Anti-CC motif chemokine receptor 8 (CCR8) antibodies and methods of use

Abstract

The present disclosure provides anti-CCR8 antibodies and compositions, as well as methods for their preparation and use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 253,676, filed October 8, 2021, and U.S. Provisional Application No. 63 / 221,734, filed July 14, 2021, both of which are incorporated by reference in their entireties and claim priority.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in xml format via EFS-Web and is incorporated herein by reference in its entirety. The xml copy created on July 12, 2022 is named 00B206.1290.xml. [Background technology]

[0003] background Regulatory T (Treg) cells, which express the transcription factor Foxp3, are important for maintaining peripheral immune tolerance and preventing autoimmunity. See, e.g., Sakaguchi et al., Cell (2008) 133:775-787. Treg cells also constitute a major component of the immune infiltrate of solid cancers, promoting tumor initiation and progression by establishing an immunosuppressive tumor microenvironment and attenuating antitumor immune responses. See, e.g., Plitas and Rudensky, Annu. Rev. Cancer Biol. (2020) 4:459-477. Treg cells also impede the efficacy of immunotherapy. See, e.g., Nishikawa and Sakaguchi, Curr. Opin. Immunol. (2014) 27:1-7. An increased proportion of Treg cells among tumor-infiltrating lymphocytes is associated with poorer outcomes in several cancer indications. See, e.g., Fu et al., Gastroenterology (2007) 132:2328-2339; Petersen et al., Cancer (2006) 107:2866-2872; Shang et al., Nature-Scientific Reports (2015) 5:15179 (9 pages); Shen et al., J. Cancer Res. Clin. Oncol. (2010) 136:1585-1595; and Tanaka and Sakaguchi, Eur. J. Immunol. (2019) 49:1140-1146.

[0004] Several strategies aimed at depleting or inhibiting Treg cells have been shown to enhance antitumor immunity and result in tumor growth inhibition in preclinical breast cancer, melanoma and colon cancer models. See, e.g., Bos et al., J. Exp. Med. (2013) 2435-2446; Klages et al., Cancer Res. (2010) 70: 7788-7799; and Pastille et al., Cancer Res. (2014) 74: 4258-4269. However, strategies targeting surface receptors expressed on both Treg cells and effector T cells (e.g., CD25) have shown limited efficacy in established tumors, likely due to the concomitant depletion of effector T cells that are important for antitumor immunity. See, e.g., Onizuka et al. Cancer Res. (1999) 59: 3128-3133.

[0005] The chemokine receptor CCR8 is a seven-transmembrane G protein-coupled receptor (GPCR) that is ligated with high affinity by human / mouse CCL1 and is selectively and highly expressed by Treg cells in the tumor microenvironment, but is almost absent from peripheral Treg cells or effector T cells. High expression of CCR8 on Treg cells is associated with advanced disease stage and poor overall survival in breast cancer patients. See, for example, Plitas et al., Immunity (2016) 45: 1122-1134. Thus, CCR8 is a promising and safer target for Treg cell depletion in cancer treatment. Thus, agents that recognize CCR8 and methods of using such agents are desirable. Summary of the Invention

[0006] overview The present disclosure provides anti-CCR8 antibodies, compositions, and methods for preparing and using them.

[0007] Embodiment 1. In certain non-limiting embodiments, the subject matter of the present disclosure provides a monoclonal antibody that binds to CC motif chemokine receptor 8 (CCR8), comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:31; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:26; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:27; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:28.

[0008] Embodiment 2. The foregoing antibody of embodiment 1, which binds to CCR8 independently of CCR8 sulfation.

[0009] Embodiment 3. The antibody of embodiment 1 or 2, as described above, which binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID NO:106.

[0010] Embodiment 4. The antibody according to any one of embodiments 1 to 3, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 47; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0011] Embodiment 5. The antibody according to any one of embodiments 1 to 4, comprising a VH sequence selected from the group consisting of SEQ ID NOs: 35 to 47 and a VL sequence selected from the group consisting of SEQ ID NOs: 48 to 52.

[0012] Embodiment 6. The antibody described in any one of embodiments 1 to 5, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 47; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 48; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0013] Embodiment 7. The antibody according to any one of embodiments 1 to 6, comprising a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 47, and a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 48.

[0014] Embodiment 8. The antibody of any one of embodiments 1 to 7, wherein the VL comprises a V4M mutation, a P43A mutation, a F46L mutation, a C90Q mutation, or a combination thereof.

[0015] Embodiment 9. The antibody of any one of embodiments 1 to 8, wherein the VH comprises a G49S mutation, a K71R mutation, an S73N mutation, or a combination thereof.

[0016] Embodiment 10. The antibody according to any one of embodiments 1 to 9, comprising a heavy chain amino acid sequence of SEQ ID NO:55 and a light chain amino acid sequence of SEQ ID NO:56.

[0017] Embodiment 11. The antibody according to any one of embodiments 1 to 9, comprising a heavy chain amino acid sequence of SEQ ID NO: 60 and a light chain amino acid sequence of SEQ ID NO: 56.

[0018] Embodiment 12. The antibody according to any one of embodiments 1 to 9, comprising a heavy chain amino acid sequence of SEQ ID NO: 111 and a light chain amino acid sequence of SEQ ID NO: 56.

[0019] Embodiment 13. The antibody according to any one of embodiments 1 to 9, comprising a heavy chain amino acid sequence of SEQ ID NO: 113 and a light chain amino acid sequence of SEQ ID NO: 56.

[0020] Embodiment 14. In a specific non-limiting embodiment, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, comprising a VH sequence selected from the group consisting of SEQ ID NOs: 35-47 and a VL sequence selected from the group consisting of SEQ ID NOs: 48-52.

[0021] Embodiment 15. In a specific non-limiting embodiment, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, comprising a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

[0022] Embodiment 16. In certain non-limiting embodiments, the subject matter of the present disclosure provides a monoclonal antibody that binds to CCR8, comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3.

[0023] Embodiment 17. The antibody of embodiment 16, as described above, which binds to CCR8 independently of CCR8 sulfation.

[0024] Embodiment 18. The antibody of embodiment 16 or 17, which binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID NO: 106.

[0025] Embodiment 19. The antibody described in any one of embodiments 16 to 18, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0026] Embodiment 20. The antibody according to any one of embodiments 16 to 19, comprising a VH sequence selected from the group consisting of SEQ ID NOs: 10 to 21 and a VL sequence selected from the group consisting of SEQ ID NOs: 22 to 25.

[0027] Embodiment 21. The antibody described in any one of embodiments 16 to 20, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 21; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 24; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0028] Embodiment 22. The antibody described in any one of embodiments 16 to 21, comprising a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 21, and a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 24.

[0029] Embodiment 23. The antibody of any one of embodiments 16 to 22, wherein the VL comprises a Y2I mutation.

[0030] Embodiment 24. The antibody of any one of embodiments 16 to 23, wherein the VH comprises an S73N mutation, a V78L mutation, a T76N mutation, an F91Y mutation, and a P105Q mutation, or a combination thereof.

[0031] Embodiment 25. The antibody described in any one of embodiments 16 to 24, comprising a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58.

[0032] Embodiment 26. The antibody described in any one of embodiments 16 to 24, comprising a heavy chain amino acid sequence of SEQ ID NO: 61 and a light chain amino acid sequence of SEQ ID NO: 58.

[0033] Embodiment 27. The antibody described in any one of embodiments 16 to 24, comprising a heavy chain amino acid sequence of SEQ ID NO: 112 and a light chain amino acid sequence of SEQ ID NO: 58.

[0034] Embodiment 28. The antibody described in any one of embodiments 16 to 24, comprising a heavy chain amino acid sequence of SEQ ID NO: 114 and a light chain amino acid sequence of SEQ ID NO: 58.

[0035] Embodiment 29. In a specific non-limiting embodiment, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, comprising a VH sequence selected from the group consisting of SEQ ID NOs: 10-21 and a VL sequence selected from the group consisting of SEQ ID NOs: 22-25.

[0036] Embodiment 30. In a specific non-limiting embodiment, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, comprising the VH sequence of SEQ ID NO:21 and the VL sequence of SEQ ID NO:24.

[0037] Embodiment 31. In certain non-limiting embodiments, the subject matter of the present disclosure provides a monoclonal antibody that binds to CCR8, comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0038] Embodiment 32. The aforementioned antibody described in embodiment 31, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 95; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 94; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0039] Embodiment 33. The antibody of embodiment 31 or 32, comprising a VH sequence of SEQ ID NO: 95 and a VL sequence of SEQ ID NO: 94.

[0040] Embodiment 34. The antibody described in any one of embodiments 31 to 33, comprising a heavy chain amino acid sequence of SEQ ID NO: 101 and a light chain amino acid sequence of SEQ ID NO: 100.

[0041] Embodiment 35. The antibody described in any one of embodiments 31 to 33, comprising a heavy chain amino acid sequence of SEQ ID NO: 115 and a light chain amino acid sequence of SEQ ID NO: 100.

[0042] Embodiment 36. In certain non-limiting embodiments, the subject matter of the present disclosure provides a monoclonal antibody that binds to CCR8, comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0043] Embodiment 37. The antibody described in embodiment 36, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 97; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 96; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0044] Embodiment 38. The antibody of embodiment 36 or 37, comprising a VH sequence of SEQ ID NO: 97 and a VL sequence of SEQ ID NO: 96.

[0045] Embodiment 39. The antibody described in any one of embodiments 36 to 38, comprising a heavy chain amino acid sequence of SEQ ID NO: 103 and a light chain amino acid sequence of SEQ ID NO: 102.

[0046] Embodiment 40. The antibody described in any one of embodiments 36 to 38, comprising a heavy chain amino acid sequence of SEQ ID NO: 116 and a light chain amino acid sequence of SEQ ID NO: 102.

[0047] Embodiment 41. In certain non-limiting embodiments, the subject matter of the present disclosure provides a monoclonal antibody that binds to CCR8, comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81.

[0048] Embodiment 42. The aforementioned antibody described in embodiment 41, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 99; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 98; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0049] Embodiment 43. The antibody of embodiment 41 or 42, comprising a VH sequence of SEQ ID NO: 99 and a VL sequence of SEQ ID NO: 98.

[0050] Embodiment 44. The antibody described in any one of embodiments 41 to 43, comprising a heavy chain amino acid sequence of SEQ ID NO: 105 and a light chain amino acid sequence of SEQ ID NO: 104.

[0051] Embodiment 45. The antibody described in any one of embodiments 41 to 44, comprising a heavy chain amino acid sequence of SEQ ID NO: 117 and a light chain amino acid sequence of SEQ ID NO: 104.

[0052] Embodiment 46. In certain non-limiting embodiments, the presently disclosed subject matter provides monoclonal antibodies that bind to CCR8, wherein the monoclonal antibodies bind to CCR8 regardless of CCR8 sulfation.

[0053] Embodiment 47. The antibody of embodiment 46, which binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID NO: 106.

[0054] Embodiment 48. The antibody of embodiment 46, which binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID NO: 106.

[0055] Embodiment 49. In a specific, non-limiting embodiment, the subject matter of the present disclosure provides a monoclonal antibody that binds to mouse CCR8, comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0056] Embodiment 50. The aforementioned antibody described in embodiment 49, comprising: (a) a VH sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 70; (b) a VL sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 69; and (c) a sequence selected from the group consisting of the VH sequence defined in (a) and the VL sequence defined in (b).

[0057] Embodiment 51. The antibody of embodiment 49 or 50, comprising a VH sequence of SEQ ID NO: 70 and a VL sequence of SEQ ID NO: 69.

[0058] Embodiment 52. The antibody described in any one of embodiments 49 to 51, comprising a heavy chain amino acid sequence of SEQ ID NO: 72 and a light chain amino acid sequence of SEQ ID NO: 71.

[0059] Embodiment 53. The antibody of any one of embodiments 1 to 48, which is a human antibody.

[0060] Embodiment 54. The antibody of any one of embodiments 1 to 48, which is a humanized antibody.

[0061] Embodiment 55. The antibody of any one of embodiments 1 to 52, which is a chimeric antibody.

[0062] Embodiment 56. The antibody of any one of embodiments 1 to 55, which is an antibody fragment that binds to CCR8.

[0063] Embodiment 57. The antibody of any one of embodiments 1 to 56, which is a full-length antibody.

[0064] Embodiment 58. The antibody of embodiment 57, which is a full-length IgG1 antibody.

[0065] Embodiment 59. The antibody described in any one of embodiments 1 to 58, comprising an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:59.

[0066] Embodiment 60. The antibody of any one of embodiments 1 to 59, comprising a kappa constant domain comprising the amino acid sequence of SEQ ID NO:54.

[0067] Embodiment 61. The antibody described in any one of embodiments 1 to 60, which binds to CCR8 with a binding affinity (Kd) of about 1×10 M to about 1×10 M.

[0068] Embodiment 62. The antibody of any one of embodiments 1 to 48, wherein CCR8 is human CCR8.

[0069] Embodiment 63. The antibody of any one of embodiments 1 to 62, wherein the antibody is afucosylated.

[0070] Embodiment 64. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated nucleic acid encoding the aforementioned antibody of any of embodiments 1-63.

[0071] Embodiment 65. In certain non-limiting embodiments, the presently disclosed subject matter provides a host cell comprising the aforementioned nucleic acid described in embodiment 64.

[0072] Embodiment 66. In a specific, non-limiting embodiment, the presently disclosed subject matter provides a method for producing an antibody that binds to CCR8, comprising culturing the aforementioned host cell of embodiment 65 under conditions suitable for expression of the antibody.

[0073] Embodiment 67 The method of embodiment 66, further comprising recovering the antibody from the host cell.

[0074] Embodiment 68. In a specific, non-limiting embodiment, the presently disclosed subject matter provides an antibody produced by the aforementioned method of embodiment 67.

[0075] Embodiment 69. In certain non-limiting embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising the aforementioned antibody of any of embodiments 1-63 and a pharma- ceutically acceptable carrier.

[0076] Embodiment 70. The aforementioned pharmaceutical composition of embodiment 69, further comprising an additional therapeutic agent.

[0077] Embodiment 71. The antibody as defined in any one of embodiments 1 to 63 or the pharmaceutical composition as defined in any one of embodiments 69 to 70 for use as a medicament.

[0078] Embodiment 72. The antibody as described in any one of embodiments 1 to 63 or the pharmaceutical composition as described in any one of embodiments 69 to 70 for use in the treatment of cancer.

[0079] Embodiment 73. In a specific non-limiting embodiment, the presently disclosed subject matter provides the use of any one of the aforementioned antibodies of embodiments 1-63 or any of the aforementioned pharmaceutical compositions of embodiments 69-70 in the manufacture of a medicament for treating cancer.

[0080] Embodiment 74. In a specific, non-limiting embodiment, the presently disclosed subject matter provides the use of any one of the aforementioned antibodies of embodiments 1-63 or any of the aforementioned pharmaceutical compositions of embodiments 69-70 in the manufacture of a medicament for depleting regulatory T cells.

[0081] Embodiment 75. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody of any one of embodiments 1-63 or the pharmaceutical composition of any one of embodiments 69-70.

[0082] Embodiment 76. In certain non-limiting embodiments, the subject matter of the present disclosure provides a method for depleting regulatory T cells in a tumor microenvironment in a subject having cancer, comprising administering to the subject an effective amount of the antibody described in any one of embodiments 1-63 or the pharmaceutical composition described in any one of embodiments 69-70 sufficient to deplete regulatory T cells in the tumor microenvironment.

[0083] Embodiment 77. In certain non-limiting embodiments, the subject matter of the present disclosure provides a method for depleting regulatory T cells outside of a tumor microenvironment in a subject having cancer, comprising administering to the subject an effective amount of the antibody described in any one of embodiments 1-63 or the pharmaceutical composition described in any one of embodiments 69-70 sufficient to deplete regulatory T cells outside of the tumor microenvironment.

[0084] Embodiment 78. In certain non-limiting embodiments, the presently disclosed subject matter provides an in vitro method for depleting regulatory T cells from a cancer cell population, comprising contacting the cell population with an aforementioned antibody described in any one of embodiments 1-63 or an aforementioned pharmaceutical composition described in any one of embodiments 69-70 in an amount sufficient to deplete regulatory T cells from the cell population.

[0085] Embodiment 79. The use or method described above in any one of embodiments 73 to 78, wherein the cancer is selected from the group consisting of bladder cancer, blastoma, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, testicular cancer and uterine cancer.

[0086] Embodiment 80. The use or method of any one of embodiments 74, 76, 78 and 79, wherein regulatory T cells present in the tumor microenvironment of the cancer are depleted.

[0087] Embodiment 81. The use or method according to any one of embodiments 74, 77, 78 and 79, wherein regulatory T cells outside the tumor microenvironment of the cancer are depleted.

