Anti-CCR8 antibodies and methods of use

Abstract

Provided are antibodies and antigen-binding fragments thereof that bind to human CCR8, pharmaceutical compositions comprising said antibodies, and the use of the antibodies or the compositions for the treatment of 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 for treating cancer. Background Art

[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 shorter extracellular loops (ECL1-3). The N-terminus and ECL2 are important for ligand binding and activation of CCR8 (Gadhe et al., J Biomol Struct Dyn. [Journal of Biomolecular Structure and Dynamics] 2015; 33(11): 2491-510; Barington L. et al., J Biol Chem. [Journal of Biological Chemistry] July 29, 2016; 291(31): 16208-20). Human CCR8 has four ligands, including CCL1, CCL8, CCL16 and CCL18, and CCL1 is the main ligand of CCR8 in T regulatory cells (Treg) and Th2 cells (Islam, SA et al., J Exp Med. [Journal of Experimental Medicine] 2013 Sep 23;210(10):1889-98; Barsheshet et al., Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences of the United States of America] 2017 Jun 6;114(23):6086-6091; Sokol et al., 2018). CCR8 is normally expressed in some Treg and Th2 cells in the blood (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 positive 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). In addition, CCR8 is also expressed in 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, colorectal 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 USA. 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 is also associated 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 mark an activated tumor Treg population, as these cells have elevated activation markers and are more suppressive than their CCR8-negative counterparts (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 IL10 (Barsheshet et al., Proc Natl Acad Sci USA. 2017 Jun 6; 114(23):6086-6091). Therefore, CCR8 is a promising therapeutic target to specifically target tumor Tregs, which play an important suppressive role in the tumor microenvironment.

[0004] Recent studies in CCR8-null mice suggest that CCR8 is not required for tumor Treg recruitment or function; however, depletion of CCR8 Tregs using an Fc-intact but unmutated anti-mouse CCR8 antibody has anti-tumor 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). In addition to single-agent activity, anti-mouse CCR8 antibodies have also been shown to work in combination with anti-PD1 antibodies. In these studies, anti-mouse CCR8 antibodies did not induce toxicity in tumor-bearing mice and could preserve spleen, thymus, and skin Tregs (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 USA. 2022 Feb 15;119(7):e2114282119). CCR8- / - mice are 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 suggest that CCR8+ tumor Treg depletion is a promising strategy and is unlikely to cause deleterious effects. However, there are currently no CCR8 antibodies approved for use in the treatment of human cancer. Therefore, anti-CCR8 antibodies as disclosed herein may be useful in the treatment of human cancer. Summary of the invention

[0005] The present disclosure relates 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 an antigen-binding fragment thereof that binds to human CCR8.

[0007] This disclosure encompasses the following embodiments.

[0008] An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment specifically binds to human CCR8.

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

[0010] An antibody or an antigen-binding fragment thereof that binds to human CCR8, the antibody or the antibody-binding fragment thereof comprising: (i) a heavy chain variable region comprising (a) HCDR1 (heavy chain complementary 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 complementary 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 complementary 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 complementary 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) a heavy chain variable region comprising (a) HCDR1 (heavy chain complementary 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 complementary determining region 1) of SEQ ID NO:7, (e) LCDR2 of SEQ ID NO:8, and (f) LCDR3 of SEQ ID NO:9.

[0011] An antibody or antigen-binding fragment comprising: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23.

[0012] An antibody or antigen-binding fragment, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids have been inserted, deleted or substituted in 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.

[0013] An antibody or antigen-binding fragment comprising: (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) 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 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] An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is hypofucosylated or afucosylated.

[0017] The antibody or antigen-binding fragment thereof comprises an added bisecting GlcNac structure.

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

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

[0020] An antibody or antigen-binding fragment that binds to an epitope comprising at least one, two or three amino acid residues at positions selected from the group consisting of positions 172, 177 and 269 of the HuCCR8 amino acid sequence.

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

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

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

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

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

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

[0027] The method, wherein the cancer is head and neck cancer, nasopharyngeal cancer, colon cancer, stomach 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.

[0028] The method, 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 inhibitor is BGB-A1217 and BGB-A317.

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

[0036] A vector comprising the nucleic acid.

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

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

[0039] Antibodies or antigen-binding fragments 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), which are HCDR1 comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:14, HCDR2 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:15, and HCDR3 comprising the amino acid sequence of SEQ ID NO:6, and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), which are LCDR1 comprising the amino acid sequence of SEQ ID NO:7, LCDR2 comprising the amino acid sequence of SEQ ID NO:8, and LCDR3 comprising 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), which are HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:5, and HCDR3 comprising the amino acid sequence of SEQ ID NO:6; or HCDR1 comprising the amino acid sequence of SEQ ID NO:14, HCDR2 comprising the amino acid sequence of SEQ ID NO:15, and HCDR3 comprising the amino acid sequence of SEQ ID NO:6; or HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:15, and HCDR3 comprising the amino acid sequence of SEQ ID NO:6; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), which are LCDR1 comprising the amino acid sequence of SEQ ID NO:7, LCDR2 comprising the amino acid sequence of SEQ ID NO:8, and LCDR3 comprising the amino acid sequence of SEQ ID NO:9. LCDR3 of the amino acid sequence of NO:9.

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

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

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

[0047] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region comprising an 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 that is 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 an 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 that is 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, the antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region comprising an 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 in 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 an 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 in 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 comprises: (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, the antibodies of the present disclosure are of IgG1, IgG2, IgG3 or IgG4 isotype. In more specific embodiments, the antibodies of the present disclosure comprise the Fc domain of wild-type human IgG1 (also referred to as human IgG1 wt or huIgG1).

[0051] In one embodiment, the antibodies of the present disclosure bind to human or cyno CCR8 with a binding affinity (EC50) of 0.1 nM to 100 nM. In another embodiment, the antibodies of the present disclosure bind to human CCR8 or Cyno CCR8 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, 1 nM.

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

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

[0054] The present disclosure relates to isolated nucleic acids comprising nucleotide sequences encoding the amino acid sequences of 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 having 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 the antibodies or antigen-binding fragments of the present 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 is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 25, and encodes the VL region of the antibody or antigen-binding fragment of the present disclosure.

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

[0056] In yet another aspect, the disclosure relates to a method for treating a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a CCR8 antibody or antigen-binding fragment thereof, or a CCR8 antibody pharmaceutical composition. In another embodiment, the disease to be 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 DESCRIPTION OF THE DRAWINGS

[0058] FIG1 shows the immunogen human CCR8 protein sequence, a comparison between human, monkey and mouse CCR8, and the composition of human CCR8 N-terminal-Fc protein. Figure 1A The immunogen human CCR8 protein sequence is shown, as well as a comparison between human, monkey and mouse CCR8. Figure 1B The composition of human CCR8-N-terminal-Fc protein is shown.

[0059] Figure 2 Comparison of blocking activity between representative chimeric anti-CCR8 antibodies is shown.

[0060] Figure 3Shown is a comparison of CCR8 binding activity between representative chimeric anti-CCR8 antibodies using Jurkat human CCR8 overexpressing cells.

[0061] Figures 4A-4I Glycan profiles of Ch305-AF, hu305-4F-21-AF, and hu305-4F-2m-AF analyzed by intact and reduced mass spectrometry are shown. Figures 4A-4C The intact and reduced mass spectra of Ch305-AF are shown: Figure 4A The full quality results are shown, Figure 4B and Figure 4C The reduced mass results for HC and LC are shown separately. Figures 4D-4F Intact and reduced mass spectrometry results for hu305-4F-21-AF are shown: Figure 4D The full quality results are shown, Figure 4E and Figure 4F The reduced mass results for HC and LC are shown separately. Figure 4G-4I Intact and reduced mass spectrometry results of hu305-4F-2m-AF are shown. Figure 4G The full quality results are shown, Figure 4H and Fig. 4I The reduced mass results for HC and LC are shown separately. Together, these results indicate that the measured masses are consistent with the theoretical masses and no fucose-related N-glycosylation masses were detected.

