CN117693527A

CN117693527A - anti-C motif chemokine receptor 8 (CCR 8) antibodies and methods of use

Info

Publication number: CN117693527A
Application number: CN202280049269.1A
Authority: CN
Inventors: G·加姆帕; I·侯赛尼; M·胡塞尼; J·T·科尔伯; J·M-D·佩恩德; S·鲁兹; 孙永莲; C·P·C·赵; T·A·德尔菲诺
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2021-07-14
Filing date: 2022-07-13
Publication date: 2024-03-12

Abstract

The present disclosure provides anti-CCR 8 antibodies, compositions, and methods of making and using the same.

Description

anti-C motif chemokine receptor 8 (CCR 8) antibodies and methods of use

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/253,676 filed on 8 10 2021 and U.S. provisional application number 63/221,734 filed on 14 7 2021, both of which are incorporated herein by reference in their entirety and claim priority.

Sequence listing

The present application contains a sequence listing that has been submitted via EFS-Web in xml format and is incorporated by reference herein in its entirety. The xml copy was created at 2022, 7, 12, and was named 00b206.1290.Xml.

Background

Regulatory T (Treg) cells expressing 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 immune infiltration of solid cancers, promoting tumor development and progression by establishing an immunosuppressive tumor microenvironment and suppressing anti-tumor immune responses. See, e.g., plitas and Rudensky, annu.rev.cancer biol (2020) 4:459-477.Treg cells also hinder the efficacy of immunotherapy. See, e.g., nishikawa and Sakaguchi, curr. Opin. Immunol. (2014) 27:1-7. An increase in the proportion of Treg cells in tumor infiltrating lymphocytes is associated with poor outcome for 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 (page 9); shen et al, j.cancer res.clin.oncol. (2010) 136:1585-1595; and Tanaka and Sakaguchi, eur.j. Immunol. (2019) 49:1140-1146.

Several strategies involving Treg cell depletion or inhibition have been shown to enhance anti-tumor immunity and cause tumor growth inhibition in preclinical breast, 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 (such as CD 25) show limited efficacy in established tumors, probably due to the simultaneous depletion of effector T cells critical for anti-tumor immunity. See, e.g., onizuka et al Cancer Res (1999) 59:3128-3133.

Chemokine receptor CCR8 is a seven-transmembrane G Protein Coupled Receptor (GPCR) and is linked with high affinity by human/mouse CCL1 and is selectively and highly expressed by Treg cells within the tumor microenvironment, but not substantially peripheral Treg cells or effector T cells. High CCR8 expression on Treg cells is associated with reduced advanced disease stage and overall survival in patients with breast cancer. See, e.g., plitas et al, immunity (2016) 45:1122-1134. CCR8 thus represents a promising and safer target for Treg cell depletion in cancer treatment. Thus, there is a need for agents that recognize CCR8 and methods of using such agents.

Disclosure of Invention

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to the C-C motif chemokine receptor 8 (CCR 8), wherein the antibody comprises: 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.

Embodiment 2. The antibody of embodiment 1 above, which binds to CCR8 independent of sulfation of CCR 8.

Embodiment 3. The antibody of embodiment 1 or 2 above, wherein the antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID No. 106.

Embodiment 4. The antibody according to any one of the preceding embodiments 1 to 3, comprising a sequence selected from the group consisting of: (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: 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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 5 the antibody according to any one of the preceding 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.

Embodiment 6 the antibody according to any one of the preceding embodiments 1 to 5, comprising a sequence selected from the group consisting of: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 7. The antibody of 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.

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

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

Embodiment 10. The antibody of any of embodiments 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID NO. 55 and the light chain amino acid sequence of SEQ ID NO. 56.

Embodiment 11. The antibody of any of embodiments 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID NO. 60 and the light chain amino acid sequence of SEQ ID NO. 56.

Embodiment 12. The antibody of any of embodiments 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID NO. 111 and the light chain amino acid sequence of SEQ ID NO. 56.

Embodiment 13. The antibody of any one of embodiments 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID NO. 113 and the light chain amino acid sequence of SEQ ID NO. 56.

In certain non-limiting embodiments, 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 to 47; and a VL sequence selected from the group consisting of: SEQ ID NOS: 48 to 52.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, comprising the VH sequence of SEQ ID No. 47 and the VL sequence of SEQ ID No. 48.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, wherein the antibody comprises: a heavy chain variable domain (VH) comprising (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; and a light chain variable domain (VL) comprising: (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.

Embodiment 17 the antibody of embodiment 16 above, which binds to CCR8 independent of sulfation of CCR 8.

Embodiment 18. The antibody of embodiment 16 or 17, wherein the antibody binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID No. 106.

Embodiment 19 the antibody of any one of the preceding embodiments 16 to 18 comprising a sequence selected from the group consisting of seq id no: (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: 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: SEQ ID NOS.22 to 25; and (c) a VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 20 the antibody according to any one of the preceding 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.

Embodiment 21 the antibody of any one of the preceding embodiments 16 to 20 comprising a sequence selected from the group consisting of seq id no: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 22. The antibody of 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.

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

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

Embodiment 25. The antibody of any one of embodiments 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID NO. 57 and the light chain amino acid sequence of SEQ ID NO. 58.

Embodiment 26. The antibody of any one of embodiments 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID NO. 61 and the light chain amino acid sequence of SEQ ID NO. 58.

Embodiment 27. The antibody of any one of embodiments 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID NO. 112 and the light chain amino acid sequence of SEQ ID NO. 58.

Embodiment 28. The antibody of any one of embodiments 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID NO. 114 and the light chain amino acid sequence of SEQ ID NO. 58.

In certain non-limiting embodiments, 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 to 21; and a VL sequence selected from the group consisting of: SEQ ID NOS.22 to 25.

In certain non-limiting embodiments, 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.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, wherein the antibody comprises: a heavy chain variable domain (VH) comprising (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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID No. 85; and a light chain variable domain (VL) comprising: (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.

Embodiment 32. The foregoing antibody according to embodiment 31, comprising a sequence selected from the group consisting of: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 33. The antibody of embodiment 31 or 32, comprising the VH sequence of SEQ ID NO. 95 and the VL sequence of SEQ ID NO. 94.

Embodiment 34. The antibody of any one of embodiments 31 to 33, comprising the heavy chain amino acid sequence of SEQ ID NO. 101 and the light chain amino acid sequence of SEQ ID NO. 100.

Embodiment 35. The antibody of any one of embodiments 31 to 33, comprising the heavy chain amino acid sequence of SEQ ID NO. 115 and the light chain amino acid sequence of SEQ ID NO. 100.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, wherein the antibody comprises: 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.

Embodiment 37. The foregoing antibody of embodiment 36, comprising a sequence selected from the group consisting of: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 38. The antibody of embodiment 36 or 37, comprising the VH sequence of SEQ ID NO. 97 and the VL sequence of SEQ ID NO. 96.

Embodiment 39. The antibody of any one of embodiments 36 to 38, comprising the heavy chain amino acid sequence of SEQ ID NO. 103 and the light chain amino acid sequence of SEQ ID NO. 102.

Embodiment 40. The antibody of any of embodiments 36 to 38, comprising the heavy chain amino acid sequence of SEQ ID NO. 116 and the light chain amino acid sequence of SEQ ID NO. 102.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, wherein the antibody comprises: 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.

Embodiment 42. The foregoing antibody of embodiment 41, comprising a sequence selected from the group consisting of: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 43 the antibody of embodiment 41 or 42 comprising the VH sequence of SEQ ID NO 99 and the VL sequence of SEQ ID NO 98.

Embodiment 44. The antibody of any one of embodiments 41 to 43, comprising the heavy chain amino acid sequence of SEQ ID NO. 105 and the light chain amino acid sequence of SEQ ID NO. 104.

Embodiment 45 the antibody of any one of embodiments 41 to 44 comprising the heavy chain amino acid sequence of SEQ ID NO. 117 and the light chain amino acid sequence of SEQ ID NO. 104.

In certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to CCR8, wherein the antibody binds to CCR8 independent of sulfation of CCR 8.

Embodiment 47. The antibody of embodiment 46, wherein the antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID NO. 106.

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

Embodiment 49 in certain non-limiting embodiments, the presently disclosed subject matter provides a monoclonal antibody that binds to mouse CCR8, wherein the antibody comprises: 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.

Embodiment 50. The foregoing antibody of embodiment 49, comprising a sequence selected from the group consisting of: (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 VH sequence as defined in (a) and a VL sequence as defined in (b).

Embodiment 51 the antibody of embodiment 49 or 50 comprising the VH sequence of SEQ ID NO. 70 and the VL sequence of SEQ ID NO. 69.

Embodiment 52. The antibody of any of embodiments 49 to 51 comprising the heavy chain amino acid sequence of SEQ ID NO:72 and the light chain amino acid sequence of SEQ ID NO: 71.

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

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

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

Embodiment 56. The antibody of any one of embodiments 1 to 55, which is an antibody fragment that binds to CCR 8.

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

Embodiment 58. The antibody of embodiment 57, which is a full length IgG1 antibody.

Embodiment 59. The antibody of 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.

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.

Embodiment 61. The antibody of any one of embodiments 1 to 60, wherein the antibody binds to CCR8 with a binding affinity (Kd) of about 1 x 10 "12M to about 1 x 10" 11M.

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

Embodiment 63. The antibody of any one of embodiments 1 to 62, wherein the antibody is defucosylated.

Embodiment 64 in certain non-limiting embodiments, the presently disclosed subject matter provides an isolated nucleic acid encoding the foregoing antibody according to any one of embodiments 1-63.

In certain non-limiting embodiments, the presently disclosed subject matter provides a host cell comprising the foregoing nucleic acid according to embodiment 64.

In certain non-limiting embodiments, the presently disclosed subject matter provides a method of producing an antibody that binds to CCR8, comprising culturing the host cell of embodiment 65 under conditions suitable for expression of the antibody.

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

In certain non-limiting embodiments, the presently disclosed subject matter provides an antibody produced by the foregoing method of embodiment 67.

Embodiment 69 in certain non-limiting embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising the foregoing antibody according to any one of embodiments 1-63 and a pharmaceutically acceptable carrier.

Embodiment 70 the foregoing pharmaceutical composition of embodiment 69, further comprising an additional therapeutic agent.

Embodiment 71 the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of embodiments 69-70 for use as a medicament.

Embodiment 72 the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of claims 69-70 for use in the treatment of cancer.

Embodiment 73 in certain non-limiting embodiments, the presently disclosed subject matter provides the use of the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of embodiments 69-70 in the manufacture of a medicament for treating cancer.

Embodiment 74 in certain non-limiting embodiments, the presently disclosed subject matter provides the use of the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of embodiments 69-70 in the manufacture of a medicament for depleting regulatory T cells.

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

In certain non-limiting embodiments, the presently disclosed subject matter provides a method of depleting regulatory T cells in a tumor microenvironment of a subject having cancer, the method comprising administering to the subject an effective amount of the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of embodiments 69-70 sufficient to deplete the regulatory T cells in the tumor microenvironment.

In certain non-limiting embodiments, the presently disclosed subject matter provides a method of depleting regulatory T cells outside a tumor microenvironment of a subject having cancer, the method comprising administering to the subject an effective amount of the foregoing antibody according to any one of embodiments 1-63 or the foregoing pharmaceutical composition according to any one of embodiments 69-70 sufficient to deplete the regulatory T cells outside the tumor microenvironment.

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

Embodiment 79 the foregoing use or method of any one of embodiments 73-78, 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.

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

Embodiment 81 the foregoing use or method of any one of embodiments 74, 77, 78 and 79, wherein said regulatory T cells outside of said tumor microenvironment of said cancer are depleted.

Embodiment 82 the foregoing use or method according to any one of embodiments 73-81, further comprising administering an additional therapeutic agent.

Embodiment 83. The foregoing use or method of embodiment 82, wherein the additional therapeutic agent is an anti-cancer agent.

Embodiment 84. The foregoing use or method of embodiment 83, wherein the anti-cancer agent is selected from the group consisting of: microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormone therapies, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cytostatic agents, apoptosis activators, 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 therapies and immunocytoplasmic agents.

Embodiment 85. The foregoing use or method of embodiment 83 or 84, wherein the anti-cancer agent is a PD-L1 binding antagonist.

Embodiment 86. The foregoing use or method of embodiment 85, wherein the PD-L1 binding antagonist is alemtuzumab.

Embodiment 87 the foregoing use or method according to any one of embodiments 73 to 85, wherein the subject is a human.

Embodiment 88 the foregoing use or method of any of embodiments 73-85, wherein the subject is a mouse.

Embodiment 89 in certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating a disease in a mouse, the method comprising administering to the mouse an effective amount of the foregoing monoclonal antibody according to any one of embodiments 49 to 52 to treat the disease.

Embodiment 90. The foregoing method of embodiment 89, wherein the mouse comprises a xenograft.

Embodiment 91. The antibody of embodiment 63, wherein the proportion of defucosylation is between about 80% to about 95%.

Embodiment 92. The antibody of any one of embodiments 1 to 15 and 46 to 48, wherein the average clearance after intravenous administration of a single 10mg/kg dose on day 1 is about 3 to about 5 mL/day/kg over a period of 35 days.

Drawings

Fig. 1 depicts the results of a screen for anti-CCR 8 monoclonal antibodies (mabs) that selectively bind to Treg cells (tregs) from human colorectal cancer Dissociating Tumor Cells (DTCs) (obtained from Discovery Life Sciences). Shown are the Mean Fluorescence Intensity (MFI) values for CD 8T cells (defined as cd45+cd14-cd3+cd8+cd4-) (circle,) regular CD 4T cells (defined as cd45+cd14-cd3+cd8-cd4+foxp3-) (square, +%) and Treg cells (defined as cd45+cd14-cd3+cd8-cd4+foxp3+) (triangle, respectively). Three of the five anti-CCR 8 mAb clones Ab1-Ab5 clone specific for intratumoral Treg cells, rather than conventional CD4 or CD 8T cells, and were ranked according to CCR8 MFI: hu.ab4.h1l1> hu.ab5.h1l1> hu.ab3.h1l1.

Figures 2A to 2B depict the proposed mechanism of action of Natural Killer (NK) cell mediated antibody dependent cytotoxicity (ADCC), leading to depletion of tregs that tumor-invasively express CCR8 (figure 2A) and suggesting ADCC activity of human/cynomolgus monkey cross-reactive anti-CCR 8 mabs for further study (figure 2B). EC against CCR8 mabs hu.ab3.h1l1, hu.ab5.h1l1 and hu.ab4.h1l1 ₅₀ The values were determined to be 0.02nM, 0.02nM and 0.08nM, respectively.

FIGS. 3A through 3D depict agonist and antagonist activities of human/cynomolgus monkey cross-reactive anti-CCR 8 mAbhu.Ab4.H1L1, hu.Ab5.H1L1 and hu.Ab3.H1L1 and the comparative anti-CCR 8mAb (humanized anti-human Yoshida anti-CCR 8 antibody, murine anti-human CCR8mAb 433H (BD Biosciences) and murine anti-human CCR8mAb L263G8 (bioleged). As shown in FIG. 3A, CCL1 (known ligand of CCR 8) showed agonist activity, but none of the anti-CCR 8 test mAbs showed antagonist activity, the data in FIG. 3B showed antagonistic (neutralizing) activity against CCR8mAb hu.Ab4.H1L1 (20 nM ligand) whereas anti-CCR 8mAb hu.Ab5.H1L1 and hu.Ab1L1 did not show antagonistic (neutralizing) activity against the non-human CCR8 ligand CCR8 (20 nM ligand) at the concentrations studied, and the data in FIG. 3B showed no antagonistic activity against murine anti-human CCR8mAb (37C 8 mAb) and the comparative anti-human CCR8mAb (37C 8 mAb) nd)) showed no agonism, whereas CCR8 ligand CCL1 showed agonism. The data in fig. 3D shows that the comparison anti-CCR 8 mAb (humanized anti-human Yoshida anti-CCR 8 antibody, murine anti-human CCR8 mAb433H (BD Biosciences), and murine anti-human bioleged L263G8 (bioleged)) exhibited antagonistic (neutralizing) activity against CCR8 ligand CCL 1. In examples, ICs with ligand blocking activity are provided ₅₀ Values.

Figures 4A to 4F depict binding data of hu.ab3.h1l1 (figure 4A), hu.ab4.h1l1 (figure 4B) and hu.ab5.h1l1 (figure 4C), as well as commercial anti-CCR 8 mAb murine anti-human CCR8 mAb433H (BD Biosciences) (figure 4D) and murine anti-human CCR8 mAb L263G8 (bioleged) (figure 4E), and humanized anti-human Yoshida anti-CCR 8 mAb (figure 4F) to HEK293 cells transiently transfected with N-terminal FLAG tagged plasmids encoding human GPCRs (CCR 2, CCR3, CCR4, CCR5, CCR8, CXCR4, ACKR2 and ACKR 4), hCCR8 constructs or with a mimetic construct using transIT X2 (reagent: dna=3:1). Cell surface expression of each GPCR was confirmed by staining with anti-FLAG antibody control (5 ug/mL). mabs hu.ab4.h1l1 and hu.ab5.h1l1 stained only hCCR 8-containing cells, confirming their specificity for hCCR 8. mAb hu.ab3.h1l1 showed staining for a variety of other GPCRs, indicating lack of specificity. CCR8 selective hu.ab4.h1l1 and hu.ab5.h1l1 mabs showed optimal ADCC activity (as shown in figure 2) were used for further study.

FIGS. 5A to 5D depict the light chain variable region (FIG. 5A) and heavy chain variable region (FIGS. 5B to 5D) alignments of the sequences of the rabbit (rb. Ab 4) and humanized Ab4 (L1-L4 and H1-H12) CCR8 mAbs studied. Based on the binding assessment of the variant antibodies, Y2 on the light chain (L3) and S73, T76, V78, F91 and P105 on the heavy chain (H12) were determined to be the key rabbit cursor residues. CDRs, variable regions, constant regions, and full-length sequences are provided in the examples.

FIGS. 6A to 6D depict the light chain variable region (FIG. 6A) and heavy chain variable region (FIGS. 6B to 6D) alignments of the sequences of the rabbit (rb. Ab 5) and humanized Ab5 (L1-L5 and H1-H13) CCR8 mAbs studied. The C90Q mutation in CDR L3 was introduced to remove unpaired cysteines which can be detrimental during manufacture. V4, P43 and F46 on the light chain (L1) and G49, K71 and S73 (H13) on the heavy chain were determined to be key rabbit cursor residues based on the binding assessment of the variant antibodies. CDRs, variable regions, constant regions, and full-length sequences are provided in the examples.

Fig. 7A to 7D depict the results of cell-based affinity measurements on hu.ab5.h13l1 and hu.ab4.h12l3 mabs using radiolabeled IgG and CHO cell lines stably expressing human CCR8 or cynomolgus monkey ("cyno") CCR 8. The data show that hu.ab4.h12l3 and hu.ab5.h13l1 mabs have similar affinities for human and cynomolgus CCR8, indicating the desired cross-reactivity (compare fig. 7A and 7B, and compare fig. 7C and 7D). Kd (nM) affinity data from these studies are provided in the examples.

Figures 8A to 8B depict the binding data of hu.ab4.h12l3 (figure 8A) and hu.ab5.h13l1 (figure 8B) mabs to a panel of sulfated GPCRs and again confirm that these Ab4 and Ab5 variants are similar to Ab4 and Ab5 data provided in figures 4B and 4C, showing selectivity to CCR8. CCR8 constructs with C-terminal FLAG are also provided in FIG. 4C, due to weak binding to the N-terminal FLAG tag of hCR 8 (which affects binding to the N-terminal epitope of Ab 5; see FIG. 16).

FIGS. 9A to 9B depict the effect of anti-CCR 8 mAbs hu.Ab4.H12L3 and hu.Ab5.H13L1 on CCR8 activation, e.g., by Ca ²⁺ Inflow assay (fig. 9A) and CCR8 CCL1 ligand binding (fig. 9B). Similar to the data of fig. 3A, fig. 9A again demonstrates that neither Ab4 nor Ab5 anti-CCR 8mAb variants show agonism in the absence of CCR8 ligand CCL 1. Similar to the data of fig. 3B, fig. 9B again demonstrates that Ab4 variants exhibit antagonism against CCR8 ligand CCL1 (20 nM ligand), whereas Ab5 variants do not exhibit ligand blocking activity at the concentrations studied. In examples, ICs with ligand blocking activity are provided ₅₀ Values.

FIGS. 10A to 10E depict the difference in staining of hu.Ab4.H12L3 and hu.Ab5.H13L1 versus CCR8+HEK293 cells with (hCR 8.TPST1/2 NTC) and without Tyrosyl Protein Sulfotransferase (TPST) 1 and Tyrosyl Protein Sulfotransferase (TPST) 2 (hCR 8.TPST1/2 KO) compared to humanized anti-human Yoshida CCR8mAb and the commercial antibodies murine anti-human CCR8mAb 433H (BD Biosciences) and murine anti-human CCR8mAb L263G8 (Biolegend). hu.ab4.h12l3 (fig. 10A) and hu.ab5.h13l1 (fig. 10B) showed similar binding/staining to the two cell lines (hccr 8.tpst1/2NTC and hccr8.tpst1/2 KO), indicating that they bind to CCR8 independent of tyrosine sulfation ("independent of sulfation"). In contrast, humanized anti-human Yoshida CCR8 antibodies (fig. 10C) and the commercial antibodies murine anti-human CCR8mAb 433H (BD Biosciences) (fig. 10D) and murine anti-human CCR8mAb L263G8 (Biolegend) (fig. 10E) failed to bind to TPST1/2KO cells, indicating that they required tyrosine sulfation of CCR8 for binding and are therefore considered "sulfation-dependent".

FIGS. 11A through 11D depict that the defucosylated CCR8 mAbs Afuc.hu.AB5.H13L1 and Afuc.hu.AB4.H12L3 show enhanced (> 10-fold increase) ADCC activity compared to their fucosylated CCR8 counterparts Hu.AB5.H13L1 and Hu.AB4.H12L3 using NK-92F158 (FIG. 11A) and NK-92V158 (FIG. 11B) as effector cells against CHO cells stably expressing hCR 8 and also show 10-20-fold improvement in ADCC activity compared to humanized anti-human Yoshida anti-CCR 8 antibodies (FIG. 11C). Commercial anti-CCR 8 mAb murine anti-human CCR8 mAb 433H (BD Biosciences) and murine anti-human CCR8 mAb L263G8 (Biolegend) demonstrated (as expected) no ADCC activity, as the assay used was primarily associated with antibodies comprising a human Fc region (fig. 11C). Fig. 11D shows that murine anti-human CCR8 mAb 433H (BD Biosciences) and murine anti-human CCR8 mAb L263G8 (Biolegend) have ADCC activity and human anti-CCR 8 activity using assays specific for antibodies comprising a murine Fc region. Activity data is also provided in the examples.