[0088] Embodiment 82. The use or method of any one of embodiments 73 to 81, further comprising administering an additional therapeutic agent.

[0089] Embodiment 83. The use or method described in embodiment 82, wherein the additional therapeutic agent is an anti-cancer agent.

[0090] Embodiment 84. The aforementioned use or method of embodiment 83, wherein the anticancer agent is selected from the group consisting of microtubule disrupting agents, metabolic antagonists, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormonal therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, immunomodulatory agents, inhibitors of cell adhesion, cytotoxic or cytostatic agents, activators of cell apoptosis, agents that increase the sensitivity of cells to apoptosis inducers, cytokines, anticancer vaccines or oncolytic viruses, Toll-like receptor (TLR) agents, bispecific antibodies, cell therapy, and immune cell engagers.

[0091] Embodiment 85. The use or method of embodiment 83 or 84, wherein the anticancer agent is a PD-L1 binding antagonist.

[0092] Embodiment 86. The aforementioned use or method of embodiment 85, wherein the PD-L1 binding antagonist is atezolizumab.

[0093] Embodiment 87. The use or method as described in any one of embodiments 73 to 85, wherein the subject is a human.

[0094] Embodiment 88. The use or method described in any one of embodiments 73 to 85, wherein the subject is a mouse.

[0095] Embodiment 89. In a specific, non-limiting embodiment, the presently disclosed subject matter provides a method for treating a disease in a mouse, comprising administering to the mouse an effective amount of a monoclonal antibody of any one of the preceding embodiments 49-52 to treat the disease.

[0096] Embodiment 90 The method of embodiment 89, wherein the mouse comprises a xenograft.

[0097] Embodiment 91. The antibody of embodiment 63, wherein the afucosylation rate is about 80% to about 95%.

[0098] Embodiment 92. The antibody of any one of embodiments 1 to 15 and 46 to 48, wherein the mean clearance after a single 10 mg / kg dose administered intravenously on day 1 is about 3 to about 5 mL / day / kg over 35 days. [Brief description of the drawings]

[0099] [Figure 1] Figure 1 shows the results of screening for anti-CCR8 monoclonal antibodies (mAbs) that selectively bind to Treg cells (Treg) from human colorectal cancer dissociated tumor cells (DTC) (obtained from Discovery Life Sciences). Mean fluorescence intensity (MFI) values ​​are shown for CD8 T cells (defined as CD45+CD14-CD3+CD8+CD4-) (circles, ), conventional CD4 T cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3-) (squares, ), and Treg cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3+) (triangles, ▲). Three of the five anti-CCR8 mAb clones Ab1-Ab5 specifically stained intratumoral Treg cells but not conventional CD4 or CD8 T cells and were ranked based on CCR8 MFI: hu.Ab4.H1L1>hu.Ab5.H1L1>hu.Ab3.H1L1. [Diagram 2] 2A-2B show the proposed mechanism of action of natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) leading to depletion of tumor-infiltrating CCR8-expressing Tregs (FIG. 2A) and the ADCC activity of human / cyno cross-reactive anti-CCR8 mAbs proposed for further study (FIG. 2B). EC50 values ​​were determined to be 0.02 nM, 0.02 nM, and 0.08 nM for anti-CCR8 mAbs hu.Ab3.H1L1, hu.Ab5.H1L1, and hu.Ab4.H1L1, respectively. [Diagram 3]Figures 3A-3D show the agonist and antagonist activities of human / cyno cross-reactive anti-CCR8 mAbs hu.Ab4.H1L1, hu.Ab5.H1L1, and hu.Ab3.H1L1, and comparator anti-CCR8 mAbs (humanized anti-human Yoshida anti-CCR8 antibody, mouse anti-human CCR8 mAb 433H (BD Biosciences), and mouse anti-human CCR8 mAb L263G8 (Biolegend)). As shown in Figure 3A, CCL1, a known ligand for CCR8, shows agonist activity, whereas none of the anti-CCR8 tested mAbs show agonist effects. The data in Figure 3B show that anti-CCR8 mAb hu.Ab4.H1L1 exhibits antagonist (neutralizing) activity against the CCR8 ligand CCL1 (20 nM ligand), whereas anti-CCR8 mAbs hu.Ab5.H1L1 and hu.Ab3.H1L1 show no ligand blocking activity (non-neutralizing) at the concentrations tested. The data in Figure 3C show that comparator anti-CCR8 mAbs (humanized anti-human Yoshida anti-CCR8 antibody, mouse anti-human CCR8 mAb 433H (BD Biosciences), and mouse anti-human CCR8 mAb L263G8 (Biolegend)) show no agonist effect, whereas the CCR8 ligand CCL1 shows an agonist effect. The data in FIG. 3D show that the comparator anti-CCR8 mAbs (humanized anti-human Yoshida anti-CCR8 antibody, mouse anti-human CCR8 mAb 433H (BD Biosciences), and mouse anti-human Biolegend L263G8 (Biolegend)) exhibit antagonist (neutralizing) activity against the CCR8 ligand CCL1. IC50 values ​​for ligand blocking activity are provided in the Examples. [Figure 4]4A-4F show binding data for hu.Ab3.H1L1 (FIG. 4A), hu.Ab4.H1L1 (FIG. 4B), and hu.Ab5.H1L1 (FIG. 4C), as well as the commercially available anti-CCR8 mAbs mouse anti-human CCR8 mAb 433H (BD Biosciences) (FIG. 4D), and mouse anti-human CCR8 mAb L263G8 (Biolegend) (FIG. 4E), and humanized anti-human Yoshida anti-CCR8 mAb (FIG. 4F), to HEK293 cells transiently transfected with N-terminally FLAG-tagged plasmids encoding human GPCRs (CCR2, CCR3, CCR4, CCR5, CCR8, CXCR4, ACKR2, and ACKR4), hCCR8 constructs, or mock constructs using transIT X2 (reagent:DNA=3:1). Cell surface expression of each GPCR was confirmed by staining with an anti-FLAG antibody control (5ug / mL). mAbs hu.Ab4.H1L1 and hu.Ab5.H1L1 stained only cells containing hCCR8, confirming their specificity for hCCR8. mAb hu.Ab3.H1L1 stained multiple other GPCRs, indicating a lack of specificity. The CCR8-selective hu.Ab4.H1L1 and hu.Ab5.H1L1 mAbs that showed the best ADCC activity (listed in Figure 2) were carried forward for further studies. [Diagram 5] Figures 5A-5D show alignments of the light chain variable region (Figure 5A) and heavy chain variable region (Figures 5B-5D) sequences of the rabbit (rb.Ab4) and humanized Ab4 (L1-L4 and H1-H12) CCR8 mAbs studied. Y2 on the light chain (L3) and S73, T76, V78, F91 and P105 on the heavy chain (H12) were determined to be important rabbit Vernier residues based on binding evaluation of the variant antibodies. The CDRs, variable regions, constant regions and full length sequences are provided in the Examples. [Figure 6]Figures 6A-6D show alignment of the light chain variable region (Figure 6A) and heavy chain variable region (Figure 6B-6D) of the studied rabbit (rb.Ab5) and humanized Ab5 (L1-L5 and H1-H13) CCR8 mAb sequences. The C90Q mutation in CDR L3 was introduced to remove an unpaired cysteine ​​that could be a cause during manufacturing. V4, P43 and F46 on light chain 6 (L1) and G49, K71 and S73 on heavy chain (H13) were determined to be critical rabbit Vernier residues based on binding evaluation of the variant antibodies. The CDRs, variable regions, constant regions and full-length sequences are provided in the Examples. [Figure 7] Figures 7A-7D show the results of cell-based affinity measurements of hu.Ab5.H13L1 and hu.Ab4.H12L3 mAbs using radiolabeled IgG and CHO cell lines stably expressing human CCR8 or cynomolgus monkey ("cyno") CCR8. The data show that hu.Ab4.H12L3 and hu.Ab5.H13L1 mAbs have similar affinity for both human and cyno CCR8 and demonstrate desirable cross-reactivity (compare Figure 7A with Figure 7B and Figure 7C with Figure 7D). Kd (nM) affinity data from these studies are provided in the Examples. [Figure 8] Figures 8A-8B show binding data for hu.Ab4.H12L3 (Figure 8A) and hu.Ab5.H13L1 (Figure 8B) mAbs to a panel of sulfated GPCRs, reaffirming that these Ab4 and Ab5 variants exhibit selectivity for CCR8, similar to the Ab4 and Ab5 data provided in Figures 4B and 4C. Due to poor binding of hCCR8 to the N-terminal FLAG tag (which affects Ab5 binding to the N-terminal epitope; see Figure 16), a CCR8 construct with a C-terminal FLAG is also shown in Figure 4C. [Figure 9]Figures 9A-9B show the effect of anti-CCR8 mAbs hu.Ab4.H12L3 and hu.Ab5.H13L1 on CCR8 activation as determined by Ca2+ influx assay (Figure 9A) and CCR8 CCL1 ligand binding (Figure 9B). Similar to the data in Figure 3A, Figure 9A reaffirms that neither the Ab4 nor the Ab5 anti-CCR8 mAb variants show agonist effects in the absence of CCR8 ligand CCL1. Similar to the data in Figure 3B, Figure 9B reaffirms that the Ab4 variants show antagonist effects on the CCR8 ligand CCL1 (20 nM ligand), whereas the Ab5 variants show no ligand blocking activity at the concentrations tested. IC50 values ​​for ligand blocking activity are provided in the Examples. [Figure 10] Figures 10A-10E show the difference in staining of hu.Ab4.H12L3 and hu.Ab5.H13L1 compared to humanized anti-human Yoshida CCR8 mAb and commercial antibodies mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend) on CCR8+ HEK293 cells with (hCCR8.TPST1 / 2 NTC) and without (hCCR8.TPST1 / 2 KO) tyrosyl-protein sulfotransferase (TPST)1 and tyrosyl-protein sulfotransferase (TPST)2. hu.Ab4.H12L3 (FIG. 10A) and hu.Ab5.H13L1 (FIG. 10B) showed similar binding / staining to both cell lines (hCCR8.TPST1 / 2 NTC and hCCR8.TPST1 / 2 KO), indicating that they bind CCR8 independent of tyrosine sulfation ("sulfation-independent"). In contrast, the humanized anti-human Yoshida CCR8 antibody (FIG. 10C) and the commercial antibodies mouse anti-human CCR8 mAb 433H (BD Biosciences) (FIG. 10D) and mouse anti-human CCR8 mAb L263G8 (Biolegend) (FIG. 10E) were unable to bind TPST1 / 2 KO cells, indicating that they require tyrosine sulfation of CCR8 for binding and are therefore considered "sulfation-dependent". [Figure 11]Figures 11A-11D show enhanced ADCC activity (>10-fold improvement) of afucosylated CCR8 mAbs Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 compared to their fucosylated CCR8 counterparts hu.Ab5.H13L1 and hu.Ab4.H12L3 against CHO cells stably expressing hCCR8 using NK-92 F158 (Figure 11A) and NK-92 V158 (Figure 11B) as effector cells, with 10-20-fold improved ADCC activity compared to the humanized anti-human Yoshida anti-CCR8 antibody (Figure 11C). The commercially available anti-CCR8 mAbs mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend) did not show ADCC activity (as expected) since the assay used is mainly related to antibodies containing human Fc regions (Figure 11C). Figure 11D shows that mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend) have ADCC activity using an assay specific for antibodies containing mouse Fc regions and human anti-CCR8 activity. Activity data is also provided in the examples. [Figure 12]12A-12D show selective ADCC activity against human Treg cells compared to conventional human CD4 T cells from peripheral blood mononuclear cells (PBMCs) harvested after transfer into NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice to induce CCR8 expression when incubated with afucosylated, fucosylated (hIgG1) and afucosylated isotype control mAb ("gD.afuc") and primary NK cells as effector cells. ADCC activity against Treg cells was measured by calculating the ratio of harvested Treg cells to harvested CD8 cells (Treg / CD8) or conventional CD4 T cells to harvested CD8 T cells (CD4conv / CD8). CCR8 mAbs Afuc.hu.Ab4.H12L3 and hu.Ab4.H12L3 selectively mediated ADCC activity against Treg cells (FIG. 12A) compared to conventional CD4 T cells (FIG. 12B), with the afucosylated variants exhibiting increased ADCC activity. Similarly, CCR8 mAbs Afuc.hu.Ab5.H13L1 and hu.Ab5.H13L1 selectively mediated ADCC activity against Treg cells (FIG. 12C) compared to conventional CD4 T cells (FIG. 12D), with the afucosylated variants exhibiting increased ADCC activity. [Figure 13]Figures 13A-13D show selective ADCC activity towards Treg cells compared to conventional CD4 T cells when human dissociated renal cell carcinoma (RCC) cells were incubated with afucosylated, fucosylated (hIgG1) and afucosylated isotype control mAb ("gD.afuc") and primary NK cells as effector cells. ADCC activity towards Treg cells was measured by calculating the ratio of recovered Treg cells to recovered CD8 cells (Treg / CD8) or conventional CD4 T cells to recovered CD8 T cells (CD4conv / CD8). CCR8 mAbs Afuc.hu.Ab4.H12L3 and hu.Ab4.H12L3 selectively mediated ADCC activity towards Treg cells (Figure 13A) compared to conventional CD4 T cells (Figure 13B), with the afucosylated variants showing increased ADCC activity. Similarly, CCR8 mAbs Afuc.hu.Ab5.H13L1 and hu.Ab5.H13L1 selectively mediated ADCC activity against Treg cells (FIG. 13C) compared to conventional CD4 T cells (FIG. 13D), with the afucosylated variants showing increased ADCC activity. [Figure 14] Figures 14A-14E show that afucosylated anti-CCR8 mAbs Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 exhibit enhanced ADCP activity compared to fucosylated mAbs hu.Ab5.H13L1 and hu.Ab4.H12L3 in CD14+ monocyte-derived macrophages from four different donors with FcgRIIa(H131R) / FcgRIIIa(V158F) genotypes of HR / FF (Figure 14A), RR / FF (Figure 14B), HR / VF (Figure 14C) and RR / VF (Figure 14D), and also show a 3-4 fold improvement in ADCP activity compared to humanized anti-human Yoshida anti-CCR8 antibody (Figure 14E). Activity data are also provided in the Examples. [Figure 15]Figures 15A-15D show that the afucosylated anti-CCR8 mAb Afuc.hu.Ab5.H13L1 exhibits similar improved ADCP activity compared to the FcgRIIa-enhanced G236A.I332E variant Afuc.hu.Ab5.H13L1.G236A.I332E in CD14+ monocyte-derived macrophages from four different donors with the FcgRIIa(H131R) / FcgRIIIa(V158F) genotypes: HR / FF (Figure 15A), RR / FF (Figure 15B), HR / VF (Figure 15C), and RR / VF (Figure 15D). [Figure 16] Figures 16A-16B are epitope maps of the hu.Ab5.H13L1 (Figure 16A) and hu.Ab4.H12L3 (Figure 16B) mAbs. As shown in Figure 16A, constructs were made encoding individual alanine point mutations at positions 2-24 of hCCR8 with a C-terminal FLAG tag, and hu.Ab5.H13L1 did not bind to D2A, Y3A, L5A, or D6A, indicating that the epitope includes at least the DYTLD region of the human CCR8 N-terminus. As shown in Figure 16B, constructs were made encoding human CCR8.CCR5 chimeras (N-term1, N-term2, ECL1, ECL2, and ECL3) in which different extracellular regions of hCCR8 were replaced with the corresponding regions from CCR5 with a C-terminal FLAG tag; hu.Ab4.H12L3 did not bind to the ECL1 and ECL2 chimeras, indicating that the epitope of this antibody includes at least the ECL1 and ECL2 regions of CCR8. huCCR8 N-term: MDYTLDLSVTTVTDYYYPDIFSSP (SEQ ID NO: 110). [Figure 17]Figures 17A-17I show progressive depletion of Treg cells (measured as the percentage of Treg cells with CD45+ leukocytes) in the tumor (Figure 17A), but not in the spleen (Figure 17B) or tumor-draining lymph nodes (Figure 17C) in CT26 tumor-bearing mice 3 days after injection of a single dose of increasing concentrations of mouse surrogate anti-CCR8 mAb from 0.003 to 5 mg / kg. Anti-CCD8 mAb treatment did not result in depletion of CD4 conventional T cells (Figures 17D-17F) or CD8 T cells (Figures 17G-I). An isotype control antibody (anti-gp120) was used. [Figure 18] Figures 18A-18D show tumor growth inhibition after treatment with a single dose (Figure 18B) or twice weekly (Figure 18C) of murine surrogate anti-CCR8 mAb in mice bearing established CT26 syngeneic tumors, compared to treatment with anti-CD25 mAb (Figure 18D) or isotype control mAb (anti-gp120) (Figure 18A). Treatment was initiated when tumors reached a volume of 150-250 mm3. Tumor volumes are measured over time. Grey lines represent individual mice, black lines represent group fitting. [Figure 19] Figures 19A-19E show CT26 tumor growth inhibition observed with effector-competent mouse surrogate anti-CCR8 mAb administered at the time of tumor inoculation (Figure 19B) or tumors reaching 150-250 mm3 (Figure 19D). No tumor growth inhibition is observed with an effector-incompetent LALAPG variant of the same ligand-blocking anti-CCR8 mAb (Figures 19C and 19E). Tumor volumes are measured over time. Grey lines represent individual mice and black lines represent group fitting. An isotype control mAb (anti-gp120) was used (Figure 19A). [Figure 20] Figures 20A-D show that the combination of mouse surrogate anti-CCR8 mAb with anti-PDL1 mAb (Figure 20D) is unexpectedly more effective in inhibiting EMT6 tumor growth than anti-CCR8 mAb alone (Figure 20B) or anti-PDL1 mAb alone (Figure 20C). Treatment was initiated when tumors reached 150-250 mm3. Tumor volumes are measured over time. Grey lines represent individual mice and black lines represent group fitting. An isotype control mAb (anti-gp120) was used (Figure 20A). [Figure 21] Figure 21 shows the serum pharmacokinetic profiles (mean ± SD) of anti-gD (control) and test anti-CCR8 mAbs Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 in cynomolgus monkeys after a single 10 mg / kg intravenous bolus injection. Afuc.hu.Ab5.H13L1 demonstrated desirable sustained serum concentration levels over 35 days post-dosing, which is expected to elicit more sustained target engagement that may translate into better anti-cancer activity and less frequent dosing. [Figure 22] Figures 22A-22C show the results of whole blood flow cytometry analysis of total Treg cell numbers from nine male cynos administered 10 mg / kg afucosylated anti-gD (control; group 1 designated 1001, 1002, 1003; Figure 22A), Afuc.hu.Ab5.H13L1 (group 2 designated 2001, 2002, 2003; Figure 22B), or Afuc.hu.Ab4.H12L3 (group 3 designated 3001, 3002, 3003; Figure 22C) by intravenous injection. Both test anti-CCR8 mAbs did not substantially reduce absolute total T-reg cell numbers in whole blood up to 840 hours after administration. [Figure 23]Figures 23A-23I show the results of whole blood flow cytometry analysis for the depletion of CCR8+FoxP3+ Treg cells in nine male cynos administered afucosylated anti-gD (control; group 1 designated as 1001 (Figure 23A), 1002 (Figure 23B), 1003 (Figure 23C)), Afuc.hu.Ab4.H12L3 (group 3 designated as 3001 (Figure 23D), 3002 (Figure 23E), 3003 (Figure 23F)), or Afuc.hu.Ab5.H13L1 (group 2 designated as 2001 (Figure 23G), 2002 (Figure 23H), 2003 (Figure 23I)). Blood was collected from each animal prior to dosing ("Pre-study") and at 0 hours on Day 1 ("Pre-dose"). Each animal was then administered a single dose of 10 mg / kg of afucosylated anti-gD (control group), Afuc.hu.Ab5.H13L1 (group 2) or Afuc.hu.Ab4.H12L3 (group 3) by intravenous injection. Blood was then collected from the animals and subjected to the following treatments prior to flow cytometric analysis: (i) blood samples not spiked with any of the tested CCR8 mAbs ("non-spiked"), (ii) blood samples further spiked with a saturating concentration of Afuc.hu.Ab5.H13L1, and (iii) blood samples further spiked with a saturating concentration of Afuc.hu.Ab4.H12L3. Each of the non-spiked and spiked samples was then treated with a labeled goat anti-human IgG antibody and analyzed by flow cytometry. As seen in Figures 23A-23C, flow cytometry of blood initially treated with control (group 1) but not spiked showed no modulation of total CCR8+ T-reg cells. Furthermore, flow cytometry of spiked blood also had little effect on total CCR8+ T-reg cell numbers. For group 3, as seen in Figures 23D-23F, flow cytometry of blood analyzed in each of the three animals showed a reduction in CCR8+ T-reg cells up to 168 hours post-dose. For group 2, as seen in Figures 23G-23I, flow cytometry of blood analyzed showed a reduction in CCR8+ T-reg cells in animals 2002 and 2003.Both Group 2 and Group 3 animals showed little or no effect on total Treg cell numbers (Figures 22A-22C), but showed a reduction in the number of peripheral blood CCR8+ T-reg cells after spiked or unspiked dosing (Figures 23D-23I), consistent with the proposed mechanism of action (see Figure 2A). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0100] Detailed Description of Specific Embodiments I. Definition For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0101] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the specific binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by methods common in the art, including those described herein. Specific illustrative, exemplary methods for measuring binding affinity are also described herein.