[0062] FIG. 5 shows a comparison of CCR8 binding activities of different humanized anti-CCR8 antibodies using Jurkat human CCR8 overexpressing cells. Figure 5A Comparisons between Ch305, hu305-4F-2l, and hu305-4F-2m are shown, while Figure 5B Comparison between Ch305, hu305-5W-3a, and hu305-5P-3a is shown.

[0063] FIG. 6 shows a comparison of the binding activity of humanized Ch305 anti-CCR8 antibodies to CCR8+ primary Tregs ( Fig. 6A Comparison of CCR8 blocking activity between ) and humanized Ch305 anti-CCR8 antibodies ( Figure 6B ).

[0064] Figure 7 A comparison of cyno CCR8 cross-reactivity between different anti-CCR8 antibodies is shown.

[0065] Figure 8 A comparison of NK cell-mediated cytotoxicity of different anti-CCR8 antibodies is shown.

[0066] FIG. 9 shows epitope identification of Ch305 by domain swapping and epitope identification of hu305-5W-3a by alanine scanning. Fig. 9A Results for Ch305 are shown, and Fig. 9B Results for the reference antibody by domain swapping are shown. Fig. 9C Results of key epitope residues of hu305-5W-3a identified by alanine scanning are shown.

[0067] Fig.10 The efficacy of the combination of anti-mouse CCR8 antibody and anti-mouse PD1 antibody in the CT26 syngeneic mouse model is shown.

[0068] Fig.11 Shown are the tumor Treg depletion effects of representative different anti-CCR8 antibodies in the MC38 syngeneic model of human CCR8 KI mice. definition

[0069] Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0070] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0071] Unless the context clearly indicates otherwise, the term "or" means and is used interchangeably with the term "and / or".

[0072] As used herein, the term "anti-cancer agent" refers to any agent useful for treating a cell proliferative disorder such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0073] 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 in Accession No. P51685 (CCR8_HUMAN) or NP_005192.1. The amino acid sequence of cynomolgus monkey ("Cyno") CCR8 (SEQ ID NO: 2) can also be found in Accession No. A0A8J8XUI3_MACFA or XP_015300839.1. The amino acid sequence of mouse CCR8 (SEQ ID NO: 3) can also be found in Accession No. P56484 (CCR8_MOUSE) or NP_031746.1.

[0074] As used herein, the terms "administration / administering" and "treating / treatment", when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ or a biological fluid, mean that an exogenous drug, therapeutic agent, diagnostic agent or composition is in contact with the animal, human, subject, cell, tissue, organ or biological fluid. The treatment of cells encompasses the contact of an agent with a cell and the contact of an agent with a fluid, wherein the fluid is in contact with the cell. The terms "administration" and "treatment" also mean in vitro and ex vivo treatment, for example, treatment of cells by an agent, a diagnostic agent, a binding compound or by another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., a rat, a mouse, a dog, a cat, a rabbit), and most preferably a human. On the one hand, treating any disease or disorder refers to alleviating the disease or disorder (i.e., slowing down or preventing or reducing the development of a disease or at least one clinical symptom thereof). On the other hand, "treat / treating / treatment" refers to alleviating or alleviating at least one physical parameter, including those that the patient may not be able to discern. In yet another aspect, "treat," "treating," or "treatment" refers to regulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), 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.

[0075] In the context of the present disclosure, the term "subject" is a mammal, eg, a primate, preferably a higher primate, such as a human (eg, a patient having or at risk of having a disorder described herein).

[0076] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of an antibody interacts with the antigen at many sites through non-covalent forces. Generally, the more interactions, the stronger the affinity.

[0077] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen non-covalently, reversibly and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), with more conservative regions interspersed therebetween, called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs) arranged in the following order from the amino terminus to the carboxyl terminus: 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 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.

[0078] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. The antibody 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).

[0079] In some embodiments, the anti-CCR8 antibody comprises at least one antigen binding site and at least one 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.

[0080] The term "monoclonal antibody" or "mAb" or "Mab" herein means a population of substantially homogeneous antibodies, that is, the antibody molecules contained in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies comprising different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epitopes. The modifier "monoclonal" indicates the characteristics of antibodies obtained from a substantially homogeneous antibody population and should not be construed as requiring antibodies to be produced by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, e.g., Kohler et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: ALABORATORY 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 (including IgG, IgM, IgD, IgE, IgA), and any subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4). Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained in vivo by injecting cells from a single hybridoma intraperitoneally into mice, such as primary primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluid, or from culture supernatants, using column chromatography methods well known to those skilled in the art.

[0081] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light chain" (about 25 kDa) and a "heavy chain" (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can be defined as a constant region that is primarily responsible for effector function. Typically, human light chains are classified as κ and λ light chains. In addition, human heavy chains are typically classified as α, δ, ε, γ or μ, and the isotype of the antibody is 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 about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

[0082] The variable regions of each light chain / heavy chain (VL / VH) pair form an antibody binding site. Thus, typically, a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical in primary sequence.

[0083] Typically, the variable domains of the heavy and light chains contain three hypervariable regions, also referred to as "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, both the 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 positions of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (see, 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) ("IMGT" numbering scheme).The definition of antigen binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res. [Nucleic Acids Research], 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res. [Nucleic Acids Research], 29:207-209 (2001); MacCallum et al., J. Mol. Biol. [Journal of Molecular Biology], 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 86:9268-9272 (1989); Martin et al., Methods Enzymol. [Methods in Enzymology], 203:121-153 (1991); and Rees et al., in Sternberg MJE (ed.), Protein Structure Prediction [Protein Structure Prediction], Oxford University Press. Press [Oxford University Press], Oxford, 141-172 (1996). For example, according to 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). According to Josiah, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Josiah, 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. According to IMGT, the CDR amino acid residues in VH are numbered as approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered as approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (according to Kabat numbering). According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0084] The term "hypervariable region" means the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from "CDR" (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 Edition Public Health Service, National Institutes of Health, Bethesda, Maryland (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0085] Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as 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.

[0086] As used herein, an antibody "specifically binds" a target protein, meaning that the antibody exhibits preferential binding to the target protein compared to other proteins, but such specificity does not require absolute binding specificity. Antibody "specific binding" or "selective binding" used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological agents (e.g., in a biological sample, blood, serum, plasma, or tissue sample). Thus, under certain specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a specific antigen at least twice the background level and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a specific antigen at least ten (10) times the background binding level and does not specifically bind to other antigens present in the sample in significant amounts.

[0087] The term "human antibody" as used herein means an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine carbohydrate chains. Similarly, a "mouse antibody" or a "rat antibody" means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0088] The term "humanization" or "humanized antibody" means an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain the minimum sequence derived from non-human immunoglobulins. Generally, humanized antibodies will include substantially at least one and typically all of two variable domains, all or substantially all of its hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of FRs are those of human immunoglobulin sequences. Humanized antibodies will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish humanized antibodies from parent rodent antibodies, prefixes "hum", "hu", "Hu" or "h" are added to the antibody clone name. The rodent antibody of humanized form will generally include the same CDR sequence of the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications or for other reasons.

[0089] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has 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. A 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 only a framework region, only a complementary determining region, a framework and a complementary determining region, a variable segment (as defined above), or other combinations of sequences or subsequences comprising a variable region. Sequence identity can be determined using the methods described herein, for example, using BLAST, ALIGN, or another alignment algorithm known in the art to align two sequences. A corresponding human germline nucleic acid or amino acid sequence may have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a reference variable region nucleic acid or amino acid sequence. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutated forms of human germline sequences or antibodies containing consensus framework sequences derived from analysis of human framework sequences, such as described in Knappik et al., J. Mol. Biol. [Journal of Molecular Biology] 296: 57-86, 2000.

