Anti-CCR8 antibodies and uses thereof

Abstract

The present invention provides anti-CCR8 antibodies and antigen-binding fragments thereof, as well as methods for making and using said anti-CCR8 antibodies and antigen-binding fragments thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 197,271, filed June 4, 2021, and U.S. Provisional Patent Application No. 63 / 236,551, filed August 24, 2021, each of which is incorporated by reference in its entirety herein.

[0002] FIELD OF THEINVENTION The present invention relates to the field of oncology. The present invention relates to anti-CCR8 antibodies with ADCC activity and the treatment of cancer patients with said antibodies. The anti-CCR8 antibodies of the present invention bind to a unique epitope and do not inhibit ligand binding to CCR8. The present invention also relates to Treg-depleting antibodies and methods of treatment with one or more of bispecific T cell engager molecules, agonists of T cell costimulatory receptors, and antagonists of the PD-1 / PD-L1 pathway. [Background technology]

[0003] 2. Background of the Invention CC chemokine receptor type 8 (CCR8) is a member of the β-chemokine receptor family and is a seven-transmembrane G protein-coupled receptor with an extracellular N-terminus of 35 amino acids. CCL1 is the ligand for CCR8, ccr8, and CCL1-induced CCR8 signaling occurs via G proteins. Binding of CCL1 to CCR8 results in intracellular calcium flux that can be inhibited by pertussis toxin. Downstream activation of the RAS / ERK1 / 2 MAP kinase pathway has been demonstrated in CCR8-expressing cell lines (see, for example, Louahed et al. (2003) “CCR8-dependent activation of the RAS / MAPK pathway mediates anti-apoptotic activity of I-309 / CCL1 and vMIP-I”, European J. of Immunology; 33(2): 494-501).

[0004] Chemokines and their receptors are important for the migration of various cell types to sites of inflammation. Previous studies of CCR8 and its ligands suggest a role in the proper positioning of activated T cells at the site of antigen challenge and within specific regions of peripheral and lymphoid tissues. CCR8 may also contribute to the regulation of monocyte chemotaxis and thymocyte apoptosis (Tiffany et al.(1997) “Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309”, J Exp Med; Jul 7; 186(1):165-70).

[0005] Recent data in multiple tumor types have demonstrated that CCR8 expression is a marker for tumor-specific T regulatory (Treg) cells (see, for example, Plitas et al. (2016) “Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer”, Immunity; 45(5):1122-1134; Villarreal et al. (Sept. 2018) “Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced Responses in Colon Cancer” Tumor Biol. And Immun.). CCR8 is expressed at much higher frequencies and levels on the surface of tumor-resident Tregs compared to circulating or normal tissue Tregs and conventional T effector (Teff) cells. Infiltration of Treg cells in solid tumors is associated with poor clinical outcomes, and Tregs suppress anticancer immune responses by inhibiting the cytotoxicity of Teff cells.

[0006] Some data suggest that inhibiting CCR8 function can reduce Treg suppression of immune responses in tumors, thereby promoting inflammatory responses and reducing tumor volume. Another therapeutic approach is to deplete tumor Treg cells by anti-CCR8 antibody-dependent cell death (e.g., ADCC). In ADCC, anti-CCR8 antibodies preferentially bind to CCR8 on tumor-resident Tregs, and depleting these tumor-resident Tregs by ADCC can induce redirected T cell lysis of tumor-resident CCR8+ Tregs while sparing Tregs in normal tissues that express little or no CCR8. (e.g., Tanaka et al. (2019) “Targeting Treg cells in cancer immunotherapy” European J. of Immun.; 49(8)1140-1146).

[0007] Treg depleting antibodies, such as anti-CCR8 antibodies, are known in the art. For example, PCT application publication WO 2018 / 181425 describes antibodies against CCR8 with ADCC activity for use in treating cancer, and discloses a commercially available rat anti-mouse CCR8 antibody (SA214G2).

[0008] There is a need for alternative anti-CCR8 antibodies that 1) can bind to human and cynomolgus monkey CCR8 on tumor-resident Treg cells, 2) result in specific depletion of tumor-resident Treg cells, 3) exhibit an acceptable pharmacokinetic profile (compared to anti-CCR8 antibodies that bind to different epitopes), and / or 4) exhibit sufficient efficacy for the treatment of cancer.

[0009] There is also a need for anti-CCR8 antibodies that do not inhibit the binding of a ligand (such as CCL1) to CCR8 and / or that bind to an epitope of CCR8 that includes at least one of residues 1-12 of SEQ ID NO:31. Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention The present invention provides an antibody or antigen-binding fragment thereof that binds to human CC chemokine receptor type 8 (CCR8), the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SEQ ID NO: 1; (b) an HCDR2 amino acid sequence of SEQ ID NO: 2; (c) an HCDR3 amino acid sequence of SEQ ID NO: 3; (d) a light chain complementarity determining region (LCDR) 1 amino acid sequence of KSSQSVLYSSNNX1NYLA (SEQ ID NO: 1235), where X1 is K or R; (e) an LCDR2 amino acid sequence of SEQ ID NO: 5; and (f) an LCDR3 amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment comprises an LCDR1 amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 13 and the following amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNX1NYLAWYX2QKPGQX3PKLLISWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYSIPITFGGGTKVEIKR (SEQ ID NO: 1236), where X1 is K or R, X2 is H or Q, and / or X3 is S or P. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 13, and a light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 363. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain (HC) amino acid sequence of SEQ ID NO: 15, and a light chain (LC) amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 365. In other embodiments, the antibody or antigen-binding fragment comprises two HCs and two LCs, both HCs comprise the amino acid sequence of SEQ ID NO: 15, and both LCs comprise the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises two HCs and two LCs, both HCs comprise the amino acid sequence of SEQ ID NO: 15, and both LCs comprise the amino acid sequence of SEQ ID NO: 365. In one embodiment, the antibody or antigen-binding fragment is an antibody.