[0102] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not possess the alterations, which alterations improve the affinity of the antibody for antigen.

[0103] The terms "anti-CCR8 antibody" and "antibody that binds to CCR8" refer to an antibody that is capable of binding to CCR8 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CCR8. In one embodiment, the extent of binding of an anti-CCR8 antibody to an unrelated, non-CCR8 protein is less than about 10% of the binding of the antibody to CCR8, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to CCR8 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -13 M~10 -8 M, for example, 10 -13 M~10 -9 Dissociation constant (K D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 1×10 -12 M ~ approx. 1×10 -10 M, about 1 x 10 -12 M ~ approx. 1×10 -11 M, or approximately 1 x 10 -11 M ~ approx. 5×10 -11 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 2×10 -11 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 5×10 -12 K of M D The antibody has a K of 1 μM or less. D An antibody is said to "specifically bind" to CCR8 if it has the following structure: In certain embodiments, an anti-CCR8 antibody binds to an epitope of CCR8 of at least two different species (e.g., human and cyno).

[0104] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0105] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology (2005) 23:1126-1136.

[0106] The term "epitope" refers to a site on an antigen, either proteinaceous or non-proteinaceous, to which an anti-CCR8 antibody binds. Epitopes can be formed from a continuous stretch of amino acids (linear epitopes) or can include non-contiguous amino acids (conformational epitopes), and are formed in spatial proximity, for example, due to antigen folding (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound by anti-CCR8 antibodies after exposure of the proteinaceous antigen to a denaturing agent, whereas conformational epitopes are typically destroyed by treatment with a denaturing agent. An epitope includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 30, or at least 35, or 3-25, 3-20, 3-15, 3-10, 3-5, 30-40, 35-40, or 5-10 amino acids in a unique spatial conformation.

[0107] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind the same epitope) can be performed using methods routine in the art, such as, for example, but not limited to, alanine scanning, peptide blotting (see, e.g., Kobeissy et al., Meth. Mol. Biol. (2004) 248:443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Hochleitner et al., Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0108] Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows multiple monoclonal antibodies that specifically bind to CCR8 to be classified based on their respective binding profiles to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinct from epitopes represented in other classifications or may be a unique epitope that overlaps in part.

[0109] Competitive binding can also be used to easily determine whether an antibody binds to the same epitope of CCR8 as a reference antibody or competes for binding with a reference anti-CCR8 antibody. For example, an "antibody that binds to the same epitope" as a reference anti-CCR8 antibody refers to an antibody that inhibits the binding of the reference anti-CCR8 antibody to the antigen by 50% or more in a competitive assay, and conversely, the reference antibody inhibits the binding of the antibody to the antigen by 50% or more in a competitive assay. Also, for example, to determine whether an antibody binds to the same epitope as a reference anti-CCR8 antibody, the reference antibody can be bound to CCR8 under saturation conditions. After removing excess reference anti-CCR8 antibody, the ability of the anti-CCR8 antibody in question to bind to CCR8 is evaluated. If the anti-CCR8 antibody can bind to CCR8 after saturation binding of the reference anti-CCR8 antibody, it can be concluded that the anti-CCR8 antibody binds to a different epitope than the reference anti-CCR8 antibody. However, if the anti-CCR8 antibody cannot bind to CCR8 after saturation binding of the reference anti-CCR8 antibody, this anti-CCR8 antibody may bind to the same epitope as the reference anti-CCR8 antibody binds. To confirm whether the antibody in question binds to the same epitope or only steric reasons prevent binding, routine experiments can be used (e.g. peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two setups, i.e., both antibodies are saturating antibodies. In both setups, if only the first (saturating) antibody can bind to CCR8, it can be concluded that this anti-CCR8 antibody and the reference anti-CCR8 antibody compete for binding to CCR8.

[0110] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0111] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0112] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portions of the heavy and / or light chain are derived from a different source or species.

[0113] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0114] As used in this application, the term "constant region of human origin" or "human constant region" refers to the constant heavy chain region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4, and / or the constant light chain kappa or lambda region. Such constant regions are known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system (also called the EU index of Kabat) as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0115] "Effector functions" refer to biological activities attributable to the Fc region of an antibody and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0116] For example, an "effective amount" of an agent in a pharmaceutical composition refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0117] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, the antibody produced by the host cell may comprise a full-length heavy chain or may comprise a cleaved variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. In one aspect, the heavy chain comprising an Fc region as specified herein comprised in an antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, the heavy chain comprising an Fc region as specified herein comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0118] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally appear in the following order in a VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0119] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc region as defined herein. A full length antibody is understood to include a heavy chain variable domain and a light chain variable domain as defined herein, and an Fc region as defined herein.

[0120] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. As used herein, included are progeny of mutants that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0121] A "human antibody" is an antibody having an amino acid sequence that corresponds to an antibody produced by a human or a human cell, or to an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0122] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0123] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0124] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, e.g., each of the "complementarity determining regions" (CDRs).

[0125] In certain embodiments, the antibody comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). In certain embodiments, the antibody comprising six CDRs is a full-length antibody. In certain embodiments, the antibody comprising six CDRs is an antibody fragment.

[0126] Exemplary CDRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0127] Unless otherwise indicated, CDRs are determined according to Kabat et al. (supra) and Chothia (supra). One of skill in the art will understand that the designations of CDRs may also be determined according to McCallum (supra) or any other scientifically accepted nomenclature system.

[0128] In one aspect, the CDR residues include those identified in Figures 5A-5D and 6A-6D and Tables C1, C2, D1 and D2. In another aspect, the CDR residues include those identified in Tables N1, N2, O1 and O2.

[0129] A "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the subject is a human.

[0130] An "isolated" antibody is one that is separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0131] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands and single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies described herein in vitro and / or in vivo, e.g., in a host or subject. Such DNA vectors (e.g., cDNA) or RNA vectors (e.g., mRNA) can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo. (See, e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2101823 B1).

[0132] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from components of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from the natural chromosomal location.

[0133] "Isolated nucleic acid encoding an anti-CCR8 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-CCR8 antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations within a host cell.

[0134] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, except for variant antibodies that may, for example, contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation, such variants being generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the present disclosure may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or a portion of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0135] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0136] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, containing two identical light chains and two identical heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0137] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which may contain information regarding the indications, use, dosage, administration, concomitant therapy, contraindications and / or warnings pertaining to such therapeutic product.

[0138] "Percentage of amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity for the purpose of alignment. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation at the US Copyright Office, Washington DC, 20559, registered under US Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0139] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package is described by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global rather than local alignment. The percent amino acid identity is given in the output alignment header.

[0140] The terms "pharmaceutical composition" or "pharmaceutical formulation" are used interchangeably herein and refer to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0141] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0142] The term "CCR8" as used herein refers to any native CCR8 from any vertebrate source, including mammals such as primates (e.g., humans, monkeys (cyno)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CCR8, and any form of CCR8 resulting from processing within a cell. The term also encompasses naturally occurring variants of CCR8, such as splice variants or allelic variants. In certain embodiments, the CCR8 is human CCR8 ("hCCR8" or "huCCR8"). An exemplary human CCR8 amino acid sequence is set forth in SEQ ID NO: 106, as shown in the table below. In certain embodiments, the CCR8 is cynomolgus monkey ("cyno") CCR8. An exemplary cyno CCR8 amino acid sequence is set forth in SEQ ID NO: 107, as shown in the table below. In certain embodiments, the CCR8 is mouse CCR8 ("mCCR8"). The amino acid sequence of an exemplary mouse CCR8 is set forth in SEQ ID NO: 108, as shown in the table below. [Table 1]

[0143] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease (e.g., cancer) in the subject being treated, and can be performed for prevention ("prophylactic treatment" or "prophylactically treating") or during the course of clinical pathology ("therapeutic treatment" or "therapeutic treating"). Desirable effects of therapeutic treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis of cancer, reduction in the rate of disease progression, amelioration or remission of the disease, and remission or improvement of prognosis. Desirable effects of preventative treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease. In some aspects, the antibodies described herein are used to delay the onset of the disease or to slow the progression of the disease.

[0144] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using the VH or VL domain of an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0145] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0146] II. Compositions and Methods In one aspect, the present disclosure is based in part on the discovery of novel anti-CCR8 antibodies with unique and improved binding and selectivity to CCR8. The anti-CCR8 antibodies of the present disclosure also have improved antibody stability (e.g., low aggregation, good solubility, and low viscosity). The present disclosure is further based in part on the discovery that afucosylated forms of the antibodies of the present disclosure have increased antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) activity. In certain aspects, antibodies that bind to CCR8 are provided. Antibodies as described herein are useful, for example, for the treatment of cancer.

[0147] A. Exemplary Anti-CCR8 Antibodies In one aspect, the disclosure provides an antibody that binds to CCR8. In one aspect, the antibody provided is an isolated antibody that binds to CCR8. In one aspect, the disclosure provides an antibody that specifically binds to CCR8. In certain aspects, the anti-CCR8 antibody binds to an epitope that includes one or more of amino acid residues 2-6 of SEQ ID NO: 106. In certain aspects, the anti-CCR8 antibody binds to an epitope that includes one or more of amino acid residues 91-104 and 172-193 of SEQ ID NO: 106. In certain aspects, the CCR8 is human CCR8, mouse CCR8, or cyno CCR8. In certain aspects, the CCR8 is human CCR8. In one aspect, the disclosure provides an antibody that binds to CCR8 independent of tyrosine sulfation of CCR8 ("sulfation-independent"). Exemplary antibodies that the inventors have discovered to be sulfation-independent include Ab4 and Ab5, and are further described in more detail below.

[0148] In certain aspects, the antibodies provided herein have a potency of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 1×10 -12M ~ approx. 1×10 -10 M, about 1 x 10 -12 M ~ approx. 1×10 -11 M, or approximately 1 x 10 -11 M ~ approx. 5×10 -11 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 2×10 -11 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 5×10 -12 K of M D In one embodiment, K D is measured using radiolabeled IgG and a CHO cell line stably expressing the antigen. Stable CHO cells expressing the antigen are seeded at 50,000 cells / well in cold binding buffer (Opti-MEM + 2% FBS + 50 mM HEPES, pH 7.2 + 0.1% sodium azide). Binding of a fixed concentration of the desired IgG using the NEX244 Iodogen method (Perkin Elmer) is performed. 125 I Radiolabeled antigen is mixed with serially diluted antibodies of interest, starting at 20 nM or 50 nM. The antibody mixture is added to the cells and incubated at room temperature for 12 hours with gentle agitation. The cells and antibodies are then transferred to a Millipore multiscreen filter plate. The filter plate is washed 4 times with 250 μL cold binding buffer, dried for at least 30 minutes, and the filters are punched into 5 mL polystyrene tubes. Radioactivity is measured using a Perkin Elmer Wallac Wizard 2470 Gamma Counter set at 1 count / min with a counting efficiency of 0.8. Data is fitted using a heterogeneous one-site fit Ki competitive binding model in GraphPad Prism.

[0149] In certain embodiments, the antibodies provided herein exhibit a mean clearance of about 3 to about 5 mL / day / kg over 35 days after a single 10 mg / kg dose administered intravenously on day 1. For example, but not by way of limitation, such administration can include a single 10 mg / kg IV bolus of mAb. Blood samples for analysis can be collected, for example, at 0.25, 2, 6 hours; 1, 2, 7, 14, 21, 28, and 35 days after administration; serum can be assayed for concentration of mAb using various means, for example, a qualified ELISA analytical method. In certain embodiments, administration is to a mammal. In certain embodiments, administration is to a primate. In certain embodiments, administration is to a non-human primate, e.g., a cyno. In certain embodiments, administration is to a human.

[0150] (i) Ab5 and Fragment Embodiments In one aspect, the present disclosure provides an anti-CCR8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:31; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:32; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:26; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:27; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:28. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to both human CCR8 and cyno CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0151] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:31, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:28. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:28, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO:31. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32.

