BR122023027378A2 - USE OF AN ANTIBODY OR ANTIGEN-BINDING PORTION THEREOF THAT SPECIFICALLY BINDS TO ONE OR MORE AMINO ACIDS WITHIN THE N-TERMINAL EXTRACELLULAR DOMAIN OF HUMAN CCR8, VECTOR AND METHODS FOR REDUCE, DEPLETE OR KILL TUMOR-INFILTRATING REGULATORY T CELLS AND FOR ACTIVATE NK CELLS OR INDUCE NK CELL-MEDIATED DEATH OF TUMOR-INFILTRATING REGULATORY T CELLS - Google Patents

Abstract

The present invention relates to antibodies or antigen-binding portions thereof that specifically bind to human CCR8, polynucleotides and vectors encoding the same, and pharmaceutical compositions comprising the same. Some aspects of the invention are directed to methods of treating a disease or condition comprising administering the anti-CCR8 antibody to a subject in need.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 62/957,758, filed on January 6, 2020; 62/985,152, filed on March 4, 2020; and 63/198,803, filed November 13, 2020, each of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The content of the electronically submitted sequence listing (Name: 4416_010PC03_Seqlisting_ST25; Size: 91,925 bytes; Creation Date: January 4, 2021) filed with this application is incorporated into this document by reference in its entirety.

FIELD OF INVENTION

[0003] The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to human CCR8.

BACKGROUND OF THE INVENTION

[0004] Immunotherapy is a rapidly advancing and very promising treatment for various forms of cancer, with many recent successes. However, some patients have shown no response to current immunotherapies and others exhibit relapse after initial response.

[0005] The human immune system includes checks and balances that serve to prevent an overactive immune system from harming the body. Regulatory T cells ("Tregs") play a vital role in maintaining a functional immune system by suppressing the immune response. However, the ability of Tregs, and especially tumor-infiltrating Tregs, to dampen an immune response may block a natural immune response to a tumor.

[0006] In part, due to the essential role of immune cells, it has been very difficult to generate therapies that specifically target Tregs and, more particularly, tumor-infiltrating Tregs. Thus, there remains a need for therapies that are specifically capable of targeting and inhibiting the activity of Tregs in the tumor microenvironment.

SUMMARY OF THE INVENTION

[0007] Certain aspects of the present invention are directed to an antibody or an antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8. In some aspects, the antibody is capable of: (a) enhancing an immune response to a tumor; (b) reduce, deplete, or kill tumor-infiltrating regulatory T cells ("Treg"); (c) inducing internalization of CCR8 in tumor-infiltrating regulatory T cells ("Treg"); (d) activate NK cells; (e) inducing NK cell-mediated killing of tumor-infiltrating regulatory T cells ("Treg"); (f) bind to CCR8 of cynomolgus monkey ("cyno"); (g) bind to human CCR8 with a KD of 10 nM or less as measured by BIACORE™; or (h) any combination thereof.

[0008] In some aspects, the N-terminal extracellular domain of human CCR8 comprises the amino acid sequence set forth in SEQ ID NO: 172. In some aspects, the antibody binds to at least two, at least three, at least at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten amino acids set forth in SEQ ID NO: 172. In some aspects, the antibody binds to at least two, at least at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten contiguous amino acids set forth in SEQ ID NO: 172. In some aspects, the antibody binds to an amino acid sequence selected from SEQ ID NOs: 180-200.

[0009] In some aspects, the antibody further binds to cyno CCR8.

[0010] In some aspects, the antibody binds to human CCR8 with a KD of 10 nM or less, as measured by BIACORE™. In some aspects, the antibody binds to human CCR8 with a KD of 1 nM or less, as measured by BIACORE™.

[0011] In some aspects, the antibody induces antibody-dependent cellular cytotoxicity (ADCC) in a subject after administration of the anti-CCR8 antibody. In some aspects, the ADCC comprises an EC50 of 1 μg/mL or less after administration of the antibody or antigen-binding portion thereof. In some aspects, the ADCC comprises an EC50 of 0.1 μg/mL or less after administration of the antibody or antigen-binding portion thereof.

[0012] In some aspects, the antibody is capable of inducing the activation of NK cells. In some aspects, the antibody is capable of inducing upregulation of 4-1BB, ICAM-1, or 4-1BB and ICAM-1 on the surface of NK cells. In some aspects, the antibody is capable of inducing downregulation of CD16 on the surface of NK cells in the subject.

[0013] In some aspects, the antibody is capable of inducing NK cell-mediated killing of tumor-infiltrating Treg cells in the subject. In some aspects, the antibody induces depletion in the number of tumor-infiltrating Treg cells in a subject after administration of the antibody or antigen-binding portion thereof, relative to the number of tumor-infiltrating Treg cells before administration. In some aspects, the number of tumor-infiltrating Treg cells is depleted by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% relative to the number of tumor-infiltrating Treg cells prior to administration. In some aspects, the antibody induces internalization of CCR8 by tumor-infiltrating Treg cells.

[0014] In some aspects, the antibody comprises a variable heavy chain (VH), comprising a VH complementarity determining region (CDR) 1, a VH CDR2 and a VH CDR3; wherein the VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157 and 167.

[0015] In some aspects, the VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96 , 106, 116, 126, 136, 146, 156 and 166.

[0016] In some aspects, VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95 , 105, 115, 125, 135, 145, 155 and 165.

[0017] In some aspects, the VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 , 110, 120, 130, 140, 150, 160 and 170.

[0018] In some aspects, the VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99 , 109, 119, 129, 139, 149, 159 and 169.

[0019] In some aspects, the VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98 , 108, 118, 128, 138, 148, 158 and 168.

[0020] In some aspects, the antibody does not bind to cyno CCR8.

[0021] In some aspects, the antibody comprises a variable heavy chain (VH), comprising a VH complementarity determining region (CDR) 1, a VH CDR2 and a VH CDR3; wherein the VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137 and 147.

[0022] In some aspects, the VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136 and 146.

[0023] In some aspects, VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 45, 105, 115, 135 and 145.

[0024] In some aspects, the VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140 and 150.

[0025] In some aspects, the VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139 and 149.

[0026] In some aspects, the VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 48, 108, 118, 138 and 148.

[0027] In some aspects, the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50; (b) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 and a VL CDR3 comprising the sequence of amino acids established in SEQ ID NO: 110; (c) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 116, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 117, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 and a VL CDR3 comprising the established amino acid sequence in SEQ ID NO: 120; (d) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 135, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 136, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID No.: 137, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID No.: 138, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID No.: 139 and a VL CDR3 comprising the amino acid sequence established in SEQ ID N°: 140; or (e) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 and a VL CDR3 comprising the sequence of amino acids established in SEQ ID NO: 150.

[0028] In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 41, 101, 111, 131 and 141. In some aspects, the VL chain comprises a amino acid sequence selected from the amino acid sequence established in SEQ ID NOs: 42, 102, 112, 132 and 142.

[0029] In some aspects, the antibody comprises: (a) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 42 ; (b) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 101 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 102; (c) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 111 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 112; (d) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 131 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 132; or (e) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 141 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 142.

[0030] In some aspects, the antibody binds to human CCR8 and cyno CCR8.

[0031] In some aspects, the antibody comprises a variable heavy chain (VH), comprising a VH complementarity determining region (CDR) 1, a VH CDR2 and a VH CDR3; wherein the VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157 and 167.

[0032] In some aspects, the VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126 , 156 and 166.

[0033] In some aspects, VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125 , 155 and 165.

[0034] In some aspects, the VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130 , 160 and 170.

[0035] In some aspects, the VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129 , 159 and 169.

[0036] In some aspects, the VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98, 128 , 158 and 168.

[0037] In some aspects, the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 9 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 19 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 20; (c) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 30; (d) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 38, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 39 and a VL CDR3 comprising the amino acid sequence established in SEQ ID NO: 40; (e) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 58, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 59 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 60; (f) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 69 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 70; (g) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 78, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 79 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 80; (h) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 89 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 90; (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97, a VL CDR1 comprising the amino acid sequence established in SEQ ID NO: 98, a VL CDR2 comprising the amino acid sequence established in SEQ ID NO: 99 and a VL CDR3 comprising the sequence amino acid copy established in SEQ ID NO: 100; (j) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 125, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 127, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130; (k) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 155, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 157, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 and a VL CDR3 comprising the amino acid sequence established in SEQ ID N°: 160; or (l) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 165, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 166, a VH CDR3 comprising the amino acid sequence established in SEQ ID NO: 167, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 168, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO: 169 and a VL CDR3 comprising the sequence of amino acids established in SEQ ID NO: 170.

[0038] In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11,21,31,51,61, 71,81,91, 121 , 151 and 161. In some aspects, the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152 and 162.

[0039] In some aspects, the antibody comprises: (a) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 2 ; (b) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 11 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 12; (c) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 22; (d) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 31 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 32; (e) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 51 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 52; (f) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 62; (g) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 71 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 72; (h) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 82; (i) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 91 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 92; (j) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 122; (k) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 151 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 152; or (l) a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 162.

[0040] In some aspects, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some aspects, the antibody comprises a single chain variable fragment (scFv) of an antibody.

[0041] In some aspects, the antibody or antigen-binding portion thereof is afucosylated.

[0042] In some aspects, the antibody is a bispecific antibody, a bispecific T cell engager (BiTE), a multispecific antibody, a biparatopic antibody, an immunoconjugate, an antibody-drug conjugate or any combination thereof.

[0043] Certain aspects of the present invention are directed to a bispecific antibody comprising an antibody or an antigen-binding portion thereof disclosed herein.

[0044] Certain aspects of the present invention are directed to a BiTe comprising an antibody or an antigen-binding portion thereof disclosed herein.

[0045] Certain aspects of the present invention are directed to a multispecific antibody comprising an antibody or an antigen-binding portion thereof disclosed herein.

[0046] Certain aspects of the present invention are directed to a biparatopic antibody comprising an antibody or an antigen-binding portion thereof disclosed herein.

[0047] In some aspects, the bispecific antibody, BiTe, multispecific antibody or biparatopic antibody comprises a first VH domain, comprising a first VH CDR1, a first VH CDR2 and a first VH CDR3; a first VL domain, comprising a first VL CDR1, a first VL CDR2 and a first VL CDR3; a second VH domain, comprising a second VH CDR1, a second VH CDR2 and a second VH CDR3; and a second VL domain, comprising a second VL CDR1, a second VL CDR2 and a second VL CDR3; wherein (a) the first VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 45, 105, 115, 135 and 145; (b) the first VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136 and 146; and (c) the first VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137 and 147.

[0048] In some aspects, (a) the first VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 48, 108, 118, 138 and 148; (b) the first VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139 and 149; and (c) the first VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140 and 150. In some aspects, (a) the second VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125, 155 and 165; (b) the second VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156 and 166; and (c) the second VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157 and 167. In some aspects, (a) the second VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98 , 128, 158 and 168; (b) the second VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159 and 169 ; and (c) the second VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160 and 170.

[0049] Certain aspects of the present invention are directed to an immunoconjugate comprising an antibody or antigen-binding portion thereof disclosed herein. In some aspects, the immunoconjugate is an antibody-drug conjugate.

[0050] Certain aspects of the present invention are directed to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding portion thereof disclosed herein.

[0051] Certain aspects of the present invention are directed to a T cell receptor (TCR) comprising an antibody or antigen-binding portion thereof disclosed herein.

[0052] Certain aspects of the present invention are directed to a nucleic acid molecule or a set of nucleic acid molecules that encode the antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein document, a multispecific antibody disclosed in this document, a biparatopic antibody disclosed in this document, an immunoconjugate disclosed in this document, a CAR disclosed in this document or a TCR disclosed in this document.

[0053] Certain aspects of the present invention are directed to a vector or a set of vectors comprising a nucleic acid molecule or a set of nucleic acid molecules disclosed herein.

[0054] Certain aspects of the present invention are directed to a cell comprising a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein, or a vector or a set of vectors disclosed in this document. In some respects, the cell is a host cell. In some aspects, the cell is an immune cell. In some aspects, the cell is a T cell.

[0055] Certain aspects of the present invention are directed to a pharmaceutical composition comprising an antibody or an antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein -ment, a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein document, a vector or set of vectors disclosed in this document, or a cell disclosed in this document and a pharmaceutically acceptable carrier.

[0056] Certain aspects of the present invention are directed to a method of treating a tumor in a subject in need, comprising administering to the subject an antibody or an antigen-binding portion thereof disclosed herein, a bi-specific antibody disclosed herein , a bispecific antibody disclosed in this document, a BiTE disclosed in this document, a multispecific antibody disclosed in this document, a biparatopic antibody disclosed in this document, an immunoconjugate disclosed in this document, a CAR disclosed in this document, a TCR disclosed in this document , a nucleic acid molecule or a set of nucleic acid molecules disclosed in this document, a vector or a set of vectors disclosed in this document, a cell disclosed in this document or a pharmaceutical composition disclosed in this document.

[0057] Certain aspects of the present invention are directed to a method of reducing, depleting or killing tumor-infiltrating regulatory T cells ("Treg"), comprising contacting Treg cells with an antibody or antigen-binding moiety disclosed herein. document, a bispecific antibody disclosed in this document, a bispecific antibody disclosed in this document, a BiTE disclosed in this document, a multispecific antibody disclosed in this document, a biparatopic antibody disclosed in this document, an immunoconjugate disclosed in this document, a CAR disclosed in this document herein, a TCR disclosed herein, a nucleic acid molecule or set of nucleic acid molecules disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed in this document.

[0058] Certain aspects of the present invention are directed to a method of activating NK cells or inducing NK cell-mediated killing of tumor-infiltrating regulatory T cells ("Treg"), comprising contacting Treg cells with an antibody or an antigen-binding moiety disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein , an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein, a vector or a set of vectors disclosed herein, a cell -claimed herein or a pharmaceutical composition disclosed herein.

[0059] In some aspects, the contact is in vitro or ex vivo. In some aspects, the contact is in vivo.

[0060] In some aspects, the antibody or antigen-binding portion thereof induces activation of NK cells. In some aspects, the antibody or antigen-binding portion thereof induces upregulation of 4-1BB, ICAM-1 or 4-1BB and ICAM-1 on the surface of NK cells.

[0061] In some aspects, the antibody or antigen-binding portion thereof induces downregulation of CD16 on the surface of NK cells. In some aspects, the antibody or antigen-binding portion thereof induces NK cell-mediated killing of tumor-infiltrating Treg cells. In some aspects, the antibody or antigen-binding portion thereof is depleted, the number of tumor-infiltrating Treg cells relative to the number of tumor-infiltrating Treg cells in the absence of the antibody or antigen-binding portion thereof. In some aspects, the antibody or antigen-binding portion thereof is depleted, the number of tumor-infiltrating Treg cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% relative to the number of tumor-infiltrating Treg cells in the absence of the antibody or antigen-binding portion thereof. In some aspects, the antibody or antigen-binding portion thereof induces internalization of CCR8 by tumor-infiltrating Treg cells.

[0062] In some aspects, the tumor is selected from the group consisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts, promyelocyte erythroleukemia, myelomonocytic monocytic leukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, lymphocytic leukemia chronic, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, Lymphoma Polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease , solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteoarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma , colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma , papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma , oligodendro glioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; cancer of the oral cavity (e.g. lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer, or any combination of these. In some aspects, the tumor is refractory or recurrent. In some aspects, the tumor is advanced, locally advanced, or metastatic.

[0063] In some aspects, the method further comprises administering an additional anti-cancer agent. In some aspects, the additional anticancer agent is selected from a small molecule, a polypeptide, a radiation therapy, a surgery, and a combination thereof. In some aspects, the additional anticancer agent comprises a chemotherapy. In some aspects, the chemotherapy comprises a platinum-based chemotherapy. In some aspects, the additional anti-cancer agent comprises a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TI-GIT inhibitor, a CD112R inhibitor. , a TAM inhibitor, a STING agonist, a 4-1BB agonist, a CCL22 inhibitor, an agent that induces NK cell activation, or a combination thereof. In some aspects, the additional anticancer agent comprises a PD-1 antagonist. In some aspects, the PD-1 antagonist is selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224. In some aspects, the additional anticancer agent comprises a PD-L1 inhibitor. In some aspects, the PD-L1 inhibitor is selected from the group consisting of FAZ053, Atezolizumab, Avelumab, Durvalumab and BMS-936559. In some aspects, the additional anticancer agent comprises an anticancer agent selected from the group consisting of Sunitinib (SUTENT®), Cabozantinib (CABO- METYX®), Axitinib (INLYTA®), Lenvatinib (LENVIMA®), Everolimus (A- FINITOR® ), Bevacizumab (AVASTIN®), epacadostat, NKTR-214 (CD-122 biased agonist), Tivozanib (FOTIVDA®), abexinostat, Ipilimumab (YERVOY®), tremelimumab, Pazopanib (VOTRIENT®), So - rafenib (NEXAVAR®), Temsirolimus (TORISEL®), Ramucirumab (CYRAMZA®), niraparib, savolitinib, vorolanib (X-82), Regorafenib (STIVARGO®), Donafenib (multikinase inhibitor), Camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), Ramucirumab (CYRAMZA®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), Tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate, nintedanib, lirilumab, Nivolumab (OPDIVO® ), Pembrolizumab (KEYTRUDA®), Atezolizumab (TECENTRIQ®), Avelumab (BAVENCIO®), Durvalumab (IMFIMZI®), Cemiplimab-rwlc (LIBTAYO®), tislelizumab, spartalizumab and any combination of these. In some aspects, the additional anticancer agent comprises a TIM-3 inhibitor. In some aspects, the TIM-3 inhibitor is MGB453 or TSR-022. In some aspects, the additional anticancer agent comprises a LAG-3 inhibitor. In some aspects, the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033. In some aspects, the additional anticancer agent comprises a TIGIT inhibitor. In some aspects, the additional anticancer agent comprises a CD112R inhibitor. In some aspects, the additional anticancer agent comprises a TAM inhibitor (Axl, Mer, Tyro). In some aspects, the additional anticancer agent comprises a 4-1BB agonist. In some aspects, the additional anticancer agent comprises a Tyrosine Kinase Inhibitor (TKI). In some aspects, the additional anticancer agent comprises an agent that induces activation of NK cells and therefore increases ADCC activity. In some aspects, the additional anticancer agent comprises a CCL2 inhibitor. In some aspects, the additional anticancer agent comprises an agent that induces activation of NK cells.

[0064] In some aspects, the antibody or antigen-binding portion thereof is administered before the additional anticancer agent. In some aspects, the antibody or antigen-binding portion thereof is administered after the additional anticancer agent. In some aspects, the antibody or antigen-binding portion thereof and the additional anticancer agent are co-administered.