FIGS. 12A through 12D depict selective ADCC activity against human Treg cells when incubated with defucosylated, fucosylated (hIgG 1), and defucosylated isotype control mAb ("gD.afuc") and primary NK cells as effector cells, as compared to conventional human CD 4T cells from Peripheral Blood Mononuclear Cells (PBMC) that are transferred to NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl Recovery after induction of CCR8 expression in SzJ (NSG) mice. ADCC activity against Treg cells was measured by calculating the ratio of recovered Treg cells to recovered CD8 cells (Treg/CD 8) or conventional CD4T cells to recovered CD 8T cells (CD 4conv/CD 8). In contrast to conventional CD4T cells (fig. 12B), CCR8 mAb afuc.hu.ab4H12L3 and hu.ab4.h12l3 selectively mediate ADCC activity against Treg cells (fig. 12A), wherein the defucosylated variants exhibit increased ADCC activity. Similarly, CCR8 mAb afuc.hu.ab5.h13l1 and hu.ab5.h13l1 selectively mediate ADCC activity against Treg cells (fig. 12C) compared to conventional CD4T cells (fig. 12D), wherein the defucosylated variants exhibit increased ADCC activity.

Figures 13A to 13D depict selective ADCC activity against Treg cells compared to conventional CD4T cells when human dissociated Renal Cell Carcinoma (RCC) cells are incubated with defucosylated, fucosylated (hIgG 1) and defucosylated isotype control mAb ("gd.afuc") and primary NK cells as effector cells. ADCC activity against Treg cells was measured by calculating the ratio of recovered Treg cells to recovered CD8 cells (Treg/CD 8) or conventional CD4T cells to recovered CD 8T cells (CD 4conv/CD 8). CCR8 mAb afuc.hu.ab4.h12l3 and hu.ab4.h12l3 selectively mediate ADCC activity against Treg cells (fig. 13A) compared to conventional CD4T cells (fig. 13B), wherein the defucosylated variants exhibit increased ADCC activity. Similarly, CCR8 mAb afuc.hu.ab5.h13l1 and hu.ab5.h13l1 selectively mediate ADCC activity against Treg cells (fig. 13C) compared to conventional CD4T cells (fig. 13D), wherein the defucosylated variants exhibit increased ADCC activity.

FIGS. 14A through 14E show that defucosylated anti-CCR 8 mAb Afuc.hu.Ab5.H13L1 and Afuc.hu.Ab4.H12L3 are in CD14 from four different donors with FcgRIIA (H131R)/FcgRIIIa (V158F) genotypes as compared to fucosylated mAbs hu.Ab5.H13L1 and hu.Ab4.H12L3 ⁺ Monocyte-derived macrophages exhibited enhanced ADCP activity, these genotypes HR/FF (fig. 14A), RR/FF (fig. 14B), HR/VF (fig. 14C) and RR/VF (fig. 14D), and also exhibited 3-4 fold increases in ADCP activity compared to the humanized anti-human Yoshida anti-CCR 8 antibody (fig. 14E). Activity data is also provided in the examples.

FIGS. 15A through 15D show that defucosylated anti-CCR 8 mAb Afuc.hu.Ab5.H13L1.G236A.I332E compared to FcgRIIa-enhanced G236A.I332E variant Afuc.hu.Ab5.H13L1 in four different genotypes from having FcgRIIa (H131R)/FcgRIIIa (V158F)Donor CD14 ⁺ Monocyte-derived macrophages showed similar improved ADCP activity, with genotypes HR/FF (FIG. 15A), RR/FF (FIG. 15B), HR/VF (FIG. 15C) and RR/VF (FIG. 15D).

Fig. 16A to 16B depict table maps of hu.ab5.h13l1 (fig. 16A) and hu.ab4.h12l3 (fig. 16B) mabs. As shown in fig. 16A, wherein a construct encoding individual alanine point mutations at positions 2-24 in hCCR8 with a C-terminal FLAG tag was generated, hu.ab5.h13l1 did not bind to D2A, Y3A, L5A and D6A, indicating that the epitope includes at least the DYTLD region of the N-terminus of human CCR8. As shown in fig. 16B, where a construct was generated encoding a human CCR8.CCR5 chimera (N-term 1, N-term2, ECL1, ECL2 and ECL 3) in which the different extracellular regions of hCCR8 were replaced by corresponding regions from CCR5 with a C-terminal FLAG tag, hu.ab4.h12l3 did not bind to ECL1 and ECL2 chimeras, indicating that the epitope of the antibody includes at least ECL1 and ECL2 regions at the N-terminus of CCR8.Huccr 8: MDYTLDLSVTTVTDYYYPDIFSSP (SEQ ID NO: 110).

Figures 17A to 17I depict progressive depletion of Treg cells (measured as fraction of Treg cells with cd45+ leukocytes) in tumors (figure 17A) but not in spleens (figure 17B) or tumor draining lymph nodes (figure 17C) in CT26 tumor-bearing mice three days after injection of single dose mice with increasing concentrations between 0.003-5mg/kg instead of anti-CCR 8 mAb. anti-CCD 8 mAb treatment did not produce CD4 normal T cells (fig. 17D to 17F) or CD 8T cell depletion (fig. 17G-I). Isotype control antibodies (anti-gp 120) were used.

Fig. 18A to 18D depict tumor growth inhibition following treatment with a single dose (fig. 18B) or twice weekly dosing (fig. 18C) of mice substituted for anti-CCR 8 mAb in mice with established CT26 syngeneic tumors compared to treatment with anti-CD 25 mAb (fig. 18D) or isotype control mAb (anti-gp 120) (fig. 18A). When the tumor volume reaches 150-250mm ³ Treatment was started at that time. Tumor volumes were measured over time. Gray lines represent individual mice and black lines represent group fits.

FIGS. 19A to 19E depict replacement of anti-CCR 8 mAb (FIG. 19B) or tumor up to 150-250mm with effector mice administered at tumor inoculation ³ (figure)19D) Observed growth inhibition of CT26 tumors. No tumor growth inhibition was observed with the same ligand blocking anti-CCR 8 mAb effect disabled LALAPG variants (fig. 19C and 19E). Tumor volumes were measured over time. Gray lines represent individual mice and black lines represent group fits. Isotype control mAb (anti-gp 120) was used (fig. 19A).

Figures 20A to 20D show that in the growth inhibition of EMT6 tumors, mice replaced the combination of anti-CCR 8mAb and anti-PDL 1 mAb (figure 20D) compared to either anti-CCR 8mAb alone (figure 20B) or anti-PDL 1 mAb alone (figure 20C). When the tumor reaches 150-250mm ³ At that time, treatment was started. Tumor volumes were measured over time. Gray lines represent individual mice and black lines represent group fits. Isotype control mAb (anti-gp 120) was used (fig. 20A).

Figure 21 depicts serum pharmacokinetic profiles (mean ± SD) of anti-gD (control) and test anti-CCR 8mAb afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 in cynomolgus monkeys after a single dose of 10mg/kg IV bolus. Afuc.hu.ab5.h13l1 showed the required sustained serum concentration levels during the 35 days post-dose, which is expected to trigger more sustained target engagement, which may translate into better anti-cancer activity and lower dosing frequency.

Fig. 22A to 22C depict the results of whole blood flow cytometry analysis of total Treg cell counts of 9 male cynomolgus monkeys, to which 10mg/kg of defucosylated anti-gD (control; group 1, designated 1001, 1002, 1003; fig. 22A), afuc.hu.ab5.h13l1 (group 2, designated 2001, 2002, 2003; fig. 22B), or afuc.hu.ab4.h12l3 (group 3, designated 3001, 3002, 3003; fig. 22C) was administered via intravenous injection. Neither test anti-CCR 8mAb significantly reduced the absolute count of total T-reg cells in whole blood for up to 840 hours after dosing.

Fig. 23A to 23I depict the results of total blood flow cytometry analysis of the reduction of ccr8+foxp3+ Treg cells in 9 male cynomolgus monkeys, to which defucosylated anti-gD (control; group 1, designated 1001 (fig. 23A), 1002 (fig. 23B), 1003 (fig. 23C)) afuc.hu.ab4.h12l3 (group 3, designated 3001 (fig. 23D), 3002 (fig. 23E), 3003 (fig. 23F)), or afuc.hu.ab5.h13l1 (group 2, designated 2001 (fig. 23G), 2002 (fig. 23H), 2003 (fig. 23I)). Blood was collected from each animal prior to dosing ("pre-study") and 0 hours on day 1 ("pre-dosing"). Each animal was then given a single dose of 10mg/kg of defucosylated anti-gD (control group), afuc.hu.ab5.h13l1 (group 2) or afuc.hu.ab4.h12l3 (group 3) via intravenous injection. Blood was then collected from the animals and subjected to the following treatments prior to flow cytometry analysis: (i) a blood sample not labeled with any of the test CCR8 mabs ("unlabeled"), (ii) a blood sample further labeled with a saturated concentration of afuc.hu.ab5.h13l1, and (iii) a blood sample further labeled with a saturated concentration of afuc.hu.ab4.h12l3. Each of the unlabeled and labeled samples was then treated with a labeled goat anti-human IgG antibody and analyzed by flow cytometry. As can be seen in fig. 23A to 23C, flow cytometry of the initial treatment with control (group 1), but non-labeled blood, indicated that total ccr8+t-reg cells were not regulated. In addition, flow cytometry of the added blood had little effect on total CCR8+T-reg cell count. As can be seen in figures 23D to 23F, flow cytometry analysis of the blood of each of the three animals indicated that ccr8+t-reg cells decreased up to 168 hours post-dose. With respect to group 2, as can be seen in FIGS. 23G through 23I, flow cytometry of blood analysis indicated that CCR8+T-reg cells were depleted in animals 2002 and 2003. Animals in groups 2 and 3 showed little to no effect on total Treg cell count (fig. 22A to 22C), but showed a decrease in peripheral blood ccr8+ T-reg cell numbers after administration (fig. 23D to 23I), either labeled or unlabeled, consistent with the proposed mechanism of action (see fig. 2A).

Detailed description of certain aspects

I. Definition of the definition

For purposes herein, a "recipient human framework" is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as the human immunoglobulin framework or human consensus framework, or it may comprise amino acid sequence changes. In some aspects, 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 aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K _D ) And (3) representing. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity will be described herein.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more Complementarity Determining Regions (CDRs) that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not have such alterations.

The terms "anti-CCR 8 antibody" and "CCR 8 binding antibody" refer to antibodies that are capable of binding CCR8 with sufficient affinity such that the antibodies are useful as diagnostic and/or therapeutic agents for targeting CCR 8. In one aspect, the anti-CCR 8 antibody binds to an unrelated, non-CCR 8 protein to less than about 10% of the binding of the antibody to CCR8, as measured, for example, by Surface Plasmon Resonance (SPR). In certain aspects, the dissociation constant (K _D ) Is less than or equal to 1. Mu.M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10) ^-8 M or less, e.g. 10 ^-13 M to 10 ^-8 M, e.g. 10 ^-13 M to 10 ^-9 M). In certain aspects, antibodies that bind CCR8 have a binding capacity of about 1 x 10 ^-12 M to about 1X 10 ^-10 M, about 1X 10 ^-12 M to about 1X 10 ^-11 M, or about 1X 10 ^-11 M to about 5X 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a binding capacity of about 2 x 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a size of about 5 x 10 ^-12 Kd of M. When the antibody has _D . When the antibody has a K of 1. Mu.M or less _D The antibody is said to "specifically bind" to CCR 8. In certain embodiments, the anti-CCR 8 antibody binds to an epitope of CCR8 in at least two different species (e.g., human and cynomolgus monkey).

The term "antibody" is used herein in its broadest sense and includes 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.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds to an 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 The method comprises the steps of carrying out a first treatment on the surface of the A diabody antibody; a linear antibody; single chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dabs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, nature Biotechnology (2005) 23:1126-1136.

The term "epitope" refers to a site on a protein or non-protein antigen to which an anti-CCR 8 antibody binds. Epitopes can be formed either by continuous stretches of amino acids (linear epitopes) or by inclusion of non-continuous amino acids (conformational epitopes), for example due to antigen folding, i.e. due to tertiary folding of protein antigens, being spatially close. The linear epitope is typically still bound by the anti-CCR 8 antibody after exposure of the protein antigen to the denaturing agent, while the conformational epitope is typically destroyed after treatment with the denaturing agent. An epitope comprises 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 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 30 to 40, 35 to 40, or 5 to 10 amino acids in a unique spatial configuration.

Screening for Antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using methods conventional in the art, such as, but not limited to, alanine scanning, peptide blotting (see, e.g., kobeisy et al, meth.mol. Biol. (2004) 248:443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see hochletitner et al, prot. Sci.9 (2000) 487-496), and cross blocking (see "Antibodies", harlow and Lane (Cold Spring Harbor Press, cold Spring harbor., NY).

Antibody Profiling (ASAP), also known as Modification Assisted Profiling (MAP), based on antigen structure allows the binning of a large number of monoclonal antibodies that bind specifically to CCR8 based on their binding profile to a chemically or enzymatically modified antigen surface (see, e.g., US 2004/0101920). The antibodies in each group bind to the same epitope, which may be a unique epitope that is significantly different from or partially overlaps with the epitope represented by the other group.

Competitive binding can also be used to easily determine whether an antibody binds to the same epitope of CCR8 or competes for binding with a reference anti-CCR 8 antibody. For example, an antibody that "binds to the same epitope" as a reference anti-CCR 8 antibody refers to an antibody that blocks binding of the reference anti-CCR 8 antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Also, for example, to determine whether an antibody binds to the same epitope as a reference anti-CCR 8 antibody, the reference antibody is allowed to bind to CCR8 under saturation conditions. After removal of excess reference anti-CCR 8 antibody, the ability of the anti-CCR 8 antibody in question to bind CCR8 was assessed. If the anti-CCR 8 antibody is capable of binding to CCR8 after saturation binding of the reference anti-CCR 8 antibody, it can be concluded that the anti-CCR 8 antibody in question binds to a different epitope than the reference anti-CCR 8 antibody. However, if the anti-CCR 8 antibody in question does not bind to CCR8 after saturation binding of the reference anti-CCR 8 antibody, the anti-CCR 8 antibody in question may bind to the same epitope as the reference anti-CCR 8 antibody. To confirm whether the antibody in question binds to the same epitope or is blocked for steric reasons, routine experimentation (e.g., peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. The assay should be performed in two settings, i.e., both antibodies are saturated antibodies. If in both settings only the first (saturated) antibody is able to bind to CCR8, it can be concluded that the anti-CCR 8 antibody in question and the reference anti-CCR 8 antibody compete for binding to CCR8.

In some aspects, two antibodies are considered to bind to the same or overlapping epitope if one antibody inhibits binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more by a factor of 1, 5, 10, 20, or 100 as measured in a competitive binding assay (see, e.g., junghans et al, cancer Res50 (1990) 1495-1502).

In some aspects, 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 an "overlapping epitope" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and some of them may be further classified into subclasses (isotypes), for example, igG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ . In certain aspects, the antibody is an IgG ₁ An isoform. In certain aspects, the antibody is a polypeptide having P329G, L234IgG mutated with A and L235A to reduce effector function in Fc region ₁ An isoform. In other aspects, the antibody is an IgG ₂ An isoform. In certain aspects, the antibody is an IgG having an S228P mutation in the hinge region ₄ Isotype to improve IgG ₄ Stability of the 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 can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.

The term "constant region derived from human" or "human constant region" as used herein refers to the constant heavy chain region and/or constant light chain kappa or lambda region of a human antibody of subclass IgG1, igG2, igG3 or IgG 4. Such constant regions are well known in the art and are described, for example, by Kabat, E.A., et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Servce, national Institutes of Health, bethesda, MD (1991) (see, also, e.g., johnson, G., and Wu, T.T., nucleic Acids Res.28 (2000) 214-218; kabat, E.A., et al, proc.Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise indicated herein, numbering of the amino acid regions in the constant regions is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, E.A. et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991), NIH publication No. 91-3242.

"effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the variation of 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; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve a desired therapeutic or prophylactic result at the necessary dosage and for the necessary period of time.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The 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 carboxy terminus of the heavy chain. However, antibodies produced by the host cell may undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain, or the antibody may comprise a cleaved variant of a full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Thus, the C-terminal lysine (Lys 447) or C-terminal glycine (Gly 446) and lysine (Lys 447) of the Fc region may or may not be present. In one aspect, a heavy chain comprising an Fc region as specified herein, said heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system) is comprised in an antibody according to the invention. In one aspect, a heavy chain comprising an Fc region as specified herein, said heavy chain comprising an additional C-terminal glycine residue (G446, numbering according to the EU index) is comprised in an antibody according to the invention. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al (Sequences of Proteins of Immunological Interest, 5 th edition, U.S. department of health and public service, national institutes of health, besseda, 1991).

"framework" or "FR" refers to the variable domain residues other than the Complementarity Determining Regions (CDRs). The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, CDR and FR sequences typically occur in VH (or VL) with the following sequences: FR1-CDR-H1 (CDR-L1) -FR2-CDR-H2 (CDR-L2) -FR3-CDR-H3 (CDR-L3) -FR4.

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein. It is understood that a full length antibody comprises a heavy chain variable domain and a light chain variable domain as defined herein, and an Fc region as defined herein.

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 progeny derived from the primary transformed cell, regardless of the number of passages. The progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell.

A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human antibody that utilizes a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

A "human consensus framework" is a framework that represents the amino acid residues that are most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subset of sequences is as described in Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, bethesda MD (1991), volumes 1-3. In one aspect, for VL, the subgroup is subgroup κI as described in Kabat et al, supra. In one aspect, for VH, the subgroup is subgroup III as described in Kabat et al, supra.

"humanized" antibody refers to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FR. In certain aspects, the 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. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a non-human antibody, in "humanized form" refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein refers to the individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, e.g., the "complementarity determining regions" ("CDRs").

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

Exemplary CDRs herein include:

(a) A highly variable loop present at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J.mol. Biol.196:901-917 (1987));

(b) CDRs present at the following amino acid residues: 24 to 34 (L1), 50 to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2) and 95 to 102 (H3) (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991)); and

(c) Antigen contact points occur at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2) and 93 to 101 (H3) (MacCallum et al, J.mol. Biol.262:732-745 (1996)).

CDRs are determined according to the above-mentioned document by Kabat et al and the above-mentioned document by Chothia, unless otherwise indicated. Those skilled in the art will appreciate that CDR names may also be determined based on mccall's above-mentioned literature or any other scientifically accepted naming system.

In one aspect, CDR residues include those identified in fig. 5A to 5D and 6A to 6D, as well as tables C1, C2, D1 and D2. In other aspects, CDR residues comprise those identified in tables N1, N2, O1 and O2.

A "subject" is a mammal. Mammals include, but are not limited to, domesticated 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.

An "isolated" antibody is an antibody that has been isolated from a component of its natural environment. In some aspects, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a nucleotide polymer. Each nucleotide consists of a base, in particular 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. In general, nucleic acid molecules are described by a sequence of bases, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually expressed from 5 'to 3'. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single and double stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having 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 antibodies described herein in vitro and/or in vivo (e.g., in a host or subject). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the coding molecule such that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., stadler et al, nature Medicine 2017, published online at 2017, 6/12, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"isolated nucleic acid encoding an anti-CCR 8 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the anti-CCR 8 antibody, including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more positions in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of a monoclonal antibody preparation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the present disclosure can be prepared 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 part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

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

"Natural antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminal to the C-terminal, each heavy chain has a variable domain (VH), also known as a variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH 1, CH2 and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also known as a variable light chain domain or light chain variable region, followed by a constant light Chain (CL) domain.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information concerning the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the candidate sequence to the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity for the purposes of the alignment. The alignment for determining the 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 FASTA packages. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. Alternatively, the percent identity value may be generated using the sequence comparison computer program ALIGN-2. ALIGN-2 sequence comparison computer program was written by GeneTek corporation and the source code has been submitted with the user document to U.S. Copyright Office, washington D.C.,20559, registered there with U.S. copyright accession number TXU510087 and described in WO 2001/007511.

For purposes herein, the BLOSUM50 comparison matrix is used to generate values for percent amino acid sequence identity using the ggsearch program of FASTA package version 36.3.8c or higher, unless otherwise specified. FASTA packages are described by W.R.Pearson and D.J.Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; R.Pearson (1996) "Effective protein sequence comparison" meth.enzymol.266:227-258; and Pearson et al (1997) Genomics 46:24-36 and are publicly available from fasta. Bioch. Virginia. Edu/fasta_www2/fasta_down. Shtml or ebi.ac/Tools/sss/fasta. Alternatively, sequences may be compared using a public server accessible at fasta. Bioch. Virginia. Edu/fasta_www2/index. Cgi, using a ggsearch (global protein: protein) program and default options (BLOSUM 50; open: -10; ext: -2; ktup=2) to ensure that global rather than local alignment is performed. The percentage amino acid identity is given in the output alignment header.

The terms "pharmaceutical composition" and "pharmaceutical formulation" are used interchangeably herein and refer to a preparation in a form that allows for the biological activity of the active ingredient contained in the preparation to be effective, and that is free of additional components that have unacceptable toxicity to the subject to whom the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to ingredients of a pharmaceutical composition or formulation other than the active ingredient, which are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, unless otherwise indicated, the term "CCR8" refers to any natural CCR8 from any vertebrate source, including mammals such as primates (e.g., humans, monkeys ("cynomolgus monkeys")) and rodents (e.g., mice and rats). The term includes "full length" unprocessed CCR8, as well as any form of CCR8 produced by processing in a cell. The term also encompasses naturally occurring variants of CCR8, such as splice variants or allelic variants. In certain aspects, CCR8 is human CCR8 ("hCCR 8" or "huCCR 8"). The amino acid sequence of an exemplary human CCR8 is shown in SEQ ID NO. 106, as shown in the following table. In certain aspects, CCR8 is a cynomolgus monkey ("cyno") CCR8. The amino acid sequence of an exemplary cynomolgus monkey CCR8 is shown in SEQ ID NO:107 as set forth in the following table. In certain aspects, CCR8 is a mouse CCR8 ("CCR 8"). The amino acid sequence of exemplary mouse CCR8 is shown in SEQ ID NO. 108, as shown in the following table.

