Anti-CCR8 antibodies and methods of use

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human CCR8, pharmaceutical compositions comprising the antibodies, and uses of such antibodies or compositions to treat diseases such as cancer.

Description

[Technical Field]

[0001] Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human CCR8, compositions comprising the antibodies, and methods of use for the treatment of cancer. [Background technology]

[0002] CC motif chemokine receptor 8 (CCR8) is a seven-transmembrane G protein-coupled receptor (GPCR). Human CCR8 consists of 355 amino acids, and its extracellular domain includes a 35-amino acid (aa) N-terminus and three short extracellular loops (ECL1-ECL3). The N-terminus and ECL2 are important for ligand binding and activation of CCR8 (Gadhe et al., J Biomol Struct Dyn. 2015;33(11):2491-510; Barington L., et al., J Biol Chem. 2016 Jul 29;291(31):16208-20). Human CCR8 has four ligands, including CCL1, CCL8, CCL16, and CCL18, and CCL1 is the major ligand for CCR8 in regulatory T cells (Treg) and Th2 cells (Islam, SA, et al., J Exp Med. 2013 Sep 23; 210(10): 1889-98; Barsheshet et al., Proc Natl Acad Sci US A. 2017 Jun 6; 114(23): 6086-6091; Sokol et al., 2018). CCR8 is normally expressed in the blood by Treg and Th2 cell subpopulations (Schaerli et al., J Exp Med. 2004 May 3;199(9):1265-75; Soler et al., J Immunol. 2006 Nov 15;177(10):6940-51), and is also expressed in the thymus and spleen (Francesco Annunziato et al., J Exp Med. 2002 Aug 5;196(3):379-87; Lee et al., J Immunol. 2007 Jan 1;178(1):301-11; Thyagarajan et al., PLoS One. 2018 Jul 19;13(7):e0200765).Furthermore, CCR8 is also expressed on skin T cells (Schaerli et al., J Exp Med. 2004 May 3;199(9):1265-75., Ebert et al., J Immunol. 2006 Apr 1;176(7):4331-6., Campbell et al., Cancer Res. 2021 Jun 1;81(11):2983-2994).

[0003] It has been well documented that CCR8 is significantly upregulated in tumor Tregs in different types of human cancers, including but not limited to head and neck cancer, colon cancer, lung cancer, breast cancer, bladder cancer, and esophageal cancer (Plitas et al., Immunity. 2016 Nov 15; 45(5): 1122-1134; Magnuson et al., Proc Natl Acad Sci US A. 2018 Nov 6; 115(45): E10672-E10681.; Wang et al., Cancer Immunol Immunother. 2020 Sep; 69(9): 1855-1867; Van Damme et al., J Immunother Cancer. 2021 Feb; 9(2): e001749). High CCR8 expression also correlates with poor prognosis in patients with breast, bladder, and lung cancer (Plitas et al., Immunity. 2016 Nov 15;45(5):1122-1134; Alvisi et al., J Clin Invest. 2020 Jun 1;130(6):3137-3150; Wang et al., Cancer Immunol Immunother. 2020 Sep;69(9):1855-1867). CCR8 expression appears to define an activated population of tumor Tregs, as these cells display elevated activation markers and are more suppressive than CCR8-negative cells (Villarreal et al., Cancer Res. 2018 Sep 15;78(18):5340-5348; Wang et al., Nat Immunol. 2019 Sep;20(9):1220-1230; Whiteside et al., Immunology. 2021 Aug;163(4):512-520). Activation of CCR8 by CCL1 can further enhance the suppressive activity of CCR8+ Tregs by upregulating FOXp3, CCR8, CD39, and IL-10 (Barsheshet et al., Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):6086-6091).Therefore, CCR8 is a promising therapeutic target for specifically targeting tumor Tregs, which play an important suppressive role in the tumor microenvironment.

[0004] Recent studies in CCR8-deficient mice have shown that CCR8 is not required for tumor Treg recruitment or function, but depletion of CCR8 Tregs using Fc-intact, non-mutated anti-mouse CCR8 antibodies has antitumor effects (Van Damme et al., J Immunother Cancer. 2021 Feb;9(2):e001749; Bhatt et al., J Exp Med. 2021 Jun 7;218(6):e20201329; Campbell et al., Cancer Res. 2021 Jun 1;81(11):2983-2994; Kidani et al., Proc Natl Acad Sci US A. 2022 Feb 15;119(7):e2114282119). Anti-mouse CCR8 antibodies have demonstrated both single-agent activity and combined efficacy with anti-PD1 antibodies. In these studies, anti-mouse CCR8 antibodies did not induce toxicity in tumor-bearing mice and could preserve Tregs in the spleen, thymus, and skin (Villarreal et al., Cancer Res. 2018 Sep 15;78(18):5340-5348; Campbell et al., Cancer Res. 2021 Jun 1;81(11):2983-2994; Kidani et al., Proc Natl Acad Sci US A. 2022 Feb 15;119(7):e2114282119). CCR8- / - mice were also viable and fertile (Chung et al., J Immunol. 2003 Jan 1;170(1):581-7; Yabe et al., Int Immunol. 2015 Apr 27(4):169-81). Taken together, these data show that eliminating CCR8+ tumor Tregs is a promising strategy and is unlikely to induce adverse effects.However, at present, no CCR8 antibody has been approved for use in human cancer treatment.Therefore, the anti-CCR8 antibody disclosed herein is useful for human cancer treatment. Summary of the Invention

[0005] The present disclosure is directed to anti-CCR8 antibodies and antigen-binding fragments thereof that specifically bind to CCR8.

[0006] In one embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding fragment thereof, that binds to human CCR8.

[0007] The present disclosure encompasses the following embodiments.

[0008] An antibody or an antigen-binding fragment thereof, which specifically binds to human CCR8.

[0009] An antibody or antigen-binding fragment thereof that specifically binds to human CCR8 (SEQ ID NO: 1).

[0010] (i) a heavy chain variable region comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; (ii) a heavy chain variable region comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; or (iii) An antibody or antigen-binding fragment thereof that binds to human CCR8, comprising a heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 14, (b) HCDR2 of SEQ ID NO: 15, and (c) HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) LCDR2 of SEQ ID NO: 8, and (f) LCDR3 of SEQ ID NO: 9.

[0011] (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% identical to SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 26, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 23; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 16, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 17; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 16, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 20; or (v) the antibody or the antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 22, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 23.

[0012] The antibody or antigen-binding fragment thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NOs: 10 and 11, SEQ ID NOs: 26 and 23, SEQ ID NOs: 16 and 17, SEQ ID NOs: 16 and 20, or SEQ ID NOs: 22 and 23 have been inserted, deleted, or substituted.

[0013] (i) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 26, and a light chain variable region (VL) comprising SEQ ID NO: 23; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 16 and a light chain variable region (VL) comprising SEQ ID NO: 17; (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 16 and a light chain variable region (VL) comprising SEQ ID NO: 20; or (v) The antibody or the antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 22, and a light chain variable region (VL) comprising SEQ ID NO: 23.

[0014] The antibody or the antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0015] The antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0016] The antibody or antigen-binding fragment thereof has reduced glycosylation or is hypofucosylated or defucosylated.

[0017] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an increase in bisecting GlcNac structures.

[0018] The antibody or antigen-binding fragment thereof, wherein the Fc domain is that of IgG1.

[0019] The antibody or antigen-binding fragment binds to an epitope comprising at least one, two, three, four, five, or six amino acid residues at positions selected from 26, 172, 177, 178, 266, and 269 of the amino acid sequence of HuCCR8.

[0020] The antibody or antigen-binding fragment binds to an epitope comprising at least one, two or three amino acid residues (may be multiple) at positions selected from 172, 177 and 269 of the amino acid sequence of HuCCR8.

[0021] The antibody or antigen-binding fragment thereof binds to an epitope comprising at least one, two, or three amino acid residues at positions selected from 26, 178, and 266 of the amino acid sequence of HuCCR8.