[0065] Certain aspects of the present invention are directed to a method of producing an antibody or an antigen-binding portion thereof, comprising culturing a cell disclosed herein under suitable conditions. In some aspects, the method further comprises isolating the antibody or antigen-binding portion thereof.

[0066] In some aspects, the antibody or antigen-binding portion thereof is afucosylated.

[0067] Certain aspects of the present invention are directed to a use of an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein , a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules. cleico disclosed in this document, a vector or set of vectors disclosed in this document, a cell disclosed in this document or a pharmaceutical composition disclosed in this document for the manufacture of a medicament in the treatment of a tumor in a subject in need.

[0068] Certain aspects of the present invention are directed to a use of an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein , a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules. cleico disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein for the manufacture of a medicament for reducing, depleting, or killing tumor-infiltrating Treg cells in a subject. in need.

[0069] Certain aspects of the present invention are directed to a use of an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein , a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules. cleico disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein for the manufacture of a medicament in activating NK cells or inducing NK cell-mediated death of cells Tumor-infiltrating Treg in a subject in need.

[0070] Certain aspects of the present invention are directed to an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein -ment, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein, a vector or a set of vectors disclosed in this document, a cell disclosed in this document or a pharmaceutical composition disclosed in this document for use in a method of treating a tumor in a subject in need.

[0071] Certain aspects of the present invention are directed to an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein -ment, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein, a vector or a set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein for use in a method of reducing, depleting, or killing tumor-infiltrating Treg cells in a subject in need. .

[0072] Certain aspects of the present invention are directed to an antibody or antigen-binding portion thereof disclosed herein, a bispecific antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein -ment, a biparatopic antibody disclosed herein, an immunoconjugate disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule or a set of nucleic acid molecules disclosed herein, a vector or a set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein for use in activating NK cells or inducing NK cell-mediated killing of tumor-infiltrating Treg cells in a subject in need. BRIEF DESCRIPTION OF FIGURES

[0073] FIGS. 1A-1L are graphical representations of binding to human CCR8 (HuCCR8-ECD-Fc) or cyno CCR8 (CyCCR8-ECD-Fc) as measured by fluorescent absorbance (FIGS. 1A, 1C, 1E, 1G, 1I and 1K) and relative binding along a secondary antibody to 293T cells expressing human CCR8, cyno CCR8, human CCR2, mouse CCR8, and negative control 293T cells (as indicated; FIGs. 1B, 1D, 1F, 1H, 1J and 1L) illustrating the binding of anti-CCR8-1 (FIGS. 1A-1B), anti-CCR8-1-1 (FIGS. 1C-1D), anti-CCR8-1-2 (FIGS. 1E-1F), anti-CCR8-1-3 (FIGS. 1G-1H), anti-CCR8-1-4 (FIGS. 1I-1J) and anti-CCR8-1-5 antibodies (FIGS. 1K-1L).

[0074] FIGS. 2A-2R are graphical representations of binding to human CCR8 (HuCCR8-ECD-Fc) or cyno CCR8 (CyCCR8-ECD-Fc) as measured by fluorescent absorbance (FIGS. 2A, 2C, 2E, 2G, 2I, 2K, 2M , 2O, 2Q, 2S, and 2U) and the relative binding along a secondary antibody to 293T cells expressing human CCR8, cyno CCR8, human CCR2, mouse CCR8, and negative control 293T cells (as indicated; FIGs. 2B , 2D, 2F, 2H, 2J, 2L, 2N, 2P, 2R, 2T and 2V) illustrating the binding of anti-CCR8-2 (FIGS. 2A-2B), anti-CCR8 -2-1 (FIGS. 2C- 2D), anti-CCR8-2-2 (FIGS. 2E-2F), anti-CCR8-2-3 (FIGS. 2G-2H), anti-CCR8-2-4 (FIGS. 2I-2J), anti- CCR8-2-5 (FIGS. 2K-2L), anti-CCR8-2-6 (FIGS. 2M-2N), anti-CCR8-2-7 (FIGS. 2O-2P), anti-CCR8-2-8 (FIGS. 2Q-2R), anti-CCR8-2-9 (FIGS. 2S-2T) and anti-CCR8-2-10 antibodies (FIGS. 2U-2V).

[0075] FIGS. 3A-3B are bar graphs illustrating the in-cell affinity (KD) for anti-CCR8-1 antibody (FIG. 3A) or anti-CCR8-2 antibody (FIG. 3B) for human CCR8, cyno CCR8 and several negative controls (amyloid precursor-like protein 2 (APLP2), alpha-antichymotrypsin 1 (SERPINA3), solute carrier family member 9 (SLC6A9) and phospholipid phosphatase 3 (PLPP3)).

[0076] FIG. 4A is a graphical representation showing the binding of anti-CCR8-1 and anti-CCR8-2 antibodies to isolated tumor-infiltrating leukocytes (TILs) from breast and kidney, square and triangle tumor samples, respectively. FIG. 4B is a graphical representation showing the relative binding of an isotype control, anti-CCR8-1, anti-CCR8-2, and a positive control anti-CCR8 antibody to isolated regulatory T cells (Treg cells) from samples of renal cell carcinoma, as indicated.

[0077] FIG. 5 is a bar graph illustrating ADCC signaling in 293T cells with forced expression of human or cynomolgus monkey (cyno) CCR8 after contact with anti-CCR8-1 and anti-CCR8-2 antibodies, as indicated.

[0078] FIGS. 6A-6B are bar graphs illustrating ADCC as evidenced by the percentage of 293T cells, expressing human CCR8 (FIG. 6A) or cyno CCR8 (FIG. 6B), remaining after contact with an anti-CCR8-1 or anti-CCR8 antibody. -CCR8-2, as indicated, in samples from two donors (D1 and D2).

[0079] FIGS. 7A-7B are line graphs illustrating the percentage of dead target Raji cells expressing human CCR8 (FIG. 7A) or empty vector (EV; FIG. 7B), following contact with an anti-CCR8-1 or anti-CCR8 antibody. -CCR8-2 as indicated.

[0080] FIG. 8A is a line graph illustrating the percentage of 4-1BB+/CD16- cells (NK cells) in culture with Raji cells forced to express CCR8, after contacting the culture with an anti-CCR8-1 or anti-CCR8 antibody. CCR8-2 as indicated. FIG. 8B is a bar graph illustrating the number of Raji cells expressing the CCR8 surface target (black bars) following exposure to anti-CCR8-1 antibody or anti-CCR8-2 antibody relative to an isotype control. . FIG. 8C is a bar graph illustrating the expression level of 4-1BB (relative to isotype controls) in CD3-NKp46+ NK cells in culture with Raji cells forced to express CCR8 (left bars). Control Raji cells that do not express the CCR8 surface target are represented by right bars (FIGS. 8B-8C).

[0081] FIG. 9 is a graphical representation of the relative number (%) of FOXP3+ cells of total CD3+/CD4+ TILs isolated from freshly resected human tumors and incubated with an anti-CCR8-1 or anti-CCR8-2 antibody, as indicated.

[0082] FIG. 10 is a bar graph illustrating CCR8 internalization in 293T cells with forced expression of human CCR8 or empty vector.

[0083] FIG. 11 is a line graph illustrating competitive binding of a monoclonal antibody that binds to human CCR8 (purchased from BIOLEGEND®; catalog number 360603) and the anti-CCR8-1 antibody or the anti-CCR8-1 antibody. 2 as measured by flow cytometry.

[0084] FIGS. 12A-12B are line graphs showing ADCC activity in relation to antibody concentration in Jurkat ADCC CD16VV (FIG. 12A) or CD16FF (FIG. 12B) reporter cells following incubation with increasing concentrations of the anti-wild-type antibody. -CCR8-1, an afucosylated anti-CCR8-1 antibody, the wild-type anti-CCR8-2 antibody, an afucosylated anti-CCR8-2 antibody, and an isotype control as indicated.

[0085] FIGS. 13A-13B are graphical representations illustrating anti-CCR8-1 antibody binding to tumor Tregs. Tregs were identified using flow cytometry in TIL isolated from tumors as CD3+/FoxP3+. Anti-CCR8-1 antibody binding was measured on blocked kidney and breast tumor cells using an APC-conjugated secondary antibody. FIG. 13A is a scatter plot showing the antibody to positive control binding ratio on Treg in contact with the anti-CCR8-1 antibody, a positive control and only secondary (negative control). FIG. 13B shows two histogram plots representing APC-A staining in gated Tregs from human kidney tumors with frequency normalized to the mode for an IgG1 isotype control (gray trace) or anti-CCR8-1 (black trace). FIG. 13C is a scatterplot showing the percentage of CD3+ TIL cells isolated from tumors incubated with allogeneic natural killer (NK) cells and contacted with control antibody, anti-CCR8-1 antibody, or a positive control antibody. . For each antibody condition, the cluster on the left is the percentage of CD3+ cells that are FoxP3- cells (e.g., not Tregs), while the cluster on the right is the normalized percentage of CD3+ cells that are FoxP3+ cells (e.g., non-Tregs). example, Tregs). DETAILED DESCRIPTION OF THE INVENTION

[0086] Certain aspects of the present invention are directed to antibodies or antigen-binding portions thereof that specifically bind to CCR8 ("anti-CCR8 antibody"). In certain aspects, the anti-CCR8 antibody specifically binds to the N-terminal extracellular domain of human CCR8. Other aspects of the present invention are directed to methods of treating a subject in need, comprising administering the anti-CCR8 antibodies disclosed herein.

I. Terms

[0087] So that the present invention can be more readily fulfilled, certain terms are defined first. As used in this order, unless expressly provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the order.

[0088] It should be noted that the term "one" entity refers to one or more of these entities; for example, "a nucleotide sequence" is meant to represent one or more nucleotide sequences. As such, the terms "a" (or "one"), "one or more" and "at least one" may be used interchangeably in this document.

[0089] Additionally, "and/or", when used herein, should be considered as a specific invention of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A" (per se alone) and "B" (by itself). Likewise, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (by itself); B (by itself); and C (by itself).

[0090] The term "about" is used in this document to mean approximately, more or less, around or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range, extending the limits above and below the established numerical values. In general, the term "about" is used in this document to modify a numerical value above and below the established value by a 10 percent variance, up or down (greater or smaller).

[0091] It is understood that wherever aspects are described in this document with the language "comprising", otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one skilled in the art with a general dictionary of many of the terms used in this invention.

[0093] Units, prefixes and symbols are denoted in their form accepted by the Système International de Unites (International System of Units - SI). Numeric ranges include the numbers that define the range. Unless otherwise noted, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in the amino to carboxy orientation. The titles provided herein are not limitations on the various aspects of the invention, which may be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0094] As used herein, the term "amount" or "level" is used in the broadest sense and refers to an amount, concentration, or abundance of a substance (e.g., a metabolite, a small molecule, a protein, an mRNA, a pain marker). When referring to a metabolite or small molecule (e.g., a drug), the terms "amount", "level" and "concentration" are generally used interchangeably and generally refer to a detectable amount in a sample. biological. "Elevated levels" or "enhanced levels" refer to an increase in the amount, concentration, or abundance of a substance in a sample relative to a control sample, such as from an individual or individuals who do not suffer from the disease or disorder. (e.g. cancer) or an internal control. In some embodiments, the elevated level of a substance (e.g., a drug) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% in relation to the amount of the substance in a control sample, as determined by techniques known in the art (for example, HPLC). "Decreased levels" refers to a decrease in the amount, concentration, or abundance of a substance (e.g., a drug) in an individual relative to a control, such as an individual or individuals who do not suffer from the disease or disorder (e.g., cancer) or an internal control. In some aspects, a reduced level is little or no detectable quantity, concentration or abundance. In some aspects, the reduced level of a substance (e.g., a drug) in a sample refers to a decrease in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., HPLC).

[0095] When referring to a protein, mRNA or a marker, such as those described herein, the terms "expression level" or "expression level" are generally used interchangeably and generally refer to an amount detectable of a protein, mRNA, or marker in a biological sample. In some aspects, a detectable amount or detectable level of a protein, mRNA or a marker is associated with a probability of response to an agent, such as those described herein. "Expression" generally refers to the process by which the information contained in a gene is converted into structures (e.g., a protein marker, such as PD-L1) present and operating in the cell. Therefore, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modification of a polypeptide). Fragments of the transcribed polynucleotide, translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., post-translational modification of a polypeptide) should also be considered expressed if they originate from a transcript generated by alternative splicing or a degraded transcript, or of post-translational processing of the polypeptide, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide such as mRNA and then translated into a polypeptide and also those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNAs). "High expression", "high expression levels" or "elevated levels" refers to increased expression or increased levels of a substance within a sample relative to a control sample, such as an individual or individuals who do not suffer from the disease or disorder (e.g. cancer) or an internal control. In some aspects, elevated expression of a substance (e.g., a protein marker such as PD-L1) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20 %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS). "Reduced expression", "reduced expression levels" or "decreased levels" refers to a decreased expression or decreased levels of a substance (e.g., a protein marker) in an individual relative to a control, as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some respects, reduced expression is little or no expression. In some aspects, reduced expression of a substance (e.g., a protein marker) in a sample refers to a reduction in the amount of the substance of about 5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS).

[0096] As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits or neutralizes a biological activity of a native polypeptide disclosed in this document. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides or proteins. In some aspects, inhibition in the presence of the antagonist is observed in a dose-dependent manner. In some aspects, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25% , at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about at least about 100% lower than the signal measured with a negative control under comparable conditions. Also disclosed herein are methods for identifying suitable antagonists for use in the methods of the invention. For example, such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), ForteBio® systems, radioimmunoassay (RIA), Meso Scale discovery assay (e.g., Meso Scale Discovery Electrochemiluminescence (MSD-ECL) and bead-based Luminex® assay These assays determine the ability of an antagonist to bind the polypeptide of interest (e.g., a receptor or ligand) and therefore indicate the ability of the antagonist. antagonist to inhibit, neutralize or block the activity of the polypeptide. The effectiveness of an antagonist can also be determined using functional assays, such as the ability of an antagonist to inhibit the function of the polypeptide or an agonist. a functional assay may comprise contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The potency of an agonist is generally defined by its IC50 value (required concentration. to inhibit 50% of the agonist response). The lower the IC50 value, the greater the potency of the antagonist and the lower the concentration required to inhibit the maximum biological response.

[0097] As used herein, the term "anti-CCR8 antibody" refers to an antibody that specifically binds to CCR8. In some aspects, the anti-CCR8 antibody inhibits a biological activity of CCR8 and/or a downstream pathway(s) mediated by CCR8 signaling or another CCR8-mediated function. An anti-CCR8 antibody includes, but is not limited to, antibodies that block, antagonize, suppress, inhibit, or reduce a biological activity of CCR8 (e.g., ligand binding, activation of G protein signaling), including pathways to downstream mediated signaling or function of CCR8, such as binding to the receptor and/or triggering a cellular response to CCR8 or its metabolites (e.g., immunosuppression). In some aspects, an anti-CCR8 antibody provided by the invention binds to human CCR8 and prevents, blocks or inhibits the binding of human CCR8 to a ligand (e.g., CCL1) or interaction between CCR8 and G protein. anti-CCR8 antibody prevents, blocks or inhibits the binding of human CCR8 to CCL1. In some aspects, the anti-CCR8 antibody prevents, blocks or inhibits the binding of human CCR8 to CCL8. In some aspects, the anti-CCR8 antibody prevents, blocks or inhibits the binding of human CCR8 to CCL16. In some aspects, the anti-CCR8 antibody prevents, blocks or inhibits the binding of human CCR8 to CCL18.

[0098] As used herein, the term "antibody" refers to an entire antibody, which comprises two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody and a fully human antibody. The antibody may be produced or derived from any of a variety of species, for example, mammals such as humans, non-human primates (e.g., orangutans, baboons or chimpanzees), horses, cattle, pigs, sheep, goats , dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody may be a purified or recombinant antibody. As used herein, the term "antibody fragment", "antigen-binding fragment" or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., CCR8) and inhibits the activity of the target antigen. Such fragments include, for example, a single-chain antibody, a single-chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab' fragment or an F(ab')2 fragment. A scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, for example, Todorovska et al., (2001) J. Immunol. Methods 248(1):47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1):177-189; Poljak, (1994) Structure 2(12):1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol. 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety.

[0099] As used herein, the term "antibody fragment" also includes, for example, single-domain antibodies, such as camelized single-domain antibodies. See, for example, Muyldermans et al., (2001) Trends Biochem. Sci. 26:230-235; Nuttall et al., (2000) Curr. Pharm. Biotech. 1:253-263; Reichmann et al., (1999) J. Immunol. Met. 231:25-38; publication of PCT order nos. WO 94/04678 and WO 94/25591; and US Patent No. 6,005,079, all of which are incorporated herein by reference in their entirety. In some aspects, the invention provides single-domain antibodies comprising two VH domains with modifications such that single-domain antibodies are formed.

[0100] In some aspects, an antigen-binding fragment includes the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some aspects, an antigen-binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.

[0101] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody with two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment ligation. See, for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

[0102] Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chain/light chain pairs have different specificities (Milstein and Cuello , (1983) Nature 305:537-539). Antibody variable domains with desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy chain constant domain, including at least part of the CH2 and CH3 hinge regions. For details of currently known illustrative methods for generating bispecific antibodies, see, for example, Suresh et al., (1986) Methods Enzymol. 121:210; PCT Publication No. WO 96/27011; Brennan et al., (1985) Science 229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et al., (1992) J. Immunol. 148(5):1547- 1553; Hollinger et al., (1993) Proc. Natl. Academic. Sci. USA 90:6444-6448; Gruber et al., (1994) J. Immunol. 152:5368; and Tutt et al., (1991) J. Immu nol. 147:60. Bispecific antibodies also include cross-linked or heteroconjugated antibodies. Heteroconjugate antibodies can be produced using any convenient cross-linking methods. Suitable crosslinking agents are well known in the art and are disclosed in US Patent No. 4,676,980, along with various crosslinking techniques.

[0103] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptides of the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provided an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy to produce bispecific antibody fragments through the use of single-chain Fv(scFv) dimers has also been reported. See, for example, Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies may be "linear antibodies" as described in, for example, Zapata et al. (1995) Protein Eng 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0104] Antibodies with more than two valences (e.g., trispecific antibodies) are contemplated and described in, for example, Tutt et al. (1991) J Immunol 147:60.

[0105] As used herein, the term "biparatopic" refers to an antibody that is capable of binding to two epitopes on a single antigen, e.g., polypeptide, target. In some aspects, the biparatopic antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to a first epitope and the second antigen-binding region binds to a second epitope on the same antigen.