[0152] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

[0153] In another aspect, the antibody described herein comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:31, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:32; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:26, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:27, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:28. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to both human CCR8 and cyno CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0154] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

[0155] In another aspect, the anti-CCR8 antibody comprises one or more CDR sequences of a VH sequence selected from the group consisting of SEQ ID NOs: 35 to 47. In another embodiment, the anti-CCR8 antibody comprises one or more CDR sequences of a VL sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In another embodiment, the anti-CCR8 antibody comprises a CDR sequence of a VH sequence selected from the group consisting of SEQ ID NOs: 35 to 47 and a CDR sequence of a VL sequence selected from the group consisting of SEQ ID NOs: 48 to 52.

[0156] In another aspect, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 47. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 48. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 47. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 48.

[0157] In a further aspect, the anti-CCR8 antibody comprises CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain selected from the group consisting of SEQ ID NOs: 35 to 47, and CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain selected from the group consisting of SEQ ID NOs: 48 to 52.

[0158] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 47 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 48.

[0159] In one embodiment, the anti-CCR8 antibody comprises one or more heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain selected from the group consisting of SEQ ID NOs: 35 to 47. In one embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain selected from the group consisting of SEQ ID NOs: 35 to 47. In one embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47, and a framework having at least 95% sequence identity to a framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47. In another embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47, and a framework having at least 98% sequence identity to a framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 35 to 47.

[0160] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 47 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 47. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 47 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 47. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 47 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 47. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 47 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 47.

[0161] In one embodiment, the anti-CCR8 antibody comprises one or more light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52 and a framework of at least 95% sequence identity to a framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52. In another embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52 and a framework of at least, particularly at least 98% sequence identity to a framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 48 to 52.

[0162] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 48 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 48. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 48 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 48. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 48 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 48. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 48 and a framework of at least 98% sequence identity in particular to the framework amino acid sequence of the VL domain of SEQ ID NO: 48.

[0163] In one embodiment, the anti-CCR8 antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35 to 47. and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In one embodiment, the VH domain has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 47. In one embodiment, the VL domain has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In one embodiment, the dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0164] In one embodiment, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:31, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:26, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:27; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:28, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:47, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:48. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0165] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 47. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 47. In a particular embodiment, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 47. In a particular embodiment, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., FR). Optionally, the anti-CCR8 antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 35 to 47, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In another aspect, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In one aspect, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In a particular aspect, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8.In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 52. In a particular embodiment, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., FR). Optionally, the anti-CCR8 antibody comprises a VL sequence selected from the group consisting of SEQ ID NOs: 48 to 52, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

[0166] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO: 47, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In another embodiment, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., FRs).Optionally, the anti-CCR8 antibody comprises the VL sequence of SEQ ID NO: 48, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

[0167] In another aspect, there is provided an anti-CCR8 antibody, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 35-47 and a VL sequence selected from the group consisting of SEQ ID NOs: 48-52, including post-translational modifications of these sequences. In one aspect, the antibody comprises a VH sequence of SEQ ID NO: 47 and a VL sequence of SEQ ID NO: 48, including post-translational modifications of these sequences.

[0168] In one embodiment, the VL sequence comprises a V4M mutation, a P43A mutation, a F46L mutation, a C90Q mutation, or a combination thereof. In one embodiment, the VH comprises a G49S mutation, a K71R mutation, a S73N mutation, or a combination thereof.

[0169] In another aspect, an anti-CCR8 antibody is provided that comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59. In one aspect, the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54. In another aspect, an anti-CCR8 antibody is provided that comprises (a) an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59, and (b) a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54.

[0170] In another aspect, an anti-CCR8 antibody is provided comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In one aspect, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 48.

[0171] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56.

[0172] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:60 and a light chain of SEQ ID NO:56.

[0173] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided, wherein the heavy chain of the antibody comprises a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one aspect, the C-terminus of the heavy chain is PG terminating in the truncated C-terminus. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 111 and a light chain of SEQ ID NO: 56. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 113 and a light chain of SEQ ID NO: 56.

[0174] In another aspect of any of the above embodiments, anti-CCR8 antibody is provided that does not bind to CCR8 ligand.In one aspect, anti-CCR8 antibody does not have CCR8 ligand blocking activity.In one aspect, anti-CCR8 antibody is a non-neutralizing antibody.In one aspect, CCR8 ligand is CCL1.

[0175] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that binds to CCR8 independently of tyrosine sulfation of CCR8 for binding (ie, sulfation-independent).

[0176] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that is an afucosylated antibody variant. In one aspect, the afucosylated antibody variant has enhanced FcγRIIIa receptor binding. In one aspect, the afucosylated anti-CCR8 antibody variant has enhanced antibody-dependent cellular cytotoxicity (ADCC). In one aspect, the anti-CCR8 afucosylated antibody variant has antibody-dependent cellular phagocytosis (ADCP) activity.

[0177] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that has improved antibody stability. In one aspect, the anti-CCR8 antibody has low aggregation, good solubility, and / or low viscosity. In a particular aspect of any of the above embodiments, the anti-CCR8 antibody has a concentration of about 1×10 -12 M ~ approx. 1×10 -11 K of M D In certain embodiments, an anti-CCR8 antibody that binds to CCR8 has a CCR8 antibody concentration of about 5×10 -12 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 4×10 -12 K of M D In certain embodiments, the antibody that binds to CCR8 has a concentration of about 3×10 -12 K of M D has.

[0178] In one aspect, the anti-CCR8 antibody is designated in the present disclosure as "hu.Ab5.H13L1," which can be fucosylated or afucosylated, optionally contains one or more heavy chain mutations at G236A and I331E, and optionally comprises a truncated C-terminus of the heavy chain in which one or two of the C-terminal amino acid residues have been removed.

[0179] In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0180] (ii) Ab4 and Fragment Embodiments In one aspect, the present disclosure provides an anti-CCR8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:3. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to both human CCR8 and cyno CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0181] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7.

[0182] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0183] In another aspect, the antibody described herein comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to both human CCR8 and cyno CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0184] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3.

[0185] In another aspect, the anti-CCR8 antibody comprises one or more CDR sequences of a VH sequence selected from the group consisting of SEQ ID NOs: 10 to 21. In another aspect, the anti-CCR8 antibody comprises one or more CDR sequences of a VL sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In another aspect, the anti-CCR8 antibody comprises a CDR sequence of a VH sequence selected from the group consisting of SEQ ID NOs: 10 to 21 and a CDR sequence of a VL sequence selected from the group consisting of SEQ ID NOs: 22 to 25.

[0186] In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 21. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 24. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 21. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 24.

[0187] In a further aspect, the anti-CCR8 antibody comprises CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21, and CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25.

[0188] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO:21 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO:24.

[0189] In one embodiment, the anti-CCR8 antibody comprises one or more heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21. In one embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21. In one embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21, and a framework having at least 95% sequence identity to the framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21. In another embodiment, the anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21, and a framework having at least 98% sequence identity to the framework amino acid sequence of a VH domain selected from the group consisting of SEQ ID NOs: 10 to 21.

[0190] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 21 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 21. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 21 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 21. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 21 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 21. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 21 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 21.

[0191] In one embodiment, the anti-CCR8 antibody comprises one or more light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25 and a framework having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25 and a framework of at least 95% sequence identity to a framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25. In another embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25 and a framework of at least, particularly at least 98% sequence identity to a framework amino acid sequence of a VL domain selected from the group consisting of SEQ ID NOs: 22 to 25.

[0192] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 24 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 24. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 24 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 24. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 24 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 24. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 24 and a framework of particularly at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 24.

[0193] In one embodiment, the anti-CCR8 antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In one embodiment, the VH domain has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21. In one embodiment, the VL domain has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In one embodiment, the dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0194] In one aspect, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:21, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0195] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 21. In a particular embodiment, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., FR). Optionally, the anti-CCR8 antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 10 to 21, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7. In another aspect, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In one aspect, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8.In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25. In a particular embodiment, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., FR). Optionally, the anti-CCR8 antibody comprises a VL sequence selected from the group consisting of SEQ ID NOs: 22 to 25, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0196] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO:21, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:7. In another embodiment, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., FRs).Optionally, the anti-CCR8 antibody comprises the VL sequence of SEQ ID NO: 24, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0197] In another aspect, there is provided an anti-CCR8 antibody, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 10-21 and a VL sequence selected from the group consisting of SEQ ID NOs: 22-25, including post-translational modifications of these sequences. In one aspect, the antibody comprises a VH sequence of SEQ ID NO: 21 and a VL sequence of SEQ ID NO: 24, including post-translational modifications of these sequences.

[0198] In one embodiment, the VL sequence comprises a Y2I mutation, hi one embodiment, the VH sequence comprises a S73N mutation, a V78L mutation, a T76N mutation, a F91Y mutation, and a P105Q mutation, or a combination thereof.

[0199] In another aspect, an anti-CCR8 antibody is provided that comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59. In one aspect, the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54. In another aspect, an anti-CCR8 antibody is provided that comprises (a) an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59, and (b) a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54.

[0200] In another aspect, an anti-CCR8 antibody is provided comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In one aspect, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO: 21 and the VL sequence of SEQ ID NO: 24.

[0201] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58.

[0202] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:61 and a light chain of SEQ ID NO:58.

[0203] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided, wherein the heavy chain of the antibody comprises a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one aspect, the C-terminus of the heavy chain is PG terminating in the truncated C-terminus. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 112 and a light chain of SEQ ID NO: 58. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 114 and a light chain of SEQ ID NO: 58.

[0204] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that binds to CCR8 ligand.In one aspect, the anti-CCR8 antibody has antagonistic effect on CCR8 ligand.In one aspect, the anti-CCR8 antibody has CCR8 ligand blocking activity.In one aspect, the anti-CCR8 antibody is a neutralizing antibody.In one aspect, the CCR8 ligand is CCL1.

[0205] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that binds to CCR8 independently of tyrosine sulfation of CCR8 for binding (ie, sulfation-independent).

[0206] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that is an afucosylated antibody variant. In one aspect, the afucosylated antibody variant has enhanced FcγRIIIa receptor binding. In one aspect, the afucosylated anti-CCR8 antibody variant has enhanced antibody-dependent cellular cytotoxicity (ADCC). In one aspect, the anti-CCR8 afucosylated antibody variant has antibody-dependent cellular phagocytosis (ADCP) activity.

[0207] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided that has improved antibody stability. In one aspect, the anti-CCR8 antibody has low aggregation, good solubility, and / or low viscosity. In a particular aspect of any of the above embodiments, the anti-CCR8 antibody has a concentration of about 1×10 -11 M ~ approx. 5×10 -11 K of M D In certain embodiments, an anti-CCR8 antibody that binds to CCR8 has a CCR8 antibody concentration of about 2×10 -11 K of M D has.

[0208] In one aspect, the anti-CCR8 antibody is designated in the present disclosure as "hu.Ab4.H12L3," which can be fucosylated or afucosylated, optionally contains one or more heavy chain mutations at G236A and I331E, and optionally comprises a truncated C-terminus of the heavy chain in which one or two of the C-terminal amino acid residues have been removed.

[0209] In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0210] (iii) Embodiments of Ab1 and Fragments Thereof In one aspect, the present disclosure provides an anti-CCR8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0211] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85.

[0212] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0213] In another aspect, the antibody described herein comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0214] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0215] In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 95. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 94. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 95. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 94.

[0216] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 95 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 94.

[0217] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 95 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 95. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 95 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 95. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 95 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 95. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 95 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 95.

[0218] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 94 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 94. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 94 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 94. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 94 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 94. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 94 and a framework of at least 98% sequence identity in particular to the framework amino acid sequence of the VL domain of SEQ ID NO: 94.

[0219] In one embodiment, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 95. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0220] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 95. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 95. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO: 95, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. In another embodiment, an anti-CCR8 antibody is provided, the antibody comprising a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 94.In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VL sequence of SEQ ID NO: 94, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0221] In another aspect, there is provided an anti-CCR8 antibody, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises a VH sequence of SEQ ID NO: 95 and a VL sequence of SEQ ID NO: 94, including post-translational modifications of these sequences.

[0222] In another aspect, an anti-CCR8 antibody is provided that comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59. In one aspect, the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54. In another aspect, an anti-CCR8 antibody is provided that comprises (a) an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59, and (b) a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54.

[0223] In another aspect, an anti-CCR8 antibody is provided, comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In one aspect, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO: 95 and the VL sequence of SEQ ID NO: 94.

[0224] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:101 and a light chain of SEQ ID NO:100.

[0225] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided, wherein the heavy chain of the antibody comprises a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one aspect, the C-terminus of the heavy chain is PG terminating in the truncated C-terminus. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 115 and a light chain of SEQ ID NO: 100.

[0226] In one embodiment, the anti-CCR8 antibody is designated in the present disclosure as "hu.Ab1.H1L1", which may be fucosylated or afucosylated, optionally contains one or more heavy chain mutations at G236A and I331E, and optionally includes a truncated C-terminus of the heavy chain in which one or two of the C-terminal amino acid residues have been removed. In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0227] (iv) Embodiments of Ab2 and Fragments Thereof In one aspect, the present disclosure provides an anti-CCR8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:86 or SEQ ID NO:87; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:88; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:89; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:76; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:77; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:78. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0228] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89.

[0229] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0230] In another embodiment, the antibody described herein comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In certain embodiments, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain embodiments, the anti-CCR8 antibody is a full-length antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0231] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0232] In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 97. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 96. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 97. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 96.

[0233] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO:97 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO:96.

[0234] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 97 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 97. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 97 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 97. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 97 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 97. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 97 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 97.

[0235] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 96 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 96. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 96 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 96. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 96 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 96. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 96 and a framework of at least 98% sequence identity in particular to the framework amino acid sequence of the VL domain of SEQ ID NO: 96.

[0236] In one embodiment, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 97. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 96. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0237] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 97. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 97. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO: 97, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89. In another embodiment, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 96. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 96.In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VL sequence of SEQ ID NO: 96, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0238] In another aspect, an anti-CCR8 antibody is provided, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises a VH sequence of SEQ ID NO: 97 and a VL sequence of SEQ ID NO: 96, including post-translational modifications of these sequences.

[0239] In another aspect, an anti-CCR8 antibody is provided that comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59. In one aspect, the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54. In another aspect, an anti-CCR8 antibody is provided that comprises (a) an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59, and (b) a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54.

[0240] In another aspect, an anti-CCR8 antibody is provided, comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0241] In one embodiment, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO:97 and the VL sequence of SEQ ID NO:96.

[0242] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:103 and a light chain of SEQ ID NO:102.

[0243] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided, wherein the heavy chain of the antibody comprises a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one aspect, the C-terminus of the heavy chain is PG terminating in the truncated C-terminus. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 116 and a light chain of SEQ ID NO: 102.

[0244] In one aspect, the anti-CCR8 antibody is designated in the present disclosure as "hu.Ab2.H1L1," which can be fucosylated or afucosylated, optionally contains one or more heavy chain mutations at G236A and I331E, and optionally comprises a truncated C-terminus of the heavy chain in which one or two of the C-terminal amino acid residues have been removed.

[0245] In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0246] (v) Ab3 and Fragment Embodiments In one aspect, the present disclosure provides an anti-CCR8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0247] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93.

[0248] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81.

[0249] In another aspect, the antibody described herein comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In certain aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a human antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a humanized antibody. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to human CCR8 and is a chimeric antibody.

[0250] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81.

[0251] In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 99. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 98. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 99. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 98.

[0252] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO:99 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO:98.

[0253] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 99 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 99. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 99 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 99. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 99 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 99. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 99 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 99.

[0254] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 98 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 98. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 98 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 98. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 98 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 98. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 98 and a framework of at least 98% sequence identity in particular to the framework amino acid sequence of the VL domain of SEQ ID NO: 98.

[0255] In one embodiment, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 99. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 98. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to CCR8.

[0256] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 99. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 99. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO: 99, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: SEQ ID NO: 90 or SEQ ID NO: 91, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93. In another embodiment, an anti-CCR8 antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 98. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 98. In certain embodiments, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., FRs).Optionally, the anti-CCR8 antibody comprises the VL sequence of SEQ ID NO: 98, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81.

[0257] In another aspect, an anti-CCR8 antibody is provided, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises a VH sequence of SEQ ID NO: 99 and a VL sequence of SEQ ID NO: 98, including post-translational modifications of these sequences.

[0258] In another aspect, an anti-CCR8 antibody is provided that comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59. In one aspect, the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54. In another aspect, an anti-CCR8 antibody is provided that comprises (a) an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59, and (b) a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54.

[0259] In another aspect, an anti-CCR8 antibody is provided, comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO: 99 and the VL sequence of SEQ ID NO: 98.

[0260] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:105 and a light chain of SEQ ID NO:104.

[0261] In another aspect of any of the above embodiments, an anti-CCR8 antibody is provided, wherein the heavy chain of the antibody comprises a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one aspect, the C-terminus of the heavy chain is PG terminating in the truncated C-terminus. In one aspect, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO: 117 and a light chain of SEQ ID NO: 104.