[0011] The present invention provides an antibody, or antigen-binding fragment thereof, that binds to human CCR8, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR) and the LC comprises a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 13, and the LCVR comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 365. In one embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 365. In one embodiment, the HC has the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 573, and the LC has the amino acid sequence given by SEQ ID NO: 16. In another embodiment, the antibody comprises two HCs and two LCs, each HC having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 573, and each LC having the amino acid sequence of SEQ ID NO: 16. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0012] The invention also provides an antibody, or antigen-binding fragment thereof, that binds to human CCR8, comprising an HCDR1 amino acid sequence of SEQ ID NO: 839, an HCDR2 amino acid sequence of SEQ ID NO: 840, an HCDR3 amino acid sequence of SEQ ID NO: 841, an LCDR1 amino acid sequence of SEQ ID NO: 842, an LCDR2 amino acid sequence of SEQ ID NO: 843, and an LCDR3 amino acid sequence of SEQ ID NO: 844. In some embodiments, the antibody or antigen-binding fragment comprises an HCVR amino acid sequence of SEQ ID NO: 1017 and an LCVR amino acid sequence of SEQ ID NO: 1018. In some embodiments, the antibody comprises an HC amino acid sequence of SEQ ID NO: 1125 or SEQ ID NO: 1237 and an LC amino acid sequence of SEQ ID NO: 1126. In some embodiments, the antibody comprises an HC amino acid sequence of SEQ ID NO: 1125 and an LC amino acid sequence of SEQ ID NO: 1126. In some embodiments, the antibody comprises an HC amino acid sequence of SEQ ID NO: 1237 and an LC amino acid sequence of SEQ ID NO: 1126. For example, an antibody may comprise two HCs and two LCs, where both HCs comprise the amino acid sequence of SEQ ID NO: 1125 or SEQ ID NO: 1237, and both LCs comprise the amino acid sequence of SEQ ID NO: 1126. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0013] The invention also provides an antibody, or antigen-binding fragment thereof, that binds human CCR8, comprising an HCDR1 amino acid sequence of SEQ ID NO: 845, an HCDR2 amino acid sequence of SEQ ID NO: 846, an HCDR3 amino acid sequence of SEQ ID NO: 847, an LCDR1 amino acid sequence of SEQ ID NO: 848, an LCDR2 amino acid sequence of SEQ ID NO: 849, and an LCDR3 amino acid sequence of SEQ ID NO: 850. In some embodiments, the antibody or antigen-binding fragment comprises an HCVR amino acid sequence of SEQ ID NO: 1019 and an LCVR amino acid sequence of SEQ ID NO: 1020. In some embodiments, the antibody comprises an HC amino acid sequence of SEQ ID NO: 1127 or SEQ ID NO: 1238 and an LC amino acid sequence of SEQ ID NO: 1128.