[0106] The invention also encompasses variant forms of multispecific antibodies, such as the double variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25 (11): 1290-1297. DVD-Ig molecules are designed so that two different light chain variable domains (VL) from two antibodies of different origin are linked in sequence (in tandem) directly or through a short linker, using techniques of Recombinant DNA, followed by the light chain constant domain. Likewise, the heavy chain comprises two different heavy chain (VH) variable domains linked in tandem, followed by the CH1 constant domain and Fc region. Methods for producing DVD-Ig molecules from two parent antibodies are further described, for example, in PCT publications No. WO 08/024188 and WO 07/024715. In some aspects, the bispecific antibody is a Fabs-in-Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a full-length antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO 2015/103072.

[0107] As used herein, "cancer antigen" or "tumor antigen" refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells that express tumor-specific antigens, (iv ) cells expressing tumor-associated antigens, (v) embryonic antigens in tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix ) growth factor receptors, (x) growth factor ligands and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.

[0108] As used herein, the term "cancer-specific immune response" refers to the immune response induced by the presence of tumors, cancer cells or cancer antigens. In certain aspects, the response includes the proliferation of cancer antigen-specific lymphocytes. In certain aspects, the response includes expression and upregulation of antibodies and T cell receptors and the formation and release of lymphokines, chemokines and cytokines. Both the innate and acquired immune systems interact to initiate antigenic responses against tumors, cancer cells, or cancer antigens. In certain respects, the cancer-specific immune response is a T cell response.

[0109] The term "carcinoma" is recognized in the art and refers to malignancies of epithelial or endocrine tissues, including carcinomas of the respiratory system, carcinomas of the gastrointestinal system, carcinomas of the genitourinary system, testicular carcinomas, breast carcinomas, prostatic carcinomas , carcinomas of the endocrine system and melanomas. The anti-CCR8 antibodies described herein can be used to treat patients who have, are suspected of having, or who may be at high risk of developing any type of cancer, including renal carcinoma or melanoma, or any viral disease. Exemplary carcinomas include those formed from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcino-sarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which tumor cells form recognizable glandular structures.

[0110] As used herein, the term "CCR8" or "CC chemokine receptor type 8" refers to a G protein-coupled receptor. CCR8 is known to have at least four ligands: CCL1, CCL8, CCL16 and CCL18. CCL1 is believed to potentiate human Treg cells by inducing the expression of CCR8, FOXp3, CD39, Granzyme B and IL-10, in a STAT3-dependent manner. See, for example, Barsheshet et al., PNAS 114(23): 6086-91 (6 June 2017). CCR8 is expressed mainly in Treg cells and, to a lesser extent, in small fractions of TH2 cells, monocytic cells, NK cells and CD8+ cells. CCR8 is a transmembrane receptor with seven transmembrane domains, an extracellular N-terminal domain (SEQ ID NO: 172) and an intracellular C-terminal domain, which interacts with the G protein. The amino acid sequence for human CCR8 (UniProt P51685; SEQ ID NO: 171) is shown in Table 1, below. Table 1: Human CCR8 sequence. The N-terminal extracellular domain (SEQ ID NO: 172) is underlined.

[0111] As used herein, the term "compete", when used in the context of antigen-binding proteins (e.g., immunoglobulins, antibodies, antigen-binding fragments thereof) that compete for binding to the same epitope, refers to is an interaction between antigen-binding proteins as determined by an assay (e.g., a competitive binding assay; a cross-blocking assay), in which a test antigen-binding protein (e.g., a test antibody) inhibits (e.g., reduces or blocks) the specific binding of a reference antigen-binding protein (e.g., a reference antibody) to a common antigen (e.g., CCR8 or a fragment thereof).

[0112] A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence that is derived from a specific sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable by one of ordinary skill in the art, as having its origin in the sequence. Polypeptides derived from another peptide may have one or more mutations with respect to the initial polypeptide, for example, one or more amino acid residues that have been replaced by another amino acid residue or that have one or more insertions or deletions of amino acid residues.

[0113] A polypeptide may comprise an amino acid sequence that does not occur naturally. Such variants necessarily have less than 100% sequence identity or similarity to the initial molecule. In certain aspects, the variant will have an amino acid sequence of about 75% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide, more preferably about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and more preferably from about 95% to less than 100%, for example, along the length of the variant molecule.

[0114] In certain aspects, the antibodies of the invention are encoded by a nucleotide sequence. The nucleotide sequences of the invention may be useful for various applications, including cloning, gene therapy, protein expression and purification, introduction of mutation, DNA vaccination of a host in need thereof, generation of antibodies for, for example, immunization. passive, PCR, primer and probe generation and the like.

[0115] It will also be understood by one of ordinary skill in the art that antibodies suitable for use in the methods disclosed herein may be altered so that they vary in sequence from the naturally occurring native sequences from which they were derived, while maintaining the desirable activity of native sequences. For example, nucleotide or amino acid substitutions may be made that lead to conservative substitutions or changes to "non-essential" amino acid residues. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0116] Antibodies suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions in one or more amino acid residues, for example, in essential or non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta side chains -branched (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in a linker polypeptide is preferentially replaced by another amino acid residue from the same side chain family. In certain aspects, a string of amino acids can be replaced by a structurally similar string that differs in the order and/or composition of side chain family members. Alternatively, in certain aspects, mutations may be introduced randomly throughout all or part of a coding sequence, such as saturation mutagenesis, and the resulting mutants may be incorporated into the binding polypeptides of the invention and screened for their ability to bind to the desired target.

[0117] As used herein, the term "cross-reaction" refers to the ability of an antibody of the invention to bind to CCR8 from a different species. For example, an antibody of the present invention that binds to human CCR8 can also bind to another species of CCR8. As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or functionally interacting with, cells that physiologically express CCR8. Methods for determining cross-reactivity include standard binding assays as described in this document, for example, by BIACORETM surface plasmon resonance (SPR) analysis using a BIACORETM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry techniques.

[0118] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mediated mainly by CD8+ T cells.

[0119] As used herein, the term "EC50" refers to the concentration of an antibody, or an antigen-binding portion thereof, that induces a response, whether in an in vitro or in vivo assay, that is 50% of the maximum response, that is, halfway between the maximum response and the initial assessment.

[0120] As used herein, the term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Effective values for this use will depend on the severity of the disorder treated and the general state of the patient's own immune system.

[0121] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. The term "epitope mapping" refers to a process or method of identifying the binding site, or epitope, of an antibody, or antigen-binding fragment thereof, on its target protein antigen. Epitope mapping methods and techniques are provided herein. Epitopes can be formed by both contiguous amino acids and non-contiguous amino acids juxtaposed by a tertiary fold of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by a tertiary fold are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which contiguous or overlapping CCR8 peptides are tested for reactivity with the provided anti-CCR8 antibody. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology , Vol. 66, G. E. Morris, Ed. (1996)).

[0122] Also encompassed by the present invention are antibodies that bind to an epitope in CCR8 that comprises all or a portion of an epitope recognized by the specific antibodies described herein (e.g., the same or an overlapping region or a region between or encompassing the region).

[0123] Also covered by the present invention are antibodies that bind to the same epitope and/or antibodies that compete for binding to human CCR8 with the antibodies described in this document. Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of an antibody to block the binding of another antibody to a target antigen, that is, a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as CCR8. Numerous types of competitive binding assays are known, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Me -thods in Enzymology 9:242 (1983)); Solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); direct solid phase labeled assay, direct solid phase labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); direct solid phase RIA label using I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); Solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and RIA directly marked. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, this assay involves the use of purified antigen bound to a solid surface or cells containing one of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of marker bound to the surface or solid cells in the presence of the test immunoglobulin. Generally, the test immunoglobulin is present in excess. Generally, when a competing antibody is present in excess, it inhibits the specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75 % or more.

[0124] Other techniques include, for example, epitope mapping methods such as x-ray analyzes of antigen crystals: antibody complexes that provide atomic resolution of the epitope and mass spectrometry combined with hydrogen/deuterium exchange ( H/D) that studies the conformation and dynamics of antigen:antibody interactions. Other methods monitor antibody binding to antigen fragments or mutated variations of the antigen, where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. . Furthermore, computational combinatorial methods for epitope mapping can also be used. These methods depend on the ability of the antibody of interest to affinity isolate specific short peptides from expression peptide libraries in combinatorial phages. The peptides are then considered drivers for defining the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that demonstrate the mapping of discontinuous conformational epitopes.

[0125] As used herein, the term "Fc-mediated effector functions" or "Fc effector functions" refers to the debiological activities of an antibody other than the primary function and purpose of the antibody. For example, the effector functions of an agnostic therapeutic antibody are biological activities other than activation of the target protein or pathway. Examples of antibody effect functions include binding to C1q and complement-dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g. B cell receptor); lack of activation of platelets expressing the Fc receptor; and B cell activation. Many effector functions begin with Fc binding to an Fc receptor. In some aspects, the antibody directed to the tumor antigen has effector function, for example, ADCC activity. In some aspects, an antibody targeting the tumor antigen described herein comprises a variant constant region with increased effector function (e.g., increased ability to mediate ADCC) relative to the unmodified form of the constant region.

[0126] As used herein, the term "Fc receptor" refers to a polypeptide found on the surface of immune effector cells, which is bound by the Fc region of an antibody. In some aspects, the Fc receptor is an FCY receptor. There are three subclasses of FCY receptors, FCYRI(CD64), FCYRII(CD32) and FYCRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind to and activate Fc, FcyRI, FcyRIIA, and FcyRIIIA receptors. FcyRIIB is an inhibitory receptor and, therefore, antibody binding to this receptor does not activate complement and cellular responses. FcyRI is a high-affinity receptor that binds to IgG in monomeric form, while FcyRIIA and FcyRIIA are lower affinity receptors that bind to IgG only in multimeric form and have slightly lower affinity. The binding of an antibody to an Fc and/or C1q receptor is governed by specific residues or domains in the Fc regions. Binding also depends on residues located in the hinge region and the CH2 portion of the antibody. In some embodiments, the agonistic and/or therapeutic activity of the antibodies described herein is dependent on the binding of the Fc region to the Fc receptor (e.g., FcyR). In some aspects, the agonistic and/or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., FcyR).

[0127] As used herein, the term "human antibody" includes antibodies with variable and constant regions (if present) of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (See, for example, example, Lonberg et al., (1994) Nature 368(6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93 and Harding & Lonberg, (1995) Ann. N.Y. Acad. Sci. 764:536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies).

[0128] As used herein, the term "humanized" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. In some embodiments of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted, as long as they do not abrogate the antibody's ability to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0129] A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0130] As used herein, the term a "heterologous antibody" is defined in relation to the non-human transgenic organism that produces such an antibody. This term refers to an antibody with an amino acid sequence or a coding nucleic acid sequence corresponding to that found in an organism other than the transgenic non-human animal and generally from a species other than the transgenic non-human animal.

[0131] The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a particular antigen. The terms "induce", as used in relation to the induction of CDC or ADCC, refer to the stimulation of certain direct cell death mechanisms.

[0132] As used herein, the term "immunogenic cell death" (alternatively known as "immunogenic apoptosis" refers to a modality of cell death associated with the activation of one or more signaling pathways that induce the expression of pre- death and emission of damage-associated molecular patterns (DAMPs) (e.g., adenosine triphosphate, ATP) from the tumor cell, resulting in increased immunogenicity of the tumor cell and death of the tumor cell in an immunogenic manner (e.g., by phagocytosis). As used herein, the term "immunogenic cell death-inducing agent" refers to a chemical, biological or pharmacological agent that induces a process, pathway or modality of immunogenic cell death.

[0133] As used herein, the terms "inhibits", "reduces" or "blocks" (e.g., referring to the inhibition or reduction of human CCR8-mediated phosphorylation of STAT1 and/or STAT3 in a cell). squid) are used interchangeably and encompass both complete and partial inhibition/blockade. CCR8 inhibition/blockade reduces or alters the normal level or type of activity that occurs without inhibition or blockade. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of CCR8 when in contact with an anti-CCR8 antibody compared to CCR8 that is not in contact with an anti-CCR8 antibody, e.g., inhibits the CCR8 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0134] As used herein, the term "inhibits growth" (e.g., referring to a tumor or cells, e.g., tumor cells) is intended to include any measurable decrease in the growth of a tumor or cell, for example, inhibiting the growth of a tumor by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[0135] As used herein, a subject "in need of prevention", "in need of treatment" or "in need thereof", refers to someone who, in the judgment of an appropriate physician (e.g. , a doctor, nurse or a practicing nurse in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a certain treatment (such as treatment with a composition comprising an anti-CCR8 antibody).

[0136] The term "in vivo" refers to processes that occur in a living organism.

[0137] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (for example, an isolated antibody that specifically binds to human CCR8 is substantially free of antibodies that specifically bind antigens other than CCR8). An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity with other CCR8 proteins from different species. However, the antibody continues to exhibit specific binding to human CCR8 in a specific binding assay as described herein. Furthermore, an isolated antibody is typically substantially free of other cellular material and/or chemicals. In some aspects, a combination of "isolated" antibodies with different CCR8 specificities is combined into a well-defined composition.

[0138] As used herein, the term "isolated nucleic acid molecule" refers to nucleic acids that encode antibodies or portions of antibodies (e.g., VH, VL, CDR3) that bind to CCR8, referring to is a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free from other nucleotide sequences encoding antibodies or antibody portions that bind to antigens other than CCR8, which other sequences may naturally flank the nucleic acid in human genomic DNA. For example, a sequence selected from a sequence set forth in Table 8 corresponds to the nucleotide sequences comprising the heavy chain (VH) and light chain (VL) variable regions of anti-CCR8 antibody monoclonal antibodies described herein. document.

[0139] As used herein, "isotype" refers to the class of antibody (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes. In some aspects, a human monoclonal antibody of the invention is of the IgG1 isotype. In some aspects, a human monoclonal antibody of the invention is of the IgG2 isotype. In some aspects, a human monoclonal antibody of the invention is of the IgG3 isotype. In some aspects, a human monoclonal antibody of the invention is of the IgG4 isotype. As is apparent to one skilled in the art, the identification of antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) is routine in the art and commonly involves a combination of alignments. sequence with known antibodies, published Fc variant sequences and conserved sequences.

[0140] As used herein, the term "KD" or "KD" refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen. The KD value is a numerical representation of the ratio between the antibody off rate constant (kd) and the antibody on rate constant (ka). The KD value is inversely related to the binding affinity of an antibody to an antigen. The lower the KD value, the greater the affinity of the antibody for its antigen. Affinity is the binding strength of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and classify the order strengths of bimolecular interactions.

[0141] As used herein, the term "kd" or "kd" (alternatively "koff" or "koff") refers to the off rate constant for the dissociation of an antibody from an antibody/antigen complex. The kd value is a numerical representation of the fraction of complexes that decay or dissociate per second and is expressed in sec-1 units.

[0142] As used herein, the term "ka" or "ka" (alternatively "kon" or "kon") refers to the rate constant for the association of an antibody with an antigen. The value of ka is a numerical representation of the number of antibody/antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen and is expressed in M-1sec-1 units.

[0143] As used herein, the term "lymphocytes" refers to a type of leukocyte or white blood cell that is involved in the body's immunological defenses. There are two main types of lymphocytes: B cells and T cells. The term "tumor-infiltrating lymphocyte" (abbreviated "TIL") or "tumor-infiltrating Treg" as used herein refers to a lymphocyte or a Treg, respectively, that is associated with tumor cells, for example, that is located within a tumor mass.

[0144] As used herein, the terms "linked", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two or more elements, components, or domains, by any means, including chemical conjugation or recombinant means. Chemical conjugation methods (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0145] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a unique binding specificity and affinity for a particular epitope. Therefore, the term "human monoclonal antibody" refers to an antibody that exhibits a unique binding specificity and that has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse with a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0146] As used herein, the term "natural killer (NK) cell" refers to a type of cytotoxic lymphocyte. These are large, usually granular, non-T, non-B lymphocytes that kill certain tumor cells and play an important role in innate immunity toward viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity. (ADCC).

[0147] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory occurs naturally.

[0148] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and also the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected codons (or all) is replaced with mixed base and/or deoxynosine residues (Batzer et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al., Biol. Chem. 8:91-98, 1994). For arginine and leucine, modifications to the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0149] The polynucleotides used in this document can be composed of any polyribonucleotide, or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions. , hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. Furthermore, the polynucleotide may be composed of triple-stranded regions comprising RNA or DNA or RNA and DNA. A polynucleotide may also contain one or more modified bases, or modified DNA or RNA backbones for stability, or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; therefore, the "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms.

[0150] A nucleic acid is "operatively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, when necessary, to join two protein-coding regions, contiguous and in the reading frame. For switch sequences, operationally linked indicates that the sequences are capable of performing switch recombination.

[0151] As used herein, "parental administration", "parenterally administered" and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, generally by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural injection or infusion , intracerebral, intracranial, intracarotid and intrasternal.

[0152] As used herein, the term "patient" includes human subjects and other mammals receiving prophylactic or therapeutic treatment.

[0153] The term "percent identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of equal nucleotides or amino acid residues, when compared and aligned to obtain maximum match, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to skilled persons) or by visual inspection. Depending on the application, the "percent identity" may exist in a region of the sequence being compared, for example, in a functional domain or, alternatively, in the entire length of the two sequences being compared. For sequence comparison, typically a sequence acts as a reference sequence, against which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and the sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the percent sequence identity for the assay sequence(s) relative to the reference sequence, based on the program's designated parameters.

[0154] Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Academic. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0155] An example of an algorithm that is suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). The software for performing BLAST analyzes is available to the public through the National Center for Biotechnology Information (NCBI) website.

[0156] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are, within the scope of reasonable medical judgment, suitable for use in contact with tissues, organs and/or body fluids of humans and animals without excessive toxicity, irritation, allergic reaction or other problems, or proportional complications with a reasonable benefit/risk ratio.

[0157] As used herein, a "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delaying and isotonic agents and the like that are physiologically compatible. The compositions may include a pharmaceutically acceptable salt, for example, an acid addition salt or a base addition salt (see, for example, Berge et al. (1977) J Pharm Sci 66:1-19).

[0158] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and polymers of non-occurring amino acids. -Natural.

[0159] As used herein, the term "avoidance" when used in relation to a condition, refers to the administration of a composition that reduces the frequency or delays the emergence of symptoms of a medical condition in a subject in relation to a the way you don't receive the composition.

[0160] As used herein, the term "purified" or "isolated" applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally occurring organic or biological molecules) that naturally accompany it, for example, other proteins, lipids and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99%) , by weight, of the total protein in a sample.

[0161] As used herein, the term "rearranged" refers to a configuration of a heavy chain or light chain immunoglobulin locus, in which a V segment is positioned immediately adjacent to a D-J or J segment, in a conformation that essentially encodes a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison with germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

[0162] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It must be understood that such terms are intended to refer not only to the specific object cell, but to the progeny of that cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, this progeny may not actually be identical to the cell of origin, but is still included within the scope of the term "host cell". as used in this document.