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to a natural course of a disease (e.g., cancer) in a subject attempting to alter the treatment, and may be performed for prophylaxis ("prophylactic treatment" or "in prophylactic treatment") or may be performed during a clinical pathology ("therapeutic treatment" or "in therapeutic treatment") procedure. 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 the cancer, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Desirable effects of prophylactic treatment include, but are not limited to, preventing the occurrence or recurrence of disease. In some aspects, the antibodies described herein are used to delay the progression of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). (see, e.g., kit et al Kuby Immunology, 6 th edition, w.h. freeman and co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated using VH or VL domains, respectively, from antibodies that bind that antigen to screen libraries of complementary VL or VH domains. See, e.g., portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).

The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been 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".

II compositions and methods

In one aspect, the present disclosure is based in part on the discovery of novel anti-CCR 8 antibodies that have unique and improved binding and selectivity for CCR8. The presently disclosed anti-CCR 8 antibodies 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 the defucosylated forms of the presently disclosed antibodies have increased Antibody Dependent Cellular Cytotoxicity (ADCC) and Antibody Dependent Cellular Phagocytosis (ADCP) activity. In certain aspects, antibodies that bind to CCR8 are provided. The antibodies described herein are useful, for example, in the treatment of cancer.

A. Exemplary anti-CCR 8 antibodies

In one aspect, the disclosure provides antibodies that bind to CCR8. In one aspect, the provided antibodies are isolated antibodies that bind to CCR8. In one aspect, the disclosure provides antibodies that specifically bind to CCR8. In certain aspects, the anti-CCR 8 antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID No. 106. In certain aspects, the anti-CCR 8 antibody binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID No. 106. In certain aspects, CCR8 is human CCR8, mouse CCR8, or cynomolgus CCR8. In certain aspects, CCR8 is human CCR8. In one aspect, the disclosure provides antibodies that bind CCR8 independent of tyrosine sulfation of CCR8 ("sulfation independent"). Exemplary antibodies that the inventors have discovered that are independent of sulfation include Ab4 and Ab5, as described in further detail below.

In certain aspects, the seed antibodies provided herein have a concentration of 1. Mu.M, 100nM, 10nM, 1nM, 0.1nM, 0.01nM or 0.001nM (e.g., 10 nM) ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 Dissociation constant (K) of M) _D ). In certain aspects, antibodies that bind CCR8 have a binding capacity of about 1 x 10 ^-12 M to about 1X 10 ^-10 M, about 1X 10 ^-12 M to about 1X 10 ^-11 M, or about 1X 10 ^-11 M to about 5X 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a binding capacity of about 2 x 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a size of about 5 x 10 ^-12 K of M _d . In one aspect, K _D Measurement was performed using radiolabeled IgG and CHO cell lines stably expressing the antigen. Stable CHO cells expressing antigen were seeded at 50,000 cells per well in cold binding buffer (Opti-mem+2% fbs+50mM HEPES,pH 7.2+0.1% sodium azide). Fixed concentrations were performed using NEX244 Iodogen method (Perkin Elmer) ¹²⁵ I radiolabeled target antigen is mixed with target antibody in serial dilutions starting at 20nM or 50 nM. The antibody mixture was added to the cells and incubated at room temperature for 12 hours with gentle agitation. Cells and antibodies were then transferred to Millipore multi-layer screen filter plates. The filter plate was washed 4 times with 250 μl of cold binding buffer and dried for at least 30 minutes, and the filter was driven into a 5mL polystyrene tube. Radioactivity was measured using a Perkin Elmer Wallac Wizard 2470 gamma counter set to 1 count per minute with a count efficiency of 0.8. The Ki competitive binding model was fitted to the data using heterologous single site fitting in GraphPad Prism.

In certain aspects, the antibodies provided herein have an average clearance of about 3 to about 5 mL/day/kg over a period of 35 days following intravenous administration of a single 10mg/kg dose on day 1. For example, but not limited to, such administration may include a single 10mg/kg IV bolus of mAb. Blood samples for analysis can be collected, for example, at 0.25, 2, 6 hours; the concentration of mAb in serum can be determined 1, 2, 7, 14, 21, 28, and 35 days after administration using a variety of methods (e.g., a qualified ELISA assay). In certain aspects, the administration is to a mammal. In certain aspects, the administration is to a primate. In certain aspects, the administration is to a non-human primate, such as a cynomolgus monkey. In certain aspects, the administration is to a human.

(i) Embodiment of Ab5 and fragments thereof

In one aspect, the present disclosure provides an anti-CCR 8 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to both human CCR8 and cynomolgus CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 32. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 32. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 32; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 28. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 32; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 28; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 31. In a further aspect, the 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 32.

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) 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. In one aspect, the antibody comprises: (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.

In another aspect, an 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to both human CCR8 and cynomolgus CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In another aspect, the present disclosure provides an antibody 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.

In another aspect, the anti-CCR 8 antibody comprises one or more of the CDR sequences of a VH sequence selected from the group consisting of: SEQ ID NOS.35 to 47. In another embodiment, an anti-CCR 8 antibody comprises one or more of the CDR sequences of a VL sequence selected from the group consisting of: SEQ ID NOS: 48 to 52. In another embodiment, an anti-CCR 8 antibody comprising: CDR sequences of VH sequences 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.

In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 47. In another embodiment, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 48. In another embodiment, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID NO: 47. In another embodiment, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 48.

In a further aspect, an anti-CCR 8 antibody comprising: CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of VH domains selected from the group consisting of: SEQ ID NOS.35 to 47; and CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of VL domains selected from the group consisting of: SEQ ID NOS: 48 to 52.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of VH domains 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 a framework amino acid sequence of a VH domain selected from the group consisting of: SEQ ID NOS.35 to 47. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains 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 a framework amino acid sequence of a VH domain selected from the group consisting of: SEQ ID NOS.35 to 47. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains 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 aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 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 of SEQ ID No. 47. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 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 of SEQ ID No. 47. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 47; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 47. In another aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 47; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 47.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domains selected from the group consisting of: 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 a framework amino acid sequence of a VL domain selected from the group consisting of: SEQ ID NOS: 48 to 52. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains selected from the group consisting of: 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 a framework amino acid sequence of a VL domain selected from the group consisting of: SEQ ID NOS: 48 to 52. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains selected from the group consisting of: 48 to 52; and a framework having 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 aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains selected from the group consisting of: 48 to 52; and a framework having specifically 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 48; 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 VL domain of SEQ ID No. 48. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 48; 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 VL domain of SEQ ID No. 48. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 48; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 48. In another aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 48; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 48.

In one aspect, an anti-CCR 8 antibody 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, (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 a VH domain having at least 90%, 91%, 92%, 93%, 94% with an amino acid sequence selected from the group consisting of% 95%, 96%, 97%, 98%, 99% or 100% sequence identity: SEQ ID NOS.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 aspect, 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 aspect, 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 aspect, the dissociation constant (K _D ) And a VH sequence comprising a sequence selected from the group consisting of: 35 to 47 and a VL sequence selected from the group consisting of: dissociation constants (K) of antibodies of SEQ ID NOS.48 to 52 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In one aspect, an anti-CCR 8 antibody 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, (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 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 binds to CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 no: SEQ ID NOS.35 to 47. In one aspect, an anti-CCR 8 antibody comprising: 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 certain aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in an amino acid sequence selected from the group consisting of: SEQ ID NOS.35 to 47. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, an anti-CCR 8 antibody comprising: 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 aspect, 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 aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 no: SEQ ID NOS: 48 to 52. In one aspect, an anti-CCR 8 antibody comprising: 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 certain aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in an amino acid sequence selected from the group consisting of: SEQ ID NOS: 48 to 52. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, an anti-CCR 8 antibody comprising: 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 aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 47. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 47, including post-translational modifications of the sequence. In a particular aspect, 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 aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 48. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 48, including post-translational modifications of the sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as provided above. In one aspect, the antibody comprises: a VH sequence selected from the group consisting of: SEQ ID NOS.35 to 47; a VL sequence selected from the group consisting of: SEQ ID NOS.48 to 52, including post-translational modifications of those sequences. In one aspect, the antibody comprises the VH sequence of SEQ ID NO. 47 and the VL sequence of SEQ ID NO. 48, including post-translational modifications of those sequences.

In one aspect, the VL sequence comprises a V4M mutation, a P43A mutation, an F46L mutation, a C90Q mutation, or a combination thereof. In one aspect, the VH comprises a G49S mutation, a K71R mutation, an S73N mutation, or a combination thereof.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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-CCR 8 antibody is provided, wherein the antibody 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.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody comprises: 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. In one aspect, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO. 47 and the VL sequence of SEQ ID NO. 48.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO: 56.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 60 and a light chain of SEQ ID NO. 56.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the heavy chain of the antibody comprises a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO:111 and a light chain of SEQ ID NO: 56. In one aspect, an anti-CCR 8 antibody comprises the heavy chain of SEQ ID NO. 113 and the light chain of SEQ ID NO. 56.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody does not bind to a CCR8 ligand. In one aspect, the anti-CCR 8 antibody does not have CCR8 ligand blocking activity. In one aspect, the anti-CCR 8 antibody is a full length antibody. In one aspect, the CCR8 ligand is CCL1.

In another aspect of any of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody binds to CCR8 independent of tyrosine sulfation of CCR8 (i.e., independent of sulfation).

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody is a defucosylated antibody variant. In one aspect, the defucosylated antibody variants have enhanced fcyriiia receptor binding. In one aspect, the defucosylated anti-CCR 8 antibody variant has enhanced Antibody Dependent Cellular Cytotoxicity (ADCC). In one aspect, the anti-CCR 8 defucosylated antibody variant has Antibody Dependent Cell Phagocytosis (ADCP) activity.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody has improved antibody stability. In one aspect, the anti-CCR 8 antibody has low aggregation, good solubility, and/or low viscosity. In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody has a binding capacity of about 1 x 10 ^-12 M to about 1X 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a size of about 5 x 10 ^-12 K of M _D . In certain aspects, antibodies that bind CCR8 have a binding capacity of about 4 x 10 ^-12 K of M _D . In certain aspects, antibodies that bind CCR8 have a binding capacity of about 3 x 10 ^-12 K of M _D 。

In one aspect, the anti-CCR 8 antibody is named "hu.ab5.h13l1" in the present disclosure, which may be fucosylated or defucosylated, optionally containing one or more heavy chain mutations at G236A and I331E, and optionally comprising a shortened C-terminus of the heavy chain, wherein one or both of the C-terminal amino acid residues have been removed.

In a further aspect, the anti-CCR 8 antibody according to any of the preceding aspects is a monoclonal antibody, including chimeric, humanized or human antibodies. In one aspect, the anti-CCR 8 antibody is anti-Body fragments, e.g. Fv, fab, fab ', scFv, diabodies or F (ab') ₂ Fragments.

(ii) Embodiment of Ab4 and fragments thereof

In one aspect, the present disclosure provides an anti-CCR 8 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to both human CCR8 and cynomolgus CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 7. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 7. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 7; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 3. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 7; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 3; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 6. In a further aspect, the antibody comprises: (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, 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) 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. In one aspect, the antibody comprises: (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.

In another aspect, an 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to both human CCR8 and cynomolgus CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In another aspect, the present disclosure provides an antibody comprising: (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; and a light chain variable domain (VL) comprising: (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 another aspect, the anti-CCR 8 antibody comprises one or more of the CDR sequences of a VH sequence selected from the group consisting of: SEQ ID NOS 10 to 21. In another aspect, the anti-CCR 8 antibody comprises one or more of the CDR sequences of a VL sequence selected from the group consisting of: SEQ ID NOS.22 to 25. In another aspect, an anti-CCR 8 antibody comprising: CDR sequences of VH sequences 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.

In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 21. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 24. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 21. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 24.

In a further aspect, an anti-CCR 8 antibody comprising: CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of VH domains selected from the group consisting of: SEQ ID NOS 10 to 21; and CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of VL domains selected from the group consisting of: SEQ ID NOS.22 to 25.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of VH domains 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 a framework amino acid sequence of a VH domain selected from the group consisting of: SEQ ID NOS 10 to 21. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains 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 a framework amino acid sequence of a VH domain selected from the group consisting of: SEQ ID NOS 10 to 21. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains selected from the group consisting of: SEQ ID NOS 10 to 21; 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 10 to 21. In another aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of VH domains selected from the group consisting of: SEQ ID NOS 10 to 21; 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 10 to 21.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 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 the VH domain of SEQ ID No. 21. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 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 the VH domain of SEQ ID No. 21. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 21; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 21. In another aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 21; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 21.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domains 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 a framework amino acid sequence of a VL domain selected from the group consisting of: SEQ ID NOS.22 to 25. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains 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 a framework amino acid sequence of a VL domain selected from the group consisting of: SEQ ID NOS.22 to 25. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains selected from the group consisting of: SEQ ID NOS.22 to 25; and a framework having 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 aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of VL domains selected from the group consisting of: SEQ ID NOS.22 to 25; and a framework having specifically 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 24; 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 VL domain of SEQ ID No. 24. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 24; 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 VL domain of SEQ ID No. 24. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 24; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 24. In another aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 24; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 24.

In one aspect, an anti-CCR 8 antibody comprising: (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; 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 aspect, the VH domain has at least 95% sequence identity to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOS 10 to 21. In one aspect, 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 aspect, the dissociation constant (K _D ) And a VH sequence comprising a sequence selected from the group consisting of: SEQ ID NOS 10 to 21; and a VL sequence selected from the group consisting of: the dissociation constants (K) of the antibodies of SEQ ID NOS.22 to 25 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In one aspect, an anti-CCR 8 antibody comprising: (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; and 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 binds to CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO. 21 and the VL sequence of SEQ ID NO. 24 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 no: SEQ ID NOS 10 to 21. In one aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in an amino acid sequence selected from the group consisting of: SEQ ID NOS 10 to 21. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, an anti-CCR 8 antibody comprising: 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 aspect, 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 7. In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 no: SEQ ID NOS.22 to 25. In one aspect, an anti-CCR 8 antibody comprising: 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, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in an amino acid sequence selected from the group consisting of: SEQ ID NOS.22 to 25. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, an anti-CCR 8 antibody comprising: a VL sequence selected from the group consisting of: SEQ ID NOS.22 to 25, including post-translational modifications of the sequences. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 21. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 21, including post-translational modifications of the sequence. In a particular aspect, 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 aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 24. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 24, including post-translational modifications of the sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as provided above. In one aspect, the antibody comprises: a VH sequence selected from the group consisting of: SEQ ID NOS 10 to 21; a VL sequence selected from the group consisting of: SEQ ID NOS.22 to 25, including post-translational modifications of those sequences. In one aspect, the antibody comprises the VH sequence of SEQ ID NO. 21 and the VL sequence of SEQ ID NO. 24, including post-translational modifications of those sequences.

In one aspect, the VL sequence comprises a Y2I mutation. In another aspect, the VH sequence comprises an S73N mutation, a V78L mutation, a T76N mutation, an F91Y mutation, and a P105Q mutation, or a combination thereof.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody comprises: a heavy chain variable domain (VH) comprising: (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; and a light chain variable domain (VL) comprising: (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 one aspect, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO. 21 and the VL sequence of SEQ ID NO. 24.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 57 and a light chain of SEQ ID NO. 58.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 61 and a light chain of SEQ ID NO. 58.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the heavy chain of the antibody comprises a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 112 and a light chain of SEQ ID NO. 58. In one aspect, an anti-CCR 8 antibody comprises the heavy chain of SEQ ID NO. 114 and the light chain of SEQ ID NO. 58.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody binds to a CCR8 ligand. In one aspect, the anti-CCR 8 antibody has antagonism against a CCR8 ligand. In one aspect, the anti-CCR 8 antibody has CCR8 ligand blocking activity. In one aspect, the anti-CCR 8 antibody is a neutralizing antibody. In one aspect, the CCR8 ligand is CCL1.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody has improved antibody stability. In one aspect, the anti-CCR 8 antibody has low aggregation, good solubility, and/or low viscosity. In certain aspects of any of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the antibody has a binding capacity of about 1 x 10 ^-11 M to about 5X 10 ^-11 K of M _D . In certain aspects, antibodies that bind CCR8 have a binding capacity of about 2 x 10 ^-11 K of M _D 。

In one aspect, the anti-CCR 8 antibody is named "hu.ab4.h12l3" in the present disclosure, which may be fucosylated or defucosylated, optionally containing one or more heavy chain mutations at G236A and I331E, and optionally comprising a shortened C-terminus of the heavy chain, wherein one or both of the C-terminal amino acid residues have been removed.

In a further aspect, the anti-CCR 8 antibody according to any of the preceding aspects is a monoclonal antibody, including chimeric, humanized or human antibodies. In one aspect, the anti-CCR 8 antibody is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody, or F (ab') ₂ Fragments.

(iii) Embodiment of Ab1 and fragments thereof

In one aspect, the present disclosure provides an anti-CCR 8 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 85; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 75. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 75; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 84. In a further aspect, the antibody comprises: (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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85.

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) 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. In one aspect, the antibody comprises: (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.

In another aspect, an 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In another aspect, the present disclosure provides an antibody comprising: (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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85; and a light chain variable domain (VL) comprising: (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 another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 95. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 94. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 95. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 94.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 95; 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 of SEQ ID No. 95. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 95; 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 of SEQ ID No. 95. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 95; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 95. In another aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 95; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 95.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 94; 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 VL domain of SEQ ID No. 94. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 94; 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 VL domain of SEQ ID No. 94. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 94; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 94. In another aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 94; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 94.

In one aspect, an anti-CCR 8 antibody comprising: (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; and 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 binds to CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO:95 and the VL sequence of SEQ ID NO:94 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 95. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 95, including post-translational modifications of the sequence. In a particular aspect, 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 aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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: 94. In one aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 94. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 94, including post-translational modifications of the sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as provided above. In one aspect, the antibody comprises the VH sequence of SEQ ID NO. 95 and the VL sequence of SEQ ID NO. 94, including post-translational modifications of those sequences.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody comprises: a heavy chain variable domain (VH) comprising: (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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85; and a light chain variable domain (VL) comprising: (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 one aspect, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO. 95 and the VL sequence of SEQ ID NO. 94.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 101 and a light chain of SEQ ID NO. 100.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the heavy chain of the antibody comprises a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 115 and a light chain of SEQ ID NO. 100.

In one aspect, the anti-CCR 8 antibody is named "hu.ab1.h1l1" in the present disclosure, which may be fucosylated or defucosylated, optionally containing one or more heavy chain mutations at G236A and I331E, and optionally comprising a shortened C-terminus of the heavy chain, wherein one or both of the C-terminal amino acid residues have been removed. In a further aspect, the anti-CCR 8 antibody according to any of the preceding aspects is a monoclonal antibody, including chimeric, humanized or human antibodies. In one aspect, the anti-CCR 8 antibody is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody, or F (ab') ₂ Fragments.

(iv) Embodiment of Ab2 and fragments thereof

In one aspect, the present disclosure provides an anti-CCR 8 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 89. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 89; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 78. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 89; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 78; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 88. In a further aspect, the antibody comprises: (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.

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) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 76; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In one aspect, the antibody comprises: (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. 77; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

In another aspect, an 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 aspects, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In another aspect, the present disclosure provides an antibody 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.

In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 97. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 96. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 97. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID NO: 96.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 97; 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 of SEQ ID No. 97. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 97; 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 of SEQ ID No. 97. In one aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 97; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 97. In another aspect, an anti-CCR 8 antibody comprising: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 97; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 97.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 96; 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 VL domain of SEQ ID No. 96. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 96; 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 VL domain of SEQ ID No. 96. In one aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 96; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 96. In another aspect, an anti-CCR 8 antibody comprising: three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 96; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 96.

In one aspect, an anti-CCR 8 antibody 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, (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; and 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 binds to CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO:97 and the VL sequence of SEQ ID NO:96 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 97. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 97, including post-translational modifications of the sequence. In a particular aspect, 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 aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 96. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 96, including post-translational modifications of the sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as provided above. In one aspect, the antibody comprises the VH sequence of SEQ ID NO. 97 and the VL sequence of SEQ ID NO. 96, including post-translational modifications of those sequences.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody comprises: 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.

In one aspect, an anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO. 97 and the amino acid sequence of SEQ ID NO. 96.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 103 and a light chain of SEQ ID NO. 102.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the heavy chain of the antibody comprises a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect, an anti-CCR 8 antibody comprises the heavy chain of SEQ ID NO. 116 and the light chain of SEQ ID NO. 102.

In one aspect, the anti-CCR 8 antibody is named "hu.ab2.h1l1" in the present disclosure, which may be fucosylated or defucosylated, optionally containing one or more heavy chain mutations at G236A and I331E, and optionally comprising a shortened C-terminus of the heavy chain, wherein one or both of the C-terminal amino acid residues have been removed.

(v) Embodiment of Ab3 and fragments thereof

In one aspect, the present disclosure provides an anti-CCR 8 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 93. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 93; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 81. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 93; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 81; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 92. In a further aspect, the antibody comprises: (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.

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) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 79; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 80; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 81. In one aspect, the antibody comprises: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 79; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 80; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 81.

In another aspect, an 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a human antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a humanized antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to human CCR8, and is a chimeric antibody.

In another aspect, the present disclosure provides an antibody 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.

In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 99. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 98. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 99. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 98.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 99; 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 of SEQ ID No. 99. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 99; 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 of SEQ ID No. 99. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 99; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 99. In another aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 99; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 99.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 98; 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 VL domain of SEQ ID No. 98. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 98; 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 VL domain of SEQ ID No. 98. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 98; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 98. In another aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 98; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 98.

In one aspect, an anti-CCR 8 antibody 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, (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; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence of SEQ ID NO 99Sequence identity; 99, an amino acid sequence of seq id no; 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 binds to CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO 99 and the VL sequence of SEQ ID NO 98 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 99. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 99, including post-translational modifications of the sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: an 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. In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 98. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 98, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:79, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:80, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as provided above. In one aspect, the antibody comprises the VH sequence of SEQ ID NO:99 and the VL sequence of SEQ ID NO:98, including post-translational modifications of those sequences.

In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody comprises: 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. In one aspect, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO 99 and the VL sequence of SEQ ID NO 98.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO. 105 and a light chain of SEQ ID NO. 104.