[0090] The term "equilibrium dissociation constant (K D , M)” refers to the dissociation rate constant (kd, time -1 ) divided by the association rate constant (ka, time -1 , M -l The equilibrium dissociation constant can be measured using any method known in the art. The antibodies disclosed herein will generally have a dissociation constant of less than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some aspects, is less than about 10 -11 M, 10 -12 M or 10 -13 The equilibrium dissociation constant of M.

[0091] The term "cancer" or "tumor" herein has the broadest meaning as understood in the art, and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to a certain type or location.

[0092] 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, such as its binding affinity to CCR8. In particular, common conservative substitutions of amino acids are well known in the art.

[0093] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., Nuc. Acids Res. [Nucleic Acids Research] 25: 3389-3402, 1977; and Altschul et al., J. Mol. Biol. [Molecular Biology] 215: 403-410, 1990, respectively. Software for performing BLAST analysis is available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or meet some positive threshold score T when aligned with the same word length in the database sequence. T is called the neighborhood word score threshold. These initial neighborhood word hits serve as values ​​for starting a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched 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 will be stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score approaches zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either 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 a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 by default and compares two chains. For amino acid sequences, the BLAST program uses as defaults a wordlength of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) 50, expectation (E) 10, M=5, N=-4 and a comparison of both strands.

[0094] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 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 nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0095] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. [Computer Applications in Biological Sciences] 4: 11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48: 444-453 (1970), which has been incorporated into the GAP program in the GCG software package, using the BLOSUM62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0096] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0097] In the context of nucleic acid, the term "operably connected" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcription regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably connected to a coding sequence if it stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system. Typically, a promoter transcription regulatory sequence operably connected to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcription regulatory sequences (e.g., enhancers) do not need to be physically adjacent to or next to the coding sequence that enhances its transcription.

[0098] In some aspects, the disclosure provides compositions, such as pharmaceutically acceptable compositions, comprising an anti-CCR8 antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

[0099] The compositions disclosed herein can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. A suitable mode 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.

[0100] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that is sufficient to affect the treatment of a disease, disorder, or symptom when administered to a subject to treat at least one clinical symptom of a disease or disorder. A "therapeutically effective amount" may vary with the antibody, disease, disorder, and / or symptoms of a disease or disorder, the severity of the disease, disorder, and / or symptoms of a disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. The appropriate amount in any given case will be apparent to those skilled in the art, or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of a combination of subjects used to effectively treat a disease, disorder, or condition.

[0101] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic conditions or disorders 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 or in separate containers (e.g., capsules, powders, and liquids) of each active ingredient. Powders and / or liquids can be reconstituted or diluted to a desired dose before administration. In addition, such administration also encompasses the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in terms of treating the conditions or disorders described herein.

[0102] As used herein, the phrase "in combination with" means that the anti-CCR8 antibody is administered to a subject simultaneously with, just before, or just after administration of the additional therapeutic agent. In certain embodiments, the anti-CCR8 antibody is administered as a co-formulation with the additional therapeutic agent. DETAILED DESCRIPTION

[0103] The disclosure provides antibodies and antigen binding fragments that specifically bind to human CCR8. In addition, the disclosure provides antibodies with desired pharmacokinetic characteristics and other desired properties, which can therefore be used to reduce the possibility of cancer or treat cancer. The disclosure further provides pharmaceutical compositions comprising antibodies and methods for preparing and using such pharmaceutical compositions for preventing and treating cancer and related disorders. Anti-CCR8 Antibody

[0104] 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, antibodies or antigen-binding fragments thereof generated as described below.

[0105] The present disclosure provides antibodies or antigen-binding fragments that specifically bind 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 antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibody or antigen-binding fragment comprises a HCDR comprising an amino acid sequence of any one of the HCDRs listed in Table 1. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibody comprises (or alternatively consists of) one, two, three or more HCDRs comprising an amino acid sequence of any one of the HCDRs listed in Table 1.

[0106] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CCR8, wherein the antibodies or antigen-binding fragments comprise a VL domain comprising an 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 a LCDR comprising an 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 an amino acid sequence of any one of the LCDRs listed in Table 1.

[0107] Other antibodies or antigen-binding fragments thereof disclosed herein include amino acids that have been altered but have at least 60%, 70%, 80%, 90%, 95% or 99% identity percentage in the CDR regions with the CDR regions disclosed in Table 1. In some aspects, they include amino acid alterations wherein no more than 1, 2, 3, 4 or 5 amino acids are altered in the CDR regions when compared to the CDR regions depicted in the sequences described in Table 1.

[0108] Other antibodies of the disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered, but have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences set forth in Table 1. In some aspects, it includes changes in the amino acid sequence, wherein no more than 1, 2, 3, 4 or 5 amino acids are altered in the variable region when compared to the variable region depicted in the sequence described in Table 1, while maintaining substantially the same therapeutic activity.

[0109] The present disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chains and full-length light chains of antibodies that specifically bind to CCR8. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0110] A sequence listing of the present disclosure is provided in Table 1 below. Table 1. Sequence list Identification of epitopes and antibodies binding to the same epitopes

[0111] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human CCR8 epitopes. In certain aspects, the antibodies and antigen-binding fragments can bind to the same epitope of CCR8.

[0112] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CCR8 antibodies described in Table 1. Therefore, 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 their binding in a statistically significant manner). The ability of the test antibody to inhibit the binding of the antibodies and antigen-binding fragments thereof disclosed herein to CCR8 proves that the test antibody can compete with the antibody or its antigen-binding fragment for binding to CCR8. Without being bound by any one theory, such an antibody can bind to an epitope on CCR8 that is identical or related (e.g., structurally similar or spatially adjacent) to the antibody or its antigen-binding fragment that competes with it. In certain aspects, an antibody that binds to the same epitope on CCR8 as an antibody or its antigen-binding fragment disclosed herein is a human or humanized monoclonal antibody. Such a human or humanized monoclonal antibody can be prepared and separated as described herein. Further changes to the Fc region framework

[0113] In yet other aspects, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change 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 a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0114] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered CIq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0115] On the other hand, one or more amino acid residues are changed to change the ability of the antibody to fix complement. This method is described in, for example, the disclosure WO 94 / 29351 of Bodmer et al. In a specific aspect, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced by one or more allotype amino acid residues of IgG1 subclass and κ isotype. Allotype amino acid residues also include but are not limited to the heavy chain constant region of IgG1, IgG2 and IgG3 subclasses and the light chain constant region of κ isotype, as described in Jefferis et al., MAbs [monoclonal antibodies] 1: 332-338 (2009).

[0116] In another aspect, the Fc region is modified by modifying one or more amino acids to increase 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 the publication WO 00 / 42072 by Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. [Journal of Biological Chemistry] 276: 6591-6604, 2001).

[0117] In yet another aspect, the glycosylation of the modified antibody. For example, an aglycosylated antibody (i.e., the antibody lacks or has reduced glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody to the "antigen". This carbohydrate modification can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation of the site. This aglycosylation can increase the affinity of the antibody to the antigen. Such methods are described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 of Co et al.

[0118] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, 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 increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation pathways. Cells with altered glycosylation pathways have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. Presta, in publication WO 03 / 035835, describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al., WO 99 / 54342, describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999). CCR8 Antibody Production

[0119] 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, while 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 mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0120] The disclosure further provides polynucleotides encoding antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or segments comprising complementary determining regions described herein. In some aspects, the polynucleotides encoding the heavy chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotides 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 aspects, the polynucleotides encoding the light chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotides selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 20 or SEQ ID NO: 23.