[0022] The antibody or antigen-binding fragment binds to an epitope including (1) amino acid residues at positions 20 to 30, (2) amino acid(s) at positions 170 to 180, and (3) amino acid(s) at positions 260 to 270 of the HuCCR8 amino acid sequence.

[0023] The antibody or antigen-binding fragment binds to an epitope including (1) the amino acid residue at position 26, (2) one, two, or three amino acids (or more) at positions 172, 177, and 178, and (3) one or two amino acids (or more) at positions 266 and 269 of the HuCCR8 amino acid sequence.

[0024] The antibody or antigen-binding fragment binds to an epitope including (1) one, two, or three amino acid residues at the N-terminal position, (2) one, two, or three amino acid(s) at the ECL2 region position, and (3) one, two, or three amino acid(s) at the ECL3 region position of the HuCCR8 amino acid sequence.

[0025] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, further comprising a pharmaceutically acceptable carrier.

[0026] A method of treating cancer comprising administering to a patient in need thereof an effective amount of said antibody or said antigen-binding fragment.

[0027] The method, wherein the cancer is head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, or small cell lung cancer.

[0028] The foregoing methods, wherein the antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

[0029] The method, wherein the therapeutic agent is an immune checkpoint inhibitor.

[0030] The method, wherein the immune checkpoint inhibitor is an anti-PD1 antibody.

[0031] The method, wherein the anti-PD1 antibody is BGB-A317.

[0032] The method, wherein the immune checkpoint inhibitor is an anti-TIGIT antibody.

[0033] The method, wherein the anti-TIGIT antibody is BGB-A1217.

[0034] The method, wherein the checkpoint inhibitors are BGB-A1217 and BGB-A317.

[0035] An isolated nucleic acid encoding said antibody or said antigen-binding fragment.

[0036] A vector comprising the nucleic acid.

[0037] A host cell comprising said nucleic acid or said vector.

[0038] A process for producing an antibody or antigen-binding fragment thereof, comprising culturing said host cell and recovering said antibody or antigen-binding fragment from the culture.

[0039] The antibody or antigen-binding fragment thereof for use in treating or reducing the likelihood of head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, and small cell lung cancer.

[0040] In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, or SEQ ID NO:15.

[0041] In another embodiment, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising one or more complementarity determining regions (HCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, or SEQ ID NO:15, and / or (b) a light chain variable region comprising one or more complementarity determining regions (LCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0042] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs), where HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 14, HCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 15, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), where LCDR1 comprises the amino acid sequence of SEQ ID NO: 7, LCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0043] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, or the HCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, or the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0044] In another embodiment, an antibody or antigen-binding fragment of the present disclosure comprises a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0045] In another embodiment, an antibody or antigen-binding fragment of the present disclosure comprises a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0046] In another embodiment, an antibody or antigen-binding fragment of the present disclosure comprises a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0047] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, or SEQ ID NO:26, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, or SEQ ID NO:26; and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:23, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:23.

[0048] In another embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 26, or an amino acid sequence comprising one, two, or three amino acid substitutions within the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 26; and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23, or an amino acid sequence comprising one, two, three, four, or five amino acid substitutions within the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0049] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure: (a) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; (b) a heavy chain variable region (VH) comprising SEQ ID NO: 16, and a light chain variable region (VL) comprising SEQ ID NO: 17; (c) a heavy chain variable region (VH) comprising SEQ ID NO: 16, and a light chain variable region (VL) comprising SEQ ID NO: 20; (d) a heavy chain variable region (VH) comprising SEQ ID NO: 22 and a light chain variable region (VL) comprising SEQ ID NO: 23; or (e) a heavy chain variable region (VH) comprising SEQ ID NO: 26, and a light chain variable region (VL) comprising SEQ ID NO: 23.

[0050] In one embodiment, an antibody of the disclosure is of the IgG1, IgG2, IgG3, or IgG4 isotype. In a more specific embodiment, an antibody of the disclosure comprises the Fc domain of wild-type human IgG1 (also called human IgG1wt or huIgG1).

[0051] In one embodiment, an antibody of the disclosure binds to human CCR8 or cynoCCR8 with a binding affinity (EC50) of 0.1 nM to 100 nM, hi another embodiment, an antibody of the disclosure binds to human CCR8 or CynoCCR8 with a binding affinity (EC50) of less than 100 nM, 50 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

[0052] In another embodiment, the anti-human CCR8 antibodies of the present disclosure exhibit cross-species binding activity to cynomolgus monkey CCR8.

[0053] In one embodiment, the antibodies of the present disclosure have strong Fc-mediated effector function: such antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing CCR8.

[0054] The present disclosure relates to isolated nucleic acids comprising nucleotide sequences encoding the amino acid sequences of such antibodies or antigen-binding fragments. In one embodiment, the isolated nucleic acid comprises the VH nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, or SEQ ID NO: 27, or a nucleotide sequence that constitutes at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, or SEQ ID NO: 27, and encodes the VH region of an antibody or antigen-binding fragment of the disclosure. Alternatively or additionally, the isolated nucleic acid comprises the VL nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 25, or a nucleotide sequence that constitutes at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 25, and encodes the VL region of an antibody or antigen-binding fragment of the disclosure.

[0055] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a CCR8 antibody or antigen-binding fragment thereof, and optionally a pharmaceutically acceptable excipient.

[0056] In yet another aspect, the present disclosure relates to a method for treating a disease in a subject, comprising administering a therapeutically effective amount of a CCR8 antibody or antigen-binding fragment thereof, or a CCR8 antibody pharmaceutical composition to a subject in need thereof. In another embodiment, the disease treated by the antibody or antigen-binding fragment is cancer.

[0057] The present disclosure relates to the use of antibodies or antigen-binding fragments thereof, or CCR8 antibody pharmaceutical compositions for treating diseases such as cancer. [Brief explanation of the drawings]

[0058] [Figure 1]Figure 1 shows the sequence of the immunogen human CCR8 protein, a comparison between human, monkey, and mouse CCR8, and the composition of the N-terminus-Fc protein of human CCR8. (A) shows the sequence of the immunogen human CCR8 protein and a comparison between human, monkey, and mouse CCR8. (B) shows the composition of the human CCR8-N-terminus-Fc protein. [Figure 2] 1 shows a comparison of blocking activity between representative chimeric anti-CCR8 antibodies. [Figure 3] 1 shows a comparison of CCR8 binding activity between representative chimeric anti-CCR8 antibodies using Jurkat human CCR8-overexpressing cells. [Figure 4A] Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4B]Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4C] Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4D]Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4E] Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4F]Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4G] Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4H]Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 4I] Figure 1 shows the glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF analyzed by mass spectrometry in intact and reduced forms. Figures A–C show the results of mass spectrometry for intact and reduced forms of Ch305-AF; Figure A shows the intact mass; Figures B and C show the masses of the reduced forms of HC and LC, respectively. Figures D–F show the results of mass spectrometry for intact and reduced forms of hu305-4F-2l-AF; Figure D shows the intact mass; Figures E and F show the masses of the reduced forms of HC and LC, respectively. Figures G–I show the results of mass spectrometry for intact and reduced forms of hu305-4F-2m-AF; Figure G shows the intact mass; Figures H and I show the masses of the reduced forms of HC and LC, respectively. These results, taken together, indicate that the measured masses are consistent with the theoretical masses and that no fucose-related N-glycosylation was detected. [Figure 5] Figure 1 shows a comparison of the CCR8 binding activity of different humanized anti-CCR8 antibodies using Jurkat human CCR8-overexpressing cells. (A) Comparison between Ch305, hu305-4F-2l, and hu305-4F-2m; (B) Comparison between Ch305, hu305-5W-3a, and hu305-5P-3a. [Figure 6] Comparison of binding activity to CCR8+ primary Tregs (A) and CCR8 blocking activity (B) between humanized Ch305 anti-CCR8 antibodies. [Figure 7] 1 shows a comparison of the cross-reactivity of cynoCCR8 between different anti-CCR8 antibodies. [Figure 8] Figure 1 shows a comparison of NK cell-mediated cytotoxicity of different anti-CCR8 antibodies, identifying the epitope of Ch305 by domain swapping, and identifying the epitope of hu305-5W-3a by alanine scanning. [Figure 9A] 1 shows the epitope identification of Ch305 by domain swapping and the epitope identification of hu305-5W-3a by alanine scanning. [Figure 9B] 1 shows the epitope identification of Ch305 by domain swapping and the epitope identification of hu305-5W-3a by alanine scanning. 1 shows the results of domain swapping with a reference antibody. [Figure 9C] 1 shows the identification of epitopes in Ch305 by domain swapping and in hu305-5W-3a by alanine scanning. The results of the major epitope residues in hu305-5W-3a identified by alanine scanning are shown. [Figure 10] 1 shows the efficacy of a combination of anti-mouse CCR8 antibody and anti-mouse PD1 antibody in a CT26 syngeneic mouse model. [Figure 11] 1 shows the tumor Treg-depleting effects of different representative anti-CCR8 antibodies in the MC38 syngeneic model in human CCR8KI mice.

