 55 N-terminal biotinylation 5 termNYNDTSLVENHLXPATEGPLMASFK (e.g. via TTDS_Lysin linker); AVFVP C38S(to avoid aggregation) 101 CXCR MACFA N Y3, Y27MNYPLMLEMDLENLEDLFLEFDKFD 55 N-terminal biotinylation 5 termNYNDTSLVENHLXPATEGPLMASFK (e.g. via TTDS_Lysin linker); AVFVP C38S(to avoid aggregation) 102 CXCR MOUSE N Y3, Y14,MNYPLTLDMGSITYNMDDLYKELAF 57 N-terminal biotinylation 5 term Y20,Y26 YSNSTEIPLQDSNFXSTVEGPLLTSFK (e.g. via TTDS_Lysin linker); AVFMPC40S (to avoid aggregation) 103 CXCR HUMAN TRD Y6, Y10MAEHDYHEDYGFSSFNDSSQEEHQD 32 N-terminal biotinylation 6 FLQFSKV(e.g. via TTDS_Lysin linker) 104 CXCR MACFA TRD Y4 Y7,MAEYDHYEDDGFLNSFNDSSQEEHQ 33 N-terminal biotinylation 6 Y39DFLQFRKV (e.g. via TTDS_Lysin linker) 105 CXCR MOUSE TRD Y11, (Y15)MDDGHQESALYDGHYEGDFWLFNN 41 N-terminal biotinylation 6SSDNSQENKRFLKFKEV (e.g. via TTDS_Lysin linker) 106 CXCR HUMAN N Y6,Y10 MAEHDYHEDYGFSSFNDSSQEEHQD 43 N-terminal biotinylation 6 termFLQFSKVFLPXMYLVVFV (e.g. via TTDS_Lysin linker); C33S (to avoidaggregation) 107 CXCR MACFA N Y4, Y7, MAEYDHYEDDGFLNSFNDSSQEEHQ 44N-terminal biotinylation 6 term Y39 DFLQFRKVFLPXMYLVVFV (e.g. viaTTDS_Lysin linker); C34S (to avoid aggregation) 108 CXCR MOUSE NY11, (Y15) MDDGHQESALYDGHYEGDFWLFNN 45 N-terminal biotinylation 6term SSDNSQENKRFLKFKEVFLPX (e.g. via TTDS_Lysin linker); C42S (toavoid aggregation) 157 CX3C HUMAN TRD Y14 MDQFPESVTENFEYDDLAEA 20N-terminal biotinylation R1 (e.g. via TTDS_Lysin linker) 158 CX3CMACFA TRD Y20 LRNVEANFLGFLLPLLIMSY 20 N-terminal biotinylation R1(e.g. via TTDS_Lysin linker) 159 CX3C MOUSE TRD Y15MSTSFPELDLENFEYDDSAEA 21 N-terminal biotinylation R1 (e.g. viaTTDS_Lysin linker) 160 CX3C HUMAN N Y14, Y22MDQFPESVTENFEYDDLAEAXYIGDI 31 N-terminal biotinylation R1 termVVFGT (e.g. via TTDS_Lysin linker); C21S (to avoid aggregation) 161CX3C MACFA N Y20, Y22 LRNVEANFLGFLLPLLIMSYXYFRIIQ 31 N-terminalbiotinylation

R1 term TLFS (e.g. via TTDS_Lysin linker); C21S (to avoidaggregation) 162 CX3C MOUSE N Y15, Y23 MSTSFPELDLENFEYDDSAEAXYLGD32 N-terminal biotinylation R1 term IVAFGT (e.g. via TTDS_Lysinlinker); C22S (to avoid aggregation) 163 CXCR HUMAN TRD Y27MSNITDPQMWDFDDLNFTGMPPADE 29 N-terminal biotinylation 1 DYSP (e.g.via TTDS_Lysin linker) 164 CXCR MACMU TRD Y14, Y28MSNATDPQMGDDDYDLNFTGMPPT 30 N-terminal biotinylation 1 DEDYSP (e.g.via TTDS_Lysin linker) 165 CXCR MOUSE TRD Y6, Y32MAEAEYFIWTNPEGDFEKEFGNITGM 35 N-terminal biotinylation 1 LPTGDYFIP(e.g. via TTDS_Lysin linker) 166 CXCR HUMAN N Y27MSNITDPQMWDFDDLNFTGMPPADE 39 N-terminal biotinylation 1 termDYSPXMLETETLNK (e.g. via TTDS_Lysin linker); C30S (to avoidaggregation) 167 CXCR MACMU N Y14, Y28, MSNATDPQMGDDDYDLNFTGMPPT 46N-terminal biotinylation 1 term Y41 DEDYSPXRLETQSLNKYVVIVT (e.g.via TTDS_Lysin linker); C31S (to avoid aggregation) 168 CXCR MOUSEN Y6, Y32 MAEAEYFIWTNPEGDFEKEFGNITGM 44 N-terminal biotinylation 1term LPTGDYFIPXKRVPITNR (e.g. via TTDS_Lysin linker); C36S (toavoid aggregation)

TABLE-US-00005 TABLE 5.1 Polypeptides synthesized for CCR8 astarget ID Anti-gen Species Domain Sulfation Additionalmodifications TPP-13798 CCR8 cyno Lid -- C-terminal biotinylation(e.g. via TTDS linker) TPP-13796 CCR8 human Lid -- C-terminalbiotinylation (e.g. via TTDS linker) TPP-13797 CCR8 mouse Lid --C-terminal biotinylation (e.g. via TTDS linker) TPP-13794 CCR8 cynoTRD -- N-terminal biotinylation (e.g. via TTDS_Lysin linker)TPP-13795 CCR8 cyno TRD Y3, Y15, Y17 N-terminal biotinylation (e.g.via TTDS_Lysin linker) TPP-13791 CCR8 human TRD -- N-terminalbiotinylation (e.g. via TTDS_Lysin linker) TPP-13792 CCR8 mouse TRD-- N-terminal biotinylation (e.g. via TTDS_Lysin linker) TPP-13793CCR8 mouse TRD Y3, Y14, Y15 N-terminal biotinylation (e.g. viaTTDS_Lysin linker) TPP-13965 CCR8 cyno N-term -- N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C25S (to avoidaggregation) TPP-14828 CCR8 cyno N term Y3, Y15, Y17 N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C25S (to avoidaggregation) TPP-13963 CCR8 human N term -- N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C25S (to avoidaggregation) TPP-14827 CCR8 human N term Y3, Y15, Y17 N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C25S (to avoidaggregation) TPP-13964 CCR8 mouse N term -- N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C23S (to avoidaggregation) TPP-14830 CCR8 mouse N term Y3, Y14, N-terminalbiotinylation (e.g. via TTDS_Lysin linker); C23S (to avoidaggregation)

To test if posttranslational modifications of proteins were causingthe difficulties for the antibody generation, peptides comprisingTRD and/or N term sequences were used, which were either unmodifiedor sulfated. Sulfation on the cynomolgus and human TRD of CCR8 wereintroduced on at least 50% of the tyrosines, e.g. on the positions3, 15 and 17 of the respective peptides. For the mouse TRD and Nterm peptides, sulfations were introduced on the tyrosines atposition 3, 14 and 15 of the respective peptides (cf. Table 6.1).In addition, peptides comprising the LID where engineered byintroduction of a C-terminal biotinylation that was attached via aTTDS (Trioxatridecan-succinamic acid) linker. Other tags andlinkers can also be used. The peptides for the TRD or N term weremodified by introduction of a N-terminal biotinylation attached viaa TTDS-Lysin linker. Again, further known linker and/or tags canlikewise be used to facilitate immobilization of the peptides.

Peptide Synthesis

Peptides can be obtained from different commercial sources and canbe prepared as known in the art, for example using standard Fmocsolid-phase peptide synthesis (SPPS) chemistry. The efficientsynthesis of sulfated peptides is technically challenging astyrosine-sulfated peptides are stable under basic conditions, whilethe sulfotyrosines undergo desulfation under acidic conditionsrendering the simple stepwise incorporation of FmocTyr(SO3Na)OHinto the growing peptide unsuitable. Global sulfation of peptidesis possible, for example using sulfur trioxide-pyridine, but doesnot allow for specific sulfation of selected tyrosines. However,Fmoc-based SSPS can nevertheless be used for synthesis of sulfatedpeptides. To this end orthogonally protected tyrosine derivativeslike fluorosulfated tyrosine, azidomethyl protected tyrosine orneopentyl-protected tyrosine may be used for incorporation. Theprotected tyrosine can be subsequently unmasked in a selective andquantitative way.

Chen et al., Angew Chem, 2016, have reported a one-step synthesisof Fmoc-fluorosulfated tyrosine. An efficient Fmoc solid-phasepeptide synthesis strategy is then introduced for incorporating thefluorosulfated tyrosine residue into peptides of interest. Standardsimultaneous peptide-resin cleavage and removal of the acid-labileside-chain protecting groups affords the crude peptides containingfluorosulfated tyrosine. Basic ethylene glycol, serving as solventand reactant, transforms the fluorosulfated tyrosine peptides intosulfotyrosine peptides in high yield.

The resulting peptides can be subsequently purified as known in theart, e.g. by HPLC, e.g. on a BEH C-18 (Waters) or a Kinetex C-18(Phenomenex) column and can be analyzed by mass spectrometry(IonSpray and Positive Ion Detector), e.g. for quality control.

In literature, two disulfide bridges within the full length humanCCR8 protein have been described to connect the extracellular loop1 (ECL1) and extracellular loop 2 (ECL2) via the cysteines onpositions 106 and 183. A further disulfide bridge between cysteines25 and 272 is assumed to connect the 7.sup.th transmembrane helixwith the N-terminus. However, the peptides according to the exampleare not expected to form any disulfide bridges betweencysteines.

Example 6: Phage Display and Human Anti-CCR8 Antibody Screening

A fully human antibody phage display library (Biolnvent n-CoDeR Fablambda library) was used to isolate human monoclonal antibodies byselection against soluble biotinylated peptides. Peptides wereprovided by Pepscan. The synthesis of peptides was performed asdescribed in example 5. For the panning procedure the followingprotocol was applied.

TABLE-US-00006 TABLE 6.1 List of peptides including modificationsused for antibody selection by phage display and ELISA screening.Anti- D.sub.0- ID gen Species main Sulfation Additionalmodifications TPP-14828 CCR8 cyno N Y3, Y15, N-terminalbiotinylation term Y17 via TTDS_Lysin linker; C25S (to avoidaggregation) TPP-14827 CCR8 human N Y3, Y15, N-terminalbiotinylation term Y17 via TTDS_Lysin linker; C25S (to avoidaggregation) TPP-14829 CCR4 human N Y22 N-terminal biotinylation(off-target) term via TTDS_Lysin linker; C29S (to avoidaggregation)

Streptavidin-coupled Dynabeads M-280 (Invitrogen.TM.) were coatedfor one hour at room temperature (RT) with the biotinylated peptide(1 tube) and the biotinylated off-target peptide (3 tubes),respectively. Dynabeads were washed and subsequently blocked for 1h at RT with end-over-end rotation.

For depletion of off-target binders, the blocked phage library wasadded to the blocked off-target loaded Dynabeads and incubated for10 min at room temperature with end-over-end rotation. Thisdepletion step was repeated 2 times. The depleted phage library wasadded to the blocked target loaded Dynabeads and incubated for 60min at RT with end-over-end rotation.

After stringent washing (3.times. in blocking buffer (PBS-T, 3%milk powder) and 9.times. in PBS-T (150 mM NaCl; 8 mM Na2HPO4; 1.5mM KH2PO4; adjusted to pH=7.4-7.6, 0.05% Tween-20)), Dynabeads withFab-phages binding specifically to the coated target were directlyused to infect E. coli strain HB101. Subsequently the phages wereamplified in E. coli strain HB101 using M13K07 Helper Phage(Invitrogen).

In the following selection rounds the target concentration wasdecreased to augment the selection pressure for high affinitybinders. During panning of the library, two different selectionstrategies were carried out (FIG. 4). The strategies were designedto identify antibodies exhibiting binding activity towards thehuman CCR8 N-terminal region comprising the TRD and/or thecynomolgus CCR8 N-terminal region comprising the TRD. All peptidesused as antigens or for off target panning were sulfated in atleast one position, cf. Table 4.1. and Table 6.1.

For both strategies, a depletion step was included usingbiotinylated human CCR4 N-terminal region comprising the TRD(TPP-14829, SEQ ID NO:22, with Y22 being sulfated) as off-targetpeptide. Strategy I comprised a first panning round on the humanCCR8 N-terminal, sulfated peptide (TPP-14827, SEQ ID NO:46, withY3, Y15, Y17 being sulfated) followed by either 3 rounds on thesame peptide (TPP-14827, SEQ ID NO:46 with Y3, Y15, Y17 beingsulfated) or on the cynomolgus CCR8 N-terminal, sulfated peptide(TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated).

Strategy II comprised a first panning round on the cynomolgus CCR8N-terminal, sulfated peptide (TPP-14828, SEQ ID NO:47 with Y3, Y15,Y17 being sulfated) followed by either 3 rounds on the same peptide(TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated) or threerounds on the human CCR8 N-terminal, sulfated peptide (TPP-14827,SEQ ID NO:46 with Y3, Y15, Y17 being sulfated), as depicted in FIG.4.

For a first qualitative assessment, for each clone pool monoclonalcultivation and expression of 88 randomly picked Fab-on-phageclones was performed and clones were subsequently tested forbinding to the respective target previously used for panning. Inaddition, binding to human and cynomolgus CCR8 expressing cells wasdetermined by flow cytometry (FACS).

A specific binder is defined in the particular context as amolecule showing (i) in the ELISA assay a signal intensity for thetarget which is at least 10 times higher than the signal intensityfor the off-target, (ii) in FACS assay a signal for the CCR8expressing cell lines which is at least 2 times higher than thesignal for a control cell line not expressing CCR8, and (iii) inFACS assay a signal on the control cell line which is smaller than10000.

Plasmid-DNA from 12 pools with significant hit rates in at leastone of the two assays was submitted to gene III removal and theresulting soluble Fabs were screened in a high throughput ELISA andFACS screening.

Example 7: High Throughput Screening for Human Anti-Human CCR8Antibodies

From the 12 clone pools generated during phage display, 17000different sFab clones were screened in a high throughput screeningby ELISA (HTS-ELISA) to determine their binding to the peptidesthat were previously used for phage display panning, namely (i)TPP-14827, SEQ ID NO:46, with Y3, Y15, Y17 being sulfated, (ii)TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated and (iii)the respective off-target peptide TPP-14829, SEQ ID NO:22, with Y22being sulfated. For ELISA screening, the peptides were immobilizedon streptavidin coated plates (Greiner bio-one 781997) with aconcentration of 0.1 .mu.g/ml at 4.degree. C. in coating buffer(Carbonat-Basis, Candor 121125). After washing the plates 3 timeswith 60 .mu.l PBS 0.05% Tween and blocking with 50 .mu.l SmartBlock.RTM. (Candor 113500) for 1 h at 20.degree. C., 10 .mu.l sFabsamples were added to the plates and incubated at 20.degree. C. for1 h. After subsequent washing with 60 .mu.l PBS 0.05% Tween for 3times, 20 .mu.l of an anti-c-Myc HRP antibody were added andincubated for 1 h at 20.degree. C. followed by subsequent washing 3times with 60 .mu.l PBS 0.05% Tween and addition of 20 .mu.l AmplexRed solution (Invitrogen A12222, 1:1000 in NaP-buffer 50 mM pH7.6with 1:10000 of 30% H2O2). After final incubation for 20 min at20.degree. C. the signal was determined using an emissionwavelength of 595 nm and an excitation wavelength of 530 nm. Foranalysis, the signal to background ratio of the single sFab cloneswas used whereas the background is defined by the average value of48 wells on each plate, not containing any sFab culture but therespective sample medium.

In total 2193 different sFab clones showed significant binding toboth human and cynomolgus N-terminal, sulfated peptides TPP-14827(SEQ ID NO:46, with Y3, Y15, Y17 being sulfated) and TPP-14828 (SEQID NO:47 with Y3, Y15, Y17 being sulfated) as indicated by a signalto background ratio bigger than 5 while not demonstrating anysignificant binding to the off target TPP-14829 (SEQ ID NO:22 withY22 being sulfated). These sFab clones were reformatted into fulllength human IgG1 antibodies and applied to FACS screening on humanand cynomolgus CCR8 expressing cell lines.

Based on binding to both, cynomolgus as well as human CCR8expressing cells as well as not demonstrating any significantunspecific binding to a parental cell line not expressing CCR8, tenantibody clones were selected for production and furthercharacterization. These initial hits were TPP-17575 to TPP-17581and TPP-18205 to TPP-18207, the CDRs are shown in Table 7.1.Antibodies were further manually optimized for applicability intherapy to yield the set of antibodies shown in Table 7.2 a andTable 7.2 b, cf. also sequence listing.

Furthermore, in order to obtain antibody TPP-27495, the sequencesof TPP-23411 were engineered to comprise YTE mutations M252Y, S254Tand T256E. TPP-27495 is 70% afucosylated.

Furthermore, in order to obtain antibody TPP-27496, the sequencesof TPP-23411 were engineered to comprise LS mutations M428L andN434S. TPP-27496 is 70% afucosylated.

Example 8: Generation of Anti-Murine CCR8 Antibodies

Phage Display and Antibody Screening

A fully human antibody phage display library (Biolnvent n-CoDeR Fablambda library) was used to isolate human monoclonal antibodiesrecognizing murine CCR8 by selection against soluble biotinylatedpeptides. Peptides were provided by Pepscan. The synthesis ofpeptides was performed as described elsewhere herein. For thepanning procedure the following protocol was applied.

Streptavidin-coupled Dynabeads M-280 (Invitrogen.TM.) were coatedfor one hour at room temperature (RT) with the biotinylated peptide(1 tube) and the biotinylated off-target peptide (3 tubes),respectively. Dynabeads were washed and subsequently blocked for 1h at RT with end-over-end rotation. For depletion of off-targetbinders, the blocked phage library was added to the blockedoff-target loaded Dynabeads and incubated for 10 min at roomtemperature with end-over-end rotation. This depletion step wasrepeated 2 times. The depleted phage library was added to theblocked target loaded Dynabeads and incubated for 60 min at RT withend-over-end rotation.

After stringent washing (3.times. in blocking buffer (PBS-T, 3%milk powder) and 9.times. in PBS (150 mM NaCl; 8 mM Na2HPO4; 1.5 mMKH2PO4; adjusted to pH=7.4-7.6) with 0.05% Tween-20)), Dynabeadswith Fab-phages binding specifically to the coated target weredirectly used to infect E. coli strain HB101. Subsequently thephages were amplified in E. coli strain HB101 using M13KO7 HelperPhage (Invitrogen.TM.).

In the following selection rounds the target concentration wasdecreased to augment the selection pressure for high affinitybinders.

TABLE-US-00007 TABLE 7.1 CDRs for specific human anti-human CCR8candidate antibodies obtained with the method according to thecurrent invention. Identifier LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3TPP-17575 SGSSSNIGNNYVS GNSNRPS QTWGTGIRV SYAMH LISWDGGSTYYADSVKGGGIGRRPG- LEY (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 277) (SEQID NO: 280) NO: NO: 282) NO: NO: 278) 281) 276) TPP-17576SGSSSNIGNNAVN RNNQRSS QSYDNSLSASV SYAMS RIRSKANSYATAYAASVKG PLDS(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 295) (SEQ ID NO: 298)NO: NO: 300) NO: NO: 296) 299) 294) TPP-17577 SGSSSNIGSNTVN RNNQRPSAAWDDSLKALV NAWMS VISYDGRNKYSADSVKG GLPYGY (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 313) (SEQ ID NO: 316) NO: NO: 318) NO: NO: 314)317) 312) TPP-17578 SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV NAWMSGINWNGGSTGYADSVKG TYSGHY- GPYFDN (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 331) (SEQ ID NO: 334) NO: NO: 336) NO: NO: 332) 335)330) TPP-17579 SGSWSNIGNDNVY RNNQRPS QSYDRSLSGSV TYWMTGVSWNGSRTHYVDSVKR YSGYPD- YYGMDV (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 349) (SEQ ID NO: 352) NO: NO: 354) NO: NO: 350) 353)348) TPP-17580 SGSSSNIGSYPVN RNNQRPS SSYSVTDNLI SYGMHGVSWNGSRTRYADSVKG GSYNSGW- YAVS (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 367) (SEQ ID NO: 370) NO: NO: 372) NO: NO: 368) 371)366) TPP-17581 TGSSSNIGAGYDVH SHNQRPS SAWDSSLSAWV NYRMTGINWNGGSTGYADSVKG GRFDTRGFYGFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 385) (SEQ ID NO: 388) NO: NO: 390) NO: NO: 386) 389)384) TPP-18205 SGGNSNIGTYFVS TNNQRPS ETWDSNTRV SYAMSGIRWNSGSKGYAGSVKG SGNYYVGYHGMDV (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 403) (SEQ ID NO: 406) NO: NO: 408) NO: NO: 404) 407)402) TPP-18206 TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV HYGMHGINWNGGSTGYADSVKG GHHSG- YDGRFFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 421) (SEQ ID NO: 424) NO: NO: 426) NO: NO: 422) 425)420) TPP-18207 TGSSSNIGAGYDVH KNNQRPS AAWDDSLRGWV NAWMSGINWNGGSTGYADSVKG TYTGN- YGPYFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 439) (SEQ ID NO: 442) NO: NO: 444) NO: NO: 440) 443)438)

TABLE-US-00008 TABLE 7.2 a CDRs for specific human anti-human CCR8candidate antibodies according to the current invention havingsuperior therapeutic profile. Identifier LCDR1 LCDR2 LCDR3 HCDR1HCDR2 HCDR3 TPP- SGSSSNIGSHIVN GNTNRPS SSYTSISTLV SYSMNGVSWAGSRTHYADSVKG AAAGTRGFDY 16966 (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 259) (SEQ ID NO: 262) NO: 263) NO: 264) NO: 258) NO:260) TPP- SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV SAWMS GISWSGGSTGYADSVKGTYSGHYGPYFDY 17546 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 457)(SEQ ID NO: 532) NO: 533) NO: 534) NO: 456) NO: 458) TPP-SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV NAWMS GINWNGGSTGYADSVKGTYSGHYGPYFD- N 17578 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:331) (SEQ ID NO: 334) NO: 335) NO: 336) NO: 330) NO: 332) TPP-SGGNSNIGTYFVS TNNQRPS ETWDSNTRV SYAMS GIRWNSGSKGYAGSVKGSGNYYVGYHGMDV- 18205 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:403) (SEQ ID NO: 406) NO: 407) NO: 408) NO: 402) NO: 404) TPP-TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV HYGMH GINWNGGSTGYADSVKGGHHSGYDGRFFDY 18206 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:421) (SEQ ID NO: 424) NO: 425) NO: 426) NO: 420) NO: 422) TPP-SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV SAWMS GISWSGGSTGYADSVKGTYSGHYGPYFD- Y 19546 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:457) (SEQ ID NO: 460) NO: 461) NO: 462) NO: 456) NO: 458) TPP-TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AIKWGGGSHGYADSVKGGHHSGKDGRF- FDY 20950 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:475) (SEQ ID NO: 478) NO: 479) NO: 480) NO: 474) NO: 476) TPP-TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AIKWGGGSHGYADSVKGGHHKGYDGRF- FDY 20955 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:493) (SEQ ID NO: 496) NO: 497) NO: 498) NO: 492) NO: 494) TPP-TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AIKWGGGSTGYADSVKGGHHSGKDGRF- FDY 20965 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:511) (SEQ ID NO: 514) NO: 515) NO: 516) NO: 510) NO: 512) TPP-SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV SAWMS GISWSGGSTGYALSVKGTYSGHYGPFFD- Y 21045 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:529) (SEQ ID NO: 532) NO: 533) NO: 534) NO: 528) NO: 530) TPP-SGSSSNIGSHTVN RNNQRPS AAWDDSLNGWV SAWMS GISWSGGRTGYALSVKGTYSGHYGPYFD- Y 21047 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:547) (SEQ ID NO: 550) NO: 551) NO: 552) NO: 546) NO: 548) TPP-SGGNSNIGTYFVS TNNQROS ETWDSNTRV SYAMS GIRWNNGSKGYAGSVKGSGNYYYGYHGMDV- 21181 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:565) (SEQ ID NO: 568) NO: 569) NO: 570) NO: 564) NO: 566) TPP-SGGNSNIGTYFVS TNNQRPS ETWDSNTRV SYAMS GIRWNNGSKGYAGSVKGSGNEYYGYHGMDV- 21183 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:583) (SEQ ID NO: 586) NO: 587) NO: 588) NO: 582) NO: 584) TPP-TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AINWNGGSTGYADSVKGGHHSGYDGRF- FDY 23411 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:619) (SEQ ID NO: 622) NO: 623) NO: 624) NO: 618) NO: 620)

TABLE-US-00009 TABLE 7.2 b CDRs for anti-human CCR8 candidateantibodies derived from TPP-23411. Iden- tifier Mutation LCDR1LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 TPP- VL: TGSSSNIGAGYNVH TNNRRPSAAWDASLSGWV SYGMH GINWNGGSTGYADSVK GHIHSGY- DGRFFDY 29596 A10V,(SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID T13A; NO: 665) NO: 666)NO: 667) NO: 661) (SEQ ID NO: 662) NO: 663) VH: H31S; C- terminal KTPP- VL: T13; TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMHAINWNGGSTGYADSVK GH- IHSGYDGRFFDY 29597 VH: (SEQ ID (SEQ ID (SEQ ID(SEQ ID G (SEQ ID H31S, NO: 685) NO: 686) NO: 687) NO: 681) (SEQ IDNO: 682) NO: 683) G50A; C- terminal K TPP- VL: TGSSSNIGAGYNVHTNNRRPS AAWDASLSGWV HYGMH GINWNGGSTGYADSVK GHIHSGY- DGRFFDY 18429A10V (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID NO: 707) NO: 708)NO: 709) NO: 703) (SEQ ID NO: 704) NO: 705) TPP- VL: TGSSSNIGAGYNVHTNNRRPS AAWDASLSGWV HYGMH GINWNGGSTGYADSVK GHHSGYD- GRFFDY 18430T13A (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID NO: 727) NO: 728)NO: 729) NO: 723) (SEQ ID NO: 724) NO: 725) TPP- VL: TGSSSNIGAGYNVHTNNRRPS AAWDASLSGWV HYGMH GINWNGGSTGYADSVK GHHSGYD- GRFFDY 18432A10V, (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID T13A NO: 747) NO:748) NO: 749) NO: 743) (SEQ ID NO: 744) NO: 745) TPP- VH:TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH GINWNGGSTGYADSVK GHHSGYD-GRFFDY 18433 H31S (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID NO:767) NO: 768) NO: 769) NO: 763) (SEQ ID NO: 764) NO: 765) TPP- VH:TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV HYGMH AINWNGGSTGYADSVK GHHSGYD-GRFFDY 18436 G50A (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID NO:787) NO: 788) NO: 789) NO: 783) (SEQ ID NO: 784) NO: 785) TPP- VH:TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AINWNGGSTGYADSVK GHHSGYD-GRFFDY 19571 H31S, (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID G50ANO: 807) NO: 808) NO: 809) NO: 803) (SEQ ID NO: 804) NO: 805) TPP-VL: TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AINWNGGSTGYADSVKGHHSGYD- GRFFDY 27477 A10V; (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQID VH: NO: 831) NO: 832) NO: 833) NO: 827) (SEQ ID NO: 828) NO:829) H31S, G50A TPP- VL: TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMHAINWNGGSTGYADSVK GHHSGYD- GRFFDY 27478 T13A; (SEQ ID (SEQ ID (SEQID (SEQ ID G (SEQ ID VH: NO: 851) NO: 852) NO: 853) NO: 847) (SEQID NO: 848) NO: 849) H31S, G50A TPP- VL: TGSSSNIGAGYNVH TNNRRPSAAWDASLSGWV SYGMH GINWNGGSTGYADSVK GHHSGYD- GRFFDY 27479 A10V, (SEQID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID T13A; NO: 871) NO: 872) NO:873) NO: 867) (SEQ ID NO: 868) NO: 869) VH: H31S TPP- VL:TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV HYGMH AINWNGGSTGYADSVK GHHSGYD-GRFFDY 27480 A10V, (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID T13A;NO: 891) NO: 892) NO: 893) NO:8 87) (SEQ ID NO: 888) NO: 889) VH:G50A TPP- C- TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV HYGMHGINWNGGSTGYADSVK GHIHSGYD- GRFFDY 29367 terminal (SEQ ID (SEQ ID(SEQ ID (SEQ ID G (SEQ ID K NO: 911) NO: 912) NO: 913) NO: 907)(SEQ ID NO: 908) NO: 909) TPP- VL: TGSSSNIGAGYNVH TNNRRPSAAWDASLSGWV HYGMH GINWNGGSTGYADSVK GHIHSGY- DGRFFDY 29368 A10V,(SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID T13A; NO: 931) NO: 932)NO: 933) NO: 927) (SEQ ID NO: 928) NO: 929) C- terminal K TPP- VH:TGSSSNIGAGYNVH TNNRRPS AAWDASLSGWV SYGMH AINWNGGSTGYADSVK GHIHSGY-DGRFFDY 29369 H31S, (SEQ ID (SEQ ID (SEQ ID (SEQ ID G (SEQ ID G50A;NO: 951) NO: 952) NO: 953) NO: 947) (SEQ ID NO: 948) NO: 949) C-terminal K

TABLE-US-00010 TABLE 7.3 CDRs for specific anti-murine CCR8candidate antibodies derived with a mixed approach. IdentifierLCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 TPP-14099 SGSSFNIGSHFVY KNNQRPSAAWDDSLNGPV DYGVH GVSWNGSRTHYADSVKG RGA (SEQ ID (SEQ ID (SEQ ID(SEQ (SEQ ID NO: 217) (SEQ NO: 220) NO: 221) NO: 222) ID NO: ID NO:216) 218) TPP-14095 SGSSSNIGSNYVY GNNNRPS AAWDDSLNGWV SYGMHAISGSGGSTYYADSVKG GRQLGS- (SEQ ID (SEQ ID (SEQ ID (SEQ (SEQ ID NO:203) (SEQ NO: 206) NO: 207) NO: 208) ID NO: ID NO: 202) 204)TPP-15285 SGSSFNIGSHFVY KNNQRPS AAWDDSLNGPV DYGVH GVSWNGSRTHYADSVKGRGA (SEQ ID (SEQ ID (SEQ ID (SEQ (SEQ ID NO: 231) (SEQ NO: 234) NO:235) NO: 236) ID NO: ID NO: 230) 232) TPP-15286 SGSSSNIGSNYVYGNNNRPS AAWDDSLNGWV SYGMH AISGSGGSTYYADSVKG GRQLGS- (SEQ ID (SEQ ID(SEQ ID (SEQ (SEQ ID NO: 245) (SEQ NO: 248) NO: 249) NO: 250) IDNO: ID NO: 244) 246)

TABLE-US-00011 TABLE 8.1 List of peptides used for antibodyselection by phage display and ELISA screening Additional IDAntigen Species Domain Sulfation modifications TPP-13792 CCR8 mouseTRD no N-terminal (SEQ ID biotinylation NO: 45) via TTDS_Lysinlinker TPP-13793 CCR8 mouse TRD Y3, Y14, N-terminal (SEQ ID Y15biotinylation NO: 45) via TTDS_Lysin linker TPP-13799 CCR4 humanTRD no N-terminal (off-target) (SEQ ID biotinylation NO: 19) viaTTDS_Lysin linker TPP-13800 CCR4 human TRD Y19, Y22 N-terminal (SEQID biotinylation NO: 19) via TTDS_Lysin linker

TABLE-US-00012 TABLE 9.1 Structural analysis of HCDR3 for specifichuman anti-human CCR8 candidate antibodies obtained a methodaccording to the current invention. % % % Iden- % % % % % % (H +Posi- Nega- % tifier HCDR3 H K R Y D E H K R Y D E Y) tive tiveCharged Length TPP- GGIGRRPGLEY 0,0 0,0 2,0 1,0 0,0 1,0 0,0 0,018,2 9,1 0,0 9,1 9,1 18,2- 9,1 27,3 11,0 17575 (SEQ ID NO: 278)TPP- PLDS 0,0 0,0 0,0 0,0 1,0 0,0 0,0 0,0 0,0 0,0 25,0 0,0 0,0 0,025,0 25- ,0 4,0 17576 (SEQ ID NO: 296) TPP- GLPYGY 0,0 0,0 0,0 2,00,0 0,0 0,0 0,0 0,0 33,3 0,0 0,0 33,3 0,0 0,0 - 0,0 6,0 17577 (SEQID NO: 314) TPP- TYSGHYGPYFDN 1,0 0,0 0,0 3,0 1,0 0,0 8,3 0,0 0,025,0 8,3 0,0 33,3 8,- 3 8,3 16,7 12,0 17578 (SEQ ID NO: 332) TPP-YSGYPDYYGMDV 0,0 0,0 0,0 4,0 2,0 0,0 0,0 0,0 0,0 33,3 16,7 0,0 33,30- ,0 16,7 16,7 12,0 17579 (SEQ ID NO: 350) TPP- GSYNSGWYAVS 0,00,0 0,0 2,0 0,0 0,0 0,0 0,0 0,0 18,2 0,0 0,0 18,2 0,0 0,0 0,0 11,017580 (SEQ ID NO: 368) TPP- GRFDTRGFYGFDY 0,0 0,0 2,0 2,0 2,0 0,00,0 0,0 15,4 15,4 15,4 0,0 15,4- 15,4 15,4 30,8 13,0 17581 (SEQ IDNO: 386) TPP- SGNYYVGYHGMDV 1,0 0,0 0,0 3,0 1,0 0,0 7,7 0,0 0,023,1 7,7 0,0 30,8 7- ,7 7,7 15,4 13,0 18205 (SEQ ID NO: 404) TPP-GEIHSGYDGRFFDY 2,0 0,0 1,0 2,0 2,0 0,0 15,4 0,0 7,7 15,4 15,4 0,030,- 8 23,1 15,4 38,5 13,0 18206 (SEQ ID NO: 422) TPP- TYTGNYGPYFDY0,0 0,0 0,0 4,0 1,0 0,0 0,0 0,0 0,0 33,3 8,3 0,0 33,3 0,- 0 8,3 8,312,0 18207 (SEQ ID NO: 440) Aver- 0,4 0,0 0,5 2,3 1,0 0,1 3,1 0,04,1 20,6 9,7 0,9 23,8 7,3 10,6 17,9- 10,7 age

TABLE-US-00013 TABLE 9.2 Structural analysis of HCDR3 for specifichuman anti-human CCR8 candidate antibodies with superiortherapeutic profile, e.g. low internalization. % % Iden- % % % % %% % Posi- Nega- % tifier HCDR3 H K R Y D E H K R Y D E (H + Y) tivetive Charged Length TPP- AAAGTRGFDY 0,0 0,0 1,0 1,0 1,0 0,0 0,0 0,010,0 10,0 10,0 0,0 10,0 10- ,0 10,0 20,0 10,0 16966 (SEQ ID NO:260) TPP- TYSGHYGPYFDY 1,0 0,0 0,0 4,0 1,0 0,0 8,3 0,0 0,0 33,3 8,30,0 41,7 8,- 3 8,3 16,7 12,0 17546 (SEQ ID NO: 458) TPP-TYSGHYGPYFDN 1,0 0,0 0,0 3,0 1,0 0,0 8,3 0,0 0,0 25,0 8,3 0,0 33,38,- 3 8,3 16,7 12,0 17578 (SEQ ID NO: 332) TPP- SGNYYVGYHGMDV 1,00,0 0,0 3,0 1,0 0,0 7,7 0,0 0,0 23,1 7,7 0,0 30,8 7- ,7 7,7 15,413,0 18205 (SEQ ID NO: 404) TPP- GEIHSGYDGRFFDY 2,0 0,0 1,0 2,0 2,00,0 15,4 0,0 7,7 15,4 15,4 0,0 30,- 8 23,1 15,4 38,5 13,0 18206(SEQ ID NO: 422) TPP- TYSGHYGPYFDY 1,0 0,0 0,0 4,0 1,0 0,0 8,3 0,00,0 33,3 8,3 0,0 41,7 8,- 3 8,3 16,7 12,0 19546 (SEQ ID NO: 458)TPP- GEIHSGKDGRFFDY 2,0 1,0 1,0 1,0 2,0 0,0 15,4 7,7 7,7 7,7 15,40,0 23,1- 30,8 15,4 46,2 13,0 20950 (SEQ ID NO: 476) TPP-GEIHKGYDGRFFDY 2,0 1,0 1,0 2,0 2,0 0,0 15,4 7,7 7,7 15,4 15,4 0,030,- 8 30,8 15,4 46,2 13,0 20955 (SEQ ID NO: 494) TPP-GEIHSGKDGRFFDY 2,0 1,0 1,0 1,0 2,0 0,0 15,4 7,7 7,7 7,7 15,4 0,023,1- 30,8 15,4 46,2 13,0 20965 (SEQ ID NO: 512) TPP- TYSGHYGPFFDY1,0 0,0 0,0 3,0 1,0 0,0 8,3 0,0 0,0 25,0 8,3 0,0 33,3 8,- 3 8,316,7 12,0 21045 (SEQ ID NO: 530) TPP- TYSGHYGPYFDY 1,0 0,0 0,0 4,01,0 0,0 8,3 0,0 0,0 33,3 8,3 0,0 41,7 8,- 3 8,3 16,7 12,0 21047(SEQ ID NO: 548) TPP- SGNYYYGYHGMDV 1,0 0,0 0,0 4,0 1,0 0,0 7,7 0,00,0 30,8 7,7 0,0 38,5 7- ,7 7,7 15,4 13,0 21181 (SEQ ID NO: 566)TPP- SGNEYYGYHGMDV 1,0 0,0 0,0 3,0 1,0 1,0 7,7 0,0 0,0 23,1 7,7 7,730,8 7- ,7 15,4 23,1 13,0 21183 (SEQ ID NO: 584) TPP-GEIHSGYDGRFFDY 2,0 0,0 1,0 2,0 2,0 0,0 15,4 0,0 7,7 15,4 15,4 0,030,- 8 23,1 15,4 38,5 13,0 23411 (SEQ ID NO: 620) Aver- 1,3 0,2 0,42,6 1,4 0,1 10,1 1,6 3,5 21,3 10,8 0,5 31,4 15,2 11,4 2- 6,6 12,4age

For the generation of antibodies binding mouse CCR8, a selectionstrategy was designed to identify antibodies exhibiting bindingactivity towards the mouse CCR8 tyrosine rich domain (TRD) withinthe N-terminal region. For positive selection on the mouse CCR8 TRDpeptides, a mixture containing 50% normal peptide and 50% sulfatedpeptide was used (Seq ID NO:45 non-sulfated (TPP-13792) or with Y3,Y14, Y15 being sulfated (TPP-13793)). A depletion step was includedusing a biotinylated off-target peptide being the human CCR4 TRDwithin the N-terminal region. Also, the depletion step on humanCCR4 was conducted on a mixture of sulfated and non-sulfatedpeptide (Seq ID No: 19 non-sulfated (TPP-13799) or with Y19 and Y22being sulfated (TPP-13800)).