[0121] The polynucleotides disclosed herein can encode variable region sequences of anti-CCR8 antibodies. They can also encode variable regions and constant regions of antibodies. Some polynucleotide sequences encode polypeptides comprising the variable regions of the heavy and light chains of one of the exemplary anti-CCR8 antibodies.

[0122] The present disclosure also provides expression vectors and host cells for producing anti-CCR8 antibodies. The selection of expression vectors depends on the expected host cells of the expression vectors. Generally, the expression vectors include promoters and other regulatory sequences (e.g., enhancers) operably connected to polynucleotides encoding anti-CCR8 antibody chains or antigen-binding fragments. In some aspects, in addition to under the control of inducing conditions, inducible promoters are used to prevent the expression of inserted sequences. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The culture of transformed organisms can be expanded under non-inducing conditions, but not in the case of a group of coding sequences that are biased towards host cells to better tolerate their expression products. In addition to promoters, other regulatory elements may also be required or desired for effective expression of anti-CCR8 antibodies or antigen-binding fragments. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be increased by including an enhancer appropriate for the cell system in use (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 the CMV enhancer can be used to increase expression in mammalian host cells.

[0123] The host cell for carrying and expressing the anti-CCR8 antibody chain can be prokaryotic or eukaryotic. Escherichia coli is a prokaryotic host that can be used to clone and express the polynucleotides disclosed herein. Other applicable microbial hosts include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (e.g., replication origins) compatible with host cells. In addition, there will be any number of well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems from bacteriophage λ. Promoters are typically optionally expressed with operator sequences, and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express anti-CCR8 polypeptides. A combination of insect cells and baculovirus vectors may also be used.

[0124] In other aspects, mammalian host cells are used to express and produce the anti-CCR8 polypeptides disclosed herein. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include any normal dead or normal or abnormal immortalized animal or human cells. For example, several suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Press, New York, New York, 1987. Expression vectors for mammalian host cells may include expression control sequences, such as replication origins, promoters and enhancers (see, for example, Queen et al., Immunol. Rev. [Immunology Review] 89: 49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulatable or adjustable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art. Testing and diagnostic methods

[0125] The antibodies or antigen binding fragments disclosed herein can be used for a variety of applications, including but not limited to methods for detecting CCR8. In one aspect, the antibodies or antigen binding fragments can be used to detect the presence of CCR8 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In some aspects, the biological sample includes cells or tissues. In other aspects, such tissues include normal and / or cancerous tissues that express CCR8 at higher levels relative to other tissues.

[0126] In one aspect, the present disclosure provides a method for detecting the presence of CCR8 in a biological sample. In some aspects, the method includes contacting the biological sample with an anti-CCR8 antibody under conditions that allow the antibody to bind 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, urine, tissue, sputum or blood samples.

[0127] Also included is a method for diagnosing an obstacle associated with CCR8 expression. In some aspects, the method includes contacting the test cell with an anti-CCR8 antibody; determining the expression level (quantitative or qualitative) of CCR8 expressed by the test cell by detecting the combination of the anti-CCR8 antibody and the CCR8 polypeptide; and comparing the expression level of the test cell with the CCR8 expression level in a control cell (e.g., a normal cell or non-CCR8 expressing cell of the same tissue source as the test cell), wherein a higher level of CCR8 expression in the test cell compared to the control cell indicates the presence of an obstacle associated with CCR8 expression. Treatment

[0128] The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods of treating CCR8-related disorders or diseases. In one aspect, the CCR8-related disorder or disease is cancer.

[0129] On the one hand, the disclosure provides a method for treating cancer. In some aspects, the method includes applying an effective amount of anti-CCR8 antibodies or antigen binding fragments to patients in need. Cancer may include but is not limited to head and neck cancer, nasopharyngeal carcinoma, 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. More particularly, cancer may include head and neck squamous cell carcinoma, nasopharyngeal carcinoma, colorectal cancer with high microsatellite instability, lung adenocarcinoma, lung squamous cell carcinoma, stomach / gastric adenocarcinoma, triple negative breast cancer, human epidermal growth factor receptor 2 (her2) + breast cancer, pancreatic adenocarcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, bladder urothelial carcinoma, renal cell carcinoma (renal clear cell carcinoma), microsatellite stable colorectal cancer (CRC_MSS), esophageal squamous cell carcinoma, esophageal adenocarcinoma, progesterone / estrogen receptor positive breast cancer, liver hepatocellular carcinoma.

[0130] Antibodies or antigen-binding fragments as disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-lived or long-term. A variety of dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at different time points, push injection administration and pulse infusion.

[0131] The antibody or antigen-binding fragment of the present disclosure can be formulated, administered and applied in a manner consistent with good medical practice. Factors considered in this context include the specific obstacle for treatment, the specific mammal for treatment, the clinical condition of the individual patient, the cause of the obstacle, the delivery site of the medicament, the method of administration, the administration regimen and other factors known to medical practitioners. The antibody does not need to be formulated with one or more medicaments currently used to prevent or treat the obstacle under study. The effective amount of such other medicaments depends on the amount of the antibody present in the formulation, the type of obstacle or treatment and other factors discussed above. These are usually used with the same dosage and route of administration as described herein, or with about 1%-99% of the dosage described herein, or with any dosage and any route determined to be suitable by experience / clinical use.

[0132] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment of the present disclosure will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient once or over a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 100 mg / kg can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose can be about 1 μg / kg to 100 mg / kg or more. For repeated administration over a few days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about twenty, or, for example, about six doses of the antibody). An initial higher loading dose is administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of the therapy can be easily monitored by conventional techniques and assays. Combination therapy

[0133] In one aspect, the CCR8 antibodies of the present disclosure may be used in combination with other therapeutic agents. Other therapeutic agents that may be used with the CCR8 antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents; (e.g., ), 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), CD-20 targeted agents (e.g., rituximab, ofatumumab, 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), CD-19 targeted agents (e.g., MEDI-551, MOR208), 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., BI672).

[0134] The anti-CCR8 antibodies disclosed herein can be used in combination with other therapeutic agents (e.g., other immune checkpoint antibodies). Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD1 antibodies may include, but are not limited to, antibodies disclosed in U.S. Patent No.: 8,735,553. Pembrolizumab (formerly known as 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 the PD1 receptor ligand PD-L1 to PD-L2. Pembrolizumab has been approved for indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is in clinical studies for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Pat. No. US 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin lymphoma.

[0135] Other immune checkpoint antibodies for use in combination with anti-CCR8 antibodies may include anti-TIGIT antibodies. Such anti-TIGIT antibodies may include, but are not limited to, anti-TIGIT antibodies as disclosed in WO 2019 / 129261. Pharmaceutical compositions and formulations

[0136] Also provided are compositions comprising anti-CCR8 antibodies or antigen-binding fragments thereof or polynucleotides comprising sequences encoding anti-CCR8 antibodies or antigen-binding fragments, including pharmaceutical formulations. 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 may further comprise a suitable carrier, such as a pharmaceutically acceptable excipient well known in the art, including a buffer.

[0137] Pharmaceutical formulations of the anti-CCR8 antibodies or antigen-binding fragments described herein are prepared 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)), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 1% to 2% hydroxybenzoic acid) and / or hydroxybenzoic acid. about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including 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 nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc. Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Pat. Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0138] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter including a histidine-acetate buffer.

[0139] Sustained release preparations may 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, eg films, or microcapsules.