[0059] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0060] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0061] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0062] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0063] The term "CC motif chemokine receptor 8" or "CCR8" or "CD198" refers to a cellular receptor. The amino acid sequence of human CCR8 (SEQ ID NO: 1) can also be found under accession number P51685 (CCR8_human) or NP_005192.1. The amino acid sequence of cynomolgus monkey ("Cyno") CCR8 (SEQ ID NO: 2) can also be found under accession number A0A8J8XUI3_MACFA or XP_015300839.1. The amino acid sequence of mouse CCR8 (SEQ ID NO: 3) can also be found under accession number P56484 (CCR8_mouse) or NP_031746.1.

[0064] As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, or diagnostic agent or composition with such animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell and, when a fluid is in contact with the cell, contact of a reagent with a fluid. The terms "administration" and "treatment" also refer to in vitro and ex vivo treatment of, e.g., a cell, with a reagent, diagnostic compound, binding compound, or with another cell. The term "subject" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., a rat, mouse, dog, cat, rabbit), and most preferably a human. Treating any disease or disorder, in one aspect, refers to ameliorating such disease or disorder (i.e., slowing, arresting, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, such as a physical parameter that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another aspect, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0065] The term "subject" in the context of this disclosure is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein).

[0066] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen through non-covalent forces at multiple sites. Generally, the more interactions, the stronger the affinity.

[0067] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0068] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0069] In some embodiments, the anti-CCR8 antibody comprises at least one antigen-binding site, at least the variable region. In some embodiments, the anti-CCR8 antibody comprises an antigen-binding fragment from a CCR8 antibody described herein. In some embodiments, the anti-CCR8 antibody is isolated or recombinant.

[0070] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies that contain different amino acid sequences within the variable domains, particularly within the complementarity-determining regions (CDRs), and are often specific for different epitopes. 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. Monoclonal antibodies (mAbs) can be obtained by methods known to those of skill in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, etc., and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0071] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Human heavy chains are further typically classified as α, δ, ε, γ, or μ, and antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and heavy chains also contain a "D" region of approximately 10 or more amino acids.

[0072] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0073] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering scheme).Definitions of antigen-binding sites are also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000), and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001), MacCallum et al., J. Mol. Biol., 262:732-745 (1996), and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989), Martin et al., Methods Enzymol., 203:121-153 (1991), and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0074] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from the "CDRs" (e.g., LCDR1, LCDR2, and LCDR3 in the light-chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy-chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (which defines antibody CDR regions by sequence). See also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (which defines antibody CDR regions by structure). The terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0075] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv); nanobodies; and multispecific antibodies formed from antibody fragments.

[0076] As used herein, an antibody "specifically binds" to a target protein means that the antibody exhibits selective binding to that target relative to other proteins, although this specificity does not require absolute binding specificity. When used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, an antibody "specifically binds" or "selectively binds" refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, e.g., a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as the background level and does not specifically bind to significant amounts of other antigens present in the sample. In one aspect, under specified immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as the background binding level and does not specifically bind to significant amounts of other antigens present in the sample.

[0077] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0078] The terms "humanized" or "humanized antibody" refer to forms of antibodies that contain sequences from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). When necessary to distinguish a humanized antibody from a rodent parent antibody, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences of the rodent parent antibody but can include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0079] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence may be framework regions only, complementarity-determining regions only, framework regions and complementarity-determining regions, variable segments (as defined above), or other combinations of sequences or subsequences contained in the variable region. Sequence identity may be determined by aligning two sequences using methods described herein, for example, BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence or a variant of a human germline sequence, or from an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0080] “Equilibrium dissociation constant (K D The term "dissociation rate constant (kd, time -1 ) is the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8Less than m, e.g., about 10 -9 Less than M or 10 -10 M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0081] The terms "cancer" or "tumor" as used herein have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to any particular type or location.

[0082] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" means replacing an original amino acid with a new amino acid that does not substantially change the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to CCR8. In particular, general conservative substitutions of amino acids are well known in the art.

[0083] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence for as long as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value; if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and both strands are compared.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) of 50, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0084] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0085] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), as incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0086] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0087] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous to or located in close proximity to the coding sequence whose transcription they enhance.

[0088] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, that include an anti-CCR8 antibody described herein and are formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0089] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid formulations, such as solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended method of administration and therapeutic application. Typical preferred compositions are in the form of an injectable or infusible solution. One preferred method of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0090] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art and can also be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components for effective treatment of the disease, disorder, or condition.

[0091] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in this disclosure. Such administration encompasses the co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administration in multiple containers for each active ingredient, or in separate containers (e.g., capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration also encompasses the use of various therapeutic agents in a sequential manner, at about the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0092] As used herein, the term "in combination with" means that the anti-CCR8 antibody is administered to a subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, the anti-CCR8 antibody is administered as a combination with an additional therapeutic agent. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present disclosure provides antibodies, antigen-binding fragments, that specifically bind to human CCR8. Additionally, the present disclosure provides antibodies that have desirable pharmacokinetic properties and other desirable attributes, and thus can be used to reduce the likelihood of or treat cancer. The present disclosure also provides pharmaceutical compositions comprising the antibodies, as well as methods for making and using such pharmaceutical compositions, for the prevention and treatment of cancer and related disorders.

[0094] Anti-CCR8 antibody The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CCR8. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, the antibodies or antigen-binding fragments thereof prepared as described below.

[0095] The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CCR8, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 26 in Table 1. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CCR8, wherein the antibody or antigen-binding fragment comprises an HCDR comprising the amino acid sequence of any one of the HCDRs listed in Table 1. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CCR8, wherein the antibody comprises (or alternatively consists of) one, two, three, or more HCDRs comprising the amino acid sequence of any of the HCDRs listed in Table 1.

[0096] The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibodies or antigen-binding fragments comprise a VL domain comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:23 (Table 1). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibodies or antigen-binding fragments comprise an LCDR comprising the amino acid sequence of any one of the LCDRs listed in Table 1. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibodies or antigen-binding fragments comprise (or alternatively consist of) one, two, three, or more LCDRs comprising the amino acid sequence of any of the LCDRs listed in Table 1.

[0097] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acid alterations in the CDR regions that have a percent identity of at least 60%, 70%, 80%, 90%, 95%, or 99% with the CDR regions disclosed in Table 1. In some embodiments, this includes amino acid alterations in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the CDR regions when compared to the CDR regions set forth in the sequences set forth in Table 1.

[0098] Other antibodies of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered but have a percent identity of at least 60%, 70%, 80%, 90%, 95%, or 99% to the sequences set forth in Table 1. In some embodiments, this includes amino acid sequence alterations in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the variable regions when compared to the variable regions set forth in the sequences set forth in Table 1, while retaining substantially the same therapeutic activity.