[0163] As used herein, the term "recombinant antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g. a mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectoma, (c) antibodies isolated from a combinatorial library of recombinant human antibodies and (d) antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize certain human germline immunoglobulin sequences, are encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), the variable region contains the antigen-binding domain, which is encoded by several genes that rearrange to form using an antibody specific to a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (called somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change in further response to an antigen (i.e., isotype switch). Therefore, the rearranged and somatically mutated nucleic acid molecules encoding the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but will be substantially identical or similar (that is, they have at least 80% identity).

[0164] As used herein, the term "reference antibody" (used interchangeably with "reference mAb") or "reference antigen-binding protein" refers to an antibody, or a fragment of binding to the antigen thereof, which binds to a specific epitope on CCR8 and is used to establish a relationship between itself and one or more distinct antibodies, wherein the relationship is the binding of the reference antibody and one or more distinct antibodies to the same epitope in CCR8. As used herein, the term connotes an anti-CCR8 antibody that is useful in a test or assay, such as those described herein (e.g., a competitive binding assay), as a competitor, wherein the assay is useful for the discovery, identification or development of one or more distinct antibodies that bind to the same epitope.

[0165] As used herein, the terms "specific binding", "selective binding", "selective binding" and "specific binding" refer to the binding of the antibody to an epitope on a predetermined antigen. Typically, the antibody binds at an equilibrium dissociation constant (KD) of approximately less than 10-6 M, such as approximately less than 10-7, 10-8 M, 10-9 M, or 10-10 M or even less. when determined by surface plasmon resonance (SPR) technology on a BIACORE™ 2000 instrument using recombinant human CCR8 as the analyte and antibody as the ligand and binds to the predetermined antigen with an affinity that is at least two times greater than that its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. In certain aspects, an antibody that specifically binds to CCR8 binds with an equilibrium dissociation constant (KD) of approximately less than 100 nM (10-7 M), optionally approximately less than 50 nM (5 x 10-8 M), optionally approximately less than 15 nM (1.5 x 10-8 M), optionally approximately less than 10 nM (10-8 M), optionally approximately less than 5 nM (5 x 10-9 M), optionally optionally approximately less than 1 nM (10-9 M), optionally approximately less than 0.1 nM (10-10 M), optionally approximately less than 0.01 nM (10-11 M), or even less , when determined by surface plasmon resonance (SPR) technology on a BIACORE™ 2000 instrument using recombinant human CCR8 as the analyte and the antibody as the ligand, where binding to the predetermined antigen occurs with an affinity that is at least twice as high as the antibody's affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen The phrases "an antibody that recognizes an antigen" and "a specific antibody to an antigen" are used interchangeably in this document with the term "an antibody that specifically binds to an antigen".

[0166] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immunological disorder. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0167] For nucleic acids, the term "substantial homology" indicates that two nucleic acids or designated sequences thereof, when ideally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to 95% and at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments hybridize under selective hybridization conditions, for strand complement.

[0168] The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of homology = # of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced for the ideal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

[0169] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using an NWSgapdna.CMP matrix and a weight of gap of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the E algorithm . Meyers and W. Miller (CABIOS, 4: 11 17 (1989)) which was incorporated into the ALIGN program (version 2.0), using a PAM120 weight residual table, a gap length penalty of 12 and a penalty. gap of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) which was incorporated into the GAP program in GCG software package (available at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a of length 1, 2, 3, 4, 5 or 6.

[0170] The nucleic acid and protein sequences of the present invention can additionally be used as a "query sequence" to perform a search in public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST (ver. 2.0) programs by Altschul, et al. (1990) J. Mol. Biol. 215:403-10. Nucleotide searches in BLAST can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the program XBLAST, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0171] Nucleic acids may be present in whole cells, in a cell lysate or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl bands, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0172] The nucleic acid compositions of the present invention, although often in a native sequence (except modified restriction sites and the like), cDNA, genomic or mixtures thereof can be mutated, in accordance with standard techniques for providing sequences genes. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from V, D, J switches, native constants and other sequences described herein are contemplated (where "derived" indicates that a sequence is identical to or modified from another sequence) .

[0173] The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (also known as TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFH cells, T cells. cytotoxic cells (also known as TC cells, CD8+ T cells, cytotoxic T lymphocytes, cytotoxic T cells, cytotoxic T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (also known as Treg cells or suppressor T cells) and subtypes, including CD4+ FOXP3+ Treg cells, CD4+FOXP3- Treg, Tr1 cells, Th3 cells, and Treg17 cells, natural killer T cells (also known as NKT cells), mucosa-associated invariant T cells (MAITs), and gamma delta T cells (Yδ T cells), including VY9/Vδ2 T cells. Any one or more of mentioned or unmentioned T cells can be the target cell type for a method of using the invention.

[0174] As used herein, the term "T cell-mediated response" refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and T helper cells (e.g. CD4+ cells). T cell-mediated responses include, for example, cytotoxicity and T cell proliferation.

[0175] As used herein, the term "regulatory T cell", "regulatory T cell", "Treg" or "Treg", used interchangeably herein, refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or negatively regulate the induction and proliferation of effector T cells. Tregs are known to direct the lysis of effector T cells, support the formation of tolerogenic dendritic cells, support the formation of M2 macrophages, produce immunosuppressive metabolites and cytokines, serve as an IL-2 sink, and promote the formation of of neovasculature. Although there are many types of Tregs, many Tregs express CD4 and FOXP3, with FOXP3 serving as a marker for Tregs in many cases.

[0176] As used herein, the terms "therapeutically effective amount" or "therapeutically effective dose", or similar terms used herein, are intended to mean an amount of an agent (e.g., an anti-CCR8 antibody or a fraction -antigen-binding segment thereof) that will elicit the desired biological or medical response (for example, an improvement in one or more symptoms of a cancer).

[0177] The terms "treat", "treating" and "treatment", as used in this document, refer to therapeutic or preventive measures described in this document. Methods of "treatment" employ administering to a subject in need of such treatment a human antibody of the present invention, e.g., a subject in need of an enhanced immune response against a specific antigen or a subject who ultimately has may acquire such a disorder in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0178] As used herein, the term "tumor microenvironment" (alternatively "cancer microenvironment"; abbreviated TME) refers to the cellular environment or medium in which the tumor or neoplasm exists, including surrounding blood vessels, as well as non-cancerous cells, including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and the surrounding microenvironment are closely related and constantly interact. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0179] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a loop of circular double-stranded DNA whose additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operationally linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0180] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practice, or in testing the methods and compositions presently disclosed. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0181] Various aspects of the invention are described in more detail in the following subsections.

II. Compositions of the invention

[0182] Certain aspects of the present invention are directed to antibodies or antigen-binding portions thereof that specifically bind to CCR8 ("anti-CCR8 antibody"). In certain aspects, the anti-CCR8 antibody specifically binds to the N-terminal extracellular domain of human CCR8. Historically, it has been very difficult to generate therapeutic antibodies to CCR8. CCR8, like other GPCRs, is challenging to raise antibodies against due to its strong membrane association, lack of sequence exposure on the cell surface, and difficulty in expressing the full CCR8 protein itself. Numerous previous attempts to leverage multiple antibody generating platforms have failed. (See also Jo and Jung, Experimental & Molecular Medicine 48:e207 (2016).) The present invention solves this problem by specifically targeting the extracellular N-terminal domain of human CCR8. By raising antibodies specifically targeting the N-terminal domain, the present invention focused on generating antibodies that target the longer extracellular portion of CCR8. This allowed the obtaining of previously unattainable anti-CCR8 antibodies. In doing so, the antibodies described in this document are capable of inhibiting CCR8 activity in a manner not previously described. In particular, the antibodies disclosed herein are capable of (a) enhancing an immune response to a tumor; (b) reduce, deplete, or kill tumor-infiltrating regulatory T cells ("Treg"); (c) inducing internalization of CCR8 in tumor-infiltrating regulatory T cells ("Treg"); (d) activate NK cells, (e) induce NK cell-mediated killing of tumor-infiltrating regulatory T cells ("Treg"); (f) bind to CCR8 of cynomolgus monkey ("cyno"); (g) bind to human CCR8 with a KD of 10 nM or less as measured by BIACORE™; or (h) any combination thereof.

[0183] In some aspects, the antibody or antigen-binding portion thereof is further manipulated by removing one or more post-translational modifications. In some aspects, the antibody or antigen-binding portion thereof is engineered to remove one or more fucose sugar units. In some aspects, the antibody or antigen-binding portion thereof is modified to remove one or more fucose sugar units from the Fc (IgG1) region of the antibody. In some aspects, the antibody or antigen-binding portion thereof is afucosylated. In some aspects, removal of one or more fucose sugar units increases the ADCC of the antibody or antigen-binding fragment thereof. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units (e.g., afucosylated antibody) is at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, or at least about 5.0 times greater than anti-CCR8 antibody that is not modified to remove one or more fucose sugar units (e.g., fucosylated antibody). In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 3.0 times greater than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. fucose sugar. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 3.5 times greater than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. fucose sugar. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 4.0 times greater than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. or more units of fucose sugar.

[0184] In certain aspects, the anti-CCR8 antibody is capable of inducing an immune response to a tumor. Treg cells serve to regulate the immune response by downregulating T cell activity as a means of keeping the immune system in check. Tumor-infiltrating Tregs may act to prevent a tumor-directed immune response, thereby allowing the tumor to avoid destruction by a subject's immune system. The antibodies described in these documents are capable of inhibiting tumor-infiltrating Tregs, thus reducing this barrier to an anti-tumor immune response. In some aspects, the anti-CCR8 antibody increases an immune response to a tumor in a subject in need by at least about 50%, at least about 100%, at least about 150%, at least about of 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450% or at least about 500% in relation to a response immune system in the absence of anti-CCR8 antibody.

[0185] An antitumor immune response can be measured using any indicators known in the art. In some aspects, the antitumor immune response is determined by comparing the number of tumor-infiltrating T cells (TILs) in a tumor sample obtained from a subject before and after contacting the tumor with the anti-CCR8 antibody. In some aspects, the number of TILs is measured by immunohistochemistry or quantitative polymerase chain reaction (qPCR). In some aspects, the number of TILs in the tumor sample is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about of 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, or at least about 20 times, relative to the number of TILs in a tumor sample obtained from the subject before contacting the tumor with the antibody anti-CCR8.

[0186] In certain aspects, the anti-CCR8 antibody is capable of reducing, depleting or killing tumor-infiltrating Treg cells. In some aspects, the anti-CCR8 antibody induces depletion in the number of tumor-infiltrating Treg cells in a subject following administration of the antibody or antigen-binding portion thereof, relative to the number of tumor-infiltrating Treg cells. before administration. In some aspects, the number of tumor-infiltrating Treg cells is depleted by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or about 100% relative to the number of tumor-infiltrating Treg cells before administration. In some aspects, anti-CCR8 preferentially reduces, depletes, or kills tumor-infiltrating Treg cells over peripheral Treg cells.

[0187] In some aspects, the anti-CCR8 antibody induces antibody-dependent cellular cytotoxicity (ADCC) in a subject following administration of the anti-CCR8 antibody. In some aspects, the ADCC comprises an EC50 of about 100 μg/mL or less after administration of the antibody or antigen-binding portion thereof. In some aspects, ADCC comprises an EC50 of about 100 μg/mL or less, about 90 μg/mL or less, about 80 μg/mL or less, about 70 μg/mL or less, about 60 μg/mL or less, about 50 μg/mL or less, about 45 μg/mL or less, about 40 μg/mL or less, about 35 μg/mL or less, about 30 μg/mL or less, about 30 μg/mL or less, about 25 μg/mL or less, about 20 μg/mL or less, about 15 μg/mL or less, about 10 μg/mL or less, about 5 µg/mL or less, about 1 µg/mL or less, about 0.5 µg/mL or less, about µg/mL or less, about 0.1 µg/mL or less, or about 0, 01 μg/mL or less after administration of the anti-CCR8 antibody. In some aspects, the ADCC comprises an EC50 of about 1 μg/mL or less after administration of the antibody or antigen-binding portion thereof. In some aspects, the ADCC comprises an EC50 of about 0.1 μg/mL or less after administration of the antibody or antigen-binding portion thereof.

[0188] Without being limited by any particular theory or mechanism, it is hypothesized that inhibition of CCR8 signaling in tumor-infiltrating Tregs leads to the activation of NK cells in the tumor microenvironment. Activated NK cells are then able to target and kill tumor-infiltrating Tregs, reducing their number and increasing the immune response to the tumor. Consequently, in some aspects, the anti-CCR8 antibody is capable of activating NK cells. In some aspects, NK cells are activated in the tumor microenvironment. In some aspects, NK cells are tumor-infiltrating NK cells. NK cell activation can be measured using any techniques known in the art. In some aspects, NK cell activation is determined by measuring the percentage of cells that express one or more markers of activated NK cells. In certain aspects, NK cell activation is determined by measuring the percentage of cells in the tumor microenvironment that express NKp46 but do not express CD3 (e.g., NKp46+/CD3- cells).

[0189] In some aspects, NK cell activation is characterized by increased expression of one or more target genes by NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of 4-1BB on the surface of NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of ICAM-1 on the surface of NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of 4-1BB and ICAM-1 on the surface of NK cells. In some aspects, the level of 4-1BB and/or ICAM-1 on the surface of NK cells following contact of the anti-CCR8 antibody with a tumor is upregulated by at least about 1.5-fold, 2-fold. , 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times, relative to the level of 4 -1BB and/or ICAM-1 on the surface of NK cells in a tumor sample collected before contact.

[0190] In some aspects, NK cell activation is characterized by decreased expression of one or more target genes by NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing downregulation of CD16 on the surface of NK cells. In some aspects, the level of CD16 on the surface of NK cells upon contact of anti-CCR8 antibody with a tumor is downregulated by at least about 1.5-fold, 2-fold, 2.5-fold, 3.0-fold, 3.5 times, 4.0 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times, relative to the level of CD16 on the surface of NK cells in a tumor sample taken before contact.

[0191] In some aspects, the number of activated NK cells in the tumor microenvironment is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about of 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, at least about 98%, at least about 99%, or about 100% relative to the percentage of activated NK cells in a tumor sample obtained from the subject prior to contact of the tumor with the anti-CCR8 antibody. In some aspects, the percentage of activated NK cells in the tumor microenvironment is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold , at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about of 8 times, at least about 9 times, at least about 10 times, at least about 15 times, or at least about 20 times, relative to the number of activated NK cells in a tumor sample obtained from the subject before contact the tumor with the anti-CCR8 antibody. In some aspects, anti-CCR8 antibody is capable of inducing NK cell-mediated killing of tumor-infiltrating Tregs.

[0192] The anti-CCR8 antibodies described in this document are capable of specific binding to human CCR8. However, in some aspects, anti-CCR8 antibodies are capable of binding to CCR8 from non-human animals. In some aspects, the anti-CCR8 antibody is capable of specifically binding to human CCR8 and human primate CCR8. In some aspects, the anti-CCR8 antibody is capable of binding to human CCR8 and cynomolgus (cyno) CCR8. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a higher affinity than non-human CC8 (e.g., CCR8 cyno). In some aspects, the anti-CCR8 antibody binds to human CCR8, but does not bind to cyno CCR8.

[0193] In some aspects, the anti-CCR8 antibody binds to human CCR8 with an equilibrium dissociation constant (KD) of about 100 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 50 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 25 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 20 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 15 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 10 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 5 nM or less. In some aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 1 nM or less. In some aspects, KD is measured by BI-ACORE™. In certain aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 10 nM or less, as measured by BIACORE™. In certain aspects, the anti-CCR8 antibody binds to human CCR8 with a KD of about 1 nM or less, as measured by BIACORE™.

[0194] Inhibition of CCR8 by the antibodies and antigen-binding portions thereof disclosed herein may occur through any mechanism. Without being linked by any particular mechanism, in some aspects, the anti-CCR8 antibody induces the internalization of CCR8 by tumor-infiltrating Treg cells. Internalization of the surface CCR8 receptor eliminates the receptor's ability to bind its ligand and enhance intracellular signaling, effectively inhibiting CCR8 activity in tumor-infiltrating Treg cells. In certain aspects, the anti-CCR8 antibody binds to CCR8 expressed by tumor-infiltrating Treg cells.

[0195] In some aspects, the anti-CCR8 antibody blocks the interaction between CCR8 and its ligand, for example, through steric hindrance, a confirmation change, internalization of the CCR8 receptor, or any combination thereof. In some aspects, binding of anti-CCR8 antibody to the N-terminal extracellular domain of CCR8 inhibits the ability of the CCR8 receptor to interact with the G protein, for example, through a conformational change and/or through internalization of the receptor. of CCR8. I.A. Epitopes

[0196] The antibodies described herein specifically bind to the N-terminal extracellular domain of CCR8 or a fragment thereof. The N-terminal extracellular domain of human CCR8 is generally defined as consisting of amino acids 1-35 of the complete CCR8 sequence (e.g., amino acids 1-35 of SEQ ID NO: 171) (veruniprot.org/uniprot/P51685). The amino acid sequence of the N-terminal extracellular domain of human CCR8 comprises the amino acid sequence MDYTLLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK (SEQ ID NO: 172).

[0197] In some aspects, the anti-CCR8 antibody binds to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten amino acids within the N-terminal extracellular domain of human CCR8, for example, as set forth in SEQ ID NO: 172. In some aspects, the at least two, at least three, at least four, at least at least five, at least six, at least seven, at least eight, at least nine or at least ten amino acids within the N-terminal extracellular domain of human CCR8, for example, as set forth in SEQ ID NO: 172 are contiguous . In some aspects, the anti-CCR8 antibody binds to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten contiguous amino acids within the N-terminal extracellular domain of human CCR8, for example, as set forth in SEQ ID NO: 172. In some aspects, the at least two, at least three, at least four, at least five, at least at least six, at least seven, at least eight, at least nine or at least ten amino acids within the N-terminal extracellular domain of human CCR8, for example, as set forth in SEQ ID NO: 172 are not contiguous. In some aspects, the anti-CCR8 antibody binds to at least one amino acid in the extracellular N-terminal domain of human CCR8 and at least one amino acid of human CCR8 that is not within the extracellular N-terminal domain of human CCR8.

[0198] In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-10 of SEQ ID NO: 172. In some aspects, The anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on the human CCR8, comprising one or more amino acids selected from amino acid residues 1-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 5-10 of SEQ ID NO: 172. In some the In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to a epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids. acids selected from amino acid residues 5-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 5-35 of SEQ ID NO: 172. In some aspects, The anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on the Human CCR8, comprising one or more amino acids selected from amino acid residues 10-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from of amino acid residues 10-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 10-30 of SEQ ID No.: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-35 of SEQ ID NO: 172. In some aspects, the antibody anti-CCR8 binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 15-35 of SEQ ID NO : 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 20-25 of SEQ ID NO: 172. In some aspects, the antibody anti-CCR8 binds to an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 20-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on CCR8 human, comprising one or more amino acids selected from amino acid residues 20-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8, comprising one or more amino acids selected from residues of amino acids 25-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 25-35 of SEQ ID NO : 172. In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising one or more amino acids selected from amino acid residues 30-35 of SEQ ID NO: 172.