The panning strategy was to conduct four panning rounds in total,each on the specified mixture of sulfated and non-sulfatedpeptides, see FIG. 5.

For each clone pool monoclonal cultivation and expression of 88randomly picked Fab-phage clones was performed and clones weresubsequently tested for binding to the respective target usedbefore for panning. However, separate ELISA measurements wereconducted for the sulfated and the non-sulfated peptides.

In addition, binding to cells expressing human, mouse andcynomolgus CCR8 was determined by flow cytometry (FACS).

A specific binder is defined in the particular context as amolecule showing (i) in the ELISA assay a signal intensity for thetarget which is at least 10 times higher than signal intensity forthe off-target, and (ii) in FACS assay a signal for the CCR8expressing cell lines which is at least 10 times higher than thesignal for a control cell line not expressing CCR8.

Within these measurements, the antibodies TPP-14095 and TPP-14099were identified demonstrating binding to mouse CCR8 expressingcells but no binding to control cells, control cells expressinghuman CCR8, or control cells expressing cynomolgus CCR8. Further,the antibodies were found to bind to the sulfated TRD peptide butnot to the non-sulfated peptide.

Example 9: Structural Characterization of the CCR8 Antibodies

Out of the six CDR loops, the H3 loop shows the greatest structuraldiversity and is located in the center of the binding site. It alsogains the most mutations through affinity maturation and has onaverage the largest number of contacts with the antigen. Ittherefore plays a crucial role in antigen binding. Upon analysis ofthe specific structure of the antibodies obtained in the previousexamples, it was surprisingly found that the composition of theHCDR3 was structurally different from usual human HCDR3 domains. Inmore detail, the HCDR3 domain of the human anti-human CCR8antibodies had a substantially higher number of tyrosine residues(.about.20% for the initial set of specific human CCR8 binders,.about.21% for the optimized candidates) than would be expected fora random fully human HCDR3 with matched length (.about.10%, cf.Zemlin, Michael, et al. "Expressed murine and human HCDR3 intervalsof equal length exhibit distinct repertoires that differ in theiramino acid composition and predicted range of structures." Journalof molecular biology 334.4 (2003): 733-749.).

These data suggest a beneficial impact of tyrosine presence forspecific binding to the sulfated antigen. The optimized antibodieswere furthermore characterized by a substantial increase inhistidine frequency. For these antibodies, the histidine contentwas on average 10%. The average occurrence of histidine in HCDR3 ofa matched length is approximately 2% in murine HCDR3 and even lowerin human HCDR3, cf. Zemlin, Michael, et al. (2003).

Because the antigens used for selection are characterized bynegative charges provided in the form of sulfated tyrosines andnegatively charged amino acids, the inventors hypothesized that theoccurrence of positive charges in HCDR3 improves the binding to thesulfated antigen but is less essential for binding to thenon-sulfated antigen. While the frequency of positively chargedresidues (H, K, R) in the HCDR3s of the initially obtained set ofantibodies was approximately 7%, the improved antibodies had onaverage 15% of positive amino acids per HCDR3 (e.g. up to.about.31%).

The antibodies according to the current invention are characterizedby a tyrosine frequency which is higher than for usual fully humanHCDR3s. While these high tyrosine frequencies are more easilyobtained with murine CDRs this may explain why previous attempts toobtain human anti-human antibodies specifically recognizing CCchemokine receptors such as CCR8 have not been successful.

Example 10: Functional Characterization of the Anti-CCR8Antibodies

Cell Lines

Stable HEK 293 and CHO cell lines expressing human, cynomolgus ormurine CCR8 were generated using Inscreenex, as known in the art.Thymoma cell lines Hut-78 and lymphoma cell line TALL-1 wereobtained from ATCC and were confirmed to show endogenous CCR8expression. Because CCR8 expression may vary, expression levelswere monitored.

Human T Regulatory Cells

FACS sorted CD4+ and CD25+ cells from PBMCs of healthy donors werepurchased from either BioIVT, AllCells, or StemCell Technologies.After thawing the cells and recovery in culture overnight, thecells were activated with anti-CD3 and anti-CD28 beads designed forTreg expansion from Miltenyi and supplemented with IL-2 (R&DSystems) as directed by the Treg expansion kit protocol. Typically,after activation of naive PBMCs, CCR8 expression was the highestbetween day 5-7, and cells can be restimulated 7-9 days afterprimary activation. Upon restimulation, CCR8 expression was thehighest between day 2-4.

Example 10.1.1: Evaluation of Affinities for Binding CCR8 fromDifferent Species

FACS Experiments

The affinity of the antibodies for human, cynomolgus and murineCCR8 and further chemokine receptors was determined by FACS asknown in the art. In brief, CHO cells were engineered to expressthe CCR8 from the respective species. In the alternative, activatedhuman Tregs from a human donor were used to determine the affinityof the antibodies for activated Tregs. EC50 values were determinedby FACS. All candidate antibodies had not only a superior affinityfor human CCR8 with an EC50 in the low digit nanomolar range, butalso a superior affinity for cynomolgus CCR8 in the same order ofmagnitude. Similar affinities in these species are important tofacilitate studies for prediction of later safety issues in humans.This is a major issue in the search for therapeutic antibodies inimmune oncology, because mouse models have major limitations in theprediction of immunological side effects in humans. The EC50determined for binding human Tregs were between 10 and 25 nM forthe inventive antibodies shown in

Table 10.1.1.1

Affinities of inventive antibodies in CHO cells expressing humanCCR8, CHO cells expressing cynomolgus CCR8, or activated humanTregs (donor 1) are shown in FIGS. 6, 7 and 8, respectively.

TABLE-US-00014 TABLE 10.1.1.1 EC50 values of inventive antibodiesin nM. FACS staining was performed on CHO cells stably expressingeither human CCR8 or cynomolgus CCR8, or on human activatedCD4+CD25+ T cells. These cells were activated with the Tregexpansion kit from Miltenyi. TPP-17578 TPP-19546 TPP-18206TPP-21360 CHO with hCCR8: 1.66 0.78 0.64 0.40 EC50 in nM CHO withcynoCCR8: 1.04 0.52 0.67 0.34 EC50 in nM human Treg: 25.12 15.2523.1 10.35 EC50 in nM

Antibodies TPP-23411 and TPP-21360 deviate from each other by onelysine residue added at the C-term of the heavy chain, i.e.TPP-23411 comprises the lysine. TPP-23411 and TPP-21360 were testedin a separate FACS experiment for binding to human and cynomolgusCCR8. The EC50 curves for binding of human CCR8 as well ascynomolgus CCR8 were perfectly aligned between the two antibodies(data not shown, cf. Table 10.1.1.2 for IC50 values).

TABLE-US-00015 TABLE 10.1.1.2 EC50 values of candidates measured innM. Staining was performed on CHO cells stably expressing eitherhuman CCR8 or cynomolgus CCR8. TPP-21360 TPP-23411 CHO with hCCR8:EC50 in nM 1.4 1.7 CHO with cynoCCR8: EC50 in nM 0.8 0.9

TPP-21181 and TPP-23411 were tested in a separate FACS experimentfor binding to human and cynomolgus CCR8, cf. FIG. 9, 10.Respective EC50 values are listed in Table 10.1.1.3.

TABLE-US-00016 TABLE 10.1.1.3 EC50 values of antibodies measured innM. Staining was performed on CHO cells stably expressing eitherhuman CCR8 or cynomolgus CCR8. TPP-21181 TPP-23411 CHO with hCCR8:EC50 in nM 4.8 4.4 CHO with cynoCCR8: EC50 in nM 1.8 1.3

In a further FACS experiment, prior art antibodies L263G8(Biolegend) and 433H (ICOS, BD) were compared to TPP-21360 withregards to binding to human and cynomolgus CCR8, cf. FIG. 11, 12and Table 10.1.1.4. Prior art antibodies showed only low orsubstantially no overall binding to cynomolgus CCR8 (lowsaturation, here MFI<<1000). Only the antibodies according tothe current invention specifically recognized human and cynomolgusCCR8 with affinities in the same order of magnitude, i.e. in somecases even in the sub nanomolar range.

TABLE-US-00017 TABLE 10.1.1.4 EC50 values of TPP-21360 compared toL263G8 and 433H. Staining was performed on CHO cells stablyexpressing either human CCR8 or cynomolgus CCR8. TPP-21360 L263G8433H CHO with hCCR8: EC50 in nM 0.58 0.31 2.8 CHO with cynoCCR8:EC50 in nM 0.51 132 12.1

These results show that the use of the sulfated antigen facilitatesthe generation of cross-reactive antibodies such ascynomolgus/human for difficult targets such as CCR8, which arespecific for their intended target. As a proof of concept,activated human Tregs were stained with inventive antibodyTPP-23411 or prior art antibody L263G8, as shown in FIG. 13. Theinventive antibodies which recognize murine CCR8 have likewiseexcellent affinities for their target, as shown in Table10.1.1.5.

TABLE-US-00018 TABLE 10.1.1.5 EC50 values of TPP-14095 andTPP-14099. FACS staining was performed on CHO cells expressingmurine CCR8. TPP-14095 TPP-14099 EC50 in nM 0.7 3

Table 10.1.1.6 shows EC50 values for binding of various furtherinventive anti-human CCR8 antibodies to CHO cells transfected withhuman CCR8 or cynomolgus CCR8, or to Hut78 cells expressingendogenous human CCR8. Interestingly, antibodies having a G50Mmutation had a higher binding affinity to CHO cells expressingcynomolgus CCR8. Anti-CCR8 antibodies comprising this frameworkmutation are therefore preferred.

TABLE-US-00019 TABLE 10.1.1.6 EC50 values in M characterizing thebinding of inventive anti-human CCR8 antibodies to CHO cellstransfected with human CCR8 or cynomolgus CCR8, as describedelsewhere herein, or to cell line HuT78, which endogenouslyexpresses human CCR8. hCCR8:CHO cynoCCR8:CHO HuT78 Antibody EC50[M] EC50 [M] EC50 [M] TPP-17577 1.11E-08 9.81E-09 TPP-175781.15E-08 2.72E-09 1.78E-07 TPP-18205 8.33E-09 8.59E-08 1.41E-07TPP-18206 2.63E-09 2.23E-09 4.70E-08 TPP-20955 4.69E-10 4.75E-104.56E-10 TPP-21047 2.56E-09 3.77E-10 5.02E-09 TPP-23411 1.38E-097.76E-10 2.78E-09 TPP-27495 1.32E-09 5.35E-10 3.25E-09 TPP-274961.51E-09 4.55E-10 3.68E-09 TPP-18429 1.48E-09 2.25E-09 2.93E-08TPP-18430 1.69E-09 1.37E-09 5.24E-08 TPP-18432 2.21E-09 3.33E-091.91E-08 TPP-18433 1.48E-09 1.92E-09 3.30E-08 TPP-18436 1.12E-094.42E-10 3.76E-09 TPP-19571 1.48E-09 8.79E-10 1.49E-09 TPP-274782.74E-09 1.02E-09 TPP-27477 1.01E-09 4.18E-10 2.10E-09 TPP-274792.22E-09 5.88E-09 1.31E-08 TPP-27480 1.14E-09 7.70E-10 4.71E-09

Example 10.1.2: SPR Experiments for Characterization of BindingAffinities of Antibodies for Unmodified TRD Domains of DifferentSpecies

To analyze the affinity of the antibodies for their unmodified(i.e. non-sulfated) antigen, surface plasmon resonance (SPR)binding assays were performed on a Biacore T200 instrument at25.degree. C. with assay buffer HBS-EP+1.times., 1 mg/ml BSA, 300mM NaCl. IgGs were captured using anti-human Fc IgGs covalentlyamine coupled to a CM5 sensor chip. N terminally His6-tagged humanCCR8 TRD (TPP-19950: MDYTLDLSVTTVTDYYYPDIFSSP, SEQ ID NO:43),cynomolgus CCR8 TRD (TPP-19952: MDYTLDPSMTTMTDYYYPDSLSSP, SEQ IDNO:44) or murine CCR8 TRD (TPP-19951: MDYTMEPNVTMTDYYPDFFTAP, SEQID NO:45) was C-terminally fused with human serum albumin (HSA) togenerate the CCR8-HSA fusion proteins. The respective CCR8-HSAfusion protein was used as analyte with a concentration range of1.56-200 nM in multicycle kinetics mode. Obtained sensorgrams werefitted to a 1:1 Langmuir binding model. For human anti-human CCR8antibodies TPP-23411 and TPP-21360, binding was low (160 nM) andbinding to cynomolgus CCR8 TRD was even lower, e.g. in the .mu.Mrange, supporting the importance of sulfation for the antibodyrecognition for the inventive antibodies.

Table 10.1.2.1: Binding affinities of inventive antibodies for theHis6-tagged HSA-fused TRD of human, cynomolgus or murine CCR8determined by SPR. Binding to cynomolgus CCR8 peptide is in the.mu.M range and is an approximation as the highest concentrationtested was 200 nM. HSA alone and isotype controls did not show anybinding (data not shown).

TABLE-US-00020 TABLE 10.1.2.1 Binding affinities of inventiveantibodies for the His6-tagged HSA-fused TRD of human, cynomolgusor murine CCR8 determined by SPR. Binding to cynomolgus CCR8peptide is in the .mu.M range and is an approximation as thehighest concentration tested was 200 nM. HSA alone and isotypecontrols did not show any binding (data not shown). Ligand Analytek.sub.a [1/Ms] k.sub.d [1/s] K.sub.D [M] TPP-21360 cyno 3.5E+041.3E-01 3.8E-06 human 1.2E+05 1.8E-02 1.6E-07 mouse no bindingTPP-14099 cyno no binding human no binding mouse 2.2E+03 1.1E-035.2E-07 TPP-23411 cyno 6.2E+03 1.2E-01 2.0E-05 human 1.1E+051.7E-02 1.6E-07 mouse no binding

Example 10.1.3: SPR Experiments for Systematic Characterization ofBinding Affinities

SPR binding assays were performed to systematically evaluateantibody binding to a) non-sulfated vs sulfated antigen, b) TRD vsN term and c) human vs cynomolgus CCR8. SPR binding assays wereperformed on a Biacore T200 instrument (Cytiva) at 25.degree. C.with assay buffer lx HBS-EP+(order no. BR100826, Cytiva). Peptideswere used as ligands and were captured using their C-terminalbiotin label on to a streptavidin coated sensor chip ("Sensor ChipSA", Order No. BR100398, Cytiva). To this end, a terminal lysine(K) was added at the C-term of each peptide for biotinylation. Forthe N term of CCR8, the naturally occurring cysteine between TRDand LID domain was replaced by a serine. Antibodies were used asanalytes (TPP-19546, TPP-21181, TPP-23411, hIgG1 isotype controlTPP-9809 and mIgG2a isotype control TPP-10748). The concentrationof analyte in assay buffer was 0.05-10 nM and the measurement wasperformed in multicycle kinetics mode. The sensor surface wasregenerated with glycine pH 2.0 after each cycle. Obtainedsensorgrams were double referenced (subtraction of reference flowcell signal and buffer injection) and were fitted to a 1:1 Langmuirbinding model to derive kinetic data using the Biacore T200Evaluation software.

Table 10.1.3.1: List of peptides used to characterize the epitopeof the antibodies. Peptides comprising the TRD sequence (#1 to 4)or N term (#5 to 8) of CCR8, derived from cynomolgus (#1, 2, 5, 6)or human (#3, 4, 7, 8) CCR8, either sulfated (#2, 4, 6, 8) ornon-sulfated (#1, 3, 5, 7). SO3: sulfate residue. Biot: biotinmodification for immobilization.

TABLE-US-00021 TABLE 10.1.3.1 List of peptides used to characterizethe epitope of the antibodies. Peptides comprising the TRD sequence(# 1 to 4) or N term (# 5 to 8) of CCR8, derived from cynomolgus (#1, 2, 5, 6) or human (# 3, 4, 7, 8) CCR8, either sulfated (# 2, 4,6, 8) or non-sulfated (# 1, 3, 5, 7). SO3: sulfate residue. Biot:biotin modification for immobilization. # Type Peptide Sequence 1Cyno_TRD H-CMDYTLDPSMTTMTDYYYPDSLSSPK(Biot)-OH (SEQ ID NO: 966) 2Cyno_TRD_Ys H-CMDY(SO3)TLDPSMTTMTDY(SO3)YY(SO3)PDSLSSPK(Biot)-OH(SEQ ID NO: 967) 3 HUMAN_TRD H-CMDYTLDLSVTTVTDYYYPDIFSSPK(Biot)-OH(SEQ ID NO: 968) 4 HUMAN_TRD_YsH-CMDY(SO3)TLDLSVTTVTDY(SO3)YY(SO3)PDIFSSPK(Biot)-OH (SEQ ID NO:969) 5 Cyno_NTerm H-CMDYTLDPSMTTMTDYYYPDSLSSPSDGELIQRNDKK(Biot)-OH(SEQ ID NO: 970) 6 Cyno_NTerm_YsH-CMDY(SO3)TLDPSMTTMTDY(SO3)YY(SO3)PDSLSSPSDGELIQRNDK K(Biot)-OH(SEQ ID NO: 971) 7 HUMAN_NTermH-CMDYTLDLSVTTVTDYYYPDIFSSPSDAELIQTNGKK(Biot)-OH (SEQ ID NO: 972) 8HUMAN_NTerm_YsH-CMDY(SO3)TLDLSVTTVTDY(SO3)YY(SO3)PDIFSSPSDAELIQTNGK K(Biot)-OH(SEQ ID NO: 973)

For the analyzed inventive antibodies TPP-19546, TPP-21181, andTPP-23411, no binding to the non-sulfated TRD or non-sulfated Nterm of CCR8 was detected, independent of the species. In contrast,excellent affinities with K.sub.D values in the pM range weredetermined for sulfated TRD and sulfated N-term, e.g. of cynomolgusand/or human CCR8. In some cases, the K.sub.D value for binding tothe TRD and to the N term of human CCR8 was substantially the same,i.e. within the error margin. In other cases, the binding to the Nterm was slightly improved.

In summary, these results demonstrate that sulfated tyrosine iscausal for the binding. They furthermore suggest, that the sulfatedTRD is necessary and sufficient for binding, but that the presenceof the complete N term as epitope may in some cases further improvethe binding. Finally, these results also demonstrate thecross-reactivity for human and cynomolgus CCR8.

TABLE-US-00022 TABLE 10.1.2.3 SPR binding affinities of inventiveantibodies for the TRD or N term of cynomolgus or human CCR8.Peptides with odd numbers are characterized by unmodifiedtyrosines, peptides with even numbers are characterized by sulfatedtyrosines (Ys). Isotype controls did not show binding to any of thepeptides (data not shown). 1) mp = multiphasic: approximatedvalues. Ligand Activity Anti- k.sub.a k.sub.d K.sub.D R.sub.MaxLevel in Peptide # body (1/Ms) (1/s) (M) (RU) (RU) % 1) Cyno_TRD 1IgG01_ no binding 12 Cyno_TRD_Ys 2 TPP- 4.92E+06 8.44E-04 1.71E-1046 12.4 81.8 mp HUMAN_TRD 3 19546 no binding 17 HUMAN_TRD_Ys 46.69E+06 1.62E-03 2.42E-10 36 14.5 54.8 mp Cyno_NTerm 5 no binding27 Cyno_NTerm_Ys 6 1.67E+07 1.54E-03 9.22E-11 58 12 106.9 mpHUMAN_NTerm 7 no binding 14 HUMAN_NTerm_Ys 8 2.61E+07 4.19E-031.60E-10 24 21 25.2 mp Cyno_TRD 1 IgG02_ no binding 12 Cyno_TRD_Ys2 TPP- 4.23E+09 5.87E+00 1.39E-09 30 12.4 52.9 mp HUMAN_TRD 3 21181no binding 17 HUMAN_TRD_Ys 4 5.56E+09 1.60E+01 2.87E-09 16 14.523.8 mp Cyno_NTerm 5 no binding 27 Cyno_NTerm_Ys 6 1.23E+101.07E+01 8.68E-10 43 12 78.5 mp HUMAN_NTerm 7 no binding 14HUMAN_NTerm_Ys 8 7.69E+06 2.22E-02 2.88E-09 16 21 17.0 mp Cyno_TRD1 IgG03_ no binding 12 Cyno_TRD_Ys 2 TPP- 4.38E+06 4.54E-041.04E-10 46 12.4 82.1 mp HUMAN_TRD 3 23411 no binding 17HUMAN_TRD_Ys 4 2.86E+06 4.40E-04 1.54E-10 47 14.5 71.0 mpCyno_NTerm 5 no binding 27 Cyno_NTerm_Ys 6 8.21E+06 4.64E-045.65E-11 62 12 112.9 mp HUMAN_NTerm 7 no binding 14 HUMAN_NTerm_Ys8 9.19E+06 8.05E-04 8.76E-11 32 21 33.6 mp

Example 10.2: Specificity of Binding

FACS analysis was performed as described elsewhere herein todetermine unspecific binding to target negative human cell lines.No unspecific binding of anti-human CCR8 antibody TPP-23160 wasobserved for CHO cells with mock transfectants (FIG. 14).

In addition, HEK cells were transiently transfected with human CCR1or human CCR4. Off target binding was low for most of the inventiveantibodies (FIG. 15, 16).

A cell panel with cell lines from different tumor tissues was usedto characterize unspecific binding in these tissues (Table 10.2.1).The overall profile was favorable for most antibodies, while acertain degree of polyreactivity could be observed for someantibodies. Overall, the degree of polyreactivity was in anacceptable range for therapeutic applications for all inventiveantibodies. However, TPP-20950, TPP-18206, TPP-18429, TPP-18430,and TPP-18433 showed a higher degree of polyreactivity and showed acertain degree of binding in at least two or three out of the sevencell lines shown below.

TABLE-US-00023 TABLE 10.2.1 Polyreactivity of inventive antibodiesin a cell panel. If applicable, EC50 is shown in M. Burkitt LiverLiver Kidney Kidney Lung Colon lymphoma Antibody HepG2 SKHep1 786-OA498 H292 HT29 Raji TPP-19546 none none none none none noneTPP-20950 none none none 8.35E-07 7.59E-07 none TPP-20955 none nonenone none none none TPP-21045 1.48E-07 none none none none noneTPP-21360 none none none none none none TPP-5657 none none nonenone none none (Isotype) TPP-17577 none none none none none nonenone TPP-17578 none none none none none none none TPP-18205 nonenone none none binding none none TPP-18206 binding binding bindingnone none none none TPP-20955 none none none none none none noneTPP-21047 none none none none none none none TPP-23411 none nonenone none none none none TPP-27478 none none none none none nonenone TPP-27479 none none none none none none none TPP-27480 nonenone none none none none none TPP-27477 none none none none nonenone none TPP-27477 none none none none none none none TPP-27460none none none none none none none TPP-27461 none none none nonenone none none TPP-27496 none none none none none none noneTPP-27495 none none none none none none none TPP-18429 bindingbinding binding none none none none TPP-18430 binding binding nonenone none none none TPP-18432 none none none none none none noneTPP-18433 binding binding none none none none none TPP-18436 nonenone none none none none none TPP-23411 (afuco) none none none nonenone none none

A first ELISA based experiment was performed to analyze unspecificbinding to BVP, insulin or DNA for antibodies TPP-18206, TPP-17578,TPP-19546 and TPP-23411 and two reference antibodies. Unspecificbinding of the inventive antibodies to insulin, DNA and/or BVP waslower than unspecific binding of the first reference antibody(Gantenerumab, Roche) to these antigens, and was also lower thanunspecific binding of the second reference antibody (Remicade,Janssen Biotech) to these antigens.

Among the tested inventive antibodies, TPP-23411 showed the lowestpolyreactivity for each of DNA, BVP and insulin.

A second ELISA based experiment was performed to analyze unspecificbinding to BVP, insulin or DNA for antibodies TPP-18206 (wt orafuco), TPP-21360 afuco, TPP-23411 (wt or afuco), TPP-20955,TPP-21047, and references TPP-9809 (isotype control), Gantenerumab(TPP-12151), Lenzilumab (TPP-12166), Remicade (TPP-12160) andAvastin (TPP-17586). Unspecific binding of the tested inventiveantibodies to insulin, DNA as well as BVP was lower than unspecificbinding of the first reference antibody (Gantenerumab, Roche).TPP-20955 and TPP-21047 showed overall more unspecific binding thanthe other inventive antibodies (data not shown). Overall, thedegree of polyreactivity was in an acceptable range for therapeuticapplications for all inventive antibodies, independent of theirafucosylation status.

A third ELISA based experiment was performed to analyze unspecificbinding to BVP, insulin or DNA for antibodies TPP-27495 (YTE),TPP-27496 (LS), TPP-18429, TPP-18430, TPP-18432, TPP-18433,TPP-18436, TPP-27479, TPP-27480 and control antibodies. Overall,the degree of polyreactivity was in an acceptable range fortherapeutic applications for all inventive antibodies, independentof YTE or LS mutations.

Unwanted binding of inventive CCR8 antibodies to immune cellpopulations different from human Tregs was analyzed by staining ofthese cell populations, cf. FIG. 17.

FIG. 18 shows upregulation of CCR8 on human Tregs afteractivation.

Example 10.3: Evaluation of ADCC and ADCP Induction

Example 10.3.1: Afucosylation of Antibodies

The antibodies according to the current invention wereglycoengineered using GlymaxX technology, cf. U.S. Pat. No.8,642,292, to improve the interaction between the antibodies FCregion and the FC receptor expressed by the effector cells. Inbrief, the technology is based on the heterologous, cytosolicco-expression of bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulosereductase that redirects the de novo fucose synthesis pathwaytowards sugar-nucleotide GDP-Rhamnose that cannot be metabolized byeukaryotic cells. This depletes the fucose pool and leads to theproduction of afucosylated antibodies, both on the core part (nocore fucose) and on the variable part (no antennary fucose) of anN-glycan. It also inhibits the fucosylation of O-glycans andprotein-O-fucosylation. Removal of fucose from N-glycans on N297resulted in an increased affinity to Fc.gamma.Rs, as shown inexample 10.3.2.

Example 10.3.2: Human Fc.gamma. Receptor Binding Analysis Based onSPR

To determine the ability of the inventive antibodies and theirafucosylated versions to bind the respective FC receptors expressedon the human effector cells involved in ADCC or ADCP, SPR bindingassays were performed on a T200 instrument at 25.degree. C. with aCM5 sensor chip and assay buffer HBS-EP+500 mM NaCl. Fc.gamma.Rvariants were captured via an amine coupled anti-His captureantibody (ab) and IgGs were used as analyte at concentrations up to25 .mu.M. KD values were derived from a steady state affinityanalysis or from kinetic data fitted to a 1:1 Langmuir isotherm.Afucosylated versions of the antibodies showed increased binding toboth human and cynomolgus FcgRIIIa, suggesting an improved ADCCinduction and thus Treg depletion for the inventive antibodies intheir afucoslyated form in both species (Table 10.3.2.1).

Furthermore, a comparable affinity for both, cynomolgus and humanFc.gamma.RIII is important to model the ADCC efficacy in acynomolgus animal model. These model systems are particularlyimportant in tumor immunology because rodent models are often notsuited to reflect immunological side effects of a therapeuticantibody. The afucosylated antibodies according to the currentinvention showed highly comparable affinities for the respective FCreceptors in cynomolgus and human.

TABLE-US-00024 TABLE 10.3.2.1 SPR analysis of affinities ofinventive antibodies for different FC receptors. Italics:approximation only as highest concentration was 25 .mu.M and nosaturation was reached; n.e.: not evaluable; n.b.: no binding; res.bdg.: residual binding, not quantitatively evaluable; KD >25.mu.M: fitted value outside of saturation. curve. TPP-9809: isotypecontrol. Human Human Human Fc Fc Human Human Fc gamma Cyno CynoCyno Human gamma Fc Fc gamma RIIIA/ Fc Fc Fc Fc RIIA/ gamma gammaRIIIA/ CD16a gamma gamma gamma Engi- gamma CD32a RIIIB/ RIIB/CCD16a (F176) RIII/ RIIA/ RIIB/ KD [M] neered RI/CD64 (R167) CD16b(CD32b/c) (V167) (Acro) CD16 CD32a CD32- b TPP-19546 Afuco 1.10E-082.20E-05 4.30E-06 n.b. 3.40E-07 4.20E-07 1.40E-07 KD >25 .mu.MKD >25 .mu.M TPP-19546 1.80E-07 n.b n.b. n.b. 4.80E-06 KD >10.mu.M 3.10E-06 n.b. n.b. TPP-20950 Afuco 1.10E-07 1.70E-05 4.60E-06KD >25 .mu.M 2.80E-07 3.70E-07 1.70E-07 KD >25 .mu.M KD>25 .mu.M TPP-20950 1.80E-07 n.b. n.b. n.b. 7.60E-06 KD >20.mu.M 5.80E-06 n.b. n.b. TPP-20955 Afuco 1.40E-07 6.20E-06 3.10E-069.80E-06 3.40E-07 4.20E-07 1.80E-07 KD >25 .mu.M KD >25 .mu.MTPP-20955 2.80E-07 n.b. n.b. n.b. 8.90E-06 KD >25 .mu.M 5.60E-06n.b. n.b. TPP-20965 Afuco 1.10E-07 1.60E-05 4.60E-06 n.b. 4.30E-075.20E-07 1.90E-07 KD >25 .mu.M KD >25 .mu.M TPP-209652.00E-07 KD >25 .mu.M n.b. n.b. 3.10E-06 7.60E-06 2.30E-05 n.b.n.b. TPP-21045 Afuco 7.95E-09 1.06E-05 3.31E-06 n.b. 2.88E-073.16E-07 1.17E-07 KD >25 .mu.M KD >25 .mu.M TPP-210457.62E-08 KD >25 .mu.M n.b. n.b. 3.84E-06 KD >25 .mu.M2.23E-06 n.b. n.b. TPP-21047 Afuco 7.73E-08 9.00E-06 2.97E-06 KD>25 .mu.M 2.44E-07 2.68E-07 9.39E-08 KD >25 .mu.M KD >25.mu.M TPP-21047 7.26E-08 1.51E-05 n.b. KD >25 .mu.M 2.79E-061.01E-05 1.46E-06 KD >25 .mu.M KD >25 .mu.M TPP-21360 Afuco1.32E-07 1.22E-05 4.63E-06 KD >25 .mu.M 4.01E-07 4.78E-071.74E-07 KD >25 .mu.M KD >25 .mu.M TPP-21360 2.72E-07 n.bn.b. n.b. 5.76E-06 KD >25 .mu.M 3.39E-06 n.b. n.b. TPP-9809Afuco 1.32E-07 1.29E-05 5.03E-06 KD >25 .mu.M 4.59E-07 5.68E-072.13E-07 KD >25 .mu.M KD >25 .mu.M TPP-9809 1.69E-07 1.74E-05KD >25 .mu.M KD >25 .mu.M 5.43E-06 1.38E-05 4.28E-06 KD>25 .mu.M KD >25 .mu.M TPP-9809 wild 2.30E-07 1.77E-051.96E-05 KD >25 .mu.M 5.57E-06 1.32E-05 2.26E-06 KD >25 .mu.M1.84E-05

Example 10.3.3: ADCC Induction Mediated by CCR8 Antibodies

Example 10.3.3.1: ADCC Induction Mediated by Anti-Human CCR8Antibodies

For functional ADCC assays, either HEK cells expressing human CCR8or activated human Tregs were used as target cells, andNK92v-GPF-CD16 176V (NantKwest) were used as effector cells. In a96 well tissue culture treated plate, a co-culture of therespective target cells expressing CCR8 with effector cells at aratio of 4:1 in the presence of various concentration oftherapeutic antibody were incubated in RPMI 1640 with 1% heatinactivated FBS, 100 U/ml Pen/Strep, 1 mN Na-pyruvate, and1.times.NEAA. Digitonin was used for maximum lysis, and CytoTox-Glo(Promega) was added to the assay at the end of a 2 hr incubation.Raw luminescent values were determined using the CytoTox-gloPromega program. The Mean Max Background, MMB=(Target Max)-(TargetSpontaneous) is used to calculate % lysis. % Lysis=(RawLuminescent-no ab)/MMB.times.100.

For antibody candidates TPP-19546 (upper panel) and TPP-21360(lower panel), afucosylation increased ADCC induced cytotoxicity to.about.50 and .about.70% respectively in HEK cells expressing humanCCR8 (cf. FIG. 19). On activated human Tregs with about 85% CCR8expression, ADCC derived cytotoxicity resulting from afucosylatedTPP-21360 or TPP-23411 was as high as 59% or 52% (cf. FIG. 20,Table 10.3.3.1.3). For afucosylated TPP-21360 the average EC50 of %cytotoxicity for activated human Tregs with about 85% CCR8expression using NK92v as effector cells was .about.20 pM. Tables10.3.3.1.1 to 10.3.3.1.6 show the results for different inventiveantibodies and/or different donors.

TABLE-US-00025 TABLE 10.3.3.1.1 ADCC assay summary: EC50 and Maxresponse using activated Tregs as target cells and NK92v cell lineas effector cells. ADCC Potency Donor 1730 Donor 1213 EC.sub.50 MaxResponse EC.sub.50 Max Response Antibody Isotype (nM) (%) (nM) (%)TPP-17577 WT 5.76E+11 13 ~7.98e+014 10 Afuco 4.57 40 1.85 41TPP-18342 WT 1.50 15 0.37 25 Afuco 0.28 40 0.295 62 TPP-17578 WT1.97E+05 8 5 Afuco 19.0 38 9.2 25 TPP-18205 WT ~1.8e-2 24 ~5.58e-320 Afuco 1.05 58 0.23 52 TPP-18206 WT 4.68 12 8.59E+15 4 Afuco 1.6851 0.23 47 TPP-9809 Afuco 6 4 Mogamulizumab 1.09 65 0.31 58

TABLE-US-00026 TABLE 10.3.3.1.2 ADCC assay summary: EC50 and Maxresponse using activated Tregs as target cells and NK92v cell lineas effector cells. All TPPs used in this assay were afucosylated.ADCC potency (Donor 1100) TPP- TPP- TPP- TPP- TPP- Iso 19546 2136020945 20950 20955 afuco Moga hTreg 8840 4.41 3.2 9.51 5.82 NA 35.2EC.sub.50 (pM) Max 54 98 96 97 91 36 96 Response (%) R.sup.2 0.750.80 0.61 0.89 0.50 NA 0.94

TABLE-US-00027 TABLE 10.3.3.1.3 ADCC assay summary: EC50 and Maxresponse using activated Tregs as target cells and NK92v cell lineas effector cells. The activated human Treg used in this study hadabout 85% CCR8 expression. ADCC potency (Donor 1100) TPP-21360TPP-23411 TPP-9809 WT Afuco WT Afuco WT Afuco Moga hTreg EC.sub.50(pM) 271 18.5 10763 22.1 NA NA 193.4 Max Response 26 59 27 52 8 874 (%) R2 0.97 0.98 0.61 0.96 NA NA 0.89

TABLE-US-00028 TABLE 10.3.3.1.4 ADCC assay summary: EC50 and Maxresponse using activated Tregs as target cells and NK92v cell lineas effector cells. The activated human Treg used in this study hadonly about 31% CCR8 expression. ADCC potency (Donor 1261) TPP-21360TPP-23411 TPP-9809 WT Afuco WT Afuco WT Afuco Moga hTreg 81.3 65.6~157 55.9 NA NA 86.4 EC.sub.50 (pM) Max Response 13 25 8 24 2 3 71(%) R.sup.2 0.27 0.90 0.30 0.64 NA NA 0.97

TABLE-US-00029 TABLE 10.3.3.1.5 ADCC assays: EC50 and Max responseusing HEK cells expressing human CCR8 (>95% CCR8) as targetcells and NK92v cell line as effector cells. ADCC Potency:HEK-hCCR8 + NK92v Afucosylated TPP-27479 TPP-27478 TPP-23411TPP-9809 EC50 (pM) 363 110 141 N/A Max Response 32 35 35 17 (%)R.sup.2 0.94 0.82 0.85 N/A ADCC Potency: HEK-hCCR8 + NK92vAfucosylated TPP-29369 TPP-29367 TPP-23411 TPP-9809 EC50 (pM) 26.3137 68.7 N/A Max Response 38 22 34 7 (%) R.sup.2 0.65 0.36 0.71N/A

TABLE-US-00030 TABLE 10.3.3.1.6 ADCC assays: EC50 and Max responseusing HEK cells expressing human CCR8 (>95% CCR8) as targetcells and NK92v cell line as effector cells. ADCC Potency:HEK-hCCR8 + NK92v Afucosylated TPP-29369 TPP-29367 TPP-23411TPP-29368 TPP-9809 EC50 (pM) 99 460 167 2892 N/A Max 36 31 41 25 20Response (%) R.sup.2 0.91 0.83 0.89 0.72 N/A ADCC Potency:HEK-hCCR8 + NK92v n = 2 TPP-27479 TPP-27478 TPP-23411 TPP-29368TPP-9809 EC50 (pM) 823 11 182 2292 N/A Max 25 32 25 26 8 Response(%) R.sup.2 0.67 0.57 0.66 0.36 N/A

Example 10.3.3.2: ADCC Induction Mediated by Anti-Mouse CCR8Antibodies (Surrogate Antibodies)

To compare the behavior of the inventive human anti-human/cyno CCR8antibodies with the inventive human anti-murine CCR8 surrogateantibodies, the latter were likewise characterized for ADCCinduction. The functional ADCC assay for the surrogate antibodieswas performed using primary murine NK cells as effector cells andmurine CCR8 expressing HEK293 cells and BW 5417.3 (data not shown)as target cells. Ultra-low IgG containing medium was used duringcoculturing to avoid unspecific binding of CCR8 antibodies to serumIgGs. Furthermore, to more closely reflect the in vivo situation,target cells were first preincubated with surrogate CCR8 antibodiesbefore effector cells were added, allowing for better Fc.gamma.RIIIclustering on effector cells, that is the early step of ADCCmode-of-action. Effector and target cells were cocultured at aratio of 10:1 in RPMI 1640 with 1% Ultra-low IgG FBS One Shotmedium for 20 hours using U-bottom 96-well plates. Surrogateanti-murine CCR8 antibody TPP-15285 and isotype control antibodyTPP-10748 were used in various concentration steps. Cytotoxicitywas determined using the CytoTox-Glo.TM. Cytotoxicity Assay(Promega) (% Cytotoxicity=[(Experimental value-Control w/oantibody/(Maximal lysis of target cells-Spontaneous lysis of targetcells)].times.100%). Experimental values were determined bycoculture of target cells with effector cells after addition ofsurrogate CCR8 antibodies. Control values were determined bycoculture of target cells with effector cells without addition ofinventive antibodies. Maximal lysis values were determined fortarget cells where Digitonin had been added at the start of theassay. Spontaneous lysis values were determined by measuring thespontaneous lysis of target cells in assay medium without additionof any further reagents. 92% to 97% of target cells showedexpression of murine CCR8 (determined by FACS). The ADCC-relatedcytotoxicity for TPP-15285 was .about.39% and the mean EC50 was.about.111 .mu.M (Table 10.3.3.2.1). The highest inventive antibodyconcentration resulted in a hook effect, further supporting thesuggested ADCC-based mode-of-action. The optimized functional ADCCassay thus confirmed that ADCC is a relevant mode of action for thein vivo efficacy of the inventive surrogate CCR8 antibodies andthus confirm the suitability of anti-murine CCR8 antibody TPP-15285as surrogate antibody for the anti-human CCR8 antibodies.