[0099] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to CCR8. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0100] The sequence listing of the present disclosure is shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0101] Identification of epitopes and antibodies that bind to the same epitopes The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CCR8. In certain embodiments, the antibodies and antigen-binding fragments can bind to the same epitope of CCR8.

[0102] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CCR8 antibodies listed in Table 1. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibit the binding of other antibodies in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody of the present disclosure and its antigen-binding fragment to CCR8 indicates that the test antibody can compete with that antibody or its antigen-binding fragment for binding to CCR8. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on CCR8 as the competing antibody or its antigen-binding fragment. In certain embodiments, an antibody that binds to the same epitope on CCR8 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0103] Further modifications to the Fc region framework In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This technique is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0104] In another embodiment, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0105] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0106] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0107] In yet another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks or has reduced glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an "antigen." Such sugar modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such techniques are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0108] Additionally or alternatively, antibodies can be generated with altered glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line in which the FUT8 gene, encoding fucosyltransferase, has been functionally disrupted so that the expressed antibodies exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked sugars, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increase in bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0109] CCR8 antibody production Anti-CCR8 antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including, but not limited to, recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, and full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0110] The disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 26. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23.

[0111] The polynucleotides of the present disclosure can encode the variable region sequences of anti-CCR8 antibodies. They can also encode both the variable and constant regions of the antibodies. Some polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of one of the exemplified anti-CCR8 antibodies.

[0112] The present disclosure also provides expression vectors and host cells for producing anti-CCR8 antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-CCR8 antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of anti-CCR8 antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by including enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0113] Host cells for harboring and expressing anti-CCR8 antibody chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, as well as other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be made in these prokaryotic hosts, which typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. Additionally, any number of well-known promoters are present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems from phage lambda. Promoters typically control expression (optionally with operator sequences) and contain ribosome binding site sequences for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-CCR8 polypeptides. Insect cells can also be used in combination with baculovirus vectors.

[0114] In other embodiments, mammalian host cells are used to express and produce the anti-CCR8 polypeptides of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is reviewed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, temporally specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0115] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for detecting CCR8. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of CCR8 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal and / or cancerous tissues that express CCR8 at higher levels than other tissues.

[0116] In one aspect, the present disclosure provides a method for detecting the presence of CCR8 in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-CCR8 antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine, tissue, sputum, or blood sample.

[0117] Also included are methods for diagnosing disorders associated with CCR8 expression. In certain embodiments, the methods include contacting test cells with an anti-CCR8 antibody; determining (quantitatively or qualitatively) the expression level of CCR8 expressed by the test cells by detecting binding of the anti-CCR8 antibody to a CCR8 polypeptide; and comparing the expression level by the test cells with the CCR8 expression level in control cells (e.g., normal cells or non-CCR8-expressing cells of the same tissue origin as the test cells), wherein a higher level of CCR8 expression in the test cells compared to the control cells indicates the presence of a disorder associated with CCR8 expression.

[0118] Treatment method The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for treating CCR8-associated disorders or diseases. In one embodiment, the CCR8-associated disorder or disease is cancer.

[0119] In one aspect, the present disclosure provides a method for treating cancer. In certain aspects, the method comprises administering an effective amount of an anti-CCR8 antibody or antigen-binding fragment to a patient in need thereof. The cancer may include, but is not limited to, head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, and small cell lung cancer. Specifically, the cancers may include head and neck squamous cell carcinoma, nasopharyngeal carcinoma, microsatellite instability-high colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric / stomach adenocarcinoma, triple-negative breast cancer, human epidermal growth factor receptor 2 (HER2)+ breast cancer, pancreatic adenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma of the endocervical tract, urothelial bladder cancer, renal cell carcinoma (kidney clear cell carcinoma), microsatellite-stable colorectal cancer (CRC_MSS), esophageal squamous cell carcinoma, esophageal adenocarcinoma, progesterone / estrogen receptor-positive breast cancer, and hepatocellular carcinoma of the liver.

[0120] The antibodies or antigen-binding fragments disclosed herein can be administered by any suitable means, for example, parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if localized treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time periods, bolus administration, and pulse infusion.

[0121] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used in the same dosages and via the same routes of administration as those described herein, or between about 1 and 99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0122] For disease prevention or treatment, the appropriate dosage of the antibody or antigen-binding fragment of the present disclosure will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to the patient may be about 1 μg / kg to 100 mg / kg of antibody, whether by one or more individual administrations or by continuous infusion, for example. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is generally maintained until a desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives from about 2 to about 20 doses, or for example, about 6 doses, of the antibody). An initial, higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0123] Combination therapy In one embodiment, the CCR8 antibodies of the present disclosure can be used in combination with other therapeutic agents. Other therapeutic agents that can be used in combination with the CCR8 antibodies of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations; (e.g., Abraxane®), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20-targeted agents (e.g., rituximab, opioids, tumumab, RO5072759, LFB-R603), CD52-targeted agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19-targeted agents (e.g., MEDI-551, MOR20 8), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, polo-like kinase 1 inhibitors (e.g., BI 672).

[0124] The anti-CCR8 antibody of the present disclosure can be used in combination with other therapeutic agents, such as other immune checkpoint antibodies. Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD1 antibodies may include, without limitation, antibodies disclosed in U.S. Patent No. 8,735,553. Pembrolizumab (formerly MK-3475), disclosed by Merck, is a humanized IgG4-K immunoglobulin with a molecular weight of approximately 149 kDa that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the treatment of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is currently undergoing clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0125] Other immune checkpoint antibodies for use in combination with anti-CCR8 antibodies may include anti-TIGIT antibodies, including, but not limited to, the anti-TIGIT antibodies disclosed in WO2019 / 129261.

[0126] Pharmaceutical Compositions and Formulations Also provided are compositions, such as pharmaceutical preparations, comprising anti-CCR8 antibodies or antigen-binding fragments thereof, or polynucleotides comprising sequences encoding anti-CCR8 antibodies or antigen-binding fragments. In certain embodiments, the compositions comprise one or more antibodies or antigen-binding fragments that bind to CCR8, or one or more polynucleotides comprising sequences encoding one or more antibodies or antigen-binding fragments that bind to CCR8. These compositions can further comprise suitable carriers, for example, pharmaceutically acceptable excipients such as buffers, which are well known in the art.

[0127] Pharmaceutical formulations of the anti-CCR8 antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, 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 such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0128] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0129] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0130] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes. [Example]

[0131] Example 1. Preparation of mouse anti-CCR8 antibody Immunization of mice To generate antibodies against CCR8, a total of 50 adult female mice from different strains (BALB / c, C57BL / 6, SJL, and MRL / lpr) were immunized with different combinations of immunogens, including a human CCR8 expression plasmid (huCCR8 DNA), L929 cells overexpressing human CCR8 (L929-huCCR8), and L929 cells overexpressing cynomolgus monkey CCR8 (L929-cynoCCR8). The human, cynomolgus monkey, and mouse CCR8 protein sequences are listed as SEQ ID NOS: 1 to 3 in Table 1, respectively. A comparison between human, monkey, and mouse CCR8 and the composition of the N-terminal Fc protein of human CCR8 are shown in Figure 1. Human CCR8 expression plasmid (huCCR8 DNA) (50 μg per mouse) was delivered intramuscularly by in vivo electroporation using an EPT-I delivery device (TERESA, Shanghai, China) as previously described (Jiang et al., J Virol. 2017 Apr 13;91(9):e02052-16). L929-huCCR8 and L929-cynoCCR8 cells (5 × 10 per mouse) were then injected into the muscle. 6 The mice were intraperitoneally injected with 293T cells (293T-huCCR8) and cynomolgus monkey CCR8 (293T-cynoCCR8) cells. Typically, 5 to 7 immunizations were administered at 3-week intervals to induce a strong anti-CCR8 humoral immune response. On day 14 after each immunization, blood was collected and serum antibody titers against 293T cells overexpressing human CCR8 (293T-huCCR8) and cynomolgus monkey CCR8 (293T-cynoCCR8) were determined by FACS. Animals with sufficient levels of anti-CCR8 serum antibodies were selected for the final boost.