[0199] In some aspects, the anti-CCR8 antibody binds to an epitope on human CCR8 comprising an amino acid sequence selected from SEQ ID NOs: 180-200.

II.B. Antibody Sequences

[0200] In some aspects, the anti-CCR8 antibody comprises an entire antibody, for example, an antibody comprising two light chain polypeptides and two heavy chain polypeptides. In some aspects, the anti-CCR8 antibody comprises a fragment of a full-length antibody that retains the ability to bind CCR8. In some aspects, the anti-CCR8 antibody is a single chain antibody. In some aspects, the anti-CCR8 antibody is a single-chain Fv fragment (scFv). In some aspects, the anti-CCR8 antibody is an Fd fragment. In some aspects, the anti-CCR8 antibody is a Fab fragment. In some aspects, the anti-CCR8 antibody is a Fab' fragment. In some aspects, the anti-CCR8 antibody is an F(ab')2 fragment. In some aspects, the anti-CCR8 antibody is selected from an intrabody, a minibody, a tribody, or a diabody.

[0201] In some aspects, the anti-CCR8 antibody comprises a variable heavy chain (VH) and a variable light chain (VL). In some aspects, VH comprises a VH complementarity determining region (CDR) 1, a VH CDR2 and a VH CDR3; and the VL comprises a VL CDR1, a VL CDR2 and a VL CDR3. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in Table 2A. In some aspects, VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 201. In some aspects, VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 202. In some aspects, VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 203. In some aspects, VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204. Table 2A: VH CDR1 Consensus Sequences Note: amino acid residue listed in parentheses above (and elsewhere in the present invention) alternatively designates the amino acid at that particular position. For example, Y(Y/A)MH means that the sequence can be YYMH or YAMH.

[0202] In some aspects, the VH CDR2 comprises the amino acid sequence set forth in Table 2B. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 205. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 207. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 208. Table 2B: VH CDR2 Consensus Sequences

[0203] In some aspects, the VH CDR3 comprises the amino acid sequence set forth in Table 2C. In some aspects, VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 210. Table 2C: VH CDR3 consensus sequences

[0204] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: 205, and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 204, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: 205 and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: 208 and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set out in SEQ ID NO: 204, a VH CDR2 with the amino acid sequence set out in SEQ ID NO: 208 and a VH CDR3 with the amino acid sequence set out in SEQ ID No.: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO. °: 208 and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: 208 and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the sequence of amino acids set forth in SEQ ID NO: 202, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: 206, and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the antibody anti-CCR8 comprises a VH CDR1 with the amino acid sequence established in SEQ ID NO: 202, a VH CDR2 with the amino acid sequence established in SEQ ID NO: 207 and a VH CDR3 with the amino acid sequence established in SEQ ID NO: 210. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 203, a VH CDR2 with the amino acid sequence set forth in SEQ ID NO: : 206 and a VH CDR3 with the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 with the amino acid sequence set forth in SEQ ID NO: 203 , a VH CDR2 with the amino acid sequence established in SEQ ID NO: 207 and a VH CDR3 with the amino acid sequence established in SEQ ID NO: 210.

[0205] In some aspects, the VL CDR1 comprises the amino acid sequence set forth in Table 3A. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 211. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 213. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 214. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 215. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 216. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 217. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 218. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 219. Table 3A: Sequences of VL CDR1 consensus

[0206] In some aspects, the VL CDR2 comprises the amino acid sequence set forth in Table 3B. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 220. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 221. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 222. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 223. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 223. set forth in SEQ ID NO: 224. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 225. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: : 226. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 227. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 228. In In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 229. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 230. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 231. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 232. Table 3B: VL CDR2 consensus sequences

[0207] In some aspects, the VL CDR3 comprises the amino acid sequence set forth in Table 3C. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 233. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 234. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 235. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 237. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 238. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 239. In some aspects, the VL CDR3 comprises the amino acid sequence established in SEQ ID NO: 240. Table 3C: VL CDR3 consensus sequences

[0208] In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 211, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 220 and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 233. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 212, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 221 and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 234. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 with the amino acid sequence set forth in SEQ ID NO: 222 and a VL CDR3 with the amino acid sequence set forth in SEQ ID NO: 235. In some aspects, the anti- -CCR8 comprises a VL CDR1 with the amino acid sequence established in SEQ ID NO: 213, a VL CDR2 with the amino acid sequence established in SEQ ID NO: 222 and a VL CDR3 with the amino acid sequence established in SEQ ID NO: 236. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 with the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 with the amino acid sequence set forth in SEQ ID No.: 223 and a VL CDR3 with the amino acid sequence set forth in SEQ ID No.: 235. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 with the amino acid sequence set forth in SEQ ID No.: 213, a VL CDR2 with the amino acid sequence set forth in SEQ ID NO: 223 and a VL CDR3 with the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 with the sequence of amino acids set forth in SEQ ID NO: 217, a VL CDR2 with the amino acid sequence set forth in SEQ ID NO: 227 and a VL CDR3 with the amino acid sequence set forth in SEQ ID NO: 238. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 with the amino acid sequence established in SEQ ID NO: 218, a VL CDR2 with the amino acid sequence established in SEQ ID NO: 231 and a VL CDR3 with the amino acid sequence established in SEQ ID NO: 239. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 with the amino acid sequence set forth in SEQ ID NO: 219, a VL CDR2 with the amino acid sequence set forth in SEQ ID NO: : 232 and a VL CDR3 with the amino acid sequence established in SEQ ID NO: 240.

[0209] In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87 , 97, 107, 117, 127, 137, 147, 157 and 167. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6 , 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156 and 166. In some aspects, the VH CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence established in SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155 and 165.

[0210] In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137 and 147. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136 and 146. In some aspects, the VH CDR1 of the anti-CCR8 antibody comprises a sequence - amino acid sequence selected from the amino acid sequence established in SEQ ID NOs: 45, 105, 115, 135 and 145.

[0211] In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97 , 127, 157 and 167. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76 , 86, 96, 126, 156 and 166. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35 , 55, 65, 75, 85, 95, 125, 155 and 165.

[0212] In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 110, 120, 130, 140, 150, 160 and 170. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9 , 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159 and 169. In some aspects, the VL CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence established in SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 148, 158 and 168.

[0213] In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140 and 150. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139 and 149. In some aspects, the VL CDR1 of the anti-CCR8 antibody comprises a sequence - amino acid sequence selected from the amino acid sequence established in SEQ ID NOs: 48, 108, 118, 138 and 148.

[0214] In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100 , 130, 160 and 170. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79 , 89, 99, 129, 159 and 169. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38 , 58, 68, 78, 88, 98, 128, 158 and 168.

[0215] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 47, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 48, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 49 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0216] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 107, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 108, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 109 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110.

[0217] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 116, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 117, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 118, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 119 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120.

[0218] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 135, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 136, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 137, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 138, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 139 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140.

[0219] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 147, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 148, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 149 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150.

[0220] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 9 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0221] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 19 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0222] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 29 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0223] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 38, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 39 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0224] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 58, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 59 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60.

[0225] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 67, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 69 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0226] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 77, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 78, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 79 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80.

[0227] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 87, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 89 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90.

[0228] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 97, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 98, a VL CDR2 comprising the amino acid sequence set out in SEQ ID NO : 99 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0229] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 125, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 127, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 128, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 129 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130.

[0230] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 155, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 157, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 158, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 159 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160.

[0231] In some aspects, the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 165, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 166, a VH CDR3 comprising the amino acid sequence set out in SEQ ID NO: 167, a VL CDR1 comprising the amino acid sequence set out in SEQ ID NO: 168, a VL CDR2 comprising the amino acid sequence set out in SEQ ID N °: 169 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170.

[0232] In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 %, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid selected from SEQ ID NOs: 41, 101, 111, 131 and 141. In some aspects , the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 41, 101, 111, 131 and 141. In some aspects, the VL chain comprises an amino acid sequence with at least at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid selected from SEQ ID NOs: 42, 102, 112, 132 and 142. In some aspects, the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 42, 102, 112, 132 and 142. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90% , at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid selected from SEQ ID NOs: 41, 101, 111, 131 and 141; and the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97 %, at least about 98% or at least about 99% sequence identity having an amino acid selected from SEQ ID NOs: 42, 102, 112, 132 and 142; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 41, 101, 111, 131 and 141; and the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 42, 102, 112, 132 and 142; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0233] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 41 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 42; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 42; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0234] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity having the amino acid sequence set forth in SEQ ID NO: 101 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 102; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 101 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 102; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0235] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 111 and a VL chain comprising a established amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity having the amino acid sequence set forth in SEQ ID NO: 112; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 111 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 112; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0236] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 131 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 132; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 131 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 132; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0237] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 141 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 142; wherein the anti-CCR8 antibody does not bind to cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 141 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 142; wherein the anti-CCR8 antibody does not bind to cyno CCR8.

[0238] In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 %, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11,21, 31,51,61, 71,81,91, 121, 151 and 161. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31 51, 61, 71, 81, 91, 121, 151 and 161. In some aspects, the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid sequence selected from the sequence of amino acids set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152 and 162. In some aspects, the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152 and 162. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about of 99% sequence identity with an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11,21,31,51,61,71,81,91, 121, 151 and 161 ; and the VL chain comprises an amino acid sequence of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97 %, at least about 98% or at least about 99% sequence identity having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152 and 162; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, 51, 61, 71, 81, 91, 121, 151 and 161; and the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152 and 162; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0239] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0240] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 11 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 12; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 11 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 12; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0241] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with a sequence identity set forth in SEQ ID NO: 21 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 22; wherein the anti-CCR8 antibody binds to human CCR8 cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 22; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0242] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 31 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 32; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 31 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0243] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 51 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 52; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 51 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 52; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0244] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with an amino acid sequence set forth in SEQ ID NO: 61 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with an amino acid sequence set forth in SEQ ID NO: 62; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 62; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0245] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 71 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity having the amino acid sequence set forth in SEQ ID NO: 72; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 71 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 72; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0246] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 81 and a VL chain comprising a amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 82; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 82; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0247] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 91 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 92; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 91 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 92; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0248] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 121 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 122; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 122; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0249] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 151 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity having the amino acid sequence set forth in SEQ ID NO: 152; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 151 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 152; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0250] In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 161 and a VL chain comprising a amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 162; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 162; wherein the anti-CCR8 antibody binds to human CCR8 and cyno CCR8.

[0251] In some aspects, the anti-CCR8 antibody is a human antibody. In some aspects, the anti-CCR8 antibody is a humanized antibody. In some aspects, anti-CCR8 is a chimeric antibody.

[0252] In some aspects, the anti-CCR8 antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1 and IgA2, an IgD and an IgE antibody. In some aspects, the anti-CCR8 antibody is an IgG1 antibody. In some aspects, the anti-CCR8 antibody is an IgG4 antibody. In some aspects, the anti-CCR8 antibody comprises a wild-type IgG1 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises a wild-type IgG4 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises an Fc domain comprising at least one mutation. In some aspects, the anti-CCR8 antibody comprises a mutant IgG1 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises a mutant IgG4 heavy chain constant region. In some aspects, the mutant IgG4 heavy chain constant region comprises any of the substitutions S228P, L235E, L235A, or a combination thereof, in accordance with EU numbering.

[0253] In some aspects, the invention provides an antibody or antigen-binding portion thereof that binds to substantially the same epitope on human CCR8 as the anti-CCR8 antibody, in accordance with any of the aspects mentioned above. In some aspects, the invention provides an antibody or antigen-binding portion thereof that cross-competes for binding to human CCR8 as the anti-CCR8 antibody, in accordance with any of the aspects mentioned above.

[0254] In some aspects, the anti-CCR8 antibody comprises an altered heavy chain constant region that has modified effector function relative to its corresponding unchanged constant region. Effector functions involving the constant region of the anti-CCR8 antibody can be modulated by altering the properties of the constant region or Fc. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more receptors. Fc receptors and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody containing an altered constant region compared to the activity of the unchanged form of the constant region. In particular aspects, modulation includes situations in which an activity is abolished or completely absent.

[0255] In one aspect, the anti-CCR8 antibody comprises a constant region of the IgG4 heavy chain. In one aspect, the IgG4 heavy chain constant region is a wild-type IgG4 heavy chain constant region. In another aspect, the constant region of IgG4 comprises a mutation, for example, one or both of S228P and L235E or L235A, for example, according to EU numbering (Kabat, E.A., et al., Supra). In one aspect, the anti-CCR8 antibody described herein comprises an IgG1 constant region. In one aspect, the IgG1 heavy chain constant region is a wild-type IgG1 heavy chain constant region. In another aspect, the heavy chain constant region of IgG1 comprises a mutation.

[0256] An altered constant region with altered FcR binding affinity and/or altered ADCC activity and/or altered CDC activity is a polypeptide that has either enhanced or decreased FcR binding activity, and/or ADCC activity and/or CDC activity compared to the unchanged form of the constant region. An altered constant region that exhibits greater binding to an FcR binds to at least one FcR with greater affinity than the unchanged polypeptide. An altered constant region that exhibits decreased binding to an FcR binds to at least one FcR with lower affinity than the unchanged form of the constant region. Such variants that exhibit reduced binding to an FcR may have little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42 , 41, 40, 39, 38, 37, 36, 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, 8, 7, 6, 5, 4, 3, 2, or 1%) of binding to the FcR compared to the binding level of an immunoglobulin constant of native sequence or Fc region to the FcR. Likewise, an altered constant region that exhibits modulated ADCC and/or CDC activity may exhibit increased or reduced ADCC and/or CDC activity compared to the unchanged constant region.

[0257] In some aspects, the anti-CCR8 antibody exhibits increased effector function. In some aspects, an anti-CCR8 antibody comprises a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see, for example, Burton et al. (1992) Adv Immun 51:1- 18; Canfield et al. (1991) J Exp Med 173:1483-1491; and Mueller et al. (1997) Mol Immunol 34(6):441-452).

[0258] In some aspects, the anti-CCR8 antibody comprises an altered constant region exhibiting enhanced complement-dependent cytotoxicity (CDC). Modulated CDC activity can be achieved by introducing one or more amino acid substitutions, insertions or deletions into an Fc region of the antibody. See, for example, US Patent No. 6,194,551. Alternatively or additionally, cysteine residues can be introduced into the Fc region, thus allowing the formation of interchain disulfide bonds in this region. The homodimeric antibody thus generated may have improved internalization capacity and/or increased complement-mediated cell killing. See, for example, Caron et al. (1992) J Exp Med176:1191-1195 and Shopes (1992) Immunol148:2918-2922; PCT publication no. WO 99/51642 and WO 94/29351; Duncan and Winter (1988) Nature 322:738-40; and US Patent Nos. 5,648,260 and 5,624,821.

[0259] In some aspects, the anti-CCR8 antibody is a bispecific antibody, a bispecific T cell engager (BiTE), a multispecific antibody, a biparatopic antibody, an immunoconjugate, an antibody-drug conjugate or any combination thereof. II.C. Antibody and Immunoconjugate Variants

[0260] Certain aspects of the present invention are directed to antibodies comprising a first antigen-binding region that binds to human CCR8 and a second antigen-binding region that binds to a second antigen, wherein the first antigen-binding region antigen binding comprises an anti-CCR8 antibody described herein. In some aspects, the antibody is a bispecific antibody, for example, capable of binding to only two antigens. In some aspects, the antibody is a multispecific antibody, for example, capable of binding to more than two antigens. In some aspects, the multispecific antibody is capable of binding to at least about 3 antigens, at least about 4 antigens, at least about 5 antigens, or at least about 6 antigens.

[0261] In some aspects, the antibody is a biparatopic antibody. Biparatopic antibodies are capable of binding to two epitopes on a single polypeptide target. In some aspects, the biparatopic antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region and/or the second antigen-binding region comprises an anti-antigen antibody. -CCR8 disclosed in this document.

[0262] In some aspects, the multispecific antibody is a bispecific T cell coupler (BiTE). BiTE constructs comprise a first antigen-binding region that binds to the CD3 receptor on the T cell and a second antigen-specific binding region. In some aspects, the BiTE comprises a first antigen-binding region that binds CD3 and a second antigen-binding region that binds human CCR8, wherein the second antigen-binding region comprises an anti-CCR8 antibody. disclosed in this document.

[0263] In some aspects, the bispecific antibody, the multispecific antibody, the BiTE or the biparatopic antibody comprises a first VH CDR1, a first VH CDR2 and a first VH CDR3; a first VL domain, comprising a first VL CDR1, a first VL CDR2 and a first VL CDR3; a second VH domain, comprising a second VH CDR1, a second VH CDR2 and a second VH CDR3; and a second VL domain, comprising a second VL CDR1, a second VL CDR2 and a second VL CDR3; wherein (a) the first VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 45, 105, 115, 135 and 145; (b) the first VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136 and 146; and (c) the first VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137 and 147. In some aspects, (a) the first VL CDR1 comprises a sequence of amino acids selected from the amino acid sequence established in SEQ ID NOs: 48, 108, 118, 138 and 148; (b) the first VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139 and 149; and (c) the first VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140 and 150.

[0264] In some aspects, (a) the VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125, 155 and 165; (b) the VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156 and 166; and (c) the VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157 and 167. In some aspects, (a) the VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98 , 128, 158 and 168; (b) the VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159 and 169; and (c) the VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160 and 170.

[0265] In some aspects, the antibody is a biparatopic antibody and (a) the second VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65 , 75, 85, 95, 125, 155 and 165; (b) the second VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156 and 166 ; and (c) the second VH CDR3 comprises a sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157 and 167. In some aspects, the antibody is a biparatopic antibody and (a) the second VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38 , 58, 68, 78, 88, 98, 128, 158 and 168; (b) the second VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159 and 169; and (c) the second VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160 and 170.

[0266] Certain aspects of the present invention are directed to an immunoconjugate comprising an anti-CCR8 antibody disclosed herein. In some aspects, the immunoconjugate is an antibody-drug conjugate. The immunoconjugate may include any cytotoxic agent known in the art linked to an anti-CCR8 antibody disclosed herein. In some aspects, the antibody-drug conjugate comprises a cytotoxic agent selected from the group consisting of a maytansinoid (e.g., maytansine), a dolastatin, an auristatin drug analog, cryptophycin, a duocarmycin derivative ( for example, an analogue of CC-1065 and duocarmycin), an enediyne antibiotic (e.g., esperamycin and calicheamycin), pyrolobenodiazepine (PBD), and any combination thereof. An antibody-drug conjugate comprising an anti-CCR8 antibody and a cytotoxic agent may enable efficacy in tumor indications with low numbers of effector cells, including NK cells and macrophages.