TABLE-US-00031 TABLE 10.3.3.2.1 ADCC assay summary usingfucosylated (wild type) surrogate CCR8 antibodies. ADCC potency(wild type) TPP-15285 TPP-10748 EC.sub.50 (pM) 111 NA Max Response(%) 39 7 R.sup.2 0.89 NA

TABLE-US-00032 TABLE 10.3.3.2.2 ADCC assays showing cytotoxicity in% induced by wild type surrogate CCR8 antibody TPP-15285 (4replicates). ng/ml TPP-15285 TPP-10748 10000 15.02 36.48 33.4220.33 -1.62 5.23 6.95 -8.48 1000 15.04 38.89 39.03 35.56 -0.77-2.64 3.93 -0.76 100 22.44 29.84 28.11 43.39 3.27 3.99 -0.64 -1.4110 -2.14 8.68 3.88 35.69 11.76 -1.64 -7.4 1.79 1 -8.82 -7.13 5.0211.33 5.46 -3.06 -5.7 -0.72 0.1 -8.4 -2.87 5.78 9.85 10.44 -8.694.64 -0.8 0.01 -12.68 -2.77 10.75 3.8 1 5.51

Example 10.3.4: ADCP Induction Mediated by Anti-CCR8 Antibodies

Example 10.3.4.1: ADCP Induction Mediated by Anti-Human CCR8Antibodies

To generate macrophage (M2c) effector cells, CD14+ cells werenegatively selected (Miltenyi) from PBMC of healthy donors(AllCells). M2c macrophage differentiation was performed accordingto an eight days differentiation protocol provided by StemCell Tech(see FIG. 21). On the assay day, M2c were stained for expression ofCD16, CD32, CD64, CD163, CD206, CD11b, CD80, and CD14 using FACS.The target cells were either HEK cells expressing human CCR8 oractivated human Treg cells. Briefly, the target cells were labeledwith carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher)according to the manufacturer's instructions. The target cells wereincubated in a co-culture in 96 well plates with differentconcentrations of antibodies and effector cells at target toeffector ratio of 4:1. In these assays, anti-CD47 (positivecontrol) was added at 1 .mu.g/ml; this gives enhanced response asit blocks do not eat me signal. After 3-4 hours incubation, thesamples were run on MACSQuant or Attune flow cytometer. %phagocytosis was determined by CD206+CFSE+ double positive cells.CD206 is a marker expressed on macrophages.

M1 macrophages were generated similarly and from the same sourcematerial (fresh PBMC). In brief, CD14+ cells were isolated bynegative selection and cultured for four days in serum-free media(StemCell Tech Immnocult) with 50 ng/mL M-CSF. However, at day 5,M1 macrophages were polarized by addition of 1 Ong/mL LPS+50 ng/mLIFN-g whereas M2c macrophage were polarized with the addition of 10ng/mL IL-10.

FIG. 22 shows induction of phagocytosis in HEK cells expressinghuman CCR8 as target cells with in vitro differentiated macrophagesM2c as effector cells. Both, wild type and afucosylated versions ofinventive antibodies TPP-19546, TPP-21360 and TPP-23411 inducedADCP. FIGS. 23 and 24 and Tables 10.3.4.1.1 to 10.3.4.1.7 show theADCP induction as characterized by EC50 and maximal response formultiple inventive antibodies, multiple donors and differentmacrophage populations. For all antibodies, a substantial degree ofADCP was observed.

TABLE-US-00033 TABLE 10.3.4.1.1 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. All the TPPs used in this assay wereafucosylated. TPP-9809 is the isotype control. For human Treg donor1212 CCR8 was expressed by 40% of cells. ADCP Potency (hTreg Donor1212 + M2c Donor) TPP- TPP- TPP- TPP- TPP- TPP- 19546 21045 2136020950 20955 9809 EC50 (pM) NA 1220 577 128 408 NA Max Response 2030 39 32 29 15 (%) R.sup.2 NA 0.49 0.88 0.71 0.83 NA

TABLE-US-00034 TABLE 10.3.4.1.2 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. For human Tregdonor 1163 CCR8 was expressed by 67% of cells. ADCP Potency (hTregDonor 1163 + M2c Donor 1) TPP- TPP- TPP-19546 TPP-21045 21360 20965TPP-9809 WT Afuco WT Afuco Afuco Afuco WT Afuco EC50 NA NA 130 30.1205 766 NA NA (pM) Max 14 15 27 28 39 44 11 10 Response (%) R.sup.2NA NA 0.95 0.88 0.96 1.0 NA NA

TABLE-US-00035 TABLE 10.3.4.1.3 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. For human Tregdonor 1163 CCR8 was expressed by 67% of cells. ADCP Potency (hTregDonor 1163 + M2c Donor 2) TPP-19546 TPP-21045 TPP-21360 TPP-20965TPP-9809 WT Afuco WT Afuco Afuco Afuco WT Afuco EC50 (pM)~1.688e+007 404.4 162 52.9 105 300 NA NA Max Response (%) 18 18 2728 37 36 11 14 R.sup.2 0.94 0.87 0.98 0.95 0.97 0.98 NA NA

TABLE-US-00036 TABLE 10.3.4.1.4 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. For human Tregdonor 1212 CCR8 was expressed by 40% of cells. ADCP Potency (humanTreg Donor 1212 + M2c Donor 3) TPP- TPP- TPP- TPP- TPP- TPP-Afucosylated 20965 21360 21045 20950 20955 9809 EC50 (pM) 163 519.2 23 14 NA Max Response 14 14 13 14 13 7 (%) R.sup.2 0.99 0.960.79 0.93 0.96 NA

TABLE-US-00037 TABLE 10.3.4.1.5 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. For human Tregdonor 1212 CCR8 was expressed by 40% of cells. ADCP Potency (humanTreg Donor 1212 + M2c Donor 4) TPP- TPP- TPP- TPP- TPP- TPP-Afucosylated 20965 21360 21045 20950 20955 9809 EC50 (pM) 563 27.517.1 10.7 13.2 NA Max Response (%) 23 23 23 24 23 14 R.sup.2 0.930.99 0.99 0.97 0.98 NA

TABLE-US-00038 TABLE 10.3.4.1.6 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. ADCP Potency (humanTreg Donor 1100 + M2c Donor 5) TPP-21360 TPP-23411 TPP-9809 WTAfuco WT Afuco WT Afuco EC50 (pM) 1855 2003 1607 2188 NA NA MaxResponse (%) 25 25 26 26 19 21 R.sup.2 0.94 0.90 0.87 0.90 NANA

TABLE-US-00039 TABLE 10.3.4.1.7 ADCP assay summary: EC50 and Maxresponse using activated Tregs as target cells and M2c macrophagesas effector cells. TPP-9809 is isotype control. ADCP Potency (humanTreg Donor 261 + M2c Donor 6) TPP-21360 TPP-23411 TPP-9809 WT AfucoWT Afuco WT Afuco EC50 (pM) 870 664 1257 613 NA NA Max Response (%)24 23 23 23 17 18 R.sup.2 0.99 0.97 0.95 0.97 NA NA

TABLE-US-00040 TABLE 10.3.4.1.7 ADCP assay summary with Incucytelive-imaging: EC50 and Max response using macrophages as effectorcells and human CCR8 expressing HEK cells as target cells at ratioof 4:1. Human CCR8 expression was 80%. TPP-9809 is isotype control.M2c macrophages were plated into ultralow attachment96-well-plates. After addition of the therapeutic antibodies invarious concentrations the pHrodo red dye (Sartorius #4649) labeledtarget cells were added and plates were incubated at 37.degree. C.and 5% CO.sub.2. Phase contrast and red fluorescence images wereautomatically acquired and analyzed every 30 min for 12 hours using20.times. objective in Incucyte S3 (4 pictures/ well intriplicates). ADCP induction varied for tested therapeutic wildtype antibodies. ADCP Potency (human CCR8-HEK + M2c Donor) TPP-TPP- TPP- TPP- TPP- TPP- 23411 29368 29369 27478 27479 9809 EC50(pM) 227 326 102 151 79.5 NA Max 13.7 15.0 13.3 10.9 9.8 6.7Response (%) R.sup.2 0.97 0.97 0.96 0.93 0.95 NA

Example 10.3.4.2: ADCP Induction Mediated by Anti-Murine CCR8Antibodies (Surrogate Antibodies)

To compare the behavior of the inventive human anti-human/cyno CCR8antibodies with the inventive human anti-murine CCR8 surrogateantibodies, the latter were likewise characterized for ADCPinduction. For the functional ADCP assay primary murine bone marrowderived M2 macrophages were used as effector cells and murine CCR8expressing HEK293 cells and BW 5417.3 cells (data not shown) astarget cells. To generate the effector cells, murine bone marrowderived macrophages were plated into 24-well plate and polarizedinto M2 macrophages by addition of 20 ng/ml M-CSF, 0.05 .mu.g/mlIL-4 and 0.05 .mu.g/ml IL-13. Target cells were labeled withcarboxyfluorescein succinimidyl ester dye (CFSE, ThermoFisher) andwere added to effector cells in effector to target ratio of 2:1.Finally, surrogate anti-murine CCR8 antibody TPP-15285 and isotypecontrol TPP-10748 were added in various concentrations. After 2hours of coculturing cells were stained for murine M2 macrophagemarker F4/80 and % of phagocytosis was determined using flowcytometry measuring the fraction of F4/80+ and CFSE+ doublepositive macrophages. 92% to 97% of target cells showed expressionof murine CCR8 (as determined by FACS). Phagocytosis induced byTPP-15285 was .about.11% and the mean EC50 was .about.402 .mu.M(Table 10.3.4.2.1). The highest antibody concentration resulted ina hook effect, further supporting the suggested mode of action. Theoptimized functional ADCP assay further confirmed that ADCP is arelevant mode of action for the in vivo efficacy of the inventivesurrogate CCR8 antibodies and thus confirm the suitability ofanti-murine CCR8 antibody TPP-15285 as surrogate antibody for theanti-human CCR8 antibodies.

TABLE-US-00041 TABLE 10.3.4.2.1 ADCP assay summary usingfucosylated (wild type) surrogate CCR8 antibodies. TPP-15285TPP-10748 EC50 (pM) 402 NA Max Response (%) 11 5 R.sup.2 0.93NA

TABLE-US-00042 TABLE 10.3.4.2.2 ADCP assay using surrogate CCR8antibodies. ng/ml TPP-15285 TPP-10748 10000 9 9.28 4.26 4.47 100010.33 11.61 4.96 4.12 100 8.3 8.79 4.93 5.61 10 4.99 4.49 3.6 4.121 4.34 4.28 3.59 4.54

Example 10.4: Modulation of CCR8 Signaling by CCR8 Antibodies

CCL1 is a specific ligand of CCR8. Upon binding to CCR8, CCL1 caninduce calcium (Ca) flux, chemotaxis, and--via .beta.-arrestinsignaling--receptor internalization, the latter being possiblyindependent of G protein signaling. There are at least 6 differentways an antibody can modulate a GPCR: 1. An inverse agonist, uponbinding, quiets the potential endogenous activation of the GPCR.For CCR8 the endogenous activation can be e.g. .about.10%. 2. Anantagonist biased to .beta.-arrestin or G-protein blocks eitherG-protein dependent signaling or .beta.-arrestin signaling and hasno effect on the respective other pathway. 3. A neutral antagonistcan block both G-protein and .beta.-arrestin signaling pathways. 4.A full agonist activates both G-protein and .beta.-arrestinsignaling pathways. 5. An agonist biased to .beta.-arrestin orG-protein activates either G-protein signaling or .beta.-arrestinsignaling pathways, but not both. 6. A dimerization promotingagonist crosslinks two GPCRs, e.g. with the different arms of theantibody, resulting in various signaling activities

For an ADCC and ADCP based approach for depleting intra-tumoralTregs, it would be ideal to exclude any potential signaling effectsthat an antibody can have on CCR8. To assess this, the inventorsused Ca flux assay as a readout for G-protein dependent signaling,and .beta.-arrestin activation and phospho signaling as a readoutfor G-protein independent signaling pathways. Phospho Erk1/2 isinvolved in the MAP kinase pathway and regulates a variety ofcellular responses, while phospho AKT is well known to beassociated with the PI3 kinase pathway and has been shown in CCR8to be involved in promoting chemotaxis. AKT pathway is alsoinvolved in promoting cell survival and growth, and recently, CCL1has been shown to promote survival of CCR8 expressing cells(Barsheshet, Yiftah, et al. "CCR8+ FOXp3+ Treg cells as masterdrivers of immune regulation." Proceedings of the National Academyof Sciences 114.23 (2017): 6086-6091.).

Example 10.4.1: DiscoverX Assay for Monitoring of .beta.-ArrestinSignaling

In response to a stimulus, i.e. the binding of a ligand--such asCCL1 for CCR8--GPCRs can activate G-protein independent signaling,such as .beta.-arrestin signaling. This can result in theinternalization of the chemokine receptor (Fox, James M., et al."Structure/Function Relationships of CCR8 Agonists and Antagonists.Amino-terminal extension of CCL1 by a single amino acid generates apartial agonist." Journal of Biological Chemistry 281.48 (2006):36652-36661). The .beta.-arrestin assay was purchased fromDiscoverX. In brief, CCR8 is fused in frame with a small enzymedonor fragment ProLink.TM. (PK) and co-expressed in cells stablyexpressing a fusion protein of .beta.-arrestin and the larger,N-terminal deletion mutant of .beta.-galactosidase (called enzymeacceptor or EA). Activation of CCR8 stimulates binding of.beta.-arrestin to the PK-tagged CCR8 and forces complementation ofthe two enzyme fragments, resulting in the formation of an active.beta.-galactosidase enzyme. This interaction leads to an increasein enzyme activity that can be measured using chemiluminescentPathHunter Detection Reagents.

In a first experiment, antibody or CCL1 was incubated with the CHOcells co-expressing human CCR8 tagged with ProLink and EA for 90min before adding detection agent. Neither the prior art antibodiesnor the inventive antibodies induced .beta.-arrestin signaling(FIG. 25).

In a next experiment, the cells were stimulated with CCL1 at EC80to activate .beta.-arrestin signaling, and inventive antibodies orprior art antibodies were added to evaluate their ability to blockactivated .beta.-arrestin signaling. Surprisingly, .beta.-arrestinsignaling was blocked by both prior art antibodies L263G8 and 433H,while inventive antibody TP-23411 showed no significant effect on.beta.-arrestin signaling, at least up to a concentration of 100 nM(FIG. 26).

TABLE-US-00043 TABLE 10.4.1.1 IC50 values for inhibition of CCL1induced .beta.-arrestin signaling of TPP-23411 and prior artantibodies L263G8 and 433H measured in nM. Staining was performedon CHO cells stably expressing human CCR8 tagged with ProLink. NoIC50 could be determined for the inventive antibody, suggestingthat TPP-23411 does not block CCL1 induced .beta.-arrestinsignaling. TPP-23411 L263G8 433H CHO with hCCR8: IC50 in nM N/A13.84 0.78

Example 10.4.2: Phospho ELISA

Phospho AKT signaling promotes survival and growth, while phosphoErk1/2 signaling regulates a variety of cellular responses. PhosphoErk1/2 and phospho AKT antibodies were purchased from CellSignalingand ELISA assays were performed according to the manufacturersprotocol. CHO cells expressing human CCR8 or activated human Tregswere treated with CCL1, TPP-23411, Biolegend L263G8 or BD antibody433H, or remained untreated for the negative control. CCL1 servedas positive control. Interestingly, prior art antibodies BiolegendL263G8 and BD antibody 433H both induced a significant increase ofphosphorylated Erk1/2 levels (FIG. 27, 28) as well asphosphorylated AKT levels (FIG. 29, 30), e.g. after 15 minutes inactivated human Tregs, while this was not the case for inventiveantibodies. In summary, both prior art antibodies are agonistic forG protein independent pathways, such as AKT phosphorylation orERK1/2 phosphorylation.

Example 10.4.3: Calcium Flux Assay

Measurement of intracellular calcium mobilization is a robust assaythat can be performed in a high-throughput manner to study theeffect of compounds or antibodies on potential drug targets such asCCR8. To this end, the activity of receptors that signal throughrelease of Ca2+ can be monitored using the fluorescence-based FLIPRcalcium assay (Molecular Devices, Sunnyvale, Calif.). In brief, CHOcells expressing human CCR8 were seeded into assay plates andincubated overnight. Cells were then loaded with Ca+ sensitive dyeBUV396/496.

For the measurement of antagonistic function, monitoring of Ca fluxsignal via FLIPR Tera started in parallel to the addition ofantibodies or reference compound (MC148) at differentconcentrations to the cells. After 1 min incubation of the cellswith the antibodies or reference compound, CCL1 agonist was addedand calcium flux signal was recorded for an additional 3 min viaFLIPR Tetra. To measure agonistic function, antibodies/referencecompound were added in parallel to monitoring Ca flux signal viaFLIPR for 3 min.

Tables 10.4.3.1 to 10.4.3.4 show results of the calcium flux assayfor anti-murine or anti-human CCR8 antibodies. No IC50 could bedetermined for TPP-14099 and TPP-21047. IC50 for further inventiveantibodies varied. In general, majority of inventive antibodies andalso the prior art antibodies efficiently blocked CCL1 induced Gprotein dependent signaling.

TABLE-US-00044 TABLE 10.4.3.1 Ca Flux assay. CHO cells expressingmurine CCR8 were used to test the ability of the antibodies toblock CCL1 induced G-protein dependent Ca signaling. CCL1 at 40ng/ml was used for induction. Both TPP-14095 and TPP-14099 bind tomouse CCR8. No agonistic effect on G-protein dependent Ca signalingwas observed for the inventive antibodies or the prior artantibodies (data not shown). Ca Flux Assay using CHO cellsexpressing murine CCR8 TPP-14095 TPP-14099 IC50 (nM) 1.65 N/A

TABLE-US-00045 TABLE 10.4.3.2 Ca Flux assay. CHO cells expressinghuman CCR8 were used to test the ability of the antibodies to blockCCL1 induced G-protein dependent Ca signaling. CCL1 was used at aconcentration of 80 ng/ml. TPP-9809 TPP-18205 TPP-18206 TPP-17577TPP-17578 IC50 (nM) N/A 1.6 3 93.8 4.53

TABLE-US-00046 TABLE 10.4.3.3 Ca Flux assay. CHO cells expressinghuman CCR8 were used to test the ability of the antibodies to blockCCL1 induced G-protein dependent Ca signaling. CCL1 was used at aconcentration of 60 ng/ml. TPP-9809 TPP-20955 TPP-21045 TPP-21047TPP-21360 IC50 (nM) N/A 0.52 0.29 N/A 0.50

TABLE-US-00047 TABLE 10.4.3.4 Ca Flux assay. CHO cells expressinghuman CCR8 were used to test the ability of the antibodies to blockCCL1 induced G-protein dependent Ca signaling. CCL1 was used at aconcentration of 60 ng/ml. TPP- TPP- TPP-9809 TPP-20955 21360 23411443H L263G8 Hill Slope ~-1.815e+046 ~-0.03928 -2.208 -0.8122 -1.048-0.6795 IC50 (nM) N/A ~1.02e-025 0.417 1.22 1.3 1.26 Span ratio(from ~93.49 ~11357 433.9 400.1 995.9 921.2 max to min)

Example 10.5: Internalization of Inventive Antibodies

In order to evaluate internalization of CCR8, imaging technologywas used to visualize this process. Specific anti-CCR8 antibodiesTPP-21360 and TPP-23411 and the corresponding isotype controlantibody TPP-5657 were labelled with a fluorescent dye.Additionally, commercial antibodies L263G8 and 433H as well as themurine antibody SA214G2 were also conjugated to the permanent dyeBODIPY. These antibodies were lysine-conjugated with a two to sixmolar excess of BODIPY.RTM. FL dye (ester, D2184, ThermoFisher) atpH 8.3. After the conjugation, the reaction mixture was purified bychromatography (PD10 desalting column 1-2.5 ml; GE Healthcare) toeliminate excess dye and to adjust the pH-value. Afterwards, theprotein solution was concentrated (VIVASPIN 500, Fa. Sartoriusstedim biotec). Determination of the dye load of the antibody wascarried out with a spectrophotometer (NanoDrop) and calculated withthe formula D:P=A.sub.dye .epsilon..sub.protein: (A.sub.280-0,16A.sub.dye) .epsilon..sub.dye. The dye load of target-specificantibodies TPP-21360 and TPP-23411 and of the isotype controlantibody were shown to be in a similar range (dye load/ab: 4.0 and4.1). The commercial antibodies elicit dye load/antibody of 3.8 and3.9. Conjugation might elicit a negative impact on antibodyaffinity thus testing of conjugated antibodies in a cellbinding-assay (FACS) is essential to ensure that the labelling didnot alter the binding to CCR8 (CD198) (cf. FIG. 31A, FIG. 32A, FIG.34A, FIG. 34B). The conjugated antibodies were then ready to usefor internalization assays performed with the human CCR8-positivecell line HuT78 and the murine CCR8-positive cell line BW5147.3.Prior to treatment, the CCR8-expressing cell lines(2.times.10.sup.4/well) were seeded in 100 .mu.l medium in a 96-MTP(CellCarrier Ultra, PerkinElmer). After 18 h incubation at37.degree. C./5% CO.sub.2, medium was changed and labelled antibodywas added in various concentrations (2.5, 1 .mu.g/ml; triplicates).The same treatment protocol was applied for the labelled isotypecontrol antibody (negative control). Furthermore, the parentalCHO-K1 cell line (CCR8 negative cells) was treated alike serving ascounter screen (specificity control). The internalization studywith the target-specific antibodies was performed in a kineticfashion. Images were taken after different time points (0 h, 0.5 h,2 h, 6 h and 24 h) and internalization efficacy was determined bymeasuring total internalized fluorescence intensity/cell. Themeasurement was performed using the Operetta CLS (PerkinElmer) andthe data analyses of images were performed with the Harmonyhigh-content analysis software (PerkinElmer).

FIGS. 31B and 32B show a substantial internalization of thecommercial anti-human CCR8 antibodies 433H and L263G8 in HuT78cells and commercial anti-murine CCR8 antibody SA214G2 in BW5147.3cells, while FIG. 33 demonstrates a low or no internalization ofthe inventive antibodies TPP-21360 and TPP-23411, which issubstantially comparable to the isotype control.

TABLE-US-00048 TABLE 10.5.1 Summary of internalization data forinventive antibodies and prior art antibodies. FACS FACS HuT78Internalization mBW5147.3 Internalization antibody [MFI] [foldisotype] [MFI] [fold isotype] TPP-23411 600 0 n.a. n.a. TPP-21360600 0 n.a. n.a. 433H 50 22 n.a. n.a. L263G8 40 22 n.a. n.a. SA214G2n.a. n.a. 48 11 (murine) n.a: not tested.

To confirm that the low internalization was indeed a featurecharacterizing all inventive anti-CCR8 antibodies obtained with asulfated peptide antigen as described herein, the inventiveantibodies listed in Table 10.5.2 (see also FIG. 89) were analyzedwith respect to their internalization behavior with vesiclearea/cell as readout. An anti-CD71 antibody with known substantialinternalization was used as positive control. Indeed, all testedinventive anti-human CCR8 antibodies were non-internalizingantibodies, i.e. antibodies showing no substantial degree ofinternalization. Interestingly, TPP-17577, the only tested humananti human anti-CCR8 antibody without histidine within the HCDR3showed a somewhat higher internalization than all tested human antihuman anti-CCR8 antibody comprising histidine within the HCDR3.

TABLE-US-00049 TABLE 10.5.2 Summary of internalization data forinventive antibodies. Internalization Internalization HuT78 HUVECTPP-17577 n.a. none TPP-17578 n.a. none TPP-18205 n.a. noneTPP-18206 n.a. none TPP-20955 none none TPP-21047 none noneTPP-23411 none none TPP-27495 none none TPP-27496 none noneTPP-18429 none none TPP-18430 none none TPP-18432 n.a. noneTPP-18433 n.a. none TPP-18436 none none TPP-19571 n.a. noneTPP-27478 none none TPP-27477 none n.a. TPP-27479 none noneTPP-27480 none none n.a: not tested.

Example 10.6.1: FcRn Affinity Chromatography of Anti-CCR8Antibodies

The neonatal Fc receptor (FcRn) is linked to the regulation ofmetabolic processing of IgG antibodies in vivo. Binding to FcRn inacidic environment of endosomes of vascular endothelial cellsprotects IgGs from lysosomal degradation. Release of IgGs from FcRninto the blood stream occurs at physiological pH 7.4. Thus, FcRnbinds to IgGs at pH<6.5 and has no binding at physiological pH7.4 Interaction between IgGs and FcRn has an impact on thepharmacokinetics of IgGs in vivo. Characterization of pH dependentIgG binding to FcRn by mimicking the antibody recycling interactionvia immobilized FcRn can reveal IgG properties that contribute totheir pharmacokinetics.

FcRn affinity runs were performed on an Akta Pure 25 chromatographysystem at 25.degree. C. A FcRn column (Roche, Order No. 8128057001;prepacked with approximately 1 mL resin; binding capacity:.gtoreq.100 .mu.g IgG) was equilibrated with running buffer A: 20mM MES/HCl pH 5.5 140 mM NaCl. 30 .mu.L of each antibody (1 mg/mLin equilibration buffer) was applied to the column. Linear pHgradient was generated via increasing the percent of running bufferB 20 mM Tris/HCl pH 8.8 140 mM NaCl as followed: 0 min--20% B, 10min--20% B, 80 min--100% B, 90 min--100% B, 93 min--20% B, 103min--20% B. Flow rate was 0.5 mL/min. pH value at point of elutionwas monitored and read from chromatograms generated with Unicorn7.1.

As can be seen from Table 10.6.1.1, prior art antibody Ustekinumabserved as control for standard pharmacokinetics and prior artantibody Briakinumab served as a control for rapid pharmacokinetics(cf. Schoch, Angela, et al. "Charge-mediated influence of theantibody variable domain on FcRn-dependent pharmacokinetics."Proceedings of the National Academy of Sciences 112.19 (2015):5997-6002.). TPP-17577, TPP-18205, TPP-27495, TPP-21047 andTPP-27496 show an elution pH greater than pH 7.5 and can beconsidered to show binding to FcRn also at pH 7.4 to pH 7.5. Themeasured pH may be used to predict the clearance and half-life ofan antibody.

TABLE-US-00050 TABLE 10.6.1.1 FcRn affinity chromatography fordifferent antibodies: pH at elution. Two pH values indicate adouble peak. TPP Nr. pH at Elution Ustekinumab 7.22 27478 7.2527477 7.25 19571 7.25 23411 7.38 20955 7.38 18436 7.38 18432 7.3827479 7.43 23480 7.5 18433 7.5 18430 7.5 18429 7.5 18206 7.5 175787.5 17577 7.50/7.69 18205 7.50/7.77 Briakinumab 7.85 27495 7.8821047 8 27496 8.13

Example 10.6.2: FcRn Binding Analysis Based on SPR

To assess the binding affinity of anti-CCR8 antibodies to FcRn, thebinding was investigated using SPR at pH 6.0 and pH 7.4. Bindingassays were performed on a Biacore T200 instrument at 25.degree. C.with a CM5 sensor chip and assay buffer PBST. For FcRn bindingassays, human, mouse or cynomolgus FcRn were amine-coupled to theCM5 sensor chip surface (.about.300 RU) and IgGs were injected atconcentrations ranging from 15.6-2.000 nM in PBST, pH 6.Regeneration was performed with PBST pH 7.4. In another experimentat pH 7.4 only, IgGs were tested with one concentration of 2 .mu.M,thus it was only qualitatively assessed if a binding occurs. KDvalues were derived from a steady state affinity analysis as wellas from kinetic analysis by fitting to a 1:1 binding isotherm.

As can be seen from Table 10.6.2.1, and as discussed before,Ustekinumab served as positive control and Briakinumab as negativecontrol as published in Schoch et al, 2015. These two antibodies donot significantly differ in their affinity to FcRn, but in theirbinding response to FcRn at pH 7.4. Thus, it can be assumed thatantibodies showing a binding response at pH 7.4 might have anaccelerated half-life in vivo.

Example 10.7: Heparin Affinity Chromatography of Anti-CCR8Antibodies

For the inventive antibodies recognizing human CCR8,charge-mediated interaction is highly likely, because theantibodies recognize a charged antigen tyrosine motif and have acertain structural composition, e.g. with regards to their HCDR3.Charge mediated interactions are however a driving force todegradation of IgGs besides FcRn binding at pH 7.4. Binding to anegatively charged glycocalyx of monocytes or macrophages may leadto pinocytosis and subsequent proteolytic degradation. Kraft et al.showed that antibody behavior in vivo can be predicted moreefficiently and accurately when conducting heparine affinitychromatography together with FcRn chromatography (Kraft, Thomas E.,et al. "Heparin chromatography as an in vitro predictor forantibody clearance rate through pinocytosis." MAbs. Vol. 12. No. 1.Taylor & Francis, 2020.).

TABLE-US-00051 TABLE 10.6.2.1 SPR analysis of affinities ofinventive antibodies for different FCRN receptors. Small bindingresponse is defined as a response <30 RU, binding response as>30 RU. Binding at Binding at Binding at Binding at Binding atBinding at Ligand: pH 6.0 pH 7.4 Ligand: at pH 6.0 pH 7.4 Ligand:pH 6.0 pH 7.4 [KD Antibody FcRn [KD in M] [KD in M] FcRn [KD in M][KD in M] FcRn [KD in M] in M] TPP-27496 human 1.4E-08 bindingresponse cyno 8.9E-09 binding response mouse 1.6E-08 bindingresponse TPP-27495 human 2.9E-08 binding response cyno 3.0E-08binding response mouse 3.3E-08 binding response TPP-27480 human6.1E-08 binding response cyno 6.2E-08 binding response mouse1.2E-07 binding response TPP-27479 human 8.3E-08 binding responsecyno 9.0E-08 binding response mouse 5.0E-08 binding responseTPP-27478 human 9.7E07 No response cyno 8.5E-07 No response mouse1.3E-07 No response TPP-27477 human 1.1E-06 small/no binding cyno1.0E-06 small/no binding mouse 1.7E-07 small/no binding responseresponse response Ustekinumab human 2.0E-07 No response cyno2.6E-07 No response mouse 7.1E-08 small binding responseBriakinumab human 2.3E-07 binding response cyno 1.6E-07 bindingresponse mouse 5.6E-07 binding response TPP-21047 human 6.5E-08small binding response cyno 4.0E-08 small binding response mouse1.7E-07 small binding response TPP-18436 human 5.7E-08 bindingresponse cyno 5.3E-08 binding response mouse 3.3E-08 bindingresponse TPP-18433 human 5.8E-08 binding response cyno 5.4E-08binding response mouse 4.2E-08 binding response TPP-18432 human8.0E-08 binding response cyno 8.5E-08 binding response mouse2.7E-08 binding response TPP-18430 human 7.4E-08 binding responsecyno 6.9E-08 binding response mouse 5.2E-08 binding responseTPP-18429 human 8.1E-08 binding response cyno 7.6E-08 bindingresponse mouse 6.1E-08 binding response TPP-18205 human 9.6E-08 Noresponse cyno 7.9E-08 No response mouse 1.0E-07 No responseTPP-17577 human 5.5E-08 binding response cyno 6.6E-08 bindingresponse mouse 4.3E-08 binding response TPP-18206 human 1.4E-07binding response cyno 2.7E-07 binding response mouse 2.1E-07binding response TPP-17578 human 1.0E-07 small binding responsecyno 1.3E-07 small binding response mouse 3.8E-08 small bindingresponse TPP-20955 human 5.4E-08 small binding response cyno4.0E-08 small binding response mouse 5.8E-08 small binding responseTPP-23411 human 5.1E-08 small binding response cyno 7.0E-08 smallbinding response mouse TPP-9809 human 1.4E-07 No response cyno4.0E-07 No response mouse 2.8E-08 No response TPP-9809 human1.6E-07 No response cyno 4.1E-07 No response mouse 3.9E-08 Noresponse TPP-19571 human 1.3E-06 small binding response cyno1.2E-06 small binding response mouse 1.9E-07 binding response

High affinity, high level of interaction and therefore highretention times may predict a shorter half-life of the testedantibodies. Heparine affinity runs were performed on an Akta Pure25 chromatography system at 25.degree. C. A heparane column (Tosoh)was equilibrated with running buffer A: 50 mM TRIS, pH 7.4. 50.mu.L of each antibody (1 mg/mL in 20 mM Histidin pH 5.5) wasapplied to the column. A linear salt gradient was generated byincreasing the percent of running buffer B 20 mM Tris pH 7.4 1MNaCl as followed: 2 min post injection--0% B, 0-55% B over 16.5minutes, 100% over 0.5 minutes, 100% B for four minutes. Flow ratewas 0.8 mL/min. Time of elution was monitored and read fromchromatograms generated with Unicorn 7.1. As can be seen from Table10.7.1, and as discussed before, Ustekinumab served as positivecontrol and Briakinumab as negative control. Most tested inventiveantibodies center around a retention time of 15 to 18 min. Thus,these antibodies might show a reduced half-life if electrostaticinteractions contribute in vivo.

TABLE-US-00052 TABLE 10.7.1 Heparine affinity chromatography fordifferent antibodies: pH at elution. TPP-Nr. Retention time(minutes) Ustekinumab 11.95 TPP-17578 13.53 TPP-23411 (afuco) 15.02TPP-27478 15.05 TPP-27495 15.29 TPP-19571 15.29 TPP-27496 15.34TPP-27477 15.35 Briakinumab 15.78 TPP-23411 (wt) 15.86 TPP-1820516.02 TPP-21047 16.06 TPP-27480 16.09 TPP-18436 16.21 TPP-2095516.73 TPP-18432 17.01 TPP-18206 17.24 TPP-18206 17.51 TPP-1843017.54 TPP-18429 17.54 TPP-18433 17.56 TPP-17577 18.06

TABLE-US-00053 Mean log2 CCR8 SD log2 CCR8 T-test p-value Nexpression expression compared to isotype Isotype 2 7.386 0.128 --TPP-23411 2 5.459 0.346 0.0537

Example 11: Therapeutic Applicability of the Target

For therapeutic use of a certain target in immune oncology, thespecific expression of a target is of utmost importance forminimizing systemic side effects. The target shall be specific forthe target tissue or cells, e.g. for intra-tumoral Tregs and shallnot be expressed on healthy tissues. Furthermore, increasedexpression of the target in a certain tumor tissue may pointtowards the medical use for that indication. For example, CCR8 isalso expressed on B cell lymphoma and T cell lymphoma tumor celllines.

Systemic removal of Treg cells may evoke and enhance not only tumorimmunity, but also autoimmunity as evidenced by patients sufferingfrom Immune dysregulation-polyendocrinopathy-enteropathy-X-linked(IPEX) syndrome. In this disease, patients lack Tregs due to agenetic alteration and die within the first 2 years of life due tosystemic autoimmunity if not treated with appropriateimmunosuppression. The high specificity of a target aimed atremoval or suppression of intra-tumoral Tregs is thus assumed to bevery important to avoid side effects of a therapeutic antibody,such as those observed for CCR4 targeting antibodies.

Example 11.1: Specificity of the Target CCR8

In a first analysis, the inventors evaluated the expression of CCR8mRNA in different human tissues and cell types. Significantexpression of CCR8 mRNA was only observed in activated regulatory Tcells, as well as tumor infiltrating lymphocytes (FIG. 35).

In a second analysis, the inventors evaluated the CCR8 mRNAexpression in 50 different tumor indications. Indications withhighest CCR8 expression are e.g. breast cancer, lung adenocarcinoma(ADC), testis cancer, stomach cancer and squamous cell carcinoma(SCC), head and neck malignancies as well as esophageal tumors, buthigh expression was also found in colorectal cancer, ovarian cancerand cervical cancer. In all indications except pancreaticadenocarcinoma and melanoma, expression was found to be higher inthe tumor compared to the corresponding normal tissue (FIG.36).

In a third analysis, the inventors evaluated the co-expression ofCCR8 mRNA with FOXP3 mRNA in various tumor indications.Co-expression with Treg marker FOXP3 is important to demonstratethat CCR8 is indeed mainly expressed on Tregs, cf. Table 11.1.1.IHC stainings confirmed these findings, see FIG. 37.

TABLE-US-00054 TABLE 11.1.1 CCR8 and the regulatory T cell definingmarker FOXP3 are the most closely correlated genes across all 9588TCGA tumor samples (excluding Thymoma as indication) pointingtowards these genes being co-expressed in the same cell type. Thetable shows the 25 genes (out of 35021 genes) most stronglycorrelated with CCR8 and FOXP3 mRNA expression levels and theirrespective Pearson correlation coefficients, r. CCR8 FOXP3 CCR81.000 FOXP3 1.000 FOXP3 0.828 CCR8 0.828 ICOS 0.814 ICOS 0.821 CCR40.813 TIGIT 0.804 TIGIT 0.762 CTLA4 0.766 P2RY10 0.760 SIRPG 0.747CD80 0.748 SLAMF1 0.741 TNFRSF9 0.746 CCR4 0.737 CD3G 0.741 IL2RB0.737 SLAMF1 0.726 CD5 0.733 IL7R 0.724 P2RY10 0.727 IL2RB 0.716CD3G 0.724 CTLA4 0.715 TNFRSF9 0.723 CD5 0.713 CXCL9 0.721 ITK0.713 CD2 0.720 IL2RA 0.711 GBP5 0.718 LAX1 0.707 UBASH3A 0.714IKZF3 0.699 CD3E 0.713 GBP5 0.699 CXCR6 0.713 CXCR6 0.693 CXCR30.712 SIRPG 0.690 LTA 0.712 CD2 0.689 CD80 0.710 CSF2RB 0.688 CD60.708 SLAMF7 0.683 ITK 0.706 CXCL9 0.681 CXCL13 0.704

In addition, the correlation of CCR8 mRNA with pan-T cell markerCD3, cytotoxic T cell marker CD8, macrophage marker MS4A7, generalinflammation marker IFNg, and B cell markers CD19, CD20, CD22 wasevaluated for 50 tumor indications, cf. Table 11.1.2.Interestingly, for nearly all indications, the correlation washighly significant, suggesting increased expression of CCR8 uponimmune cell infiltration into the tumor per se, and T reginfiltration in particular. From this it can be expected thatimmune cell infiltration and in particular T cell infiltration arelikewise valuable biomarkers for patient stratification to identifypatients which are more likely to profit from anti-CCR8therapy.