[0132] Plasma cell screening using the Beacon Optofluidic System Three to five days after the final boost, spleens were harvested and mashed to a single-cell suspension. Plasma cells were isolated using a mouse CD138-positive selection kit (STEMCELL) according to the manufacturer's instructions. Plasma cells were isolated at a density of 6.25 × 10 61 × 10 / ml of enriched plasma cells were imported into the channel and loaded into the NanoPen chamber of an OptoSelect 14K chip™ (Berkeley Lights) according to the manufacturer's instructions. For screening of huCCR8-specific plasma cells, a density of 1 × 10 / ml was used. 8 293T-huCCR8 cells at 100µg / ml and Alexa Fluor 647 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) at 5µg / ml were imported into the channel. After import, the freeze valve was turned on, and the exposure time of the CY5 channel to the Alexa Fluor 647 fluorophore was set to 3000ms. Positive signals were captured by time-lapse photography at a setting of 6 minutes and 10 cycles. After screening for huCCR8-specific plasma cells using 293T-huCCR8 cells, cynoCCR8-specific plasma cells were screened using 293T-cynoCCR8 cells. Plasma cells that showed positive signals for both 293T-huCCR8 and 293T-cynoCCR8 cells were individually exported to a 96-well plate filled with lysis buffer.

[0133] Example 2. Cloning and sequencing of antibody VH and VL genes and chimeric antibody expression First-strand cDNA was synthesized, and the entire cDNA was amplified using the Opto Plasma B Discovery cDNA Synthesis Kit™ (Berkeley Lights) according to the manufacturer's instructions. The antibody VH and VL genes were amplified using the Opto Plasma B Discovery Sanger Prep Kit™ (Berkeley Lights) according to the manufacturer's instructions. The amplified VH and VL genes were cloned into a mammalian expression vector containing a human IgG1 gene and a kappa chain constant region gene, respectively. The amino acid sequences of the three HCDRs, three LCDRs, VH and VL, and the DNA sequences of the VH and VL of a representative antibody, Ch305, from clone PBG04-305 (also referred to as clone 305) are listed in Table 1 as SEQ ID NOS: 4-13. The chimeric antibody was expressed in Expi293™ cells and purified by affinity chromatography.

[0134] Example 3. Determination of binding affinity and specificity of anti-CCR8 antibodies To determine binding affinity, purified chimeric anti-CCR8 antibodies were serially diluted and incubated with 293T-huCCR8 or 293T-cynoCCR8 cells at 4°C for 30 minutes. After washing twice with FACS buffer, diluted Alexa Fluor 647 goat anti-human IgG secondary antibody was added and incubated with 293T-huCCR8 or 293T-cynoCCR8 cells in the dark at 4°C for 30 minutes. After washing twice with FACS buffer, cells were resuspended in FACS buffer and acquired on a Beckton Dickinson LSR Fortessa™. Titration curves were generated using a nonlinear sigmoidal dose-response fit from GraphPad, and the EC50 values of representative antibodies are shown in Table 2. As shown in Table 2, all selected clones exhibited high affinity for both 293T-huCCR8 and 293T-cynoCCR8 cells, with EC50 values of approximately 10 nM or less. [Table 2]

[0135] To determine nonspecific binding, purified chimeric anti-CCR8 antibodies were diluted to 50 nM and incubated with 293T-huCCR1, 293T-huCCR4, and 293T-parental cells for 30 minutes at 4°C. After two washes with FACS buffer, diluted Alexa Fluor 647™ goat anti-human IgG secondary antibody was added and incubated with 293T-huCCR1, 293T-huCCR4, and 293T-parental cells in the dark for 30 minutes at 4°C. After two washes with FACS buffer, cells were resuspended in FACS buffer and acquired on a BD LSR Fortessa™ Cell Analyzer. Only a few clones showed nonspecific binding to 293T-huCCR1, 293T-huCCR4, or 293T-parental cells; they were excluded from further characterization. Only antibodies specifically binding to human and monkey CCR8 were subsequently characterized.

[0136] Example 4. Blocking and cell binding of purified chimeric anti-CCR8 antibodies Anti-CCR8 antibodies block the interaction between the CCL1 ligand and the CCR8 receptor, inhibiting downstream signaling. The DiscoverX Bioassay™ (Eurofins) was used as a cell-based assay to identify the blocking activity of the generated anti-CCR8 antibodies. Briefly, human CCR8 expressed in CHO cells from the PathHunter β-Arrestin eXpress GPCR Assay Kit™ (Eurofins) was thawed and seeded into a 96-well assay plate and incubated at 37°C with 5% CO2. After 48 hours, serial dilutions of anti-CCR8 antibodies were added to each well and incubated at 37°C for 30 minutes. 13.7 nM human CCL1 was added to each well and incubated at 37°C for 90 minutes. Subsequently, the detection working solution was added to the assay plate and incubated at room temperature in the dark for 1 hour. The plate was read using a luminescence plate reader. IC50 values were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Table 3 and Figure 2, the CCR8 chimeric antibody Ch305 exhibited the strongest ligand-blocking activity among all the variants shown. [Table 3]

[0137] To evaluate the binding activity of anti-CCR8 antibodies to CCR8 expressed on live cells, Jurkat cells were engineered to overexpress human CCR8. Jurkat-human CCR8 cells were seeded into 96-well plates and incubated with serial dilutions of anti-CCR8 antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC50 values for dose-dependent binding to human CCR8 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Table 4 and Figure 3, the CCR8 antibodies exhibited high binding affinity to human CCR8. [Table 4]

[0138] Example 5. Humanization of anti-human CCR8 mAb Ch305 For humanization of Ch305, human germline IgG genes were searched for sequences that shared high homology with the cDNA sequence of the variable region of Ch305 by blasting the human immunoglobulin gene databases on the IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) websites. Human IGVH and IGVK genes, which are frequently present in the human antibody repertoire (Glanville et al., Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20216-21) and share high homology with Ch305, were selected as templates for humanization.

[0139] Humanization was performed using CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and a humanized antibody (hu305) was engineered into a human IgG1 format using an in-house developed expression vector. In the first round of humanization, mutations from murine to human amino acid residues in the framework regions were guided by simulated 3D structures, and structurally important murine framework residues for maintaining the canonical structure of the CDRs were retained in the initial version of humanized hu305. Specifically, the CDRs of Ch305 Vκ (SEQ ID NOS: 7-9) were grafted into the framework of the human germline variable region gene IGVκ4-1, while retaining some murine framework residues. The H-CDRs of Ch305 Vh (SEQ ID NOS: 4-6) were grafted into the framework of the human germline variable region gene IGVH4-59, while retaining some murine framework residues. Simulated 3D structures and mutational analysis showed that only the N-terminal half is important for antigen binding, so in the subsequent humanized variant of Ch305 (humanized 305 antibody, or humanized 305), only the N-terminal half of Kabat HCDR2 was maintained.

[0140] Humanized antibodies were constructed in a full-length human antibody format using an in-house developed expression vector containing the human IgG1 and kappa chain constant regions, respectively, with adaptable subcloning sites. Expression and preparation of humanized antibodies derived from clone 305 can be achieved by cotransfection of the heavy chain construct and the corresponding light chain construct into ExpiCHO cells and purification using a Protein A column. The purified antibodies were concentrated to 0.5-5 mg / mL in PBS, aliquoted, and stored in a -80°C freezer.

[0141] Based on the first-round humanized 305 template, humanized 305 was further engineered by introducing mutations into the CDR and framework regions to improve binding affinity to human CCR8, humanness, and biophysical properties for therapeutic use in humans.