[0262] In one aspect, the anti-CCR8 antibody is designated in the present disclosure as "u.Ab3.H1L1," which can be fucosylated or afucosylated, optionally contains one or more heavy chain mutations at G236A and I331E, and optionally comprises a truncated C-terminus of the heavy chain in which one or two of the C-terminal amino acid residues have been removed.

[0263] In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0264] (vi) Mouse Surrogate Embodiments In one aspect, the disclosure provides an anti-CCR8 antibody that binds to mouse CCR8, comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64. In certain aspects, the anti-CCR8 antibody comprises all six of the aforementioned CDRs. In some aspects, the anti-CCR8 antibody is a full-length antibody. In certain aspects, the anti-CCR8 antibody is a full-length antibody that binds to mouse CCR8. In certain embodiments, the anti-CCR8 antibody is a full-length antibody that binds to mouse CCR8 and is a chimeric antibody (eg, a rabbit-mouse chimera).

[0265] In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:65 or SEQ ID NO:66, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:68. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:68. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:68 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:68, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68.

[0266] In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0267] In another aspect, the antibody described herein comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0268] In another aspect, the disclosure provides an antibody comprising a light chain variable domain (VL) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0269] In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 70. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 69. In another embodiment, the anti-CCR8 antibody comprises the CDR sequences of the VH sequence of SEQ ID NO: 70. In another embodiment, the anti-CCR8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 69.

[0270] In a further aspect, the anti-CCR8 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO:70 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO:69.

[0271] In one embodiment, an anti-CCR8 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 70 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 70. In one embodiment, an anti-CCR8 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 70 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 70. In one embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 70 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 70. In another embodiment, the anti-CCR8 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 70 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 70.

[0272] In one embodiment, the anti-CCR8 antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 69 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 69. In one embodiment, the anti-CCR8 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 69 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 69. In one embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 69 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 69. In another embodiment, the anti-CCR8 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 69 and a framework of at least 98% sequence identity in particular to the framework amino acid sequence of the VL domain of SEQ ID NO: 69.

[0273] In one embodiment, the anti-CCR8 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 70. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 69. In one aspect, the antibody has a dissociation constant (K D ) with up to 10-fold decrease or increase in dissociation constant (K D ) binds to mouse CCR8.

[0274] In another embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70. In one embodiment, the anti-CCR8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 70. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CCR8 antibody comprising the sequence retains the ability to bind to mouse CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 70. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VH sequence of SEQ ID NO: 70, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: SEQ ID NO: 65 or SEQ ID NO: 66, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68. In another embodiment, an anti-CCR8 antibody is provided, the antibody comprising a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69. In one embodiment, the anti-CCR8 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 69. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CCR8 antibody comprising that sequence retains the ability to bind to CCR8. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 69.In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the anti-CCR8 antibody comprises a VL sequence of SEQ ID NO: 69, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0275] In another aspect, there is provided an anti-CCR8 antibody, the antibody comprising the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH sequence of SEQ ID NO: 70 and the VL sequence of SEQ ID NO: 69, including post-translational modifications of these sequences.

[0276] In another aspect, an anti-CCR8 antibody is provided that binds to mouse CCR8, the antibody comprising a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64. In one aspect, the anti-CCR8 antibody comprises the VH sequence of SEQ ID NO: 70 and the VL sequence of SEQ ID NO: 69.

[0277] In one embodiment, the anti-CCR8 antibody comprises a heavy chain of SEQ ID NO:72 and a light chain of SEQ ID NO:71.

[0278] In a further embodiment, the anti-CCR8 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody. In one embodiment, the anti-CCR8 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0279] (vii) Other embodiments In a further aspect, an anti-CCR8 antibody according to any of the above aspects may incorporate any of the features described in Sections 1-5 below, either alone or in combination.

[0280] 1. Antibody fragment In certain aspects, the antibodies provided herein are antibody fragments.

[0281] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each of which contains the variable domains of the heavy and light chains (VH and VL, respectively) as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Thus, a "Fab fragment" is an antibody fragment having a light chain containing a VL domain and a CL domain, and a heavy chain fragment containing a VH domain and a CH1 domain. A "Fab' fragment" differs from a Fab fragment by the addition of residues at the carboxy terminus of the CH1 domain, which contains one or more cysteines from the antibody hinge region. A Fab'-SH is a Fab' fragment in which the cysteine ​​residues (multivalent) of the constant domains bear a free thiol group. Pepsin treatment results in a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and part of the Fc region. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0282] In another embodiment, the antibody fragment is a diabody, triabody or tetrabody. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0283] In a further embodiment, the antibody fragment is a single chain Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments may be further stabilized by the creation of an interchain disulfide bond through the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0284] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, which can connect either the N-terminus of VH to the C-terminus of VL, or vice versa. The protein can retain the specificity of the original antibody, even though the constant regions have been removed and the linker has been introduced. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0285] In another embodiment, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0286] Antibody fragments can be produced by a variety of techniques, including but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., E. coli), as described herein.

[0287] 2. Chimeric and humanized antibodies In certain aspects, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0288] In certain aspects, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains in which the CDRs (or a portion thereof) are derived from a non-human antibody, and the FRs (or a portion thereof) are derived from human antibody sequences. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0289] Humanized antibodies and methods for producing same are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0290] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., BBaca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0291] 3. Human antibodies In certain aspects, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0292] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0293] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0294] Human antibodies can also be generated by isolating variable domain sequences selected from a phage display library of human origin. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0295] 4. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for CCR8 and another specificity is for any other antigen. In certain embodiments, a bispecific antibody can bind to two (or more) different epitopes of CCR8. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing CCR8. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0296] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced using techniques such as the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); production of bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); use of general light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431); use of "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al. al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0297] 5. Antibody Variants In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0298] a) Substitution, insertion and deletion variants In certain aspects, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include the CDRs and FRs.

[0299] In one aspect, the VL sequence disclosed herein comprises a V4M mutation, a P43A mutation, a F46L mutation, a C90Q mutation, or a combination thereof. In one aspect, the VH sequence of an antibody disclosed herein comprises a G49S mutation, a K71R mutation, a S73N mutation, or a combination thereof. In one aspect, the VL sequence of an antibody disclosed herein comprises a Y2I mutation. In one aspect, the VH sequence of an antibody disclosed herein comprises a S73N mutation, a V78L mutation, a T76N mutation, a F91Y mutation, and a P105Q mutation, or a combination thereof.

[0300] Conservative substitutions are shown in Table 2 under the heading of "Conservative Substitutions." More substantial changes are provided in Table 2 under the heading of "Exemplary Substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 2]

[0301] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0302] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0303] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., improvement) in a particular biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have a particular biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more. CDR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0304] For example, to improve antibody affinity, changes (e.g., substitutions) can be made in the CDRs. Such changes can be made within CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting from a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity is the CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0305] In certain aspects, substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside of the antigen contact residues in the CDRs. In the particular variant VH and VL sequences described above, each CDR is either unaltered or has no more than one, two, or three amino acid substitutions.

[0306] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0307] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0308] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0309] If the antibody comprises an Fc region, the oligosaccharides attached to the antibody may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally linked to Asn297 in the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNac), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the bisecting oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies described herein may be made to generate antibody variants with specific improved properties.

[0310] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose linkages (direct or indirect) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc at the stem of the biantennary oligosaccharide structure, and such antibodies are further referred to herein as "afucosylated antibodies". In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). In certain embodiments, the percentage of afucosylation is about 65% to about 100%, about 80% to about 100%, or about 80% to about 95%. The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at about position 297 (Fc region residues in EU numbering) within the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300 (e.g., Asn299), due to slight sequence variations in antibodies. Such antibodies having an increased proportion of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function, see e.g., U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0311] In one aspect, the present disclosure provides an afucosylated antibody variant with enhanced FcγRIIIa receptor binding. In one aspect, the present disclosure provides an afucosylated antibody variant with enhanced antibody-dependent cellular cytotoxicity (ADCC). In one aspect, the present disclosure provides an afucosylated antibody variant with antibody-dependent cellular phagocytosis (ADCP) activity.

[0312] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or eliminated GDP-fucose synthesis or transport proteins (see, e.g., U.S. Patent Publications 2004259150, 2005031613, 2004132140, 2004110282). See also Pereira et al., MABS (2018) 693-711.

[0313] In a further aspect, antibody variants are provided that have bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342, WO 2004 / 065540, WO 2003 / 011878.

[0314] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to the Fc region. Such an antibody variant may have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0315] c) Fc domain variants In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0316] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on 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 assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., 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); U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the desired ADCC activity can be assessed in vitro, for example in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0317] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. 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 in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0318] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0319] In a particular embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0320] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., at positions 234 and 235 (residue EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0321] In certain embodiments, the antibody variants include an Fc region with one or more amino acid substitutions, e.g., substitutions at positions, that improve FcγR binding (and thereby improve effector function). In certain embodiments, the antibody variants include an Fc region with at least one of the following amino acid substitutions: G236A, I332E, S298A, E333A, K334A, S239D, A330L, F243L, R292P, Y300L, V305I, P396L, L235V, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E (see, e.g., Liu et al., Antibodies (Basel) (2020); 9(4): 64).

[0322] In some aspects, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as disclosed in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0323] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in U.S. Patent Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 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, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0324] The Fc region residues important for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were found to be important for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are important for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671), various mutants of residues 248-259, 301-317, 376-382, and 424-437 are reported and investigated.

[0325] In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253, and / or 310, and / or 435 (EU numbering of residues) of the Fc region. In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one aspect, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0326] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310, and / or 433, and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460).

[0327] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252, and / or 254, and / or 256 (EU numbering of residues). In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region. See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0328] The C-terminus of the heavy chain of the antibody as reported herein may be a complete C-terminus terminating in amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In one embodiment, the C-terminus of the heavy chain is PG terminating in a shortened C-terminus. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal proline residue (P445, EU index numbering of amino acid positions).

[0329] d) Cysteine ​​Engineered Antibody Variants In certain aspects, it may be desirable to generate cysteine ​​engineered antibodies, e.g., THIOMAB™, in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain aspects, the replaced residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies are described, for example, in U.S. Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0330] e) Antibody derivatives In certain aspects, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0331] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0332] In the case of a natural antibody or natural antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors.

[0333] In the case of a bispecific antibody with heterodimeric heavy chains, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH comprising the first heteromonomeric Fc region and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH comprising the second heteromonomeric Fc region of the antibody (e.g., the first and / or second light chain and / or the first and / or second heavy chain of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors, and usually these nucleic acids are located on two or three expression vectors, i.e. one vector may contain two or more of these nucleic acids. An example of these bispecific antibodies is CrossMab (see, for example, Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomer heavy chains contains a so-called "knob mutation" (T366W and, optionally, one of S354C or Y349C) according to the EU index numbering, and the other contains a so-called "hole mutation" (T366S, L368A and Y407V and, optionally, Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0334] In one aspect, an isolated nucleic acid encoding an antibody for use in the methods reported herein is provided.

[0335] In one aspect, a method of making an anti-CCR8 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture).

[0336] For recombinant production of anti-CCR8 antibodies, for example, nucleic acids encoding the above-mentioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0337] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in appropriate fractions and may be further purified.

[0338] In addition to prokaryotes, eukaryotic organisms such as filamentous fungi and yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0339] Suitable host cells for expressing (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0340] Plant cell cultures can also be used as hosts, see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0341] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0342] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, a Y0, NS0, Sp20 cell).

[0343] C. Assay The anti-CCR8 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0344] 1. Binding and other assays In one embodiment, the antibodies described herein are tested for their antigen binding activity by known methods such as, for example, ELISA, Western blot, etc.

[0345] In another embodiment, a competitive assay may be used to identify antibodies that compete with the anti-CCR8 antibodies of the presently disclosed subject matter, e.g., Ab1, Ab2, Ab3, Ab4, and Ab5, for binding to CCR8. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) bound by the anti-CCR8 antibodies of the presently disclosed subject matter, e.g., Ab1, Ab2, Ab3, Ab4, and Ab5. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ).

[0346] In an exemplary competitive assay, immobilized CCR8 is incubated in a solution containing a first labeled antibody that binds to CCR8 (e.g., an anti-CCR8 antibody of the presently disclosed subject matter, e.g., Ab1, Ab2, Ab3, Ab4, and Ab5) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CCR8. The second antibody may be present in a hybridoma supernatant. As a control, immobilized CCR8 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CCR8, excess unbound antibody is removed and the amount of label bound to immobilized CCR8 is measured. If the amount of label bound to immobilized CCR8 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to CCR8. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0347] 2. Activity assay In one embodiment, an assay is provided to identify the anti-CCR8 antibody having biological activity. Biological activity may include, for example, antibody-dependent cellular cytotoxicity (ADCC), ADCC against Treg, antibody-dependent cellular phagocytosis (ADCP), depletion of Treg. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0348] In certain embodiments, the anti-CCR8 antibodies described herein are tested to measure the ADCC of the antibodies. The ADCC assay is performed using CD16 engineered NK-92_F158 as effector cells and CHO cells stably expressing human CCR8 and Ga 15 subunits (CHO / hCCR8.Gna15) as target cells, as previously reported in Kamen, L., et al., Development of a kinetic antibody-dependent cellular cytotoxicity assay. J Immunol Methods, 2019. 468: p. 49-54, and Schnueriger, A., et al., Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies. Mol Immunol, 2011. 48(12-13): p. 1512-17, with some modifications. Briefly, the lysis of target cells by ADCC is measured by calcein release method. Target cells were labeled with calcein-AM, then washed and incubated for 3000 h. Cells are seeded in 384-well plates at a density of 10 ... The degree of specific ADCC activity is calculated as follows: %ADCC=100×(Average experimental release - Average AICC) / (Average maximum release - Average spontaneous release)

[0349] ADCC activity is plotted as a function of antibody concentration and the data is fitted to a asymmetric sigmoidal 4 parameter logistic (4PL) model.

[0350] In a particular embodiment, the anti-CCR8 antibodies described herein are tested to measure ADCC against Treg cells. 7 Human PBMCs were cultured using NOD.Cg-Prkdc scid Il2rg tm1Wjl T cells are transferred intraperitoneally into 10 / SzJ (NSG) mice (JAX) and spleens are harvested 2-3 weeks after transfer. Human T cells are enriched from single cell suspensions of NSG splenocytes and primary NK cells are enriched from human PBMCs. Human T cells are incubated with 0.001-1 μg / mL anti-CCR8 antibody for 30 min at room temperature, followed by addition of primary NK cells at an effector:target ratio of 2:1. After overnight incubation at 37 °C, cells are collected and surface stained and intracellularly stained. Antibodies used to define T cell populations are CD45 (HI30), CD3 (SK7), CD8 (RPA-T8), and CD14 (63D3), CD4 (RPA-T4), and FOXP3 (236A / E7). CountBright Absolute Counting Beads are added to each sample prior to acquisition. Flow cytometry is performed. Absolute cell counts are calculated. ADCC activity towards Treg cells is measured by calculating the ratio of recovered Treg cells to recovered CD8 cells (Treg / CD8) or conventional CD4 T cells to recovered CD8 T cells (CD4conv / CD8).

[0351] In a particular embodiment, the anti-CCR8 antibodies described herein are tested to measure their binding to regulatory T cells (Treg cells or Tregs) by fluorescence-activated cell sorting (FACS) flow cytometry. Thaw human colorectal dissociated tumor cells (DTCs). Surface stain the cells with eFluor 780-conjugated Fixable Viability Dye and 2ug / mL mAb specific for CCR8, OX40 (positive control), Herceptin (negative control), or anti-hIgG (negative control) for 20 minutes at 4°C, followed by secondary detection with AF647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcg fragment specific for 10 minutes at 4°C. Cells are then stained intracellularly. Antibodies used to define T cell populations are CD45 (HI30), CD3 (SK7), CD8 (RPA-T8), and CD14 (63D3) (BD Biosciences), CD4 (RPA-T4), and FOXP3 (236A / E7). Flow cytometry is performed and analyzed.

[0352] In certain embodiments, the anti-CCR8 antibodies described herein are tested to measure the ADCP of the antibodies. + Monocytes are first isolated from the blood of donors with known FcgRIIa and FcgRIIIa genotype information. +Monocytes are differentiated into macrophages. 50ng / mL hIL-10 is then added to polarize macrophages for 24 hours prior to the ADCP assay. NucLight Red transfected CHO / hCCR8.Gna15 target cells are pre-incubated with anti-CCR8 antibody for 20 minutes in the presence of 20mg / mL non-specific human IgG. The above cell mixture is then added to the macrophage (effector cell) plate at an E:T ratio of 1:1. Cell images are obtained with bright field and red laser settings every hour for a period of 6 hours. The number of red blood cells (remaining target cells) in each well is normalized by the number of macrophages. ADCP activity is calculated as the percentage reduction in normalized red blood cell count in each sample compared to the negative control in the presence of an isotype control antibody. ADCP activity is plotted as a function of antibody concentration and the data is fitted to an asymmetric sigmoidal 4-parameter logistic (4PL) model. The EC of each antibody is calculated using the EC values ​​of 100-1500 μg / mL. 50 Values ​​are determined as the concentration that reaches 50% target cell killing.