TABLE-US-00055 TABLE 11.1.2 Pearson correlation coefficients, r,between 1og2 mRNA levels of CCR8 and Treg marker FOXP3, pan-T cellmarker CD3, cytotoxic T cell marker CD8, macrophage marker MS4A7,general inflammation marker IFNg, or B cell markers CD19, CD20,CD22. Results suggest a highly significant co- expression of CCR8with immune cell and in particular T reg infiltration into tumorsacross 48 out of 50 indications from TCGA. It can therefore beexpected that e.g. T cell infiltration constitutes a valuablestratification biomarker for identifying patients likely profitingfrom anti-CCR8 therapy. pan T cells cytotoxic T regulatory T cells(CD3E + CD3D + cells (CD8A + (FOXP3) CD3G) CD8B) Indication N r pvr pv r pv Head and Neck cancer - Oral 79 0.916 1.87E-83 0.7911.22E-45 0.661 2.51E-27 cancer-SCC Breast cancer - ADC-Ductal- 1500.641 1.70E-09 0.584 8.82E-08 0.448 8.80E-05 TPBC Head and Neckcancer - Laryngeal 276 0.928 1.51E-36 0.717 2.42E-14 0.576 1.20E-08cancer-SCC Lung cancer - NSCLC-ADC- 132 0.901 3.77E-40 0.7639.07E-22 0.580 4.79E-11 Mixed Breast cancer - ADC-Ductal- 197 0.8701.30E-31 0.729 1.13E-17 0.602 4.20E-11 TNBC Gastric cancer - ADC165 0.853 2.87E-45 0.747 4.64E-29 0.677 3.04E-22 Lung cancer -NSCLC-ADC 134 0.829 4.51E-82 0.689 2.92E-46 0.486 2.83E-20 Gastriccancer - ADC-Intestinal 198 0.726 1.65E-13 0.684 1.40E-11 0.5961.70E-08 Gastric cancer - ADC-Diffuse 63 0.825 2.90E-18 0.6654.52E-10 0.597 6.23E-08 Testicular cancer - Germ cell 619 0.3284.35E-03 0.367 1.32E-03 0.255 2.85E-02 tumor-Seminoma Lung cancer -NSCLC-SCC 99 0.895 2.21E-168 0.775 1.43E-96 0.589 6.11E-46 Thymoma71 0.351 8.31E-05 0.372 2.81E-05 0.357 6.14E-05 Esophageal cancer -ADC 177 0.909 9.69E-35 0.780 2.17E-19 0.697 3.21E-14 Breast cancer- ADC-Ductal 336 0.783 4.02E-129 0.675 1.58E-83 0.570 1.28E-54Gastric cancer - ADC-Intestinal- 89 0.895 1.11E-27 0.812 5.44E-190.594 1.57E-08 Tubular Breast cancer - ADC-Lobular 96 0.8483.26E-50 0.730 9.15E-31 0.642 5.98E-22 Pancreatic cancer -ADC-Ductal 156 0.864 5.89E-45 0.697 1.14E-22 0.621 4.96E-17Colorectal cancer - ADC 69 0.902 7.12E-124 0.703 2.54E-51 0.5136.34E-24 Esophageal cancer - SCC 75 0.832 7.93E-26 0.653 5.31E-130.578 7.03E-10 Head and Neck cancer - SCC 76 0.836 7.80E-53 0.5487.42E-17 0.435 1.68E-10 Breast cancer - ADC 83 0.781 4.14E-14 0.6875.12E-10 0.549 3.20E-06 Uterine cancer - Cervical-SCC- 207 0.8091.19E-27 0.536 7.66E-10 0.368 5.71E-05 Non-keratinizing Uterinecancer - Cervical-SCC- 197 0.853 1.29E-16 0.637 1.75E-07 0.5951.70E-06 Keratinizing Bladder cancer - Transitional cell 534 0.8287.54E-71 0.680 7.75E-39 0.501 5.68E-19 carcinoma Ovarian cancer -ADC- 66 0.545 4.22E-25 0.491 4.53E-20 0.381 5.07E-12Cystadenocarcinoma-Papillary serous Uterine cancer - Cervical-SCC127 0.804 3.16E-20 0.681 1.02E-12 0.644 3.76E-11 Kidney cancer -ccRCC 77 0.835 5.60E-140 0.655 8.13E-67 0.553 4.86E-44 Skin cancer- Melanoma 86 0.797 5.44E-105 0.798 1.66E-105 0.721 7.80E-77Thyroid cancer - Papillary 356 0.829 3.81E-93 0.610 2.53E-38 0.3657.48E-13 carcinoma-Classical variant Mesothelioma - Epithelioid 3190.843 1.98E-16 0.760 7.00E-12 0.691 2.65E-09 Sarcoma - Malignantfibrous 108 0.828 6.76E-14 0.621 1.15E-06 0.569 1.31E-05histiocytoma Prostate cancer - ADC-Acinar 477 0.773 7.89E-97 0.7081.88E-74 0.559 5.85E-41 type Bladder cancer - Transitional cell 570.889 6.39E-46 0.743 1.97E-24 0.662 5.43E-18 carcinoma-PapillaryUterine cancer - Endometrial- 307 0.686 2.58E-16 0.498 4.11E-080.381 4.82E-05 ADC-Endometrioid Uterine cancer - Endometrial- 1470.405 1.60E-03 0.381 3.15E-03 0.301 2.17E-02 ADC-Papillary serousSarcoma - Liposarcoma- 482 0.799 5.61E-14 0.649 3.66E-08 0.5604.97E-06 Dedifferentiated Sarcoma - Leiomyosarcoma 106 0.8384.58E-29 0.754 1.04E-20 0.710 1.53E-17 Liver cancer - HCC 58 0.4051.77E-15 0.674 1.52E-48 0.558 1.64E-30 Thyroid cancer - Follicular51 0.398 3.47E-05 0.251 1.08E-02 0.041 6.80E-01 carcinoma Braincancer - Glioma- 472 0.616 1.34E-18 0.612 2.31E-18 0.492 1.86E-11Astrocytoma-Glioblastoma Kidney cancer - Papillary-Type II 74 0.7554.86E-17 0.688 2.38E-13 0.648 1.54E-11 Kidney cancer - Papillary120 0.858 4.77E-38 0.618 1.04E-14 0.618 9.58E-15 Uterine cancer -Endometrial- 102 0.716 3.73E-10 0.573 3.20E-06 0.378 3.79E-03Carcinosarcoma-Malignant mixed mullerian tumor Kidney cancer -Papillary-Type I 363 0.781 5.15E-17 0.460 2.58E-05 0.482 9.16E-06Kidney cancer - Chromophobe 84 0.227 6.63E-02 0.179 1.50E-01 0.1771.55E-01 Adrenal cancer - Adrenocortical 55 0.176 1.21E-01 0.3631.01E-03 0.333 2.72E-03 carcinoma Adrenal cancer - 114 0.4554.92E-09 0.377 1.99E-06 0.340 2.09E-05 Pheochromocytoma Braincancer - Glioma-Astrocytoma 108 0.620 2.70E-22 0.541 2.29E-16 0.4732.32E-12 Brain cancer - Glioma- 58 0.479 4.70E-09 0.553 4.19E-120.427 2.70E-07 Oligoastrocytoma Brain cancer - Glioma- 57 0.2172.11E-03 0.295 2.46E-05 0.236 7.98E-04 Oligodendroglioma M2 B cells(CD19 + macrophages macrophages Inflammation MS4A1 + (MS4A7) (MRC1)(IFNg) CD22) Indication N r pv r pv r pv r pv Head and Neck cancer- 79 0.742 1.62E-37 0.578 7.11E-20 0.505 8.98E-15 0.610 1.82E-22Oral cancer-SCC Breast cancer - ADC- 150 0.107 3.75E-01 0.3711.44E-03 0.552 5.92E-07 0.404 4.78E-04 Ductal-TPBC Head and Neckcancer - 276 0.740 1.40E-15 0.687 7.76E-13 0.317 3.52E-03 0.4344.20E-05 Laryngeal cancer-SCC Lung cancer - NSCLC- 132 0.5342.74E-09 0.623 5.84E-13 0.464 4.25E-07 0.520 7.74E-09 ADC-MixedBreast cancer - ADC- 197 0.622 6.43E-12 0.607 2.82E-11 0.4463.70E-06 0.563 1.30E-09 Ductal-TNBC Gastric cancer - ADC 165 0.6754.00E-22 0.704 1.26E-24 0.629 1.52E-18 0.435 1.42E-08 Lung cancer -NSCLC- 134 0.594 7.28E-32 0.642 1.75E-38 0.415 1.03E-14 0.4919.47E-21 ADC Gastric cancer - ADC- 198 0.531 9.78E-07 0.5338.65E-07 0.436 9.37E-05 0.501 4.68E-06 Intestinal Gastric cancer -ADC- 63 0.684 9.45E-11 0.593 7.99E-08 0.403 5.93E-04 0.405 5.55E-04Diffuse Testicular cancer - Germ 619 -0.040 7.37E-01 0.429 1.35E-040.203 8.20E-02 0.035 7.67E-01 cell tumor-Seminoma Lung cancer -NSCLC-SCC 99 0.658 2.03E-60 0.633 8.10E-55 0.494 1.09E-30 0.5744.27E-43 Thymoma 71 0.147 1.09E-01 0.039 6.69E-01 -0.055 5.47E-010.228 1.23E-02 Esophageal cancer - ADC 177 0.713 4.46E-15 0.7192.23E-15 0.598 5.98E-10 0.662 1.61E-12 Breast cancer - ADC-Ductal336 0.232 5.09E-09 0.538 9.58E-48 0.606 3.17E-63 0.465 1.62E-3- 4Gastric cancer - ADC- 89 0.703 1.44E-12 0.743 1.50E-14 0.6037.94E-09 0.596 1.38E-08 Intestinal-Tubular Breast cancer - ADC- 960.264 3.78E-04 0.483 9.41E-12 0.653 7.46E-23 0.495 2.60E-12 LobularPancreatic cancer - ADC- 156 0.757 1.55E-28 0.766 1.34E-29 0.3891.13E-06 0.423 9.66E-08 Ductal Colorectal cancer - ADC 69 0.7521.72E-62 0.770 2.78E-67 0.436 5.18E-17 0.479 1.09E-20 Esophagealcancer - SCC 75 0.678 3.14E-14 0.431 1.18E-05 0.550 6.51E-09 0.3573.57E-04 Head and Neck cancer - 76 0.594 3.86E-20 0.516 8.40E-150.424 5.27E-10 0.381 3.41E-08 SCC Breast cancer - ADC 83 0.2713.15E-02 0.505 2.41E-05 0.603 1.73E-07 0.468 1.11E-04 Uterinecancer - Cervical- 207 0.555 1.54E-10 0.527 1.71E-09 0.346 1.59E-040.559 9.91E-11- SCC-Non-keratinizing Uterine cancer - Cervical- 1970.730 2.64E-10 0.498 1.10E-04 0.456 4.65E-04 0.591 2.03E-06-SCC-Keratinizing Bladder cancer - 534 0.509 1.36E-19 0.590 2.75E-270.427 1.14E-13 0.409 1.53E-12 Transitional cell carcinoma Ovariancancer - ADC- 66 0.309 3.22E-08 0.293 1.64E-07 0.410 7.12E-14 0.1391.49E-02 Cystadenocarcinoma- Papillary serous Uterine cancer -Cervical- 127 0.629 1.51E-10 0.577 9.49E-09 0.516 5.17E-07 0.6453.57E-11- SCC Kidney cancer - ccRCC 77 0.520 2.71E-38 0.2451.04E-08 0.577 8.41E-49 0.476 1.71E-31 Skin cancer - Melanoma 860.614 2.96E-50 0.639 1.74E-55 0.665 1.54E-61 0.653 9.36E-59 Thyroidcancer - Papillary 356 0.440 1.41E-18 0.649 7.94E-45 0.446 3.63E-190.365 7.05E-13- carcinoma-Classical variantMesothelioma-Epithelioid 319 0.465 2.68E-04 0.317 1.64E-02 0.5969.94E-07 - 0.364 5.35E-03 Sarcoma - Malignant 108 0.160 2.63E-010.125 3.84E-01 0.527 6.99E-05 0.524- 8.03E-05 fibrous histiocytomaProstate cancer - ADC- 477 0.594 3.24E-47 0.596 1.25E-47 0.5287.01E-36 0.595 1.78E-47 Acinar type Bladder cancer - 57 0.6071.14E-14 0.709 1.80E-21 0.586 1.57E-13 0.628 7.43E-16 Transitionalcell carcinoma- Papillary Uterine cancer - 307 0.456 7.25E-07 0.4225.28E-06 0.425 4.61E-06 0.402 1.59E-05 Endometrial-ADC-Endometrioid Uterine cancer - 147 0.369 4.36E-03 0.347 7.66E-030.122 3.61E-01 0.044 7.43E-01 Endometrial-ADC-Papillary serousSarcoma - Liposarcoma- 482 0.206 1.21E-01 0.118 3.79E-01 0.4821.26E-04 0.- 526 2.25E-05 Dedifferentiated Sarcoma - Leiomyosarcoma106 0.569 1.97E-10 0.558 5.09E-10 0.674 2.30E-15 - 0.491 8.84E-08Liver cancer - HCC 58 0.524 1.54E-26 0.263 5.06E-07 0.555 3.42E-300.575 1.09E-32 Thyroid cancer - Follicular 51 0.243 1.39E-02 0.3345.99E-04 0.110 2.69E-01 0.171 8.61E-02- carcinoma Brain cancer-

Glioma- 472 0.418 2.23E-08 0.402 8.97E-08 0.365 1.44E-06 0.3124.52E-05 Astrocytoma-Glioblastoma Kidney cancer - Papillary- 740.443 1.91E-05 0.510 5.24E-07 0.575 6.83E-09 0.451 1.33E-05- TypeII Kidney cancer - Papillary 120 0.457 6.48E-08 0.492 4.23E-090.651 1.13E-16 0.512 7.97E-10- Uterine cancer - 102 0.370 4.58E-030.213 1.12E-01 0.519 3.49E-05 0.277 3.69E-02 Endometrial-Carcinosarcoma-Malignant mixed mullerian tumor Kidney cancer -Papillary- 363 0.206 7.19E-02 0.252 2.70E-02 0.609 4.04E-09 0.5278.67E-0- 7 Type I Kidney cancer - 84 0.326 7.52E-03 0.262 3.36E-020.246 4.68E-02 0.165 1.86E-01 Chromophobe Adrenal cancer - 55 0.3943.32E-04 0.327 3.24E-03 0.273 1.48E-02 0.084 4.59E-01Adrenocortical carcinoma Adrenal cancer - 114 0.221 6.66E-03 0.2255.71E-03 0.315 8.80E-05 0.191 1.94E-02 Pheochromocytoma Braincancer - Glioma- 108 0.348 5.53E-07 -0.023 7.48E-01 0.543 1.57E-160.502 6.07E-14 Astrocytoma Brain cancer - Glioma- 58 0.212 1.40E-020.133 1.26E-01 0.627 5.05E-16 0.214 1.30E-02 Oligoastrocytoma Braincancer - Glioma- 57 0.148 3.77E-02 0.029 6.82E-01 0.338 1.07E-060.097 1.76E-01 Oligodendroglioma

Example 11.2: Specificity of CCR8 for Intra-Tumoral Tregs Comparedwith T Effector Cells

There are several issues to consider with regards to Treg depletionfor cancer immunotherapy in humans. Treg and activated effector Tcells most often share the expression of the same types of cellsurface molecules, including CD25 and CTLA-4, making it difficultto selectively deplete Tregs without also affecting effector Tcells by antibodies specific for these molecules.

Foxp3+CD25+CD4+ Treg cells in tumors (tumor infiltrating orintra-tumoral Tregs) express higher levels of cell surfacemolecules associated with T-cell activation, such as CD25, CTLA-4,PD-1, LAG3, TIGIT, ICOS, and TNF receptor super family membersincluding 4-1BB, OX-40, and GITR, compared with Tregs in lymphoidor non-lymphoid tissues or the blood. In addition, tumorinfiltrating Tregs express high levels of specific chemokinereceptors including CCR4 and CCR8.

CD4+CD25+ sorted and unsorted PBMCs were activated with anti-CD3and anti-CD28 beads+IL2 for 6 days. FACS analysis showed CCR8protein expression preferentially on stimulated Tregs(CD4+CD25+FoxP3+CD127dim), see FIG. 38. Alternative targets OX40,GITR, and CD25 but not CCR8 are significantly expressed onstimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). CCR4shows rather Treg specific expression but no intratumor T regspecificity. These findings are consistent with the observationthat CCR4 antibodies have shown immunological side effects, whichhave been attributed to the systemic depletion of Tregs. In termsof specificity, CCR8 seems to be superior to support the specificdepletion of intra-tumoral Tregs.

To confirm the findings, raw data for NSCLC, CRC and liver cancerpatients were retrieved from Guo, Xinyi, et al. "Globalcharacterization of T cells in non-small-cell lung cancer bysingle-cell sequencing." Nature medicine 24.7 (2018): 978-985,Zhang, Yuanyuan, et al. "Deep single-cell RNA sequencing data ofindividual T cells from treatment-naive colorectal cancerpatients." Scientific data 6.1 (2019): 1-15, and Zheng, Chunhong,et al. "Landscape of infiltrating T cells in liver cancer revealedby single-cell sequencing." Cell 169.7 (2017): 1342-1356,respectively and used for analysis of CCR8 specificity, see FIG.39. Indeed, for these tumors, CCR8 mRNA expression is largelyrestricted to regulatory T cells (light grey boxes) residing intumor tissue while largely being absent in regulatory T cells fromnormal tissues as well as from CD4 helper T and CD8 cytotoxic Tcells (medium and dark gray boxes, respectively).

FACS analysis of human tumor lysate samples confirmed that CCR8protein is specifically expressed on human tumor-infiltrating Tregsin non-small-cell lung cancer (NSCLC), colorectal carcinoma (CRC),renal cell carcinoma (RCC) and lymph nodes (LNs, data not shown),FIG. 40. Up to 90% of tumor-infiltrated Tregs are CCR8 positive,e.g. in NSCLC, 40-90% in stage III+ and 40-70% in stage I/II, inCRC 20-40% in stage III+, and in RCC 40-50% in stage III+. Based onthese data, stratification according to staging is assumed to leadto a further benefit for patients treated with an anti-CCR8antibody.

Example 12: In Vivo Experiments

Example 12.1.1: Surrogate Antibodies & Syngeneic TumorModels

In mouse experiments surrogate antibodies binding to mouse-CCR8were used to demonstrate anti-tumor efficacy and to characterizethe mode-of-action, namely Treg depletion within the tumordepending on ADCC (antibody-dependent cellular cytotoxicity) andADCP (antibody-dependent cellular phagocytosis) potency.

TABLE-US-00056 TABLE 12.1.1.1 List of surrogate anti-mouse CCR8antibodies used for in vivo studies. ADCC was determined usingHEK293 cells overexpressing murine CCR8 as target cells and NK92cells as effector cells. ADCC activity is measured by lactatedehydrogenase (LDH) release. Anti-CCR8 antibody Isotype ADCCpotency in vitro TPP-14095 Human IgG1 Not determined TPP-14099EC50: 3.14 nM TPP-18208_aglyco EC50: >98 nM TPP-18209_aglyco Notdetermined TPP-15285 Mouse IgG2a EC50: 0.5 nM TPP-15286 Notdetermined TPP-9809 Human IgG1 Non-binding isotype controlTPP-10748 Mouse IgG2a Non-binding isotype control TPP-15726 HumanIgG1 Aglycosylated isotype control

Anti-murine CCR8 antibody TPP-14099 binds CHO cells expressingmurine CCR8 with an EC50 of 3 nM and murine iTregs with an EC50 of13.2 nM, as determined by FACS (data not shown).

Different syngeneic tumor models were used to model tumors with ahigh degree of immune infiltration as well as tumors with a lowdegree of immune cell infiltration. Furthermore, the used modelsdeviate in their responses to treatment with knowncheckpoint-inhibitors, e.g. treatment with anti-CTLA4, anti-PD1 oranti-PD-L1 antibodies. An overview of tested tumor models with theefficacy data for the respective inventive anti-CCR8 antibody isshown in Table 12.1.1.2. Remarkably, efficacy was demonstrated invarious syngeneic tumor models.

In brief, a suspension containing usually 500.000-1.000.000 tumorcells per 100 .mu.l PBS, medium or a mixture of 50% Matrigel wassubcutaneously injected into the flank of female immunocompetentmice (e.g. Balb/c, C57B16). At palpable tumor sizes of about 60-100mm.sup.3 (usually 100 mm.sup.3) CCR8-antibody administrationstarted by intraperitoneal injection of typically 10 mg/kg in avolume of 5-10 ml/kg. In dose-titration studies lower doses weretested. Non-binding antibodies of the respective isotype served ascontrol. Groups comprised n=10 mice. Therapy was applied twice perweek, typically as a q3/4d schedule, and tumor size and body weightwere measured three times per week. Tumor growth data are plottedas mean volume over time.

Tumor and tissue samples were taken from identically treatedsatellite animals (n=5 per group) 24 hours after the secondantibody treatment and were analyzed by flow cytometry afterdissociation into single cells and lysis of erythrocytes withparticular focus on changes in absolute numbers of regulatory Tcells (Tregs) per 100 mg tissue. Used markers for determiningactivated Tregs were CD45+CD4+CD25+FoxP3+. Other FACS markerscomprised CD8, NKp46, 4-1BB, F4/80, CD11c, Grl and MHCII.

TABLE-US-00057 TABLE 12.1.1.2 Overview of different tumor modelswith parameters to characterize treatment outcome. Efficacy wasmeasured by Treg depletion, Overall Response Rate andTumor-to-Control ratio based on final tumor volumes of anti-CCR8surrogate antibodies in syngeneic mouse tumor models. Treg CD8+ TTPP depletion cell/Treg ORR.sup.2 T/C Model (10 mg/kg) (%).sup.1ratio (% CRs) vol.sup.3 CT26.sup.4 14099 78.3 56 58 (30) 0.15 H22n.d. n.d. 0 (0) 0.30 4T1 n.d. n.d. 20 (0) 0.42 Hepa1-6 n.d. n.d. 10(0) 0.45 C38 45.5 17 4.5 (0) 0.55 MC38 n.d. n.d. 0 (0) 0.78 LL/cn.d. n.d. 0 (0) 0.85 MBT2 n.d. n.d. 10 (10) 0.86 RM-1 n.d. n.d. 0(0) 0.99 B16BI6 n.d. n.d. 0 (0) 1.06 CT26.sup.4 15285 82.3 64 72(30) 0.18 EMT-6 61.6 3 90 (0) 0.28 F9 73.2 54 60 (30) 0.21 C38 68.216 40 (30) 0.23 B16F10-OVA 59.0 77 0 (0) 0.37 A20 28.0 3 40 (30)0.46 4T1 37.1 1 0 (0) 0.90 B16F10 7.5 3 0 (0) 0.95 EG7-ova n.d.n.d. 0 (0) 0.86 PANC02 n.d. n.d. 10 (0) 0.51 MBT2 n.d. n.d. 30 (20)0.40 MC38 n.d. n.d. 30 (0) 0.35 MC38 n.d. n.d. 0 (0) 0.58 J558 n.d.n.d. 0 (0) 0.89 RENCA n.d. n.d. 0 (0) 0.81 Colon26 n.d. n.d. 0 (0)0.50 KLN205 n.d. n.d. 0 (0) 0.79 .sup.1Treg depletion wascalculated as percent difference between the intra-tumoral Tregs inthe isotype control and the intra-tumoral Tregs in tumors treatedwith the therapeutic antibody. .sup.2Overall response rate (ORR)and Complete response (CR) are based on an adaption of the RECISTcriteria to the fast tumor growth in a mouse model (CR: completeresponder (<10% of initial tumor volume); PR: partial responder(max 300% of initial tumor volume); NR: non-responder (>300% ofinitial tumor volume)). .sup.3T/C refers to treatment tumorvolume/control tumor volume at study end. n.d.: not determined..sup.4Averages of several studies in this tumor model.

For each syngeneic mouse tumor model, 10 tumors in both the isotypecontrol group as well as the anti-CCR8 treatment group wereharvested at the end of the treatment cycle, 24 h after lasttreatment (bi-weekly treatment until tumors in either group reachedan average volume of 3000 mm.sup.3) and genome wide mRNA expressionwas measured via RNA-seq. Of 17 models assessed via RNA-seq 13models showed elevated CD8 mRNA levels in the anti-CCR8 treatmentgroup compared to the control group with fold changes as shown inTable 12.1.1.3. For models CT26, H22, RM1, MBT2, and Hepa1-6 theincrease in CD8 levels was significant according to a T-teststatistic indicating significant increases in cytotoxic T cellsinfiltration at end of the treatment cycle. These data stronglysuggest that the CD8+ cells were involved in effecting the tumorresponse.

TABLE-US-00058 TABLE 12.1.1.3 Changes in CD8 mRNA levels induced byanti-CCR8 treatment. Model Fold change T test p-value CT26 8.6048.7E-08 H22 7.160 1.7E-09 RM1 3.105 3.1E-03 MBT2 2.716 1.3E-09WEHI-164 1.680 1.4E-01 Hepa1-6 1.621 2.7E-02 LL2 1.315 1.3E-01PANC02 1.242 5.4E-01 KLN205 1.194 5.2E-01 RENCA 1.188 6.7E-01Colon26 1.137 7.6E-01 B16BL6 1.022 9.3E-01 J558 0.924 8.6E-01 MC380.898 6.2E-01 4T1 0.499 8.8E-02 EG7-OVA 0.449 4.3E-02

Example 12.1.2: Syngeneic Model Characterization for BiomarkerIdentification

Based on genome wide RNA-seq data of early tumors from thesyngeneic models, increased levels of the following genes werefound to strongly correlate with tumor response: Eif3j2, Eno1b,Ifi441, Hist1h2a1, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b,Catsperg2, Zfp868, Serpina1b, Prss41, Clrb, Cyld, Ccnblip1, Masp2,Acaa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151,Rbm46, Arg2, Mir8099-2, Igsf21, O1fr373, C1s2, Crym, Arv1, Hddc3,Plppr4, Ppplr11, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d,Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1,Trim43b, Cc2d1a, Serpina1d, Cacna1a, Kcnj14, Ttc13, Farsa,O1fr1217, Jam1, H2-B1, Tnpo2, Rims3, Dock9, Car5b, Atpla4, H2-Q1,Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2, Nsd3, Wt1, Nap112, Ranbp9,Gtpbp3, AY761185, Rnaset2a, Serpina3i, E112, Gal3st2b, Urb2, F12,Klk1, Ifi214, Cstl1, Agtpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4,H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, H2-DMb2. Interestingly, thisset is highly enriched for early complement factors, complementregulating factors such as Serpins, and MHC components. Thesemarkers or a combination thereof may be used for stratification orto predict or monitor tumor response. In particular, the presenceof high levels of Complement C1/C4 might contribute to Treg lysis.Where depletion/consumption of complement factors reduces theefficacy of Treg depletion, supplementing the complement system,e.g. in a combination treatment may be an option.

The different syngeneic models were further characterized withregards to absolute numbers of immune cell subsets and frequencywithin total CD45+ immune cells in the tumors. For the modelsanalyzed via flow cytometry (Table 12.1.2.1), B16F10 ischaracterized by the lowest number of immune cells--and showed theleast reduction in tumor volume, cf. Table 12.1.2.1. Thus, immunecell infiltration may be used for stratification to predictresponse to treatment with CCR8 antibodies. This hypothesis couldlater be confirmed using B16B16.

TABLE-US-00059 TABLE 12.1.2.1 Characterization of tumormicroenvironment by absolute numbers of immune cell subsets andfrequency within total CD45+ immune cells in the tumors of thevehicle group analyzed at an early time- point (24 hours after thesecond treatment). T/C vol describes the ratio of tumor volume ofthe CCR8-antibody treated group (10 mg/kg twice weekly) versuscontrol group at the end of the study. Absolute immune cell numbersT/C CD4+ Model vol CD45+ CD8+ conv Treg NK cells Mph CT26 0.181368322 192436 74946 66189 271952 54108 F9 0.21 535626 19741 2106813965 50021 33206 EMT-6 0.28 1178255 9201 26504 18767 10016 290251Bl6F10- 0.37 388299 80015 26337 9948 68771 33597 OVA 4T1 0.42399181 17992 11636 32076 16386 52023 A20 0.46 548792 144609 7789480907 26629 85936 C38 0.55 525455 176380 57803 37518 36523 17514B16F10 0.95 57459 2633 3269 653 6558 20057 Relative immune cellnumbers % CD4+ T/C % CD8+ conv of % Treg % NK cells % Mph of Modelvol.sup.1 of CD45+ CD45+ of CD45+ of CD45+ CD45+ CT26 0.18 14.1 5.54.8 19.9 4.0 F9 0.21 3.7 3.9 2.6 9.3 6.2 EMT-6 0.28 0.8 2.2 1.6 0.924.6 B16F10- 0.37 20.6 6.8 2.6 17.7 8.7 OVA 4T1 0.42 4.5 2.9 8.04.1 13.0 A20 0.46 26.4 14.2 14.7 4.9 15.7 C38 0.55 33.6 11.0 7.17.0 3.3 B16F10 0.95 4.6 5.7 1.1 11.4 34.9

Example 12.2: Efficacy in CT26-Tumor Bearing Mice

Treatment with anti-CCR8 antibodies TPP-15285 and TPP-15286 showedstrong anti-tumor efficacy in CT26 tumor bearing mice (FIG. 41).TPP-15285 showed reduced efficacy at the lowest dose of 0.1 mg/kg,other dose-dependent differences were not significant. As expected,an anti-PD-L1 antibody ("PDL1") demonstrated medium efficacy in themodel. Non-binding antibodies of the respective isotypes did notshow any efficacy.

Spider plots (tumor growth of individual mice over time) of thecorresponding treatment groups (FIG. 42) clearly demonstratedhomogenously strong efficacy with complete responses occurring inall dose-groups with both anti-CCR8 antibodies.

Treg analysis of CT26 tumor samples 24 hours after the secondantibody treatment by flow cytometry showed strongly reducednumbers of Tregs in the anti-CCR8 antibody treated group versus theisotype control group. Treg depletion was clearly dose-dependent incase of TPP-15285 (FIG. 43).

TABLE-US-00060 TABLE 12.2.1 Corresponding data from FIG. 43. Mean %Tregs Mean # % CD8+ T Ratio Ratio # relative to CD8+ T cellsrelative to CD8+ T CD4+ conv CT26 Tregs iso ctrl cells iso ctrlcells:Tregs cells:Tregs mIgG2a Iso 162741 100 437223 100 3 1TPP-15285, 10 mg/kg 20872 13 1239497 283 64 9 TPP-15285, 1 mg/kg53303 33 716253 164 16 4 TPP-15285, 0.1 mg/kg 76360 47 684058 15610 2 TPP-15286, 10 mg/kg 16321 10 624631 143 44 7 TPP-15286, 1mg/kg 14349 9 765654 175 87 11 TPP-15286, 0.1 mg/kg 23202 141421674 325 68 11 mIgG1 iso 87365 100 326799 100 4 1 anti-PDL1, 10mg/kg 107029 123 313229 96 3 1

Example 12.3: Mode of Action Studies

Example 12.3.1: ADCC/ADCP Mode of Action Studies in CT26-TumorBearing Mice

Aglycosylated antibodies, which can be produced in prokaryotichosts show almost identical antigen-binding affinity, stability ata physiological temperature, and in vivo serum persistence comparedwith glycosylated counterparts. However, the absence of N-linkedglycans abrogates nearly all Fc.gamma.R-binding affinity and immuneeffector functions that are essential for clearingantibody-opsonized target cells via ADCC or ADCP. To provideevidence for the ADCC/ADCP based mode of action, aglycosylatedversions of the antibodies were generated as a negativecontrol.

TPP-18208 and TPP-18209 are aglycosylated human IgG1 versions ofanti-CCR8 surrogate antibodies and are unable to bind effectorcells, such as NK cells and/or macrophages, via Fc-gamma-receptors.These antibodies were tested for anti-tumor efficacy in CT26 tumorbearing mice and were compared with the respectivewild-type/glycosylated counterparts comprising the same sequence(TPP-14095, TPP-14099). In addition, anti-CCR8 antibodies TPP-15285and TPP-15286 (glycosylated, mIgG2 isotype) were included in thestudy.

The wild-type antibodies showed a strong anti-tumor efficacy, whilethe aglycosylated antibodies did not show any efficacy compared tothe isotype control (FIG. 44). Spider plots illustrate the resultsfor individual mice in each treatment group (FIG. 45). Accordingly,ex vivo tumor analysis by flow cytometry demonstrated Tregdepletion only after treatment with the wild-type (glycosylated)antibodies whereas the aglycosylated antibodies showed no Tregdepletion compared to the respective non-binding isotype control(FIG. 46). These results confirmed ADCC and/or ADCP as predominantmode of action for Treg depletion by anti-CCR8 antibodies in CT26tumors.

Example 12.3.2: B Cell or T Cell Involvement and Impact of CD8+Cell Depletion or CD19+ Cell Depletion on Anti-Tumor Efficacy ofAnti-CCR8 Antibodies

To test the relevance of the presence of tumor infiltrating CD8+ Tcells or CD19+ B cells on the anti-tumor efficacy of anti-CCR8antibodies, the inventors used genetically modified C57BL6 miceexpressing diphtheria toxin receptor (DTR) under the control ofeither the lineage specific Cd8a+ or the lineage specific Cd19+promoter. Upon injection of diphtheria toxin (DT) CD8+ T cells orCD19+ B cells were quantitatively depleted from both the blood ofthe mice (data not shown) as well as from subcutaneously carriedsyngeneic MC38 tumors (Table 12.3.2.1).

As expected, depletion of CD8+ T cells completely abrogated theanti-tumor effect of anti-CCR8 treatment, resulting in tumor growthidentical to that of the isotype control group (FIG. 87). Theseresults confirmed the dependency of the anti-tumor efficacy of theinventive anti-CCR8 antibodies on the presence of CD8+ T cells. Incontrast, and very surprisingly, depleting CD19+ B cellssignificantly increased the efficacy of TPP15285 (FIG. 88),resulting in a reduction of the T/C ratio from 0.35 to 0.16 (T-testp-value=0.032). Combining anti-CCR8 treatment with B cell depletionor B cell depleting agents such as anti-CD19 antibodies oranti-CD20 antibodies such as Rituximab is therefore suggested tofurther improve the cancer treatment in the clinic.

TABLE-US-00061 TABLE 12.3.2.1 Depletion of CD8a and CD19 expressionin diphtheria toxin treated DTR mice. Mean log2 Cd8a Mean log2 Cd19Treatment (N = 10 in each group) (mRNA levels) (mRNA levels) DTRmice + Isotype [TPP-10748] 6.919 1.333 DTR mice + anti-CCR8 7.3802.002 [TPP-15285, 10 mg/kg] CD19-DTR + DT + Isotype 7.466 0.406CD19-DTR + DT + anti-CCR8 9.039 0.000 [TPP-15285, 10 mg/kg] CD8-DTR+ DT + Isotype 0.711 1.766 CD8-DTR + DT + anti-CCR8 2.736 1.592[TPP-15285, 10 mg/kg]

TABLE-US-00062 TABLE 12.3.2.2 Treatment groups to evaluateinfluence of CD19+ B cell depletion or CD8+ T cell depletion. T/CDose (CD8 T/C (CD19 Group Treatment Dose Route scheme depleted)depleted) 01a Isotype control 10 mg/kg i.p. BIW 1 1 01b Anti-CCR8antibody TPP15285 10 mg/kg i.p. BIW 0.58 0.35 02a Isotype control10 mg/kg i.p. BIW 1.39 0.75 with 0.015 mg/kg Diphtheria toxin 02bAnti-CCR8 antibody TPP15285 10 mg/kg i.p. BIW 1.01 0.2 with 0.015mg/kg Diphtheria toxin

Example 12.3.3: Time Course of Increasing CD8/FOXP3 Ratios in CT26Syngeneic Tumor Bearing Mice

To gain some mechanistic insights into the immune cell levels, mRNAlevels of CD8 and FOXP3 were monitored in CT26 syngeneic tumorbearing mice treated with TPP15285 or isotype control. 10 tumorswere harvested for RNA-seq analysis directly before administeringthe first dose as well as 12 h, 24 h, 36 h, 48 h, 72 h after thefirst dose was administered. Similarly, tumors were harvested 24 hafter administration of a second, third and fourth dose ofTPP15285. All tumors were subjected to RNA-seq analysis to measuremurine Foxp3, CD8a, and CD8b1 mRNA levels. Ratios of mean Cd8a andCd8b1 to Fopx3 ratios were computed and the significance of thedifferences in the ratios between the control and TPP15285 treatedgroups was computed via a T-test.

CD8 to FOXP3 mRNA ratios varied between a value of 0.9 and a valueof around 2.0 in the isotype control and the earliest time pointsafter TPP15285 treatment, but then significantly increased in theTPP15285 groups to values of 5.8, 3.9, and 4.4 at 72 h after 1stdose, 24 h after 2nd dose, and 24 h after 3rd dose, respectively,demonstrating significant depletion of regulatory T cells byTPP15285. Ratios fell back to a value of 2.6 at 24 h after the 4thdose was administered.