[0140] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Examples Example 1. Generation of mouse anti-CCR8 antibodies Mouse immunization

[0141] 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 immunogen combinations including human CCR8 expression plasmid (huCCR8 DNA), L929 cells overexpressing human CCR8 (L929-huCCR8) and L929 cells overexpressing cynomolgus monkey CCR8 (L929-cynoCCR8). Human, cynomolgus monkey and mouse CCR8 protein sequences are listed in Table 1 as SEQ ID NOs: 1 to 3, respectively, and the comparison of human, monkey and mouse CCR8 and the composition of human CCR8 N-terminal-Fc protein are shown in Figure 1. Human CCR8 expression plasmid (huCCR8 DNA) (50 μg per mouse) was delivered intramuscularly by in vivo electroporation using the 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 injected intraperitoneally. 6Cells). Typically, 5-7 immunizations were performed at three-week intervals to induce an effective anti-CCR8 humoral immune response. On the 14th day after each immunization, blood was collected and serum titers against 293T cells overexpressing human CCR8 (293T-huCCR8) and 293T cells overexpressing cynomolgus monkey CCR8 (293T-cynoCCR8) were determined by FACS. Animals with sufficient anti-CCR8 serum antibody levels were selected for final booster immunization. Plasma Cell Screening Using the Beacon Optofluidic System

[0142] 3-5 days after the final boost, spleens were harvested and mashed into single cell suspensions. Plasma cells were isolated using a mouse CD138 positive selection kit (STEMCELL) according to the manufacturer's instructions. 6 / ml of enriched plasma cells were introduced into the channel and injected into the OptoSelect 14KChip TM In order to select huCCR8-specific plasma cells, a density of 1×10 8 293T-huCCR8 cells of 5 μg / ml and Alexa Fluor 647 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) at a concentration of 5 μg / ml were introduced into the channel. After introduction, the freezing valve was opened, and the exposure time of the CY5 channel of the Alexa Fluor647 fluorescent group was set to 3000 ms. Bloom-like positive signals were captured by setting a 6-minute time period and 10 cycles of time-lapse imaging. After completing huCCR8-specific plasma cell screening with 293T-huCCR8 cells, cynoCCR8-specific plasma cells were screened with 293T-cynoCCR8 cells. Plasma cells showing positive signals for 293T-huCCR8 and 293T-cynoCCR8 cells were respectively output to 96-well plates filled with lysis buffer. Example 2. Antibody VH and VL gene cloning, sequencing and chimeric antibody expression

[0143] First-strand cDNA was synthesized according to the manufacturer's instructions and PCR amplified using the Opto Plasma B Discovery cDNA Synthesis Kit. TM Total cDNA was amplified using the Opto PlasmaB Discovery Sanger Prep Kit (Berkeley Optical Technologies) according to the manufacturer's instructions. TM(Berkeley Optical Technologies, Inc.) was used to amplify antibody VH and VL genes. The amplified VH and VL genes were cloned into mammalian expression vectors containing human IgG1 and κ chain constant region genes, respectively. The three HCDRs, three LCDRs, amino acid sequences of VH and VL of the representative antibody Ch305 from clone PBG04-305 (also referred to as clone 305) and the DNA sequences of VH and VL are listed in Table 1 as SEQ ID NOs: 4 to 13. The chimeric antibodies were expressed by Expi293 TM cells and purified by affinity chromatography. Example 3. Determination of Anti-CCR8 Antibody Binding Affinity and Specificity

[0144] To determine binding affinity, purified chimeric anti-CCR8 antibodies were serially diluted and incubated with 293T-huCCR8 or 293T-cynoCCR8 cells for 30 min at 4°C. After washing twice with FACS buffer, diluted AlexaFluor 647 goat anti-human IgG secondary antibody was added and incubated with 293T-huCCR8 or 293T-cynoCCR8 cells in the dark for 30 min at 4°C. After washing twice with FACS buffer, cells were resuspended in FACS buffer and incubated with a Beckton Dickinson LSRFortessa TM Titration curves were generated using sigmoidal dose-response with nonlinear fitting using GraphPad, and the EC50s of representative antibodies are shown in Table 2. As shown in Table 2, all selected clones showed high affinity for 293T-huCCR8 and 293T-cynoCCR8 cells, with EC50s of approximately or below 10 nM. Table 2. EC50 of anti-CCR8 antibodies against 293T-huCCR8 and 293T-cynoCCR8 cells Antibody EC50 (nM) against 293T-huCCR8 EC50 (nM) against 293T-cynoCCR8 Ch210 2.73 2.79 Ch211 1.75 1.57 Ch216-4 1.73 1.77 Ch224 1.00 0.85 Ch230-2 1.71 1.72 Ch231 1.80 1.90 Ch233 0.91 0.71 Ch305 3.67 4.10 Ch327 4.32 4.76 Ch342-2 9.03 12.09 Ch357 7.12 9.96 Ch366-2 11.18 10.93 Ch404 9.08 8.19 Ch407 2.09 2.24 Ch127 4.18 4.44 Ch326 1.36 1.39

[0145] 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 at 4°C for 30 min. After washing twice with FACS buffer, diluted Alexa Fluor 647 TM Goat anti-human IgG secondary antibody was used and incubated with 293T-huCCR1, 293T-huCCR4 and 293T parental cells in the dark at 4°C for 30 min. After washing twice with FACS buffer, the cells were resuspended in FACS buffer and detected by BD LSRFortessa TMOnly very few clones showed non-specific binding to 293T-huCCR1, 293T-huCCR4 or 293T parental cells and were excluded for further characterization, and only antibodies that specifically bound to human and monkey CCR8 were subsequently characterized. Example 4. Blocking and cell binding of purified chimeric anti-CCR8 antibodies

[0146] Anti-CCR8 antibodies block the interaction between CCL1 ligand and CCR8 receptor and inhibit downstream signaling. TM (Eurofins) as a cell-based assay to identify the blocking activity of generated anti-CCR8 antibodies. Briefly, PathHunter β-Arrestin eXpress GPCR Assay Kit TM Human CCR8 expressed on CHO cells of (Eurofin Group) was thawed and seeded into a 96-well assay plate and incubated at 37°C in 5% CO2. After 48 hours, a series of dilutions of anti-CCR8 antibody were added to each well and incubated at 37°C for 30 minutes. 13.7nM human CCL1 was added to each well and incubated at 37°C for 90 minutes. Then, the working detection solution was added to the assay plate and incubated at room temperature for 1 hour in the dark. The plate was read on a luminescence microplate reader. IC50 values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Tables 3 and Figure 2 As shown in , CCR8 chimeric antibody Ch305 showed the strongest ligand blocking activity among all the variants shown. Table 3. Comparison of ligand blocking activity of anti-CCR8 antibodies

[0147] To evaluate the binding activity of anti-CCR8 antibodies to CCR8 expressed on living cells, Jurkat cells were engineered to overexpress human CCR8. Jurkat-human CCR8 cells were seeded into 96-well plates and incubated with a series of 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 with a four-parameter logistic model using GraphPad Prism. As shown in Table 4 and Figure 3 As shown in , CCR8 antibodies have high binding affinity to human CCR8. Table 4. Comparison of binding affinity of anti-CCR8 antibodies Example 5. Humanization of anti-human CCR8 mAb Ch305

[0148] In order to humanize Ch305, the human immunoglobulin gene database was blasted in the IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) websites to search for human germline IgG genes with high homology to the cDNA sequence of the Ch305 variable region. Human IGVH and IGVH that are present at high frequencies in the human antibody library (Glanville et al., Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences of the United States] December 1, 2009; 106(48):20216-21) and are highly homologous to Ch305 were selected. Gene as a template for humanization.