In another aspect of any one of the above embodiments, there is provided an anti-CCR 8 antibody, wherein the heavy chain of the antibody comprises a shortened C-terminus, wherein one or both of the C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect, the anti-CCR 8 antibody comprises the heavy chain of SEQ ID NO. 117 and the light chain of SEQ ID NO. 104.

In one aspect, the anti-CCR 8 antibody is named "hu.ab3.h1l1" in the present disclosure, which may be fucosylated or defucosylated, optionally containing one or more heavy chain mutations at G236A and I331E, and optionally comprising a shortened C-terminus of the heavy chain, wherein one or both of the C-terminal amino acid residues have been removed.

(vi) Embodiments of mouse substitutes

In one aspect, the present disclosure provides an anti-CCR 8 antibody that binds to mouse CCR8 and comprises 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, an anti-CCR 8 antibody includes all six of the CDRs described above. In certain aspects, the anti-CCR 8 antibody is a full length antibody. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to mouse CCR 8. In certain aspects, the anti-CCR 8 antibody is a full length antibody that binds to mouse CCR8, and is a chimeric antibody (e.g., rabbit and mouse chimeras).

In one aspect, the present disclosure provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 68. In another aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID No. 68; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 64. In a further aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO. 68; CDR-L3 comprising the amino acid sequence of SEQ ID NO. 64; and CDR-H2 comprising the amino acid sequence of SEQ ID NO. 6. In a further aspect, the antibody comprises: (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.

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) 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. In one aspect, the antibody comprises: (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.

In another aspect, an 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:65 or SEQ ID NO:66, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, and (iii) 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) CDR-L1 comprising the amino acid sequence of SEQ ID NO:62, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:63, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

In another aspect, the present disclosure provides an antibody 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.

In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 70. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 69. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VH sequence of SEQ ID No. 70. In another aspect, an anti-CCR 8 antibody comprises one or more of the CDR sequences of the VL sequence of SEQ ID No. 69.

In a further aspect, an anti-CCR 8 antibody comprising: 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.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO 70; 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 of SEQ ID No. 70. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 70; 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 of SEQ ID No. 70. In one aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 70; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 70. In another aspect, an anti-CCR 8 antibody comprising: three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO. 70; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID No. 70.

In one aspect, an anti-CCR 8 antibody comprising: one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 69; 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 VL domain of SEQ ID No. 69. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 69; 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 VL domain of SEQ ID No. 69. In one aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 69; and a framework having at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 69. In another aspect, an anti-CCR 8 antibody comprising: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO. 69; and a framework having at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID No. 69.

In one aspect, an anti-CCR 8 antibody 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, (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; and 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; 70, an amino acid sequence of seq id no; 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 binds to mouse CCR8 with a dissociation constant (K _D ) Dissociation constant (K) with an antibody comprising the VH sequence of SEQ ID NO:70 and the VL sequence of SEQ ID NO:69 _D ) Up to a 10-fold reduction or up to a 10-fold increase compared to the above.

In another aspect, an anti-CCR 8 antibody comprising: 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 aspect, an anti-CCR 8 antibody comprising: 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 aspects, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequence retain the ability to bind to mouse CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 70. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID No. 70, including post-translational modifications of the sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: an 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. In another aspect, an anti-CCR 8 antibody is provided, wherein the antibody 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: 69. In one aspect, an anti-CCR 8 antibody comprising: 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 aspects, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-CCR 8 antibodies comprising the sequences retain the ability to bind to CCR 8. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO. 69. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR). Optionally, the anti-CCR 8 antibody comprises the VL sequence of SEQ ID No. 69, including post-translational modifications of the sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from the group consisting of: (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.

In another aspect, there is provided an anti-CCR 8 antibody, wherein the antibody comprises a VH sequence of any one of the aspects as provided above and a VL sequence of any one of the aspects as 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 those sequences.

In another aspect, an anti-CCR 8 antibody that binds to mouse CCR8 is provided, wherein the antibody comprises: 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. In one aspect, the anti-CCR 8 antibody comprises the VH sequence of SEQ ID NO. 70 and the VL sequence of SEQ ID NO. 69.

In one aspect, an anti-CCR 8 antibody comprises a heavy chain of SEQ ID NO:72 and a light chain of SEQ ID NO: 71.

In a further aspect, the anti-CCR 8 antibody according to any of the preceding aspects is a monoclonal antibody, including a chimeric antibody. In one aspect, the anti-CCR 8 antibody is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody, or F (ab') ₂ Fragments.

(vii) Other embodiments

In a further aspect, an anti-CCR 8 antibody according to any of the above aspects may incorporate any of the features described in paragraphs 1 to 5 below, alone or in combination.

1.Antibody fragments

In certain aspects, the antibodies provided herein are antibody fragments.

In one aspect, the antibody fragment is Fab ', fab ' -SH or F (ab ') ₂ Fragments, in particular Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments, termed "Fab" fragments, each containing a heavy chain variable domain and a light chain variable domain (VH and VL, respectively) as well as a constant domain of the light Chain (CL) and a first constant domain of the heavy chain (CH 1). Thus, the term "Fab fragment" is intended to include fragments comprising the VL structureAn antibody fragment comprising a heavy chain fragment of a VH domain and a CH1 domain. Fab 'fragments differ from Fab fragments in that the Fab' fragment has added at the carboxy terminus of the CH1 domain residues including one or more cysteines from the antibody hinge region. Fab '-SH is a Fab' fragment in which the cysteine residues of the constant domain have free sulfhydryl groups. Pepsin treatment to produce F (ab') ₂ A fragment having two antigen binding sites (two Fab fragments) and a portion of the Fc region. Fab and F (ab') which contain salvage receptor binding epitope residues and have increased in vivo half-lives ₂ See U.S. Pat. No. 5,869,046 for a discussion of fragments.

In another aspect, the antibody fragment is a diabody, a triabody, or a tetrabody. A "diabody antibody" is an antibody fragment having two antigen binding sites, which may be bivalent or bispecific. See, for example, 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). For a description of trisomy and tetrasomy antibodies see also Hudson et al, nat. Med.9:129-134 (2003).

In a further aspect, 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 (CH 1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) a VH-CH 1-linker-VL-CL, b) a VL-CL-linker-VH-CH 1, c) a VH-CL-linker-VL-CH 1, or d) a VL-CH 1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single chain Fab fragments can be further stabilized by generating interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

In another aspect, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment" or "scFv" is a fusion protein of the heavy chain variable domain (VH) and the light chain variable domain (VL) of an antibody, linked by a linker. In particular, linkers are short polypeptides of 10 to about 25 amino acids and are typically rich in glycine to obtain flexibility, and serine or threonine to obtain solubility, and the N-terminus of VH can be linked to the C-terminus of VL, or vice versa. The protein retains the original antibody specificity despite removal of the constant region and introduction of the linker. For reviews of scFv fragments, see, e.g., pluckthun, volume The Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore editions, (Springer-Verlag, new York), pages 269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain aspects, single domain antibodies are human single domain antibodies (domatis, inc., waltham, MA; see, e.g., U.S. patent No. 6,248,516B1).

Antibody fragments may be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, recombinantly produced by recombinant host cells (e.g., E.coli), as described herein.

2.Chimeric and humanized antibodies

In certain aspects, the antibodies provided herein are chimeric antibodies. Some chimeric antibodies are described in the following documents: for example, U.S. Pat. No. 4,816,567 and Morrison et al, proc.Natl. Acad.Sci.USA,81:6851-6855 (1984). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain aspects, the chimeric antibody is a humanized antibody. Typically, the non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FR (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed as described in: for example, almagro and Franson, front. Biosci.13:1619-1633 (2008), and further described, for example, in the following references: for example, riechmann et al Nature 332:323-329 (1988); queen et al, proc.Nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. 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) transplantation); padlan, mol. Immunol.28:489-498 (1991) (description "surface remolding"); dall' Acqua et al Methods 36:43-60 (2005) (description "FR shuffling"); and Osbourn et al, methods 36:61-68 (2005) and Klimka et al, br.J.cancer,83:252-260 (2000) (describes the "guided selection" approach to FR shuffling).

Human framework regions useful for humanization include, but are not limited to: the framework regions were selected using the "best fit" method (see, e.g., sims et al J. Immunol.151:2296 (1993)); framework regions of consensus sequences of human antibodies derived from specific subsets 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 (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)); and framework regions from screening FR libraries (see, e.g., baca et al, J. Biol. Chem.272:10678-10684 (1997) and Rosok et al, J. Biol. Chem.271:22611-22618 (1996)).

3.Human antibodies

In certain aspects, the antibodies provided herein are human antibodies. Various techniques known in the art may be used to produce human antibodies. For a general description of human antibodies, see: van Dijk and van de Winkel, curr. Opin. Pharmacol.5:368-74 (2001) and Lonberg, curr. Opin. Immunol.20:450-459 (2008).

Human antibodies can be prepared by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci, either present extrachromosomal to the animal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, e.g., descriptions xenomouise ^TM Technical U.S. Pat. nos. 6,075,181 and 6,150,584; description of the inventionTechnical U.S. patent No. 5,770,429; description of K-M- >Technical U.S. Pat. No. 7,041,870 and description->Technical U.S. patent application publication No. US 2007/0061900). Human variable regions from whole antibodies produced by such animals may be further modified, for example by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor J.Immunol.,133:3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, inc., new York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in: li et al, proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. 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).

Human antibodies can also be produced by isolating variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the intended human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

4. Multispecific antibodies

In certain aspects, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificity for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for CCR8 and the other specificity is for any other antigen. In certain aspects, the bispecific antibody can bind to two (or more) different epitopes of CCR 8. Multispecific (e.g., bispecific) antibodies can also be used to localize a cytotoxic agent or cell to a CCR 8-expressing cell. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, milstein and Cuello, nature 305:537 (1983)) and "mortar and pestle" 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 prepared by the following method: engineering electrostatic steering effects for the preparation of antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al Science,229:81 (1985)); the use of leucine zippers to produce bispecific antibodies (see, e.g., kostelny et al, j. Immunol.,148 (5): 1547-1553 (1992) and WO 2011/034605); use of universal light chain technology for circumventing the light chain mismatch problem (see, e.g., WO 98/50431); the "diabody" technique is used for the preparation of bispecific antibody fragments (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)); and the use of single chain Fv (sFv) dimers (see, e.g., gruber et al, j. Immunol.,152:5368 (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al J.Immunol.147:60 (1991).

5. Antibody variants

In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of antibodies can 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 an antibody. Any combination of deletions, insertions, and substitutions may be made to achieve the final construct, provided that the final construct has the desired characteristics, e.g., antigen binding.

a)Substitution, insertion and deletion variants

In certain aspects, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FR.

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

Conservative substitutions are shown under the heading "conservative substitutions" in table 2. Further substantial changes are provided under the heading "exemplary substitutions" of table 2, and are further described below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity (e.g., maintained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will require the exchange of members of one of these classes for members of the other class.

One type of substitution variant involves replacement of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more of the resulting variants selected for further investigation will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

For example, changes (e.g., substitutions) can be made in the CDRs to improve antibody affinity. Such changes may be made in CDR "hot spots", i.e. residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., chordhury, methods afol. Biol.207:179-196 (2008)), and/or residues that contact antigen, the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, by Hoogenboom et al, edited in Methods in Molecular Biology 178:1-37 (O' Brien et al, human Press, totowa, N.J. (2001)). In certain aspects of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4 to 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.

In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not substantially reduce the antigen binding capacity of the antibody binding molecule. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not substantially reduce binding affinity. Such alterations may be, for example, external to the antigen-contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either remains unchanged or comprises no more than one, two or three amino acid substitutions.

A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or a set of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants may be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N-terminus or C-terminus of the antibody with an enzyme that increases the serum half-life of the antibody (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide.

b)Glycosylation variants

In certain aspects, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody comprises an Fc region, the oligosaccharides attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched-chain double-antenna oligosaccharides, which are typically linked by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of a double-antennary oligosaccharide structure. In some aspects, oligosaccharides in the antibodies described herein can be modified to produce antibody variants with certain improved properties.

In one aspect, antibody variants having non-fucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose (directly or indirectly) attached to the Fc region, are provided. Such nonfucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack fucose residues that link the first GlcNAc in the stem of the double antennary oligosaccharide structure, and such antibodies are further referred to herein as "defucosylated antibodies. In one aspect, antibody variants are provided having an increased proportion of nonfucosylated oligosaccharides in the Fc region as 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 proportion of defucosylation is from about 65% to about 100%, from about 80% to about 100%, or from about 80% to about 95%. The percentage of nonfucosylated oligosaccharides, as described for example in WO 2006/082515, is the sum of the (average) amount of oligosaccharides lacking fucose residues relative to all oligosaccharides (e.g. complex, hybrid and high mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to an asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between position 294 and position 300, e.g. Asn 299, due to minor sequence variations in antibodies. Such antibodies with increased proportion of nonfucosylated oligosaccharides in the Fc region may have improved fcyriiia receptor binding and/or improved effector function, in particular improved ADCC function. See, for example, US2003/0157108 and US2004/0093621.

In one aspect, the present disclosure provides defucosylated antibody variants with enhanced fcyriiia receptor binding. In one aspect, the present disclosure provides defucosylated antibody variants with enhanced Antibody Dependent Cellular Cytotoxicity (ADCC). In one aspect, the disclosure provides defucosylated antibody variants with Antibody Dependent Cellular Phagocytosis (ADCP) activity.

Examples of cell lines capable of producing antibodies with reduced fucosylation include cells lacking the protein fucosylation Lec13 CHO (Ripka et al arch. Biochem. Biophys.249:533-545 (1986), US2003/0157108, and WO 2004/056312, especially example 11), and knockout cell lines such as alpha-1, 6-fucosyltransferase genes, FUT8, knockout CHO cells (see, e.g., yamane-Ohnuki et al biotech. Bioeng.87:614-622 (2004), kanda et al, biotechnol bioeng, 94 (4): 680-688 (2006), and WO 2003/085107), or cells with reduced or abolished GDP fucose synthesis or transporter activity (see, e.g., US2004259150, US2005031613, US2004132140, US 2004110282). See also Pereira et al, MABS (2018) 693-711.

In a further aspect, the antibody variant provides bisected oligosaccharides, e.g., wherein a double antennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. As described above, such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in Umana et al, nat Biotechnol 17,176-180 (1999); ferrara et al, biotech Bioeng 93,851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764.

c)Variant Fc region

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG ₁ 、IgG ₂ 、IgG ₃ Or IgG ₄ Fc region), whichAmino acid modifications (e.g., substitutions) are included at one or more amino acid positions.

In certain aspects, the invention contemplates antibody variants having some, but not all, effector functions, which make them ideal candidates for applications in which the in vivo half-life of the antibody is important, while certain effector functions, such as Complement Dependent Cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC), are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm a reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in page table 3, of Ravetch and Kinet, annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. 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); 5,821,337 (see Bruggemann, M. Et al, J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods (see, e.g., ACTI for flow cytometry ^TM Nonradioactive cytotoxicity assay (CellTechnology, inc.Mountain View, CA); cytoToxNon-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 additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al Proc. Nat' l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3C binding ELISA in WO 2006/029879 and WO 2005/100402. In order to assess the activation of complement,CDC assays may be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996); cragg, M.S. et al, blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., petkova, s.b. et Al, int' l.immunol.18 (12): 1759-1769 (2006); WO 2013/120929 Al).

Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants having improved or reduced binding to FcR are described. ( See, for example, U.S. Pat. nos. 6,737,056; WO 2004/056312, shields et al J.biol.chem.9 (2): 6591-6604 (2001). )

In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce fcγr binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant is further comprised in a polypeptide derived from human IgG ₁ D265A and/or P329G in the Fc region of the Fc region. In one aspect, the polypeptide is derived from human IgG ₁ In the Fc region of the Fc region, the substitutions were L234A, L235A and P329G (LALA-PG). (see, e.g., WO 2012/130831). In another aspect, the polypeptide is derived from human IgG ₁ Substitutions in the Fc region of the Fc region were L234A, L A and D265A (LALA-DA).

In certain aspects, the antibody variants comprise an Fc region having one or more amino acid substitutions (e.g., substitutions at positions) that improve fcγr binding (and thereby improve effector function). In certain aspects, the antibody variant comprises an Fc region having at least one amino acid substitution of G236A, I332E, S298A, E333A, K334A, S239D, A L, F243L, R292P, Y300L, V I, P396L, L235V, L234 235Q, G236W, S239M, H268 35270E, K326D, A330M, K E (see, e.g., liu et al Antibodies (Basel) (2020); 9 (4): 64).

In some examples, alterations are made in the Fc region resulting in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), such as, for example, U.S. Pat. No. 6194551, WO 99/51642 and Idusogie et al J.Immunol.164:4178-4184 (2000).

Antibodies responsible for transferring maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994)) with increased half-life and improved binding to neonatal Fc receptor (FcRn) are described in US2005/0014934 (Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants having substitutions 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, for example, substitution of the Fc region residue 434 (see, e.g., U.S. Pat. nos. 7371826; dall' acqua, w.f. et al j. Biol. Chem.281 (2006) 23514-23524).

Residues of the Fc region that are critical for mouse Fc-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., dall' Acqua, W.F. et al J.Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU numbering of residues) are involved in interactions (Medesan, C. Et al, eur. J. Immunol.26 (1996) 2533; finan, M. Et al, int. Immunol.13 (2001) 993; kim, J. K. Et al, eur. J. Immunol.24 (1994) 542). Residues I253, H310 and H435 were found to be critical for human Fc interactions with murine FcRn (Kim, j.k. Et al, eur.j.immunol.29 (1999) 2819). Studies on the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are critical for this interaction (Finan, M.et al, int. Immunol.13 (2001) 993; shields, R.L., et al, J.biol. Chem.276 (2001) 6591-6604). Various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined in Yeung, y.a. et al (j.immunol.182 (2009) 7667-7671).

In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253, and/or 310 and/or 435 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are I253A, H310A and H435A. See, e.g., greys, a. Et al, j.immunol.194 (2015) 5497-5508.

In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310, and/or 433 and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are H310A, H433A and Y436A. (see, e.g., WO 2014/177460 Al).

In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues). In certain aspects, the antibody variants comprise an Fc region having amino acid substitutions at positions 252, 254, and 256. In one aspect, the polypeptide is derived from human IgG ₁ Substitutions in the Fc region of the Fc region were M252Y, S T and T256E. For other examples of variants of the Fc region, see additionally: duncan and Winter, nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; WO 94/29351.

The C-terminus of the heavy chain of an antibody as reported herein may be the complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one of all aspects 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 aspect of all aspects 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 aspect of all aspects 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).

d)Cysteine engineered antibody variants

In certain aspects, it may be desirable to generate cysteine engineered antibodies, e.g., THIOMAB ^TM An antibody, wherein one or more residues of the antibody are substituted with cysteine residues. In certain embodiments, the substituted residue is present at an accessible site of the antibody. As further described herein, reactive thiol groups are located at accessible sites of antibodies by substitution of those residues with cysteines, and can be used to conjugate antibodies with other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates. Cysteine engineered antibodies may be produced as described, for example, in U.S. patent nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO 2016040856.

e)Antibody derivatives

In certain aspects, the antibodies provided herein can be further modified to include additional non-protein moieties known and readily available in the art. Moieties suitable 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers) and dextran or poly (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may or may not have branching. The number of polymers attached to the antibody may vary, and if more than one polymer is 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 including, but not limited to, the particular characteristics or functions of the antibody to be improved, whether the antibody derivative will be used in a defined-condition therapy, and the like.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to produce antibodies, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding an antibody are provided.

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

In the case of certain bispecific antibodies with heterodimeric heavy chains, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising a first heteromonomer (heteromonomer) Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heteromonomer 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 that constitutes a first VL of the antibody and/or an amino acid sequence that constitutes a first VH of the antibody comprising a first heteromonomer Fc region and/or an amino acid sequence that constitutes a second VL of the antibody and/or an amino acid sequence that constitutes a second VH of the antibody comprising a second heteromonomer Fc region (e.g., a first light chain and/or a second light chain and/or a first heavy chain and/or a second heavy chain of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors, typically these nucleic acids are located on two or three expression vectors, i.e., one vector may contain more than one of these nucleic acids. Examples of such bispecific antibodies are cross mabs (see, e.g., schaefer, w. et al, PNAS,108 (2011) 11187-1191). For example, one of the heteromonomer heavy chains comprises a so-called "knob mutation" (T366W, and optionally one of S354C or Y349C), and the other of the heteromonomer heavy chains comprises a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see, e.g., carter, p. Et al, immunotechnol.2 (1996) 73), numbered according to the EU index.

In one aspect, there is provided an isolated nucleic acid encoding an antibody as used in the methods reported herein.

In one aspect, a method of producing an anti-CCR 8 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-CCR 8 antibodies, the nucleic acid encoding the antibody (e.g., as described above) is 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 conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody), or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237, U.S. Pat. No. 5, 5,789,199, and U.S. Pat. No. 5,840,523. (see also Charlton, K.A., in: methods in Molecular Biology, volume 248, lo, B.K.C. (editions), humana Press, totowa, NJ (2003), pages 245-254, describing expression of antibody fragments in E.coli.) antibodies can be isolated from bacterial cell pastes in soluble fractions after expression, and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with a partially or fully human glycosylation pattern, are also suitable cloning or expression hosts for vectors encoding antibodies. See gerngros, T.U., nat.Biotech.22 (2004) 1409-1414; and Li, H.et al, nat. Biotech.24 (2006) 210-215.

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 can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S. Pat. No. 7,125,978 and U.S. Pat. No. 6,417,429 (describes PLANTIBODIES STM technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth 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 (as described, for example, in 293 or 293T cells: graham, F.L. et al, J.Gen. Virol.36 (1977) 59-74); baby hamster kidney cells (BHK); mouse support cells (such as, for example, TM4 cells described in Mather, J.P., biol.Reprod.23 (1980) 243-252); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells as described, for example, in Mather, J.P. et al, annals N.Y. Acad.Sci.383 (1982) 44-68); MRC 5 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 cell lines suitable for antibody production, see, e.g., yazaki, p. And Wu, a.m., methods in Molecular Biology, volume 248, lo, b.k.c. (editions), humana Press, totowa, NJ (2004), pages 255-268.

In one aspect, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphocyte (e.g., Y0, NS0, sp20 cell).