TABLE-US-00063 TABLE 12.3.3.1 CD8/FOXP3 ratio of mRNA levelsmeasured at different time points after administration of TPP15285or isotype control. TPP15285 CD8/ Isotype CD8/ T-test Time pointFOXP3 ratio FOXP3 ratio p-value Day 11, 0 h after 1st dose 1.4331.822 4.0E-01 Day 11, 12 h after 1st dose 0.900 1.468 1.7E-01 Day12, 24 h after 1st dose 1.589 0.911 2.5E-01 Day 12, 36 h after 1stdose 1.965 1.012 1.3E-01 Day 13, 48 h after 1st dose 2.386 0.9765.5E-03 Day 14, 72 h after 1st dose 5.755 1.721 4.3E-02 Day 15, 24h after 2nd dose 3.882 2.028 9.4E-02 Day 18, 24 h after 3rd dose4.424 1.727 3.7E-03 Day 22, 24 h after 4th dose 2.579 1.8701.6E-01

Example 12.4: Efficacy of CCR8-Antibody TPP-15285 in EMT6-TumorBearing Mice

Example 12.4.1: Efficacy of CCR8-Antibody TPP-15285 in EMT6-TumorBearing Mice--Different Dosing

The anti-CCR8 surrogate antibody TPP-15285 showed dose-dependentefficacy in EMT6 tumor bearing mice with the strongest effect inthe 10 mg/kg dose group. For this dose, TPP-15285 had a superiorefficacy over anti-CTLA4 antibody (FIG. 47, FIG. 48). Strongefficacy was still observed in the 1 mg/kg dose group whereas the0.1 and 0.01 mg/kg dose groups showed almost no efficacy. Spiderplots illustrate these results on an individual mouse level (FIG.49).

Treg analysis of EMT6-tumor samples 24 hours after the secondantibody treatment by flow cytometry showed strongly reducednumbers of Tregs in the anti-CCR8 antibody treated versus theisotype control group. Treg depletion was clearly dose-dependent(FIG. 50). Treg depletion was also observed for anti-CTLA4, aspreviously described in literature. Additionally, a strong increaseof CD8+ T cells in the tumors sampled at study end weredemonstrated for the efficacious dose groups 10 mg/kg and 1 mg/kgof TPP-15285 as well as for anti-CTLA4 (FIG. 51). Furtheralterations of immune cell populations are shown in Table 12.4.1.1and Table 12.4.1.2.

TABLE-US-00064 TABLE 12.4.1.1 Relative percentage of immune cellpopulations determined by FACS analysis of immune cells 24 hoursafter second treatment of EMT6-tumor bearing mice with differentdoses of CCR8-antibody TPP-15285, or at the end of the study. TPP-TPP- TPP- TPP- 15285 15285, aCTLA4 mIgG2a 15285, 15285, 0.1 mg/0.01 mg/ HamIgG 10 mg/ EMT6 Iso 10 mg/kg 1 mg/kg kg kg iso kg FACSMean % CD45+ relative to iso 100 94 76 93 102 100 87 24h Mean %CD4conv+ relative to iso 100 91 120 121 155 100 52 after Mean %CD4+ relative to iso 100 59 69 95 135 100 44 2nd Mean % activatedCD8+ relative to 100 52 93 70 105 100 47 treat- iso ment Mean % NKcells relative to iso 100 95 104 135 134 100 82 Mean % CD8+ T cellsrelative 100 61 99 90 113 100 48 FACS Mean % CD45+ relative to iso100 153 116 108 107 100 91 at Mean % CD4conv+ relative to iso 100245 169 115 112 100 262 study Mean % CD4+ relative to iso 100 209150 121 119 100 152 end Mean % activated CD8+relative to 100 532377 96 67 100 314 iso Mean % NK cells relative to iso 100 310 179100 86 100 158 Mean % CD8+30 T cells relative 100 529 406 119 88100 309

TABLE-US-00065 TABLE 12.4.1.2 Relative percentage of Tregs,CD8:Treg ratio or CD4conv:Treg ratio determined by FACS analysis ofimmune cells 24 hours after second treatment of EMT6-tumor bearingmice with different doses of CCR8-antibody TPP-15285, or at the endof the study. TPP- TPP- TPP- TPP- 15285 15285, aCTLA4 mIgG2a 15285,15285, 0.1 mg/ 0.01 mg/ HamIgG 10 mg/ EMT6 Iso 10 mg/kg 1 mg/kg kgkg iso kg FACS Mean % Treg 10 26 22 75 126 100 18 analysis relativeto iso 24h after CD8:Treg ratio 1.1 3.2 4.8 1.4 0.9 1.2 4.1 secondCD4conv:Treg ratio 0.9 3.6 4.6 1.4 1 1.6 5.8 treat- ment FACS Mean% Treg 100 160 125 122 122 100 86 analysis relative to iso at studyCD8:Treg ratio 1.3 4.6 4.2 1.2 0.9 1.1 4.3 end CD4conv:Treg ratio1.2 1 0.6 0.7 0.6 1.7 1.7

Example 12.4.2: Efficacy of CCR8-Antibody TPP-15285 in EMT6-TumorBearing Mice--Single or Multiple Dosing

Murine breast cancer model EMT-6 was used with TPP-15285 to testthe impact of single vs. multiple dosing. Four different treatmentschemes were tested: single treatment, two times BIW treatment,three times BIW treatment, or four times BIW treatment. Each groupwas treated with vehicle control or 0.1 mg/kg, 1 mg/kg, or 5 mg/kgof TPP-15285. In addition to the efficacy study, satellite animalswere sacrificed for ex vivo phenotyping at 24 hr, 48 hr, and 120 hrafter treatments. Antibody administration occurred i.p. but otherroutes can be used.

Efficacy data for single treatment, two times BIW treatment, threetimes BIW treatment, or four times BIW treatment are shown in FIG.85. Interestingly, single dose treatment was suitable to obtain anexcellent treatment efficacy. At the study end, there was anincrease in frequency, absolute number and activation of CD8+intra-tumoral T cells, cf. Table 12.4.2.1. However, at the studyend there was no difference in intra-tumoral Treg number andfrequency or CCR8 expression, cf. Table 12.4.2.2. In addition,there was no difference in intra-tumoral NK cells or macrophagescell number at the end of the study or during the study (data notshown).

However, in the time course, there was a dose-dependent reductionof intra-tumoral Tregs (CD4+CD25+Foxp3+) after 1st and 2ndtreatments, but less pronounced after 3.sup.rd and 4th treatments,cf. Table 12.4.2.3. This was also observed in CCR8+ Tregs (data notshown). Furthermore, absolute numbers of CD8+ T cells increasedover time, cf. Table 12.4.2.4. Accordingly, CD8 to Treg ratiosincreased post first dose and remained high until after the fourthdose compared to isotype controls. Characterization of bloodsamples did not show substantial decrease of Tregs or increase inCD8 T cells in the blood upon different treatment schedule and overtime (data not shown). In addition, no change was observed in NKand macrophages in blood (data not shown).

TABLE-US-00066 TABLE 12.4.2.1 Absolute CD8+ cells per 100 mg tumoras determined by FACS at study end. FACS analysis revealed increaseof frequency, absolute number and activation status of Cytotoxic Tcells. Vehicle (PBS, four treatment) TPP-15285 0.1 mg/kg TPP-152851 mg/kg TPP-15285 5 mg/kg Treatment Mean SD n Mean SD n Mean SD nMean SD n single dose 1802 1066 4 3793 1300 5 4435 2787 5 179951209 3 2 treatments 3661 1826 5 6609 3373 5 7752 5470 5 3treatments 7800 8399 5 19189 21883 4 7358 4575 5 4 treatments 105698227 5 9099 3561 5 17604 10357 4

TABLE-US-00067 TABLE 12.4.2.3 Absolute CD4+ CD25+ FoxP3+ Treg cellsper 100 mg tumor as determined by FACS at study end. Vehicle (PBS,four treatment) TPP-15285 0.1 mg/kg TPP-15285 1 mg/kg TPP-15285 5mg/kg Treatment Mean SD n Mean SD n Mean SD n Mean SD n single dose2751 1624 4 2976 1507 5 2190 892 5 9366 594 3 2 treatments 26861472 5 5011 3179 5 3413 1855 5 3 treatments 3187 2399 5 5517 5406 42214 1508 5 4 treatments 3041 1449 5 3349 1427 5 7745 6271 5

TABLE-US-00068 TABLE 12.4.2.3 Absolute CD4+ CD25+ FoxP3+ Treg cellsper 100 mg tumor as determined by FACS in satellite animals. Time(after Vehicle TPP-15285/ 0.1 mg/kg TPP-15285 1 mg/kg TPP-15285 5mg/kg treatment no.) Mean SD n Mean SD n Mean SD n Mean SD n 24h(1) 13013 2878 4 7526 4116 5 11395 9162 6 10149 8020 5 48h (1)12187 8549 5 10241 3097 6 4521 2593 6 11149 14745 6 120h (1) 137646040 5 9884 5660 6 8975 3810 6 3008 2067 6 24h (2) 14315 11220 37836 5964 6 2893 1347 4 1951 1041 6 48h (2) 15022 7702 5 9377 50106 3478 2374 6 1540 1495 6 120h (2) 5134 619 5 9567 5948 6 8922 82455 3837 2598 6 24h (3) 11789 5191 5 6619 2723 4 4429 3400 6 34621954 6 48h (3) 15616 7260 5 7258 3704 4 5879 3175 6 3750 1558 6120h (3) 24h (4) 48h (4) 7011 2911 5 8137 4271 6 12037 7024 5 42933191 5

TABLE-US-00069 TABLE 12.4.2.4 Absolute CD8+ cells per 100 mg tumoras determined by FACS in satellite animals Time (after VehicleTPP-15285/ 0.1 mg/kg TPP-15285 1 mg/kg TPP-15285 5 mg/kg treatmentno.) Mean SD n Mean SD n Mean SD n Mean SD n 24h (1) 12743 4875 47549 4679 5 17691 18215 6 13238 7023 5 48h (1) 9626 4564 5 118804806 6 8949 5814 6 16745 15525 6 120h (1) 25694 24727 5 13604 77786 56974 26915 6 19107 14265 6 24h (2) 15518 6383 3 19344 10833 616597 6630 4 13271 8470 6 48h (2) 18726 8477 4 34655 15707 6 3303918954 6 14310 7167 6 120h (2) 8122 4436 5 46685 34541 6 38352 382355 53765 26430 6 24h (3) 25371 22860 5 18750 8436 4 34955 10345 641932 30806 5 48h (3) 32308 23581 5 24993 21998 4 39662 17864 669106 23874 6 120h (3) 24h (4) 48h (4) 17885 14712 5 32814 19935 650298 13584 5 34091 10942 5

Example 12.5: Efficacy of Anti-CCR8 Antibody TPP-15285 in F9 TumorBearing Mice

The anti-CCR8 surrogate antibody TPP-15285 showed dose-dependentefficacy in F9 tumor bearing mice with strong effects in the 10mg/kg and 1 mg/kg dose groups, but reduced efficacy in the 0.4mg/kg dose group, comparable to the efficacy of anti-PD-L1 antibody(FIG. 52, FIG. 53). Spider plots illustrate these results on anindividual mouse base (FIG. 54).

Treg analysis of F9 tumor samples 24 hours after the secondantibody treatment was performed by flow cytometry and showedstrongly reduced numbers of Tregs in the 10 mg/kg dose anti-CCR8antibody treated group versus the isotype control group. Tregdepletion was clearly dose-dependent (FIG. 55). Treg depletion wasalso observed for anti-PD-L1 antibody treatment. Additionally, astrong and dose-dependent increase of CD8+ T cells in the tumorswas demonstrated for all dose groups of TPP-15285 as well as foranti-PD-L1 antibody (FIG. 55). Further effects on various immunecell populations are shown in FIG. 55, FIG. 56 and Table12.5.1.

TABLE-US-00070 TABLE 12.5.1 Effects of anti-CCR8 antibodies oranti-PDL1 antibody on immune cell populations and their ratios at24 hours after second treatment. mIgG2a TPP15285, TPP15285,TPP15285, iso 10 mg/kg 1 mg/kg 0.4 mg/kg mIgG1 iso PDL1 Mean #Tregs 25324 10642 22734 41111 26435 17105 % Tregs relative 100 4290 162 100 65 to iso ctrl Mean # CD8+ T 113947 553074 388731 29160399850 246400 cells % CD8+T cells 100 485 341 256 100 247 relativeto iso ctrl Ratio CD8+ T 5.1 54.4 15.9 7.7 3.3 15.1 cells:TregsRatio CD4+ conv 2.2 28.5 9.6 4.6 3.0 5.7 cells:Tregs Mean # CD45+435118 1191853 1080867 1042860 578616 615440 % CD45+ cells 100 274248 240 100 106 relative to iso ctrl Mean # CD4 cony 49812 294714230463 179896 89198 98995 cells % CD4 cony cells 100 592 463 361100 111 relative to iso ctrl Mean # CD4+ 81866 335500 279725 240886128434 126134 cells % CD4+ T cells 100 410 342 294 100 98 relativeto iso ctrl Mean # NK cells 83353 75576 119531 172201 121590 79803% NK cells 100 91 143 207 100 66 relative to iso ctrl

Example 12.6: Efficacy of Anti-CCR8 Antibodies in CombinationTherapy

Various experiments were performed to analyze the efficacy ofanti-CCR8 antibodies alone or in combination with checkpointtargeting antibodies, such as anti-PD-L1, anti-PD1, and anti-CTLA4antibodies. Combination with these checkpoint targeting moleculeswas found to provide additional efficacy. In addition, furthercombination treatments were evaluated, e.g. with chemotherapeuticagents such as oxaliplatin, doxorubicin, gemcitabine or radiationtherapy. Table 12.6.1 summarizes the results for differentcombination treatments. Additional benefit was observed, if thetreatment with the second therapeutic agent was started only afterthe start of treatment with anti-CCR8 antibody.

TABLE-US-00071 TABLE 12.6.1 Anti-CCR8 antibody combinationtreatments and results. Tumor model Further [Example] Combinationtreatment Results benefit C38 TPP-15285 (10 mg/kg) CR combo: 8/10;++ [12.6.1] Anti-PD-L1 ab (3 mg/kg) CR anti-CCR8 ab mono: 3/10; CRanti-PD-L1 ab mono: 0/10 CT26 TPP-15285 (10 mg/kg) T/C combo: 0.45;+ (large tumors) Anti-PD-L1 ab (10 mg/kg) T/C anti-CCR8 ab mono:0.57; [data not shown] T/C anti-PD-L1 ab mono: 0.79 B16F10-OVATPP-15285 (10 mg/kg) T/C combo: 0.13; ++ [12.6.2] Anti-CTLA4 ab (10mg/kg) T/C anti-CCR8 ab mono: 0.37; T/C anti-CTLA4 ab mono: 0.30EMT-6 TPP-15285 (1 mg/kg) Synergistic efficacy in combination; ++[12.6.3] Anti-PD-1 ab (10 mg/kg) no efficacy in monotherapy C38(survival) TPP-15285 (5 mg/kg) CR combo: 10/10; ++ [12.6.4]Anti-PD-1 ab (5 mg/kg) CR anti-CCR8 ab mono: 9/10; CR anti-PD-1 abmono: 4/10 MB49 TPP-15285 (10 mg/kg) T/C combo: 0.16; ++ [12.6.5]Anti-PD-1 ab (10 mg/kg) T/C anti-CCR8 ab mono: 0.37; combo startafter CCR8 T/C anti-PD1 ab mono: 0.63 treatment EMT-6 TPP-15285 (5mg/kg) T/C (day: 19) + [12.6.6] Oxaliplatin (5 mg/kg) anti-CCR8 abmono: 0.20 Doxorubicin (6 mg/kg) Oxaliplatin mono: 0.61 Docetaxel(10 mg/kg) Doxorubicin mono: 0.43 Docetaxel mono: 0.50 Combo withOxaliplatin: 0.26 Combo with Doxorubicin: 0.19 Combo withDocetaxel: 0.20 Combo: synergistic effects in final tumoralCD8/Treg ratio as well as in frequency of activated CD8+ CD25+ Tcells in the blood Lewis Lung TPP-15285 (10 mg/kg) T/C (day: 25) +[12.6.7] Anti-PD-1 ab (10 mg/kg) Combo with aPD-1: 0.67* Anti-PD-L1ab (10 mg/kg) Combo with aPD-Ll: 0.93 Anti-CTLA4 ab (10 mg/kg)anti-CCR8 ab mono: 0.78 combo start after anti-PD-1 mono: 0.91anti-CCR8 ab treatment anti-PD-Ll mono: 0.96 anti-CTLA4 mono: 1.10* increased CD8+ frequency and CD8/Treg ratio at study end MB49TPP-15285 (10 mg/kg) T/C (day: 14) ++ [FIG. 68] Anti-PD-1 ab (10mg/kg) Combo with aPD-L1: 0.35 Anti-PD-L1 ab (10 mg/kg) Combo withaPD-1: 0.37 Gemcitabine (100 mg/kg) Combo with Gemcitabine: 0.56combo start after CCR8 anti-CCR8 ab mono: 0.61 treatment anti-PD-Llab mono: 0.67 anti-PD-1 ab mono: 0.79 Gemcitabine mono: 0.73 EMT-6TPP-15285 (3 mg/kg) Combo: 0.42 ++ [12.6.8] 3 .times. 2 GyRadiotherapy 3 .times. 2 Gy alone: 0.80 anti-CCR8 ab mono: 0.54MBT2 TPP-15285 (10 mg/kg) T/C (day: 24) ++ [12.6.9] Anti-PD-L1 ab(10 mg/kg) Combo with aPD-L1 ab: 0.41 Anti-PD-1 ab (10 mg/kg) Combowith aPD-1 ab: 0.15 Paclitaxel (10 mg/kg, q4d, i.v.) Combo withPaclitaxel: 0.39 anti-CCR8 ab mono: 0.5 anti-PD-L1 ab mono: 0.89anti-PD-1 ab mono: 0.66 Paclitaxel mono: 0.83

These results demonstrate the beneficial impact of combininganti-CCR8 antibody treatment with further therapeutically activeagents. The inventors furthermore conclude that the sequentialadministration of the therapeutic agents adds additional benefit,if the anti-CCR8 antibody is administered first and if a furthertherapeutic agent is administered only after an initial reductionof Treg cells (e.g. by at least 50%) has taken place, e.g. asdemonstrated in examples 12.6.5 and 12.6.7.

In view of these experiments and results the inventors areconvinced that triple combination, e.g. with an anti-CCR8 antibody,an antibody targeting a checkpoint protein and a targetedtherapeutic agent (small molecules as well as antibodies) providesfurther benefit in survival due to the differences in the modes ofaction of these treatments. Therefore, the combination of ananti-CCR8 antibody with a checkpoint targeting antibody and a tumorcell targeting antibody or agent is suggested for tumor treatment.In a preferred embodiment, the combination is a combination of aCCR8 antibody, an anti-PD(L)-1 antibody and paclitaxel.

Example 12.6.1: Efficacy of Anti-CCR8 Antibodies in C38 TumorBearing Mice & Combination Therapy with Anti-PD-L1 Antibody

The anti-CCR8 surrogate antibodies TPP-14099 and TPP-15285 showedstrong efficacy in C38 tumor bearing mice, comparable to theefficacy of anti-PD-L1 antibody (FIG. 57 a). Combination ofTPP-15285 with 3 mg/kg anti-PD-L1 antibody showed further improvedefficacy. Both antibodies were formulated separately in phosphatebuffered saline and were applied as in total four individualintraperitoneal injections twice weekly, i.e. in combination at thesame day with a simultaneous treatment start.

Mice were monitored in a survival study over 94 days (FIG. 57 b).Remarkably, 8 out of 10 mice treated with combination of anti-CCR8antibody and anti-PD-L1 antibody survived day 94, 4 out of 10 micetreated with CCR8 antibody TPP-15285 alone survived day 94, andnone of the mice treated with anti-PD-L1 antibody alone survivedday 94, demonstrating a synergistic efficacy of anti-CCR8 antibodyand PD-L1 antibody. C38 is a low infiltrated syngeneic mouse modelwhich uses C38 colon cancer cells for tumor induction. The model isgenerally considered responsive to anti PD-L1 antibody treatment.In view of the available data described herein, the stratificationof patients based on T cell infiltration and/or response to PD-L1therapy is suggested to provide additional benefit.

Treg analysis of C38 tumor samples 24 hours after the secondantibody treatment by flow cytometry showed strongly reducednumbers of Tregs in the anti-CCR8 antibody treated groups versusisotype control group (FIG. 58). The strongest Treg depletion wasobserved for the combination of TPP-15285 with anti-PD-L1 antibody.Additionally, the highest CD8+/Treg ratio was observed for thecombination of TPP-15285 with anti-PD-L1 antibody. Interestingly,analysis of macrophages showed an increase at day 24 of the study(FIG. 59).

Example 12.6.2: Efficacy of Anti-CCR8 Antibodies in B16F10-OVATumor Bearing Mice & Combination Therapy with Anti-CTLA4Antibody

The anti-CCR8 antibody TPP-15285 (10 mg/kg) showed strong efficacyin B16F10-OVA bearing mice comparable to the efficacy of ananti-CTLA4 antibody (10 mg/kg) (FIG. 60). Combination of TPP-15285with anti-CTLA4 antibody showed synergistic efficacy, correlatingwith an increase of CD8+ T cells and IFNg/TNFa levels.

Both antibodies were formulated in PBS and were applied as in totalthree individual intraperitoneal injections twice weekly, incombination at the same day with a simultaneous treatmentstart.

Treg analysis of B16F10-OVA tumor samples 24 hours after the secondantibody treatment (day 12) by flow cytometry showed stronglyreduced numbers of Tregs in the CCR8-antibody treated groups versusthe isotype control group (Table 12.6.2.1). The strongest Tregdepletion was observed for the combination of TPP-15285 withanti-CTLA4 antibody. Additionally, the strongest CD8+ T cellincrease was observed for the combination of TPP-15285 withanti-CTLA4 antibody.

FACS analysis of B16F10-OVA-tumors 24 hours after second antibodytreatment with TPP-15285+isotype control, anti-CTLA4antibody+isotype control or TPP-15285+anti-CTLA4 antibodydemonstrated a decrease of intra-tumoral Treg frequency and anincrease of absolute numbers of CD8+ T cells.

TABLE-US-00072 TABLE 12.6.2.1 Intra-tumoral cell populationsdetermined by FACS and IFNg determined by ELISA at the study end.mulgG2a + TPP15285 + aCTLA4 + TPP15285 + hulgG1 isotypes hulgG1isotype mulgG2a isotype aCTLA4 absolute CD45 199442.1 437004.9611423.6 717324.5 absolute CD8 25327.0 155532.5 220147.5 312653.9absolute Treg 6261.1 9148.6 15413.2 11448.3 abs NK+ 11066.6 36950.065954.0 57789.1 % NK+ 5.9 8.1 10.1 7.6 % CD8 14.6 34.8 36.5 42.7 %Treg 31.58 18.86 23.2 8.98 IFNg (pg/ml per mg protein) 3.80 28.8318.40 35.70 *mean for n = samples.

Example 12.6.3: Combination Treatment with Anti-PD-1 Antibody andAnti-CCR8 Antibody in EMT-6 Tumor Bearing Mice: SynergisticEfficacy

The therapeutic efficacies of anti-murine CCR8 antibody TPP-15285(1 mg/kg) alone, anti-murine PD-1 antibody (CDRs: atezolizumab, 10mg/kg) alone or combination of TPP-15285 (1 mg/kg) and anti-murinePD-1 antibody (10 mg/kg) were evaluated in EMT-6 tumor bearingmice. Both antibodies were formulated in PBS and applied as intotal four individual intraperitoneal injections twice weekly, incombination at the same day with a simultaneous treatmentstart.

The anti-CCR8 antibody TPP-15285 showed weak efficacy in EMT-6bearing mice at a low dose of 1 mg/kg, comparable to the weakefficacy of anti-PD-1 antibody at 10 mg/kg (FIG. 61). Combinationof 1 mg/kg TPP-15285 with 10 mg/kg anti-PD-1 showed synergisticefficacy.

Treg analysis of EMT-6 tumor samples 24 hours after the secondantibody treatment by flow cytometry showed strongly reducednumbers of Tregs in the CCR8-antibody treated groups versus theisotype control group or anti-PD-1 monotherapy (FIG. 62).

TABLE-US-00073 TABLE 12.6.3.1 Intra-tumoral cell populationsdetermined by FACS in EMT-6 tumors at the study end. isotypeTPP15285 + Anti-PD1 ab + TPP15285 + Intra-tumoral immune cellscontrol isotype control isotype control anti-PD1 ab CD4+ (*)48583.4 27646.1 54791.8 42312.3 Tregs 30686.4 7998.8 28781.711506.9 (CD4+ CD25+ FoxP3+) (*) CD8+ T-cells (*) 13825.7 16398.819233.3 23132.7 NK cells (CD45+ CD3+ NKp46+) (*) 29119.6 44152.151372.0 67155.1 CD8: Treg ratio 0.5 2.1 0.7 2.0 * mean for n =samples, cells/100 mg tumor.

Example 12.6.4: Combination Treatment with Anti-PD-1 Antibody andAnti-CCR8 Antibody in C38 Tumor Bearing Mice

The therapeutic efficacies of anti-murine CCR8 antibody TPP-15285(5 mg/kg) alone, anti-murine PD-1 antibody (aPD-1, CDRs:atezolizumab, 5 mg/kg) alone or combination of TPP-15285 (5 mg/kg)and anti-murine PD-1 antibody (5 mg/kg) were evaluated in C38 tumorbearing mice. Both antibodies were formulated in PBS and applied asin total four individual intraperitoneal injections twice weekly,in combination at the same day with a simultaneous treatmentstart.

The anti-CCR8 antibody TPP-15285 showed a strong efficacy in C38tumor bearing mice which was higher than the efficacy of ananti-PD-1 antibody administered at the same dose (FIG. 63 a). After64 days, combination of 5 mg/kg TPP-15285 with 5 mg/kg anti-PD-1antibody showed improved efficacy in survival with 10/10 completeresponders compared to 9/10 for anti-CCR8 monotherapy and 4/10 foranti-PD1 monotherapy (FIG. 63 b).

Treg analysis of C38 tumor samples 24 hours after the secondantibody treatment by flow cytometry are shown in FIG. 64, Table12.6.4.1.

TABLE-US-00074 TABLE 12.6.4.1 Intra-tumoral cell populationsdetermined by FACS in C38 tumors 24 hours after the second antibodytreatment. TPP15285 + Anti-PD1 ab + TPP15285 + Intra-tumoral immunecells isotype control isotype control isotype control Anit-PD1 abCD4+ (*) 79735.2 109508.8 97352.8 189350.0 Tregs 27132.4 24495.832360.0 48581.6 (CD4+ CD25+ FoxP3+) (*) CD8+ T-cells (*) 44063.8151644.0 160225.4 291992.8 NK cells (CD45+ CD3+ NKp46+) (*)124067.0 173865.5 115642.8 295252.5 CD8: Treg ratio 1.7 6.0 4.710.2 * mean for n = samples, cells/100 mg tumor.

Example 12.6.5: Sequential Combination Treatment of Anti-CCR8Antibody with Anti-PD-1 Antibody in MB49 Tumor Bearing Mice

The therapeutic efficacy of anti-murine CCR8 antibody TPP-15285 (10mg/kg) alone, anti-murine PD-1 antibody (CDRs: atezolizumab, 10mg/kg) alone and combination of TPP-15285 (10 mg/kg) and anti-PD-1antibody (10 mg/kg) was evaluated in a study in MB49-tumor bearingmice. In this study, the first dose of anti-PD1 antibody wasadministered only after anti-CCR8 antibody, i.e. after allowing theanti-CCR8 antibody to cause an effect on Tregs leading to anincreased CD8+ T cell to Treg ratio. More specifically, treatmentwith anti-PD1 antibody was started 24 hours after the secondanti-CCR8 antibody treatment. Anti-CCR8 antibody TPP-15285 showed astrong efficacy in MB49 tumor bearing mice that was also higherthan for anti-PD1 antibody (FIG. 65). Combination of 10 mg/kgTPP-15285 with 10 mg/kg anti-PD-1 showed further improvedefficacy.

FACS analysis of MB49-derived tumor samples at the study end showedincreased CD45+ cell, CD8+ cell and NK cell frequency and reducedfrequency of Tregs in the anti-CCR8 antibody treated groups versusvehicle control group or anti-PD-1 antibody monotherapy (FIG. 66,Table 12.6.5.1).

MB49 is a medium infiltrated syngeneic model using bladder cancercells which is responsive to immune checkpoint inhibitor (ICI)therapy. In view of these data, the stratification of subjects forlow immune infiltration and/or response to ICI therapy is suggestedto provide additional benefit for this combination therapy.

In particular, the start of combination treatment after anti-CCR8antibody treatment was found to provide a benefit. Without beingbound by theory, the inventors assume that initial depletion ofTregs by CCR8 antibodies enhances the activity ofantigen-presenting cells and priming of tumoral T cells resultingin improved sensitivity for checkpoint inhibitors such as PD-(L)1,because Tregs as tumor-cell extrinsic factors play a role forprimary and adaptive resistance to checkpoint inhibitors in cancerpatients.

TABLE-US-00075 TABLE 12.6.5.1 Intra-tumoral cell populationsdetermined by FACS in MB49 tumors at the study end. TPP15285 +Intra-tumoral immune cells isotype control TPP15285 Anti-PD1 abanti-PD1 ab CD45+ cells (in % of live) 30.1 53.2 40.9 72.7 Tregs(CD4+ CD25+ FoxP3+ 27.6 17.7 20.6 18.6 cells in % of CD4+ cells)(*) CD8+ cells (in % of CD45+) 6.3 8.4 8.3 16.3 NK cells (NKp46+cells in % 4.0 5.6 3.7 6.4 of CD45+ cells) CD8: Treg ratio 11.010.9 12.7 20.8 * mean for n = samples, cells/100 mg tumor.

Example 12.6.6: Combination Treatment Comprising Anti-CCR8 Antibodyand Chemotherapy in EMT-6 Tumor Bearing Mice

The therapeutic efficacies of anti-CCR8 antibody TPP-15285 (5mg/kg, q3/4d i.p.) alone, Oxaliplatin (5 mg/kg, q4d i.p.) alone,Doxorubicin (6 mg/kg, i.v. SD) alone, Docetaxel (10 mg/kg, q2dx5,i.v.) alone or a combination of TPP-15285 with either Oxaliplatin,Doxorubicin or Docetaxel were evaluated in EMT-6 tumor bearingmice. Therapeutics were formulated in PBS, combination treatmentstarted at the same day with a simultaneous treatment start.

While the combination of anti-CCR8 antibody with eachchemotherapeutic agent provided benefit over the monotherapy withthat chemotherapeutic agent, the combination was not superior overanti-CCR8 monotherapy (FIG. 67). The inventors are convinced, thatsequential administration of the therapeutic agents is beneficial,allowing the anti-CCR8 antibody to effectively deplete Tregs beforethe chemotherapeutic is applied to deplete fast dividing cells.

Immune cell populations were analyzed at the end of the study, bothin tumor and in blood by flow cytometry, cf. Table 12.6.6.1 andTable 12.6.6.2. For combination treatments intra-tumoral CD8/Tregratio and frequency of activated CD8+CD25+ T cells in the blood wasincreased compared to each monotherapy and control.

Furthermore, IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alphawere analyzed. Increased levels of IFN gamma, IL-1b and IL-2 wereobserved in mono and combo groups. Increase of TNF alpha wasobserved in combo groups only (data not shown).

TABLE-US-00076 TABLE 12.6.6.1 Intra-tumoral cell populationsdetermined by FACS at the end of the study. *mean for n = 5samples, cells/100 mg tumor if not specified otherwise. EMT-6,tumor TPP15285 + TPP15285 + TPP15285 + Vehicle TPP15 Oxali- Doxo-Doce- Oxali- Doxo- Doce- (PBS) 285 platin rubicin taxel platinrubicin taxel absolute CD8+ cells 19215.4 106043.2 42872.6 43123.862549.6 107959.2 61187.0 56554.2 absolute Tregs 7397.0 12816.07075.2 12995.4 20688.0 6358.0 4363.0 4693.8 CD8+ cell/Treg ratio2.9 11.1 7.3 2.7 3.1 22.3 15.9 13.0 % CD8+ cells of 1.8 14.0 5.95.6 5.4 13.1 14.6 9.4 CD45+ cells % act. CD8+ cells of 37.7 13.926.8 22.0 17.9 17.1 21.4 23.1 CD8+ cells % NK cells of CD45+ 5.37.9 4.2 5.0 4.8 4.8 8.6 6.5 cells

TABLE-US-00077 TABLE 12.6.6.2 Blood immune cell populationsdetermined by FACS at the end of the study. *mean for n = 5samples, cells/100 .mu.l blood EMT-6, blood TPP15285 + TPP15285 +TPP15285 + Vehicle TPP15 Oxali- Doxo- Doce- Oxali- Doxo- Doce-(PBS) 285 platin rubicin taxel platin rubicin taxel absolute CD8+13599.6 12491.2 5208.8 5502.0 9840.2 4094.8 4139.8 7000.8 cellsabsolute Tregs 1333.2 807.8 511.8 549.2 833.8 390.8 364.8 709.2Absolute CD45+ 188940 166472 120713 104991 105002 47850 65061121155 cells CD8+ cell/Treg 12.4 15.6 13.8 12.6 12.2 10.0 13.2 10.2ratio % CD8+ cells of 7.1 6.9 5.4 5.6 9.5 8.2 6.7 6.3 CD45+ cells %act. CD8 of 1.6 3.1 2.0 4.2 4.2 6.7 10.7 10.3 CD8+ cells

Example 12.6.7: Sequential Combination Treatment with Anti-PD-(L)1Antibody and Anti-CCR8 Antibody in Lewis Lung Carcinoma BearingMice

The therapeutic efficacies of anti-CCR8 antibody TPP-15285 (10mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10mg/kg) alone, anti-PD-L1 antibody (10 mg/kg) alone, anti-CTLA4antibody alone, TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10mg/kg) in combination, or TPP-15285 (10 mg/kg) and anti-PD-L1antibody (10 mg/kg) in combination were evaluated in Lewis lungtumor bearing mice (FIG. 69). Antibodies were formulated in PBS.For the anti-CCR8 antibody, doses were administered at day 11, 14,18 and 22 after tumor inoculation. For combination treatment,administration of anti-PD-(L)1 antibody started 24 hours after the2.sup.nd anti-CCR8-antibody dose. At this time point, FACS analysisshowed an intra-tumoral Treg depletion for the TPP-15285 mono groupof 60% relative to the isotype control (data not shown). Lewis lungwas used as a syngeneic model with a suppressive tumormicroenvironment and is known to be not responsive for anti PD-(L)1treatment and anti-CTLA4 treatment. No monotherapeutic efficacy ofTPP-15285 was observed despite effective Treg depletion measured 24hours after 2.sup.nd treatment. However, the combination treatmentwith anti-murine PD-1 antibody after anti-CCR8 antibody showedimproved efficacy and this was also associated with increasedCD8/Treg ratio at study end (Table 12.6.7.1).

TABLE-US-00078 TABLE 12.6.7.1 Intra-tumoral cell populationsdetermined by FACS at the study end. *mean for n = 5 samples.TPP15285 + TPP15285 + Vehicle +Anti- Anti-PD-Ll Intra-tumoralcells(PBS) TPP15285 Anti-PD-1 ab PD-1 ab Anti-PD-L1 ab ab absolute CD8+4682.4 7617.0 4593.8 6162.2 5684.4 7769.4 absolute Tregs 1202.61452.0 1207.0 805.8 1480.6 1355.0 CD8/ Treg ratio 4.0 7.9 6.0 7.34.7 5.1 % CD8 0.7 0.8 0.6 1.5 0.8 0.9 % activated CD8 19.8 14.911.9 12.2 24.5 18.6

Example 12.6.8: Combination Treatment Comprising Anti-CCR8 Antibodyand Radiotherapy in EMT-6 Tumor Bearing Mice

The therapeutic efficacies of anti-CCR8 antibody TPP-15285 (3mg/kg) or radiotherapy (RT, 3.times.2 Gy) were evaluated alone orin combination in EMT-6 tumor bearing mice (FIG. 70). Combinationtreatment was started with 3.times.2 Gy fractionated irradiation,followed by 3 mg/kg biw.times.2. Radiation therapy alone onlymildly delayed tumor growth. Combination of anti-CCR8 antibody andradiotherapy showed only minor improvement in efficacy but Tregdepletion at study end was more pronounced than for eachmonotherapy (data not shown).

Example 12.6.9: Combination Treatment Comprising Anti-CCR8 Antibodyand Administration of PD-1 Antibody, PD-L1 Antibody, or Paclitaxelin MBT2 Tumor Bearing Mice

The therapeutic efficacies of inventive anti-CCR8 antibody,anti-PD-1 antibody, anti-PD-L1 antibody, or Paclitaxel were testedalone or in combination in MBT2 syngeneic tumor bearing mice asshown in Table 12.6.9.1. Each group comprised 10 tumor-bearing miceand treatment started at day 10 after inoculation, when tumors hadreached a size of about 100 mm.sup.3. Among the single treatmentgroups, anti-CCR8 showed the strongest and a significant anti-tumoreffect. The anti-tumor effect of anti-PD-1 antibody, anti-PD-L1antibody, or Paclitaxel was significantly increased by combiningthese treatments with anti-CCR8 therapy. Antibodies were formulatedin PBS. T/C ratios were analyzed after three weeks of bi-weeklytreatment.

TABLE-US-00079 TABLE 12.6.9.1 Treatment groups and T/C ratiorelative to isotype control at day 24. Group Treatment Dose RouteDose scheme T/C 01 Isotype control 10 mg/kg i.p. BIW 1 02 Anti-CCR8antibody TPP15285 10 mg/kg i.p. BIW 0.50 03 anti-PDL1 antibody 10mg/kg i.p. BIW 0.89 04 anti-PD1 antibody 10 mg/kg i.p. BIW 0.66 05Paclitaxel 10 mg/kg i.v. Q4D 0.83 06 Combination: TPP15285 +anti-PDL1 ab 10 mg/kg i.p. + i.p. BIW + BIW 0.41 07 Combination ofTPP15285 + anti-PD1 10 mg/kg i.p. + i.p. BIW + BIW 0.15 ab 08Combination of TPP15285 + Paclitaxel 10 mg/kg i.p. + i.v. BIW + Q4D0.39

TABLE-US-00080 TABLE 12.6.9.2 Mean CD8 Standard Fold change toT-test Treatment expression deviation Isotype p-value Isotype[TPP10748] 7.493 1.378 1.000 1.0E+00 anti-CCR8 [TPP15285, 10 mg/kg,BIW] 9.308 1.282 3.518 2.5E-04 anti-PD-L1 [10 mg/kg, BIW] 7.9291.448 1.353 3.6E-01 anti-PD1 [10 mg/kg, BIW] 8.156 0.915 1.5831.1E-01 Paclitaxel [10 mg/kg, Q4D] 8.260 1.197 1.702 9.2E-02anti-CCR8 + PDL1 9.805 0.984 4.966 2.3E-06 anti-CCR8 + PD1 10.5241.078 8.171 9.4E-08 anti-CCR8 + Paclitaxel 9.018 1.088 2.8766.9E-04 CD8 mRNA levels are increased after anti-CCR8 combinationtreatments. CD8 mRNA levels were obtained via RNA-seq performed onMBT2 tumors obtained 24 h after the end of the treatment cycle.Average CD8 expression levels were computed for 10 tumors from eachtreatment group. Anti-CCR8 had treatment alone significantlyincreased CD8 levels compared to isotype while PD1, PDL1, andpaclitaxel no significant effect. CD8 levels were further increasedby combining anti-CCR8 treatment with the other three treatments.The most significant and strongest increase of over 8-fold wasobtained by the combination of anti-CCR8 with anti-PD1treatment.