[0014] The present invention further relates to a peptide having an HCDR1 amino acid sequence of: (a) X1X2GX4H (SEQ ID NO: 1233), where (i) X1 is N, S, D, G, T, or R, (ii) X2 is C, N, Y, S, or F, and (iii) X4 is M or F; and (b) SEQ ID NOs: 648, 654, 660, 666, 672, 678, 684, 690, 696, 702, 708, 714, 720, 726, 732, 738, 744, 750, 756, 762, 768, 774, 780, 786, 792, 798, 804, 810, 816, 822, 828, 834, 840, 842, 846, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 860, 861, 862, 863, 864, 865, 866, 870, 872, 876, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 900, 901, 902, 903, 904, 905, 906, 907, 908, 910, 912, 914, 920, 6, 852, 858, 867, 873, 879, 885, 891, 897, 903, 909, 915, 921, 927, 933, 939, or 945, or a variant thereof that contains one to four amino acid substitutions or is at least 90% identical to any one of the foregoing HCDR2 amino acid sequences; and (c) SEQ ID NOs: 649, 655, 661, 667, 673, 679, 685, 691, 697, 703, 709, 715, 721, 727, 733, 739, 745, 751, 757, 763, 769, 775, 781, 787, 790, 891, 897, 903, 909, 915, 921, 927, 933, 939, 745, 751, 757, 763, 769, 775, 781, 787, 7 ...2, 893, 894, 903, 909, 915, 921, 927, 933, 939, 945, 951, 957, 963, 969, 975, 981, 93, 799, 805, 811, 817, 823, 829, 835, 847, 853, 859, 868, 874, 880, 886, 892, 898, 904, 910, 916, 922, 928, 934, 940, or 946, or a variant thereof that contains one to four amino acid substitutions or is at least 90% identical to any one of the foregoing HCDR3 amino acid sequences; and (d) SEQ ID NOs: 650, 656, 662, 668, 674, 680, 686, 692, 698, 704, 710, 716, 722, 728, 734, 740, 746, 752, 758, 764, 770, 776, 782, 788, 794, 800, 806, 812, 818, 824, 830, 836, 848, 854, 860, 863, 869, 875, 881, 887, 893, 899, 905, 911, 917, 923, 929, 935, or 941, or a variant thereof comprising one to four amino acid substitutions or which is at least 90% identical to any one of the foregoing LCDR1 amino acid sequences; and (e) RX2X3X4RPS (SEQ ID NO: 1234),(ii) X3 is S, T, N, I, F, or A, and (iii) X4 is N or V; and (f) an LCDR2 amino acid sequence of SEQ ID NOs: 652, 658, 664, 670, 676, 682, 688, 694, 700, 706, 712, 718, 724, 730, 736, 742, 748, 754, 760, 766, 772, 778, 784, 790, 796, 802, 808, 814, 820, 826, 828, 830, 832, 834, 836, 838, 840, 842, 846, 848, 850, 852, 856, 860, 862, 864, 866, 870, 876, 878, 882, 884, 886, 888, 890, 902, 902, 904, 906, 912, 918, 924, 930, 936, 942, 948, 954, 960, 962, 970, 976, 982, 984, 990, 996, 1002, 1008, 1014, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1109, 1111, 1121, 112 The present invention provides an antibody or antigen-binding fragment thereof that binds to human CCR8, comprising an LCDR3 amino acid sequence of 32, 838, 850, 856, 862, 865, 871, 877, 883, 889, 895, 901, 907, 913, 919, 925, 931, 937, or 943, or a variant thereof that contains 1 to 4 amino acid substitutions, or is at least 90% identical to any one of the foregoing LCDR3 amino acid sequences. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 647, 653, 659, 665, 671, 677, 683, 689, 695, 701, 707, 713, 719, 725, 731, 737, 743, 749, 755, 761, 767, 773, 779, 785, 791, 797, 803, 809, 815, 821, 827, 833, 845, 851, 857, 866, 872, 878, 884, 890, 896, 902, 908, 914, 920, 926, 932, 938, or 944. In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 651, 657, 663, 669, 675, 681, 687, 693, 699, 705, 711, 717, 723, 729, 735, 741, 747, 753, 759, 765, 771, 777, 783, 789, 795, 801, 807, 813, 819, 825, 831, 837, 849, 855, 861, 864, 870, 876, 882, 888, 894, 900, 906, 912, 918, 924, 930, 936, or 942. In some embodiments, the HCVR is selected from the group consisting of SEQ ID NOs: 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013,1015, 1019, 1021, 1023, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, or 1052 amino acid sequences. In some embodiments, the LCVR comprises the amino acid sequence of SEQ ID NO: 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1020, 1022, 1024, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, or 1051. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0015] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of: (a) a HCVR comprising the amino acid sequence of SEQ ID NO: 1019 and a LCVR comprising the amino acid sequence of SEQ ID NO: 1020; (b) a HCVR comprising the amino sequence of SEQ ID NO: 1021 and a LCVR comprising the amino sequence of SEQ ID NO: 1022; (c) a HCVR comprising the amino sequence of SEQ ID NO: 1023 and a LCVR comprising the amino sequence of SEQ ID NO: 1024; (d) a HCVR comprising the amino sequence of SEQ ID NO: 1026 and a LCVR comprising the amino sequence of SEQ ID NO: 1025; (e) a HCVR comprising the amino sequence of SEQ ID NO: 1028 and a LCVR comprising the amino sequence of SEQ ID NO: 1027; (f) a HCVR comprising the amino sequence of SEQ ID NO: 1030 and a LCVR comprising the amino sequence of SEQ ID NO: 1029; (g) a HCVR comprising the amino sequence of SEQ ID NO: 1032 and a LCVR comprising the amino sequence of SEQ ID NO: 1031; (h) a HCVR comprising the amino sequence of SEQ ID NO: 1034 and a LCVR comprising the amino sequence of SEQ ID NO: 1033; (i) a HCVR comprising the amino sequence of SEQ ID NO: 1036 and LCVR comprising the amino sequence of SEQ ID NO: 1035, (j) HCVR comprising the amino sequence of SEQ ID NO: 1038 and LCVR comprising the amino sequence of SEQ ID NO: 1037, (k) HCVR comprising the amino sequence of SEQ ID NO: 1040 and LCVR comprising the amino sequence of SEQ ID NO: 1039, (l) HCVR comprising the amino sequence of SEQ ID NO: 1042 and LCVR comprising the amino sequence of SEQ ID NO: 1041, (m) HCVR comprising the amino sequence of SEQ ID NO: 1044 and LCVR comprising the amino sequence of SEQ ID NO: 1043, (n) HCVR comprising the amino sequence of SEQ ID NO: 1046 and LCVR comprising the amino sequence of SEQ ID NO: 1045, (o) HCVR comprising the amino sequence of SEQ ID NO: 1048 and LCVR comprising the amino sequence of SEQ ID NO: 1047, (p) HCVR comprising the amino sequence of SEQ ID NO: 1050 and LCVR comprising the amino sequence of SEQ ID NO: 1049, or (q) HCVR comprising the amino sequence of SEQ ID NO: 1052 and LCVR comprising the amino sequence of SEQ ID NO: 1051. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0016] In some embodiments, the antibody comprises an HC amino acid sequence of SEQ ID NO: 1127, 1129, 1131, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, or 1238-1254, and an LC amino acid sequence of SEQ ID NO: 1128, 1130, 1132, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, or 1159.For example, in some embodiments, the antibody comprises: (a) the HC amino acid sequence of SEQ ID NO:1127 or SEQ ID NO:1238 and the LC amino acid sequence of SEQ ID NO:1128; (b) the HC amino acid sequence of SEQ ID NO:1129 or SEQ ID NO:1239 and the LC amino acid sequence of SEQ ID NO:1130; (c) the HC amino acid sequence of SEQ ID NO:1131 or SEQ ID NO:1240 and the LC amino acid sequence of SEQ ID NO:1132; (d) the HC amino acid sequence of SEQ ID NO:1134 or SEQ ID NO:1241 and the LC amino acid sequence of SEQ ID NO:1133 (e) the HC amino acid sequence of SEQ ID NO:1136 or SEQ ID NO:1242 and the LC amino acid sequence of SEQ ID NO:1135, (f) the HC amino acid sequence of SEQ ID NO:1138 or SEQ ID NO:1243 and the LC amino acid sequence of SEQ ID NO:1137, (g) the HC amino acid sequence of SEQ ID NO:1140 or SEQ ID NO:1244 and the LC amino acid sequence of SEQ ID NO:1139, (h) the HC amino acid sequence of SEQ ID NO:1142 or SEQ ID NO:1245 and the LC amino acid sequence of SEQ ID NO:1141, (i) SEQ ID NO:1144 or SEQ ID NO: (j) the HC amino acid sequence of SEQ ID NO:1146 or SEQ ID NO:1247 and the LC amino acid sequence of SEQ ID NO:1145; (k) the HC amino acid sequence of SEQ ID NO:1148 or SEQ ID NO:1248 and the LC amino acid sequence of SEQ ID NO:1147; (l) the HC amino acid sequence of SEQ ID NO:1150 or SEQ ID NO:1249 and the LC amino acid sequence of SEQ ID NO:1149; (m) the HC amino acid sequence of SEQ ID NO:1152 or SEQ ID NO:1250 and the LC amino acid sequence of SEQ ID NO: (n) the HC amino acid sequence of SEQ ID NO:1154 or SEQ ID NO:1251 and the LC amino acid sequence of SEQ ID NO:1153, (o) the HC amino acid sequence of SEQ ID NO:1156 or SEQ ID NO:1252 and the LC amino acid sequence of SEQ ID NO:1155, (p) the HC amino acid sequence of SEQ ID NO:1158 or SEQ ID NO:1253 and the LC amino acid sequence of SEQ ID NO:1157, or (q) the HC amino acid sequence of SEQ ID NO:1160 or SEQ ID NO:1254 and the LC amino acid sequence of SEQ ID NO:1159. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv). In some embodiments, the antibody or antigen-binding fragment is a Fab. In certain embodiments, the antibody or antigen-binding fragment is a single chain Fab (scFab). In some embodiments, the antigen-binding fragment comprises the amino acid sequence of any one of the anti-CCR8 antibodies or antigen-binding fragments thereof of the present invention.