[0353] In certain embodiments, anti-CCR8 antibodies (e.g., mouse surrogate antibodies) described herein are tested in vivo to measure depletion of Treg cells, mice bearing established tumors are treated with anti-CCR8 antibodies (e.g., mouse surrogate antibodies disclosed herein) and the proportion of Treg cells, conventional CD4 T cells and CD8 T cells among leukocytes in the tumor, spleen and tumor-draining lymph nodes are analyzed. For this purpose, tumor cells are harvested in log phase growth and resuspended in HBSS-containing Matrigel at a 1:1 ratio. Mice are inoculated subcutaneously in the flank with 100,000 tumor cells in 100 microliters of HBSS+Matrigel. Tumors are grown in 100-200 wells at 37°C for 12-38 days after establishment with a mean tumor volume of 130-230 mm. 3Mice are then randomized into treatment groups. Treatment with anti-CCR8 or anti-gp120 isotype control Ab is administered intravenously. Three days later, mice are sacrificed and tumors, spleens, and tumor-draining lymph nodes are obtained for analysis. Tumors are minced and digested to generate single cell suspensions. Single cell suspensions are surface stained with fluorescently labeled anti-CD45, anti-CD4, and anti-CD8 antibodies, and intracellularly stained with fluorescently labeled anti-Foxp3 antibody. Flow cytometry may be performed on a Fortessa X-20 or FACSymphony and analyzed with FlowJo software.

[0354] In certain embodiments, the anti-CCR8 antibodies (e.g., mouse surrogate antibodies) described herein are tested for tumor growth inhibition following anti-CCR8-mediated depletion of tumor-infiltrating Treg cells in vivo. Mice bearing established tumors are treated with mouse surrogate anti-CCR8 antibodies and monitored for tumor growth over time.

[0355] D. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-CCR8 antibodies provided herein are useful for detecting the presence of CCR8 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as tumors.

[0356] In one embodiment, an anti-CCR8 antibody is provided for use in a method of diagnosis or detection. In a further embodiment, a method of detecting the presence of CCR8 in a biological sample is provided. In a particular embodiment, the method comprises contacting a biological sample with an anti-CCR8 antibody described herein under conditions that allow binding of the anti-CCR8 antibody to CCR8, and detecting whether a complex is formed between the anti-CCR8 antibody and CCR8. Such a method may be an in vitro method or an in vivo method. In one embodiment, the anti-CCR8 antibody is used to select subjects eligible for therapy with an anti-CCR8 antibody, for example, when CCR8 is a biomarker for subject selection.

[0357] In certain embodiments, labeled anti-CCR8 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties, such as enzymes or ligands, that are indirectly detected, for example, via enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H, and 131 These include, but are not limited to, I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to an enzyme that utilizes hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0358] E. Pharmaceutical Compositions In a further aspect, a pharmaceutical composition is provided comprising any of the antibodies provided herein, e.g., for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0359] Pharmaceutical compositions (formulations) of the anti-CCR8 antibodies described herein can be prepared by combining the antibodies with pharma- ceutically acceptable carriers or excipients known to those skilled in the art. For example, see Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1 st Ed., Woodhead Publishing (2015), §4 and Falconer RJ, Biotechnology Advances (2019), 37, 107412. Exemplary pharmaceutical compositions of the anti-CCR8 antibodies described herein are lyophilized, aqueous, frozen, and the like.

[0360] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polysaccharides, e.g., saccharides that are soluble in water, and / or soluble in water. These include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0361] The pharmaceutical compositions herein may also contain multiple active ingredients as required for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents useful for treating the same disease. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0362] Pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0363] F. Treatment Methods and Routes of Administration Any of the anti-CCR8 antibodies or immunoconjugates provided herein can be used in therapeutic methods.

[0364] In one embodiment, an anti-CCR8 antibody is provided for use as a medicament. In a further embodiment, an anti-CCR8 antibody is provided for use in the treatment of cancer. In a particular embodiment, an anti-CCR8 antibody is provided for use in a treatment method. In a particular embodiment, the present disclosure provides an anti-CCR8 antibody for use in a method of treating a subject (e.g., a human subject) in need of treatment, comprising administering to the subject an effective amount of an anti-CCR8 antibody. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In a further embodiment, the present disclosure provides an anti-CCR8 antibody for use in depleting regulatory T cells ("Tregs") in a tumor microenvironment. In a particular embodiment, the present disclosure provides an anti-CCR8 antibody for use in a method of depleting Tregs in a tumor microenvironment of a subject, the method comprising administering to the subject an effective amount of an anti-CCR8 antibody in depleting Tregs in the tumor microenvironment.

[0365] In a further aspect, the disclosure provides for the use of an anti-CCR8 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for the treatment of cancer. In a further aspect, the medicament is for use in a method of treating cancer, comprising administering an effective amount of the medicament to a subject (e.g., a human control) in need thereof. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, such as those described below. In a further aspect, the medicament is for depleting Tregs in a tumor microenvironment. In a further aspect, the medicament is for use in a method of depleting Tregs in a tumor microenvironment of a subject, comprising administering to the subject an effective amount of the medicament for depleting Tregs in the tumor microenvironment.

[0366] In a further embodiment, the present disclosure provides a method for treating cancer.In one embodiment, the method comprises administering an effective amount of anti-CCR8 antibody to a subject (e.g., a human subject) in need thereof to treat cancer.In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject, as described below.

[0367] In a further aspect, the present disclosure provides an anti-CCR8 antibody for use in depleting Treg cells, for example, outside or in a tumor microenvironment. For example, in certain embodiments, the present disclosure provides a method for depleting Treg cells in the tumor microenvironment of a subject (e.g., a human subject) having a cancer in need of depleting Treg cells, comprising administering to the subject an effective amount of an anti-CCR8 antibody sufficient to deplete Treg cells in the tumor microenvironment, thereby treating the cancer. In certain aspects, the present disclosure provides a method for depleting Treg cells outside the tumor microenvironment (e.g., in circulation) of a subject (e.g., a human subject) having a cancer in need of depleting Treg cells, comprising administering to the subject an effective amount of an anti-CCR8 antibody sufficient to deplete Treg cells outside the tumor microenvironment, thereby treating the cancer. Without wishing to be bound by any particular theory, by reducing the number of Treg cells outside the tumor microenvironment, the cancer is treated as the number of Treg cells infiltrating the tumor microenvironment is reduced, thereby reducing the number of Treg cells in the tumor microenvironment.

[0368] Exemplary cancers include, but are not limited to, bladder cancer (e.g., urothelial cancer), blastoma, blood cancer (lymphoma, e.g., non-Hodgkin's lymphoma, leukemia), bone cancer, brain cancer, breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer (e.g., colon cancer, rectal cancer), endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung carcinoma), ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer (e.g., melanoma, squamous cell carcinoma), testicular cancer, and uterine cancer.

[0369] In particular aspects, the cancer is bladder cancer, blood cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, and skin cancer.

[0370] In certain aspects, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, or skin cancer.

[0371] In certain aspects, the cancer is a solid tumor.

[0372] In certain embodiments, the cancer expresses CCR8.

[0373] In certain aspects, the cancer is a T cell inflammatory tumor or comprises a T cell inflammatory tumor microenvironment.

[0374] In certain embodiments, the cancer contains regulatory T cells in the tumor microenvironment, and exposure of the cancer to CCR8 antibodies as described herein results in depletion of regulatory T cells in the tumor microenvironment. In further embodiments, the present disclosure provides pharmaceutical compositions comprising any of the anti-CCR8 antibodies described herein, for example, for use in any of the above-mentioned therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the anti-CCR8 antibodies provided herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the anti-CCR8 antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0375] The antibodies described herein can be administered alone or can be used in combination therapy, e.g., useful for treating cancer. For example, combination therapy includes administering an antibody described herein and administering at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents).

[0376] The at least one additional therapeutic agent includes any agent that can be administered for treatment. In certain embodiments, the additional therapeutic agent is an additional anti-cancer agent. Exemplary anti-cancer agents include, but are not limited to, microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, anti-angiogenic agents, immunomodulatory agents, inhibitors of cell adhesion, cytotoxic or cytostatic agents, activators of cell apoptosis, agents that increase the sensitivity of cells to apoptosis inducers, cytokines, anti-cancer vaccines or oncolytic viruses, toll-like receptor (TLR) agents, bispecific antibodies, cell therapy, and immune cell engagers. In certain embodiments, the additional therapeutic agent is an immunomodulatory anti-cancer agent, for example, a checkpoint inhibitor (CPI) such as an anti-CTLA4 antibody (e.g., ipilimumab), a PD-L1 binding antagonist, or a PD-1 binding antagonist.

[0377] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, e.g., PD-1 and / or B7-1. In some examples, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In certain aspects, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some examples, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1 and / or B7-1. In one example, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through a cell surface protein expressed on T lymphocytes via signaling through PD-L1, such that dysfunctional T cells are rendered non-dysfunctional (e.g., enhance effector responses to antigen recognition). In some examples, the PD-L1 binding antagonist binds to PD-L1. In some examples, the PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Exemplary anti-PD-L1 antagonist antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, embafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. In some embodiments, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).In one particular embodiment, the PD-L1 binding antagonist is MDX-1105. In another particular embodiment, the PD-L1 binding antagonist is MEDI4736 (durvalumab). In another particular embodiment, the PD-L1 binding antagonist is MSB0010718C (avelumab). In other embodiments, the PD-L1 binding antagonist can be a small molecule, such as GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, and in some examples can be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003, and JS-003. In one embodiment, the PD-L1 binding antagonist is atezolizumab.

[0378] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, prevents, or prevents signaling resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1 and / or PD-L2. PD-1 (Programmed Death 1) is also referred to in the art as "Programmed Cell Death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is set forth in UniProtKB / Swiss-Prot Accession No. Q15116. In some examples, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partner molecules. In certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one example, the PD-1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes via signaling through PD-1, so as to prevent dysfunctional T cells from becoming dysfunctional (e.g., enhancing effector responses to antigen recognition). In some examples, the PD-1 binding antagonist binds to PD-1. In some examples, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody).Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, prorugolimab, canrelizumab, sintilimab, tislelizumab, toripalimab, dostarimab, retifanlimab, sasanlimab, penprimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimuzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, budicalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a particular embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another particular embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another particular embodiment, the PD-1 binding antagonist is a PD-L2 Fc fusion protein, such as AMP-224. In another particular embodiment, the PD-1 binding antagonist is MED1-0680. In another particular embodiment, the PD-1 binding antagonist is PDR001 (spartalizumab). In another particular embodiment, the PD-1 binding antagonist is REGN2810 (cemiplimab). In another particular embodiment, the PD-1 binding antagonist is BGB-108. In another particular embodiment, the PD-1 binding antagonist is prorugolimab. In another particular embodiment, the PD-1 binding antagonist is canrelizumab. In another particular embodiment, the PD-1 binding antagonist is sintilimab. In another particular embodiment, the PD-1 binding antagonist is tislelizumab. In another particular embodiment, the PD-1 binding antagonist is toripalimab. Other exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.Such combination therapy as mentioned above encompasses combined administration (when two or more therapeutic agents are included in the same or separate pharmaceutical compositions) and separate administration, where administration of an antibody described herein may occur before, simultaneously with, and / or after administration of the additional therapeutic agent. In one embodiment, administration of an anti-CCR8 antibody and administration of an additional therapeutic agent occur within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. In one embodiment, the antibody and additional therapeutic agent are administered to the subject on the first day of treatment. The antibodies described herein may also be used in combination with radiation therapy.

[0379] The antibodies (and any additional therapeutic agents) described herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be, for example, by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0380] The antibodies described herein will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular subject species being treated, the clinical symptoms of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or any dosage and by any route empirically / clinically determined to be appropriate.

[0381] The antibodies of the invention are suitably administered to the subject at one time or over a series of treatments. For repeated administration over several days or longer, depending on the condition, the treatment will generally be continued until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0382] In further embodiments, the use of mouse surrogates is contemplated, for example, for use as in vitro or in vivo tool molecules. For example, in one aspect, a method of treating a disease in a mouse is provided, comprising administering to the mouse an effective amount of a mouse surrogate antibody described herein to treat the disease. In certain embodiments, the mouse comprises a xenograft. In certain embodiments, the mouse model is a cancer model, for example a skin cancer model.

[0383] G. Manufactured products In another aspect, an article of manufacture is provided that contains a substance useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody disclosed herein. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may comprise (a) a first container containing a composition comprising an antibody disclosed herein, and (b) a second container containing a composition further comprising a cytotoxic agent or other therapeutic agent. The article of manufacture of this aspect described herein may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. EXAMPLES

[0384] The following are further non-limiting examples of the antibodies, methods and compositions described herein. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0385] Example 1. Discovery and Engineering of Anti-CCR8 Monoclonal Antibodies New Zealand White rabbits were immunized with recombinant huCCR8, huCCR8+ rabbit cell lines, extracellular vesicles containing huCCR8, and sulfated and non-sulfated peptides derived from the N-terminal region of huCCR8. Single B cells were isolated according to the protocol described in Lin et al., PLoS ONE 15(12), 2020. B cell culture supernatants were then assayed by direct flow-activated cell sorting (FACS; flow cytometry) of IgG+ B cells into single wells for binding to human and cyno CCR8+ CHO cells and control CHO cells. CCR8-specific B cells were lysed and immediately frozen at -80°C and stored until molecular cloning. The variable regions (VH and VL) of each monoclonal antibody from rabbit B cells were cloned into expression vectors from extracted mRNA as described in Lin et al., PLoS ONE 15(12), 2020. Individual recombinant rabbit antibodies were expressed in Expi 293 cells and subsequently purified with protein A.

[0386] Over 480 anti-CCR8 antibodies were obtained that bound to either human or cyno CCR8 CHO cells. Antibodies were further selected based on their relative mean fluorescence intensity (MFI) and sequence diversity in human and cyno CCR8 CHO cell lines. Five unique antibody groups were identified (designated Ab1-Ab5) from antibodies that showed less than a 5-fold difference in MFI in human and cyno CCR8 CHO cells. One representative sequence from each group was selected for humanization.

[0387] The variants constructed during humanization of rabbit monoclonal antibodies were evaluated in the human IgG1 format. Hypervariable regions from each of the rabbit antibodies (i.e., positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL domain, and positions 26-35 (H1), 50-65 (H2), and 95-102 (H3) in the VH domain) were grafted into the various acceptor frameworks. Residue numbers are from Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). All VL and VH Vernier positions from the rabbit antibodies were also grafted into the respective human germline frameworks. The graft with all rabbit amino acids at the Vernier positions is referred to as H1L1. The binding ability of humanized CCR8 antibodies to the CHO-huCCR8.Gna15 stable cell line was compared to their chimeric parent clones. The rabbit Vernier positions of version H1L1 antibody were reverted to human residues to assess the contribution of each rabbit Vernier position to binding to huCCR8.

[0388] mAbs were assessed for binding to regulatory T cells (Treg cells or Treg) by fluorescence-activated cell sorting (FACS) flow cytometry. Human colorectal dissociated tumor cells (DTC) (Discovery Life Sciences) were thawed according to the vendor's protocol. Cells were surface stained with eFluor 780-conjugated Fixable Viability Dye (ThermoFisher Scientific) and 2ug / mL mAbs specific for CCR8, OX40 (positive control), Herceptin (negative control), or anti-hIgG (negative control) for 20 minutes at 4°C, followed by secondary detection with AF647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcg fragment specific (Jackson ImmunoResearch) for 10 minutes at 4°C. Cells were then intracellularly stained using eBioscience Foxp3 / transcription factor staining buffer set (ThermoFisher Scientific) according to the manufacturer's protocol. Antibodies used to define T cell populations were CD45 (HI30), CD3 (SK7), CD8 (RPA-T8), and CD14 (63D3) (BD Biosciences), CD4 (RPA-T4) from BioLegend, and FOXP3 (236A / E7) from ThermoFisher Scientific. Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and analyzed with FlowJo software (BD Biosciences, version 10.5.3). Mean fluorescence intensity (MFI) values ​​for CD8 T cells (defined as CD45+CD14-CD3+CD8+CD4-) (circles,), conventional CD4 T cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3-) (squares,), and Treg cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3+) (triangles,▲) are shown in Figure 1. Three of the five CCR8 mAb clones specifically stained Treg cells and not conventional CD4 or CD8 T cells, ranked according to CCR8 MFI above 500 MFI: hu.Ab4.H1L1>hu.Ab5.H1L1>hu.Ab3.H1L1.These three CCR8 mAb clones, namely hu.Ab3.H1L1, hu.Ab4.H1L1, and hu.Ab5.H1L1, were also confirmed to retain human-cyno cross-reactivity (less than 5-fold difference between human and cyno CCR8 CHO cells) and therefore these antibodies were taken forward for further studies.