TABLE-US-00081 TABLE 12.6.9.3 Mean tumor SD tumor T/C T-test GroupTreatment size [mm3] size [mm3] ratio p-value 1 Isotype [TPP10748]3108.8 697.9 1 1.00E+00 2 anti-CCR8 [TPP15285 10 mg/kg, BIW] 1539.91431.4 0.50 1.38E-02 3 anti-PD-L1 [10 mg/kg, BIW] 2758.1 972.3 0.894.33E-01 4 anti-PD1 [10 mg/kg, BIW] 2051.7 1491.6 0.66 1.03E-01 5Paclitaxel [10 mg/kg, Q4D] 2566.2 963.7 0.83 2.53E-01 6 anti-CCR8 +PDL1 1274.9 1159.0 0.41 1.68E-03 7 anti-CCR8 + PD1 473.1 571.7 0.154.12E-06 8 anti-CCR8 + Paclitaxel 1197.1 826.4 0.39 3.14E-04 Tumorsizes at day 24 are significantly reduced by anti-CCR8 treatmentand further reduced by combining anti-CCR8 with either PD1 antibody(CrownVivoPremium, Catalog No RMP1-14), PDL1 antibody(CrownVivoPremium, Catalog No CVP034), or Paclitaxel. Largest andmost significant reduction in tumor size compared to isotypecontrol is achieved by the combination of anti-CCR8 with anti-PD1antibody.

Example 12.7.1: Correlation Between Anti PD-L1 Antibody Responseand Anti-CCR8 Antibody Response

In order to benchmark the efficacy of anti-CCR8 antibody treatmentagainst treatment with anti-PD-L1 antibody, the outcomes for thedifferent syngeneic tumor models were assembled, see Table12.7.1.1. Surprisingly, a good response to anti-PD-L1 antibodytreatment--as measured e.g. by T/C volume--was also predictive fora good response to anti-CCR8 antibody treatment. PD-L1 expressionis a known predictor to select responders to anti-PD-L1 treatment.From these observations, the inventors hypothesized that PD-L1expression might likewise be suitable to stratify subjects in orderto identify those which would most likely profit from anti-CCR8antibody treatment. This hypothesis could be retroactivelyvalidated by correlating the PD-L1 expression levels with tumorvolume.

TABLE-US-00082 TABLE 12.7.1.1 Comparison between responses to antiPD-Ll antibody vs. anti-CCR8 antibody for different syngeneicmodels. T/C.sub.vol General (end of % Treg Tumoral ORR .sup.# Tumorimmune cell experiment) depletion .sup.1 CD8+ change* (% CR) Modelinfiltration aCCR8 aPDL1 aCCR8 aCCR8 aCCR8 Responding CT26 high0.18 0.68 82.3 2.84 .sup.1 72 (30) models EMT-6 medium 0.28 0.6861.6 5.89 90 (0) F9 medium 0.21 0.25 73.2 4.85 .sup.1 60 (30) C38low 0.23 0.58.sup.+ 68.2 1.32 .sup.1 40 (30) B16F10- low 0.37 n.d.59.0 6.14 0 (0) OVA Non-resp. 4T1 medium 0.90 >0.9** 37.1 2.37 0(0) Models B16F10 low 0.95 >0.9** 7.5 1.39 0 (0)

Example 12.7.2: Correlation Between Anti-Tumor Response and mRNABiomarker for Stratification or Disease Control

Mean gene expression (RNA-seq) in early untreated tumors (100-200mm.sup.3 in size, N=10 per model), larger untreated tumors (500 and1000 mm.sup.3) or at the end of the study was correlated with T/Cratios of 21 syngeneic mouse models treated with TPP-14099 orTPP-15285 as described elsewhere herein.

Top correlated genes were found to be significantly enriched with Tcell and inflammation markers (pearson correlation values), cf.Table 12.7.2.1. Inflammation marker IFNg and IFNg response genes(e.g. Gbp3/4/5/8/9, Cxcl9, Acod1), PDL1 (CD276), C1 complementfactors, T cell genes such as Klra3/5, and Tray, as well as Tregmarkers CD25 (IL2RA), FOXP3, and CTLA4 are among the genes mostcorrelated with response.

Table 12.7.2.2 shows fold change of expression in responder celllines (T/C<0.6) versus non-responder cell lines (T/C>0.6) forearly untreated tumors. The 100 genes with largest expression foldchange between responders and non-responders are listed. IFNg andIFNg response genes (e.g. Gbp2/3/4/8/10/11, Cxcl9/11, Ubd),cytotoxic T cell markers (e.g. Granzymes, Prf1), as well as Mastcell markers (Tpsab1, Cma1, Tpsb2), and C1 complement factors areamong the genes most predictive of response.

Table 12.7.2.3 shows fold change of expression in responder celllines (T/C<0.6) versus non-responder cell lines (T/C >0.6)for larger untreated tumors (500 and 1000 mm.sup.3).

Biomarker candidates are listed in Table 12.7.2.1, 12.7.2.2 and12.7.2.3. Patients with high expression of the human counterpartsof these genes or a combination or signature of the humancounterparts of these genes can thus be expected to respond toanti-CCR8 treatment in the clinic. Furthermore, these markers ortheir combination can also be used to monitor the treatmentsuccess. Biomarkers derived from genes in italics/bold areparticularly preferred.

TABLE-US-00083 TABLE 12.7.2.1 Suitable biomarkers correlating withanti-tumor response achieved with inventive anti-CCR8 antibodies.These biomarkers were found upregulated in early untreated tumorsand correlated with treatment response. Biomarker derived fromgenes in italics/bold are particularly preferred. The 100 geneswith largest negative correlation coefficient (r values are shown)between expression and TIC ratios are listed. Gene R Gene R Gene RGene R Grin2d -0.658 Fkbp15 -0.583 Cfb -0.558 Dnah7a -0.539Olfr1395 -0.656 Irf1 -0.582 Prm1 -0.556 Yipf4 -0.538 Olfr1122-0.655 Eif3h -0.579 Gbp5 -0.556 C1ra -0.538 Gbp -0.638 Rab8a -0.578Stat1 -0.556 Tray9n-1 -0.534 Mir8098 -0.626 Xpa -0.578 Nfkbiz-0.554 Ccdc9 -0.533 Ifng -0.62 Suox -0.578 Klra5 -0.553 Gbp3 -0.533Eny2 -0.618 Scgb1b30 -0.578 Ano8 -0.552 Foxp3 -0.533 Hebp1 -0.614Rarres2 -0.574 Slc12a9 -0.552 Epb4114a -0.533 Cd274 -0.614 Ywhag-0.573 Nr1h2 -0.552 Klra3 -0.533 Gbp10 -0.613 Fg12 -0.573 Aip-0.552 I115 -0.532 Esyt2 -0.611 Wdtc1 -0.572 Shisa5 -0.551 Myo1e-0.529 Clic6 -0.607 Ctla4 -0.571 Bc12a1c -0.55 Wdr60 -0.529 Greb11-0.606 Batf2 -0.571 Fam32a -0.548 Trim56 -0.528 Trav7d-3 -0.604Olfr56 -0.571 Tatdn1 -0.547 Ppp1r15a -0.526 Pinc -0.603 Cnep1r1-0.571 Cd200r4 -0.547 Iigp1 -0.526 Cenpt -0.602 Emp1 -0.57 Gbp4-0.546 Fes -0.524 Cxcl9 -0.601 Klf4 -0.567 Ccl8 -0.545 Trim12c-0.523 Fam26f -0.6 Hrh4 -0.566 Ttc39c -0.544 Psmg4 -0.523 Il2ra-0.6 Gbp9 -0.566 Klk10 -0.544 Igfbp5 -0.521 Hectd2 -0.596 Nlrp9c-0.565 Crybg1 -0.543 Itgb1 -0.52 Chmp5 -0.593 Fam129b -0.564 Gnb2-0.542 Klf9 -0.518 Pcdhb22 -0.59 Tlr6 -0.562 Ctsg -0.542 Actr1a-0.518 Slitrk4 -0.589 Icos -0.562 Olfr1396 -0.542 Sh2d1a -0.517Ill8bp -0.586 Tmsb15a -0.56 Acod1 -0.54 Siah1a -0.516 Cyth2 -0.585Pla2g15 -0.559 ENS87554 -0.54 Ccl11 -0.516

TABLE-US-00084 TABLE 12.7.2. Suitable biomarkers correlating withanti-tumor response achieved with inventive anti-CCR8 antibodies.Fold change of expression in responder cell lines (TIC < 0.6)versus non-responder cell lines (T/C > 0.6). Top correlated(pearson) genes were found significantly enriched with T cell andinflammation markers. Log-fold Log-fold Log-fold Log-fold Genechange Gene change Gene change Gene change Gbp2b 4.6 Serpinb9b 2.6Prkg2 2.2 Pak3 2.0 Msln 4.4 Gbp4 2.5 Batf2 2.2 C1rb 2.0 Klk10 4.2Itgb4 2.5 Nppb 2.2 Itgb11 2.0 Mcpt2 3.5 Gzmf 2.5 C1s2 2.2 Serpina3g2.0 Gbp10 3.4 Gzmd 2.5 Dio2 2.2 Itih2 2.0 Foxg1 3.2 AW112010 2.5Bdnf 2.2 Tpsb2 2.0 Tpsab1 3.2 Ido2 2.5 Itgb6 2.2 Phex 2.0 Ifi44l3.1 Gbp2 2.4 Ar 2.2 Hal 2.0 Syt8 3.1 Iigp1 2.4 Tgtp2 2.2 Clcf1 2.0Cxcl9 3.0 Notum 2.4 Tnni2 2.2 Ifi47 2.0 Hectd2 2.9 Gbp3 2.4 Styk12.1 Fam26f 2.0 Lgals7 2.9 Tgtp1 2.4 H19 2.1 Trbc1 2.0 Ido1 2.8Cxel11 2.4 Rpl391 2.1 Il34 2.0 Zfpm2 2.8 Irx2 2.3 Inhba 2.1Serpina3f 2.0 Dynap 2.7 Tmem200a 2.3 Zfp985 2.1 Grem1 2.0 Cdh17 2.7Ifng 2.3 S100a7a 2.1 Btc 2.0 Gzmb 2.7 Klhdc8a 2.3 Olfr56 2.1 Sox92.0 Ubd 2.7 Fst 2.3 Gjb4 2.1 Klf4 1.9 Gbp11 2.6 Trpm6 2.3 Nkg7 2.1Pil5 1.9 Mcpt1 2.6 Syt12 2.3 Ankrd1 2.1 S1it2 1.9 Gzmg 2.6 Gbp5 2.3Rab27b 2.0 Gprc5a 1.9 Ifi202b 2.6 Cntnap4 2.3 Klf5 2.0 Gbp8 1.9Greb11 2.6 Gzmc 2.3 Eps812 2.0 Cma1 1.9 Brinp3 2.6 Prf1 2.3Epb4114a 2.0 Mgp 1.9

TABLE-US-00085 TABLE 12.7.2.3 Suitable biomarkers based on geneswith largest fold changes between responders and non- respondersbased on large untreated tumors (500 mm.sup.3 and 1000 mm.sup.3).Expression of granzymes & other immune cell markers is higherin responders than non-responder models. Biomarker derived fromgenes in italics/bold are particularly preferred. Log-fold Log-foldLog-fold Log-fold Log-fold Gene change Gene change Gene change Genechange Gene change Foxg1 4.6 Itgb4 2.9 Sorcs2 2.5 Aicda 2.3 Fas 2.0Msln 4.5 Rarres1 2.9 Gzmc 2.5 Lhx2 2.3 Klk4 2.0 Zfpm2 4.3 Dcn 2.9Prrx2 2.5 Dpep1 2.3 Sema5 a 2.0 Klk10 4.2 Osr1 2.8 Lef1 2.5 Lancl32.3 Lrch2 2.0 Dynap 4.0 Inhba 2.8 Ighg1 2.5 Ereg 2.2 Fam189a1 2.0Ar 3.8 Greb11 2.8 Chrna1 2.5 Foxa1 2.2 Tpsb2 2.0 Fst 3.6 Bdnf 2.8Ighg2b 2.5 Ighg2c 2.2 Shank1 2.0 Syt8 3.5 Wnt10a 2.7 Omd 2.5 Dmrta12.2 Dsc2 2.0 Hectd2 3.4 Prkg2 2.7 Sema3c 2.5 Cavin2 2.2 Gpm6b 2.0Pi15 3.4 Slc24a3 2.7 Epb4114a 2.5 Grem1 2.2 Slc12a1 2.0 Foxd1 3.4Ptgs2 2.7 Gzme 2.5 Fam83f 2.2 Plut 2.0 Gzmf 3.3 Arsj 2.7 Ngef 2.5Pak3 2.2 Gzmb 2.0 Zic5 3.2 Itgb8 2.7 Trp63 2.4 H19 2.2 Prelp 2.0Igf2 3.1 Sh3rf3 2.7 Klhdc8a 2.4 Slit2 2.2 Gbp4 2.0 Gzmg 3.1 Medag2.7 Slurp1 2.4 Bmp4 2.1 Rnf165 2.0 Itgbl1 3.1 Tnni2 2.7 Tpsab1 2.4Grin2d 2.1 Syt12 2.0 Tmem200a 3.1 Cntnap4 2.7 Pdx1 2.4 Ifi202b 2.1Btc 2.0 Dio2 3.0 Sema3a 2.6 Pdgfra 2.4 Kirrel3 2.1 Tssk6 2.0 Gzmd3.0 Ptgs2os2 2.6 Dkk2 2.4 Podnl1 2.1 Slit3 2.0 Aldh3al 3.0 Anxa82.6 Cavin4 2.4 Prf1 2.1 Gcsam 2.0 Scara3 3.0 Rpl391 2.6 Kenu1 2.4Lg als7 2.1 Il11 2.0 Il33 2.9 Pcdh19 2.6 Xlr 2.4 Gbp10 2.1 Sybu 2.0Brinp3 2.9 Ltbp1 2.6 Cdh17 2.3 Gbp8 2.1 Mrgprf 2.0 Pcdhgb1 2.9Gbp2b 2.5 Ighg3 2.3 Cdy12 2.1 Irx2 2.0

Example 12.8: Altered mRNA Expression in Syngeneic Tumor Modelsafter Administration of Anti-CCR8 Antibody

For each syngeneic tumor model, 10 mice treated with anti-CCR8antibody TPP-14099 and 10 mice treated with isotype control weresacrificed 24h past final treatment. Tumors were cut into smallpieces and immersed in RNAlater at 4.degree. C. Poly-A mRNA wasextracted and cDNA libraries were generated according to Illumina'sHi-seq protocol for subsequent RNA-sequencing. Samples weresequenced to a depth of .about.40 million 150 bp long paired endreads/sample.

FIGS. 63 to 73 show the impact of treatment with either isotypecontrol (TPP-9809) or anti-CCR8 antibody (TPP-14099) on the mRNAexpression levels of different immune cell markers in differentsyngeneic tumor models. The treatment increased inflammation markerling, macrophage markers Ms4a7, Acod1 and Mrc1, cytotoxic T cellmarkers Cd8a and Cd8b1, Natural killer (NK) cell marker Ncr1, pan Tcell markers Cd3e/d/g and B cell markers Cd19 and Cd22.

Remarkably, the inventors observed significant induction of LTta/bas well as Cxcr5 and its ligand Cxcl13 in the CT26, MBT2 and H22tumor models, as well as a general upregulation of these genes inseveral other models including RM1, Hepa1-6, and 4T1. Without beingbound by theory, an induction of tertiary lymphoid structures byanti-CCR8 antibody TPP-14099 or TPP-15285 may contribute to theanti-tumor response elicited by these antibodies.

TABLE-US-00086 TABLE 12.8.1 Anti-correlation of T cell marker,checkpoint proteins, Treg markers, NK cell marker, macrophagemarker, B cell marker and interferon gamma with anti-CCR8 antibodytreatment response. Cd274 Pdcd1 Cd8a [PD-L1] [PD-1] Ctla4 Ccr4 Ccr8Foxp3 Ncr1 Ms4a7 Fcgr2b Ifng CT26 r -0.693 -0.630 -0.600 -0.654-0.612 -0.664 -0.531 -0.775 -0.626 -0.4- 96 -0.606 pvalue 0.0260.051 0.067 0.040 0.060 0.036 0.114 0.008 0.053 0.145 0.063 H22 r-0.791 -0.782 -0.852 -0.804 -0.779 -0.888 -0.828 -0.738 -0.686-0.710 -0.896 pvalue 0.011 0.013 0.004 0.009 0.013 0.001 0.0060.023 0.041 0.032 0.001

Example 13: Preparation of Targeted Thorium Conjugate (TTC)Comprising Anti-CCR8 Antibodies

The disclosure of WO2016096843 is incorporated herein by referencein its entirety and in particular with regard to the production ofthe conjugates as described in this example.

Conjugation of the 3,2-hydroxypiridonone (3,2-HOPO) chelator or anyother suitable chelator to the antibodies TPP-23411, TPP-21360 orany other anti-CCR8 antibodies described can be conducted aspreviously described in patent application WO2016096843. Briefly,to activate the chelator, the 3,2-HOPO chelator, dissolved in DMAat a 1:1 ratio with 0.1 M MES buffer pH 5.4, NHS and EDC, bothdissolved in 0.1 M MES buffer pH 5.4, is mixed at a ratio of 1/1/3.For conjugation to the antibodies, a molar ratio of 7.5/7.5/22.5/1(chelator/NHS/EDC/mAb) of the activated chelator can be charged tomAb. After 20-60 min, the reaction is quenched with 12% v/v 0.3 Mcitric acid to adjust pH to 5.5. The protein concentration isdetermined by HPLC, integrating the peak area at an absorbance of280 nm. The solution is then buffer exchanged into 30 mM Citrate,50 mg/mlx M sucrose, 2 mM EDTA, 0.5 mg/ml pABA, pH 5.5 byTangential Flow Filtration (TFF) at constant volume. At the end ofthe diafiltration, the solution is discharged to a formulationcontainer. The product is formulated with TFF buffer (30 mMCitrate, .times.50 mg/ml M Sucrose, 2 mM EDTA, 0.5 mg/ml pABA, pH5.5) and 7% w/v polysorbate 80 to obtain 2.5 mg/ml of respectiveCCR8 antibody-chelator conjugates (CCR8-ACCs). CCR8-ACCs can befiltered through a 0.2 .mu.m filter into sterile vials.

CCR8-ACCs are radiolabeled with thorium-227 as described inWO2016096843. Briefly, 5 .mu.l of CCR8-ACCs are mixed with 32 .mu.lof thorium-227 (activity of 3.875 MBq/ml) and 13 .mu.l of citratebuffer, resulting in CCR8-targeted thorium-227 conjugates(CCR8-TTCs) at specific activities of 10 kBq/.mu.s. The sample canbe incubated for 60 min at room temperature to allow for stableradiolabeling of thorium-227 into the 3,2-HOPO chelator. An aliquotof the sample can be analyzed by instant thin layer chromatography(iTLC).

TABLE-US-00087 ANTIBODY SEQUENCES SEQ ID Sequence 201EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRQLGSWGQGTLVTVSS 202 SYGMH203 AISGSGGSTYYADSVKG 204 GRQLGS 205QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 206 SGSSSNIGSNYVY207 GNNNRPS 208 AAWDDSLNGWV 209GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACATATTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCAGACAGCTCGGCTCTTGGGGACAGGGAACACTGGTTACAGTGTCCTCA 210CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTCCAACATCGGCAGCAACTACGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACGGCAACAACAACAGACCCAGCGGCGTGCCCGATAGATTCAGCGGCTCTAAGTCTGGCACAAGCGCCAGCCTGGCCATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCTGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 211EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRQLGSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 212QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 213GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACATATTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCAGACAGCTCGGCTCTTGGGGACAGGGAACACTGGTTACAGTGTCCTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTG AGCCCTGGC214 CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTCCAACATCGGCAGCAACTACGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACGGCAACAACAACAGACCCAGCGGCGTGCCCGATAGATTCAGCGGCTCTAAGTCTGGCACAAGCGCCAGCCTGGCCATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCTGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 215EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVSGVSWNGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTRGAWGQGTLVTVSS 216 DYGVH217 GVSWNGSRTHYADSVKG 218 RGA 219QSVLTQPPSASGTPGQRVTISCSGSSFNIGSHFVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVL 220 SGSSFNIGSHFVY221 KNNQRPS 222 AAWDDSLNGPV 223GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCGATTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTTTCAGGCGTGTCCTGGAACGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGACAAGAGGCGCTTGGGGCCAGGGCACACTGGTCACAGTTTCTTCA 224CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTTCAACATCGGCAGCCACTTCGTGTACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACAAGAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCCCTGTTTTTGGCGGAGGCACCAAGCTGACAGTGCTA 225EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVSGVSWNGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTRGAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 226QSVLTQPPSASGTPGQRVTISCSGSSFNIGSHFVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 227GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCGATTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTTTCAGGCGTGTCCTGGAACGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGACAAGAGGCGCTTGGGGCCAGGGCACACTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGG C 228CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTTCAACATCGGCAGCCACTTCGTGTACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACAAGAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCCCTGTTTTTGGCGGAGGCACCAAGCTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 229EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVSGVSWNGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTRGAWGQGTLVTVSS 230 DYGVH231 GVSWNGSRTHYADSVKG 232 RGA 233QSVLTQPPSASGTPGQRVTISCSGSSFNIGSHFVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVL 234 SGSSFNIGSHFVY235 KNNQRPS 236 AAWDDSLNGPV 237GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCGATTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTTTCAGGCGTGTCCTGGAACGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGACAAGAGGCGCTTGGGGCCAGGGCACACTGGTCACAGTTTCTTCA 238CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTTCAACATCGGCAGCCACTTCGTGTACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACAAGAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCCCTGTTTTTGGCGGAGGCACCAAGCTGACAGTGCTA 239EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVSGVSWNGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTRGAWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 240QSVLTQPPSASGTPGQRVTISCSGSSFNIGSHFVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS 241GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCGATTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTTTCAGGCGTGTCCTGGAACGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGACAAGAGGCGCTTGGGGCCAGGGCACACTGGTCACAGTTTCTTCAGCCAAGACCACCGCCCCCAGCGTGTACCCTCTGGCTCCTGTGTGTGGCGATACCACCGGCAGCTCTGTGACCCTGGGCTGCCTCGTGAAGGGCTACTTCCCTGAGCCAGTGACCCTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTGACCGTGACCAGCAGCACATGGCCCAGCCAGAGCATCACCTGTAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAGATCGAGCCCAGAGGCCCCACCATCAAGCCTTGCCCCCCTTGCAAATGCCCTGCCCCCAATCTGCTGGGCGGACCCTCCGTGTTCATCTTCCCACCCAAGATCAAGGACGTGCTGATGATCAGCCTGAGCCCCATCGTGACCTGCGTGGTGGTGGACGTGTCCGAGGACGACCCCGATGTGCAGATCAGTTGGTTCGTGAACAACGTGGAAGTGCACACCGCCCAGACCCAGACACACAGAGAGGACTACAACAGCACCCTGAGAGTGGTGTCCGCCCTGCCCATCCAGCACCAGGATTGGATGAGCGGCAAAGAGTTCAAGTGCAAAGTGAACAACAAGGACCTGCCAGCCCCCATCGAGCGGACCATCTCTAAGCCTAAGGGCAGCGTGCGGGCTCCCCAGGTGTACGTGCTGCCTCCTCCAGAGGAAGAGATGACCAAGAAACAAGTGACACTGACATGCATGGTCACCGACTTCATGCCCGAGGACATCTACGTGGAATGGACCAACAACGGCAAGACCGAGCTGAACTACAAGAACACCGAGCCCGTGCTGGACAGCGACGGCAGCTACTTCATGTACAGCAAGCTGCGGGTGGAAAAGAAAAACTGGGTGGAACGGAACAGCTACAGCTGCAGCGTGGTGCACGAGGGCCTGCACAATCACCACACCACCAAGAGCTTCAGCCGGACCCC TGGCAAG242 CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTTCAACATCGGCAGCCACTTCGTGTACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACAAGAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCCCTGTTTTTGGCGGAGGCACCAAGCTGACAGTGCTAGGCCAGCCCAAGAGCA

GCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGGAAACAAACAAGGCCACCCTCGTGTGCACCATCACCGACTTCTACCCCGGCGTCGTGACCGTGGACTGGAAGGTGGACGGCACCCCAGTGACCCAGGGCATGGAAACCACCCAGCCCAGCAAGCAGAGCAACAACAAGTACATGGCCAGCAGCTACCTGACCCTGACCGCCAGAGCCTGGGAGAGACACAGCTCCTACAGCTGCCAAGTGACCCACGAGGGCCACACCGTGGAAAAGAGCCTGAGCAGAGCCGACTGCAGC 243EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRQLGSWGQGTLVTVSS 244 SYGMH245 AISGSGGSTYYADSVKG 246 GRQLGS 247QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 248 SGSSSNIGSNYVY249 GNNNRPS 250 AAWDDSLNGWV 251GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACATATTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCAGACAGCTCGGCTCTTGGGGACAGGGAACACTGGTTACAGTGTCCTCA 252CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTCCAACATCGGCAGCAACTACGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACGGCAACAACAACAGACCCAGCGGCGTGCCCGATAGATTCAGCGGCTCTAAGTCTGGCACAAGCGCCAGCCTGGCCATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCTGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 253EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRQLGSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 254QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS 255GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACATATTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCAGACAGCTCGGCTCTTGGGGACAGGGAACACTGGTTACAGTGTCCTCAGCCAAGACCACCGCCCCCAGCGTGTACCCTCTGGCTCCTGTGTGTGGCGATACCACCGGCAGCTCTGTGACCCTGGGCTGCCTCGTGAAGGGCTACTTCCCTGAGCCAGTGACCCTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTGACCGTGACCAGCAGCACATGGCCCAGCCAGAGCATCACCTGTAACGTGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAGATCGAGCCCAGAGGCCCCACCATCAAGCCTTGCCCCCCTTGCAAATGCCCTGCCCCCAATCTGCTGGGCGGACCCTCCGTGTTCATCTTCCCACCCAAGATCAAGGACGTGCTGATGATCAGCCTGAGCCCCATCGTGACCTGCGTGGTGGTGGACGTGTCCGAGGACGACCCCGATGTGCAGATCAGTTGGTTCGTGAACAACGTGGAAGTGCACACCGCCCAGACCCAGACACACAGAGAGGACTACAACAGCACCCTGAGAGTGGTGTCCGCCCTGCCCATCCAGCACCAGGATTGGATGAGCGGCAAAGAGTTCAAGTGCAAAGTGAACAACAAGGACCTGCCAGCCCCCATCGAGCGGACCATCTCTAAGCCTAAGGGCAGCGTGCGGGCTCCCCAGGTGTACGTGCTGCCTCCTCCAGAGGAAGAGATGACCAAGAAACAAGTGACACTGACATGCATGGTCACCGACTTCATGCCCGAGGACATCTACGTGGAATGGACCAACAACGGCAAGACCGAGCTGAACTACAAGAACACCGAGCCCGTGCTGGACAGCGACGGCAGCTACTTCATGTACAGCAAGCTGCGGGTGGAAAAGAAAAACTGGGTGGAACGGAACAGCTACAGCTGCAGCGTGGTGCACGAGGGCCTGCACAATCACCACACCACCAAGAGCTTCAGCCGGACCCCTGGCAAG 256CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTCCAACATCGGCAGCAACTACGTGTACTGGTATCAGCAGCTGCCCGGCACAGCCCCTAAACTGCTGATCTACGGCAACAACAACAGACCCAGCGGCGTGCCCGATAGATTCAGCGGCTCTAAGTCTGGCACAAGCGCCAGCCTGGCCATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGACGATTCTCTGAACGGCTGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCCAAGAGCAGCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGGAAACAAACAAGGCCACCCTCGTGTGCACCATCACCGACTTCTACCCCGGCGTCGTGACCGTGGACTGGAAGGTGGACGGCACCCCAGTGACCCAGGGCATGGAAACCACCCAGCCCAGCAAGCAGAGCAACAACAAGTACATGGCCAGCAGCTACCTGACCCTGACCGCCAGAGCCTGGGAGAGACACAGCTCCTACAGCTGCCAAGTGACCCACGAGGGCCACACCGTGGAAAAGAGCCTGAGCAGAGCCGACTGCAGC 257EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSGVSWAGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAGTRGFDYWGQGTLVTV SS 258SYSMN 259 GVSWAGSRTHYADSVKG 260 AAAGTRGFDY 261QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHIVNWYQQLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYTSISTLVFGGGTKLTVL 262 SGSSSNIGSHIVN263 GNTNRPS 264 SSYTSISTLV 265GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACAGCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTTTCAGGCGTGTCCTGGGCCGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGCTGCCGCCGGAACCAGAGGCTTTGATTATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCA 266 AGCTACAGCATGAAC 267GGCGTGTCCTGGGCCGGCAGCAGAACCCACTATGCCGACAGCGTGAAGGGC 268GCTGCCGCCGGAACCAGAGGCTTTGATTAT 269CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCAGCAGCTCCAATATCGGCAGCCACATCGTGAACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACGGCAACACCAACAGACCCAGCGGCGTGCCCGATAGATTTTCCGGCTCTAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTACTGCAGCAGCTACACCAGCATCAGCACCCTGGTTTTTGGCGGAGGCACCAAGCTGACAGTGCTA 270AGCGGCAGCAGCTCCAATATCGGCAGCCACATCGTGAAC 271 GGCAACACCAACAGACCCAGC272 AGCAGCTACACCAGCATCAGCACCCTGGTT 273EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSGVSWAGSRTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAGTRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 274QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHIVNWYQQLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYTSISTLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 275EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVSLISWDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGIGRRPGLEYWGQGTLVTV SS 276SYAMH 277 LISWDGGSTYYADSVKG 278 GGIGRRPGLEY 279QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQTWGTGIRVFGGGTKLTVL 280 SGSSSNIGNNYVS281 GNSNRPS 282 QTWGTGIRV 283GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGGAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTCTCTCTTATTAGTTGGGATGGTGGTAGCACCTACTATGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGGGGGTATAGGGCGTCGTCCTGGGCTTGAGTACTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 284 AGCTATGCTATGCAC 285CTTATTAGTTGGGATGGTGGTAGCACCTACTATGCAGACTCTGTGAAGGGT 286GGGGGTATAGGGCGTCGTCCTGGGCTTGAGTAC 287CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGAAATAATTATGTTTCCTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTCAGACCTGGGGCACTGGCATTCGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 288TCTGGAAGCAGCTCCAACATTGGAAATAATTATGTTTCC 289 GGTAACAGCAATCGGCCCTCA290 CAGACCTGGGGCACTGGCATTCGGGTG 291EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVSLISWDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGIGRRPGLEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 292QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQTWGTGIRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 293EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRPLDSWGQGTLVTVSS 294 SYAMS295 RIRSKANSYATAYAASVKG 296 PLDS 297QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYRNNQRSSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDNSLSASVFGGGTKLTVL 298 SGSSSNIGNNAVN299 RNNQRSS 300 QSYDNSLSASV 301GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAGAAGCAAAGCTAACAGTTACGCGACAGCATATGCTGCGTCGGTGAAAGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTACGAGGCCCTTGGACTCCTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 302AGCTATGCCATGAGC 303CGTATTAGAAGCAAAGCTAACAGTTACGCGACAGCATATGCTGCGTCGGTGAAAGGC 304CCCTTGGACTCC 305CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGAAATAATCAGCGGTCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAACAGCCTGAGTGCTTCGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 306TCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAAC 307AGAAATAATCAGCGGTCCTCA

308 CAGTCCTATGACAACAGCCTGAGTGCTTCGGTG 309EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRPLDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 310QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYRNNQRSSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDNSLSASVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 311EVQLLESGGGLLQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVAVISYDGRNKYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLPYGYWGQGTLVTVSS 312 NAWMS313 VISYDGRNKYSADSVKG 314 GLPYGY 315QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLKALVFGGGTKLTVL 316 SGSSSNIGSNTVN317 RNNQRPS 318 AAWDDSLKALV 319GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGTTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGAAATAAATACTCTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGGGGCCTCCCTTATGGCTACTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 320AACGCCTGGATGAGC 321GTTATATCATATGATGGAAGAAATAAATACTCTGCAGACTCCGTGAAGGGC 322GGCCTCCCTTATGGCTAC 323CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCTGCATGGGATGACAGTCTGAAGGCTCTGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTA 324TCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAAC 325 AGGAATAATCAGCGGCCCTCA326 GCTGCATGGGATGACAGTCTGAAGGCTCTGGTA 327EVQLLESGGGLLQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVAVISYDGRNKYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLPYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 328QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLKALVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSGINWNGGST 329GYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRTYSGHYGPYFDNWGQGTL VTVSS330 NAWMS 331 GINWNGGSTGYADSVKG 332 TYSGHYGPYFDN 333QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 334 SGSSSNIGSHTVN335 RNNQRPS 336 AAWDDSLNGWV 337GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGTTAGGACGTATAGTGGGCACTACGGACCCTACTTTGACAACTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 338 AACGCCTGGATGAGC 339GGTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCTGTGAAGGGC 340ACGTATAGTGGGCACTACGGACCCTACTTTGACAAC 341CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 342TCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAAC 343 AGGAATAATCAGCGGCCCTCA344 GCAGCATGGGATGACAGCCTGAATGGTTGGGTG 345EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRTYSGHYGPYFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 346QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 347EVQLLESGGGLVQPGGSLRLSCAASGFTFVTYWMTWVRQAPGKGLEWVSGVSWNGSRTHYVDSVKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSGYPDYYGMDVWGQGTL VTVSS348 TYWMT 349 GVSWNGSRTHYVDSVKR 350 YSGYPDYYGMDV 351QSVLTQPPSASGTPGQRVTISCSGSWSNIGNDNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDRSLSGSVFGGGTKLTVL 352 SGSWSNIGNDNVY353 RNNQRPS 354 QSYDRSLSGSV 355GAGGTGCAACTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGTTACATATTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTATCGGGTGTTAGTTGGAATGGCAGTAGGACGCACTATGTGGACTCCGTGAAGCGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGATATAGTGGCTACCCTGACTACTACGGTATGGACGTCTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 356 ACATATTGGATGACC 357GGTGTTAGTTGGAATGGCAGTAGGACGCACTATGTGGACTCCGTGAAGCGC 358TATAGTGGCTACCCTGACTACTACGGTATGGACGTC 359CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCTGGTCCAACATCGGAAATGATAATGTATACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGCCAGTCTTATGACAGGAGCCTGAGTGGTTCGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 360TCTGGAAGCTGGTCCAACATCGGAAATGATAATGTATAC 361 AGGAATAATCAGCGGCCCTCA362 CAGTCTTATGACAGGAGCCTGAGTGGTTCGGTG 363EVQLLESGGGLVQPGGSLRLSCAASGFTFVTYWMTWVRQAPGKGLEWVSGVSWNGSRTHYVDSVKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSGYPDYYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 364QSVLTQPPSASGTPGQRVTISCSGSWSNIGNDNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDRSLSGSVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGVSWNGSRTR 365YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGSYNSGWYAVSWGQGTLVT VSS 366SYGMH 367 GVSWNGSRTRYADSVKG 368 GSYNSGWYAVS 369QSVLTQPPSASGTPGQRVTISCSGSSSNIGSYPVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYSVTDNLIFGGGTKLTVL 370 SGSSSNIGSYPVN371 RNNQRPS 372 SSYSVTDNLI 373GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTATCGGGTGTTAGTTGGAATGGCAGTAGGACGCGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTACTAGAGGGTCATATAATAGTGGCTGGTACGCGGTCTCCTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 374 AGCTATGGCATGCAC 375GGTGTTAGTTGGAATGGCAGTAGGACGCGCTATGCGGACTCTGTGAAGGGC 376GGGTCATATAATAGTGGCTGGTACGCGGTCTCC 377CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCTGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGTTCCAACATCGGGAGTTATCCTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGCAGCTCATATTCTGTCACCGACAATTTGATATTCGGCGGAGGAACCAAGCTGACGGTCCTA 378TCTGGAAGCAGTTCCAACATCGGGAGTTATCCTGTAAAC 379 AGGAATAATCAGCGGCCCTCA380 AGCTCATATTCTGTCACCGACAATTTGATA 381EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGVSWNGSRTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGSYNSGWYAVSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 382QSVLTQPPSASGTPGQRVTISCSGSSSNIGSYPVNWYQQLPGTAPKLLIYRNNQRPSGVPDR