[0018] The present invention also provides an antibody or antigen-binding fragment thereof that binds to human CCR8, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR), and the LC comprises a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:7, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:367, or SEQ ID NO:377, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:9, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:369, or SEQ ID NO:379, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:11, and wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:12. In one embodiment, HCDR2 comprises the amino acid sequence of SEQ ID NO:8. In one embodiment, HCDR2 comprises the amino acid sequence of SEQ ID NO:367. In one embodiment, HCDR2 comprises the amino acid sequence of SEQ ID NO:377. In one embodiment, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, LCDR1 comprises the amino acid sequence of SEQ ID NO: 369. In one embodiment, LCDR1 comprises the amino acid sequence of SEQ ID NO: 379. In one embodiment, HCVR comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 372, or SEQ ID NO: 382, ​​and LCVR comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 373, or SEQ ID NO: 383. In one embodiment, HCVR comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, HCVR comprises the amino acid sequence of SEQ ID NO: 372. In one embodiment, HCVR comprises the amino acid sequence of SEQ ID NO: 382. In one embodiment, LCVR comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, LCVR comprises the amino acid sequence of SEQ ID NO: 373. In one embodiment, LCVR comprises the amino acid sequence of SEQ ID NO: 383. In one embodiment, HC comprises the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 374, or SEQ ID NO: 384, and LC comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 375, or SEQ ID NO: 385. In one embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 374. In one embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 384.In one embodiment, the LC comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, the LC comprises the amino acid sequence of SEQ ID NO: 375. In one embodiment, the LC comprises the amino acid sequence of SEQ ID NO: 385. In another embodiment, the antibody comprises two HCs and two LCs, each HC comprises the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 374, or SEQ ID NO: 384, and each LC comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 375, or SEQ ID NO: 385. In one embodiment, each HC comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, each HC comprises the amino acid sequence of SEQ ID NO: 374. In one embodiment, each HC comprises the amino acid sequence of SEQ ID NO: 384. In one embodiment, each LC comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, each LC comprises the amino acid sequence of SEQ ID NO: 375. In one embodiment, each LC comprises the amino acid sequence of SEQ ID NO: 385. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0019] The present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:352 and SEQ ID NO:353, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:354 and 355, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:573 and 355, respectively. In one embodiment, the antibody is an IgG1 of antibody 1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0020] The present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:362 and SEQ ID NO:363, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:364 and 365, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:574 and 365, respectively. In one embodiment, the antibody is an IgG1 of antibody 1.1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0021] The present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, the antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:370, and SEQ ID NO:371, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:372 and SEQ ID NO:373, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:374 and 375, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:575 and 375, respectively. In one embodiment, the antibody is an IgG1 of antibody 2.1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0022] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:376, SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:379, SEQ ID NO:380, and SEQ ID NO:381, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:382 and SEQ ID NO:383, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:384 and 385, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:576 and 385, respectively. In one embodiment, the antibody is an IgG1 of antibody 2.2. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0023] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, and SEQ ID NO:391, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:392 and SEQ ID NO:393, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:394 and 395, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:577 and 395, respectively. In one embodiment, the antibody is an IgG1 of antibody 3.0. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0024] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:402 and SEQ ID NO:403, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:404 and 405, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:578 and 405, respectively. In one embodiment, the antibody is an IgG1 of antibody 4.0. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0025] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:406, SEQ ID NO:407, SEQ ID NO:408, SEQ ID NO:409, SEQ ID NO:410, and SEQ ID NO:411, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:412 and SEQ ID NO:413, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:414 and 415, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:579 and 415, respectively. In one embodiment, the antibody is an IgG1 of antibody 4.1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0026] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:416, SEQ ID NO:417, SEQ ID NO:418, SEQ ID NO:419, SEQ ID NO:420, and SEQ ID NO:421, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:422 and SEQ ID NO:423, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:424 and 425, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:580 and 425, respectively. In one embodiment, the antibody is an IgG1 of antibody 4.2. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0027] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:426, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, and SEQ ID NO:431, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:432 and SEQ ID NO:433, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:434 and 435, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:581 and 435, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.0. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0028] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, and SEQ ID NO:441, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:442 and SEQ ID NO:443, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:444 and 445, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:582 and 445, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0029] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, and SEQ ID NO:451, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:452 and SEQ ID NO:453, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:454 and 455, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:583 and 455, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.2. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0030] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460, and SEQ ID NO:461, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:462 and SEQ ID NO:463, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:464 and 465, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:584 and 465, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.3. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0031] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, and SEQ ID NO:471, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:472 and SEQ ID NO:473, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:474 and 475, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:585 and 475, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.4. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0032] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, and SEQ ID NO:481, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:482 and SEQ ID NO:483, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:484 and 485, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:586 and 485, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.5. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0033] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:486, SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489, SEQ ID NO:490, and SEQ ID NO:491, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:492 and SEQ ID NO:493, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:494 and 495, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:587 and 495, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.6. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0034] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, and SEQ ID NO:501, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:502 and SEQ ID NO:503, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:504 and 505, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:588 and 505, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.7. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0035] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, and SEQ ID NO:511, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:512 and SEQ ID NO:513, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:514 and 515, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:589 and 515, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0036] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, and SEQ ID NO:521, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:522 and SEQ ID NO:523, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:524 and 525, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:590 and 525, respectively. In one embodiment, the antibody is an IgG1 of antibody 5.9. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0037] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, and SEQ ID NO:531, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:532 and SEQ ID NO:533, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:534 and 535, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:591 and 535, respectively. In one embodiment, the antibody is an IgG1 of antibody 6.0. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0038] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, and SEQ ID NO:541, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:542 and SEQ ID NO:543, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:544 and 545, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:592 and 545, respectively. In one embodiment, the antibody is an IgG1 of antibody 6.1. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0039] The present invention provides an antibody or antigen-binding fragment that binds to CCR8, the antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, and SEQ ID NO:551, respectively. In one embodiment, the antibody or antigen-binding fragment comprises HCVR and LCVR comprising the amino acid sequences of SEQ ID NO:552 and SEQ ID NO:553, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:554 and 555, respectively. In another embodiment, the antibody comprises HC and LC comprising the amino acid sequences of SEQ ID NO:593 and 555, respectively. In one embodiment, the antibody is an IgG1 of antibody 6.2. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0040] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HCVR, LCVR, HC, and / or LC amino acid residues as disclosed in Table 16, Table 17, Table 19, and / or Table 20.