II.D. Fc region variants

[0267] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating a variant of the Fc region. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more positions. of amino acid.

[0268] In certain embodiments, the invention contemplates an antibody variant that has some, but not all of the effector functions that make it a desirable candidate for applications, in which the half-life of the antibody in vivo is important, however, certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks binding to FcyR (consequently, likely lack of ADCC activity), but maintains the ability to bind to FcRn. The primary cells to mediate ADCC, NK cells, express FCYRIII only, while monocytes express FCYRI, FCYRII, and FCYRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to evaluate the ADCC activity of a molecule of interest are described in US Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 Non-Radioactive Cytotoxicity Assay ® (Promega, Madison, WI). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Sci. USA 95:652-656 (1998). that the antibody is unable to bind to C1q and consequently lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Determinations of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12):1759- 1769 (2006)).

[0269] Antibodies with reduced effector function include those with substitution of one or more of the Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine. (US Patent No. 7,332,581).

[0270] Certain antibody variants with improved or decreased binding to FcRs are described. (See, for example, US Patent No. 6,737,056; WO 2004/056312; and Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0271] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and/or 334 of the Fc region (EU residue numbering) .

[0272] In some embodiments, changes are made to the Fc region that result in altered (i.e., either improved or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC), for example, as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184, 2000.

[0273] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to the FcRn. These Fc variants include those substitutions in one or more residues of the Fc region: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376 , 378, 380, 382, 413, 424 or 434, for example, replacement of Fc region residue 434 (e.g., US Patent No. 7,371,826).

[0274] See also Duncan & Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94/29351 on other examples of Fc region variants.

[0275] In some embodiments, an antibody is provided, wherein the isotype is human IgG1. In some embodiments, an antibody is provided, wherein the isotype is human IgG4. In some embodiments, an antibody is provided, wherein the isotype is human IgG4, wherein there is a single mutation at serine 228 to proline (S228P). In some embodiments, an antibody is provided, wherein the isotype is human IgG4, wherein there are two mutations at serine 228 to proline (S228P) and leucine 235 to glutamate (L235E). The S228P mutation runs at position 228 in the literature, however, the exact location of a mutation in an antibody can vary depending on how that antibody is produced.

II.E. Chimeric antigen receptors (CAR) and T cell receptors (TCR)

[0276] Certain aspects of the present invention are directed to a chimeric antigen receptor (CAR) comprising an antigen-binding region that specifically binds to the N-terminal extracellular domain of human CCR8. In some aspects, the antigen-binding region comprises an anti-CCR8 antibody disclosed herein or an antibody, or antigen-binding fragment thereof that binds to the same epitope as an anti-CCR8 antibody disclosed herein. In some aspects, the CAR additionally comprises a transmembrane domain. In some aspects, the CAR additionally comprises an intracellular signaling domain. In some aspects, the CAR further comprises a hinge region and/or a spacer region.

[0277] Certain aspects of the present invention are directed to a T cell receptor (TCT) comprising an antigen-binding region that specifically binds to the N-terminal extracellular domain of human CCR8. In some aspects, the antigen-binding region comprises an anti-CCR8 antibody disclosed herein or an antibody, or antigen-binding fragment thereof that binds to the same epitope as an anti-CCR8 antibody disclosed herein. In some aspects, the TCR additionally comprises a transmembrane domain. In some aspects, the TCR additionally comprises an intracellular signaling domain.

II.F. Nucleic acid molecules, vectors and cells

[0278] Certain aspects of the present invention are directed to nucleic acid molecules that encode the anti-CCR8 antibodies disclosed herein. Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA that is linked to DNA isolated in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl bands, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid described herein may be, for example, DNA or RNA and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0279] The nucleic acids described in this document can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by techniques standard PCR amplification or cDNA cloning. For antibodies obtained from an immunoglobulin gene library (e.g., using phage expression techniques), the nucleic acid encoding the antibody can be retrieved from the library.

[0280] In some aspects, the nucleic acids may additionally encode a signal peptide.

[0281] The nucleic acid molecules described in this document can be modified to delete specific sequences, for example, restriction enzyme recognition sequences, or to optimize codons.

[0282] A method for producing the anti-CCR8 antibody disclosed herein may comprise expressing the heavy chain and light chains in a cell line comprising the nucleotide sequences encoding the heavy and light chains with a signal peptide. Host cells comprising these nucleotide sequences are encompassed herein.

[0283] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into genes of the complete antibody chain, in Fab fragment genes or in a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operationally linked" as used in this context is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0284] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1 , CH2 and/or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1 region. For a Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

[0285] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0286] To create a scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser)3 , so that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, for example, Bird et al., (1988) Science 242:423-426 ; Huston et al., (1988) Natl. Sci. USA 85:5879-5883;

[0287] Also provided herein are nucleic acid molecules that encode VH and VL sequences that are homologous to those of the anti-CCR8 antibodies disclosed herein. Exemplary nucleic acid molecules encode VH and VL sequences that are at least 70% identical, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleic acid molecules encoding the VH and VL sequences disclosed herein. Also provided herein are nucleic acid molecules with conservative substitutions (i.e., substitutions that do not alter the resulting amino acid sequence upon translation of the nucleic acid molecule), for example, for codon optimization.

[0288] Also provided are nucleic acids encoding the VH and/or VL regions of anti-CCR8 antibodies, such as the anti-CCR8 antibodies described herein, which nucleic acids comprise a nucleotide sequence that is at least about 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences encoding the VH and/or VL regions of anti-CCR8 antibodies described herein.

[0289] Also provided are nucleic acids that encode the heavy chain and/or light chain of anti-CCR8 antibodies, such as the anti-CCR8 antibodies described herein, which nucleic acids comprise a nucleotide sequence that is at least about of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences encoding the heavy and/or light chains of anti-CCR8 antibodies described in this document.

[0290] Certain aspects of the present invention are directed to vectors comprising a nucleic acid molecule disclosed herein. In some aspects, the vector is selected from a viral vector, a mammalian vector, and a bacterial vector. In some aspects, the vector is a viral particle or a virus. In some respects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector.

[0291] In certain aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex virus and an adeno-associated virus (AAV) vector. In specific aspects, the vector is an AAV vector. In some respects, the vector is a lentivirus. In specific aspects, the vector is an AAV vector. In some respects, the vector is a Sendai virus. In some respects, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present invention can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23 (2103), which is incorporated by reference herein in its entirety.

[0292] In some aspects, the vector further comprises one or more regulatory elements, including, but not limited to, one or more enhancers, promoters, miRNA binding sequences, polyA sequences, intronic sequences, splice acceptor sites and any combination of these. In some aspects, the vector comprises a tissue-specific enhancer. In some aspects, the vector comprises a tissue-specific promoter.

[0293] Certain aspects of the present invention are directed to cells, e.g., host cells, comprising an anti-CCR8 antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein, a bi-paratopic antibody disclosed in this document, a CAR disclosed in this document, a TCR disclosed in this document, a nucleic acid molecule disclosed in this document or a vector disclosed in this document. The cell can be any type of cell. In some aspects, the cell is selected from a mammalian cell, a bacterial cell, an insect cell, a plant cell and a yeast cell. In some aspects, the cell is selected from the group consisting of an E. coli cell, a fungus such as Saccharomyces cerevisiae and Pichia pastoris, an insect cell such as SF9, a mammalian cell line (e.g., human cell lines ) and a primary cell line.

[0294] In some aspects, the cell is an immune cell. In some aspects, the cell is a T cell. As such, certain aspects of the present invention are directed to an immune cell, for example, a T cell, comprising a CAR or a TCR disclosed herein.

II.G. Pharmaceutical Compositions

[0295] In certain aspects, the invention provides a pharmaceutical composition comprising an anti-CCR8 antibody with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

[0296] In certain aspects, acceptable formulation materials are preferably non-toxic to receptors at the dosages and concentrations employed. In certain aspects, the formulation materials are for s.c. and/or I.V. administration. In certain aspects, the pharmaceutical composition may contain formulation materials to modify, maintain or preserve, for example, pH, osmolality, viscosity, clarity, color. , isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain aspects, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); to bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluent agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); distribution vehicles; diluents; pharmaceutical excipients and/or adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In certain aspects, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% sucrose In certain aspects, the ideal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the anticipated route of administration, delivery format and desired dosage. , for example, Remington's Pharma-ceutical Sciences, supra. In certain aspects, these compositions can influence the physical state, stability, in vivo release rate and/or in vivo clearance rate of the anti-CCR8 antibody.

[0297] In certain aspects, the primary vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in certain aspects, a suitable vehicle or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parental administration. In certain aspects, the saline solution comprises isotonic phosphate buffered saline. In certain aspects, neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. In certain aspects, the pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may additionally include sorbitol or a suitable substitute. In certain aspects, a composition comprising an anti-CCR8 antibody can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulating agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Additionally, in certain aspects, a composition comprising an anti-CCR8 antibody can be formulated as a lyophilisate using appropriate excipients, such as sucrose.

[0298] In certain aspects, the pharmaceutical composition can be selected for parenteral distribution. In certain aspects, the compositions may be selected for inhalation or distribution through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0299] In certain aspects, the components of the formulation are present in concentrations acceptable to the site of administration. In certain aspects, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of about 5 to about 8.

[0300] In certain aspects, when parenteral administration is contemplated, a therapeutic composition may be in the form of a parenterally acceptable, pyrogen-free aqueous solution, comprising an anti-CCR8 antibody, in a pharmaceutically acceptable vehicle. . In certain aspects, a vehicle for parenteral injection is sterile distilled water, in which an anti-CCR8 antibody is formulated as a sterile and suitably preserved isotonic solution. In certain aspects, the preparation may involve formulating the desired molecule with an agent, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, which can provide controlled release or sustained supply of product which can then be distributed via depot injection. In certain aspects, hyaluronic acid can also be used and can have the effect of promoting sustained duration in circulation. In certain aspects, implantable drug delivery devices can be used to introduce the desired molecule.

[0301] In certain aspects, a pharmaceutical composition can be formulated for inhalation. In certain aspects, an anti-CCR8 antibody can be formulated as a dry powder for inhalation. In certain aspects, an inhalation solution comprising an anti-CCR8 antibody can be formulated with a propellant for aerosol delivery. In certain aspects, solutions may be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

[0302] In certain aspects, it is contemplated that the formulations may be administered orally. In certain aspects, an anti-CCR8 antibody that is administered in this manner can be formulated with or without carriers commonly used in solid dosage forms, such as tablets and capsules. In certain aspects, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain aspects, at least one additional agent may be included to facilitate absorption of an anti-CCR8 antibody. In certain aspects, diluents, flavors, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be employed.

[0303] In certain aspects, a pharmaceutical composition may involve an effective amount of an anti-CCR8 antibody in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. In certain aspects, by dissolving the tablets in sterile water, or other suitable vehicle, solutions can be prepared in unit dose form. In certain aspects, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.

[0304] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving an anti-CCR8 antibody in sustained or controlled delivery formulations. In certain aspects, techniques for formulating a variety of other sustained or controlled delivery media, such as liposome carriers, bioerodible microparticles or porous granules and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain aspects, sustained-release preparations may include semipermeable polymeric matrices in the form of shaped articles, for example, films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (US Patent No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech. , 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain aspects, sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al, Proc. Natl. Academic. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0305] The pharmaceutical composition to be used for in vivo administration is typically sterile. In certain aspects, this can be achieved by filtration through sterile filtration membranes. In certain aspects, where the composition is lyophilized, sterilization using this method may be performed before or after lyophilization and reconstitution. In certain aspects, the composition for parental administration may be stored in lyophilized form or in a solution. In certain aspects, parenteral compositions generally are placed in a container with a sterile access port, for example, a vial or bag of intravenous solution that has a stopper pierceable by a hypodermic injection needle.

[0306] In certain aspects, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain aspects, such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0307] In certain aspects, kits are provided for producing a single dose administration unit. In certain aspects, the kit may contain a first container with a dry protein and a second container with an aqueous formulation. In certain aspects, kits containing pre-filled single-chamber and multi-chamber syringes (e.g., liquid syringes and lyo-syringes) are included.

[0308] In certain aspects, the effective amount of a pharmaceutical composition comprising an anti-CCR8 antibody to be employed therapeutically will depend, for example, on the therapeutic context and objectives. One skilled in the art will recognize that appropriate dosage levels for treatment will, in certain respects, therefore vary depending, in part, upon the molecule delivered, the indication for which an anti-CCR8 antibody is used, the route of administration and the size (body weight, body surface or organ size) and/or condition (age and general health status) of the patient. In certain aspects, the clinician can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0309] In certain aspects, the dosage frequency will take into account the pharmacokinetic parameters of an anti-CCR8 antibody in the formulation used. In certain aspects, a physician will administer the composition until a dosage is reached that achieves the desired effect. In certain aspects, the composition may therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via a device or catheter. implantation. Further refinement of the appropriate dosage is routinely done by those skilled in the art and is within the scope of the tasks routinely performed by them. In certain aspects, appropriate dosages can be determined through the use of appropriate dose response data.

[0310] In certain aspects, the route of administration of the pharmaceutical composition is in accordance with known methods, for example, orally, by injection intravenously, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular , intra-arterial, intraportal, intralesional routes; by sustained release systems or implantation devices. In certain aspects, the compositions may be administered by bolus injection or continuously by infusion or implantation device. In certain aspects, individual elements of the combination therapy may be administered by different routes.

[0311] In certain aspects, the composition can be administered locally via implantation of a membrane, sponge or other appropriate material in which the desired molecule has been absorbed or encapsulated. In certain aspects, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus, or continuous administration. In certain aspects, it may be desirable to use a pharmaceutical composition comprising an anti-CCR8 antibody ex vivo. In such examples, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an anti-CCR8 antibody, after which the cells, tissues and/or organs are subsequently implanted in back to the patient.

[0312] In certain aspects, an anti-CCR8 antibody can be delivered by implanting certain cells that have been genetically manipulated, using methods such as those described herein, to express and secrete the polypeptides. In certain aspects, these cells may be animal or human cells, and may be autologous, heterologous or xenogenic. In certain aspects, cells can be immortalized. In certain aspects, in order to decrease the chance of an immune response, cells can be encapsulated to prevent infiltration of surrounding tissues. In certain aspects, encapsulation materials are typically biocompatible semi-permeable polymeric shells or membranes that allow release of the protein product(s) but prevent destruction of the cells by the patient's immune system, or others. damaging factors from surrounding tissues.

III. Methods of the Invention

[0313] Certain aspects of the present invention are directed to methods of producing and/or using the anti-CCR8 antibodies disclosed herein.

III.A. Usage Methods

[0314] Certain aspects of the present invention are directed to methods of reducing, depleting or eliminating tumor-infiltrating Tregs, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present invention are directed to methods for reducing, depleting or killing tumor-infiltrating Tregs, comprising administering a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR , a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell or a pharmaceutical composition disclosed herein.

[0315] Certain aspects of the present invention are directed to methods of activating NK cells or inducing NK cell-mediated death of Tregs regulating tumor infiltration, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present invention are directed to methods of activating NK cells or inducing NK cell-mediated killing of Tregs regulating tumor infiltration, comprising administering a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR, a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell or a pharmaceutical composition disclosed herein.

[0316] In some aspects, the contact is in vitro. In some respects, the contact is in vivo. In some aspects, contact treats a disease or condition in a subject in need of it. In some respects, contact promotes an immune response in a subsection. In some aspects, the subject has a tumor and contact enhances an immune response to the tumor.

[0317] Certain aspects of the present invention are directed to methods of treating a tumor in a subject in need thereof, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present invention are directed to methods of treating a tumor in a subject in need thereof, comprising the administration of a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR, a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell or a pharmaceutical composition disclosed herein.

[0318] In some aspects, the subject has a tumor. In some aspects, the tumor is selected from the group consisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblast promyelocyte erythroleukemia, myelomonocytic monocytic leukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia , mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, Lymphoma Polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease , solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteoarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, sar -colon coma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma , papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, cancer uterine, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, he-mangioblastoma, neuroma acoustic, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, colon cancer of the uterus, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer , laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; cancer of the oral cavity (e.g. lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer, or any combination of these.

[0319] In some aspects, the tumor is refractory to previous therapy. In some aspects, the tumor is refractory to a previous standard of care therapy. In some aspects, prior therapy comprises immunotherapy, chemotherapy, surgery, radiotherapy or any combination thereof. In some aspects, the tumor is refractory to previous chemotherapy. In some aspects, the tumor is refractory to previous immunotherapy. In some aspects, the tumor is recurrent.

[0320] In some aspects, the tumor is advanced. In some aspects, the tumor is locally advanced. In some aspects, the tumor is metastatic.

[0321] In some aspects, the anti-CCR8 antibody is administered in combination with an additional anticancer agent. In some aspects, the additional anticancer agent is selected from a small molecule, a polypeptide, a radiation therapy, a surgery, and a combination thereof.

[0322] In some aspects, the anti-CCR8 antibody is administered before the additional anticancer agent. In some aspects, the anti-CCR8 antibody is administered after the additional anticancer agent. In some aspects, the anti-CCR8 antibody is administered at the same time as the additional anticancer agent. In some aspects, the anti-CCR8 antibody and the additional anticancer agent are formulated into a single composition.

[0323] In some aspects, the additional anticancer agent comprises chemotherapy. The chemotherapy may be any chemotherapy known in the art. In some respects, chemotherapy is a standard of care treatment for the specific type of cancer. In some respects, chemotherapy is platinum-based chemotherapy. In some aspects, chemotherapy is selected from the group consisting of doxorubicin (ADRIAMICIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide ( REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide ( THA-LOMID®), alemtuzumab (CAMPATH®), ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), carfilzomib (KYPROLISTM) and any combination of these.

[0324] In some aspects, the additional anticancer agent comprises an immunotherapy. In some aspects, immunotherapy is selected from a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, a CCL22 inhibitor, an agent that induces NK cell activation, and any combination of these.

[0325] In some aspects, additional anticancer therapy comprises a PD-1 antagonist. Any PD-1 antagonist known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the PD-1 antagonist is an antibody or antigen-binding portion thereof that specifically binds to PD-1. Non-limiting examples of PD-1 antagonists that can be used in combination with the anti-CCR8 antibodies disclosed herein include PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224 .

[0326] In some aspects, additional anticancer therapy comprises a PD-L1 inhibitor. Any PD-L1 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the PD-L1 inhibitor is an antibody or antigen-binding portion thereof that specifically binds to PD-L1. Non-limiting examples of PD-L1 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include FAZ053, Atezolizumab, Avelumab, Durvalumab and BMS-936559.