C. Measurement

The physical/chemical properties and/or biological activity of the anti-CCR 8 antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

1.Binding assays and other assays

In one aspect, the antibodies described herein are tested for antigen binding activity by, for example, known methods such as ELISA, western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with anti-CCR 8 antibodies of the presently disclosed subject matter, e.g., ab1, ab2, ab3, ab4, and Ab5, for binding to CCR8. In certain aspects, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) to which the anti-CCR 8 antibodies of the presently disclosed subject matter (e.g., ab1, ab2, ab3, ab4, and Ab 5) bind. Detailed exemplary methods for locating the epitope to which an antibody binds are provided in: morris (1996), "Epitope Mapping Protocols", incorporated by reference in Methods in Molecular Biology, volume 66 (Humana Press, totowa, N.J.).

In an exemplary competition assay, immobilized CCR8 is incubated in a solution comprising a first labeled antibody that binds to CCR8 (e.g., anti-CCR 8 antibodies of the presently disclosed subject matter, e.g., ab1, ab2, ab3, ab4, and Ab 5) 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 the hybridoma supernatant. As a control, immobilized CCR8 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to CCR8, excess unbound antibody is removed and the amount of label associated with immobilized CCR8 is measured. If the amount of label associated with immobilized CCR8 is substantially reduced in the test sample relative to the control sample, it is indicated that the second antibody competes with the first antibody for binding to CCR8. See Harlow and Lane (1988) Antibodies, A Laboratory Manual chapter 14 (Cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

2.Activity determination

In one aspect, an assay for identifying an anti-CCR 8 antibody that is biologically active is provided. Biological activities may include, for example, antibody Dependent Cellular Cytotoxicity (ADCC), ADCC against tregs, antibody Dependent Cellular Phagocytosis (ADCP), depletion of tregs. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain aspects, the anti-CCR 8 antibodies described herein are tested to measure ADCC of the antibodies. ADCC assays were performed according to the following documents previously reported: kamen, L.et al Development of a kinetic antibody-dependent cellular cytotoxicity assay J Immunol Methods 2019.468: pages 49-54, and Schnuinger, A.et al Development of aquantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies mol Immunol,2011.48 (12-13): pages 1512-17, modified with some modifications, use CD16 engineered NK-92_F158 as effector cells and CHO cells stably expressing human CCR8 and Ga 15 subunits (CHO/hCR 8.Gna 15) as target cells. Briefly, ADCC lysis of target cells was measured by the calcein release method. Target cells were labeled with Calcein-AM, then washed and plated onto 384-well plates at a density of 3000 cells/well. anti-CCR 8 antibody was added at various concentrations of 0.004 to 1. Mu.g/mL, followed by NK-92_F158 cells at a 10:1 effector to target (E: T) ratio. Plates were then incubated at 37℃for 2.5 hours. After incubation, the plates were centrifuged at 200 Xg for 3 minutes, the supernatant was transferred to a white opaque 384 well microplate and the fluorescent signal was measured in Relative Fluorescence Units (RFU). The signal from the wells containing only target cells represents the spontaneous release of calcein from the labeled cells, whereas the wells containing target cells lysed using Triton X-100 provided the maximum available signal (maximum release). In the absence of added antibody, antibody independent cell mediated cytotoxicity (AICC) was measured in wells containing target cells and effector cells. Samples and controls were tested in at least two replicates in the same plate. The specific ADCC activity level was calculated as follows:

％ADCC＝

100x (average experimental release-average AICC)/(average maximum release-average spontaneous release)

ADCC activity was plotted as a function of antibody concentration and data fitted to an asymmetric sigmoid four parameter logic (4 PL) model.

In certain aspects, the anti-CCR 8 antibodies described herein are tested to measure ADCC against Treg cells. To induce CCR8 expression on T cells from human Peripheral Blood Mononuclear Cells (PBMCs), 10 will be ⁷ Intraperitoneal transfer of personal PBMC to NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl Mice (JAX) were SzJ (NSG) and spleens were collected 2-3 weeks after transfer. Human T cells were enriched from single cell suspensions of NSG splenocytes and primary NK cells were enriched from human PBMCs. Human T cells were incubated with 0.001-1. Mu.g/mL of anti-CCR 8 antibody for 30 minutes at room temperature, then primary NK cells were added at a 2:1 effector to target ratio. After overnight incubation at 37 ℃, cells were collected, stained for their surface and stained for intracellular. Antibodies used to define the T cell population were CD45 (HI 30), CD3 (SK 7), CD8 (RPA-T8) and CD14 (63D 3), CD4 (RPA-T4) and FOXP3 (236A/E7). The CountBright absolute count beads were added to each sample prior to collection. Flow cytometry was performed. Absolute cell counts were calculated. ADCC activity against Treg cells was measured by calculating the ratio of recovered Treg cells to recovered CD8 cells (Treg/CD 8) or conventional CD 4T cells to recovered CD 8T cells (CD 4conv/CD 8).

In certain aspects, the anti-CCR 8 antibodies described herein are tested by Fluorescence Activated Cell Sorting (FACS) flow cytometry to measure their binding to regulatory T cells (Treg cells or tregs). Human colorectal isolated tumor cells (DTCs) were thawed. Cells were surface stained with eFluor 780 conjugated fixable vital dye and 2ug/mL mAb specific for CCR8, OX40 (positive control), herceptin (negative control) or anti-hIgG (negative control) for 20 min at 4℃and then subjected to a secondary detection for 10 min at 4℃by goat anti-human IgG with AF647 conjugated AffiniPure F (ab') 2 fragment, fcg fragment specificity. The cells were then subjected to intracellular staining. Antibodies used to define the T cell population were CD45 (HI 30), CD3 (SK 7), CD8 (RPA-T8) and CD14 (63D 3), CD4 (RPA-T4) and FOXP3 (236A/E7) from BD Biosciences. Flow cytometry was performed and analyzed.

In certain aspects, the anti-CCR 8 antibodies described herein are tested to measure ADCP of the antibodies. Human CD14 is first isolated from donor blood with known FcgRIIA and FcgRIIIa genotype information ₊ Monocytes. Purified CD14 ⁺ Monocytes differentiate into macrophages. hIL-10 was then added at 50ng/mL to polarize macrophages for 24 hours, followed by an ADCP assay. The nuchight Red transfected CHO/hccr8.gna15 target cells were pre-incubated with anti-CCR 8 antibody in the presence of 20mg/mL non-specific human IgG for 20 min. The above cell mixture was then added to macrophage (effector cell) plates at a ratio of E:1. Cell images were acquired every one hour with bright field and red laser settings for a period of 6 hours. The red blood cell count (remaining target cells) in each well was normalized by the number of macrophages. ADCP activity was calculated as the percentage of reduction in normalized red blood cell count in each sample compared to the negative control in the presence of isotype control antibody. ADCP activity was then plotted as a function of antibody concentration and the data fitted to an asymmetric sigmoid four parameter logic (4 PL) model. EC of each antibody ₅₀ The value was determined to be the concentration that reached 50% target cell killing.

In certain aspects, an anti-CCR 8 antibody (e.g., a mouse replacement antibody) described herein is tested to measure the depletion of Treg cells in vivo, mice with established tumors are treated with the anti-CCR 8 antibody (e.g., a mouse replacement antibody disclosed herein), and the proportion of Treg cells, conventional CD 4T cells, and CD 8T cells in leukocytes in tumors, spleen, and tumor draining lymph nodes is analyzed. For this purpose, tumor cells were harvested in the logarithmic growth phase and resuspended in HBSS containing matrigel in a 1:1 ratio. With 100 microliters10 ten thousand tumor cells in hbss+ matrigel were inoculated subcutaneously in the flank of mice. Tumors were monitored until they were established and reached 130 to 230mm ³ Is a mean tumor volume of (3). Mice were then randomized into treatment groups. Intravenous administration of anti-CCR 8 or anti-gp 120 isotype control abs for treatment. Three days later, mice were sacrificed and tumors, spleens, and tumor draining lymph nodes were obtained for analysis. To generate a single cell suspension, tumors were minced and digested. Single cell suspensions were surface stained with fluorescently labeled anti-CD 45, anti-CD 4 and anti-CD 8 antibodies and intracellular stained with fluorescently labeled anti-Foxp 3 antibodies. Flow cytometry can be performed on Fortessa X-20 or FACSymphony and analyzed using FlowJo software.

In certain aspects, anti-CCR 8 antibodies (e.g., mouse surrogate antibodies) described herein are tested for tumor growth inhibition following in vivo anti-CCR 8 mediated tumor-infiltrating Treg cell depletion. Mice with established tumors were treated with mice in place of anti-CCR 8 antibodies and tumor growth was monitored over time.

D. Methods and compositions for diagnosis and detection

In certain aspects, any of the anti-CCR 8 antibodies provided herein can be used to detect the presence of CCR8 in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative detection. In certain aspects, the biological sample comprises a cell or tissue, such as a tumor.

In one aspect, an anti-CCR 8 antibody for use in a diagnostic or detection method is provided. In a further aspect, methods of detecting the presence of CCR8 in a biological sample are provided. In certain aspects, the method comprises contacting the biological sample with an anti-CCR 8 antibody under conditions that allow binding of both the anti-CCR 8 antibody and CCR8, and detecting whether a complex is formed between the anti-CCR 8 antibody and CCR 8. Such methods may be in vitro or in vivo. In one aspect, the anti-CCR 8 antibody is used to select subjects eligible for treatment with the anti-CCR 8 antibody, e.g., wherein CCR8 is a biomarker for selecting subjects.

In certain aspects, labeled anti-CCR 8 antibodies are provided. Markers include, but are not limited to, direct detectionLabels or moieties to be detected (such as fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as moieties (such as enzymes or ligands) to be detected indirectly (e.g., by enzymatic reactions or molecular interactions). Exemplary labels include, but are not limited to, radioisotopes ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i, a step of I; fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone; luciferases (luciferases), such as firefly luciferases and bacterial luciferases (U.S. Pat. No. 4,737,456); luciferin, 2, 3-dihydronaphthyridonedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme; sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as urate oxidase and xanthine oxidase; coupled to an enzyme (such as HRP, lactoperoxidase, or microperoxidase) that oxidizes the dye precursor with hydrogen peroxide; biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

E. Pharmaceutical composition

In a further aspect, there is provided a pharmaceutical composition comprising any one of the antibodies provided herein, for use, for example, in any one of the following methods of treatment. In one aspect, a pharmaceutical composition comprises any one of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any one of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The pharmaceutical compositions (formulations) of the anti-CCR 8 antibodies described herein may be prepared by combining the antibodies with pharmaceutically acceptable carriers or excipients known to the skilled artisan. See, e.g., remington' sPharmaceutical Sciences, 16 th edition, osol, a. Edit (1980), shire s., monoclonal Antibodies: meeting the Challenges in Manufacturing, formulation, delivery and Stability of Final Drug Product, 1 st edition, woodhead Publishing (2015), ≡4 and Falconer r.j., biotechnology Advances (2019), 37,107412. Exemplary pharmaceutical compositions of anti-CCR 8 antibodies as described herein are lyophilized, aqueous, frozen, and the like.

Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, including but not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; 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., zinc protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

The pharmaceutical compositions herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents for treating the same. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

Pharmaceutical compositions for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

F. Methods of treatment and routes of administration

Any of the anti-CCR 8 antibodies provided herein may be used in a method of treatment.

In one aspect, an anti-CCR 8 antibody for use as a medicament is provided. In a further aspect, an anti-CCR 8 antibody for use in the treatment of cancer is provided. In certain aspects, an anti-CCR 8 antibody for use in a method of treatment is provided. In certain aspects, the present disclosure provides an anti-CCR 8 antibody for use in a method of treating a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject an effective amount of the anti-CCR 8 antibody. In one such aspect, for example as described below, 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). In a further aspect, the present disclosure provides an anti-CCR 8 antibody for depleting regulatory T cells ("tregs") in a tumor microenvironment. In certain aspects, the present disclosure provides an anti-CCR 8 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 the anti-CCR 8 antibody to deplete tregs in the tumor microenvironment.

In a further aspect, the present disclosure provides the use of an anti-CCR 8 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating cancer. In a further aspect, the medicament is for use in a method of treating cancer, the method comprising administering to a subject (e.g., a human subject) in need thereof an effective amount of the medicament. In one such aspect, for example as described below, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. 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, the method comprising administering to the subject an effective amount of the medicament to eliminate tregs in the tumor microenvironment.

In a further aspect, the present disclosure provides a method for treating cancer. In one aspect, the method comprises administering to a subject (e.g., a human subject) in need thereof an effective amount of an anti-CCR 8 antibody to treat the cancer. In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, as described below.

In a further aspect, the present disclosure provides an anti-CCR 8 antibody for depleting Treg cells outside of or in a tumor microenvironment, for example. For example, in certain embodiments, the present disclosure provides a method for depleting Treg cells in a tumor microenvironment of a subject in need thereof (e.g., a human subject), the method comprising administering to the subject an effective amount of an anti-CCR 8 antibody sufficient to deplete Treg cells in the tumor microenvironment, thereby treating cancer. In certain aspects, the disclosure provides a method for depleting Treg cells outside of (e.g., in the circulation of) a tumor microenvironment in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject an effective amount of an anti-CCR 8 antibody sufficient to deplete Treg cells outside of the tumor microenvironment, thereby treating cancer. Without wishing to be bound by any particular theory, by reducing the number of Treg cells outside of the tumor microenvironment, the number of Treg cells in the tumor microenvironment is reduced as the number of Treg cells that infiltrate into the tumor microenvironment is reduced.

Exemplary cancers include, but are not limited to, bladder cancer (e.g., urothelial cancer), blastoma, blood cancer (e.g., lymphomas such as non-hodgkin's disease, 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., head and neck squamous cell cancer), kidney cancer (e.g., renal cancer) cell cancer), liver cancer (e.g., hepatocellular cancer), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer (e.g., melanoma, squamous cell cancer, cell carcinoma), testicular cancer, and uterine cancer.

In certain 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.

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.

In certain aspects, the cancer is a solid tumor.

In certain aspects, the cancer expresses CCR8.

In certain aspects, the cancer is a T-cell inflamed tumor or a tumor microenvironment comprising T-cell inflammation.

In certain aspects, the cancer comprises regulatory T cells in a tumor microenvironment, and exposing the cancer to CCR8 antibodies results in depletion of regulatory T cells in the tumor microenvironment, as described herein. In a further aspect, the present disclosure provides a pharmaceutical composition comprising any of the anti-CCR 8 antibodies described herein, e.g., for use in any of the above methods of treatment. In one aspect, a pharmaceutical composition comprising any one of the anti-CCR 8 antibodies provided herein, and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprising any one of the anti-CCR 8 antibodies provided herein, and at least one additional therapeutic agent, e.g., as described below.

The antibodies described herein may be administered alone or in combination therapy, e.g., for the treatment of cancer. For example, the combination therapy comprises administering an antibody as described herein, and administering at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents).

The at least one additional therapeutic agent encompasses any agent that can be administered for treatment. In certain aspects, 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 intercalating agents, alkylating agents, hormonal therapies, kinase inhibitors, receptor antagonists, tumor apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cytostatic agents, apoptosis activators, 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 therapies, and immune cell cements. In certain aspects, the additional therapeutic agent is an immunomodulatory anticancer agent, e.g., a checkpoint inhibitor (CPI) such as an anti-CTLA 4 antibody (e.g., ipilimumab), a PD-L1 binding antagonist, or a PD-1 binding antagonist

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 and/or B7-1). In some cases, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some cases, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from interaction of PD-L1 with one or more of its binding partners (such as PD-1 and/or B7-1). In one case, the PD-L1 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling through PD-L1 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing response of effector to antigen recognition). In some cases, the PD-L1 binding antagonist binds to PD-L1. In some cases, 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 Ab, MDX-1105, MEDI4736 (Devalumab), MSB0010718C (aviumab), SHR-1316, CS1001, en Wo Lishan antibody (envafolimab), TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, ke Xili monoclonal antibody (cosibelimab), lodaplizumab (lodaplimab), 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 aspects, the anti-PD-L1 antibody is alemtuzumab, MDX-1105, MEDI4736 (Devaluzumab), or MSB0010718C (avermectin). In a specific aspect, the PD-L1 binding antagonist is MDX-1105. In another specific aspect, the PD-L1 binding antagonist is MEDI4736 (devaluzumab). In another specific aspect, the PD-L1 binding antagonist is MSB0010718C (avilamab). In other aspects, the PD-L1 binding antagonist may be a small molecule, e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which in some cases may be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003 and JS-003. In one aspect, the PD-L1 binding antagonist is alemtuzumab.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and/or PD-L2). PD-1 (programmed death 1) is also known in the art as "programmed cell death 1", "PDCD1", "CD279" and "SLEB2". An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the 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 and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one case, the PD-1 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling by PD-1 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing effector to antigen recognition response). In some cases, the PD-1 binding antagonist binds to PD-1. In some cases, 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 Na Wu Shankang, palbociclizumab, MEDI-0680, PDR001 (Stidazumab), REGN2810 (Simipu Li Shan antibody), BGB-108, paruo Li Shan, carilizumab, xindi Li Shan antibody, tirilizumab, teripu Li Shan antibody, dutarizumab, ralfordin Li Shan antibody, sashan Li Shan antibody, pe An Puli mab, CS1003, HLX10, SCT-I10A, sapalizumab, butelimumab, jenomab, BI 754091, silimumab, YBL-006, BAT1306, HX008, bragg Li Shan antibody, AMG 404, CX-188, JTX-4014, A, sym021, LZM009, F520, SG001, ENUM 244C8, ENUM D4, STI-1110, AK-103 and hAb21. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (Nawuzumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, the PD-1 binding antagonist is a PD-L2 fusion protein, e.g., AMP-224. In another specific aspect, the PD-1 binding antagonist is MED1-0680. In another specific aspect, the PD-1 binding antagonist is PDR001 (swabber). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cimipn Li Shan antibody). In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is a palo Li Shan antagonist. In another specific aspect, the PD-1 binding antagonist is a karite Li Zhushan antagonist. In another specific aspect, the PD-1 binding antagonist is a syndesmosidic Li Shan antagonist. In another specific aspect, the PD-1 binding antagonist is tirelizumab. In another specific aspect, the PD-1 binding antagonist is terlipressin Li Shan. Other exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006. Such combination therapies described above encompass the combined administration (wherein two or more therapeutic agents are included in the same or separate pharmaceutical compositions) and the separate administration, in which case the administration of an antibody as described herein may be performed before, simultaneously with, and/or after the administration of the additional therapeutic agent or agents. In one aspect, the administration of the anti-CCR 8 antibody and the administration of the additional therapeutic agent are performed within about one month of each other, or within about one week, two weeks, or three weeks, or within about one, two, three, four, five, or six days. In one aspect, the antibody and the additional therapeutic agent are administered to the subject on day 1 of treatment. Antibodies as described herein may also be used in combination with radiation therapy.

Antibodies (and any additional therapeutic agents) as described herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various points in time, bolus administrations, and pulse infusion.

Antibodies as described herein will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this case include the particular disorder being treated, the particular subject species being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner. The antibody is not necessary, but is optionally co-formulated with one or more of the formulations currently used to treat the disorder in question. The effective amount of these other formulations depends on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.

The antibody is suitably administered to the subject once or in a series of treatments. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of the therapy can be readily monitored by conventional techniques and assays.

In a further embodiment, the use of mouse substitutes is contemplated, for example as in vitro or in vivo tool molecules. For example, in one aspect, a method of treating a disease in a mouse is provided, the method comprising administering to the mouse an effective amount of a mouse surrogate antibody as described herein to treat the disease. In certain embodiments, the mice comprise xenografts. In certain embodiments, the mouse model is a cancer model, such as a skin cancer model.

G. Article of manufacture

In another aspect, an article of manufacture is provided that contains a substance useful in the treatment, prevention and/or diagnosis of the above-described diseases. The article includes a container and a label or package insert (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 contains a composition that is effective in treating, preventing and/or diagnosing a condition, either by itself or in combination with another composition, and the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody as disclosed herein. The label or package insert indicates that the composition is to be used to treat the selected condition. In addition, the article of manufacture may comprise (a) a first container comprising a composition therein, wherein the composition comprises an antibody as disclosed herein; and (b) a second container containing a composition therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this aspect as described herein may further comprise package insert indicating that the composition may be used to treat a particular disorder. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The article of manufacture may also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Example

The following are further non-limiting examples of antibodies, methods, and compositions as described herein. It should be understood that various other embodiments may be practiced given the general description provided above.

EXAMPLE 1 discovery and engineering of anti-CCR 8 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 huCCR 8. Single B cells were isolated according to the protocol set forth in Lin et al, PLoS ONE 15 (12), 2020. The B cell culture supernatant was then analyzed for binding to human and cynomolgus CCR8+ CHO cells and control CHO cells by direct flow activated cell sorting (FACS; flow cytometry) into single wells. CCR 8-specific B cells were lysed and immediately cryopreserved at-80 ℃ until molecular cloning was performed. Variable regions (VH and VL) of various monoclonal antibodies from rabbit B cells were cloned into expression vectors from the extracted mRNA as described by Lin et al, PLoS ONE 15 (12), 2020. A single recombinant rabbit antibody was expressed in an Expi293 cell and subsequently purified with protein a.

Over 480 anti-CCR 8 antibodies were obtained that bound to human or cynomolgus monkey CCR8 CHO cells. Antibodies were further selected based on the relative Mean Fluorescence Intensity (MFI) and sequence diversity of human and cynomolgus CCR8 CHO cell lines. Five unique groups of antibodies (designated Ab1-Ab 5) were identified from antibodies that showed less than 5-fold MFI differences on human and cynomolgus CCR8 CHO cells. One representative sequence from each group was selected for humanization.

Variants constructed during humanization of rabbit monoclonal antibodies were assessed as human IgG 1. Hypervariable regions from each of the rabbit antibodies (i.e., positions 24 to 34 (L1), 50 to 56 (L2), and 89 to 97 (L3) in the VL domain and positions 26 to 35 (H1), 50 to 65 (H2), and 95 to 102 (H3) in the VH domain) were transplanted into the respective acceptor frameworks. Residue numbering is according to Kabat et al Sequences of proteins of immunological interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, md. (1991). In addition, all VL and VH cursor positions from rabbit antibodies were transplanted into their corresponding human germline frameworks. The graft with all rabbit amino acids at the vernier position is designated as H1L1. The binding capacity of the humanized CCR8 antibody to CHO-huccr8.gna15 stable cell lines was compared to its chimeric parental clone. The rabbit cursor positions of the H1L1 antibodies were converted back to human residues to assess the contribution of each rabbit cursor position to huCCR8 binding.