FSGSKSGTSASLAISGLRSEDEADYYCSSYSVTDNLIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYRMTWVRQAPGKGLEWVSGINWNGGSTG 383YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRFDTRGFYGFDYWGQGTLV TVSS384 NYRMT 385 GINWNGGSTGYADSVKG 386 GRFDTRGFYGFDY 387QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSHNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSAWDSSLSAWVFGGGTKLTVL 388TGSSSNIGAGYDVH 389 SHNQRPS 390 SAWDSSLSAWV 391GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTATAGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCCAGAGGGCGCTTTGATACTAGGGGTTTTTACGGCTTTGACTACTGGGGCCAAGGTACCCTGGTCACCGTGAGTTCA 392 AATTATAGGATGACC 393GGTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCTGTGAAGGGC 394GGGCGCTTTGATACTAGGGGTTTTTACGGCTTTGACTAC 395CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTCATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGCTCAGCATGGGACAGCAGCCTCAGTGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 396ACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACAC 397AGTCATAATCAGCGGCCCTCA 398 TCAGCATGGGACAGCAGCCTCAGTGCTTGGGTG 399EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYRMTWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRFDTRGFYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 400QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSHNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSAWDSSLSAWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 401EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNSGSKGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSGNYYVGYHGMDVWGQGTL VTVSS402 SYAMS 403 GIRWNSGSKGYAGSVKG 404 SGNYYVGYHGMDV 405QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVL 406 SGGNSNIGTYFVS407 TNNQRPS 408 ETWDSNTRV 409GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCGACAGGCTCCTGGCAAAGGCCTTGAATGGGTGTCCGGCATCAGATGGAACAGCGGCTCTAAGGGCTATGCCGGCTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGCGGAGCGGCAATTACTACGTGGGCTACCACGGCATGGATGTGTGGGGACAGGGAACCCTGGTTACCGTTTCTTCA 410 AGCTACGCCATGAGC 411GGCATCAGATGGAACAGCGGCTCTAAGGGCTATGCCGGCTCTGTGAAGGGC 412AGCGGCAATTACTACGTGGGCTACCACGGCATGGATGTG 413CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCCTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTACTGCGAGACATGGGACAGCAACACCAGAGTGTTTGGCGGAGGCACCAAGCTGACAGTGCTA 414AGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCC 415 ACCAACAACCAGCGGCCTAGC416 GAGACATGGGACAGCAACACCAGAGTG 417EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNSGSKGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSGNYYVGYHGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 418QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 419EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS420 HYGMH 421 GINWNGGSTGYADSVKG 422 GHHSGYDGRFFDY 423QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 424TGSSSNIGAGYNVH 425 TNNRRPS 426 AAWDASLSGWV 427GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 428 CACTATGGCATGCAC 429GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 430GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 431CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 432ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 433ACCAACAACAGACGGCCCAGC 434 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 435EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 436QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 437EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYTGNYGPYFDYWGQGTL VTVSS438 NAWMS 439 GINWNGGSTGYADSVKG 440 TYTGNYGPYFDY 441QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRGWVFGGGTKLTVL 442TGSSSNIGAGYDVH 443 KNNQRPS 444 AAWDDSLRGWV 445GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAATGCCTGGATGAGCTGGGTCCGACAGGCCCCTGGAAAAGGCCTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAACCTACACCGGCAACTACGGCCCCTACTTCGATTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCA 446 AATGCCTGGATGAGC 447GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 448ACCTACACCGGCAACTACGGCCCCTACTTCGATTAT 449CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTATGACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACAAGAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGATTCTCTGAGAGGCTGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 450ACCGGCAGCAGCTCCAATATCGGAGCCGGCTATGACGTGCAC 451AAGAACAACCAGCGGCCTAGC 452 GCCGCCTGGGATGATTCTCTGAGAGGCTGGGTT 453EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYTGNYGPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 454QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 455EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPYFDYWGQGTLVT VSS 456SAWMS

457 GISWSGGSTGYADSVKG 458 TYSGHYGPYFDY 459QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 460 SGSSSNIGSHTVN461 RNNQRPS 462 AAWDDSLNGWV 463GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTTTCTAGCGCCTGGATGAGCTGGGTCCGACAGGCTCCTGGAAAAGGCCTGGAATGGGTGTCCGGCATCTCTTGGAGCGGCGGCTCTACAGGCTATGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAACATACAGCGGCCACTACGGCCCCTACTTCGATTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCA 464 AGCGCCTGGATGAGC 465GGCATCTCTTGGAGCGGCGGCTCTACAGGCTATGCCGATTCTGTGAAGGGC 466ACATACAGCGGCCACTACGGCCCCTACTTCGATTAT 467CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 468TCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAAC 469 AGGAATAATCAGCGGCCCTCA470 GCAGCATGGGATGACAGCCTGAATGGTTGGGTG 471EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 472QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 473EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSHGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGKDGRFFDYWGQGTL VTVSS474 SYGMH 475 AIKWGGGSHGYADSVKG 476 GHHSGKDGRFFDY 477QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 478TGSSSNIGAGYNVH 479 TNNRRPS 480 AAWDASLSGWV 481GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATTAAGTGGGGCGGAGGCTCTCACGGCTATGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACTCTGGCAAGGACGGCAGATTCTTCGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 482 AGCTATGGCATGCAC 483GCCATTAAGTGGGGCGGAGGCTCTCACGGCTATGCCGATTCTGTGAAGGGC 484GGCCACCACTCTGGCAAGGACGGCAGATTCTTCGACTAT 485CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 486ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 487ACCAACAACAGACGGCCCAGC 488 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 489EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSHGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGKDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 490QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 491EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSHGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHKGYDGRFFDYWGQGTL VTVSS492 SYGMH 493 AIKWGGGSHGYADSVKG 494 GHHKGYDGRFFDYQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVP 495DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 496TGSSSNIGAGYNVH 497 TNNRRPS 498 AAWDASLSGWV 499GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATTAAGTGGGGCGGAGGCTCTCACGGCTATGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAAGGGCTACGACGGCAGATTCTTCGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 500 AGCTATGGCATGCAC 501GCCATTAAGTGGGGCGGAGGCTCTCACGGCTATGCCGATTCTGTGAAGGGC 502GGCCACCACAAGGGCTACGACGGCAGATTCTTCGACTAT 503CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 504ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 505ACCAACAACAGACGGCCCAGC 506 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 507EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSHGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHKGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 508QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 509EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGKDGRFFDYWGQGTL VTVSS510 SYGMH 511 AIKWGGGSTGYADSVKG 512 GHHSGKDGRFFDY 513QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 514TGSSSNIGAGYNVH 515 TNNRRPS 516 AAWDASLSGWV 517GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATTAAGTGGGGCGGAGGCTCTACAGGCTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACTCTGGCAAGGACGGCAGATTCTTCGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 518 AGCTATGGCATGCAC 519GCCATTAAGTGGGGCGGAGGCTCTACAGGCTACGCCGATTCTGTGAAGGGC 520GGCCACCACTCTGGCAAGGACGGCAGATTCTTCGACTAT 521CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 522ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 523ACCAACAACAGACGGCCCAGC 524 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 525EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAIKWGGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGKDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 526QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 527EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGSTGYALSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPFFDYWGQGTLVT VSS 528SAWMS 529 GISWSGGSTGYALSVKG 530 TYSGHYGPFFDY 531QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 532 SGSSSNIGSHTVN533 RNNQRPS

534 AAWDDSLNGWV 535GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTTTCTAGCGCCTGGATGAGCTGGGTCCGACAGGCTCCTGGAAAAGGCCTGGAATGGGTGTCCGGCATCTCTTGGAGCGGCGGCTCTACAGGATATGCCCTGTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAACATACAGCGGCCACTACGGCCCTTTCTTCGACTATTGGGGCCAGGGCACACTGGTCACAGTCTCTTCA 536 AGCGCCTGGATGAGC 537GGCATCTCTTGGAGCGGCGGCTCTACAGGATATGCCCTGTCTGTGAAGGGC 538ACATACAGCGGCCACTACGGCCCTTTCTTCGACTAT 539CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 540TCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAAC 541 AGGAATAATCAGCGGCCCTCA542 GCAGCATGGGATGACAGCCTGAATGGTTGGGTG 543EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGSTGYALSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 544QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 545EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGRTGYALSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPYFDYWGQGTLVT VSS 546SAWMS 547 GISWSGGRTGYALSVKG 548 TYSGHYGPYFDY 549QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL 550 SGSSSNIGSHTVN551 RNNQRPS 552 AAWDDSLNGWV 553GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTTTCTAGCGCCTGGATGAGCTGGGTCCGACAGGCTCCTGGAAAAGGCCTGGAATGGGTGTCCGGCATCTCTTGGAGCGGCGGCAGAACAGGATATGCCCTGTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAACATACAGCGGCCACTACGGCCCCTACTTCGATTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCA 554 AGCGCCTGGATGAGC 555GGCATCTCTTGGAGCGGCGGCAGAACAGGATATGCCCTGTCTGTGAAGGGC 556ACATACAGCGGCCACTACGGCCCCTACTTCGATTAT 557CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTA 558TCTGGAAGCAGCTCCAACATCGGAAGTCATACTGTAAAC 559 AGGAATAATCAGCGGCCCTCA560 GCAGCATGGGATGACAGCCTGAATGGTTGGGTG 561EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAWMSWVRQAPGKGLEWVSGISWSGGRTGYALSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSGHYGPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 562QSVLTQPPSASGTPGQRVTISCSGSSSNIGSHTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 563EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNNGSKGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSGNYYYGYHGMDVWGQGTL VTVSS564 SYAMS 565 GIRWNNGSKGYAGSVKG 566 SGNYYYGYHGMDV 567QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVL 568 SGGNSNIGTYFVS569 TNNQRPS 570 ETWDSNTRV 571GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCGACAGGCTCCTGGCAAAGGCCTTGAATGGGTGTCCGGCATCAGATGGAACAACGGCAGCAAGGGCTATGCCGGCTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAAGCGGCAACTACTACTACGGCTACCACGGCATGGATGTGTGGGGCCAGGGAACACTGGTTACCGTTTCTTCA 572 AGCTACGCCATGAGC 573GGCATCAGATGGAACAACGGCAGCAAGGGCTATGCCGGCTCTGTGAAGGGC 574AGCGGCAACTACTACTACGGCTACCACGGCATGGATGTG 575CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCCTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTACTGCGAGACATGGGACAGCAACACCAGAGTGTTTGGCGGAGGCACCAAGCTGACAGTGCTA 576AGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCC 577 ACCAACAACCAGCGGCCTAGC578 GAGACATGGGACAGCAACACCAGAGTG 579EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNNGSKGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSGNYYYGYHGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 580QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNNGSKG 581YAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSGNEYYGYHGMDVWGQGTL VTVSS582 SYAMS 583 GIRWNNGSKGYAGSVKG 584 SGNEYYGYHGMDV 585QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVL 586 SGGNSNIGTYFVS587 TNNQRPS 588 ETWDSNTRV 589GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCGACAGGCTCCTGGCAAAGGCCTTGAATGGGTGTCCGGCATCAGATGGAACAACGGCAGCAAGGGCTATGCCGGCTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAAGCGGCAACGAGTACTACGGCTACCACGGCATGGATGTGTGGGGCCAGGGAACACTGGTTACCGTTTCTTCA 590 AGCTACGCCATGAGC 591GGCATCAGATGGAACAACGGCAGCAAGGGCTATGCCGGCTCTGTGAAGGGC 592AGCGGCAACGAGTACTACGGCTACCACGGCATGGATGTG 593CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTAGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCCTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACCAGCGGCCTAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTACTGCGAGACATGGGACAGCAACACCAGAGTGTTTGGCGGAGGCACCAAGCTGACAGTGCTA 594AGCGGCGGCAACAGCAACATCGGCACCTACTTCGTGTCC 595 ACCAACAACCAGCGGCCTAGC596 GAGACATGGGACAGCAACACCAGAGTG 597EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRWNNGSKGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSGNEYYGYHGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 598QSVLTQPPSASGTPGQRVTISCSGGNSNIGTYFVSWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCETWDSNTRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 599EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS600 SYGMH 601 AINWNGGSTGYADSVKG 602 GHHSGYDGRFFDY 603QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 604TGSSSNIGAGYNVH 605 TNNRRPS 606 AAWDASLSGWV 607GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 608 AGCTATGGCATGCAC

609 GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 610GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 611CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 612ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 613ACCAACAACAGACGGCCCAGC 614 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 615EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 616QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 617EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS618 SYGMH 619 AINWNGGSTGYADSVKG 620 GHHSGYDGRFFDYQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVP 621DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 622TGSSSNIGAGYNVH 623 TNNRRPS 624 AAWDASLSGWV 625GAGGTGCAGCTGCTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCTTCCGGCTTCACCTTCTCCAGCTACGGAATGCACTGGGTCCGACAGGCCCCTGGCAAAGGATTGGAATGGGTGTCCGCCATCAACTGGAACGGCGGCTCTACCGGCTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTAGAGGCCACCACTCTGGCTACGACGGCAGATTCTTCGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 626 AGCTACGGAATGCAC 627GCCATCAACTGGAACGGCGGCTCTACCGGCTACGCCGATTCTGTGAAGGGC 628GGCCACCACTCTGGCTACGACGGCAGATTCTTCGACTAT 629CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 630ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 631ACCAACAACAGACGGCCCAGC 632 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 633EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 634QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 660EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS661 SYGMH 662 GINWNGGSTGYADSVKG 663 GHHSGYDGRFFDYQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVP 664DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 665TGSSSNIGAGYNVH 666 TNNRRPS 667 AAWDASLSGWV 668GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 669 AGCTATGGCATGCAC 670GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 671GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 672CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 673ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 674ACCAACAACAGACGGCCCAGC 675 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 676EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 677QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 678GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 679CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 680EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS681 SYGMH 682 AINWNGGSTGYADSVKG 683 GHHSGYDGRFFDY 684QSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 685TGSSSNIGAGYNVH 686 TNNRRPS 687 AAWDASLSGWV 688GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 689 AGCTATGGCATGCAC 690GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 691GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 692CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 693ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 694ACCAACAACAGACGGCCCAGC 695 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 696EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 697QSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 698GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGAC

TGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 699CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 700GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 701CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 702EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS703 HYGMH 704 GINWNGGSTGYADSVKG 705 GHHSGYDGRFFDY 706QSVLTQPPSVSGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 707TGSSSNIGAGYNVH 708 TNNRRPS 709 AAWDASLSGWV 710GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 711 CACTATGGCATGCAC 712GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 713GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 714CAGTCTGTTCTGACACAGCCTCCATCCGTGTCTGGCACACCTGGCCAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCTCTAAGAGCGGCACCAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 715ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 716ACCAACAACAGACGGCCCAGC 717 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 718EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 719QSVLTQPPSVSGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 720GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 721CAGTCTGTTCTGACACAGCCTCCATCCGTGTCTGGCACACCTGGCCAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCTCTAAGAGCGGCACCAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 722EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS723 HYGMH 724 GINWNGGSTGYADSVKG 725 GHHSGYDGRFFDYQSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVP 726DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 727TGSSSNIGAGYNVH 728 TNNRRPS 729 AAWDASLSGWV 730GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 731 CACTATGGCATGCAC 732GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 733GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 734CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 735ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 736ACCAACAACAGACGGCCCAGC 737 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 738EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 739QSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 740GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAG

GGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 741CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 742EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS743 HYGMH 744 GINWNGGSTGYADSVKG 745 GHHSGYDGRFFDY 746QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 747TGSSSNIGAGYNVH 748 TNNRRPS 749 AAWDASLSGWV 750GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 751 CACTATGGCATGCAC 752GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 753GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 754CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 755ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 756ACCAACAACAGACGGCCCAGC 757 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 758EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 759QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 760GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 761CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 762EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS763 SYGMH 764 GINWNGGSTGYADSVKG 765 GHHSGYDGRFFDY 766QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 767TGSSSNIGAGYNVH 768 TNNRRPS 769 AAWDASLSGWV 770GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 771 AGCTATGGCATGCAC 772GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 773GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 774CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 775ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 776ACCAACAACAGACGGCCCAGC 777 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 778EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 779QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 780GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 781CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 782EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS783 HYGMH 784 AINWNGGSTGYADSVKG 785 GHHSGYDGRFFDY 786QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 787TGSSSNIGAGYNVH 788 TNNRRPS 789 AAWDASLSGWV 790GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 791 CACTATGGCATGCAC 792GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 793GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 794CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCA

GCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 795ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 796ACCAACAACAGACGGCCCAGC 797 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 798EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 799QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 800GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 801CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 802EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS803 SYGMH 804 AINWNGGSTGYADSVKG 805 GHHSGYDGRFFDY 806QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 807TGSSSNIGAGYNVH 808 TNNRRPS 809 AAWDASLSGWV 810GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 811 AGCTATGGCATGCAC 812GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 813GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 814CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 815ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 816ACCAACAACAGACGGCCCAGC 817 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 818EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 819QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 820GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 821CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 822GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 823CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 824GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 825CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 826EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS827 SYGMH 828 AINWNGGSTGYADSVKG 829 GHHSGYDGRFFDY 830QSVLTQPPSVSGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 831TGSSSNIGAGYNVH

832 TNNRRPS 833 AAWDASLSGWV 834GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 835 AGCTATGGCATGCAC 836GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 837GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 838CAGTCTGTTCTGACACAGCCTCCATCCGTGTCTGGCACACCTGGCCAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCTCTAAGAGCGGCACCAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 839ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 840ACCAACAACAGACGGCCCAGC 841 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 842EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 843QSVLTQPPSVSGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 844GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 845CAGTCTGTTCTGACACAGCCTCCATCCGTGTCTGGCACACCTGGCCAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCTCTAAGAGCGGCACCAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 846EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS847 SYGMH 848 AINWNGGSTGYADSVKG 849 GHHSGYDGRFFDY 850QSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 851TGSSSNIGAGYNVH 852 TNNRRPS 853 AAWDASLSGWV 854GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 855 AGCTATGGCATGCAC 856GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 857GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 858CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 859ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 860ACCAACAACAGACGGCCCAGC 861 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 862EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 863QSVLTQPPSASGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 864GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 865CAGTCTGTTCTGACACAGCCTCCATCTGCTAGCGGAGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 866EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS867 SYGMH 868 GINWNGGSTGYADSVKG 869 GHHSGYDGRFFDYQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVP 870DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 871TGSSSNIGAGYNVH 872 TNNRRPS 873 AAWDASLSGWV 874GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 875 AGCTATGGCATGCAC 876GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 877GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 878CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 879ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 880ACCAACAACAGACGGCCCAGC 881 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 882EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 883QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 884GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCA

CACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 885CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 886EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS887 HYGMH 888 AINWNGGSTGYADSVKG 889 GHHSGYDGRFFDY 890QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 891TGSSSNIGAGYNVH 892 TNNRRPS 893 AAWDASLSGWV 894GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 895 CACTATGGCATGCAC 896GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 897GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 898CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 899ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 900ACCAACAACAGACGGCCCAGC 901 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 902EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 903QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 904GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC 905CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 906EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS907 HYGMH 908 GINWNGGSTGYADSVKG 909 GHHSGYDGRFFDY 910QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 911TGSSSNIGAGYNVH 912 TNNRRPS 913 AAWDASLSGWV 914GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 915 CACTATGGCATGCAC 916GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 917GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 918CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 919ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 920ACCAACAACAGACGGCCCAGC 921 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 922EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 923QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 924GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 925CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 926EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGT LVTVSS927 HYGMH 928 GINWNGGSTGYADSVKG 929 GHHSGYDGRFFDY 930QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 931TGSSSNIGAGYNVH 932 TNNRRPS 933 AAWDASLSGWV 934GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA

935 CACTATGGCATGCAC 936GGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 937GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 938CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 939ACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 940ACCAACAACAGACGGCCCAGC 941 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 942EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 943QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 944GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCCACTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGGCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 945CAGTCTGTTCTGACACAGCCTCCATCTGTGTCTGGCGCCCCTGGACAGAGAGTGACCATCAGCTGTACAGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC 946EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTL VTVSS947 SYGMH 948 AINWNGGSTGYADSVKG 949 GHHSGYDGRFFDY 950QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVL 951TGSSSNIGAGYNVH 952 TNNRRPS 953 AAWDASLSGWV 954GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCA 955 AGCTATGGCATGCAC 956GCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGC 957GGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTAT 958CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTA 959ACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCAC 960ACCAACAACAGACGGCCCAGC 961 GCCGCCTGGGATGCTTCTCTGAGCGGATGGGTT 962EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHSGYDGRFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 963QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVHWYQQLPGTAPKLLIYTNNRRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDASLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 964GAAGTTCAGCTGCTGGAATCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTATGGCATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGCCATCAACTGGAATGGCGGCTCTACAGGCTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCCACCACAGCGGCTACGACGGCAGATTCTTTGACTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCAGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCAAG 965CAGTCTGTTCTGACACAGCCTCCTAGCGCCTCTGGCACACCTGGACAGAGAGTGACCATCAGCTGTACCGGCAGCAGCTCCAATATCGGAGCCGGCTACAACGTGCACTGGTATCAGCAGCTGCCTGGCACAGCCCCTAAACTGCTGATCTACACCAACAACAGACGGCCCAGCGGCGTGCCCGATAGATTTTCTGGCAGCAAGAGCGGCACAAGCGCCAGCCTGGCTATCTCTGGACTGAGATCTGAGGACGAGGCCGACTACTATTGCGCCGCCTGGGATGCTTCTCTGAGCGGATGGGTTTTCGGCGGAGGCACCAAACTGACAGTGCTAGGCCAGCCTAAAGCCGCCCCTAGCGTGACCCTGTTCCCTCCAAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCAGCGACTTCTATCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGATAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTAGCAAGCAGAGCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAGACAGTGGCCCCTACCGAGTGCAGC

EXEMPLARY EMBODIMENTS

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, theclaims and the following embodiments: 1. An isolated polypeptide orconjugate thereof, wherein the isolated polypeptide comprises thetyrosine rich domain (TRD) of a seven transmembrane receptor,further characterized in that at least 25%, at least 50%, or atleast 75% of the tyrosine residues of the TRD are sulfated. 2. Theisolated polypeptide or conjugate according to embodiment 1,wherein the seven transmembrane receptor is human, cynomolgusand/or mouse. 3. The isolated polypeptide or conjugate according toembodiment 1 or 2, wherein the seven transmembrane receptor is achemokine receptor, preferably a. a CC chemokine receptor such asCCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b. aCXC chemokine receptor such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,or CXCR6, or CX3CR1 or CXCR1. 4. The isolated polypeptide orconjugate according to any of embodiments 1 to 3, wherein the seventransmembrane receptor is CCR8. 5. The isolated polypeptide orconjugate according to any of embodiments 1 to 4, wherein theisolated polypeptide comprises the N terminus of the seventransmembrane receptor including a TRD and a LID domain, preferablywherein at least one cysteine between the TRD and the LID domainhas been removed or has been altered into a different amino acid.6. The isolated polypeptide or conjugate according to any ofembodiments 1 to 5, wherein the polypeptide comprises a sequenceaccording to or having at least 90% sequence identity with a. SEQID NO:43, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:47, preferablywherein at least two or all of Y3, Y15 and Y17 have been sulfated,or b. SEQ ID NO:45 or SEQ ID NO:48, preferably wherein at least twoor all of Y3, Y14 and Y15 have been sulfated, or c. SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:3 or SEQ ID NO:6,preferably wherein at least Y10 and/or Y18 have been sulfated, ord. SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:8 or SEQ ID NO:11,preferably wherein at least Y26 has been sulfated, or e. SEQ IDNO:9 or SEQ ID NO:12, preferably wherein at least Y37 and/or Y39has been sulfated, or f. SEQ ID NO:13 or SEQ ID NO:16, preferablywherein Y16 and/or Y17 have been sulfated, or g. SEQ ID NO:14 orSEQ ID NO:17, preferably wherein Y16 has been sulfated, or h. SEQID NO:15 or SEQ ID NO:18 preferably wherein Y20 and/or Y22 has beensulfated, or i. SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:21 or SEQ ID NO:24, preferably wherein at leastY22 has been sulfated and preferably furthermore Y16, Y19 and/orY20 have been sulfated, or j. SEQ ID NO:25, SEQ ID NO:28, SEQ IDNO:26 or SEQ ID NO:29, preferably wherein two, three or all of Y3,Y10, Y14 and Y15 have been sulfated, or k. SEQ ID NO:27 or SEQ IDNO:30, preferably wherein two or three of Y10, Y12 and Y16 havebeen sulfated, or l. SEQ ID NO:31 or SEQ ID NO:34, preferablywherein at least two or three of Y18, Y26 and Y27 have beensulfated, or m. SEQ ID NO:32 or SEQ ID NO:35, preferably wherein atleast two or three of Y23, Y31 and Y32 have been sulfated, or n.SEQ ID NO:33 or SEQ ID NO:36, preferably wherein at least two orthree of Y13, Y18 and Y19 have been sulfated, or o. SEQ ID NO:37,SEQ ID NO:40, SEQ ID NO:38 or SEQ ID NO:41, preferably wherein oneor both of Y8 and Y17 have been sulfated, or p. SEQ ID NO:39 or SEQID NO:42, preferably wherein one or both of Y8 and Y17 andoptionally Y20 have been sulfated, or q. SEQ ID NO:61, SEQ IDNO:64, SEQ ID NO:62 or SEQ ID NO:65, preferably wherein at leastY28, and preferably also Y17 and/or Y37 has been sulfated, or r.SEQ ID NO:63 or SEQ ID NO:66, preferably wherein at least Y28 hasbeen sulfated, and preferably also Y19 has been sulfated, or s. SEQID NO:67, SEQ ID NO:70, SEQ ID NO:68 or SEQ ID NO:71, preferablywherein at least one or both of Y14 and Y22 has been sulfated, ort. SEQ ID NO:69 or SEQ ID NO:72, preferably wherein at least one,two or all of Y14, Y17 and Y22 have been sulfated, or u. SEQ IDNO:73 or SEQ ID NO:76, preferably wherein Y27 has been sulfated, orv. SEQ ID NO:74 or SEQ ID NO:77, preferably wherein at least one ofY14 and Y28 has been sulfated, or w. SEQ ID NO:75 or SEQ ID NO:78,preferably wherein at least Y6 has been sulfated, or x. SEQ IDNO:79 or SEQ ID NO:82, preferably wherein Y23 and/or Y25 have beensulfated, or y. SEQ ID NO:80 or SEQ ID NO:83, preferably whereinY20 and/or Y22 have been sulfated, or z. SEQ ID NO:81 or SEQ IDNO:84, preferably wherein Y24 has been sulfated, or aa. SEQ IDNO:85, SEQ ID NO:88, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:87 orSEQ ID NO:90, preferably wherein at least one or both of Y27 andY29 have been sulfated, or bb. SEQ ID NO:91, SEQ ID NO:94, SEQ IDNO:92 or SEQ ID NO:95, preferably wherein at least Y12 and/or Y21have been sulfated, or cc. SEQ ID NO:93 or SEQ ID NO:96, preferablywherein at least Y23 and/or Y14 have been sulfated, or dd. SEQ IDNO:97, SEQ ID NO:100, SEQ ID NO:98 or SEQ ID NO:101, preferablywherein at least one of Y3 and Y27 have been sulfated, or ee. SEQID NO:99 or SEQ ID NO:102, preferably wherein at least Y3 and/orY14 and/or Y20 and/or Y26 have been sulfated, or ff. SEQ ID NO:103or SEQ ID NO:106, preferably wherein at least one or both of Y6 andY10 have been sulfated, or gg. SEQ ID NO:104 or SEQ ID NO:107,preferably wherein at least two or all of Y4, Y7 and Y39 have beensulfated, or hh. SEQ ID NO:105 or SEQ ID NO:108, preferably whereinat least one or both of Y11 and Y15 have been sulfated, or ii. SEQID NO:157 or SEQ ID NO:160, preferably wherein at least Y14 hasbeen sulfated, or jj. SEQ ID NO:158, preferably wherein at leastY20 has been sulfated, or kk. SEQ ID NO:161, preferably wherein atleast Y20 or Y22 has been sulfated, or ll. SEQ ID NO:159 or SEQ IDNO:162, preferably wherein at least Y15 has been sulfated, or mm.SEQ ID NO:163 or SEQ ID NO:166, preferably wherein at least Y27 hasbeen sulfated, or nn. SEQ ID NO:164, preferably wherein at leastY14 has been sulfated or oo. SEQ ID NO:167, preferably wherein atleast Y14 or Y28 has been sulfated, or pp. SEQ ID NO:165 or SEQ IDNO:168, preferably wherein at least Y6 has been sulfated. 7. Theisolated polypeptide according to any of embodiments 1 to 6,wherein the isolated polypeptide is immobilized. 8. A method forproduction of the isolated polypeptide or conjugate according toany of embodiments 1 to 7, wherein the method comprises synthesisof the isolated polypeptide and sulfation of the respectivetyrosine residues. 9. Use of the isolated polypeptide or conjugateaccording to any of embodiments 1 to 7 a. for antibody generation,preferably for the generation of a fully human antibody, b. asantigen or for off-target panning, and/or c. for characterizationof an antibody. 10. A method for obtaining an antibody or binder,the method comprising the use of the isolated polypeptide orconjugate according to any of embodiments 1 to 7. 11. The methodaccording to embodiment 10, furthermore comprising the use of atleast one further isolated polypeptide or conjugate thereof,wherein the at least one further isolated polypeptide comprises aTRD a. of a seven transmembrane receptor different from said firstseven transmembrane receptor, or b. of the first seventransmembrane receptor derived from a different species, preferablywherein the at least one further isolated polypeptide is anisolated polypeptide according to any of embodiments 1 to 7. 12. Amethod for obtaining an antibody or antibody fragment whichspecifically binds to a human and/or cynomolgus and/or murine CC orCXC chemokine receptor, the method comprising a. syntheticallysulfating a polypeptide comprising a tyrosine rich domain (TRD) andb. selection of an antibody, antibody fragment or binderrecognizing the sulfated polypeptide, and c. optionally producingthe antibody, antibody fragment or binder. 13. The use or methodaccording to any of embodiments 8 to 12, wherein the antibody a.comprises human derived CDRs, and/or b. is a human, rat or murineIgG antibody, preferably a human IgG1 antibody or a murine IgG2aantibody, and/or c. is cross reactive for two different seventransmembrane receptors, and/or d. is cross reactive for a humanand a cynomolgus seven transmembrane receptor, and/or e. ischaracterized by a HCDR3 region comprising between 10 and 34% oftyrosine and/or between 2 and 20% of histidine, and/or f. does notmodulate G protein independent signaling of the chemokine receptor,and/or g. is a non-internalizing antibody or is characterized by aninternalization into a cell with endogenous target expression whichis lower than the 1.5, 2, 3, 4, 5, 6, 7, or 10-fold of theinternalization of the isotype control. 14. An isolated antibody,antigen-binding fragment thereof or binder obtained with a methodor use according to any of embodiments 8 to 13. 15. An isolatedantibody or antigen-binding fragment thereof, specifically bindingto a first isolated sulfated polypeptide which comprises thetyrosine rich domain (TRD) of a seven transmembrane receptor, andoptionally its LID domain, wherein at least 25%, at least 50% or atleast 75% of the tyrosine residues of the TRD are sulfated. 16. Theisolated antibody or antigen-binding fragment according toembodiment 15, wherein the cysteine between the TRD and the LIDdomain has been removed or has been exchanged into a differentamino acid. 17. The isolated antibody or antigen-binding fragmentaccording to embodiment 15 or 16, wherein the seven transmembranereceptor is a human, cynomolgus or mouse seven transmembranereceptor. 18. The isolated antibody or antigen-binding fragmentaccording to any of embodiments 15, 16 or 17, wherein the seventransmembrane receptor is a. a CC chemokine receptor, preferablyCCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b. aCXC chemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, or CXCR6, or c. CX3CR1 or CXCR1. 19. An isolated antibody orantigen-binding fragment according to any of embodiments 15 to 18,said first isolated sulfated polypeptide comprising a sequenceaccording to a. SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:3 or SEQ ID NO:6, preferably wherein at least Y10 and/orY18 have been sulfated, or b. SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:8 or SEQ ID NO:11, preferably wherein at least Y26 has beensulfated, or c. SEQ ID NO:9 or SEQ ID NO:12, preferably wherein atleast Y37 and/or Y39 has been sulfated, or d. SEQ ID NO:13 or SEQID NO:16, preferably wherein Y16 and/or Y17 have been sulfated, ore. SEQ ID NO:14 or SEQ ID NO:17, preferably wherein Y16 has beensulfated, or f. SEQ ID NO:15 or SEQ ID NO:18 preferably wherein Y20and/or Y22 have been sulfated, or g. SEQ ID NO:19, SEQ ID NO:22,SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:21 or SEQ ID NO:24,preferably wherein at least Y22 has been sulfated and preferablyfurthermore Y16, Y19 and/or Y20 have been sulfated, or h. SEQ IDNO:25, SEQ ID NO:28, SEQ ID NO:26 or SEQ ID NO:29, preferablywherein two, three or all of Y3, Y10, Y14 and Y15 have beensulfated, or i. SEQ ID NO:27 or SEQ ID NO:30, preferably whereintwo or three of Y10, Y12 and Y16 have been sulfated, or j. SEQ IDNO:31 or SEQ ID NO:34, preferably wherein at least two or three ofY18, Y26 and Y27 have been sulfated, or k. SEQ ID NO:32 or SEQ IDNO:35, preferably wherein at least two or three of Y23, Y31 and Y32have been sulfated, or l. SEQ ID NO:33 or SEQ ID NO:36, preferablywherein at least two or three of Y13, Y18 and Y19 have beensulfated, or m. SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:38 or SEQ IDNO:41, preferably wherein one or both of Y8 and Y17 have beensulfated, or n. SEQ ID NO:39 or SEQ ID NO:42, preferably whereinone or both of Y8 and Y17 and optionally Y20 have been sulfated, oro. SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:47,preferably wherein at least two or all of Y3, Y15 and Y17 have beensulfated, or p. SEQ ID NO:45 or SEQ ID NO:48, preferably wherein atleast two or all of Y3, Y14 and Y15 have been sulfated, or q. SEQID NO:61, SEQ ID NO:64, SEQ ID NO:62 or SEQ ID NO:65, preferablywherein at least Y28, and preferably also Y17 and/or Y37 has beensulfated, or r. SEQ ID NO:63 or SEQ ID NO:66, preferably wherein atleast Y28 has been sulfated, and preferably also Y19 has beensulfated, or s. SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:68 or SEQ IDNO:71, preferably wherein at least one or both of Y14 and Y22 hasbeen sulfated, or t. SEQ ID NO:69 or SEQ ID NO:72, preferablywherein at least one, two or all of Y14, Y17 and Y22 has beensulfated, or u. SEQ ID NO:73 or SEQ ID NO:76, preferably whereinY27 has been sulfated, or v. SEQ ID NO:74 or SEQ ID NO:77,preferably wherein at least one of Y14 and Y28 has been sulfated,or w. SEQ ID NO:75 or SEQ ID NO:78, preferably wherein at least Y6has been sulfated, or x. SEQ ID NO:79 or SEQ ID NO:82, preferablywherein Y23 and/or Y25 have been sulfated, or y. SEQ ID NO:80 orSEQ ID NO:83, preferably wherein Y20 and/or Y22 have been sulfated,or z. SEQ ID NO:81 or SEQ ID NO:84, preferably wherein Y24 has beensulfated, or aa. SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:86, SEQ IDNO:89, SEQ ID NO:87 or SEQ ID NO:90, preferably wherein at leastone or both of Y27 and Y29 have been sulfated, or bb. SEQ ID NO:91,SEQ ID NO:94, SEQ ID NO:92 or SEQ ID NO:95, preferably wherein atleast Y12 and/or Y21 have been sulfated, or cc. SEQ ID NO:93 or SEQID NO:96, preferably wherein at least Y23 and/or Y14 have beensulfated, or dd. SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:98 or SEQID NO:101, preferably wherein at least one of Y3 and Y27 have beensulfated, or ee. SEQ ID NO:99 or SEQ ID NO:102, preferably whereinat least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, orff. SEQ ID NO:103 or SEQ ID NO:106, preferably wherein at least oneor both of Y6 and Y10 has been sulfated, or gg. SEQ ID NO:104 orSEQ ID NO:107, preferably wherein at least two or all of Y4, Y7 andY39 have been sulfated, or hh. SEQ ID NO:105 or SEQ ID NO:108,preferably wherein at least one or both of Y11 and Y15 have beensulfated, or ii. SEQ ID NO:157 or SEQ ID NO:160, preferably whereinat least Y14 has been sulfated, or jj. SEQ ID NO:158, preferablywherein at least Y20 has been sulfated, or kk. SEQ ID NO:161,preferably wherein at least Y20 or Y22 has been sulfated, or ll.SEQ ID NO:159 or SEQ ID NO:162, preferably wherein at least Y15 hasbeen sulfated, or mm. SEQ ID NO:163 or SEQ ID NO:166, preferablywherein at least Y27 has been sulfated, or nn. SEQ ID NO:164,preferably wherein at least Y14 has been sulfated or oo. SEQ IDNO:167, preferably wherein at least Y14 or Y28 has been sulfated,or pp. SEQ ID NO:165 or SEQ ID NO:168, preferably wherein at leastY6 has been sulfated. 20. The isolated antibody or antigen-bindingfragment according to any of embodiments 15 to 19, wherein thedissociation constant or the EC50 of the antibody for binding thefirst isolated sulfated polypeptide and/or for said seventransmembrane receptor is below 150 nM, 100 nM, 10 nM, 5 nM, 2.5nM, 1 nM, 0.5 or 0.25 nM. 21. An isolated antibody orantigen-binding fragment according to any of embodiments 15 to 20,wherein the isolated antibody or antigen-binding fragmentspecifically binds to a second isolated sulfated polypeptide whichcomprises the TRD of a seven transmembrane receptor, preferablywherein the seven transmembrane receptor of the TRD comprised bythe second isolated sulfated polypeptide a. is different from theseven transmembrane receptor of the TRD comprised by the firstisolated sulfated polypeptide, or b. is the corresponding seventransmembrane receptor of the TRD comprised by the first isolatedsulfated polypeptide but from a different species. 22. The isolatedantibody or antigen-binding fragment according to embodiment 21,wherein the dissociation constant or the EC50 of the antibody forbinding the second isolated sulfated polypeptide and/or for bindingthe second seven transmembrane receptor is below 10 nM, 5 nM, 2.5nM, 1 nM, 0.5 or 0.25 nM. 23. An isolated antibody orantigen-binding fragment according to any of embodiments 15 to 22,wherein the dissociation constant (KD) of the antibody for bindingthe first isolated sulfated polypeptide is lower than thedissociation constant (KD) of the antibody for binding a firstisolated non-sulfated polypeptide having the same sequence as thefirst isolated sulfated polypeptide. 24. The isolated antibody orantigen-binding fragment according to embodiment 23, wherein thedissociation constant and/or EC50 of the antibody for binding thefirst isolated non-sulfated polypeptide is higher than 150 nM, 250nM, 500 nM, 1 .mu.M, 2 .mu.M or 3 .mu.M, or is not detectable. 25.The isolated antibody or antigen-binding fragment according to anyof embodiments 23 or 24, wherein the dissociation constant or theEC50 of the antibody or fragment for binding the first isolatedsulfated polypeptide is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or0.25 nM, and wherein the dissociation constant of the antibody orfragment for binding the first isolated non-sulfated polypeptide ishigher than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or isnot detectable. 26. The isolated antibody or antigen-bindingfragment according to any of embodiments 15 to 25, wherein theantibody comprises human, rat or mouse derived CDRs. 27. Theisolated antibody or antigen-binding fragment according to any ofembodiments 15 to 26, wherein the antibody i. comprises humanderived CDRs, and/or j. is cross reactive for human and cynomolgus,and/or k. is