[0327] In some aspects, additional anticancer therapy comprises a TIM-3 inhibitor. Any TIM-3 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the TIM-3 inhibitor is an antibody or antigen-binding portion thereof that specifically binds to TIM-3. Non-limiting examples of TIM-3 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include MGB453 and TSR-022.

[0328] In some aspects, additional anticancer therapy comprises a LAG-3 inhibitor. Any LAG-3 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the LAG-3 inhibitor is an antibody or antigen-binding portion thereof that specifically binds to LAG-3. Non-limiting examples of LAG-3 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include LAG525, BMS-986016 and TSR-033.

[0329] In some aspects, additional anticancer therapy comprises a TIGIT inhibitor. Any TIGIT inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the TIGIT inhibitor is an antibody or antigen-binding portion thereof that specifically binds to TIGIT.

[0330] In some aspects, additional anticancer therapy comprises a CD112R inhibitor. Any CD112R inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the CD112R inhibitor is an antibody or antigen-binding portion thereof that specifically binds to CD112R.

[0331] In some aspects, additional anticancer therapy comprises a CCL22 inhibitor. Any CCL22 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the CCL22 inhibitor is an antibody or antigen-binding portion thereof that specifically binds to CCL22.

[0332] In some aspects, additional anticancer therapy comprises an agent that induces the activation of NK cells and, therefore, enhances the ADCC activity of the CCR8 antibody. In some aspects, the agent that induces NK cell activation is an antibody or antigen-binding portion thereof, a small molecule, a cytokine, or a fusion of cytokines.

[0333] In certain aspects, the additional anticancer agent comprises an anticancer agent selected from the group consisting of Sunitinib (SUTENT®), Cabozantinib (CABOMETYX®), Axitinib (INLYTA®), Lenvatinib (LENVIMA®), Everolimus (AFINITOR ®), Bevacizumab (AVASTIN®), epacadostat, NKTR-214 (residual CD-122 agonist), Tivozanib (FOTIVDA®), abexinostat, Ipilimumab (YERVOY®), tremelimumab, Pazopanib (VOTRIENT®), Sorafenib (NEXAVAR®), Temsirolimus (TORISEL®), Ramucirumab (CYRAMZA®), niraparib, savolitinib, vorolanib (X-82), Regorafenib (STIVARGO®), Donafenib (multikinase inhibitor), Camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), Ramucirumab (CYRAMZA®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), Tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate, nintedanib, lirilumab, Nivolumab (OPDIVO ®), Pembrolizumab (KEYTRUDA®), Atezolizumab (TECENTRIQ®), Avelumab (BAVENCIO®), Durvalumab (IMFIMZI®), Cemiplimab-rwlc (LIBTAYO®), tislelizumab, spartalizumab and any combination of these.

[0334] In some aspects, the additional anticancer agent comprises a TAM inhibitor (Axl, Mer, Tyro). In some aspects, the additional anticancer agent comprises a 4-1BB agonist. In some aspects, the additional anticancer agent comprises a Tyrosine Kinase Inhibitor (TKI). Non-limiting examples of TKIs that can be used in combination with the anti-CCR8 antibodies disclosed herein include imatinib mesylate, dasatinib, nilotinib and bosutinib.

[0335] The anti-CCR8 antibodies of the present invention can be administered by any suitable route. In some aspects, the anti-CCR8 antibody is administered intravenously. In some aspects, the anti-CCR8 antibody is administered subcutaneously. In some aspects, the anti-CCR8 antibody is administered intramuscularly. In some aspects, the anti-CCR8 antibody is administered intraperitoneally. In some aspects, the anti-CCR8 antibody is administered orally.

III.B. Methods for producing anti-CCR8 antibodies

[0336] The invention also features methods for producing any of the anti-CCR8 antibodies described herein. In some embodiments, the methods for preparing an antibody described herein may include immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. Suitable immunogens for generating any of the antibodies described herein are set forth herein. For example, to generate an antibody that binds to the N-terminal extracellular domain of human CCR8, one of skill in the art can immunize a suitable subject (e.g., a non-human mammal, such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse or a non-human primate) with a fragment of human CCR8 comprising the N-terminal extracellular domain. In some aspects, a fragment polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 172 is used as the immunogen.

[0337] A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen together with subsequent booster immunizations a number of times sufficient to provoke the production of an antibody by the mammal. The immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g. whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tuberculosis bacteria , complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, for example, aluminum hydroxide and iodoacetate and cholesterol hemisuccinate. Other adjuvants that can be used in methods for inducing an immune response include, for example, cholera toxin proteins and parapoxvirus. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, for example, Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J Med Chem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.

[0338] In some aspects, the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal, such as a laboratory mouse, is immunized with a CCR8 polypeptide as described above. Antibody-producing cells (e.g., spleen B cells) from the immunized mammal can be isolated two to four days after at least one immunogen booster immunization, and then grown briefly in culture before fusion with cells of a lineage. adequate myeloma cell. Cells can be fused in the presence of a fusion promoter, such as, for example, vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned and the cell clones that secrete the desired antibodies are selected. For example, spleen cells from Balb/c mice immunized with a suitable immunogen can be fused with cells of the PAI myeloma cell line or the Sp2/0-Ag 14 myeloma cell line. After fusion, the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example, HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing into the desired hybridoma cells. The hybrid cells obtained are then screened for secretion of the desired antibodies, for example, an antibody that binds to human CCR8, and in some aspects, one of skill can identify an anti-CCR8 antibody from a non-immunological residual library, as per described in, for example, US Patent No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0339] In some aspects, the methods described herein may involve, or be used in conjunction with, for example, phage expression technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cells and cell-free antibody screening techniques (e.g., ribosomal display) (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; al. (2000) Methods Enzymology 328:430-444; (2001) J Virol75:7107 7113; Schaffitzel et al. (1999) J Immunol Methods 231:119-135;

[0340] Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences that encode them. Such phage can be used to display antigen-binding antibody domains, such as Fab, Fv, or disulfide-stabilized Fv antibody fragments, expressed from a repertoire or combinatorial library of antibodies (e.g., human or murine). The phages used in these methods are typically filamentous phages, such as fd and M13. The antigen-binding domains are expressed as a protein recombinantly fused to any of the pIII, pVIII or pIX phage coat proteins. See, for example, Shi et al. (2010) JMB 397:385-396. Examples of phage expression methods that can be used to produce the immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and PCT publications No. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982 and WO 95/20401. Suitable methods are also described in, for example, US Patent No. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0341] In some aspects, phage expression antibody libraries can be generated using mRNA collected from B cells of immunized mammals. For example, a sample of splenic cells comprising B cells can be isolated from mice immunized with CCR8 polypeptide, as described above. mRNA can be isolated from cells and converted to cDNA using standard molecular biology techniques. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995), supra. The cDNA encoding the variable regions of the immunoglobulin heavy chain and light chain polypeptides is used to construct the phage display library. Methods for generating such a library are described in, for example, Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; DiNiro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

[0342] In some aspects, a combination of selection and screening may be employed to identify an antibody of interest from, for example, a population of hybridoma-derived antibodies or a library of phage display antibodies. Suitable methods are known in the art and are described in, for example, Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen-combining region are produced using techniques. molecular biology standard and then introduced into a population of bacteria (e.g., E. coli). Bacteriophage expression in bacteria may, in some aspects, require the use of a helper phage. In some aspects, no helper phage is necessary (see, for example, Chasteen et al., (2006) Nucleic Acids Res 34(21):e145). Phages produced from bacteria are recovered and then contacted with, for example, a target antigen bound to a solid (immobilized) support. The phage can also be contacted with the antigen in solution and the complex is subsequently attached to a solid support.

[0343] A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., human CCR8) using any immunological or biochemical method known in the art. For example, the specific binding of an antibody to CCR8 can be determined, for example, using immunological or biochemical methods, such as, without limitation, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography and equilibrium dialysis. , as described above. Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme-linked immunosorbent assay) , "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

[0344] In aspects where the selected CDR amino acid sequences are short sequences (e.g., less than 10 to 15 amino acids in length), the nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and US Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced by chemically synthesized nucleic acids using standard molecular biology techniques. The 5' and 3' ends of chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody. .

III.C. Expression and Purification of Recombinant Antibodies

[0345] The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the heavy chain and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, for example, promoter sequences, ribosomal binding sites, transcription start and stop sequences, translation start and stop sequences, transcription terminator signals, polyadenylation signals and enhancer or activator sequences. Regulatory sequences include a promoter and transcription start and stop sequences. Furthermore, the expression vector can include more than one replication system, so that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. .

[0346] Several possible vector systems are available for expressing heavy chain and light chain polypeptides cloned from nucleic acids in mammalian cells. One class of vectors depends on the integration of the desired gene sequences within the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg ( 1982) Mol Appl Genet 1:327). The selectable marker gene can be linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors uses DNA elements that confer autonomous replication capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81 :1292) or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0347] Expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the target cell type, discussed below. Exemplary methods include CaPO4 precipitation liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybene-mediated transfection, protoplast fusion and direct microinjection.

[0348] Suitable host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacterial, insect, plant and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g. human cell lines) as well as primary cell lines.

[0349] In some aspects, an antibody or fragment thereof can be expressed and purified from transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk, as described in, for example, Houdebine (2002) Curr Opin Biotechnol 13(6):625- 629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157.

[0350] Antibodies and fragments thereof can be produced from cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions and for a period of time sufficient to allow expression of the proteins . Such conditions for protein expression will vary with the choice of expression vector and host cell and will be easily verified by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, for example, Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of suitable codons, expression vectors and suitable host cells will vary depending on several factors and can be easily optimized as needed. An antibody (or fragment thereof) described herein may be expressed in mammalian cells or in other expression systems, including, but not limited to, yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0351] After expression, antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in various ways known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity chromatography and reversed-phase HPLC. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein A or protein G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, for example, Scopes (1994) "Protein Purification, 3rd edition," Springer-Verlag, New York City, New York. The degree of purification required will vary depending on the intended use. In some cases, purification of the expressed antibody or fragments thereof will not be necessary.

[0352] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, black starch protein assay , high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

III.D. Modification of Antibodies or Antigen-Binding Fragments Thereof

[0353] Antibodies or antigen-binding fragments thereof can be modified after their expression and purification. Modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the antibodies or fragments, for example, by reacting targeted amino acid residues of the polypeptide with an organic shunting agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria, including, for example, structural analysis or amino acid sequence analysis of the antibodies or fragments.

[0354] In some aspects, antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety. The heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label, such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal tag, a luminescent tag, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, for example, an antigenic tag (FLAG (DYKDDDDK (SEQ ID NO: 241)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 142), hemagglutinin ( HA; YPYDVPDYA (SEQ ID NO: 242)), glutathione-S-transferase (GST) or maltose-binding protein (MBP)) for use in purifying antibodies or fragments. , enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)) or chloramphenicol acetyl transferase (CAT). include, for example, 32P, 33P, 14C, 125I, 131I, 35S and 3H. Suitable fluorescent tags include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, Phycoerythrin (PE), propidium iodide (PI), PerCP, PE-A-lexa Fluor® 700, Cy5, allophycocyanin and Cy7 Luminescent markers include, for example, any of a variety of luminescent lanthanide chelates (). e.g. europium or terbium). For example, suitable europium chelates include diethylene triamine pentaacetic acid (DTPA) europium chelate or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzyme markers include, for example, alkaline phosphatase, CAT, luciferase and horseradish peroxidase.

[0355] Two proteins (for example, an antibody and a heterologous moiety) can be cross-linked using any of several known chemical cross-linkers. Examples of such cross-linkers are those that link two amino acid residues through a linkage that includes a "hindered" disulfide bond. In these bonds, a disulfide bond within the cross-linking unit is protected (preventing groups on both sides of the disulfide bond) from reduction by the action of, for example, reduced glutathione or the enzyme disulfide reductase. A suitable reagent, 4-succinimidyloxycarbonyl- -methyl- □(2-pyridyldithio) toluene (SMPT), forms this bond between two proteins using a terminal lysine in one of the proteins and a terminal cysteine in the other. Heterobifunctional reagents that cross-link through a different coupling fraction in each protein can also be used. Other useful crosslinkers include, without limitation, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimido-butane), a amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an a-amino group and a guanidinium group which is present in the arginine side chain (e.g. p-azidophenyl-glyoxal monohydrate).

[0356] In some aspects, a radioactive label can be directly conjugated to the main amino acid structure of the antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g., 125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS) that binds to free amino groups to form meta derivatives. -iodophenyl (mIP) of relevant proteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or chelated (e.g., to DOTA or DTPA) which, in turn, is bound to the main structure of the protein. Methods of conjugating the radioactive tags or larger molecules/chelates that contain them with the antibodies, or antigen-binding fragments, described in this document are known in the art. with the radioactive label under conditions (e.g., pH, salt concentration and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, for example, US Patent No. 6,001,329).

[0357] Methods for conjugating a fluorescent label (sometimes referred to as a "fluorophore") to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., lysines) or sulfhydryl groups (e.g., cysteines) of proteins using linked succinimidyl ester (NHS) or tetrafluorophenyl ester (TFP) moieties. to fluorophores. In some aspects, the fluorophores can be conjugated with a heterobifunctional crosslinker moiety, such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein, or stereo fragment, with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications," John Wiley and Sons (ISBN 0471495603).

[0358] In some aspects, the antibodies or fragments can be modified, for example, with a moiety that improves the stabilization and/or retention of the antibodies in circulation, for example, in blood, serum or other tissues. For example, the antibody or fragment can be PEGylated as described in, for example, Lee et al. (1999) Bioconjugate Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Delivery Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Deli very Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, for example, Pavisic et al. (2010) Int J Pharm 387(1-2):110-119). The stabilizing fraction can improve the stability or retention of the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) times.

[0359] In some aspects, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some aspects, an antibody or antigen-binding fragment thereof described herein may be subjected to enzymatic or chemical treatment, or produced from a cell, so that the antibody or fragment has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described, for example, in US patent no. 6,933,368; Wright et al. (1991) EMBO J 10(10): 2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

EXAMPLES Example 1: Generation of antibodies

[0360] Seventeen monoclonal antibodies specific for human CCR8 were generated against an N-terminal fragment of CCR8.

[0361] Antibody Phage Panning, Cloning and Transfection

[0362] Recombinant proteins expressing the N-terminal extracellular domain of human or cynomolgus CCR8 fused to a 6X His tag followed by mouse IgG2a-Fc (CCR8-Fc) were cloned into mammalian expression vectors (SEQ ID NOs: 173 and 174, respectively; Table 4). For the human protein, the free cysteine at position 25 was mutated to a serine to avoid the disulfide bond. The resulting secreted proteins were expressed by transfection into CHO cells and were purified using Protein A and used as antigen for phage panning using the Fab display library. For panning, the purified protein was coupled to Tosyl M280 beads or to ELISA plate, and panning done using standard methods. Successive rounds of panning were performed on human CCR8-ECD-Fc or in alternating rounds with cynomolgus CCR8-ECD-Fc, with unbound phage being removed by washing after each round. DNA from the resulting bound phage pool was isolated and the heavy and light chain sequences were cloned into a mammalian expression vector. Individual transformed colonies were grown separately in wells of a 96-well bacterial culture plate and DNA was purified using the Qiagen Turbo Miniprep Kit. 96-well DNA mini-libraries were used to transfect CHO cells in the same 96-well format and secreted antibody supernatants were harvested after three days of incubation in a 37oC, 7% CO2 incubator. Table 4: CCR8-ECD-Fc sequences (signal peptide; CCR8 extracellular domain; 6X His tag and linker; and mouse IgG2a-Fc)

[0363] Flow cytometry of CHO supernatants

[0364] 293T cells expressing human CCR8, Cynomolgus CCR8, mouse CCR8 and human CCR2 were harvested using Accutase® and 100,000 cells were distributed to each well of a 96-well v-bottom plate. In some cases, the native human CCR8-expressing cell line Hut78 was also tested. CHO supernatants from minilibraries were then added at a final dilution of 1:2 for each cell type and incubated at 4°C for 1 hour. After pelleting, cells were incubated with anti-human Fc-biotin followed by streptavidin-APC or with anti-human Fc-APC at 4oC for 30 minutes. After washing and fixation, the cells were placed on the Canto II FACS with propidium iodide for live/dead discrimination. Live cells were analyzed and clones that showed specific binding to CCR8 (human and/or cynomologus), but not CCR2, were sent for DNA sequencing and further testing.

[0365] Development of Anti-CCR8-1

[0366] The parental antibody, anti-CCR8-parental-1, was developed for phage panning, as described above, on the human CCR8 N-terminal protein and had an affinity of 1 nM on the 293T CCR8 cell line -Human. To improve affinity, a library was created in the Vaccinia Virus, where the CDR3 of the heavy chain was randomized. A library of 12,000 CDR3 variants was created in vaccinia. After overnight infection with the heavy chain CDR3 library (one clone per cell) and the parental light chain virus construct, the full-length human IgG antibody is expressed on the cell surface (reference). Infected A431 cells were incubated with human CCR8-N-terminal protein at a final concentration of 0.1 μg/ml, washed, and stained with anti-Fc-Dylight649 to detect binding of CCR8 protein and anti-Hu-Fab- FITC to detect the expression of antibodies on the cell surface. Two thousand cells with high antibody expression and high CCR8 binding were classified and the virus was amplified. DNA was extracted from the virus pool and the new heavy chain V genes were cloned into the mammalian cellular expression vector containing a signal sequence and the human IgG1 constant domain (resulting in full-length IgG1) and co-transfected together. with the parental light chain in CHO cells for clone evaluation as described above. It was found that the anti-CCR8-1 antibody (comprising a variable heavy chain having the amino acid sequence set forth in SEQ ID NO: 41 and a variable light chain having the amino acid sequence set forth in SEQ ID NO: 43 ) has three amino acid mutations in CDR3 compared to the parental heavy chain and an affinity of 0.4 nM in the human CCR8 cell line 293T. The anti-CCR8-1 antibody was found to bind to huCC8-ECD-Fc, but not CyCCR8-ECD-Fc (Figure 1A), and the antibody preferentially bound to 293T cells expressing HuCCR8, but not cyno CCR8. , human CCR2 or mouse CCR8 (Figure 1B).