[0389] For example, hu.Ab3.H1L1, hu.Ab4.H1L1, and hu.Ab5.H1L1 were further tested for antibody-dependent cellular cytotoxicity (ADCC). The hIgG1 isotype was used as a negative control. See FIG. 2. ADCC assays were performed using CD16 engineered NK-92_F158 as effector cells and CHO cells stably expressing human CCR8 and G-alpha15 subunits (CHO / hCCR8.Gna15) as target cells, as previously reported in Kamen, L., et al., Development of a kinetic antibody-dependent cellular cytotoxicity assay. J Immunol Methods, 2019. 468: p. 49-54, and Schnueriger, A., et al., Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies. Mol Immunol, 2011. 48(12-13): p. 1512-17, with some modifications. Briefly, lysis of target cells by ADCC was measured by calcein release assay. Target cells were labeled with calcein-AM (C3100MP, ThermoFisher Scientific) according to the manufacturer's protocol, then washed and plated at 3000 x g on a 384-well plate. Cells / well were seeded at a density of 1000 x 1000 cells / well. Anti-CCR8 antibodies were added at various concentrations from 0.004 to 1 µg / mL, followed by the addition of NK-92_F158 cells at an effector:target (E:T) ratio of 10:1. The plates were then incubated for 2.5 h at 37 °C. After incubation, the plates were centrifuged at 200 x g for 3 min, the supernatants were transferred to white opaque 384-well microplates (OptiPlate-384, PerkinElmer, Waltham, MA), and the fluorescent signals were measured in relative fluorescence units (RFU) with excitation / emission at 485 / 520 nm using an EnSight Multimode Plate Reader (PerkinElmer). Signals from wells containing only target cells indicated spontaneous release of calcein from the labeled cells (spontaneous release), whereas wells containing target cells lysed with Triton X-100 (Sigma-Aldrich, St. Louis, MO) provided the maximum available signal (maximum release). Antibody-independent cell-mediated cytotoxicity (AICC) was measured in wells containing target and effector cells without the addition of antibody. Samples and controls were tested at least in duplicate on the same plate. The degree of specific ADCC activity was calculated as follows:

[0390] %ADCC=100×(Average experimental release - Average AICC) / (Average maximum release - Average spontaneous release)

[0391] ADCC activity was plotted as a function of antibody concentration and the data was fitted to a non-symmetric sigmoidal four parameter logistic (4PL) model using Prism (Graphpad; La Jolla, Calif.). See Figure 2. EC 50 EC values ​​were determined as the concentration at which 50% of the maximal ADCC activity of each individual antibody was reached. 50 The values ​​are also tabulated below. [Table A]

[0392] hu.Ab3.H1L1, hu.Ab4.H1L1, and hu.Ab5.H1L1 were further analyzed for their agonist (CCR8 activation) and antagonist (inhibition of CCL1; neutralization) activities. hIgG1 isotype was used as a negative control. CCR8 activation was measured using a Fluorescent Imaging Plate Reader (FLIPR) FDSS / μCell (Hamamatsu, Japan) using Ca2+. 2+ Briefly, CHO / hCCR8.Gna15 cells were injected with fluorescent Ca influx. 2+ Dye Fluo-8 NW (cat. no. 36307, AAT Bioquest) was loaded and incubated at 37°C for 30 min, then at room temperature for another 30 min. Serial dilutions of test anti-CCR8 antibodies were prepared in HHBS buffer in a clear 384-well plate, and hCCL1 in HHBS buffer was also aliquoted into a clear 384-well plate. The FLIPR assay on the FDSS / μCell was set up with antibody addition at 10 sec and hCCL1 addition at 300 sec, and a total of 500 sec of monitoring. Excitation and emission wavelengths were set at 485 nm and 525 nm, respectively. After the run, negative control correction was applied and data was normalized to the hCCL1 signal (100%) and plotted as a function of antibody concentration using Prism.

[0393] As shown in Figure 3A, CCL1, a known ligand for CCR8, exhibits agonist activity, whereas none of the anti-CCR8 tested antibodies exhibits an agonist effect. The data in Figure 3B show that the anti-CCR8 antibody hu.Ab4.H1L1 has antagonist (neutralizing) activity against the CCR8 ligand CCL1 (20 nM ligand), whereas the anti-CCR8 antibodies hu.Ab5.H1L1 and hu.Ab3.H1L1 do not exhibit ligand blocking activity (non-neutralizing) at the concentrations tested. The data in Figure 3C further demonstrate that the comparator anti-CCR8 antibodies (Yoshida humanized anti-human CCR8 antibody, mouse anti-human CCR8 mAb 433H (BD Biosciences), and mouse anti-human CCR8 mAb L263G8 (Biolegend)) also exhibit antagonist (neutralizing) activity by blocking activation of CCR8 by the CCR8 ligand CCL1. IC of ligand blocking activity 50 Values ​​are shown in Table B. As described in Van Damme et al., J. Immunother. Cancer (2021), 9: e001749, ligand blockade alone is insufficient for Treg cell depletion in mouse tumors. Thus, although hu.Ab5.H1L1 and hu.Ab3.H1L1 did not show ligand blockade, these two antibodies were still considered promising candidates since the goal was to find selective anti-CCR8 antibodies that bind to CCR8 and deplete Treg cells. [Table B]

[0394] To confirm selectivity for CCR8, hu.Ab3.H1L1, hu.Ab4.H1L1, and hu.Ab5.H1L1, as well as Yoshida humanized anti-human CCR8, mouse anti-human CCR8 mAb L263G8 (Biolegend, commercial Ab), and mouse anti-human CCR8 mAb 433H (BD Biosciences, commercial Ab) were characterized by flow cytometry on HEK293 cells transiently transfected with plasmids encoding FLAG-tagged other relevant human GPCRs (CCR2-5, CXCR4, ACKR2, and ACKR4). Cell surface expression of each GPCR was confirmed by staining with anti-FLAG antibody controls. See Figures 4A-4F. In particular, HEK293 cells were transfected with N-terminal FLAG tagged human CCR2, CCR3, CCR4, CCR5, CXCR4, ACKR2, ACKR4, hCCR8 constructs or mock constructs using transIT X2 (reagent:DNA=3:1) for 24 hours and surface stained with 5ug / ml of various anti-hCCR8 monoclonal antibodies or rabbit anti-Flag pAb (Sigma) followed by AF647 anti-hIgG or AF647 anti-RbIgG, respectively. Antibodies hu.Ab4.H1L1 and hu.Ab5.H1L1 stained only hCCR8 containing cells, confirming their specificity for hCCR8. Antibody hu.Ab3.H1L1 showed staining of multiple other GPCRs, indicating a lack of specificity. Therefore, we went ahead with the CCR8 selective hu.Ab4.H1L1 and hu.Ab5.H1L1 antibodies with the best ADCC activity.

[0395] Example 2. Mutational Analysis of Ab4 and Ab5 Anti-CCR8 Antibodies Variants of the hu.Ab4.H1L1 and hu.Ab5.H1L1 anti-CCR8 antibodies were further investigated and characterized. Figures 5A-5D show alignments of the light chain variable regions (Figure 5A) and heavy chain variable regions (Figures 5B-5D) of the studied rabbit (rb.Ab4) and humanized Ab4 (L1-L4 and H1-H12) CCR8 antibody sequences. Figures 6A-6D show alignments of the light chain variable regions (Figure 6A) and heavy chain variable regions (Figures 6B-6D) of the studied rabbit (rb.Ab5) and humanized Ab5 (L1-L5 and H1-H13) CCR8 antibody sequences. See also Tables C1-C3 and D1-D3 below. Table E provides the heavy and light chain constant domains. [Table C1] [Table C2] TIFF2024527606000008.tif44170 [Table C3] TIFF2024527606000010.tif251170TIFF2024527606000011.tif137170 [Table D1] [Table D2] TIFF2024527606000014.tif63170 [Table D3] TIFF2024527606000016.tif252170TIFF2024527606000017.tif203170 [Table E]

[0396] CCR8 binding of the humanized variants and hIgG1 Fc was assessed by flow cytometry screening and relative EC50 These included comparing the MFI and MFI with the parent rabbit antibody. Specifically, stable CHO-huCCR8.Gna15 cells were stained with Ab4 and Ab5 variants at various concentrations (starting at 10ug / ml or 66.66nM, serially diluted 1:4 for a total of 8 concentration points) for 30 minutes at 4°C, then washed twice with FACS buffer (PBS with 0.5% BSA and 0.2mM EDTA) followed by staining with AF647-anti-hIgG for 15 minutes at 4°C. Cells were washed twice with FACS buffer, resuspended in FACS buffer with propidium iodide (0.5ug / ml) and analyzed on an iQe3 (Sartorius).

[0397] As shown in Table F1, for Ab4 LCL variants L1 to L4, variants L2 and L4 containing the Y2I mutation were significantly associated with EC 50 or MFI. Thus, Y2 on the light chain was determined to be the critical rabbit Vernier residue. Variants L1 and L3 contain this Y2 residue, and variant L3 was selected for further analysis. [Table F1]

[0398] As shown in Table F2, for Ab4 HC variants H2 to H11, variant H6 (with S73N mutation), variant H7 (with T76N mutation), variant H8 (with V78L mutation), variant H9 (with F91Y mutation), variant H10 (with P105Q mutation), and variant H11 (with S73N, V78L, F91Y, and P105Q mutations) were EC 50 or MFI. Thus, S73, T76, V78, F91, and P105 on the heavy chain were determined to be the critical rabbit Vernier residues. These five residues were combined to construct variant H12 (hu.Ab4.H12). [Table F2]

[0399] As provided in Table F3, for Ab5 LC variants L2 to L5, variant L2 (having a V4M mutation), variant L3 (having a P43A mutation), variant L4 (having an F46L mutation), and variant L5 (having V4M, P43A, and F46L mutations) showed EC 50 or MFI. Thus, V4, P43 and F46 on the light chain were determined to be the critical rabbit Vernier residues. All variants contained a C90Q mutation in CDR L3, which was introduced to remove an unpaired cysteine ​​that could be a cause during production. Variant L1, which contained all three V4, P43 and F46 residues, was selected for further study. [Table F3]

[0400] For Ab5 HC variants H2-H12, as shown in Table F4, variants H5 (with G49S mutation), H6 (with K71R mutation), H7 (with S73N mutation), and H12 (with G49S, K71R, and S73N mutations) were resistant to EC 50 or MFI. Therefore, G49, K71, and S73 on the heavy chain were determined to be the important rabbit Vernier residues. These three residues were combined to construct variant H13. [Table F4]

[0401] Example 3. Characterization of hu.Ab4.H12L3 and hu.Ab5.H13L1 variants (a) Human-cyno cross-reactivity Cell-based affinity assays were performed for hu.Ab5.H13L1 and hu.Ab4.H12L3 using radiolabeled IgG and CHO cell lines stably expressing human or cyno CCR8. [Table G1] [Table G2]

[0402] Briefly, stable CHO cells expressing human or cyno CCR8 were seeded at 50,000 cells per well in cold binding buffer (Opti-MEM + 2% FBS + 50 mM HEPES, pH 7.2 + 0.1% sodium azide). A fixed concentration of radiolabeled IgG was added using the NEX244 Iodogen method (Perkin Elmer). 125 I-anti-CCR8 was mixed with serially diluted anti-CCR8 antibodies starting at 20 nM or 50 nM. The antibody mixture was added to the cells and incubated at room temperature for 12 hours with gentle agitation. The cells and antibodies were then transferred to a Millipore multiscreen filter plate. The filter plate was washed four times with 250 μL cold binding buffer, dried for at least 30 minutes, and the filters were punched into 5 mL polystyrene tubes. Radioactivity was measured using a Perkin Elmer Wallac Wizard 2470 Gamma Counter set at 1 count / min with a counting efficiency of 0.8. Data was fitted using a heterogeneous one-site fit Ki competitive binding model in GraphPad Prism.

[0403] As shown in Figures 7A-7D, hu.Ab4.H12L3 and hu.Ab5.H13L1 have similar affinity for both human and cyno CCR8, demonstrating desirable cross-reactivity. Tabulated affinity Kd (nM) data from these studies are provided below. [Table G3]

[0404] (b) CCR8 selectivity To reconfirm that the Ab4 and Ab5 variants remained selective for CCR8 compared to the corresponding H1L1 variants, binding was analyzed by flow cytometry following the procedures described for Figures 4A and 4B. As before, both hu.Ab4.H12L3 (Figure 8A) and hu.Ab5.H13L1 (Figure 8B) selectively bound to CCR8-expressing cells.

[0405] (c) CCR8 activation and ligand blockade To reaffirm that the Ab4 and Ab5 variants retain their properties regarding CCR8 activation and ligand blocking capacity, experiments were performed with hu.Ab4.H12L3 and hu.Ab5.H13L1 previously described in Example 1 and Figures 3A-3C. See Figures 9A-9B. Similar to Figure 3A, the data in Figure 9A reaffirm that neither the Ab4 nor the Ab5 anti-CCR8 antibody variants show agonist effects in the absence of the CCR8 ligand CCL1. Similar to the data in Figure 3B, the data in Figure 9B reaffirm that the Ab4 variants show antagonist effects by blocking the activation of CCR8 by the CCR8 ligand CCL1 (20 nM of ligand), whereas the Ab5 variants show no ligand blocking activity at the concentrations tested. IC of ligand blocking activity 50 The values ​​are provided in the table below. [Table H1]

[0406] (d) Sulfation independence Human CCR8 contains four potential sites of tyrosine sulfation within the N-terminus, and existing evidence indicates that modifications at these sites show some heterogeneity (Gutierrez et al. JBC 2004; Jen, et al. Biochemistry 2010). Thus, antibodies that recognize these sulfated tyrosines in CCR8 may exhibit variability in CCR8 binding and thus mediate variable Treg cell depletion. Human CCR8+ HEK293 cells lacking tyrosyl protein sulfotransferases (TPST) 1 and 2, the enzymes that catalyze tyrosine sulfation, were generated. The binding of various anti-CCR8 mAbs to wild-type (293T) and TPST1 / 2 NTC and KO cells was then analyzed.

[0407] In particular, HEK293, HEK293-hCCR8.TPST1 / 2 NTC and HEK293-hCCR8.TPST1 / 2 KO stable cell lines were stained with test and comparator anti-CCR8 antibodies (1ug / ml) for 30 minutes at 4° C., then washed twice with FACS buffer (PBS containing 0.5% BSA and 0.2mM EDTA) followed by staining with AF647-anti-hIgG for 15 minutes at 4° C. Cells were washed twice with FACS buffer, resuspended in FACS buffer containing propidium iodide (0.5ug / ml) and analyzed on a BD FACSCelesta Flow Cytometer or iQe3 (Sartorius).

[0408] Figures 10A-10E show the difference in staining of hu.Ab4.H12L3 and hu.Ab5.H13L1 compared to Yoshida humanized anti-human CCR8 antibody and commercial antibodies mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend) on CCR8+ HEK293 cells with (hCCR8.TPST1 / 2 NTC) and without (hCCR8.TPST1 / 2 KO) tyrosyl-protein sulfotransferase (TPST)1 and tyrosyl-protein sulfotransferase (TPST)2. hu.Ab4.H12L3 (FIG. 10A) and hu.Ab5.H13L1 (FIG. 10B) showed similar binding / staining to both cell lines (hCCR8.TPST1 / 2 NTC and hCCR8.TPST1 / 2 KO), indicating that they bind CCR8 independent of tyrosine sulfation ("sulfation-independent"). In contrast, the Yoshida humanized anti-human CCR8 antibody (FIG. 10C) and the commercial antibodies mouse anti-human CCR8 mAb 433H (BD Biosciences) (FIG. 10D) and mouse anti-human CCR8 mAb L263G8 (Biolegend) (FIG. 10E) were unable to bind TPST1 / 2 KO cells, indicating that they require tyrosine sulfation of CCR8 for binding and are therefore considered "sulfation-dependent".

[0409] Example 4. hu.Ab4.H12L3 and hu.Ab5.H13L1 afucosylation variants Afucosylated hu.Ab5.H13L1 and hu.Ab4.H12L3 variants (Fc N-glycan position Asn 299) and an afucosylated anti-gD control were prepared by expression and purification from FUT8 knockout (KO) CHO cells as described in Wong et al., Biotechnology and Bioengineering (2010) 106:751-763.