[0142] In summary, engineered versions of clone 305 resulted in humanized monoclonal antibodies hu305-4F-2m (SEQ ID NOS: 6-9, and 14-19), hu305-4F-2l (SEQ ID NOS: 6-9, 14-16, 18, and 20-21), hu305-5W-3a (SEQ ID NOS: 6-9, 14-15, and 22-25), and hu305-5P-3a (SEQ ID NOS: 4, 6-9, 15, 23, and 25-27), which were characterized for binding and functional activity. Humanized antibodies hu305-4F-2m, hu305-4F-2l, hu305-5W-3a, and hu305-5P-3a may also be referred to simply as 4F-2m, 4F-2l, 5W-3a, and 5P-3a.

[0143] Example 6. Preparation of defucosylated anti-CCR8 antibody Removal of core fucose from N-glycans attached to human IgG1 significantly enhances antibody-dependent cellular cytotoxicity (ADCC) responses (Shields, et al., (2002) J Biol Chem 277, 26733-26740; Shinkawa et al., (2003) J Biol Chem 278, 3466-3473). There are many approaches to reducing core fucosylation. In the studies presented herein, we used the approach of 2F-peracetyl-fucose, a fucosyltransferase (FUT) inhibitor.

[0144] One hundred eighty-six defucosylated (-AF) CCR8 variants, including Ch305-AF, hu305-4F-2l-AF, hu305-4F-2m-AF, hu305-5W-3a-AF, and hu305-5P-3a-AF, were produced in ExpiCHO-s cells by transient transfection of plasmids containing the heavy and kappa chains. Transfected cells were cultured in shake flasks using the MAX Titer protocol of the ExpiCHO expression system. 2F-peracetyl-fucose was added to the culture at a concentration of 100 μM. Cell supernatants were harvested on day 14 and filtered through a 0.2 μm filter for further analysis.

[0145] All of these antibodies were purified using Protein A chromatography capture and / or other polish purification steps under platform conditions. The glycan profiles of Ch305-AF, hu305-4F-2l-AF, and hu305-4F-2m-AF were analyzed by mass spectrometry in intact and reduced forms, and the results are shown in Tables 5–10 and Figures 4A–I. Glycan analysis demonstrated that at 100 μM 2F-peracetyl-fucose, no fucose-related N-glycosylated mass was detected. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0146] Example 7. Binding activity of humanized antibodies to human CCR8 To evaluate the binding activity of anti-CCR8 antibodies to native CCR8 on live cells, Jurkat cells (ATCC TIB-15) were engineered to overexpress human CCR8. Live Jurkat / huCCR8 cells were seeded into 96-well plates and incubated with serial dilutions of anti-CCR8 antibodies. Alexa Fluor® 647 anti-human IgG Fc antibody (Biolegend, catalog: 410714) was used as a secondary antibody to detect antibody binding to CCR8 expressed on the cell surface. EC of dose-dependent binding to human native CCR8 50 Values were determined by fitting the dose-response data to a 4-parameter logistic model using GraphPad Prism, as shown in Figure 5 and Table 11. The humanized antibodies retained binding affinity for native human CCR8, indicating that humanization resulted in little or no loss of ligand-blocking activity compared to the parent clones. [Table 11]

[0147] Treg binding of the humanized antibodies was measured in Treg cells from human PBMCs. Briefly, CD25+ cells were isolated from human PBMCs. The cells were stimulated with IL-2, TGFβ, and anti-CD3 / CD28 beads to produce Treg cells with high CCR8 expression levels. Treg cells were then seeded into 96-well plates and incubated with serial dilutions of anti-CCR8 antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values for dose-dependent binding to human CCR8 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 6A and Table 12, hu305-5W-3a had the highest binding affinity for CCR8 on Treg cells among the humanized antibodies. As shown in Table 12 below and Figure 6A, the hu305-5W-3a antibody also had the lowest IC50 for reducing ligand-receptor interaction. [Table 12]

[0148] The CCR8 blocking activity of the humanized antibodies was also determined and is shown in FIG. 6B and Table 13. [Table 13]

[0149] Example 8. Determination of cross-reactivity of anti-CCR8 antibodies To evaluate the cross-reactivity of humanized antibodies to cynomolgus (cyno) monkey CCR8, cynoCCR8 was overexpressed in 293T cells. 293T-cynoCCR8 cells were seeded into 96-well plates and incubated with serial dilutions of anti-CCR8 antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC50 values for dose-dependent binding to cynoCCR8 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 7 and Table 14, the humanized CCR8 antibodies exhibit good binding affinity to cynoCCR8. [Table 14]

[0150] Example 9. CCR8 antibodies enhanced NK cell-mediated ADCC Anti-CCR8 antibody-induced ADCC was determined by measuring Treg cell depletion by NK cells upon antibody treatment. Briefly, the NK92-MI-CD16a-F158 cell line was generated as effector cells. CCR8-expressing Tregs were used as target cells. An equal number (5 × 10 4 ) target cells and effector cells were added to a 96-well plate containing serial dilutions of anti-CCR8 antibody and co-cultured at 3°C for 24 hours. Cytotoxicity was assessed by flow cytometry. The results were calculated using the following formula:

number

[0151] As shown in Figure 8 and Table 15, all anti-CCR8 antibodies tested had ADCC activity, with hu305-5W-3a demonstrating the highest titer of ADCC-mediated killing of CCR8-expressing targets in a dose-dependent manner. [Table 15]

[0152] Example 10. Epitope analysis of anti-CCR8 antibodies To analyze the epitopes of anti-CCR8 antibodies, Jurkat cells overexpressing human CCR8 (Jurkat-huCCR8) were incubated with 200 nM of unlabeled anti-CCR8 antibody or human IgG1 isotype control antibody (primary antibody) at 4°C for 30 minutes. Without washing, 200 nM of APC-labeled anti-CCR8 antibody or human IgG1 isotype control antibody (secondary antibody) was added and incubated for an additional 30 minutes at 4°C. After washing twice with FACS buffer, the cells were resuspended in FACS buffer and acquired on a BD LSR Fortessa™ Cell Analyzer. Readouts were normalized as the % maximum binding of each APC-labeled antibody compared to the unlabeled human IgG1 isotype control antibody as the primary antibody. The epitope binning results are summarized in Table 16. A low value in a cell in the table indicates that the antibody pair shares a similar epitope. As shown in Table 16, Ch211 and Ch305 are in the same epitope bin and have similar epitopes, Ch366-2, reference anti-CCR8 antibodies 4A19 (disclosed in WO2021194942A1) and 433H (commercially available from BD Biosciences) are in the same epitope bin, and Ch127 and Ch326 are in two other different epitope bins. [Table 16]

[0153] To determine whether anti-CCR8 antibodies bind to the N-terminal domain of human CCR8, ELISA plates were coated overnight at 4°C with human CCR8 N-terminal domain-mouse Fc tag fusion proteins (SEQ ID NOs: 28 and 29). After washing and blocking, serially diluted anti-CCR8 antibodies were added to the plates and incubated at room temperature for 1 hour. After washing, diluted HRP goat anti-human IgG Fc or HRP goat anti-mouse kappa chain (in the case of 433H) secondary antibodies were added to the plates and incubated at room temperature for 45 minutes. After washing, TMB substrate was added to the plates to develop, followed by the addition of TMB substrate stop solution to stop the reaction. Absorbance was read at 450 nm using a plate reader. Titration curves were generated using a nonlinear sigmoidal dose-response fit from GraphPad, and the EC50s are shown in Table 17. As shown in Table 17, Ch127, Ch326, Ch366-2, 4A19, and 433H showed high binding affinity to the N-terminal protein of human CCR8, with an EC50 of less than 1 nM. In contrast, CH305 showed low binding affinity to the N-terminal protein of human CCR8, with an EC50 of 5 nM. CH211 only showed very weak binding activity to the N-terminal protein of human CCR8 at the highest concentration of 640 nM, indicating that it binds to the N-terminal loop of CCR8. In contrast, Ch305 showed low binding affinity to the N-terminal protein of human CCR8, with an EC50 of 5 nM, which may be a partial N-terminal loop binder of CCR8. Ch211 only showed very weak binding activity to the N-terminal protein of human CCR8 at the highest concentration of 640 nM, indicating that it is not a CCR8 N-terminal loop binder. [Table 17]