[0041] In another embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 of the antibody or antigen-binding fragment of the invention comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 sequence of the anti-CCR8 antibody or antigen-binding fragment of the invention listed herein. In one embodiment, the amino acid sequence is at least 70% identical. In one embodiment, the amino acid sequence is at least 80% identical. In one embodiment, the amino acid sequence is at least 90% identical. In another embodiment, the amino acid sequence is at least 95% identical. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0042] In another embodiment, the HCVR and / or LCVR comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the HCVR and / or LCVR sequence of an anti-CCR8 antibody or antigen-binding fragment of the invention listed herein. In one embodiment, the amino acid sequence is at least 70% identical. In one embodiment, the amino acid sequence is at least 80% identical. In one embodiment, the amino acid sequence is at least 90% identical. In another embodiment, the amino acid sequence is at least 95% identical. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0043] In another embodiment, the HC and / or LC comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the HC and / or LC sequence of an anti-CCR8 antibody or antigen-binding fragment of the invention listed herein. In one embodiment, the amino acid sequence is at least 70% identical. In one embodiment, the amino acid sequence is at least 80% identical. In one embodiment, the amino acid sequence is at least 90% identical. In another embodiment, the amino acid sequence is at least 95% identical. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0044] In one embodiment, the invention provides an afucosylated antibody of the invention.In one embodiment, the anti-CCR8 antibody of the invention is a human or humanized antibody.

[0045] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment of the present invention can be administered simultaneously with, prior to, or after a variety of drugs and treatments commonly used in the treatment of cancer, such as, for example, chemotherapeutic agents, non-chemotherapeutic agents (e.g., checkpoint inhibitors, including anti-PD-1 or anti-PD-L1 inhibitors, such as antagonist antibodies), antineoplastic agents, and / or radiation. For example, administration can be before, during, and / or after any of the treatments described herein. Examples of chemotherapeutic agents are described herein and include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone®), actinomycin D, cycloheximide, camptothecin (or water-soluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), antineoplastic antibiotics, such as adriamycin (doxorubicin) and daunomycin, and all chemotherapeutic agents described herein. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0046] In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the present invention may be administered simultaneously with, prior to, or subsequent to a checkpoint inhibitor, such as a PD-1 antagonist antibody or a PD-L1 antagonist antibody. The term "PD-1 antagonist antibody" refers to an antibody that specifically binds to PD-1 and reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its ligands, such as PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1 and / or PD-L2. The term "PD-L1 antagonist antibody" refers to an antibody that specifically binds to PD-L1 and reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-L1 with the PD-1 receptor. In some embodiments, a PD-L1 antagonist antibody inhibits the binding of PD-L1 to PD-1.In some embodiments, the PD-1 antagonist antibody is antibody 20C1.006 (which comprises the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 72-77, respectively; the VL and VH amino acid sequences of SEQ ID NOs: 78 and 79, respectively; and the LC and HC amino acid sequences of SEQ ID NOs: 80 and 81, respectively), zelvalimab (which comprises the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 32-37, respectively; the VL and VH amino acid sequences of SEQ ID NOs: 38 and 39, respectively; the LC amino acid sequence of SEQ ID NO: 40; and the HC amino acid sequence of SEQ ID NO: 41 or 636), antibody 20A2.003 (which comprises the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 42-47, respectively); , HCDR3 amino acid sequences; VL and VH amino acid sequences of SEQ ID NOs: 48 and 49, respectively; and LC and HC amino acid sequences of SEQ ID NOs: 50 and 51, respectively), antibody 22D4.006 (including LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 52-57, respectively; VL and VH amino acid sequences of SEQ ID NOs: 58 and 59, respectively; and LC and HC amino acid sequences of SEQ ID NOs: 60 and 61, respectively), or antibody 22D4.017 (including LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 62-67, respectively; VL and VH amino acid sequences of SEQ ID NOs: 68 and 69, respectively; and LC and HC amino acid sequences of SEQ ID NOs: 70 and 71, respectively). In one embodiment, the PD-1 antagonist antibody is pembrolizumab. In another embodiment, the PD-1 antagonist antibody is nivolumab. In yet another embodiment, the PD-1 antagonist antibody is cemiplimab. In a particular embodiment, the PD-1 antagonist antibody is zelvalimab. Zelvalimb is also known as AMG 404 and 20C1.009. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0047] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody or antigen-binding fragment, where the anti-CCR8 antibody or antigen-binding fragment does not inhibit ligand binding to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0048] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody, where the anti-CCR8 antibody has ADCC.

[0049] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody, which does not inhibit ligand binding to CCR8 and has ADCC. In one embodiment, the anti-CCR8 antibody further has acceptable PK. In one embodiment, the anti-CCR8 antibody binds to an epitope comprising at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises 8 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 9 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 31. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 22. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO: 31 is SEQ ID NO: 82. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody binding to the CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO:82.In some embodiments, the epitope is determined by an anti-CCR8 antibody that binds to a T4R mutation in cynomolgus CCR8. In one embodiment, binding to a T4R mutation is measured by a cell-based affinity assay that compares an antibody that binds to a cell expressing a cynomolgus cell CCR8 that contains a T4R mutation with an antibody that binds to a cell expressing a wild-type cynomolgus CCR8 (containing a threonine at position 4). In some embodiments, the anti-CCR8 antibody binds to the threonine at position 4 when it indicates reduced binding to a CCR8 that contains a T4R mutation. In certain embodiments, the anti-CCR8 antibody binds to the threonine at position 4 when it indicates undetectable binding to a CCR8 that contains a T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 that contains a T4R mutation comprises the amino acid sequence given by SEQ ID NO: 556.

[0050] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody, which does not inhibit ligand binding to CCR8 and has ADCC. In one embodiment, the anti-CCR8 antibody further has acceptable PK. In one embodiment, the anti-CCR8 antibody binds to an epitope consisting of at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of 8 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 9 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 31. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 22. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by antibodies that bind to the CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In some embodiments, the epitope is determined by an anti-CCR8 antibody that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to the T4R mutation is measured by a cell-based affinity assay that compares antibody binding to cells expressing cynomolgus CCR8 with T4R mutation with antibody binding to cells expressing wild-type cynomolgus CCR8 (with threonine at position 4). In some embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates reduced binding to CCR8 with T4R mutation. In certain embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates undetectable binding to CCR8 with T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 556.