[0149] Humanization was performed by CDR transplantation (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols [Molecular Biology Methods, Vol. 248: Antibody Engineering, Methods and Protocols], Humana Press), and the humanized antibody (hu305) was engineered into a human IgG1 format using an expression vector developed in-house. In the first round of humanization, mutations from mouse to human amino acid residues in the framework region were guided by simulated 3D structures, and mouse framework residues of structural importance were retained in the first version of humanized 305 to maintain the canonical structure of the CDR. In particular, the CDRs of Ch305 Vκ (SEQ ID NO: 7-9) were transplanted into the framework of the human germline variable gene IGVκ4-1 that retained several mouse framework residues. The H-CDRs of Ch305 Vh (SEQ ID NO: 4-6) were transplanted into the framework of the human germline variable gene IGVH4-59 that retained several mouse framework residues. In subsequent humanized variants of Ch305 (humanized 305 antibody or humanized 305), only the N-terminal half of Kabat HCDR2 was retained because, based on simulated 3D structures and mutational analysis, only the N-terminal half is important for antigen binding.

[0150] Humanized antibodies were constructed as human full-length antibodies using in-house developed expression vectors containing the constant regions of human IgG1 and κ chains, respectively, with easily adaptable subcloning sites. Expression and preparation of humanized antibodies derived from clone 305 were achieved by co-transfection of heavy chain and corresponding light chain constructs into ExpiCHO cells and purification using a protein A column. The purified antibodies were concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C.

[0151] Based on the first round humanized 305 template, humanized 305 was further engineered by introducing mutations in the CDR and framework regions to improve binding affinity to human CCR8, human nature, and biophysical properties for human therapeutic use.

[0152] In summary, the engineered version of clone 305 produced humanized monoclonal antibodies hu305-4F-2m (SEQ ID NO: 6-9 and 14-19), hu305-4F-2l (SEQ ID NO: 6-9, 14-16, 18 and 20-21), hu305-5W-3a (SEQ ID NO: 6-9, 14-15 and 22-25) and hu305-5P-3a (SEQ ID NO: 4, 6-9, 15, 23 and 25-27) and characterized their binding and functional activities. Humanized antibodies hu305-4F-2m, hu305-4F-2l, hu305-5W-3a and hu305-5P-3a can also be referred to as 4F-2m, 4F-2l, 5W-3a and 5P-3a. Example 6. Generation of afucosylated anti-CCR8 antibody

[0153] 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 methods to reduce core fucosylation. The work presented here utilizes a fucosyltransferase (FUT) inhibitor, 2F-peracetyl-fucose.

[0154] 186 non-fucosylated (-AF) CCR8 variants, including Ch305-AF, hu305-4F-2l-AF, hu305-4F-2m-AF, hu305-5W-3a-AF and hu305-5P-3a-AF, were generated by transient transfection of plasmids containing heavy and kappa chains in ExpiCHO-s cells. 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.

[0155] All of these antibodies were purified by protein A chromatography capture step and / or other polishing purification steps under platform conditions. The glycan profiles of Ch305-AF, hu305-4F-2l-AF and hu305-4F-2m-AF were analyzed by intact and reduced mass spectrometry, and the results are shown in Tables 5-10 and Figures 4A-4I Glycan analysis showed that no fucose-related N-glycosylation masses were detected in 100 μM 2F-peracetyl-fucose. Table 5. Intact mass results of Ch305-AF Table 6. Reduction quality results of Ch305-AF Table 7. Intact mass results of hu305-4F-21-AF Table 8. Reduction quality results of hu305-4F-21-AF Table 9. Intact mass results of hu305-4F-2m-AF Table 10. Reduction mass results of hu305-4F-2m-AF Example 7. Binding activity of humanized antibodies to human CCR8

[0156] To evaluate the binding activity of anti-CCR8 antibodies to native CCR8 on living cells, Jurkat cells (ATCCTIB-15) were engineered to overexpress human CCR8. Live Jurkat / huCCR8 cells were seeded in 96-well plates and incubated with a series of dilutions of anti-CCR8 antibodies. 647 anti-human IgG Fc antibody (Biolegend catalog number: 410714) was used as a secondary antibody to detect antibody binding to CCR8 expressed on the cell surface. The EC values ​​for dose-dependent binding to human native CCR8 were determined by fitting the dose-response data with a four-parameter logistic model using GraphPad Prism. 50 As shown in Figure 5 and Table 11, the humanized antibodies retained the binding affinity to native human CCR8, indicating that humanization resulted in almost no loss of ligand blocking activity compared to the original clone. Table 11. Jurkat / huCCR8 FACS binding affinity and Emax of different versions of 305 "-" in Table 11 means not tested.

[0157] Treg binding of humanized antibodies was measured on Treg cells from human PBMCs. Briefly, CD25+ cells were isolated from human PBMCs. Cells were stimulated with IL-2, TGFβ, and anti-CD3 / CD28 beads to obtain Treg cells with high CCR8 expression levels. Next, Treg cells were seeded into 96-well plates and incubated with a series of 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 value for dose-dependent binding to human CCR8 was determined by fitting the dose-response data with a four-parameter logistic model using GraphPad Prism. Fig. 6A As shown in Table 12, among the humanized antibodies, hu305-5W-3a has the highest binding affinity to CCR8 on Treg cells. Fig. 6A As shown in , the hu305-5W-3a antibody also had the lowest IC50 in reducing ligand-receptor interaction. Table 12. Comparison of primary Treg binding affinities of humanized 305 antibodies

[0158] The CCR8 blocking activity of the humanized antibody was also determined. Figure 6B and are shown in Table 13. Table 13. Comparison of blocking activity of humanized antibodies Example 8. Determination of cross-reactivity of anti-CCR8 antibodies

[0159] To evaluate the cross-reactivity of humanized antibodies with cynomolgus monkey (cyno) CCR8, cynoCCR8 was overexpressed in 293T. 293T-cynoCCR8 cells were seeded into 96-well plates and incubated with a series of 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 cyno CCR8 were determined by fitting the dose-response data with a four-parameter logistic model using GraphPad Prism. Figure 7 As shown in Table 14, the humanized CCR8 antibodies showed good binding affinity to cyno CCR8. Table 14. Comparison of cross-reactivity of humanized Ch305 antibodies Example 9. CCR8 Antibody Enhances NK Cell-Mediated ADCC

[0160] ADCC induced by anti-CCR8 antibodies was determined by measuring the depletion of Treg cells by NK cells after antibody treatment. Briefly, the NK92-MI-CD16a-F158 cell line was generated as effector cells. Tregs expressing CCR8 were used as target cells. Equal amounts (5x10 4 ) target cells and effector cells, and added a series of dilutions of anti-CCR8 antibodies, and co-cultured at 3°C ​​for 24 hours. Cytotoxicity was evaluated by flow cytometry. The results were calculated by the formula shown below:

[0161] like Figure 8 As shown in Table 15, all anti-CCR8 antibodies tested had ADCC activity, with hu305-5W-3a showing the highest potency in killing CCR8-expressing targets by ADCC in a dose-dependent manner. Table 15. Comparison of cytotoxicity of humanized antibodies Example 10. Epitope analysis of anti-CCR8 antibodies

[0162] To analyze the epitope of anti-CCR8 antibodies, Jurkat cells overexpressing human CCR8 (Jurkat-huCCR8) were incubated with 200 nM unlabeled anti-CCR8 antibody or human IgG1 isotype control antibody (primary antibody) at 4°C for 30 min. Without washing, 200 nM APC-labeled anti-CCR8 antibody or human IgG1 isotype control antibody (secondary antibody) was added and incubated for another 30 min at 4°C. After washing twice with FACS buffer, the cells were resuspended in FACS buffer and stained on a BD LSR FortessaTM The samples were collected on a cell analyzer. The readings were normalized to the % of maximum binding of a single APC-labeled antibody compared to an unlabeled human IgG1 isotype control antibody as the primary antibody. The epitope grouping results are summarized in Table 16. Low values ​​in the table cells indicate that their antibody pairs have similar epitopes. As shown in Table 16, Ch211 and Ch305 are in the same epitope grouping and have similar epitopes; Ch366-2, reference anti-CCR8 antibody 4A19 (as disclosed in WO 2021194942 A1) and 433H (commercially available from BD Biosciences) are in the same epitope grouping; and Ch127 and Ch326 are in two other different epitope bins. Table 16. Epitope grouping by FACS