[0410] (a) Percent afucosylation Upon titration of fucose in the medium of CHO FUT8KO, a panel of hu.Ab5.H13L1 was obtained with various levels of afucosylation, for example, from about 14% to about 93% afucosylated hu.Ab5.H13L1.

[0411] As shown in the table below, increasing the afucosylation level from 14% to 49% resulted in a more than four-fold increase in ADCC activity and a more than three-fold increase in ADCP activity.

[0412] Afucosylated hu.Ab5.H13L1 and hu.Ab4.H12L3 were studied in in vitro and in vivo experiments, tracking afucosylation content levels ranging from about 80% to about 95%. [Table H2]

[0413] (b) Enhanced FcγRIIIa binding of afucosylated variants The binding of fucosylated and afucosylated variants of hu.Ab5.H13L1 and hu.Ab4.H12L3 to both FcgR3a proteins by ELISA was studied. Briefly, anti-GST antibodies were coated onto Nunc Maxisorp plates. GST-FcgR3a.V158 and GST-FcgR3a.F158 were captured at 500ng / mL. The plates were then washed, and serially diluted anti-CCR8 antibodies starting at 100ug / mL were then incubated on the plates for 1 hour at room temperature. The plates were washed, and bound antibodies were detected by HRP-conjugated anti-human IgG secondary antibodies. Absorbance at 450nm was measured by a plate reader. Data was fitted using a 4-parameter logistic curve in Softmax Pro. As shown in the table below, the afucosylated IgG1 anti-CCR8 antibodies Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 exhibited enhanced FcγRIIIa binding activity (approximately a 10-fold increase in binding potency) compared to their fucosylated counterparts hu.Ab5.H13L1 and hu.Ab4.H12L3. [Table I]

[0414] (c) Enhanced ADCC activity of afucosylated variants Afuc.hu.Ab4.H12L3, hu.Ab4.H12L3, Afuc.hu.Ab5.H13L1, and hu.Ab5.H13L1 were analyzed for antibody-dependent cellular cytotoxicity (ADCC). ADCC assays were performed as previously reported in Kamen et al., Development of a kinetic antibody-dependent cellular cytotoxicity assay. J Immunol Methods (2019) 468: 49-54, and Schnueriger et al., Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies. Mol Immunol (2011) 48: 1512-17, with some modifications, using CD16-engineered NK-92_F158 as effector cells and CHO cells stably expressing human CCR8 and Ga15 subunits (CHO / hCCR8.Gna15) as target cells. Briefly, lysis of target cells by ADCC was measured by the calcein release method. Target cells were labeled with calcein-AM (C3100MP, ThermoFisher Scientific) according to the manufacturer's protocol, then washed and plated at 3000 x g for 384-well plates. Cells / well were seeded at a density of 1000 x 1000 cells / well. Anti-CCR8 antibodies were added at various concentrations from 0.004 to 1 µg / mL, followed by the addition of NK-92_F158 cells at an effector:target (E:T) ratio of 10:1. The plates were then incubated for 2.5 h at 37 °C. After incubation, the plates were centrifuged at 200 x g for 3 min, the supernatants were transferred to white opaque 384-well microplates (OptiPlate-384, PerkinElmer, Waltham, MA), and the fluorescent signals were measured in relative fluorescence units (RFU) with excitation / emission at 485 / 520 nm using an EnSight Multimode Plate Reader (PerkinElmer). Signals from wells containing only target cells indicated spontaneous release of calcein from the labeled cells (spontaneous release), whereas wells containing target cells lysed with Triton X-100 (Sigma-Aldrich, St. Louis, MO) provided the maximum available signal (maximum release). Antibody-independent cell-mediated cytotoxicity (AICC) was measured in wells containing target and effector cells without the addition of antibody. Samples and controls were tested at least in duplicate on the same plate. The degree of specific ADCC activity was calculated as follows:

[0415] %ADCC=100×(Average experimental release - Average AICC) / (Average maximum release - Average spontaneous release)

[0416] ADCC activity was plotted as a function of antibody concentration and the data was fitted to an asymmetric sigmoidal 4-parameter logistic (4PL) model using Prism (Graphpad; La Jolla, Calif.). Figures 11A-11B show that afucosylated CCR8 antibodies Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 have enhanced ADCC activity (>10-fold improvement) compared to their fucosylated counterparts hu.Ab5.H13L1 and hu.Ab4.H12L3 against CHO cells stably expressing hCCR8 using NK-92 F158 (Figure 11A) and NK-92 V158 (Figure 11B) as effector cells.

[0417] The ADCC activity of Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 was also measured against the Yoshida humanized anti-human CCR8 antibody and the commercial antibodies mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend). See Figure 11C. The data demonstrate that the Yoshida humanized anti-human CCR8 antibody exhibits weaker ADCC activity (10-20 times less ADCC activity) than the anti-CCR8 antibodies Afuc.Ab5.H13L1 and Afuc.Ab4.H12L3. The commercially available antibodies containing a mouse Fc domain, mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend), did not show ADCC activity, as expected, since the assay used in this example is primarily relevant for antibodies containing a human Fc domain.

[0418] Mouse anti-human CCR8 mAb 433H (BD Biosciences) and mouse anti-human CCR8 mAb L263G8 (Biolegend) were tested for ADCC activity in an assay involving antibodies containing mouse Fc regions but with anti-human CCR8 activity, namely Jurkat / mFcgR4 stable lines as effector cells and CHO / hCCR8 as target cells. Human CCR8 (hCCR8) was used to mimic the human clinical situation. Specifically, the assay consists of an engineered Jurkat T cell line expressing the mouse FcgRIV receptor and a luciferase reporter driven by the NFAT response element (NFAT-RE). When co-cultured with target cells and the relevant antibody, mFcgRIV effector cells bind to the Fc domain of the antibody, resulting in mFcgRIV signaling and NFAT-RE mediated luciferase activity. Materials and Reagents: Assay Buffer: RPMI 1640 without phenol red supplemented with 4% low IgG; 96-well white flat bottom polystyrene TC treated microplates, Corning#3601; Bio-Glo Reagent. Assay Procedure: Add 25μL / well diluted antibody in assay buffer (prepared in triplicate starting at 30ug / mL in a 10 point 1:4 serial dilution). Resuspend target cells in assay buffer to a final density of 1x10 6 Dispense 25 μL / well of Jurkat / mFcgRIV cells (5×10 6Add 100 μl of Bio-Glo™ Reagent to each well to an effector cell density of 125,000 / well; remix the cells in the reservoir periodically during the process to prevent the cells from settling to the bottom. Cover the assay plate with a lid and incubate the plate at 37° C. in a 5% CO2 incubator for 16 hours. Do not stack plates inside the incubator. Remove the assay plate from the incubator and equilibrate to ambient temperature for 15 minutes. Using a multichannel pipette, add 75 μl of Bio-Glo™ Reagent to the assay plate, taking care not to introduce air bubbles. Incubate the plate at room temperature for 15 minutes. Measure luminescence using an EnSight luminescence plate reader. mIgG2a isotypes hlgG1 and ratIgG2b were tested as controls. Human CCR8 (hCCR8) was used to mimic the human clinical situation. As can be seen from the data, each of the anti-hCCR8 mAbs tested - L263G8 (BioLegend) and 433H (BD Biosciences) - showed high fold induction results at an antibody concentration level of about 1 nM - with fold induction results of greater than about 10-fold and about 12-fold, respectively. The high fold induction for each of these antibodies reached a plateau at an antibody concentration level of about 40 nM, with fold induction values ​​of about 11 and 13, respectively. The results of these experiments are shown in Figure 11D.

[0419] Activity data from these studies are also provided in the table below. In summary, each antibody tested, regardless of whether it had a humanized or murine Fc region, demonstrated ADCC activity in assays that were species-matched to the effector reporter cells for which the antibody isotype is relevant. [Table J] *No activity = assay conditions not related to a specific Ab isotype

[0420] (d) Enhancement of ADCC against Treg cells To induce CCR8 expression on Treg cells from human peripheral blood mononuclear cells (PBMCs), 10 7Human PBMCs were cultured using NOD.Cg-Prkdc scid Il2rg tm1Wjl Mice (JAX) were intraperitoneally transferred with IgG1 and spleens were harvested 2–3 weeks after transfer. Human T cells were enriched from single cell suspensions of NSG splenocytes using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec) and primary NK cells were separately enriched from human PBMCs using the Human NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's protocol. Human T cells were incubated with 0.001–1ug / mL CCR8 mAb for 30 minutes at room temperature before adding primary NK cells at an effector:target ratio of 2:1. After overnight incubation at 37°C, cells were harvested, surface stained, and intracellularly stained using eBioscience Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific) according to the manufacturer's protocol. Antibodies used to define T cell populations were CD45 (HI30), CD3 (SK7), CD8 (RPA-T8), and CD14 (63D3) (BD Biosciences), CD4 (RPA-T4) from BioLegend, and FOXP3 (236A / E7) from ThermoFisher Scientific. CountBright Absolute Counting Beads (ThermoFisher Scientific) were added to each sample before acquisition. Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and analyzed with FlowJo software (BD Biosciences, version 10.5.3). Absolute cell numbers were calculated according to the manufacturer's protocol.

[0421] ADCC activity against Treg cells was measured by calculating the ratio of recovered regulatory T cells to recovered CD8 cells (Treg / CD8) or conventional CD4 T cells to recovered CD8 T cells (CD4conv / CD8). The number of recovered CD8 T cells was similar across all concentrations of CCR8 mAb and isotype control mAb ("gD.afuc") tested. As shown in Figures 12A-12D, afucosylated CCR8 antibodies Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 and fucosylated CCR8 antibodies hu.Ab5.H13L1 and hu.Ab4.H12L3 selectively mediated ADCC activity and increased depletion of Tregs from in vivo mixed lymp...

Claims

**Claim 1** A monoclonal antibody that binds to C-C motif chemokine receptor 8 (CCR8), comprising a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

28. **Claim 2** (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29; (b) the antibody binds to CCR8 independently of sulfation of CCR8; (c) the antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID NO: 106; (d) the antibody comprises: (i) a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 35 to 47; (ii) a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 48 to 52; or (iii) the VH defined in (i) and the VL defined in (ii); and / or (e) the antibody comprises: (i) a VL comprising: (1) a C90Q mutation and a Y2I mutation; or (2) a V4M mutation, a P43A mutation, an F46L mutation, a C90Q mutation, or a combination thereof; and / or (ii) a VH comprising: (1) a V78L mutation, a T76N mutation, an F91Y mutation, and a P105Q mutation; or (2) a G49S mutation, a K71R mutation, an S73N mutation, or a combination thereof, wherein the amino acid positions are numbered according to Kabat; The antibody according to claim 1. **Claim 3** (a) VH comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48; (b) VH comprises V78L mutation, T76N mutation, F91Y mutation, and P105Q mutation; and VL comprises C90Q mutation and Y2I mutation, wherein the amino acid positions are numbered by Kabat; and / or (c) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55, 60, 111 or 113, and a light chain comprising the amino acid sequence of SEQ ID NO: 56; The antibody according to claim 2. **Claim 4** A monoclonal antibody that binds to CCR8, (A) the antibody (1) (a) a heavy chain variable domain (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, and (d) a light chain variable domain (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (2) (a) a heavy chain variable domain (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85, and (d) a light chain variable domain (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75; a heavy chain variable domain (VH) comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89; and a light chain variable domain (VL) comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78; a heavy chain variable domain (VH) comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93; and a light chain variable domain (VL) comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81; or a heavy chain variable domain (VH) comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a light chain variable domain (VL) comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64, wherein CCR8 is murine CCR8; and / or the antibody binds to CCR8 independently of the sulfation of CCR8, an antibody. The antibody according to claim 5, wherein the antibody comprises a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; and a) the antibody binds to CCR8 independently of the sulfation of CCR8, b) the antibody binds to an epitope comprising one or more of amino acid residues 91-104 and 172-193 of SEQ ID NO: 106; and / or c) the antibody (i) a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 10 to 21; (ii) a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 22 to 25; or (iii) a VH defined in (i) and a VL defined in (ii); The antibody according to claim 4.

6. (a) The VH comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21, and the VL comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24; (b) The VL comprises a Y2I mutation, wherein the amino acid position is numbered by Kabat; (c) The VH comprises an S73N mutation, a V78L mutation, a T76N mutation, an F91Y mutation, a P105Q mutation, or a combination thereof, wherein the amino acid position is numbered by Kabat; and / or (d) The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 57, 61, 112 or 114, and a light chain comprising the amino acid sequence of SEQ ID NO: 58, The antibody according to claim 5.

7. The antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, (a) The antibody is (i) a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95; (ii)a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94; or (iii)a VH defined in (i) and a VL defined in (ii); comprising; and / or (b)The antibody according to claim 4, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 or 115, and a light chain comprising the amino acid sequence of SEQ ID NO:

100.

8. The antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 76, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78; (a)The antibody (i)a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97; (ii)a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96; or (iii)a VH defined in (i) and a VL defined in (ii); comprising; and / or (b)The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 or 116, and a light chain comprising the amino acid sequence of SEQ ID NO:

102. The antibody according to claim 4.

9. The antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81; (a)The antibody (i)a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99; (ii) a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98; and (iii) a VH defined in (i) and a VL defined in (ii); comprising; and / or (b) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 105 or 117, and a light chain comprising the amino acid sequence of SEQ ID NO: 104; The antibody according to claim 4. **Claim 10** The antibody binds to CCR8 independent of sulfation of CCR8, and the antibody (a) one or more of amino acid residues 2 to 6 of SEQ ID NO: 106; or (b) binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID NO: 106; The antibody according to claim 4. (a) the antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64, wherein CCR8 is murine CCR8; (a) the antibody (i) a VH comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70; (ii) a VL comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69; or (iii) a VH defined in (i) and a VL defined in (ii); (b) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 71; (c) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 71; (d) the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54; **Claim 12** (a) the antibody is a human antibody, a humanized antibody, or a chimeric antibody; (b) the antibody is an antibody fragment or a full-length antibody that binds to CCR8; (c) the antibody comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 59; (d) the antibody comprises a kappa constant domain comprising the amino acid sequence of SEQ ID NO: 54; (e) The antibody binds to CCR8 with a binding affinity (Kd) of about 1×10−12 M to about 1×10−11 M; (f) CCR8 is human CCR8; and / or (g) The antibody is afucosylated; , the antibody according to claim 1.

13. (a) The antibody is a humanized full-length IgG1 antibody; (b) The antibody comprises an IgG1 constant domain containing the amino acid sequence of SEQ ID NO: 53 and a kappa constant domain containing the amino acid sequence of SEQ ID NO: 54; and / or (c) The antibody is afucosylated and the degree of afucosylation is about 80% to about 95%; The antibody according to claim 12.

14. An isolated nucleic acid encoding the antibody according to any one of claims 1 to 13.

15. A host cell comprising the nucleic acid according to claim 14.

16. A method for producing an antibody that binds to CCR8, the method comprising culturing the host cell according to claim 15 under conditions suitable for the expression of the antibody.

17. The method according to claim 16, further comprising recovering the antibody from the host cell.

18. An antibody produced by the method according to claim 17.

19. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition according to claim 19, further comprising an additional therapeutic agent.

21. A medicament for treating cancer in a subject in need of treatment for cancer, comprising the antibody according to any one of claims 1 to 13.

22. A medicament for depleting regulatory T cells, comprising the antibody according to any one of claims 1 to 13, (a) The regulatory T cells are present in the tumor microenvironment in a subject having cancer; (b) The regulatory T cells are present outside the tumor microenvironment in a subject having cancer; and / or (c) The regulatory T cells are from an in vitro cell population; Medicament.

23. (a) The cancer is bladder cancer, blastoma, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, testicular cancer or uterine cancer; (b) The antibody is formulated for administration with an additional therapeutic agent; and / or (c) The subject is human or mouse; The medicament according to claim 21.

24. The medicament according to claim 23, wherein the additional therapeutic agent is an anticancer agent.

25. (a) The anticancer agent is a microtubule disrupting agent, antimetabolite, topoisomerase inhibitor, DNA intercalator, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, activator of tumor cell apoptosis, anti-angiogenic agent, immunomodulatory agent, inhibitor of cell adhesion, cytotoxic agent or cell division inhibitor, activator of cell apoptosis, agent that increases the sensitivity of cells to apoptosis-inducing substances, cytokine, anticancer vaccine or oncolytic virus, toll-like receptor (TLR) agent, bispecific antibody, cell therapy, or immune cell engager; (b) The anticancer agent is a PD-L1 binding antagonist; or (c) The anticancer agent is a PD-L1 binding antagonist, and the PD-L1 binding antagonist is atezolizumab; The medicament according to claim 24.

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