[0154] Example 11. Epitope mapping of anti-CCR8 antibodies To test the binding epitope of anti-CCR8 mAb Ch305, we generated domain-swapped CCR8s by transplanting amino acids from the extracellular region of mouse CCR8 into human CCR8, or vice versa. Specifically, the mapping strategy exploits the fact that Ch305 can bind to human CCR8 (SEQ ID NO: 1) but not to mouse CCR8 (SEQ ID NO: 3) (Figure 9A). An alignment of the amino acid sequences of human CCR8 (huCCR8) and mouse CCR8 (muCCR8) is shown in Figure 1A. Amino acid residues in the extracellular domain of CCR8 are underlined. The extracellular domains (ECDs) of both mouse and human CCR8 proteins are shown in Figure 1A, with the designations "N-terminus," "ECL1," "ECL2," and "ECL3" above the sequences of each region. A gene encoding full-length human CCR8 (SEQ ID NO: 1, UniProtKB: P51685) and a gene encoding full-length mouse CCR8 (SEQ ID NO: 3, UniProtKB: P56484) were cloned into the pcDNA3.4 vector. Chimeric constructs were prepared in which the extracellular domains of human CCR8, including the N-terminal region (amino acids 1-35 of SEQ ID NO: 1), ECL1 region (amino acids 94-107 of SEQ ID NO: 1), ECL2 region (amino acids 172-202 of SEQ ID NO: 1), and ECL3 region (amino acids 264-280 of SEQ ID NO: 1), were replaced with the corresponding regions of mouse CCR8, including the N-terminal region (amino acids 1-33 of SEQ ID NO: 3), ECL1 region (amino acids 92-105 of SEQ ID NO: 3), ECL2 region (amino acids 170-200 of SEQ ID NO: 3), and ECL3 region (amino acids 262-278 of SEQ ID NO: 3), respectively. Based on this exchange strategy, the absence or reduction of anti-CCR8 antibody for a particular construct implies that a particular domain (eg, ECL1) is at least partially required for anti-CCR8 antibody binding.

[0155] Table 18 shows in tabular form which murine regions have been exchanged into human CCR8. [Table 18]

[0156] For transient protein expression, plasmids containing these chimeric CCR8 constructs, as well as human CCR8 (CCR8-hu8) and mouse CCR8 (CCR8-mo8) as controls, were transfected into Expi293™ cells. The cells were then incubated with serial dilutions of purified Ch305 and the reference antibody 10A11 from Shionogi (US2022 / 0064312A1). Binding of each construct was detected and assessed using Alexa Fluor 647 rabbit anti-human IgG (catalog: 309-605-008 Jackson ImmunoResearch). The data in Figure 9A show that Ch305 specifically binds to human CCR8 but not to mouse CCR8. Binding was significantly reduced for CCR8 constructs containing regions substituted with mouse ECL2 or ECL3, indicating the critical role of these regions for anti-CCR8 antibody binding. Furthermore, replacing the N-terminal region of human CCR8 with the N-terminus of mouse CCR8 (CCR8_Ch1) also slightly impaired affinity, indicating that the N-terminus of human CCR8 contributes to the binding activity of human CCR8 to Ch305. On the other hand, replacing the N-terminus of mouse CCR8 with the N-terminus of human CCR8 (CCR8_Ch5) partially restored the binding activity of mouse CCR8 to Ch305, although much weaker than the binding activity of wild-type human CCR8 to Ch305, indicating that the N-terminus of human CCR8 contributes weakly, or even partially, to the binding activity of human CCR8 to Ch305. These data indicate that Ch305 binds to the N-terminal region, ECL2, and ECL3 of human CCR8, indicating that the epitope of Ch305 and its humanized antibody is composed of the N-terminal region, ECL2, and ECL3 of human CCR8. In contrast, the reference antibody 10A11 binds only to the N-terminal region of CCR8 and does not bind to ECL1, ECL2, or ECL3, i.e., the epitope of 10A11 on human CCR8 is located exclusively at the N-terminus of human CCR8 (Figure 9B).

[0157] To more precisely determine the specific amino acid residues essential for antibody binding to human CCR8, point mutations were introduced into the ECL2, ECL3, and N-terminal regions of human CCR8, as shown in column 1 of Table 19. To assess the effect of each mutation, humanized anti-CCR8 antibody hu305-5W-3a was used to react with Expi293 cells transfected as described above at concentrations of 50 nM, 10 nM, and 1 nM (results are summarized in Table 19). Amino acids critical for hu305-5W-3a binding are marked with an "X," indicating a greater than 50% reduction compared to wild-type huCCR8 and are considered significant.

[0158] Preliminary data indicate that antibody hu305-5W-3a recognizes amino acid D26 on the N-terminal domain, Y172, E177, and D178 on ECL2, and M266 and L269 on ECL3 (numbered according to SEQ ID NO: 1). Mutations at these sites significantly reduce binding of hu305-5W-3a. In contrast, reference antibody 10A11 recognizes these sites, as mutations at D14, Y17, and D19 on the N-terminal domain of huCCR8 completely abolished binding, indicating no epitope overlap between reference antibody 10A11 and hu305-5W-3a. To confirm the critical role of these sites for binding, transfected Expi293 cells were stained with serial dilutions of hu305-5W-3a starting from 200 nM, followed by detection with Alexa Fluor 647 rabbit anti-human IgG (catalog: 309-605-008 Jackson ImmunoResearch) to obtain a full binding curve. Figure 9C and the data in Table 20 show the effect of these key residues on hu305-5W-3a binding to CCR8. Among these sites, Y172, E177, and L269 appear to be the most important epitopes, and their substitution with Ala reduces the EC50 by more than fourfold. D178 and M266 are considered secondary epitopes with a moderate effect on Hu305-5W-3a binding. Their substitution with Ala reduces the EC50 by approximately threefold. Finally, D26 from the N-terminal domain was least affected compared to other key residues, and its mutation reduced the EC50 by more than 2-fold. Taken together, these data are consistent with the idea that Hu305-5W-3a recognizes a conformational epitope composed of the N-terminus, ECL2, and ECL3. [Table 19-1] [Table 19-2] [Table 20]

[0159] Example 12. Anti-mCCR8 mAb and anti-PD-1 mAb synergistically inhibit tumor growth in the CT26 mouse colon cancer model The efficacy of anti-mouse CCR8 antibodies as single agents or in combination with anti-mPD-1 Ab (Ch15mt) was tested in a CT26 mouse colon cancer model. Ch15mt is a murine monoclonal antibody against mouse PD-1, which was generated by immunizing rats with recombinant mouse PD-1 protein and then murinizing the rat Fc to a mouse IgG1 isotype (Chen X. et al., Front Immunol. 2022 Feb 22;13:828319). Asp265 on the heavy chain was further substituted with Ala to eliminate Fc receptor binding. Mouse CT26 colon cancer cells (3 × 10 4 ) were subcutaneously implanted into BALB / C mice. After tumor cell implantation, the length (L) and width (W) of the tumor were measured twice a week using an electric caliper, and the volume was calculated using the formula: V = 0.5(a × b 2 ) (where a and b are the long and short diameters of the tumor, respectively) to calculate the tumor size in mm 3 The tumor is approximately 100 mm 3 When the tumors reached an average volume of about 100 μg / mL, the mice were randomly assigned to seven groups (15 mice per group) and injected intraperitoneally with the mAbs once a week for 3 weeks. PBS was administered as a vehicle control. Tumor growth inhibition (TGI) was calculated using the formula: TGI = [1 - (Treatment group Tt - Treatment group T0) / (Vehicle group Tt - Vehicle group T0)] x 100, where Treatment group Tt = Treatment group tumor volume at time t, Treatment group T0 = Treatment group tumor volume at time 0, Vehicle Tt = Vehicle group tumor volume at time t, and Vehicle T0 = Vehicle group tumor volume at time 0.