[0163] In order to determine whether anti-CCR8 antibodies bind to the N-terminal domain of human CCR8, ELISA plates were coated with human CCR8 N-terminal domain-mouse Fc tag fusion protein (SEQ ID NO: 28 and 29) overnight at 4 ° C. After washing and blocking, serially diluted anti-CCR8 antibodies were added to the plate and incubated at room temperature for 1 hour. After washing, diluted HRP goat anti-human IgG Fc or HRP goat anti-mouse κ chain (for 433H) secondary antibodies were added to the plate and incubated at room temperature for 45 minutes. After washing, TMB substrate was added to the plate for color development and then terminated by adding TMB substrate stop solution. The absorbance was read at 450nm using an ELISA reader. Titration curves were generated using the S-shaped dose-response of GraphPad's nonlinear fitting, and EC50 is shown in Table 17. As shown in Table 17, Ch127, Ch326, Ch366-2, 4A19 and 433H exhibit high binding affinity to human CCR8 N-terminal protein, with EC50 <1nM. In contrast, CH305 exhibits low binding affinity to human CCR8 N-terminal protein, with EC50 of 5nM. CH211 exhibits very weak binding activity to human CCR8 N-terminal protein only at the highest concentration of 640nM, indicating that they bind to the CCR8 N-terminal loop. In contrast, Ch305 exhibits low binding affinity to human CCR8 N-terminal protein, with EC50 of 5nM, which may be a partial CCR8 N-terminal loop binder. Ch211 exhibits very weak binding activity to human CCR8 N-terminal protein only at the highest concentration of 640nM. It is a non-CCR8 N-terminal loop binder. Table 17. EC50 of anti-CCR8 antibodies against human CCR8 N-terminal protein Example 11. Epitope mapping of anti-CCR8 antibodies

[0164] To investigate the binding epitope of the anti-CCR8 mAb Ch305, domain-swapped CCR8s were generated by transplanting amino acids from the mouse CCR8 extracellular region into human CCR8, or vice versa. More specifically, the mapping strategy exploited the fact that Ch305 can bind to human CCR8 (SEQ ID NO: 1), but not mouse CCR8 (SEQ ID NO: 3) ( Fig. 9A ). The amino acid sequences of human CCR8 (huCCR8) and mouse CCR8 (muCCR8) are compared in Figure 1A The amino acid residues of the extracellular domain of CCR8 are underlined. The extracellular domain (ECD) of mouse and human CCR8 proteins is shown in Figure 1A The genes encoding full-length human CCR8 (SEQ ID NO: 1, UniProtKB: P51685) and mouse CCR8 (SEQ ID NO: 3, UniProtKB: P56484) were cloned into the pcDNA3.4 vector. Chimeric constructs were generated in which the extracellular domains of human CCR8, including the N-terminal region (amino acids 1-35 of SEQ ID NO: 1), the ECL1 region (amino acids 94-107 of SEQ ID NO: 1), the ECL2 region (amino acids 172-202 of SEQ ID NO: 1), and the ECL3 region (amino acids 264-280 of SEQ ID NO: 1) were replaced by the corresponding regions of mouse CCR8, namely the N-terminal region (amino acids 1-33 of SEQ ID NO: 3), the ECL1 region (amino acids 92-105 of SEQ ID NO: 3), the ECL2 region (amino acids 170-200 of SEQ ID NO: 3), and the ECL3 region (amino acids 262-278 of SEQ ID NO: 3). According to this exchange strategy, the absence or reduction of anti-CCR8 antibodies in a particular construct means that a particular domain (such as ECL1) is at least partially required for the binding of the anti-CCR8 antibody.

[0165] Table 18 shows in tabular form which mouse (murine) regions are exchanged for human CCR8 (human). Table 18. Domain-swapped chimeric CCR8 constructs

[0166] Plasmids containing these chimeric CCR8 constructs as well as human CCR8 (CCR8-hu8) and mouse CCR8 (CCR8-mo8) as controls were used to transfect Expi293 TM Cells were transiently expressed and then incubated with serially diluted purified Ch305 and reference antibody 10A11 from Shionogi (US 2022 / 0064312A1). Binding of each construct was evaluated using Alexa Fluor 647 rabbit anti-human IgG (Cat. No. 309-605-008, Jackson ImmunoResearch). Fig. 9A The data in show that Ch305 specifically binds to human CCR8 but not to mouse CCR8. In CCR8 constructs containing mouse ECL2 or ECL3 substitution regions, binding is significantly reduced, indicating that these regions play a key role in anti-CCR8 antibody binding. In addition, replacing the human CCR8 N-terminal region with the mouse CCR8 N-terminal (CCR8_Ch1) also slightly impairs affinity, which means that the human CCR8 N-terminal promotes the binding activity of human CCR8 to Ch305. At the same time, replacing the mouse CCR8 N-terminal with the human CCR8 N-terminal (CCR8_Ch5) rescues the binding activity of mouse CCR8 to Ch305 to a certain extent, and its binding activity is much lower than that of wild-type human CCR8 to Ch305, which means that the human CCR8 N-terminal promotes but weakly or partially promotes the binding activity of human CCR8 to Ch305. These data indicate that Ch305 binds to the N-terminal region of human CCR8, ECL2, and ECL3, which means that the epitope of Ch305 and its humanized antibody consists of the N-terminal region of human CCR8, ECL2, and ECL3. In contrast, the reference antibody 10A11 only binds to the N-terminal region of CCR8, but not to ECL1, ECL2, and ECL3, that is, the epitope of 10A11 on human CCR8 is only located at the N-terminus of human CCR8 ( Fig. 9B ).

[0167] In order to more precisely determine the specific amino acid residues that are critical for the binding of the antibody to human CCR8, point mutations were introduced into the ECL2, ECL3 and N-terminal regions of human CCR8, as shown in the first column of Table 19. To evaluate the effect of each mutant, humanized anti-CCR8 antibody hu305-5W-3a was used to react with the above-transfected Expi293 cells at concentrations of 50nM, 10nM and 1nM (the results are summarized in Table 19). The key amino acids bound by hu305-5W-3a are marked with "X", indicating a loss of >50% compared to wild-type huCCR8, which is considered significant.

[0168] Preliminary data indicate that antibody hu305-5W-3a recognizes amino acids D26 of the N-terminal domain, Y172, E177, D178 of ECL2, and M266, L269 of ECL3 (numbered according to SEQ ID NO: 1). Mutations at these sites significantly reduce the binding of hu305-5W-3a. In contrast, the 10A11 reference antibody recognizes D14, Y17, D19 on the N-terminal domain of huCCR8, as mutations at these sites completely abolish binding, indicating that there is no overlap in epitopes between reference antibody 10A11 and hu305-5W-3a. To further confirm the critical role of these sites for binding, transfected Expi293 cells were stained with hu305-5W-3a serially diluted from 200 nM and then detected with Alexa Fluor 647 rabbit anti-human IgG (Catalog No.: 309-605-008, Jackson ImmunoResearch) to obtain a complete binding curve. Fig. 9C The data in Table 20 show the effects of these key residues on the binding of hu305-5W-3a to CCR8. Among these sites, Y172, E177, and L269 are considered to be the most critical epitopes, and replacing them with Ala will result in a more than 4-fold decrease in EC50. D178 and M266 are considered to be secondary epitopes with a moderate effect on Hu305-5W-3a binding. Replacing them with Ala will result in an approximately 3-fold decrease in EC50. Finally, compared with other key residues, D26 in the N-terminal domain has the least effect, and its mutation results in a more than 2-fold decrease in EC50. In summary, these data are very consistent with the view that Hu305-5W-3a recognizes a conformational epitope composed of the N-terminus, ECL2, and ECL3. Table 19. Summary of epitope mapping of Hu305-5W-3a Table 20. Binding of Hu305-5W-3a to each key epitope mutant Example 12. Anti-mCCR8 mAb and anti-PD-1 mAb synergistically inhibit tumor growth in the CT26 mouse colon cancer model