[0367] Development of Anti-CCR8-1-1, Anti-CCR8-1-2, Anti-CCR8-2-3, Anti-CCR8-1-4 and Anti-CCR8-1-5

[0368] The heavy chain of the parental antibody (anti-CCR8-parental-1) was cloned into Vaccinia Virus to facilitate panning of the light chain shuffle with the Vaccinia Display human IgG libraries. Briefly, BHK 6x108 cells were infected with the heavy chain form of the anti-CCR8-parental-1 antibody (H23188) and a pool of lambda light chains from unexposed sources. After two days of incubation at 37oC, 7% of CO2, the supernatant was harvested and vaccinia virus particles expressing a human IgG library on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with purified human CCR8-Fc protein coupled to Tosil beads. M280. Unbound virus was washed away and bound virus was amplified by subsequent rounds. After performing two rounds on the human CCR8-Fc protein followed by two rounds on the cyno CCR8-Fc protein, DNA from the ligated pool was extracted and the new light chains cloned into a mammalian expression vector for CHO transfection along with flow cytometry and parental heavy chain analysis as detailed above. Anti-CCR8-1-1, anti-CCR8-1-2, anti-CCR8-2-3, anti-CCR8-1-4 and anti-CCR8-1-5 antibodies were found to bind to huCC8-ECD - Fc at a higher specificity than CyCCR8-ECD-Fc (Figures 1C, 1E, 1G, 1H and 1K, respectively) and each antibody preferentially bound to 293T cells expressing HuCCR8 over cyno CCR8, human CCR2 and CCR8 mouse (Figures 1D, 1F, 1H, 1J and 1K, respectively). Table 5: Anti-CCR8 Antibodies

[0369] Development of Anti-CCR8-2 and Anti-CCR8-2-1

[0370] The parental antibody (anti-CCR8-parental-2) was developed for phage panning, as highlighted above with two rounds on the human protein followed by two rounds on the N-terminal cynomolgus protein. It was found to bind to both human and cynomolgus CCR8 cell lines, with affinities of 19.9 nM and 11.1 nM, respectively. To improve affinity, a library was created in the phage where CDR1 and CDR2 of the heavy chain were randomized. This library was analyzed one round on the Cyno-CCR8-ECD-Fc protein followed by two consecutive rounds on the Human-CCR8-ECD-Fc protein. Individual phage clones were processed in a 96-well format and analyzed by phage ELISA on human and cynomolgus proteins as well as negative antigens. Clones that were specific for CCR8 were sent for sequencing and cloned into mammalian expression vectors for further characterization. The anti-CCR8-2 antibody had an improved affinity of 0.4 nM in 293T-HuCCR8 cells and 0.9 nM in 293T-Cyno CCR8 cells derived from two amino acid mutations each in CDR1 and CDR2. Both anti-CCR8-2 and anti-CCR8-2-1 antibodies showed binding to human and cyno CCR8 protein (Figures 2C and 2E) and cells expressing human and cyno CCR8 (Figures 2D and 2F).

[0371] Development of Anti-CCR8-2-2

[0372] The heavy chain of the parental antibody, anti-CCR8-parental-2, was cloned into Vaccinia Virus to facilitate light chain shuffle panning with Vaccinia Display human IgG libraries. Briefly, BHK 6x108 cells were infected with the heavy chain form of the anti-CCR8-parental-2 antibody (H23407) and a pool of lambda light chains from unexposed sources. After two days of incubation at 37°C, 7% CO2, the supernatant was harvested and vaccinia virus particles expressing a human IgG library on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with purified cynomolgus CCR8-Fc protein coupled to Tosil M280 beads. Unbound virus was washed away and bound virus was amplified by subsequent rounds. After performing two rounds on the cynomolgus CCR8-Fc protein, the bound pool was sorted by human/cyno cross-binders with 0.1 μg/ml biotin-cynomolgus CCR8-Fc and 0.1 μg/ml protein. Human CCR8-Fc. The highest binders for both proteins were collected, amplified, and DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis, as detailed above. The light chains that showed the best binding to the 293T-CCR8 cell lines were cross-paired with the heavy chain of the anti-CCR8-2 antibody (H23727) to verify the synergistic improvement in affinity. The anti-CCR8-2-2 antibody exhibited enhanced cross-reactive binding to human CCR8 compared to the parental (Figures 2E-2F). Table 6: Anti-CCR8 Antibodies

[0373] Development of Anti-CCR8-2-3 and Anti-CCR8-2-4

[0374] The parental antibody, anti-CCR8-parental-3 was developed for phage panning, as highlighted above with two rounds on the human protein followed by two rounds on the N-terminal cynomolgus protein. It was found to bind to human CCR8 cell lines, with an affinity of 6.8 nM. To improve affinity, a library was created in phage where the heavy chain CDR3 was randomized. This library was analyzed one round on the Cyno-CCR8-ECD-Fc protein followed by one round on the Human-CCR8-ECD-Fc protein. DNA from the ligated pool was extracted and cloned into a mammalian expression vector for CHO transfection and flow cytometry analysis as detailed above. Both anti-CCR8-2-3 and anti-CCR8-2-4 antibodies showed cross-reactive binding to human and cyno CCR8 cell lines (Figures 2G-2J).

[0375] Development of Anti-CCR8-2-5 and Anti-CCR8-2-6

[0376] A library of CDR3 variants for the anti-CCR8-parental-3 antibody heavy chain was also made in vaccinia virus. The library was sorted by human/cyno cross-linkers with 1 μg/ml biotin-cynomolgus CCR8-Fc and 1 μg/ml human CCR8-Fc protein. The highest binders for both proteins were collected, amplified, and DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis, as detailed above. Both anti-CCR8-2-5 and anti-CCR8-2-6 antibodies showed cross-reactive binding to human and cyno CCR8 cell lines (Figures 2K-2N).

[0377] Development of anti-CCR8-2-7, anti-CCR8-2-8, anti-CCR8-2-9 and anti-CCR8-2-10

[0378] The heavy chain of the anti-CCR8-parental-3 antibody was cloned into Vaccinia Virus to facilitate light chain scrambling panning with Vaccinex Vaccinia Display human IgG libraries. Briefly, BHK 6x108 cells were infected with the anti-CCR8-parental-3 antibody heavy chain (H23373) and a pool of lambda light chains from unexposed sources. After two days of incubation at 37°C, 7% CO2, the supernatant was harvested and vaccinia virus particles expressing a human IgG library on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with purified cynomolgus CCR8-Fc protein coupled to Tosil M280 beads. Unbound virus was washed away and bound virus was amplified by subsequent rounds. After performing two rounds on the cynomologous CCR8-Fc protein, the bound pool was sorted by human/cyno cross-binders with 0.1 μg/ml biotin-cynomolgus CCR8-Fc and 0.1 μg/ml CCR8- Human fc. The highest binders for both proteins were collected, amplified, and DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis, as detailed above. The light chains that showed the best binding to the 293T-CCR8 cell lines were cross-paired with the heavy chain of the anti-CCR8-2-3 antibody (H23499) and other heavy chains developed through the efforts of phage heavy chain CDR3. , in order to verify the synergistic improvement in affinity. The anti-CCR8-2-7, anti-CCR8-2-8, anti-CCR8-2-9 and anti-CCR8-2-10 antibodies showed enhanced binding to human CCR8 and cynomolgus cell lines over the anti-CCR8 antibody. -CCR8-parental-3 (Figures 2O-2V). Table 7: Anti-CCR8 Antibodies

Example 2: Antibodies bind to CCR8

[0379] Of the 17 antibodies, two were selected for additional characterization: anti-CCR8-1, comprising a variable heavy chain having the amino acid sequence established in SEQ ID NO: 41, and the variable light chain having the sequence of amino acids established in SEQ ID NO: 43; and anti-CCR8-2, comprising a variable heavy chain having the amino acid sequence set forth in SEQ ID NO: 11, and a variable light chain having the amino acid sequence set forth in SEQ ID NO: 13.

[0380] To test whether CCR8 antibodies bound to human or cynomolgus monkey CCR8, 293T and Raji cells expressed in cells were infected with lentivirus for human or cynomolgus monkey CCR8. Cell lines expressing CCR8 constructs were incubated with CCR8 antibodies in a dose-dependent manner at 4°C for 30 minutes, unbound CCR8 antibodies as removed by washing and bound CCR8 antibodies were detected using an antibody conjugated anti-human secondary with fluorescence at 4°C for 30 minutes. The results showed that both anti-CCR8-1 (Figure 3A) and anti-CCR8-2 (Figure 3B) bound to human CCR8 cell lines, while anti-CCR8-2 (Figure 3B) bound to the human CCR8 cell line. CCR8 from cynomolgus monkey. Example 3: Antibodies bind to CCR8+ tumor Tregs

[0381] To test whether CCR8 antibodies bound to CCR8+ tumor Tregs, tumor-infiltrating leukocytes (TILs) were isolated from freshly resected tumors and plated in 96-well plates. TILs were incubated with a fluorescently labeled panel of antibodies (CD3, CD4, FOXP3) to identify tumor Tregs at 4°C. In addition, CCR8 antibodies were incubated with TILs in a single concentration at 4°C for 30 minutes, unbound CCR8 antibodies were removed by washing, and bound CCR8 antibodies were detected using an anti-human secondary antibody conjugated with fluorescence at 4°C for 30 minutes. The results showed that both anti-CCR8-1 and anti-CCR8-2 bound to CCR8+ tumor Tregs (Figure 4A-4B).

Example 4: Antibodies induce ADCC signaling in the ADCC Reporter Bioassay

[0382] To test whether CCR8 antibodies induced ADCC signaling, CCR8 antibodies were incubated with 293T cells with forced expression of human or cynomolgus monkey CCR8 in a dose-dependent manner in 96-well plates for 6 hours at 37° C, according to the manufacturer's instructions. CD16 Jurkat antibody-dependent cellular cytotoxicity (ADCC) reporter cells were co-cultured with target cells complexed with CCR8 antibodies. After completion of the experiment, ADCC signaling was determined by a bioluminescent readout on CD16 Jurkat ADCC reporter cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC signaling using the human CCR8 cell line as targets, while anti-CCR8-2 also induced ADCC signaling using the CCR8 cell line. cynomolgus monkey (Figure 5).

Example 5: Antibodies induce ADCC of cells with forced expression of human and cynomolgus monkey CCR8 using PBMCs as effector cells

[0383] To test whether CCR8 antibodies induced ADCC of CCR8+ cells, CCR8 antibodies were incubated with 293T or Raji target cells with forced expression of human CCR8 and labeled with CellTrace Violet in a dose-dependent manner on 96-μm plates. wells. PBMCs were co-cultured with target cells complexed with CCR8 antibodies overnight at 37°C. After completion of the experiment, the number of CCR8+ target cells was evaluated by Flow Cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC of the human CCR8 cell lines (Figure 6A), while anti-CCR8-2 also induced ADCC of the cynomolgus monkey CCR8 cell line (Figure 6B).

Example 6: Antibodies induce ADCC of cells with forced expression of human CCR8 using NK cells as effector cells

[0384] To test whether CCR8 antibodies induced ADCC of CCR8+ cells, CCR8 antibodies were incubated with Raji target cells with forced expression of human CCR8 and labeled with Cell Trace Violet in a dose-dependent manner in 96-well plates. . NK cells were co-cultured with target cells complexed with CCR8 antibodies for 4 hours at 37°C. After completion of the experiment, the number of CCR8+ target cells was assessed by Flow Cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC of the human CCR8 cell line (Figures 7A-7B).

Example 7: Antibodies modulate markers of NK cell activation in the co-culture assay with forced human CCR8-expressing cells and kill CCR8-expressing cells

[0385] To test whether CCR8 antibodies modulated markers of NK cell activation, CCR8 antibodies were incubated with Raji target cells with forced expression of human CCR8 and labeled with CellTrace Violet in a dose-dependent manner on plates. of 96 wells. NK cells were co-cultured with target cells complexed with CCR8 antibodies overnight at 37°C. After completion of the experiment, the number of CCR8+ target cells was assessed by flow cytometry measuring the expression of 4-1BB, ICAM-1 and CD16 on the cell surface of NK cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced upregulation of 4-1BB and ICAM-1 on the cell surface while CD16 was downregulated on the cell surface of NK cells (Figure 8A , FIG 8C).

[0386] To test whether the CCR8 antibodies killed cells expressing CCR8, the antibodies were incubated with Raji cells and Raji target cells with forced expression of human CCR8 ("Raji-CCR8 cells") and labeled with CellTrace Violet of a dose-dependent manner in 96-well plates. After completion of the experiment, the number of CCR8+ target cells was assessed by flow cytometry measuring the number of CellTrace Violet positive cells remaining. The results showed that both anti-CCR8-1 and anti-CCR8-2 killed Raji-CCR8 cells and did not kill Raji cells without CCR8 (FIG. 8B).

Example 8: Antibodies induce ADCC of tumor Tregs using NK cells as effector cells

[0387] To test whether CCR8 antibodies induced ADCC of CCR8+ tumor Tregs, TILs were isolated from freshly resected human tumors and incubated with CCR8 antibodies. NK cells were co-cultured with TIL:antibody complexes in 96-well plates overnight at 37°C. After completion of the experiment, the number of tumor Tregs was assessed by flow cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced the death of human tumor Tregs (FIG. 9).

Example 9: Antibodies induce internalization of CCR8 in cells with forced expression of human CCR8

[0388] To test whether CCR8 antibodies induced internalization of the CCR8-IgG complex, CCR8 antibodies were incubated with pH-sensitive FabFluor reagent to generate a CCR8 internalization reporter antibody. This antibody conjugate was used to treat 293T cells with forced expression of human CCR8 for 30 min at 37°C. During antibody incubation in 293T cells, antibodies bind to CCR8 and induce internalization into an acidic endosome, causing a fluorescent signal from the conjugated FabFluor reagent. After completion of the experiment, the fluorescent signal can be analyzed by flow cytometry and compared between antibody conjugates. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced CCR8 internalization using the human CCR8 cell line as targets (FIG. 10).

Example 10: Retrogenix Antibody Binding Experiment

[0389] To identify CCR8 antibody targets, CCR8 antibodies were incubated with cells forced to express about 4,500 cell surface proteins in a fixed or live state. It was shown that anti-CCR8-1 binds only to CCR8, while anti-CCR8-2 also binds to amyloid precursor-like protein 2 (APLP2), but at a significantly lower concentration (data not shown).

Example 11: Characterization of anti-CCR8 antibody epitopes

[0390] To characterize the binding site of CCR8 antibodies, CCR8 antibodies were incubated with Raji-CCR8 cells in a dose-dependent manner in 96-well plates at 4C for 30 min. Unbound CCR8 antibodies were washed away and cells were incubated with a fluorescence-conjugated human monoclonal anti-CCR8 antibody (commercially available) at a single concentration for 30 min at 37°C. After completion of the experiment, binding of the commercially available CCR8 monoclonal antibody was assessed by Flow Cytometry. The results showed that anti-CCR8-1 partially blocked the commercially available CCR8 monoclonal antibody from binding to cells, while anti-CCR8-2 did not (FIG. 11).

Example 12: Afucosylated CCR8 antibodies show enhanced ADCC activity

[0391] Anti-CCR8-1 was further optimized to remove fucose sugar units from the Fc (IgG1) region of the antibody. To test the induction of ADCC signaling, CCR8 antibodies were incubated with 293T cells with forced expression of human CCR8 in a dose-dependent manner in 96-well plates for 6 h at 37°C according to the manufacturer's instructions. CD16VV or CD16FF Jurkat ADCC reporter cells were co-cultured with target cells complexed with CCR8 antibodies. After completion of the experiment, ADCC signaling was determined by a bioluminescent readout on CD16 Jurkat ADCC reporter cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC signaling using the human CCR8 cell line as targets, while anti-CCR8-2 also induced ADCC signaling using the human CCR8 cell line. lular CCR8 from cynomolgus monkey (FIGS. 12A-12B). Increased activity was observed with high and low affinity allelic polymorphisms.

Example 12: Antibodies bind tumor Tregs and induce ADCC

[0392] To test whether CCR8 antibodies bind to tumor Tregs and induce ADCC, tumor-infiltrating lymphocytes (TILs) were isolated from fresh kidney and breast tumor resections and incubated with the anti-CCR8-1 antibody. Anti-CCR8-1 antibody binding was measured using a PE-conjugated anti-human IgG antibody (FIG. 13A) and a commercially purchased purified anti-human CD213a1 antibody (IL-13-Rα1) was used as a control. positive (Biolegend catalog number 360404). As shown in FIG. 13A, triangles represent TIL from kidney tumors and circles represent TIL from breast tumors. All data were acquired by flow cytometry.

[0393] In a follow-up experiment, TILs isolated from fresh kidney and breast tumor resections were incubated with allogeneic NK cells and the anti-CCR8-1 antibody, mogamulizumab or isotype control for 24 hours. The percentage of CD3+ cells that were Tregs (FoxP3+) or other lymphocytes (FoxP3-) was measured using flow cytometry. In the presence of NK cells, the anti-CCR8-1 antibody resulted in a significant loss of Tregs while not affecting the non-Treg population.

[0394] Collectively, these data show that the anti-CCR8-1 antibody binds to tumor Tregs and causes NK cell-mediated ADCC. Table 8: Sequences

Claims (7)

1. Use of an antibody or an antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, characterized by the fact that it is for the preparation of a medicine, or pharmaceutical composition for the treatment of a tumor in an individual in need. 2. Vector or a set of vectors, characterized by the fact that it comprises the nucleic acid molecule or the set of nucleic acid molecules that encodes the antibody or antigen-binding portion thereof. 3. A method of reducing, depleting, or killing tumor-infiltrating regulatory T cells ("Treg"), comprising contacting Treg cells with the antibody or an antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, or the nucleic acid molecule encoding said antibody or antigen-binding portion thereof, or vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules encoding the antibody or antigen-binding portion thereof. 4. Method for activating NK cells or inducing NK cell-mediated killing of tumor-infiltrating regulatory T cells ("Treg"), comprising contacting Treg cells with the antibody or an antigen-binding portion thereof specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, or the nucleic acid molecule encoding said antibody or antigen-binding portion thereof, or vector or set of vectors comprising the nucleic acid molecule or the set of nucleic acid molecules that encodes the antibody or antigen-binding portion thereof. 5. Use of an antibody or antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, or the nucleic acid molecule encoding said antibody or antigen-binding portion thereof. antigen thereof, or vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules encoding the antibody or antigen-binding portion thereof, characterized by the fact that it is for the preparation of a drug to reduce, deplete, or kill tumor-infiltrating Treg cells in an individual in need. 6. Use of an antibody or antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, or the nucleic acid molecule encoding said antibody or antigen-binding portion thereof. antigen thereof, or vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules encoding the antibody or antigen-binding portion thereof, characterized by the fact that it is for the preparation of a drug to activate NK cells or induce NK cell-mediated killing of tumor-infiltrating Treg cells in an individual in need. 7. Use of an antibody or antigen-binding portion thereof that specifically binds to one or more amino acids within the N-terminal extracellular domain of human CCR8, or the nucleic acid molecule encoding said antibody or antigen-binding portion thereof. antigen thereof, or vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules encoding the antibody or antigen-binding portion thereof, characterized by the fact that it is for the preparation of a medicine, or pharmaceutical composition for the treatment of a tumor in an individual in need.