[0051] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody, which does not inhibit ligand binding to CCR8 and has ADCC. In one embodiment, the anti-CCR8 antibody further has acceptable PK. In one embodiment, the anti-CCR8 antibody binds to an epitope comprising at least one residue of SEQ ID NO:82. In one embodiment, the epitope comprises at least two residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least three residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least four residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least five residues of SEQ ID NO:82. In one embodiment, the epitope comprises six or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises seven or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises eight or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises nine or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises 10 or more residues of SEQ ID NO: 82. In one embodiment, the epitope comprises 11 or more residues of SEQ ID NO: 82. In one embodiment, the epitope comprises all 12 amino acid residues of SEQ ID NO: 82. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed in human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 22. In some embodiments, the epitope is determined by an anti-CCR8 antibody that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to the T4R mutation is measured by a cell-based affinity assay that compares antibody binding to cells expressing cynomolgus CCR8 with T4R mutation with antibody binding to cells expressing wild-type cynomolgus CCR8 (with threonine at position 4). In some embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates reduced binding to CCR8 with T4R mutation. In certain embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates undetectable binding to CCR8 with T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 556.

[0052] The present invention provides a method of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody, which does not inhibit ligand binding to CCR8 and has ADCC. In one embodiment, the anti-CCR8 antibody further has acceptable PK. In one embodiment, the anti-CCR8 antibody binds to an epitope consisting of at least one residue of SEQ ID NO:82. In one embodiment, the epitope consists of at least two residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least three residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least four residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least five residues of SEQ ID NO:82. In one embodiment, the epitope consists of six or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of seven or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of eight or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of nine or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of 10 or more residues of SEQ ID NO: 82. In one embodiment, the epitope consists of 11 or more residues of SEQ ID NO: 82. In one embodiment, the epitope consists of all 12 amino acid residues of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 22. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In some embodiments, the epitope is determined by an anti-CCR8 antibody that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to the T4R mutation is measured by a cell-based affinity assay that compares antibody binding to cells expressing cynomolgus CCR8 with T4R mutation with antibody binding to cells expressing wild-type cynomolgus CCR8 (with threonine at position 4). In some embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates reduced binding to CCR8 with T4R mutation. In certain embodiments, the anti-CCR8 antibody binds to threonine at position 4 when it indicates undetectable binding to CCR8 with T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 556.

[0053] The present invention also provides a method of treating cancer in a patient comprising administering to the patient an effective amount of an anti-CCR8 antibody or antigen-binding fragment of the present invention. In one embodiment, the cancer is a solid tumor. In more particular embodiments, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In one embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer. In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is head and neck squamous cell carcinoma. In some embodiments, the method further comprises administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody. In some such embodiments, the PD-1 antagonist antibody or PD-L1 antagonist antibody is administered before, simultaneously with, and / or after administration of the anti-CCR8 antibody or antigen-binding fragment. In certain embodiments, the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or zelvalimab. In other specific embodiments, the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0054] In some embodiments, the method further comprises administering a chemotherapeutic agent to the patient. In some embodiments, the method comprises administering to the patient an anti-CCR8 antibody or antigen-binding fragment of the invention and a chemotherapeutic agent. In some such embodiments, the chemotherapeutic agent may be administered before, simultaneously with, or after administration of the anti-CCR8 antibody or antigen-binding fragment of the invention. In some embodiments, the method comprises administering to the patient an anti-CCR8 antibody of the invention, a PD-1 or PD-L1 antagonist antibody, and a chemotherapeutic agent. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0055] The present invention provides methods of treating cancer in a patient comprising administering to the patient an effective amount of a Treg-depleting antibody, a bispecific T cell engager molecule, a T cell costimulatory receptor agonist, and / or a PD-1 / PD-L1 pathway antagonist. In some embodiments, the patient is administered two of the Treg-depleting antibody, the bispecific T cell engager molecule, the T cell costimulatory receptor agonist, and the PD-1 / PD-L1 pathway antagonist. In some embodiments, the patient is administered three of the Treg-depleting antibody, the bispecific T cell engager molecule, the T cell costimulatory receptor agonist, and the PD-1 / PD-L1 pathway antagonist. In some embodiments, the patient is administered each of the Treg-depleting antibody, the bispecific T cell engager molecule, the T cell costimulatory receptor agonist, and the PD-1 / PD-L1 pathway antagonist. In some embodiments, patients are administered a bispecific T cell engager molecule, an agonist of a T cell costimulatory receptor, and an antagonist of the PD-1 / PD-L1 pathway.

[0056] The present invention also provides a method of treating cancer in a patient comprising administering to the patient an effective amount of a Treg-depleting antibody and one or more of a bispecific T cell engager molecule, a T cell costimulatory receptor agonist, and a PD-1 / PD-L1 pathway antagonist. In one embodiment, the method comprises administering to the patient an effective amount of a Treg-depleting antibody and a bispecific T cell engager molecule. In one embodiment, the method comprises administering to the patient an effective amount of a Treg-depleting antibody and a PD-1 / PD-L1 pathway antagonist. In one embodiment, the method comprises administering to the patient an effective amount of a Treg-depleting antibody and a T cell costimulatory receptor agonist. In one embodiment, the method comprises administering to the patient an effective amount of a Treg-depleting antibody, a bispecific T cell engager molecule, and a PD-1 / PD-L1 pathway antagonist. In one embodiment, the method comprises administering to the patient effective amounts of a Treg-depleting antibody, a bispecific T cell engager molecule, an antagonist of the PD-1 / PD-L1 pathway, and an agonist of a T cell costimulatory receptor.

[0057] In some embodiments, the Treg depleting antibody is an anti-CCR8 antibody. In some embodiments, the Treg depleting antibody is an anti-CTLA4 antibody. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody and a bispecific T cell engager molecule. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody and a bispecific T cell engager molecule of the invention. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CTLA-4 antibody and a bispecific T cell engager molecule. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody and a PD-1 / PD-L1 pathway antagonist. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody and a PD-1 / PD-L1 pathway antagonist. In some such embodiments, the PD-1 / PD-L1 pathway antagonist is a PD-1 antagonist antibody.Antibody 20C1.006 (comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NOs: 72 to 77, respectively; VL and VH amino acid sequences set forth in SEQ ID NOs: 78 and 79, respectively; and LC and HC amino acid sequences set forth in SEQ ID NOs: 80 and 81, respectively), zelvalimab (comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NOs: 32 to 37, respectively; VL and VH amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively; the LC amino acid sequence of SEQ ID NO: 40; and the HC amino acid sequence of SEQ ID NO: 41 or 636), antibody 20A2.003 (comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NOs: 42 to 47, respectively); and LC and HC amino acid sequences of SEQ ID NOs: 50 and 51, respectively), antibody 22D4.006 (comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 52-57, respectively; VL and VH amino acid sequences of SEQ ID NOs: 58 and 59, respectively; and LC and HC amino acid sequences of SEQ ID NOs: 60 and 61, respectively), or antibody 22D4.017 (comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 amino acid sequences of SEQ ID NOs: 62-67, respectively; VL and VH amino acid sequences of SEQ ID NOs: 68 and 69, respectively; and LC and HC amino acid sequences of SEQ ID NOs: 70 and 71, respectively). In one embodiment, the PD-1 antagonist antibody is pembrolizumab. In another embodiment, the PD-1 antagonist antibody is nivolumab. In yet another embodiment, the PD-1 antagonist antibody is cemiplimab. In a particular embodiment, the PD-1 antagonist antibody is zelvalimab.