characterized by a HCDR3 region comprising between 10 and 34% oftyrosine and/or between 2 and 20% of histidine, and/or l. does notmodulate G protein independent signaling of the seven transmembranereceptor, and/or m. is a non-internalizing antibody or ischaracterized by an internalization into a cell with endogenoustarget expression which is lower than the 1.5, 2, 3, 4, 5, 6, 7, or10-fold of the internalization of the isotype control, and/or n.induces ADCC and/or ADCP o. is a human, rat or murine IgG antibody,preferably a human IgG1 antibody or a murine IgG2a antibody, and/orp. is an scFv, Fab, Fab' or a F(ab')2 fragment. 28. A conjugatecomprising an antibody or antigen-binding fragment according to anyof embodiments 15 to 27, preferably wherein the conjugate comprisese. a radioactive element, f. a cytotoxic agent, such as anauristatin, a maytansinoid, a kinesin-spindle protein inhibitor, anicotinamide phosphoribosyltransferase inhibitor or apyrrolobenzodiazepine derivative, g. a further antibody orantigen-binding fragment, or h. a chimeric antigen receptor. 29. Anantibody or antigen-binding fragment according to any ofembodiments 15 to 27 or a conjugate according to embodiment 28 foruse in the treatment of a tumor or a disease characterized by theinvolvement of cells expressing the seven transmembrane receptor,optionally in combination with an antibody targeting a checkpointinhibitor. 30. The antibody or antigen-binding fragment accordingto any of embodiments 15 to 27 or a conjugate according toembodiment 28 for use as a diagnostic agent in vivo or in vitro.31. A kit comprising an antibody or antigen-binding fragmentaccording to any of embodiments 15 to 27 or a conjugate accordingto embodiment 28 with instructions for use. 32. An isolatedantibody or antigen-binding fragment thereof specifically bindingto CCR8, wherein the antibody or antigen-binding fragment isnon-internalizing or is characterized by an internalization into acell with endogenous target expression which is lower than the 1.5,2, 3, 4, 5, 6, 7, or 10-fold of the internalization of the isotypecontrol. 33. An isolated antibody or antigen-binding fragmentthereof, specifically binding to CCR8, wherein the antibody orfragment is characterized by a HCDR3 region comprising between 10and 34% of tyrosine and between 2 and 20% of histidine. 34. Anisolated antibody or antigen-binding fragment thereof specificallybinding to CCR8, wherein the antibody comprises human derived CDRs.35. An isolated antibody or antigen-binding fragment thereofspecifically binding to CCR8, wherein the antibody orantigen-binding fragment is cross reactive for CCR8 from at leasttwo species, preferably selected from human, cynomolgus and mouse,most preferably wherein the antibody or antigen-binding fragment iscross reactive for human and cynomolgus CCR8. 36. An isolatedantibody or antigen-binding fragment thereof, specifically bindingto CCR8, wherein the antibody or antigen-binding fragment, d. doesnot block CCL1 induced .beta.-arrestin signaling and/or e. does notinduce ERK1/2 phosphorylation and/or f. does not induce AKTphosphorylation. 37. An isolated antibody or antigen-bindingfragment thereof specifically binding to CCR8, wherein the antibodyor antigen-binding fragment is afucosylated and d. inducesantibody-dependent cell-mediated cytotoxicity (ADCC) in targetcells expressing human CCR8 via human effector cells, such as humanNK cells, and e. induces antibody-dependent cell-mediatedphagocytosis (ADCP) in target cells expressing human CCR8 via humaneffector cells, such as human macrophages, f. wherein the maximalADCC and ADCP induced in vitro depletion of target cells expressinghuman CCR8 is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%,90%, 95% or 99%. 38. The isolated antibody or antigen-bindingfragment according to any of embodiments 33 to 37, wherein theantibody or antigen-binding fragment is non-internalizing or ischaracterized by an internalization into a cell with endogenoustarget expression which is lower than the 1.5, 2, 3, 4, 5, 6, 7, or10-fold of the internalization of the isotype control. 39. Theisolated antibody or antigen-binding fragment according to any ofembodiments 32, or 34 to 38, wherein the antibody orantigen-binding fragment is characterized by a HCDR3 regioncomprising between 10 and 34% of tyrosine and/or between 2 and 20%of histidine. 40. The isolated antibody or antigen-binding fragmentaccording to any of embodiments 32 to 33, or 35 to 39, wherein theantibody comprises human derived CDRs. 41. The isolated antibody orantigen-binding fragment according to any of embodiments 32 to 34,or 36 to 40, wherein the antibody or antigen-binding fragment iscross reactive for CCR8 from at least two species, preferablyselected from human, cynomolgus and mouse, most preferably whereinthe antibody or antigen-binding fragment is cross reactive forhuman and cynomolgus CCR8. 42. The isolated antibody orantigen-binding fragment according to any of embodiments 32 to 41,wherein the antibody or antigen-binding fragment binds the CCR8from a first species with a first dissociation constant KD andbinds the CCR8 from a second species with a second dissociationconstant KD, wherein the first and the second dissociation constantare in the same order of magnitude. 43. The isolated antibody orantigen-binding fragment according to any of embodiments 32 to 35,or 37 to 42, wherein the antibody or antigen-binding fragment, d.does not block CCL1 induced .beta.-arrestin signaling and/or e.does not induce ERK1/2 phosphorylation and/or f. does not induceAKT phosphorylation. 44. The isolated antibody or antigen-bindingfragment according to any of embodiments 32 to 36, or 38 to 43,wherein the antibody or antigen-binding fragment d. inducesantibody-dependent cell-mediated cytotoxicity (ADCC) in targetcells expressing human CCR8 via human effector cells, such as humanNK cells, and e. induces antibody-dependent cell-mediatedphagocytosis (ADCP) in target cells expressing human CCR8 via humaneffector cells, such as human macrophages, and optionally f.wherein the maximal ADCC and ADCP induced in vitro depletion oftarget cells expressing human CCR8 is at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%. 45. The isolated antibodyor antigen-binding fragment according to any of embodiments 32 to44, wherein the antibody or antigen-binding fragment specificallybinds to the sulfated tyrosine rich domain of CCR8. 46. Theisolated antibody or antigen-binding fragment according to any ofembodiments 32 to 45, wherein the antibody or antigen-bindingfragment specifically binds (i) with an EC50 of <15 nM, <10nM, <5 nM, <1 nM or <0.6 nM a. to human CCR8 and/or to anisolated polypeptide according to SEQ ID NO:46, wherein at leasttwo or all of Y3, Y15 and Y17 have been sulfated, and/or b. tocynomolgus CCR8 and/or to an isolated polypeptide according to SEQID NO:47, wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and/or c. to murine CCR8 and/or to an isolatedpolypeptide according to SEQ ID NO:48, wherein at least two or allof Y3, Y14 and Y15 have been sulfated, and/or (ii) with an EC50 of<50 nM, <25 nM, <15 nM or <10 nM to activated humanregulatory T cells. 47. The isolated antibody or antigen-bindingfragment thereof according to any of embodiments 32 to 46, c.wherein the antibody or antigen-binding fragment binds to human Fcgamma receptor IIIA variant V176 (CD16a) with a dissociationconstant (KD) lower than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or200 nM, and/or d. wherein the antibody or antigen-binding fragmentbinds to human Fc gamma RIIA (CD32a) with a dissociation constant(KD) lower than 30 .mu.M, 20 .mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M.48. The isolated antibody or antigen-binding fragment thereofaccording to any of embodiments 32 to 47, wherein the antibody orantigen-binding fragment is afucosylated. 49. The isolated antibodyor antigen-binding fragment thereof according to any of embodiments32 to 48, wherein the antibody or antigen-binding fragment f.induces antibody-dependent cell-mediated cytotoxicity (ADCC) intarget cells expressing human CCR8 via human effector cells, suchas human NK cells, and/or g. induces antibody-dependentcell-mediated phagocytosis (ADCP) in target cells expressing humanCCR8 via human effector cells, such as human macrophages,preferably, wherein h. the ADCC-induced maximal depletion ofactivated human regulatory T cells is at least 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or i. theADCP-induced maximal depletion of activated human regulatory Tcells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, and/orj. the maximal ADCC and ADCP induced in vitro depletion ofactivated human regulatory T cells is at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%. 50. The isolated antibodyor antigen-binding fragment according to any of embodiments 32 to49, wherein c. the EC50 of the antibody or antigen-binding fragmentfor ADCC-induced depletion of activated human regulatory T cells isbelow 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/or d. theEC50 of the antibody or antigen-binding fragment for ADCP-induceddepletion of activated human regulatory T cells is below 500 pM,250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM. 51. Theisolated antibody or antigen-binding fragment according to any ofembodiments 32 to 50, wherein an effective dose of the antibody orantigen-binding fragment d. decreases the number of activated orintra-tumoral regulatory T cells, in vitro or in a subject, to lessthan 50%, 40%, 30%, 25%, 20%, 10%, 5% or 1% and/or e. increases theratio of intra-tumoral CD8+ T cells to intra-tumoral Tregs, invitro or in a subject, to at least 5, 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, or higher and/or f. decreases thepercentage of regulatory T cells of intra-tumoral CD4+ T cells, invitro or in a subject, to <30%, <20%, <10% or <5%. 52.The isolated antibody or antigen-binding fragment according to anyof embodiments 32 to 51, wherein an effective dose of the antibodyor antigen-binding fragment induces formation of tertiary lymphoidstructures in a tumor. 53. The isolated antibody or antigen-bindingfragment thereof according to any of embodiments 32 to 52, whereinthe antibody is an IgG antibody, preferably a human IgG1 or amurine IgG2a, and or wherein the antigen-binding fragment is anscFv, Fab, Fab' or a F(ab')2 fragment. 54. A conjugate comprisingan antibody or antigen-binding fragment according to any ofembodiments 32 to 53, preferably wherein the conjugate comprises f.a radioactive element, g. a cytotoxic agent, such as an auristatin,a maytansinoid, a kinesin-spindle protein inhibitor, a nicotinamidephosphoribosyltransferase inhibitor or a pyrrolobenzodiazepinederivative, h. a further antibody or antigen-binding fragment, ori. a chimeric antigen receptor. 55. A pharmaceutical compositioncomprising an antibody or antigen-binding fragment according to anyof embodiments 32 to 53 or a conjugate according to embodiment 54.56. The pharmaceutical composition according to embodiment 55,comprising one or more further therapeutically active compounds,preferably selected from g. an antibody or compound targeting acheckpoint protein, such as PD1, PD-L1 or CTLA-4, preferablywherein the antibody or compound targeting the checkpoint proteinis Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab,Cemiplimab, Dostarlimab or Ipilimumab, h. an antibody targeting afurther chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CX3CR1 or CXCR1, i. an antibody or a small molecule targeting HER2and/or EGFR, j. the standard of care for any of head and neckcancer, breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma and esophageal tumor, melanoma, bladder cancer, livercancer, and/or prostate cancer, k. a chemotherapeutic agent,preferably a taxane, paclitaxel, doxorubicin, cis-platin,carboplatin, oxaliplatin, or gemcitabine, j. a B cell depletingagent, such as an anti-CD19 antibodies or an anti-CD20 antibodiesand/or 1. a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1. 57. An antibody or antigen-binding fragmentaccording to any of embodiments 32 to 53 or a conjugate accordingto embodiment 54 or a pharmaceutical composition according toembodiment 55 or 56 for use as a medicament. 58. The antibody orantigen-binding fragment according to any of embodiments 32 to 53or conjugate according to embodiment 54 or pharmaceuticalcomposition according to embodiment 55 or 56 for use in thetreatment of a tumor or a disease characterized by CCR8 positivecells, such as CCR8 positive regulatory T cells. 59. An antibody orantigen-binding fragment according to any of embodiments 32 to 53or a conjugate according to embodiment 54 or a pharmaceuticalcomposition according to embodiment 55 or 56 for use insimultaneous, separate, or sequential combination (i) with one ormore further therapeutically active compounds, preferably selectedfrom h. an antibody or a small molecule targeting a checkpointprotein, such as PD1, PD-L1 or CTLA-4, preferably wherein theantibody or small molecule targeting a checkpoint protein isNivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab,Cemiplimab, Dostarlimab or Ipilimumab, i. an antibody targeting afurther chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, CX3CR1 or CXCR1, j. an antibody targeting a protein which isspecifically expressed by the tumor cells, k. an antibody or asmall molecule targeting HER2 and/or EGFR, l. the standard of carefor any of head and neck cancer, breast cancer, gastric cancer,lung cancer, squamous cell carcinoma, esophageal tumor, melanoma,bladder cancer, liver cancer, and/or prostate cancer, m. achemotherapeutic agent, preferably a taxane, paclitaxel,doxorubicin, cis-platin, carboplatin, oxaliplatin, or gemcitabineand/or n. a targeted kinase inhibitor, such as Sorafinib,Regorafenib, or MEKi-1, and/or (ii) with radiation therapy, and/or(iii) with depletion of intra-tumoral B cells, in the treatment ofa tumor or a disease characterized by CCR8 positive cells, such asCCR8 positive regulatory T cells. 60. The antibody, fragment,conjugate or pharmaceutical composition for use according toembodiment 58 or 59, wherein the tumor is selected from the groupof T-cell acute lymphoblastic leukemia, breast cancer,triple-negative breast cancer, triple positive breast cancer,non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC),testicular cancer, gastric cancer, head and neck squamous cellcarcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer,pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acutemyeloid leukemia, kidney cancer, bladder cancer, skin cancer,melanoma, thyroid cancer, mesothelioma, sarcoma and prostatecancer, B cell lymphoma, T cell lymphoma, or any other cancerinvolving CCR8 expressing cells, preferably wherein the tumor isselected from head and neck cancer, breast cancer, gastric cancer,lung cancer, squamous cell carcinoma, esophageal tumor, melanoma,bladder cancer, liver cancer, prostate cancer, B cell lymphoma andT cell lymphoma. 61. The antibody, fragment, conjugate orpharmaceutical composition for use according to embodiment 57 to60, wherein the use comprises determining n. presence or quantityof tumor infiltrating lymphocytes, o. presence or quantity ofmacrophages and/or NK cells, p. presence or quantity of CCR8positive or FOXP3 positive regulatory T cells, q. tumor mutationalburden, r. cancer staging, s. presence, level or activation ofinterferon-stimulated genes or proteins, t. CCR8 expression, u.presence or quantity of complement factor proteins, serpins, and/orMHC components, v. presence or quantity of cytokines, such asinflammatory or suppressive cytokines, w. activation of immune geneexpression, and/or x. immune checkpoint (protein) expression, suchas PD-(L)1 or CTLA4 expression, y. presence or quantity of tumorinfiltrating CD19+ B cells, z. presence or quantity of tumorinfiltrating CD8+ T cells to predict or monitor treatment success.62. A polynucleotide encoding an antibody or antigen-bindingfragment according to any of embodiments 32 to 53. 63. A vectorcomprising a polynucleotide according to embodiment 62. 64. Anisolated cell arranged for production of an antibody orantigen-binding fragment according to any of embodiments 32 to 53.65. A method of producing an antibody or antigen-binding fragmentaccording to any of embodiments 32 to 53 or a conjugate accordingto embodiment 54, comprising culturing a cell according toembodiment 64 and optionally purification of the antibody orantigen-binding fragment. 66. The antibody or antigen-bindingfragment according to any of embodiments 32 to 53 or a conjugateaccording to embodiment 54 for use as a diagnostic agent in vivo orin vitro. 67. A kit comprising an antibody or antigen-bindingfragment according to any of embodiments 32 to 53 or a conjugateaccording to embodiment 54 or a pharmaceutical compositionaccording to embodiment 55 or 56 with

instructions for use. 68. An isolated anti-CCR8 antibody orantigen-binding fragment thereof, specifically binding with a KDvalue of <5E-8 M, <4E-8 M, <3E-8 M, <2E-8 M, <1E-8M, <9E-9 M, <8E-9 M, <7E-9 M, <6E-9 M, <5E-9 M,<4E-9 M, <3E-9 M, <2.5E-9 M, <2E-9 M, <1.5E-9 M,<1E-9 M, <9E-10 M, <8E-10 M, <7E-10 M, <6E-10 M,<5E-10 M, <4E-10 M, <3E-10 M, <2.5E-10 M, <2E-10 M,<1.5E-10 M, <1E-10 M, or <9E-11 M a. to an isolatedpolypeptide according to SEQ ID NO:43 and/or SEQ ID NO:46, whereinat least two or all of Y3, Y15 and Y17 have been sulfated, and/orb. to an isolated polypeptide according to SEQ ID NO:44 and/or SEQID NO:47, wherein at least two or all of Y3, Y15 and Y17 have beensulfated, and/or c. to an isolated polypeptide according to SEQ IDNO:45 and/or SEQ ID NO:48, wherein at least two or all of Y3, Y14and Y15 have been sulfated. 69. The isolated antibody orantigen-binding fragment according to embodiment 68, wherein theantibody or antigen-binding fragment is characterized by a HCDR3region comprising between 10 and 34% of tyrosine and/or between 2and 20% of histidine. 70. The isolated antibody or antigen-bindingfragment according to embodiment 68 or 69, wherein the antibody orantigen-binding fragment comprises human derived CDRs. 71. Theisolated antibody or antigen-binding fragment according to any ofembodiments 68 to 70, wherein the antibody or antigen-bindingfragment is cross reactive for human and cynomolgus CCR8. 72. Theisolated antibody or antigen-binding fragment according to any ofembodiments 68 to 71, wherein the antibody or antigen-bindingfragment a. does not block CCL1 induced .beta.-arrestin signaling,and/or b. does not induce ERK1/2 phosphorylation, and/or c. doesnot induce AKT phosphorylation. 73. The isolated antibody orantigen-binding fragment according to any of embodiments 68 to 72,wherein the antibody or antigen-binding fragment isnon-internalizing or is characterized by an internalization into acell with endogenous target expression which is lower than the 1.5,2, 3, 4, 5, 6, 7, or 10-fold of the internalization of the isotypecontrol. 74. The isolated antibody or antigen-binding fragmentthereof according to any of embodiments 68 to 73, a. wherein theantibody or antigen-binding fragment binds to human Fc gammareceptor IIIA variant V176 (CD16a) with a dissociation constant(KD) lower than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM,and/or b. wherein the antibody or antigen-binding fragment binds tohuman Fc gamma RIIA (CD32a) with a dissociation constant (KD) lowerthan 30 .mu.M, 20 .mu.M, 10 .mu.M, 5 .mu.M or 1 .mu.M. 75. Anisolated anti-CCR8 antibody or antigen-binding fragment thereofcomprising a HCDR3 sequence having at least 90%, 95%, 98% or 100%sequence identity with any of SEQ ID NO:260 (TPP-16966), SEQ IDNO:278 (TPP-17575), SEQ ID NO:296 (TPP-17576), SEQ ID NO:314(TPP-17577), SEQ ID NO:332 (TPP-17578), SEQ ID NO:350 (TPP-17579),SEQ ID NO:368 (TPP-17580), SEQ ID NO:386 (TPP-17581), SEQ ID NO:404(TPP-18205), SEQ ID NO:422 (TPP-18206), SEQ ID NO:440 (TPP-18207),SEQ ID NO:458 (TPP-19546), SEQ ID NO:476 (TPP-20950), SEQ ID NO:494(TPP-20955), SEQ ID NO:512 (TPP-20965), SEQ ID NO:530 (TPP-21045),SEQ ID NO:548 (TPP-21047), SEQ ID NO:566 (TPP-21181), SEQ ID NO:584(TPP-21183), SEQ ID NO:602 (21360), or SEQ ID NO:620 (TPP-23411),SEQ ID NO:663 (TPP-29596), SEQ ID NO:683 (TPP-29597), SEQ ID NO:705(TPP-18429), SEQ ID NO:725 (TPP-18430), SEQ ID NO:745 (TPP-18432),SEQ ID NO:765 (TPP-18433), SEQ ID NO:785 (TPP-18436), SEQ ID NO:805(TPP-19571), SEQ ID NO:829 (TPP-27477), SEQ ID NO:849 (TPP-27478),SEQ ID NO:869 (TPP-27479), SEQ ID NO:889 (TPP-27480), SEQ ID NO:909(TPP-29367), SEQ ID NO:929 (TPP-29368), or SEQ ID NO:949(TPP-29369). 76. An isolated anti-CCR8 antibody or antigen-bindingfragment thereof comprising at least one, two, three, four, five orsix CDR sequence(s) having at least 90%, 95%, 98% or 100% sequenceidentity with any of a. SEQ ID NO:258, SEQ ID NO:259, SEQ IDNO:260, SEQ ID NO:262, SEQ ID NO:263 or SEQ ID NO:264 (TPP-16966),b. SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:280, SEQID NO:281 or SEQ ID NO:282 (TPP-17575), c. SEQ ID NO:294, SEQ IDNO:295, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:299 or SEQ IDNO:300 (TPP-17576), d. SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:317 or SEQ ID NO:318 (TPP-17577), e. SEQID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:334, SEQ IDNO:335 or SEQ ID NO:336 (TPP-17578), f. SEQ ID NO:348, SEQ IDNO:349, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:353 or SEQ IDNO:354 (TPP-17579), g. SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368,SEQ ID NO:370, SEQ ID NO:371 or SEQ ID NO:372 (TPP-17580), h. SEQID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:388, SEQ IDNO:389 or SEQ ID NO:390 (TPP-17581), i. SEQ ID NO:402, SEQ IDNO:403, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:407 or SEQ IDNO:408 (TPP-18205), j. SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422,SEQ ID NO:424, SEQ ID NO:425 or SEQ ID NO:426 (TPP-18206), k. SEQID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:442, SEQ IDNO:443 or SEQ ID NO:444 (TPP-18207), l. SEQ ID NO:456, SEQ IDNO:457, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:461 or SEQ IDNO:462 (TPP-19546), m. SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476,SEQ ID NO:478, SEQ ID NO:479 or SEQ ID NO:480 (TPP-20950), n. SEQID NO:492, SEQ ID NO:493, SEQ ID NO:494, SEQ ID NO:496, SEQ IDNO:497 or SEQ ID NO:498 (TPP-20955), o. SEQ ID NO:510, SEQ IDNO:511, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:515, or SEQ IDNO:516 (TPP-20965), p. SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:532, SEQ ID NO:533 or SEQ ID NO:534 (TPP-21045), q. SEQID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:550, SEQ IDNO:551 or SEQ ID NO:552 (TPP-21047), r. SEQ ID NO:564, SEQ IDNO:565, SEQ ID NO:566, SEQ ID NO:568, SEQ ID NO:569 or SEQ IDNO:570 (TPP-21181), s. SEQ ID NO:582, SEQ ID NO:583, SEQ ID NO:584,SEQ ID NO:586, SEQ ID NO:587 or SEQ ID NO:588 (TPP-21183), t. SEQID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:604, SEQ IDNO:605 SEQ ID NO:606 (TPP-21360), u. SEQ ID NO:618, SEQ ID NO:619,SEQ ID NO:620, SEQ ID NO:622, SEQ ID NO:623 SEQ ID NO:624(TPP-23411), v. SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ IDNO:665, SEQ ID NO:666 SEQ ID NO:667 (TPP-29596), w. SEQ ID NO:681,SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:685, SEQ ID NO:686 SEQ IDNO:687 (TPP-29597), x. SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705,SEQ ID NO:707, SEQ ID NO:708 SEQ ID NO:709 (TPP-18429), y. SEQ IDNO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:728SEQ ID NO:729 (TPP-18430), z. SEQ ID NO:743, SEQ ID NO:744, SEQ IDNO:745, SEQ ID NO:747, SEQ ID NO:748 SEQ ID NO:749 (TPP-18432), aa.SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:767, SEQ IDNO:768 SEQ ID NO:769 (TPP-18433), bb. SEQ ID NO:783, SEQ ID NO:784,SEQ ID NO:785, SEQ ID NO:787, SEQ ID NO:788 SEQ ID NO:789(TPP-18436), cc. SEQ ID NO:803, SEQ ID NO:804, SEQ ID NO:805, SEQID NO:807, SEQ ID NO:808 SEQ ID NO:809 (TPP-19571), dd. SEQ IDNO:827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 831, SEQ IDNO:832 SEQ ID NO:833 (TPP-27477), ee. SEQ ID NO:847, SEQ ID NO:848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO:852 SEQ ID NO:853(TPP-27478), ff. SEQ ID NO:867, SEQ ID NO: 868, SEQ ID NO: 869, SEQID NO: 871, SEQ ID NO:872 SEQ ID NO:873 (TPP-27479), gg. SEQ IDNO:887, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ IDNO:892 SEQ ID NO:893 (TPP-27480), hh. SEQ ID NO:907, SEQ ID NO:908, SEQ ID NO: 909, SEQ ID NO: 911, SEQ ID NO:912 SEQ ID NO:913(TPP-29367), ii. SEQ ID NO:927, SEQ ID NO: 928, SEQ ID NO: 929, SEQID NO: 931, SEQ ID NO:932 or SEQ ID NO:933 (TPP-29368), or jj. SEQID NO:947, SEQ ID NO: 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ IDNO:952 or SEQ ID NO:953 (TPP-29369), kk. SEQ ID NO:202, SEQ IDNO:203, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:207 and SEQ IDNO:208 (TPP-14095), ll. SEQ ID NO:216, SEQ ID NO:217, SEQ IDNO:218, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:222 (TPP-14099),mm. SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:234, SEQID NO:235 and SEQ ID NO:236 (TPP-15285), or nn. SEQ ID NO:244, SEQID NO:245, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:249 and SEQ IDNO:250 (TPP-15286). 77. An isolated anti-CCR8 antibody orantigen-binding fragment thereof comprising a variable heavy chainsequence and/or a variable light chain sequence having at least90%, 95%, 98% or 100% sequence identity with a. a variable heavychain sequence according to SEQ ID NO:257 and/or a variable lightchain sequence according to SEQ ID NO:261 (TPP-16966), b. avariable heavy chain sequence according to SEQ ID NO:275 and/or avariable light chain sequence according to SEQ ID NO:279(TPP-17575), c. a variable heavy chain sequence according to SEQ IDNO:293 and/or a variable light chain sequence according to SEQ IDNO:297 (TPP-17576), d. a variable heavy chain sequence according toSEQ ID NO:311 and/or a variable light chain sequence according toSEQ ID NO:315 (TPP-17577), e. a variable heavy chain sequenceaccording to SEQ ID NO:329 and/or a variable light chain sequenceaccording to SEQ ID NO:333 (TPP-17578), f. a variable heavy chainsequence according to SEQ ID NO:347 and/or a variable light chainsequence according to SEQ ID NO:351 (TPP-17579), g. a variableheavy chain sequence according to SEQ ID NO:365 and/or a variablelight chain sequence according to SEQ ID NO:369 (TPP-17580), h. avariable heavy chain sequence according to SEQ ID NO:383 and/or avariable light chain sequence according to SEQ ID NO:387(TPP-17581), i. a variable heavy chain sequence according to SEQ IDNO:401 and/or a variable light chain sequence according to SEQ IDNO:405 (TPP-18205), j. a variable heavy chain sequence according toSEQ ID NO:419 and/or a variable light chain sequence according toSEQ ID NO:423 (TPP-18206), k. a variable heavy chain sequenceaccording to SEQ ID NO:437 and/or a variable light chain sequenceaccording to SEQ ID NO:441 (TPP-18207), l. a variable heavy chainsequence according to SEQ ID NO:455 and/or a variable light chainsequence according to SEQ ID NO:459 (TPP-19546), m. a variableheavy chain sequence according to SEQ ID NO:473 and/or a variablelight chain sequence according to SEQ ID NO:477 (TPP-20950), n. avariable heavy chain sequence according to SEQ ID NO:491 and/or avariable light chain sequence according to SEQ ID NO:495(TPP-20955), o. a variable heavy chain sequence according to SEQ IDNO:509 and/or a variable light chain sequence according to SEQ IDNO:513 (TPP-20965), p. a variable heavy chain sequence according toSEQ ID NO:527 and/or a variable light chain sequence according toSEQ ID NO:531 (TPP-21045), q. a variable heavy chain sequenceaccording to SEQ ID NO:545 and/or a variable light chain sequenceaccording to SEQ ID NO:549 (TPP-21047), r. a variable heavy chainsequence according to SEQ ID NO:563 and/or a variable light chainsequence according to SEQ ID NO:567 (TPP-21181), s. a variableheavy chain sequence according to SEQ ID NO:581 and/or a variablelight chain sequence according to SEQ ID NO:585 (TPP-21183), t. avariable heavy chain sequence according to SEQ ID NO:599 and/or avariable light chain sequence according to SEQ ID NO:603(TPP-21360), u. a variable heavy chain sequence according to SEQ IDNO:617 and/or a variable light chain sequence according to SEQ IDNO:621 (TPP-23411), v. a variable heavy chain sequence according toSEQ ID NO:660 and/or a variable light chain sequence according toSEQ ID NO:664 (TPP-29596), w. a variable heavy chain sequenceaccording to SEQ ID NO:680 and/or a variable light chain sequenceaccording to SEQ ID NO:684 (TPP-29597), x. a variable heavy chainsequence according to SEQ ID NO:702 and/or a variable light chainsequence according to SEQ ID NO:706 (TPP-18429), y. a variableheavy chain sequence according to SEQ ID NO:722 and/or a variablelight chain sequence according to SEQ ID NO:726 (TPP-18430), z. avariable heavy chain sequence according to SEQ ID NO:742 and/or avariable light chain sequence according to SEQ ID NO:746(TPP-18432), aa. a variable heavy chain sequence according to SEQID NO:762 and/or a variable light chain sequence according to SEQID NO:766 (TPP-18433), bb. a variable heavy chain sequenceaccording to SEQ ID NO:782 and/or a variable light chain sequenceaccording to SEQ ID NO:786 (TPP-18436), cc. a variable heavy chainsequence according to SEQ ID NO:802 and/or a variable light chainsequence according to SEQ ID NO:806 (TPP-19571), dd. a variableheavy chain sequence according to SEQ ID NO:826 and/or a variablelight chain sequence according to SEQ ID NO:830 (TPP-27477), ee. avariable heavy chain sequence according to SEQ ID NO:846 and/or avariable light chain sequence according to SEQ ID NO:850(TPP-27478), ff. a variable heavy chain sequence according to SEQID NO:866 and/or a variable light chain sequence according to SEQID NO:870 (TPP-27479), gg. a variable heavy chain sequenceaccording to SEQ ID NO:886 and/or a variable light chain sequenceaccording to SEQ ID NO:890 (TPP-27480), hh. a variable heavy chainsequence according to SEQ ID NO:906 and/or a variable light chainsequence according to SEQ ID NO:910 (TPP-29367), ii. a variableheavy chain sequence according to SEQ ID NO:926 and/or a variablelight chain sequence according to SEQ ID NO:930 (TPP-29368), jj. avariable heavy chain sequence according to SEQ ID NO:946 and/or avariable light chain sequence according to SEQ ID NO:950(TPP-29369), kk. a variable heavy chain sequence according to SEQID NO:201 and a variable light chain sequence according to SEQ IDNO:205 (TPP-14095), ll. a variable heavy chain sequence accordingto SEQ ID NO:215 and a variable light chain sequence according toSEQ ID NO:219 (TPP-14099), mm. a variable heavy chain sequenceaccording to SEQ ID NO:229 and a variable light chain sequenceaccording to SEQ ID NO:233 (TPP-15285), or nn. a variable heavychain sequence according to SEQ ID NO:243 and a variable lightchain sequence according to SEQ ID NO:247 (TPP-15286). 78. Anisolated anti-CCR8 antibody or antigen-binding fragment thereofcomprising a heavy chain sequence and/or a light chain sequencehaving at least 90%, 95%, 98% or 100% sequence identity with a. aheavy chain according to SEQ ID NO:273 and a light chain accordingto SEQ ID NO:274 (TPP-16966), b. a heavy chain according to SEQ IDNO:291 and a light chain according to SEQ ID NO:292 (TPP-17575), c.a heavy chain according to SEQ ID NO:309 and a light chainaccording to SEQ ID NO:310 (TPP-17576), d. a heavy chain accordingto SEQ ID NO:327 and a light chain according to SEQ ID NO:328(TPP-17577), e. a heavy chain according to SEQ ID NO:345 and alight chain according to SEQ ID NO:346 (TPP-17578), f. a heavychain according to SEQ ID NO:363 and a light chain according to SEQID NO:364 (TPP-17579), g. a heavy chain according to SEQ ID NO:381and a light chain according to SEQ ID NO:382 (TPP-17580), h. aheavy chain according to SEQ ID NO:399 and a light chain accordingto SEQ ID NO:400 (TPP-17581), i. a heavy chain according to SEQ IDNO:417 and a light chain according to SEQ ID NO:418 (TPP-18205), j.a heavy chain according to SEQ ID NO:435 and a light chainaccording to SEQ ID NO:436 (TPP-18206), k. a heavy chain accordingto SEQ ID NO:453 and a light chain according to SEQ ID NO:454(TPP-18207), l. a heavy chain according to SEQ ID NO:471 and alight chain according to SEQ ID NO:472 (TPP-19546), m. a heavychain according to SEQ ID NO:489 and a light chain according to SEQID NO:490 (TPP-20950), n. a heavy chain according to SEQ ID NO:507and a light chain according to SEQ ID NO:508 (TPP-20955), o. aheavy chain according to SEQ ID NO:525 and a light chain accordingto SEQ ID NO:526 (TPP-20965), p. a heavy chain according to SEQ IDNO:543 and a light chain according to SEQ ID NO:544 (TPP-21045), q.a heavy chain according to SEQ ID NO:561 and a light chainaccording to SEQ ID NO:562 (TPP-21047), r. a heavy chain accordingto SEQ ID NO:579 and a light chain according to SEQ ID NO:580(TPP-21181), s. a heavy chain according to SEQ ID NO:597 and alight chain according to SEQ ID NO:598 (TPP-21183), t. a heavychain according to SEQ ID NO:615 and a light chain according to SEQID NO:616 (TPP-21360), u. a heavy chain according to SEQ ID NO:633and a light chain according to SEQ ID NO:634 (TPP-23411), v. aheavy chain according to SEQ ID NO:676 and a light chain accordingto SEQ ID NO:677 (TPP-29596), w. a heavy chain according to SEQ IDNO:696 and a light chain according to SEQ ID NO:697 (TPP-29597), x.a heavy chain according to SEQ ID NO:718 and a light chainaccording to SEQ ID NO:719 (TPP-18429), y. a heavy chain accordingto SEQ ID NO:738 and a light chain according to SEQ ID NO:739(TPP-18430), z. a heavy chain according to SEQ ID NO:758 and alight chain according to SEQ ID NO:759 (TPP-18432), aa. a heavychain according to SEQ ID NO:778 and a light chain according to SEQID NO:779 (TPP-18433), bb. a heavy chain according to SEQ ID NO:798and a light chain according to SEQ ID NO:799 (TPP-18436), cc. aheavy chain according to SEQ ID NO: 818 and a light chain accordingto SEQ ID NO: 819 (TPP-19571), dd. a heavy chain according to SEQID NO: 842 and a light chain according to SEQ ID NO: 843(TPP-27477), ee. a heavy chain according to SEQ ID NO:862 and alight chain according to SEQ ID NO:863 (TPP-27478), ff. a heavychain according to SEQ ID NO:882 and a light chain according to SEQID NO:883

(TPP-27479), gg. a heavy chain according to SEQ ID NO:902 and alight chain according to SEQ ID NO:903 (TPP-27480), hh. a heavychain according to SEQ ID NO:922 and a light chain according to SEQID NO:923 (TPP-29367), ii. a heavy chain according to SEQ ID NO:942and a light chain according to SEQ ID NO:943 (TPP-29368), jj. aheavy chain according to SEQ ID NO:962 and a light chain accordingto SEQ ID NO:963 (TPP-29369), kk. a heavy chain according to SEQ IDNO:211 and a light chain according to SEQ ID NO:212 (TPP-14095),ll. a heavy chain according to SEQ ID NO:225 and a light chainaccording to SEQ ID NO:226 (TPP-14099), mm. a heavy chain accordingto SEQ ID NO:239 and a light chain according to SEQ ID NO:240(TPP-15285), or nn. a heavy chain according to SEQ ID NO:253 and alight chain according to SEQ ID NO:254 (TPP-15286). 79. Theisolated antibody or antigen-binding fragment thereof according toany of embodiments 68 to 78, wherein the antibody orantigen-binding fragment is afucosylated. 80. The isolated antibodyor antigen-binding fragment thereof according to any of embodiments68 to 79, wherein the antibody induces ADCC and/or ADCP. 81. Aconjugate comprising an antibody or antigen-binding fragmentaccording to any of embodiments 68 to 80, preferably wherein theconjugate comprises a. a radioactive element, b. a cytotoxic agent,such as an auristatin, a maytansinoid, a kinesin-spindle proteininhibitor, a nicotinamide phosphoribosyltransferase inhibitor or apyrrolobenzodiazepine derivative, c. a further antibody orantigen-binding fragment, or d. a chimeric antigen receptor. 82. Apharmaceutical composition comprising an antibody orantigen-binding fragment according to any of embodiments 68 to 80or a conjugate according to embodiment 81 and optionally comprisingone or more further therapeutically active compounds, preferablyselected from a. an antibody or a small molecule targeting acheckpoint protein, such as PD1, PD-L1 or CTLA-4, b. an antibodytargeting a further chemokine receptor, such as CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3,CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c. an antibody targeting aprotein which is specifically expressed by tumor cells, d. anantibody or a small molecule targeting HER2 and/or EGFR, e. thestandard of care for any of head and neck cancer, breast cancer,gastric cancer, lung cancer, squamous cell carcinoma, esophagealtumor, melanoma, bladder cancer, liver cancer, and/or prostatecancer, f. a chemotherapeutic agent, preferably a taxane,paclitaxel, doxorubicin, cis-platin, carboplatin, oxaliplatin, orgemcitabine, and/or g. a targeted kinase inhibitor, such asSorafinib, Regorafenib, or MEKi-1. 83. An antibody orantigen-binding fragment according to any of embodiments 68 to 80or a conjugate according to embodiment 81 or a pharmaceuticalcomposition according to embodiment 82 for use as a medicament. 84.An antibody or antigen-binding fragment according to any ofembodiments 68 to 80 or a conjugate according to embodiment 81 or apharmaceutical composition according to embodiment 82 for use inthe treatment of a tumor or a disease characterized by CCR8positive cells, such as CCR8 positive tumor cells or CCR8 positiveregulatory T cells. 85. An antibody or antigen-binding fragmentaccording to any of embodiments 68 to 80 or a conjugate accordingto embodiment 81 or a pharmaceutical composition according toembodiment 82 for use in simultaneous, separate, or sequentialcombination (i) with one or more further therapeutically activecompounds, preferably selected from a. an antibody or a smallmolecule targeting a checkpoint protein, such as PD1, PD-L1 orCTLA-4, b. an antibody targeting a further chemokine receptor, suchas CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10,CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c. anantibody targeting a protein which is specifically expressed by thetumor cells, d. an antibody or a small molecule targeting HER2and/or EGFR, e. the standard of care for any of head and neckcancer, breast cancer, gastric cancer, lung cancer, squamous cellcarcinoma, esophageal tumor, melanoma, bladder c