[0169] The efficacy of anti-murine CCR8 antibodies as single agents or in combination with anti-mPD-1 Ab (Ch15mt) was tested in the CT26 mouse colon cancer model. Ch15mt is a murinized 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 the mouse IgG1 isotype (Chen X. et al., Front Immunol. [Immunology Frontier]. 2022 Feb 22;13:828319). Asp265 on the heavy chain was further replaced with Ala to eliminate Fc receptor binding. Murine CT26 colon cancer cells (3x10 4 ) were implanted subcutaneously into BALB / C mice. After tumor cell implantation, tumor length (L) and width (W) were measured twice a week using an electronic caliper and expressed in mm using the following formula: 3 Indicates volume: V = 0.5 (axb 2 ), where a and b are the long and short diameters of the tumor, respectively. When the tumor reaches a size of approximately 100 mm 3 When the mean volume of the tumor was 1.57 × 10 / cm2, the mice were randomly assigned to 7 groups (15 per group) and injected intraperitoneally with mAb once a week for three weeks. PBS was administered as a vehicle control. Tumor growth inhibition (TGI) was calculated using the following formula: TGI = [1 - (treated Tt - treated T0) / (vehicle Tt - vehicle T0)] × 100, where treated Tt = treated tumor volume at time t, treated T0 = treated tumor volume at time 0, vehicle Tt = vehicle tumor volume at time t, and vehicle T0 = vehicle tumor volume at time 0.

[0170] The results demonstrated that single-agent anti-murine CCR8 (0.3 mpk) and anti-mPD-1 (3 mpk) treatment induced relatively strong anti-tumor efficacy, and as a single-agent therapy, anti-CCR8 antibody showed a higher TGI than anti-PD-1 antibody ( Fig.10 In addition, the combined administration of anti-CCR8 and anti-PD-1 mAbs showed a synergistic effect, as evidenced by higher TGI levels. Combination treatment induced almost complete tumor rejection in 8 of 16 mice, while the tumor-free rates in the anti-CCR8 and anti-PD-1 monotherapy groups were only 0 and 2 of 15 ( Fig.10 and Table 21). This suggests that anti-CCR8 treatment can synergistically enhance the anti-tumor activity of anti-PD-1 in a mouse colon tumor model. Table 21. Combination efficacy of anti-mouse CCR8 and anti-mouse PD-1 in the murine CT26 colon tumor mouse model Example 13. Pharmacodynamic response of anti-CCR8 mAb treatment in the MC38 mouse colon cancer model

[0171] The pharmacodynamic Treg depletion effects of anti-CCR8 antibodies hu305-4F-21-AF and hu305-5W-3a-AF were measured in the MC38 mouse colon cancer model. Murine MC38 colon tumor cells (purchased from Kerafast, ENH204-FP) (1x10 6 ) were implanted subcutaneously into human CCR8 transgenic C57 mice (Shanghai Model Organisms Technology Co., Ltd. (SMOC), Shanghai, China). 3 The length (L) and width (W) of the tumor were measured with an electronic caliper at 2 or 3-day intervals starting at 4:00 p.m. and expressed in mm using the following formula: 3 Indicates volume: V = 0.5 (axb 2 ), where a and b are the major and minor diameters of the tumor.

[0172] When the tumor reaches a size of approximately 200-250 mm 3 The mice were randomly assigned to 7 groups ( Fig.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 injected intraperitoneally ( Fig.11 PBS was used as a vehicle control. Tumors were harvested on day 3 after treatment (n=6 mice / treatment group). Single cells of the tumors were dissociated using gentle MACS and then filtered using a 40 μm cell strainer. After resuspension, cells were counted using a Count star fluorescent cell analyzer. 3×10 6 Cells were plated per well of a 96-well plate and stained with flow cytometry antibodies for immune cell subsets and functional markers. TM (BD Biosciences) by flow cytometry and analyzed by FlowJo TM Software analysis results.

[0173] The results demonstrated that administration of hu305-4F-2l-AF and hu305-5W-3a-AF induced a similar degree of intratumoral Treg depletion. Compared with the vehicle group, administration of hu305-4F-2l resulted in a 31% (1 mpk), 42% (3 mpk), 52% (10 mgk) decrease in Treg population (% Foxp3+CD4+ / CD3+), while administration of hu305-5W-3a resulted in a 24% (1 mpk), 32% (3 mpk), 48% (10 mgk) decrease in Treg population (% Foxp3+CD4+ / CD3+). Fig.11, Table 22). In conclusion, both hu305-4F-21-AF and hu305-5W-3a-AF showed effective intratumoral Treg depletion effects in vivo, indicating anti-tumor potential. Table 22. Pharmacodynamic Treg depletion effects of anti-CCR8 antibody treatment in the MC38 mouse colon cancer model treat Dose (mpk) % Treg depletion PBS - NA hu305-4F-2l-AF 0.1 31 hu305-4F-2l-AF 1 42 hu305-4F-2l-AF 3 52 hu305-5W-3a-AF 0.1 24 hu305-5W-3a-AF 1 32 hu305-5W-3a-AF 3 48

Claims

1. An antibody or antigen-binding fragment thereof that binds to human CCR8, the antibody or antibody-binding fragment thereof comprising: (i) a heavy chain variable region comprising (a) HCDR1 (heavy chain complementary 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 complementary 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 complementary 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 complementary 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) 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. The antibody or antigen-binding fragment of claim 1, comprising: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

23.

3. The antibody or antigen-binding fragment of claim 2, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids have been inserted, deleted or substituted in 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.

4. The antibody or antigen-binding fragment of claim 1, comprising: (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) a heavy chain variable region (VH) comprising SEQ ID NO:22 and a light chain variable region (VL) comprising SEQ ID NO:

23.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, which 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.

6. The antibody or antigen-binding fragment of 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 of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is aglycosylated or is hypofucosylated.

8. The antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is aglycosylated or is afucosylated.

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

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

11. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising at least one, two, three, four, five or six amino acid residues at positions selected from numbers 26, 172, 177, 178, 266 and 269 of the HuCCR8 amino acid sequence.

12. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising at least one, two or three amino acid residues at positions selected from the group consisting of numbers 172, 177 and 269 of the HuCCR8 amino acid sequence.

13. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising at least one, two or three amino acid residues at positions selected from the group consisting of numbers 26, 178 and 266 of the HuCCR8 amino acid sequence.

14. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising (1) amino acid residues at positions 20-30, (2) one or more amino acids at positions 170-180, and (3) one or more amino acids at positions 260-270 of the HuCCR8 amino acid sequence.

15. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising (1) the amino acid residue at position 26, (2) one, two or three amino acids at positions 172, 177 and 178, and (3) one or two amino acids at positions 266 and 269 of the HuCCR8 amino acid sequence.

16. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope comprising (1) one, two or three amino acid residues at the N-terminal position, (2) one, two or three amino acids at the ECL2 region, and (3) one, two or three amino acids 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, and further comprising a pharmaceutically acceptable carrier.

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

19. The method of claim 18, wherein the cancer is head and neck cancer, nasopharyngeal cancer, colon cancer, stomach 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. The method of claim 19, wherein the antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

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

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

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

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

32. An antibody or antigen-binding fragment thereof as described in 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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