[0058] In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody, a bispecific T cell engager molecule, and a PD-1 antagonist antibody. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody, a bispecific T cell engager molecule, and a PD-1 antagonist antibody of the invention. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CTLA-4 antibody, a bispecific T cell engager molecule, and a PD-1 antagonist antibody.

[0059] In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody, a bispecific T cell engager molecule, a PD-1 antagonist antibody, and a T cell costimulatory receptor agonist. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CCR8 antibody, a bispecific T cell engager molecule, a PD-1 antagonist antibody, and a T cell costimulatory receptor agonist of the invention. In one embodiment, the method comprises administering to the patient an effective amount of an anti-CTLA-4 antibody, a bispecific T cell engager molecule, a PD-1 antagonist antibody, and a T cell costimulatory receptor agonist.

[0060] In some embodiments, the agonist of a T cell costimulatory receptor is an agonist of 4-1BB.

[0061] In some embodiments, the bispecific T cell engager molecule comprises the amino acid sequence of any one of SEQ ID NOs: 87-345 in Table 15.

[0062] In some embodiments, the Treg depleting antibody is an antibody against CTLA-4, CCR8, CD25, TIGIT, CCR4, CD27, CD28, CD39, CD40, CD73, ICOS, OX40, 4-1BB, GITR, LAYN, IL1R2, or IL21R.

[0063] In some embodiments, the Treg depleting antibody is an anti-CTLA-4 antibody.

[0064] In some embodiments, the Treg-depleting antibody is an anti-CCR8 antibody. In some such embodiments, the anti-CCR8 antibody is capable of depleting Treg cells. In some embodiments, the anti-CCR8 antibody is an anti-CCR8 antibody with ADCC activity. In some embodiments, the anti-CCR8 antibody does not inhibit ligand binding to CCR8. In some embodiments, the anti-CCR8 antibody binds to human and cynomolgus CCR8 on tumor-resident Treg cells. In some embodiments, the anti-CCR8 antibody binds to an epitope of CCR8, the epitope comprising at least one residue from positions 1-12 of SEQ ID NO:31. In some embodiments, the anti-CCR8 antibody binds to an epitope of CCR8, the epitope consisting of at least one residue from positions 1-12 of SEQ ID NO:31. In certain embodiments, the epitope comprises a threonine at position 4 of SEQ ID NO:22. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO:31 is SEQ ID NO:82. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to the CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In some embodiments, the epitope is determined by an anti-CCR8 antibody that binds to a T4R mutation in cynomolgus CCR8. In one embodiment, binding to a T4R mutation is measured by a cell-based affinity assay that compares an antibody that binds to cells expressing cynomolgus CCR8 containing a T4R mutation with an antibody that binds to cells expressing wild-type cynomolgus CCR8 (containing a threonine at position 4). In some embodiments, the anti-CCR8 antibody binds to the threonine at position 4, indicating reduced binding to CCR8 containing a T4R mutation. In certain embodiments, the anti-CCR8 antibody binds to the threonine at position 4, demonstrating undetectable binding to CCR8 containing a T4R mutation.In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprising a T4R mutation comprises the amino acid sequence given by SEQ ID NO: 556. In some embodiments, the anti-CCR8 antibody exhibits an acceptable pharmacokinetic profile. In certain embodiments, the anti-CCR8 antibody is an anti-CCR8 antibody of the present invention.

[0065] In one embodiment, the agonist of the immune cell costimulatory receptor is an agonist of CD2, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB), TNFRSF14 (HVEM), TNFRSF18 (GITR), TNFR2, or ICOS. In a particular embodiment, the agonist of the immune cell costimulatory receptor is a 4-1BB agonist antibody.

[0066] In one embodiment, the PD-1 / PD-L1 pathway antagonist is a PD-1 antagonist antibody or a PD-L1 antagonist antibody as described herein. In some embodiments, the PD-1 antagonist antibody is any one of antibody 20C1.006, zelvalimab, antibody 20A2.003, antibody 22D4.006, or antibody 22D4.017. In one embodiment, the PD-1 antagonist antibody is pembrolizumab. In another embodiment, the PD-1 antagonist antibody is nivolumab. In yet another embodiment, the PD-1 antagonist antibody is cemiplimab. In certain embodiments, the PD-1 antagonist antibody is zelvalimab.

[0067] In one embodiment, the Treg-depleting antibody is administered simultaneously with the bispecific T cell engager molecule, the agonist of an immune cell costimulatory receptor, and / or the antagonist of the PD-1 / PD-L1 pathway. In one embodiment, the Treg-depleting antibody, the bispecific T cell engager molecule, the agonist of an immune cell costimulatory receptor, and / or the antagonist of the PD-1 / PD-L1 pathway are administered at different times. In a particular embodiment, the patient is administered a Treg-depleting antibody, a bispecific T cell engager molecule, an agonist of an immune cell costimulatory receptor, and an antagonist of the PD-1 / PD-L1 pathway. In another particular embodiment, the patient is administered an anti-CCR8 antibody, a bispecific T cell engager molecule, a 4-1BB agonist antibody, and an antagonist of the PD-1 / PD-L1 pathway. In a particular embodiment, the anti-CCR8 antibody is an anti-CCR8 antibody of the invention.

[0068] In one embodiment, the cancer is a solid tumor. In a more particular embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In one embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer. In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is head and neck squamous cell carcinoma.