Binding of mAb to regulatory T cells (Treg cells or Treg) was assessed by Fluorescence Activated Cell Sorting (FACS) flow cytometry. Human colorectal isolated tumor cells (DTCs) were thawed according to the supplier's protocol (Discovery Life Sciences). Cells were surface stained with eFluor 780 conjugated fixable vital dye (ThermoFisher Scientific) and 2ug/mL mAb specific for CCR8, OX40 (positive control), herceptin (negative control) or anti-hIgG (negative control) for 20 min at 4℃and then subjected to a secondary detection for 10min at 4℃using AF647 conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG, fcg fragment specificity (Jackson ImmunoResearch). The cells were then stained intra-cellularly using the eBioscience Foxp 3/transcription factor staining buffer set (ThermoFisher Scientific) according to the manufacturer's protocol. Antibodies used to define the T cell population were CD45 (HI 30), CD3 (SK 7), CD8 (RPA-T8) and CD14 (63D 3), CD4 (RPA-T4) from BioLegend and FOXP3 (236A/E7) from ThermoFisher Scientific from BD Biosciences. Flow cytometry was performed on Fortessa X-20 (BD Biosciences) and analyzed with FlowJo software (BD Biosciences, version 10.5.3). The Mean Fluorescence Intensity (MFI) values for CD 8T cells (defined as CD45+CD14-CD3+CD8+CD4-) (circle,) and conventional CD 4T cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3-) (square, -) and Treg cells (defined as CD45+CD14-CD3+CD8-CD4+FOXP3+) (triangle,) are shown in FIG. 1. Three of the five CCR8 mAb clones stained specifically for Treg cells, but not for conventional CD4 or CD 8T cells, and ranked according to CCR8 MFI, greater than 500MFI: hu.ab4.h1l1> hu.ab5.h1l1> hu.ab3.h1l1. It was confirmed that these three CCR8 mAb clones (i.e., hu.ab3.h1l1, hu.ab4.h1l1, and hu.ab 5.h1l1) also retained human-cynomolgus monkey cross-reactivity (less than 5-fold difference on human and cynomolgus monkey CCR8 CHO cells) and these antibodies continued to be used for further exploration.

For example, antibody-dependent cellular cytotoxicity (ADCC) of hu.ab3.h1l1, hu.ab4.h1l1, and hu.ab5.h1l1 was further studied. The hIgG1 isotype served as a negative control. See fig. 2.ADCC assays were performed according to the following documents previously reported: kamen, L.et al Development of a kinetic antibody-dependent cellular cytotoxicity assay J Immunol Methods,2019.468: pages 49-54, and Schnuinger, 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): pages 1512-17, modified with some modifications, use CD16 engineered NK-92_F158 as effector cells and CHO cells stably expressing human CCR8 and G-. Alpha.15 subunits (CHO/hCR 8. Gna15) as target cells. Briefly, ADCC lysis of target cells was measured by the calcein release method. Target cells were labeled with Calcein-AM (C3100 MP, thermoFisher Scientific) according to the manufacturer's protocol, then washed and plated onto 384-well plates at a density of 3000 cells/well. anti-CCR 8 antibody was added at various concentrations of 0.004 to 1. Mu.g/mL, followed by NK-92_F158 cells at a 10:1 effector to target (E: T) ratio. Plates were then incubated at 37℃for 2.5 hours. After incubation, the plates were centrifuged at 200 Xg for 3 minutes, the supernatants were transferred to white opaque 384 well microplates (OptiPlate-384, perkinelmer, waltham, mass.) and fluorescent signals were measured in Relative Fluorescence Units (RFU) using an EnSight multimode reader (Perkinelmer) with excitation/emission wavelength of 485/520 nm. The signal from the wells containing only target cells represents spontaneous release of calcein from the labeled cells (spontaneous release), while the signal from the wells containing target cells lysed using Triton X-100 (Sigma-Aldrich, st.louis, MO) represents maximum release. In the absence of added antibody, antibody independent cell mediated cytotoxicity (AICC) was measured in wells containing target cells and effector cells. Samples and controls were tested in at least two replicates in the same plate. The specific ADCC activity level was calculated as follows:

％ADCC＝

ADCC activity was plotted as a function of antibody concentration and data was fitted to an asymmetric sigmoid four parameter logic (4 PL) model using Prism (Graphpad; la Jolla, CA). See fig. 2.EC (EC) ₅₀ The value was determined as the concentration at which each individual antibody reached 50% of maximum ADCC activity. EC (EC) ₅₀ The values are also listed in the following table.

The agonist (CCR 8 activation) and antagonist (CCL 1 inhibition; neutralization) activities of hu.Ab3.H1L1, hu.Ab4.H1L1 and hu.Ab5.H1L1 were further analyzed. hIgG1 homologyThe isotype served as a negative control. Fluorescence imaging plate reader (FLIPR) FDSS/. Mu.cell (Pinus maritima, japan) was used to read through Ca ²⁺ Inflow monitors CCR8 activation. Briefly, CHO/hCR8.Gna 15 cells were loaded with fluorescent Ca ²⁺ Dye Fluo-8 NW (Cat #36307,AAT Bioquest) and incubated at 37℃for 30 minutes and then at room temperature for a further 30 minutes. Serial dilutions of test anti-CCR 8 antibodies were prepared in HHBS buffer in clear 384 well plates, and hCCL1 in HHBS buffer was aliquoted in clear 384 well plates. The FLIPR assay was set up on FDSS/μcell with antibody added at 10 seconds and hCCL1 added at 300 seconds and monitored for a total of 500 seconds. The fluorescence excitation and emission wavelengths were set to 485nm and 525nm, respectively. After run, negative control corrections were applied and the data normalized to hCCL1 signal (100%) and plotted as a function of antibody concentration using Prism.

As shown in fig. 3A, CCL1 (a known ligand for CCR 8) showed agonist activity, but none of the anti-CCR 8 test antibodies showed agonist effect. The data in fig. 3B indicate that the anti-CCR 8 antibodies hu.ab4.h1l1 have antagonistic (neutralizing) activity against CCR8 ligand CCL1 (20 nM ligand), whereas the anti-CCR 8 antibodies hu.ab5.h1l1 and hu.ab3.h1l1 do not exhibit ligand blocking (non-neutralizing) activity at the studied concentrations. The data in fig. 3C further shows that the comparative anti-CCR 8 antibodies (Yoshida humanized anti-human CCR8 antibodies, murine anti-human CCR8 mAb 433H (BD Biosciences), and murine anti-human CCR8 mAb L263G8 (bioleged)) also show antagonistic (neutralization) activity blocking activation of CCR8 by CCR8 ligand CCL 1. IC's for ligand blocking Activity are provided in Table B ₅₀ Values. Ligand blocking alone was insufficient to deplete Treg cells in mouse tumors as described in Van Damme et al, j.immunother. Cancer (2021), 9:e001749. Thus, although hu.ab5.h1l1 and hu.ab3.h1l1 did not exhibit ligand blockade, both antibodies were still considered promising candidates because the goal was to find a selective anti-CCR 8 antibody that binds to CCR8 and depletes Treg cells.

To confirm the selectivity for CCR8, hu.ab3.h1l1, hu.ab4.h1l1 and hu.ab5.h1l1 as well as Yoshida humanized anti-human CCR8, murine anti-human CCR8 mAb L263G8 (Biolegend, commercial Ab) and murine anti-human CCR8 mAb 433H (BD Biosciences, commercial Ab) were characterized by flow cytometry HEK293 cells transiently transfected with plasmids encoding FLAG tagged other related human GPCRs (CCR 2-5, CXCR4, actr 2 and actr 4). Cell surface expression of each GPCR was confirmed by control staining with anti-FLAG antibodies. See fig. 4A to 4F. In particular, HEK293 cells were transfected with N-terminal FLAG-labeled human CCR2, CCR3, CCR4, CCR5, CXCR4, actr 2, actr 4, hCCR8 constructs, or with mock constructs using transIT X2 (reagents: dna=3:1) and surface stained with 5ug/ml of various anti-hCCR 8 monoclonal antibodies or rabbit anti-FLAG pAb (Sigma), followed by AF 647-anti-hIgG or AF 647-anti-rbgg, respectively. Antibodies hu.ab4.h1l1 and hu.ab5.h1l1 stained only hCCR 8-containing cells, confirming their specificity for hCCR 8. Antibody hu.ab3.h1l1 showed staining of a number of other GPCRs, indicating lack of specificity. Thus, CCR8 selective hu.ab4.h1l1 and hu.ab5.h1l1 antibodies with optimal ADCC activity were continued to be used.

EXAMPLE 2 mutation analysis of Ab4 and Ab5 anti-CCR 8 antibodies

Variants of hu.ab4.h1l1 and hu.ab5.h1l1 anti-CCR 8 antibodies were further explored and characterized. FIGS. 5A to 5D depict the light chain variable region (FIG. 5A) and heavy chain variable region (FIGS. 5B to 5D) alignments of the sequences of rabbit (rb. Ab 4) and humanized Ab4 (L1-L4 and H1-H12) CCR8 antibodies. FIGS. 6A to 6D depict the light chain variable region (FIG. 6A) and heavy chain variable region (FIGS. 6B to 6D) alignments of the sequences of rabbit (rb.Ab5) and humanized Ab5 (L1-L5 and H1-H13) CCR8 antibodies. See also tables C1-C3 and D1-D3 below. Table E provides the heavy and light constant domains.

Assessment of CCR8 binding of humanized variants to hIgG1 Fc involved screening by flow cytometry and relative EC on human CCR8 CHO cells ₅₀ And MFI was compared to the parent rabbit antibody. Specifically, stable CHO-huccr8.gna15 cells were stained with different concentrations (starting from 10ug/ml or 66.66nM, 1:4 serial dilutions, total of 8 concentration points) of Ab4 and Ab5 variants at 4 ℃ for 30 min, then washed twice with FACS buffer (PBS containing 0.5% BSA and 0.2mM EDTA), and then stained with AF 647-anti-hIgG at 4 ℃ for 15 min. Cells were washed twice with FACS buffer, resuspended in FACS buffer containing propidium iodide (0.5 ug/ml) and analyzed with iQue3 (Sartorius).

For Ab4 LC variants L1-L4, variants L2 and L4 comprising Y2I mutations show EC as provided in Table F1 ₅₀ Or a significant change in MFI. From this, it was determined that Y2 on the light chain was the key rabbit cursor residue. Variants L1 and L3 contain this Y2 residue, and variant L3 is selected for further analysis.

For Ab4 HC variants H2-H11, variant H6 (with the S73N mutation), variant H7 (with the T76N mutation), variant H8 (with the V78L mutation), variant H9 (with the F91Y mutation), variant H10 (with the P105Q mutation) and variant H11 (with the S73N, V78L, F Y and P105Q mutations) showed EC as provided in table F2 ₅₀ Or a significant change in MFI. From this, it was determined that S73, T76, V78, F91 and P105 on the heavy chain are the key rabbit cursor residues.

These five residues combine to construct variant H12 (hu.ab 4.h12).

For Ab5 LC variants L2-L5, as provided in table F3, variant L2 (with V4M mutation), variant L3 (with P43A mutation), variant L4 (with F46L mutation) and variant L5 (with V4M, P a and F46L mutation) showed EC ₅₀ Or a significant change in MFI. From this, V4, P43 and F46 on the light chain were determined to be the key rabbit cursor residues. All variants contain a C90Q mutation in CDR L3, which is introduced to remove unpaired cysteines, which can be detrimental in the manufacturing process. Variant L1, which contains all three V4, P43 and F46 residues, was selected for further investigation.

For Ab5 HC variants H2-H12, as provided in table F4, variant H5 (with G49S mutation), variant H6 (with K71R mutation), variant H7 (with S73N mutation) and H12 (with G49S, K R and S73N mutation) showed EC ₅₀ Or a significant change in MFI. From this, it was determined that G49, K71 and S73 on the heavy chain are key rabbit cursor residues. These three residues combine to construct variant H13.

Example 3 characterization of hu.ab4.h12l3 and hu.ab5.h13l1 variants

(a) Human-cynomolgus monkey cross-reaction

Cell-based affinity measurements were performed using radiolabeled IgG and CHO cell lines stably expressing hu.ab5.h13l1 and hu.ab4.h12l3 of human or cynomolgus CCR 8.

Briefly, stable CHO cells expressing human or cynomolgus CCR8 were seeded at 50,000 cells per well in cold binding buffer (Opti-mem+2% fbs+50mM HEPES,pH7.2+0.1% sodium azide). Fixed concentrations of radiolabel using NEX244 Iodogen method (Perkin Elmer) ¹²⁵ I-anti-CCR 8 was mixed with serial dilutions of anti-CCR 8 antibodies starting at 20nM or 50 nM. The antibody mixture was added to the cells and incubated at room temperature for 12 hours with gentle agitation. Cells and antibodies were then transferred to Millipore multi-layer screen filter plates. The filter plate was washed 4 times with 250 μl of cold binding buffer and dried for at least 30 minutes, and the filter was driven into a 5mL polystyrene tube. Radioactivity was measured using a Perkin Elmer Wallac Wizard 2470 gamma counter set to 1 count per minute with a count efficiency of 0.8. The Ki competitive binding model was fitted to the data using heterologous single site fitting in GraphPad Prism.

As shown in fig. 7A to 7D, hu.ab4.h12l3 and hu.ab5.h13l1 have similar affinities for human and cynomolgus CCR8, indicating the desired cross-reactivity. Affinity Kd (nM) data from the as-made tables of these studies are provided below.

(b) CCR8 selectivity

To reconfirm that Ab4 and Ab5 variants were still selective for CCR8 compared to the corresponding H1L1 variants, binding was analyzed by flow cytometry according to the procedure described in fig. 4A and 4B. As previously described, hu.ab4.h12l3 (fig. 8A) and hu.ab5.h13l1 (fig. 8B) selectively bind CCR8 expressing cells.

(c) CCR8 activation and ligand blocking

To reconfirm that Ab4 and Ab5 variants retained their properties with respect to CCR8 activation and ligand blocking capacity, experiments were performed with the hu.ab4.h12l3 and hu.ab5.h13l1 antibodies, as previously described in example 1 and figures 3A-3C. See fig. 9A to 9B. Similar to fig. 3A, the data in fig. 9A again confirm that neither Ab4 nor Ab5 anti-CCR 8 antibody variants show agonism in the absence of CCR8 ligand CCL 1. Similar to the data of fig. 3B, fig. 9B again demonstrates that Ab4 variants exhibit antagonism of CCR8 activation by blocking CCR8 ligand CCL1 (20 nM ligand), while Ab5 variants exhibit no ligand blocking activity at the concentrations studied. The IC for ligand blocking activity is provided in the following Table ₅₀ Values.

(d) Sulfation independence

Human CCR8 contains four potential tyrosine sulfation sites at the N-terminus, and there is evidence that modification of these sites shows some heterogeneity (Gutierrez et al JBC 2004; jen et al Biochemistry 2010). Thus, antibodies recognizing these sulfated tyrosines in CCR8 may exhibit variability in CCR8 binding and thus mediate variable Treg cell depletion. The resulting human ccr8+hek293 cells lack Tyrosyl Protein Sulfotransferase (TPST) 1 and 2, both enzymes that catalyze the sulfation of tyrosine. The binding of various anti-CCR 8 mabs to wild-type (293T) and TPST1/2NTC and KO cells was then analyzed.

Specifically, HEK 293-hCR8.TPST1/2 NTC and HEK 293-hCR8.TPST1/2 KO stable cell lines were stained with test and comparative anti-CCR 8 antibody (1 ug/ml) at 4℃for 30 min, then washed twice with FACS buffer (PBS containing 0.5% BSA and 0.2mM EDTA), and then stained with AF 647-anti-hIgG at 4℃for 15 min. Cells were washed twice with FACS buffer, resuspended in FACS buffer containing propidium iodide (0.5 ug/ml) and analyzed with BD FACSCelesta flow cytometer or iQue3 (Sartorius).

FIGS. 10A to 10E depict the difference in staining of hu.Ab4.H12L3 and hu.Ab5.H13L1 versus CCR8+HEK293 cells with (hCR 8.TPST1/2 NTC) and without Tyrosyl Protein Sulfotransferase (TPST) 1 and Tyrosyl Protein Sulfotransferase (TPST) 2 (hCR 8.TPST1/2 KO) compared to the Yoshida humanized anti-human CCR8 antibody and the commercial antibodies murine anti-human CCR8mAb 433H (BD Biosciences) and murine anti-human CCR8mAb L263G8 (Biolegend). hu.ab4.h12l3 (fig. 10A) and hu.ab5.h13l1 (fig. 10B) showed similar binding/staining to the two cell lines (hccr 8.tpst1/2NTC and hccr8.tpst1/2 KO), indicating that they bind to CCR8 independent of tyrosine sulfation ("independent of sulfation"). In contrast, yoshida humanized anti-human CCR8 antibodies (fig. 10C) and the commercial antibodies murine anti-human CCR8mAb 433H (BD Biosciences) (fig. 10D) and murine anti-human CCR8mAb L263G8 (Biolegend) (fig. 10E) failed to bind to TPST1/2KO cells, indicating that they required tyrosine sulfation of CCR8 for binding and are therefore considered "sulfation-dependent".

Example 4.Hu.Ab4.H12L3 and hu.Ab5.H13L1 defucosylated variants

The defucosylated hu.Ab5.H13L1 and hu.Ab4.H12L3 variants (at Fc N-glycan position Asn 299) and the defucosylated anti-gD controls 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.

(a) Percent defucosylation

Titration of fucose in CHO FUT8KO medium produced a set of deglycosylated hu.ab5.h13l1 with different levels, e.g., between about 14% and about 93%.

As shown in the following table, increasing the level of defucosylation from 14% to 49% increased ADCC activity by more than 4-fold and ADCP activity by more than 3-fold.

The defucosylation hu.ab5.h13l1 and hu.ab4.h12l3 were studied in vitro and in vivo experiments to follow a defucosylation level containing between about 80% and about 95%.

(b) Enhanced binding of Fcgamma RIIIa to defucosylated variants

The binding of the fucosylated and defucosylated variants of hu.ab5.h13l1 and hu.ab4.h12l3 to the two FcgR3a proteins was studied by ELISA. Briefly, anti-GST antibodies were coated on Nunc Maxisorp plates. GST-FcgR3a.V158 and GST-FcgR3a.F158 were captured at 500 ng/mL. The plates were then washed and then serial dilutions of anti-CCR 8 antibody were incubated on the plates at room temperature for 1 hour starting at 100 ug/mL. Plates were washed and bound antibodies were detected by HRP conjugated anti-human IgG secondary antibody. Absorbance at 450nm was measured by a plate reader. Data were fitted using a 4-parameter logistic curve in Softmax Pro. As shown in the table below, the defucosylated IgG1 anti-CCR 8 antibodies afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 exhibited enhanced Fcgamma RIIIa binding activity (about 10-fold increase in binding potency) compared to the fucosylation counterparts hu.ab5.h13l1 and hu.ab4.h12l3.

(c) Enhanced ADCC activity of defucosylated variants

Antibody-dependent cellular cytotoxicity (ADCC) of afuc.hu.ab4.h12l3, hu.ab4.h12l3, afuc.hu.ab5.h13l1 and hu.ab5.h13l1 was analyzed. ADCC assays were performed according to the following documents previously reported: kamen, et al Development of a kinetic antibody-dependent cellular cytotoxicity assay.J Immunol Methods (2019) 468:49-54, and Schnuinger et al Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic anti-bodies.mol Immunol (2011) 48:1512-17, modified with some modifications, used CD16 engineered NK-92_F158 as effector cells, and CHO cells stably expressing human CCR8 and Ga 15 subunits (CHO/hCR 8.Gna 15) as target cells. Briefly, ADCC lysis of target cells was measured by the calcein release method. Target cells were labeled with Calcein-AM (C3100 MP, thermoFisher Scientific) according to the manufacturer's protocol, then washed and plated onto 384-well plates at a density of 3000 cells/well. anti-CCR 8 antibody was added at various concentrations of 0.004 to 1. Mu.g/mL, followed by NK-92_F158 cells at a 10:1 effector to target (E: T) ratio. Plates were then incubated at 37℃for 2.5 hours. After incubation, the plates were centrifuged at 200 Xg for 3 minutes, the supernatants were transferred to white opaque 384 well microplates (OptiPlate-384, perkinelmer, waltham, mass.) and fluorescent signals were measured in Relative Fluorescence Units (RFU) using an EnSight multimode reader (Perkinelmer) with excitation/emission wavelength of 485/520 nm. The signal from the wells containing only target cells represents spontaneous release of calcein from the labeled cells (spontaneous release), while the signal from the wells containing target cells lysed using Triton X-100 (Sigma-Aldrich, st.louis, MO) represents maximum release. In the absence of added antibody, antibody independent cell mediated cytotoxicity (AICC) was measured in wells containing target cells and effector cells. Samples and controls were tested in at least two replicates in the same plate. The specific ADCC activity level was calculated as follows:

% ADCC = 100x (average experimental release-average AICC)/(average maximum release-average spontaneous release)

ADCC activity was plotted as a function of antibody concentration and data was fitted to an asymmetric sigmoid four parameter logic (4 PL) model using Prism (Graphpad; la Jolla, CA). Fig. 11A to 11B show that the defucosylated CCR8 antibodies afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 have enhanced ADCC activity (improved > 10-fold) compared to the fucosylation counterparts hu.ab5.h13l1 and hu.ab4.h12l3, using NK-92F158 (fig. 11A) and NK-92V158 (fig. 11B) as effector cells against CHO cells stably expressing hCCR 8.

ADCC activity of afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 was also measured against Yoshida humanized anti-human CCR8 antibody and the commercial antibodies murine anti-human CCR8 mAb433H (BD Biosciences) and murine anti-human CCR8 mAb L263G8 (bioleged). See fig. 11C. The data show that Yoshida humanized anti-human CCR8 antibodies exhibited weaker ADCC activity (10-20 fold lower ADCC activity) than the anti-CCR 8 antibodies afuc.ab5.h13l1, afuc.ab 4.h12l3. The commercial antibodies murine anti-human CCR8 mAb433H (BD Biosciences) and murine anti-human CCR8 mAb L263G8 (Biolegend), which comprises a murine Fc domain, as expected, indicate no ADCC activity, as the assay employed in this example is primarily related to antibodies comprising a human Fc domain.