[0160] The results showed that single-agent anti-mouse CCR8 (0.3 mpk) and anti-mPD-1 (3 mpk) treatment induced relatively strong antitumor effects, and that as monotherapy, the anti-CCR8 antibody exhibited a higher TGI than the anti-PD-1 antibody (Figure 10). Furthermore, combined administration of anti-CCR8 and anti-PD-1 mAbs demonstrated a synergistic effect, as evidenced by higher TGI levels. Combination treatment induced nearly complete tumor rejection in 8 of 16 mice, whereas the tumor-free rates in the anti-CCR8 and anti-PD-1 single-agent treatment groups were only 0 and 2 of 15 mice, respectively (Figure 10 and Table 21). This demonstrates that anti-CCR8 treatment can synergistically enhance the antitumor activity of anti-PD-1 in a mouse colon tumor model. [Table 21]

[0161] Example 13. Pharmacodynamic response to anti-CCR8 mAb treatment in the MC38 mouse colon cancer model The pharmacodynamic Treg-depleting activity of the anti-CCR8 antibodies hu305-4F-2l-AF and hu305-5W-3a-AF was measured in an MC38 mouse colon cancer model. Mouse MC38 colon tumor cells (purchased from Kerafast, ENH204-FP) (1 × 10 6 ) were subcutaneously implanted into C57 mice transgenic for human CCR8 (SMOC, Shanghai, China). After tumor cell implantation, the length (L) and width (W) of the tumor were measured using an electric caliper on days 6 to 9 (when the tumor volume reached 100 mm). 3 The volume is calculated using the formula: V = 0.5(a × b 2 ) (where a and b are the long and short diameters of the tumor) 3 It is expressed as:

[0162] The tumor is approximately 200-250 mm 3When the mice reached an average volume of 1000 mg / mL, they were randomized into seven groups (Figure 11). After randomization, hu305-4F-2l-AF (1 mpk, 3 mpk, and 10 mpk) and hu305-5W-3a-AF (1 mpk, 3 mpk, and 10 mpk) were intraperitoneally injected (Figure 11). PBS was administered as a vehicle control. Tumors (n = 6 mice / treatment group) were harvested on day 3 after treatment. Single cells from the tumors were isolated using a gentle MACS and then filtered using a 40 μm cell strainer. After resuspension, cells were counted using a Count star Fluorescence Cell Analyzer. 3 × 10 cells were then collected. 6 Cells were seeded into each well of a 96-well plate and stained for immune cell subsets and functional markers using flow cytometry antibodies. Antibody fluorescence was detected by flow cytometry on a FACSCelesta™ (BD Biosciences), and the results were analyzed using FlowJo™ software.

[0163] The results showed that treatment with hu305-4F-2l-AF and hu305-5W-3a-AF induced similar levels of intratumoral Treg depletion. Treatment with hu305-4F-2l resulted in a 31% (1 mpk), 42% (3 mpk), and 52% (10 mgk) reduction in the Treg population (Foxp3+CD4+ / CD3+%) compared with the vehicle group, whereas treatment with hu305-5W-3a resulted in a 24% (1 mpk), 32% (3 mpk), and 48% (10 mgk) reduction (Figure 11, Table 22). Collectively, both hu305-4F-2l-AF and hu305-5W-3a-AF demonstrated potent intratumoral Treg depletion in vivo, suggesting their potential for antitumor activity. [Table 22]

Claims

1. (i) a heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 14, (b) HCDR2 of SEQ ID NO: 15, and (c) HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) LCDR2 of SEQ ID NO: 8, and (f) LCDR3 of SEQ ID NO: 9; (ii) a heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) HCDR2 of SEQ ID NO: 15, and (c) HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) LCDR2 of SEQ ID NO: 8, and (f) LCDR3 of SEQ ID NO: 9; or (iii) An antibody or antigen-binding fragment thereof that binds to human CCR8, comprising a heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) HCDR2 of SEQ ID NO: 5, and (c) HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) LCDR2 of SEQ ID NO: 8, and (f) LCDR3 of SEQ ID NO:

9.

2. (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 26, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 23; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 16, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 17; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 16, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 20; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO: 22, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% identical to SEQ ID NO:

23.

3. 3. The antibody or antigen-binding fragment of claim 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NOs: 10 and 11, SEQ ID NOs: 26 and 23, SEQ ID NOs: 16 and 17, SEQ ID NOs: 16 and 20, or SEQ ID NOs: 22 and 23 have been inserted, deleted, or substituted.

4. (i) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 26, and a light chain variable region (VL) comprising SEQ ID NO: 23; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 16, and a light chain variable region (VL) comprising SEQ ID NO: 17; (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 16, and a light chain variable region (VL) comprising SEQ ID NO: 20; or (v) the antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 22, and a light chain variable region (VL) comprising SEQ ID NO:

23.

5. Monoclonal antibodies, chimeric antibodies, humanized antibodies, human engineered antibodies, single-chain antibodies (scFv), Fab fragments, Fab' fragments, or F(ab') ２ The antibody or antigen-binding fragment of any one of claims 1 to 4, which is a fragment.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has reduced or no glycosylation or is hypofucosylated.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has reduced or no glycosylation or is defucosylated.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises an increase in bisecting GlcNac structures.

10. The antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the Fc domain is that of IgG1.

11. The antibody or antigen-binding fragment thereof binds to an epitope comprising at least one, two, three, four, five, or six amino acid residues at positions selected from 26, 172, 177, 178, 266, and 269 of the amino acid sequence of HuCCR8.

12. The antibody or antigen-binding fragment thereof binds to an epitope comprising at least one, two, or three amino acid residue(s) at positions selected from positions 172, 177, and 269 of the amino acid sequence of HuCCR8.

13. The antibody or antigen-binding fragment thereof binds to an epitope comprising at least one, two, or three amino acid residues at positions selected from 26, 178, and 266 of the amino acid sequence of HuCCR8.

14. The antibody or antigen-binding fragment thereof binds to an epitope including (1) amino acid residues at positions 20 to 30, (2) amino acid(s) at positions 170 to 180, and (3) amino acid(s) at positions 260 to 270 of the HuCCR8 amino acid sequence.

15. The antibody or antigen-binding fragment binds to an epitope including (1) the amino acid residue at position 26, (2) one, two, or three amino acids (or more) at positions 172, 177, and 178, and (3) one or two amino acids (or more) at positions 266 and 269 of the HuCCR8 amino acid sequence.

16. The antibody or antigen-binding fragment binds to an epitope including (1) one, two, or three amino acid residues at the N-terminus, (2) one, two, or three amino acid(s) at the ECL2 region, and (3) one, two, or three amino acid(s) at the ECL3 region of the HuCCR8 amino acid sequence.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a pharmaceutically acceptable carrier.

18. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment of claim 1.

19. 19. The method of claim 18, wherein the cancer is head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, and small cell lung cancer.

20. 20. The method of claim 19, wherein the antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

21. 21. The method of claim 20, wherein the therapeutic agent is an immune checkpoint inhibitor.

22. 22. The method of claim 21, wherein the immune checkpoint inhibitor is an anti-PD1 antibody.

23. The method of claim 22, wherein the anti-PD1 antibody is BGB-A317.

24. 22. The method of claim 21, wherein the immune checkpoint inhibitor is an anti-TIGIT antibody.

25. The method of claim 22, wherein the anti-TIGIT antibody is BGB-A1217.

26. 22. The method of claim 21, wherein the immune checkpoint inhibitor is a combination of BGB-A1217 and BGB-A317.

27. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 10.

28. A vector comprising the nucleic acid of claim 27.

29. 29. A host cell comprising the nucleic acid of claim 27 or the vector of claim 28.

30. 30. A process for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 29 and recovering the antibody or antigen-binding fragment from the culture.

31. 31. The process of claim 30, wherein the conditioned medium is supplemented with 2F-peracetyl-fucose to produce defucosylated antibodies.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 for use in treating or reducing the likelihood of head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, and small cell lung cancer.

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