In an assay related to antibodies comprising a murine Fc region but anti-human CCR8 activity, the ADCC activity of murine anti-human CCR8 mAb 433H (BD Biosciences) and murine anti-human CCR8 mAb L263G8 (Biolegend) was tested, i.e., using the Jurkat/mFcgR4 stable cell line as effector cells and CHO/hCCR8 as target cells. Human CCR8 (hCCR 8) was used to mimic a human clinical setting. Specifically, the assay consists of a genetically engineered Jurkat T cell line expressing the mouse FcgRIV receptor and a luciferase reporter driven by an NFAT response element (NFAT-RE). When co-cultured with target cells and related antibodies, 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: RPMI1640 without phenol red, supplemented with 4% low IgG; 96-well white flat bottom polystyrene TC treated microwell plates, corning #3601; bio-Glo reagent. Measurement procedure: 25. Mu.L/well of diluted antibody (3X was prepared starting at 30ug/mL and serially diluted at 1:4 for 10 spots) was added to the assay buffer. Resuspending the target cells in assay buffer, adjusting the final density to 1x10 ⁶ /ml; dispensing 25 μl of cells into each well, the target cell density being 25,000 cells/well; plates were incubated for 20 min at room temperature. mu.L/well Jurkat/mFcgRIV cells (at 5X 10) ⁶ Individual cells/mL) was added to each well to obtain an effector cell density of 125,000 cells/well; the cells in the reservoir are periodically remixed during this process to prevent the cells from settling to the bottom. The assay plate was covered with a lid and incubated at 37℃with 5% CO ₂ Plates were incubated in the incubator for 16 hours. The plates were not stacked in the incubator. The assay plate was removed from the incubator and equilibrated to ambient temperature for 15 minutes. Using a multichannel pipette, 75 μl Bio-Glo was performed ^TM Reagents were added to the assay plate taking care not to generate bubbles. Plates were incubated at room temperature15 minutes. Luminescence was measured using an EnSight light plate reader. The mIgG2a isotypes, hIgG1 and ratIgG2b were tested as controls. Human CCR8 (hCCR 8) was used to mimic a human clinical setting. As can be seen from the data, each of the tested anti-hCR 8 mAb-L263G8 (BioLegend) and 433H (BD Biosciences), showed high fold induction results at an antibody concentration level of about 1nM, showing fold induction results exceeding about 10 and about 12, respectively. The high fold induction of each of these antibodies reached a plateau at an antibody concentration level of about 40 nM-fold induction values of about 11 and 13, respectively. The results of these experiments are provided in fig. 11D.

The following table also provides activity data from these studies. In summary, each of the antibodies studied, whether or not having a humanized or murine Fc region, exhibited ADCC activity in assays where the antibody isotype was species matched to the relevant effector reporter cell.

* Inactivity = determination conditions are independent of specific Ab isoforms

(d) ADCC enhancement against Treg cells

To induce CCR8 expression on Treg cells from human Peripheral Blood Mononuclear Cells (PBMCs), 10 will be ⁷ Intraperitoneal transfer of personal PBMC to NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl Mice (JAX) were SzJ (NSG) and spleens were collected 2-3 weeks after transfer. Human T cells were enriched from single cell suspensions of NSG splenocytes using a mouse lineage cell depletion kit (Miltenyi Biotec) and primary NK cells were individually enriched from human PBMC using a 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, then primary NK cells were added at a 2:1 effector to target ratio. After overnight incubation at 37 ℃, cells, surface staining and intracellular staining were collected using the eBioscience Foxp 3/transcription factor staining buffer set (ThermoFisher Scientific) according to the manufacturer's protocol. Antibodies used to define the T cell population were from BD Biosci CD45 (HI 30), CD3 (SK 7), CD8 (RPA-T8) and CD14 (63D 3) from the sources, CD4 (RPA-T4) from the BioLegend and FOXP3 (236A/E7) from ThermoFisher Scientific. A CountBright absolute count bead (ThermoFisher Scientific) was added to each sample prior to collection. Flow cytometry was performed on Fortessa X-20 (BD Biosciences) and analyzed with FlowJo software (BD Biosciences, version 10.5.3). Absolute cell counts were calculated according to the manufacturer's protocol.

ADCC activity against Treg cells was measured by calculating the ratio of recovered regulatory T cells to recovered CD8 cells (Treg/CD 8) or conventional CD 4T cells to recovered CD 8T cells (CD 4conv/CD 8). The number of recovered CD 8T cells was similar across all concentrations of CCR8 mAb and the isotype control mAb tested ("gd.afuc"). As depicted in fig. 12A to 12D, the defucosylated CCR8 antibodies afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 and the fucosylated CCR8 antibodies hu.ab5.h13l1 and hu.ab4.h12l3 selectively mediate ADCC activity, wherein depletion of tregs from in vivo Mixed Lymphocyte Reaction (MLR) activated human PBMCs (fig. 12A and 12C) is increased as compared to conventional CD 4T cells (fig. 12B and 12D), and wherein the defucosylated variants mediate increased ADCC activity. Low levels of defucosylated anti-CCR 8 mediated ADCC were observed in conventional CD 4T cells, consistent with moderate upregulation of CCR8 on conventional CD 4T cells following transfer to NSG mice (data not shown).

Other data indicate that defucosylated CCR8 mabs afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 exhibit selective ADCC against tregs of RCC tumors. Briefly, human isolated tumor cells (renal cell carcinoma, discovery Life Sciences) were thawed according to the protocol of the supplier. Primary NK cells were enriched from human PBMCs using a human NK cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Human dissociated tumor cells were incubated with 0.001-1ug/mL CCR8mAb for 30 minutes at room temperature, then primary NK cells were added at a 2:1 effector to target ratio. After overnight incubation at 37 ℃, cells were treated as described above to determine absolute cell counts of CD8, conventional CD4 and regulatory T cells.

As depicted in fig. 13A to 13D, the defucosylated CCR8 antibodies afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 and the fucosylated CCR8 antibodies hu.ab5.h13l1 and hu.ab4.h12l3 selectively mediate ADCC activity, wherein Treg consumption from human dissociated tumor cells from RCCs (fig. 13A and 3C) is increased as compared to conventional CD 4T cells (fig. 13B and 13D), and wherein the defucosylated variants mediate increased ADCC activity. Consistent with the absence of CCR8 staining on conventional CD 4T cells within the tumor, CCR8 mAb-mediated ADCC activity was not observed on conventional CD 4T cells, indicating CCR8 mAb-mediated ADCC selectivity against intratumoral regulatory T cells.

(e) ADCP enhancement

There are conflicting reports of the effects of defucosylation on ADCP. See, e.g., herter et al J Immunol (2014) 192:2252-2260; silence et al, mAbs (2013) 6:523-532; and Kwiatkowski et al, mAbs (2020) 12:e1803645 (page 9). Furthermore, the G236A.I332E mutant has previously been shown to increase ADCP via enhanced FcgR2a binding. See Richards et al, molecular Cancer Therapeutics (2008) 7:2517-2527. Thus, fucosylated and defucosylated hIgG1.G236A.I332E Fc versions of hu.Ab5.H13L1 and hu.Ab4.H12L3 were prepared to investigate whether ADCP activity was observed. The following table provides the mutant hIgG1 constant domain of g236a.i332e, underlined from the mutant differences from the normal hIgG1 constant domain, and the full length heavy chain sequences of Ab4 and Ab5 g236a.i332e variants.

^a The full length sequence of the light chain of the Ab 5G 236A.I332E variant corresponds to hu.Ab5.L1 (SEQ ID NO: 56). ^b The full length sequence of the light chain of the Ab 4G 236A.I332E variant corresponds to hu.Ab4.L3 (SEQ ID NO: 58).

First from Gen with known FcgRIIa and FcgRIIIa genotype information using the EasySep human monocyte enrichment kit (Stem cell technology)Isolation of human CD14 from blood of entech donor ⁺ Monocytes. Purified CD14 ⁺ Monocytes differentiated into macrophages in RPMI+10% FBS containing 100ng/mL hM-CSF (PeproTech, inc.) for 5 days. hIL-10 (PeproTech, inc.) was then added at 50ng/mL to polarize macrophages for 24 hours prior to ADCP assay. The nuchight Red transfected CHO/hccr8.Gna15 target cells were pre-incubated with anti-CCR 8 antibody in the presence of 20mg/mL non-specific human IgG for 20 min. The above cell mixture was then added to macrophage (effector cell) plates at a ratio of E:1. After placing the plates inside an intucyte Zoom instrument (Essen Biosciences; ann Harbor, MI), cell images were acquired every one hour using bright field and red laser settings for a period of 6 hours. The red blood cell count (remaining target cells) in each well was normalized by the number of macrophages in the same well using instrument embedded software. ADCP activity was calculated as the percentage of reduction in normalized red blood cell count in each sample compared to the negative control in the presence of isotype control antibody. ADCP activity was then plotted as a function of antibody concentration and data fitted to an asymmetric sigmoid four parameter logic (4 PL) model using Prism. EC of each antibody ₅₀ The value was determined to be the concentration that reached 50% target cell killing.

As depicted in fig. 14A to 14D, the defucosylated anti-CCR 8 antibodies afuc.hu.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 were on CD14 from four different donors with fcgliia (H131R)/fcgliriiia (V158F) genotypes as compared to the fucosylated antibodies hu.ab5.h13l1 and hu.ab4.h12l3 ⁺ Monocyte-derived macrophages exhibited enhanced ADCP activity, genotypes HR/FF (FIG. 14A), RR/FF (FIG. 14B), HR/VF (FIG. 14C) and RR/VF (FIG. 14D). The results indicate that in the case of CCR 8-targeting antibodies, defucosylation results in enhanced ADCP.

The defucosylated anti-CCR 8 antibodies afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 also showed enhanced ADCP activity (3-4 fold increase) compared to the Yoshida humanized anti-human CCR8 antibody (fig. 14E).

The following table also provides activity data from these studies.

n.d. =undetermined

In addition, as depicted in figures 15A to 15D, the defucosylated anti-CCR 8 antibody afuc.hu.ab5.h13l1.g236a.i332e was found to be on CD14 from four different donors with fcgnriia (H131R)/fcgnriia (V158F) genotypes as compared to fcgnriia enhanced g236a.i332e variant afuc.hu.ab5.h13l1.g236a.i332e ⁺ Monocyte-derived macrophages showed similar improved ADCP activity, with genotypes HR/FF (FIG. 15A), RR/FF (FIG. 15B), HR/VF (FIG. 15C) and RR/VF (FIG. 15D). In view of the previous report, the incorporation of G236A.I332E can mediate significantly higher levels of ADCP despite the use of anti-EPCAM mAbs, and the similarity in ADCP activity between the defucosylated hIgG1 variant and the G236A.I332E mutant is surprising. See Richards et al, molecular Cancer Therapeutics (2008) 7:2517-2527.

(f) Physical characterization of Ab4 and Ab5 antibodies

The solubility, viscosity and behaviour under thermal stress (shelf life stability) of afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 were evaluated at high concentrations. As shown in the following table, both antibodies exhibit favorable chemical and physical properties useful in their manufacture and formulation, exhibit low aggregation, good solubility, low viscosity, and good shelf life stability.

Thermal stress conditions: antibody samples were incubated at 150mg/mL for 2 weeks at 40℃in 200mM arginine succinate, pH 5.5. Control samples were stored at-70 ℃. The control samples and stress samples were evaluated for dimensional variants using Size Exclusion Chromatography (SEC). SEC employs Waters Acquity UPLC H-Class (Waters, milford, mass.) andUP-SW3000 column, 4.6X300 mm (Tosoh Biosciences, king of Prusia, pa.)And (3) row. The mobile phase was 0.2M potassium phosphate buffer (pH 6.2) containing 0.25M potassium chloride. The separation was carried out at ambient temperature at a flow rate of 0.3mL/min and the column effluent was monitored at a UV wavelength of 280 nm.

Solubility in Phosphate Buffered Saline (PBS): antibodies were formulated at 150mg/mL in 200mM arginine succinate, pH 5.5, and dialyzed at 37 ℃ into PBS, pH 7.4 for 24 hours to determine their solubility. After dialysis, the samples were visually inspected for visible particles and turbidity was determined using a SpectraMax M2/M2e plate reader (Molecular Devices, san Jose, CA) to measure absorbance at 340, 345, 350, 355 and 360 nm. The values at 5 wavelengths are averaged to obtain the final solubility value.

Viscosity measurement: the viscosity of the samples at 100, 150 and 180mg/mL in 200mM arginine succinate, pH 5.5 was determined using an AR G2 rheometer (TA Instruments, new Castle, DE). A 20 mm cone geometry was used and measurements were made at a constant shear rate of the reciprocal of 1,000 seconds for 2.5 minutes.

(g) Epitope mapping of hu.Ab5.H13L1

To map the epitope of fucosylated hu.ab5.h13l1, binding to alanine point mutations in human CCR8 was analyzed by flow cytometry.

Constructs encoding single alanine point mutations in hCCR8 with C-terminal FLAG tags at positions 2-24 were generated. HEK 293 cells were transfected with constructs encoding mutant hCCR8 or with mock constructs using transIT X2 (reagent: dna=3:1) for 24 hours and surface stained with huCCR8 antibody hu.ab5.h13l1 (hIgG 1), then permeabilized and then FITC-anti-Flag (Sigma F4049).

As shown in fig. 16A, hu.ab5.h13l1 did not bind to D2A, Y3A, L a and D6A, indicating that the epitope includes at least one amino acid residue of the DYTLD region at the N-terminus of human CCR 8.

(h) Epitope mapping of hu.Ab4.H12L3

For epitope mapping of fucosylated hu.ab4.h12l3, binding to the chimeric form of human CCR8 was analyzed by flow cytometry.

A construct encoding human CCR8.CCR5 chimera (N-term 1 (amino acid residues 1 to 23 of human CCR 8), N-term2 (amino acid residues 1 to 36 of human CCR 8), ECL1 (amino acid residues 91 to 104 of human CCR 8), ECL2 (amino acid residues 172 to 193 of human CCR 8) and ECL3 (amino acid residues 264 to 271 of human CCR 8), wherein the different extracellular regions of CCR8 are replaced by the corresponding regions of CCR5 bearing a C-terminal FLAG tag. ECL is defined as extracellular loop. 293 cells were transfected with constructs encoding mutant hCCR8 or mock constructs using transIT X2 (reagent: dna=3:1) for 24 hours and surface stained with huCCR8-ab4.h12l3.higg1, followed by immobilization and then FITC-anti-Flag (Sigma F4049).

As shown in fig. 16B, hu.ab4.h12l3 did not bind to ECL1 and ECL2 chimeras, indicating that the epitope includes at least one amino acid residue of ECL1 and ECL2 regions of CCR8.

Example 5 mice in mouse colon cancer model CT26 replace anti-CCR 8 monoclonal antibodies (mAbs)

(a) Treg cell depletion

To demonstrate the ability of anti-CCR 8 abs to deplete tumor-infiltrating Treg cells in vivo, BALB/c mice with established CT26 tumors were treated with mice instead of anti-CCR 8 mabs, and the proportion of Treg cells, conventional CD 4T cells and CD 8T cells in the tumors, spleen and leukocytes in tumor draining lymph nodes was analyzed by flow cytometry.

The light and heavy chain CDR regions, light and heavy variable regions, and full length heavy and light chain sequences of the mouse replacement anti-CCR 8 mAb are provided in the table below.

CT26 tumor cells were harvested in the logarithmic growth phase and resuspended in HBSS containing matrigel at a 1:1 ratio. 10 ten thousand CT26 cells in 100 microliters of HBSS+ matrigel were inoculated subcutaneously on the flank of BALB/c mice. Tumors were monitored until they were established and reached 130 to 230mm ³ Is a mean tumor volume of (3). Mice were then randomized into treatment groups. Treatment with mice instead of anti-CCR 8 (mIgG 2 a) or anti-gp 120 isotype control antibodies was performed at 0.003mg/kg to 5mg/kg of anti-CCR 8 Ab in histidine buffer #08: a dose of 20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20 (Tween-20), pH5.5 was administered intravenously.

Three days later, mice were sacrificed and tumors, spleens, and tumor draining lymph nodes were obtained for analysis. To generate a single cell suspension, tumors were minced and digested with stirring at 37℃for 30 min in RPMI-1640 medium containing 1% Fetal Bovine Serum (FBS), 0.2U/mL Liberase DL (Sigma) and 0.2mg/mL DNaseI (Sigma). Tumor cells were passed through a 100mm filter and washed with RPMI-1640 medium containing 10% FBS. Single cell suspensions were surface stained with fluorescently labeled anti-CD 45, anti-CD 4 and anti-CD 8 antibodies for 15 minutes at 4℃and stained intracellular with fluorescently labeled anti-Foxp 3 using the eBioscience Foxp 3/transcription factor staining buffer kit (Thermo Fisher) according to the manufacturer's protocol. Flow cytometry was performed on Fortessa X-20 (BD Biosciences) or FACSymphony (BD Biosciences), and analyzed with FlowJo software (BD Biosciences).

Fig. 17A to 17I depict the dose-dependent depletion of Treg cells (plotted as fraction of Treg cells in cd45+ leukocytes) in tumors but not in spleen or tumor draining lymph nodes (fig. 17A to 17C) of CT26 tumor-bearing mice relative to isotype-treated groups. A decrease in the proportion of normal CD 4T cells ((fig. 17D to 17F)) or CD 8T cells (fig. 17G to 17I) was observed when observed with anti-CCR 8 treatment relative to isotype control group. These observations demonstrate specificity against CCR8 mediated intratumoral Treg cell depletion.

(b) Tumor growth inhibition

To demonstrate the tumor growth inhibition following CCR 8-mediated depletion of tumor-infiltrating Treg cells in vivo, BALB/c mice with established CT26 tumors were treated with mice instead of anti-CCR 8 mAb and the growth of the tumors was monitored over time.

CT26 tumor cells were harvested in the logarithmic growth phase and resuspended in HBSS containing matrigel at a 1:1 ratio. 10 ten thousand CT26 cells in 100 microliters of HBSS+ matrigel were inoculated subcutaneously on the flank of BALB/c mice. Tumors were monitored until they were established and reached 130 to 230mm ³ Is a mean tumor volume of (3). Mice were then randomized into treatment groups. Mice were treated intravenously with one or two weekly doses (first intravenous injection followed by intraperitoneal injection) of 0.1mg/kg of anti-CCR 8 (mIgG 2 a), 0.1mg/kg of anti-CD 25 antibody (clone PC61 mIgG2 a) or anti-gp 120 isotype control Ab (in histidine buffer #08:20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20 (Tween-20), ph 5.5). Tumor volumes were measured in two dimensions (length and width) using an Ultra Cal-IV caliper and volumes were calculated using the following formula: tumor size (mm) ³ ) = (length x width ² )x 0.5。

Fig. 18A to 18D depict tumor volumes of individual mice (grey line) and treatment groups (fitted curve, black line) over time. Effective tumor growth inhibition was observed using mice administered as a single dose (fig. 18B) or twice weekly (fig. 18C) in a CT26 colon cancer model instead of the anti-CCR 8 mAb. Both treatment regimens resulted in complete tumor regression in 8/9 mice. Treatment with anti-CCR 8mAb was more effective than anti-CD 25 Ab treatment (fig. 18D), which resulted in tumor regression in 3/9 mice. Isotype control mAb (anti-gp 120) was used (fig. 18A).

Example 6 comparison of Effect ability and Effect-disabled mice substituting for anti-CCR 8 Ab

To assess whether anti-CCR 8 Ab treatment works primarily by promoting ADCC and ADCP mediated Treg cell depletion or by inhibiting ligand dependent CCR8 activation, we compared ligand blocking effect-null mIgG2a.lalapg mutants of ligand blocking effect-competent mIgG2a mice instead of anti-CCR 8 Ab with the same anti-CCR 8 clones in CT26 tumor model.

CT26 tumor cells were harvested in the logarithmic growth phase and resuspended in HBSS containing matrigel at a 1:1 ratio. 10 ten thousand CT26 cells in 100 microliters of HBSS+ matrigel were inoculated subcutaneously on the flank of BALB/c mice. In the first treatment group, mice were administered at a dose of 5mg/kg twice weekly (first dose of intravenous injection, all subsequent doses of intraperitoneal injection) starting on the day of tumor inoculation starting at a dose of 5mg/kg instead of anti-CCR 8mAb (mIgG 2 a) or of the effector-disabled mIgG2 a.lapg mutant anti-CCR 8 or anti-gp 120 isotype control mAb, in histidine buffer #08:20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20 (Tween-20), pH 5.5. For the second treatment group, tumors were monitored until they were established and reached 130 to 230mm ³ The mice were then randomized into treatment groups and treated with anti-CCR 8 (mIgG 2 a) or effector-disabled mIgG2a.lalapg mutant anti-CCR 8 Ab twice weekly at a dose of 5mg/kg (first intravenous injection, all subsequent doses intraperitoneal injection) with histidine buffer #08:20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20 (Tween-20), pH 5.5. Tumor volumes were measured in two dimensions (length and width) using an Ultra Cal-IV caliper and volumes were calculated using the following formula: tumor size (mm) ³ ) = (length x width ² ) x 0.5. Animal body weight was measured using an Adventura Pro AV812 balance (Ohaus Corporation).

Fig. 19A to 19E depict tumor volumes of individual mice (grey line) and treatment groups (fitted curve, black line) over time. Tumor growth inhibition was observed with effector-competent mIgG2a mice instead of anti-CCR 8mAb (fig. 19B and 19D), but not with ligand blocking-effect-disabled miggg 2a.lalapg mutant anti-CCR 8mAb (fig. 19C and 19E). The mIgG2a anti-CCR 8 antibodies were effective at tumor vaccination (fig. 19B) or in established tumors (fig. 19D). These findings indicate that blocking ligand binding to CCR8 receptor is insufficient to mediate tumor growth inhibition following anti-CCR 8mAb treatment. Isotype control mAb (anti-gp 120) was used (fig. 19A).

Example 7 Combined efficacy of anti-CCR 8 and anti-PDL 1 mAb treatment

To assess the potential of anti-CCR 8 mAb and checkpoint inhibition in combination to improve tumor growth inhibition, mice with established EMT6 tumors were treated with anti-CCR 8 and anti-PDL 1 mAb alone or in combination.

EMT6 tumor cells were harvested in log phase and resuspended in HBSS containing matrigel at a 1:1 ratio. 10 ten thousand EMT6 cells in 100 microliters of HBSS+ matrigel were inoculated subcutaneously in the 5 th mammary fat pad of BALB/c mice. Tumors were monitored until they were established and reached 130 to 230mm ³ Is a mean tumor volume of (3). Mice were then randomized into treatment groups. Mice were administered intravenously at a single dose of 0.1mg/kg in place of anti-CCR 8 (mIgG 2 a) or isotype control antibodies. The first dose of the effect-disabled anti-PDL 1 (mIgG2a.LALAPG) Ab is 10mg/kg, the intravenous administration is carried out, the subsequent dose is 5mg/kg, and the intraperitoneal administration is carried out twice a week. Antibody was buffered in histidine buffer #08:20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20 (Tween-20), pH 5.5. Tumor volumes and body weights were measured twice weekly. Tumor volumes were measured in two dimensions (length and width) using an Ultra Cal-IV caliper and volumes were calculated using the following formula: tumor size (mm) ³ ) = (length x width ² ) x 0.5. Animal body weight was measured using an Adventura Pro AV812 balance (Ohaus Corporation).

Fig. 20A to 20D depict tumor volumes of individual mice (grey line) and treatment groups (fitted curve, black line) over time. However, the replacement of anti-CCR 8 and anti-PDL 1 mabs by mice as monotherapy (fig. 20B to 20C) caused partial tumor growth inhibition, but the combination of both mabs (fig. 20D) unexpectedly caused complete tumor rejection. Isotype control mAb (anti-gp 120) was used (fig. 20A).

Example 8 Ab1-Ab 3H 1L1 variant and anti-CCR 8 antibody comparator(i) Ab1-Ab 3H 1L1 variants

The light and heavy chain CDR regions, light and heavy chain variable regions, and full length heavy and light chain sequences of Ab1-Ab 3H 1L1 variants are provided in the following tables.

(ii) anti-CCR 8 antibody comparison

The full length heavy and light chain sequences of the Yoshida humanized anti-human CCR8 antibodies studied herein are disclosed in the US statement filed during the USSN 16/183,216 application at 10-30 of 2019 (published as US 2019/007173, later granted to US10,550,191). The light chain variable, light chain constant, heavy chain variable and heavy chain constant regions of this same antibody are disclosed in PCT application publication No. WO2020138489 as sequences 59, 52, 41 and 53.Yoshida antibodies were expressed as human hig 1 antibodies (i.e., with a human Fc region). Commercially available murine anti-human CCR8 antibody 433H (BD Biosciences) and murine anti-human CCR8 antibody L263G8 (Biolegend) were purchased for these studies. 433H (BD Biosciences) and L263G8 (Biolegend) are mouse monoclonal antibodies comprising the Fc region of the mouse IgG2a isotype. See also Mutalithas et al, clinical & Experimental Allergy (2010) 40:1175 (433H,BD Biosciences), mitson-Salazar et al, J.allergy Clin. Immunol. (2016) 907-918 (L263G 8, bioleged) and www.labome.com/review/gene/human/CCR8-anti body. Html (L263G 8, bioleged).

Example 9: terminal lysine variants of Ab1 to Ab5

Additional Fc variants of the anti-CCR 8 antibodies of the present disclosure are contemplated, wherein the C-terminus of the heavy chain of the parent antibody is a shortened C-terminus, wherein the C-terminal lysine has been removed, resulting in a shortened C-terminal end of PG. Terminal lysine variants of Ab1 to Ab-5 are provided in Table P below.

The full length sequence of the light chain of the Ab5 terminal lysine variant corresponds to hu.Ab5.L1 (SEQ ID NO: 56).

The full length sequence of the light chain of the Ab4 terminal lysine variant corresponds to hu.Ab4.L3 (SEQ ID NO: 58).

The full length sequence of the light chain of the Ab 5G 236A.I332E terminal lysine variant corresponds to hu.Ab5.L1 (SEQ ID NO: 56).

The full length sequence of the light chain of the Ab 4G 236A.I332E terminal lysine variant corresponds to hu.Ab4.L3 (SEQ ID NO: 58).

The full length sequence of the light chain of the Ab1 terminal lysine variant corresponds to hu.Ab1.L1 (SEQ ID NO: 100).

The full length sequence of the light chain of the Ab2 terminal lysine variant corresponds to hu.Ab2.L1 (SEQ ID NO: 102).

The full length sequence of the light chain of the Ab3 terminal lysine variant corresponds to hu.Ab3.L1 (SEQ ID NO: 104).

Example 10 testing of serum concentration of anti-CCR 8 mAb and ADA in cynomolgus monkey

The present study used anti-CCR 8 antibodies afuc.hu.ab5.h13l1, afuc.hu.ab4.h12l3 and control anti-gD. Three male cynomolgus monkeys-control were present in each of the three dose groups: designated 1001, 1002, 1003; afuc.hu.ab5.h13l1: designated 2001, 2002, 2003; afuc.hu.ab4.h12l3: designated 3001, 3002, 3003. Each cynomolgus monkey was given a single 10mg/kg IV bolus of anti-gD or tested for anti-CCR 8 mAb. Blood samples were collected for analysis at 0.25, 2 and 6 hours; the concentrations of anti-gD (control) and anti-CCR 8 antibodies in serum were determined using a qualified ELISA assay 1, 2, 7, 14, 21, 28 and 35 days after dosing. The lower limit of quantification (LLOQ) was determined to be 0.015625. Mu.g/mL. PK parameters were estimated using non-compartmental analysis consistent with intravenous bolus administration using Phoenix 1.4 (WinNonlin pharmacokinetic software version 6.4) (Certara, usa). Blood samples for anti-drug antibody (ADA) analysis were collected before dosing and on days 1, 8, 15, 22, 29 and 36 and serum was analyzed for antibodies to the test items using a qualified ELISA assay.

The serum concentration profiles of anti-gD, afuc.hu.ab5.h13l1 or afuc.hu.ab4.h12l3 in cynomolgus monkeys after a single 10mg/kg IV dose administration are shown in fig. 21. Systemic exposure was found to be comparable between the anti-gD group and the afuc.hu.ab5.h13l1 group, exhibiting sustained serum concentration levels during 35 days post-dosing, with average clearance of 3.96±0.412 mL/day/kg and 4.38±0.291 mL/day/kg, respectively. In contrast, afuc.hu.ab4.h12l3 showed lower exposure during the same 35 days post-dose, with an average clearance of 9.00±1.01 mL/day/kg. As shown by afuc.hu.ab5.h13l1, maintaining serum concentration levels over longer periods of time, the clearance rate is slower, which is expected to lead to more sustained target participation, which may translate into better anti-cancer activity and lower dosing frequency.

The observed systemic exposure differences for afuc.hu.ab4.h12l3 compared to the anti-gD and afuc.hu.ab5.h13l1 groups can be partly explained by the presence of anti-drug antibodies (ADA) in the afuc.hu.ab4.h12l3 treated groups at a later point in time. For example, animals 1001, 1002, and 1003 given anti-gD are negative for the presence of ADA. Animals 2001 were positive for ADA following administration of afuc.hu.ab5.h13l1, but the presence of ADA appeared to have no effect on exposure when compared to the other two animals administered afuc.hu.ab5.h13l1, negative for ADA (numbers 2002 and 2003). After administration of afuc.hu.ab4.h12l3, animals 3002 and 3003 were found to be ADA positive, and the presence of ADA appeared to have an effect on systemic exposure compared to ADA negative animals 3001.

Example 11 monitoring levels of CCR8+T-reg cells in cynomolgus monkey

The present study used anti-CCR 8 antibodies afuc.hu.ab5.h13l1, afuc.hu.ab4.h12l3 and control anti-gD. Three male cynomolgus monkeys-control group were present in each of the three dose groups: designated 1001, 1002, 1003; afuc.hu.ab5.h13l1 group 2: designated 2001, 2002, 2003; afuc.hu.ab4.h12l3 group 3: designated 3001, 3002, 3003. Blood was collected from each animal on day 1 prior to dosing ("pre-study") and 0 hours on day 1 ("pre-dosing"). Each animal was then given a single dose of 10mg/kg of defucosylated anti-gD (control group), afuc.hu.ab5.h13l1 (group 2) or afuc.hu.ab4.h12l3 (group 3) via intravenous injection. Starting on day 1, blood containing the initial dose of test CCR8mAb was collected at the following time points after dosing: 6. 24, 48, 168, 336, 504, 668 and 840 hours, and the following treatments were performed prior to flow cytometry analysis: (i) a blood sample not labeled with any of the test CCR8 mabs ("unlabeled"), (ii) a blood sample further labeled with a saturated concentration of afuc.hu.ab5.h13l1, and (iii) a blood sample further labeled with a saturated concentration of afuc.hu.ab4.h12l3. Each of the unlabeled and labeled samples was then treated with a labeled goat anti-human IgG antibody that detects the binding of the test anti-CCR 8mAb to cynoscr 8 and analyzed by flow cytometry.

Specific antibodies to phenotypically labeled antigens are used to identify T cell subsets. Specifically, T regulatory (T-reg) cells were identified as CD3+CD4+Foxp3+ cells. Drug-binding CCR8+ T-reg cells were identified using unlabeled blood samples.

As observed in the unlabeled samples, neither test anti-CCR 8 mAb significantly reduced the total absolute T-reg cell count in whole blood for up to 840 hours after administration. See fig. 22A to 22C. Neither test anti-CCR 8 mAb significantly reduced the total number of lymphocytes in whole blood for up to 840 hours after dosing (data not shown).

As previously described, afuc.hu.ab5.h13l1 and afuc.hu.ab4.h12l3 both bind to CCR8, afuc.hu.ab4.h12l3 and afuc.hu.ab5.h13l1 both act as respective non-competitive CCR8 binding agents, and afuc.hu.ab4.h12l3 has slightly higher affinity for human and cynomolgus monkey CCR 8. See, e.g., fig. 16A-16B, the affinity Kd (nM) data is provided in table G3. Afuc.hu.Ab4.H12L3 also has a tendency to improve ADA formation at a later time point.

(see example 10).

As shown in fig. 23A-23C, flow cytometry analysis of unlabeled blood from cynomolgus monkeys initially treated with control (group 1) indicated that total ccr8+ T-reg cells were not regulated. In addition, flow cytometry on the spiked blood (i.e., blood spiked initially with control (group 1) followed by saturated concentration of afuc.hu.ab5.h13l1, or blood spiked initially with control (group 1) followed by saturated concentration of afuc.hu.ab 4.h12l3) also had very little effect on the total number of ccr8+t-reg cells. Relative percentages refer to the percentage of ccr8+ T-reg cells detected by each test anti-CCR 8 mAb. The relative percentage detected for the spiked afuc.hu.ab4.h12l3 samples was higher due to the slightly higher affinity of afuc.hu.ab4.h12l3 compared to afuc.hu.ab5.h13l1.

With respect to group 3, as shown in fig. 23D-23F, flow cytometry of blood in each of the three animals (i) initially treated with afuc.hu.ab4.h12l3 ("unlabeled"), (ii) initially treated with afuc.hu.ab4.h12l3 followed by afuc.hu.ab5.h13l1-labeled blood, or (iii) initially treated with afuc.hu.ab4.h12l3 followed by afuc.hu.ab4.h12l3 showed a reduction in ccr8+ T-reg cells up to 168 hours post-administration. Partial recovery of ccr8+ Treg cell frequency was noted in two of the animals starting from 336 hours post-dose, probably due to the increased presence of ADA against afuc.hu.ab4.h12l3.

With respect to group 2, as in fig. 23G-23I, (I) blood initially treated with afuc.hu.ab5.h13l1 ("unlabeled"), (ii) blood initially treated with afuc.hu.ab5.h13l1 and then labeled with saturated concentrations of afuc.hu.ab5.h13l1, or (iii) blood initially treated with afuc.hu.ab5.h13l1 and then labeled with saturated concentrations of afuc.hu.ab4.h12l3 showed a reduction in ccr8+ T-reg cells in animals 2002 and 2003.

Animals in groups 2 and 3 showed little to no effect on total Treg cell count (fig. 22A to 22C), but showed a decrease in peripheral blood ccr8+ T-reg cell numbers after administration (fig. 23D to 23I), either labeled or unlabeled, consistent with the proposed mechanism of action (see fig. 2A).

Other embodiments

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, the illustration and example should not be construed as limiting the scope of the disclosure. All patent and scientific literature cited herein is expressly incorporated by reference in its entirety.

Claims

1. A monoclonal antibody that binds to C-C motif chemokine receptor 8 (CCR 8), wherein the antibody comprises: 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.

2. The antibody of claim 1, which binds to CCR8 independent of sulfation of CCR 8.

3. The antibody of claim 1 or 2, wherein the antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID No. 106.

4. The antibody of any one of claims 1 to 3, comprising a sequence selected from the group consisting of:

(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: 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 VH sequence as defined in (a) and a VL sequence as defined in (b).

5. The antibody of any one of claims 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.

6. The antibody of any one of claims 1 to 5, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

7. The antibody of any one of claims 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.

8. The antibody of any one of claims 1-7, wherein VL comprises a V4M mutation, a P43A mutation, an F46L mutation, a C90Q mutation, or a combination thereof.

9. The antibody of any one of claims 1 to 8, wherein VH comprises a G49S mutation, a K71R mutation, an S73N mutation, or a combination thereof.

10. The antibody of any one of claims 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID No. 55 and the light chain amino acid sequence of SEQ ID No. 56.

11. The antibody of any one of claims 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID No. 60 and the light chain amino acid sequence of SEQ ID No. 56.

12. The antibody according to any one of claims 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID No. 111 and the light chain amino acid sequence of SEQ ID No. 56.

13. The antibody according to any one of claims 1 to 9, comprising the heavy chain amino acid sequence of SEQ ID No. 113 and the light chain amino acid sequence of SEQ ID No. 56.

14. A monoclonal antibody that binds to CCR8, the monoclonal antibody 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.

15. A monoclonal antibody that binds to CCR8, comprising the VH sequence of SEQ ID No. 47 and the VL sequence of SEQ ID No. 48.

16. A monoclonal antibody that binds to CCR8, wherein the antibody comprises: a heavy chain variable domain (VH) comprising: (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; and

a light chain variable domain (VL) comprising: (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.

17. The antibody of claim 16, which binds to CCR8 independent of sulfation of CCR 8.

18. The antibody of claim 16 or 17, wherein the antibody binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID No. 106.

19. The antibody of any one of claims 16 to 18, comprising a sequence selected from the group consisting of:

(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: 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: SEQ ID NOS.22 to 25; and

(c) A VH sequence as defined in (a) and a VL sequence as defined in (b).

20. The antibody of any one of claims 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.

21. The antibody of any one of claims 16 to 20, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

22. The antibody of any one of claims 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.

23. The antibody of any one of claims 16 to 22, wherein VL comprises a Y2I mutation.

24. The antibody of any one of claims 16 to 23, wherein VH comprises an S73N mutation, a V78L mutation, a T76N mutation, an F91Y mutation, and a P105Q mutation, or a combination thereof.

25. The antibody of any one of claims 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID No. 57 and the light chain amino acid sequence of SEQ ID No. 58.

26. The antibody of any one of claims 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID No. 61 and the light chain amino acid sequence of SEQ ID No. 58.

27. The antibody of any one of claims 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID No. 112 and the light chain amino acid sequence of SEQ ID No. 58.

28. The antibody of any one of claims 16 to 24, comprising the heavy chain amino acid sequence of SEQ ID No. 114 and the light chain amino acid sequence of SEQ ID No. 58.

29. A monoclonal antibody that binds to CCR8, the monoclonal antibody 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.

30. 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.

31. A monoclonal antibody that binds to CCR8, wherein the antibody comprises: a heavy chain variable domain (VH) comprising: (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, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 85; and a light chain variable domain (VL) comprising: (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.

32. The antibody of claim 31, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

33. An antibody according to claim 31 or 32, comprising the VH sequence of SEQ ID No. 95 and the VL sequence of SEQ ID No. 94.

34. The antibody of any one of claims 31 to 33, comprising the heavy chain amino acid sequence of SEQ ID No. 101 and the light chain amino acid sequence of SEQ ID No. 100.

35. The antibody of any one of claims 31 to 33, comprising the heavy chain amino acid sequence of SEQ ID No. 115 and the light chain amino acid sequence of SEQ ID No. 100.

36. A monoclonal antibody that binds to CCR8, wherein the antibody comprises: 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.

37. The antibody of claim 36, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

38. An antibody according to claim 36 or 37, comprising the VH sequence of SEQ ID No. 97 and the VL sequence of SEQ ID No. 96.

39. The antibody of any one of claims 36 to 38, comprising the heavy chain amino acid sequence of SEQ ID No. 103 and the light chain amino acid sequence of SEQ ID No. 102.

40. The antibody of any one of claims 36 to 38, comprising the heavy chain amino acid sequence of SEQ ID No. 116 and the light chain amino acid sequence of SEQ ID No. 102.

41. A monoclonal antibody that binds to CCR8, wherein the antibody comprises: 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.

42. The antibody of claim 41, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

43. The antibody of claim 41 or 42, comprising the VH sequence of SEQ ID NO:99 and the VL sequence of SEQ ID NO: 98.

44. The antibody of any one of claims 41 to 43, comprising the heavy chain amino acid sequence of SEQ ID NO. 105 and the light chain amino acid sequence of SEQ ID NO. 104.

45. The antibody of any one of claims 41 to 44, comprising the heavy chain amino acid sequence of SEQ ID No. 117 and the light chain amino acid sequence of SEQ ID No. 104.

46. A monoclonal antibody that binds to CCR8, wherein the antibody binds to CCR8 independent of sulfation of CCR 8.

47. The antibody of claim 46, wherein the antibody binds to an epitope comprising one or more of amino acid residues 2 to 6 of SEQ ID No. 106.

48. The antibody of claim 46, wherein the antibody binds to an epitope comprising one or more of amino acid residues 91 to 104 and 172 to 193 of SEQ ID No. 106.

49. A monoclonal antibody that binds to mouse CCR8, wherein the antibody comprises: 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.

50. The antibody of claim 49, comprising a sequence selected from the group consisting of:

(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 VH sequence as defined in (a) and a VL sequence as defined in (b).

51. An antibody according to claim 49 or 50, comprising the VH sequence of SEQ ID NO. 70 and the VL sequence of SEQ ID NO. 69.

52. The antibody of any one of claims 49 to 51, comprising the heavy chain amino acid sequence of SEQ ID No. 72 and the light chain amino acid sequence of SEQ ID No. 71.

53. The antibody of any one of claims 1 to 48, which is a human antibody.

54. The antibody of any one of claims 1 to 48, which is a humanized antibody.

55. The antibody of any one of claims 1 to 52, which is a chimeric antibody.

56. The antibody of any one of claims 1 to 55, which is an antibody fragment that binds CCR 8.

57. The antibody of any one of claims 1 to 56, which is a full length antibody.

58. The antibody of claim 57 which is a full length IgG1 antibody.

59. The antibody of any one of claims 1 to 58, comprising an IgG1 constant domain comprising the amino acid sequence of SEQ ID No. 53 or SEQ ID No. 59.

60. The antibody of any one of claims 1 to 59, comprising a kappa constant domain comprising the amino acid sequence of SEQ ID No. 54.

61. The antibody of any one of claims 1-60, wherein the antibody is at about 1 x 10 ^-12 M to about 1X 10 ^-11 Binding affinity of M (K _d ) Binds to CCR8.

62. The antibody of any one of claims 1 to 48, wherein the CCR8 is human CCR8.

63. The antibody of any one of claims 1 to 62, wherein the antibody is defucosylated.

64. An isolated nucleic acid encoding the antibody of any one of claims 1 to 63.

65. A host cell comprising the nucleic acid of claim 64.

66. A method of producing an antibody that binds to CCR8, the method comprising culturing the host cell of claim 65 under conditions suitable for expression of the antibody.

67. The method of claim 66, further comprising recovering the antibody from the host cell.

68. An antibody produced by the method of claim 67.

69. A pharmaceutical composition comprising the antibody of any one of claims 1 to 63 and a pharmaceutically acceptable carrier.

70. The pharmaceutical composition of claim 69, further comprising an additional therapeutic agent.

71. The antibody of any one of claims 1 to 63 or the pharmaceutical composition of any one of claims 69 to 70 for use as a medicament.

72. The antibody of any one of claims 1 to 63 or the pharmaceutical composition of any one of claims 69 to 70 for use in the treatment of cancer.

73. Use of an antibody according to any one of claims 1 to 63 or a pharmaceutical composition according to any one of claims 69 to 70 in the manufacture of a medicament for the treatment of cancer.

74. Use of an antibody according to any one of claims 1 to 63 or a pharmaceutical composition according to any one of claims 69 to 70 in the manufacture of a medicament for depleting regulatory T cells.

75. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-63 or the pharmaceutical composition of any one of claims 69-70.

76. A method of depleting regulatory T cells in a tumor microenvironment of a subject having cancer, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-63 or the pharmaceutical composition of any one of claims 69-70 sufficient to deplete the regulatory T cells in the tumor microenvironment.

77. A method of depleting regulatory T cells outside of a tumor microenvironment of a subject having cancer, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-63 or the pharmaceutical composition of any one of claims 69-70 sufficient to deplete the regulatory T cells outside of the tumor microenvironment.

78. An in vitro method of depleting regulatory T cells from a population of cancer cells, the method comprising contacting the population of cells with an amount of the antibody of any one of claims 1 to 63 or the pharmaceutical composition of any one of claims 69 to 70 sufficient to deplete the regulatory T cells from the population of cells.

79. The use or method of any one of claims 73-78, 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.

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

81. The use or method of any one of claims 74, 77, 78 and 79, wherein the regulatory T cells outside the tumor microenvironment of the cancer are depleted.

82. The use or method of any one of claims 73-81, further comprising administering an additional therapeutic agent.

83. The use or method of claim 82, wherein the additional therapeutic agent is an anticancer agent.

84. The use or method of claim 83, wherein the anti-cancer agent is selected from the group consisting of: microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormone therapies, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cytostatic agents, apoptosis activators, 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 therapies and immunocytoplasmic agents.

85. The use or method of claim 83 or 84, wherein the anti-cancer agent is a PD-L1 binding antagonist.

86. The use or method of claim 85, wherein the PD-L1 binding antagonist is alemtuzumab.

87. The use or method of any one of claims 73-85, wherein the subject is a human.

88. The use or method of any one of claims 73-85, wherein the subject is a mouse.

89. A method of treating a disease in a mouse, the method comprising administering to the mouse an effective amount of the monoclonal antibody of any one of claims 49-52 to treat the disease.

90. The method of claim 89, wherein the mouse comprises a xenograft.

91. The antibody of claim 63, wherein the proportion of defucosylation is between about 80% to about 95%.

92. The antibody of any one of claims 1 to 15 and 46 to 48, wherein the average clearance after intravenous administration of a single 10mg/kg dose on day 1 is about 3 to about 5 mL/day/kg over a period of 35 days.