[0069] The present invention provides an anti-CCR8 antibody or antigen-binding fragment of the present invention for use in therapy. The present invention also provides an anti-CCR8 antibody or antigen-binding fragment of the present invention for use in the treatment of cancer. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is non-small cell lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In more particular embodiments, the cancer is non-small cell lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, or triple-negative breast cancer. In some embodiments, the use further comprises administering a PD-1 antagonist antibody or a PD-L1 antagonist antibody to the patient. In some such embodiments, the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered before, simultaneously with, and / or after administration of the anti-CCR8 antibody or antigen-binding fragment. In certain embodiments, the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or zelvalimab. In other particular embodiments, the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab. In some embodiments, the use further comprises administering a chemotherapeutic agent to the patient. In some such embodiments, the chemotherapeutic agent may be administered before, simultaneously with, or after administration of the anti-CCR8 antibody or antigen-binding fragment. In some embodiments, the use comprises administering to the patient an anti-CCR8 antibody or antigen-binding fragment of the invention and a chemotherapeutic agent. In some embodiments, the use comprises administering to the patient an anti-CCR8 antibody or antigen-binding fragment of the invention, a PD-1 or PD-L1 antagonist antibody, and a chemotherapeutic agent. In one embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof is an antibody.

[0070] The present invention provides an anti-CCR8 antibody or antigen-binding fragment of the present invention for the manufacture of a medicament for treating cancer. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In a more specific embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0071] The present invention also provides a pharmaceutical composition comprising an anti-CCR8 antibody, or antigen-binding fragment thereof, of the present invention, and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0072] In one embodiment, an anti-CCR8 antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 594 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 595. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 354 and the LC comprises the amino acid sequence of SEQ ID NO: 355. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 573 and the LC comprises the amino acid sequence of SEQ ID NO: 355.

[0073] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 596 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 597. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 364 and the LC comprises the amino acid sequence of SEQ ID NO: 365. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 574 and the LC comprises the amino acid sequence of SEQ ID NO: 365.

[0074] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 598 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 599. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 374 and the LC comprises the amino acid sequence of SEQ ID NO: 375. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 575 and the LC comprises the amino acid sequence of SEQ ID NO: 375.

[0075] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 600 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 601. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 384 and the LC comprises the amino acid sequence of SEQ ID NO: 385. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 576 and the LC comprises the amino acid sequence of SEQ ID NO: 385.

[0076] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 602 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 603. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 394 and the LC comprises the amino acid sequence of SEQ ID NO: 395. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 577 and the LC comprises the amino acid sequence of SEQ ID NO: 395.

[0077] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 604 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 605. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 404 and the LC comprises the amino acid sequence of SEQ ID NO: 405. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 578 and the LC comprises the amino acid sequence of SEQ ID NO: 405.

[0078] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 606 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 607. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 414 and the LC comprises the amino acid sequence of SEQ ID NO: 415. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 579 and the LC comprises the amino acid sequence of SEQ ID NO: 415.

[0079] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 608 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 609. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 424 and the LC comprises the amino acid sequence of SEQ ID NO: 425. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 580 and the LC comprises the amino acid sequence of SEQ ID NO: 425.

[0080] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 610 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 611. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 434 and the LC comprises the amino acid sequence of SEQ ID NO: 435. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 581 and the LC comprises the amino acid sequence of SEQ ID NO: 435.

[0081] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 612 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 613. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 444 and the LC comprises the amino acid sequence of SEQ ID NO: 445. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 582 and the LC comprises the amino acid sequence of SEQ ID NO: 445.

[0082] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 614 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 615. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 454 and the LC comprises the amino acid sequence of SEQ ID NO: 455. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 583 and the LC comprises the amino acid sequence of SEQ ID NO: 455.

[0083] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 616 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 617. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 464 and the LC comprises the amino acid sequence of SEQ ID NO: 465. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 584 and the LC comprises the amino acid sequence of SEQ ID NO: 465.

[0084] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 618 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 619. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 474 and the LC comprises the amino acid sequence of SEQ ID NO: 475. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 585 and the LC comprises the amino acid sequence of SEQ ID NO: 475.

[0085] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 620 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 621. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 484 and the LC comprises the amino acid sequence of SEQ ID NO: 485. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 586 and the LC comprises the amino acid sequence of SEQ ID NO: 485.

[0086] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 622 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 623. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 494 and the LC comprises the amino acid sequence of SEQ ID NO: 495. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 587 and the LC comprises the amino acid sequence of SEQ ID NO: 495.

[0087] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 624 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 625. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 504 and the LC comprises the amino acid sequence of SEQ ID NO: 505. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 588 and the LC comprises the amino acid sequence of SEQ ID NO: 505.

[0088] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 626 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 627. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 514 and the LC comprises the amino acid sequence of SEQ ID NO: 515. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 589 and the LC comprises the amino acid sequence of SEQ ID NO: 515.

[0089] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 628 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 629. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 524 and the LC comprises the amino acid sequence of SEQ ID NO: 525. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 590 and the LC comprises the amino acid sequence of SEQ ID NO: 525.

[0090] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 630 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 631. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 534 and the LC comprises the amino acid sequence of SEQ ID NO: 535. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 591 and the LC comprises the amino acid sequence of SEQ ID NO: 535.

[0091] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 632 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 633. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 544 and the LC comprises the amino acid sequence of SEQ ID NO: 545. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 592 and the LC comprises the amino acid sequence of SEQ ID NO: 545.

[0092] In one embodiment, an antibody of the invention comprises a HC encoded by a polynucleotide sequence comprising SEQ ID NO: 634 and a LC encoded by a polynucleotide sequence comprising SEQ ID NO: 635. In a specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 554 and the LC comprises the amino acid sequence of SEQ ID NO: 555. In another specific embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 593 and the LC comprises the amino acid sequence of SEQ ID NO: 555.

[0093] Also provided herein is one or more nucleic acid sequences encoding the anti-CCR8 antibodies or antigen-binding fragments of the invention. In some embodiments, the invention provides a DNA molecule comprising a polynucleotide encoding the HC of the antibody of the invention. The invention also provides a DNA molecule comprising a polynucleotide encoding the LC of the antibody of the invention. The invention also provides a DNA molecule comprising a polynucleotide encoding both the LC of the antibody of the invention and the HC of the antibody of the invention. In some embodiments, the invention provides a nucleic acid sequence encoding the heavy chain amino acid sequence of SEQ ID NO: 1127, 1129, 1131, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, or 1160. In other embodiments, the invention provides nucleic acid sequences encoding the light chain amino acid sequence of SEQ ID NO: 1128, 1130, 1132, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, or 1159.

[0094] The invention also provides a DNA molecule comprising a polynucleotide encoding the LC of the antibody, wherein the LC has the amino acid sequence of SEQ ID NO: 16. In one embodiment, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 28. The invention also provides a DNA molecule comprising a polynucleotide encoding the HC of the antibody having the amino acid sequence of SEQ ID NO: 15, and another DNA molecule comprising a polynucleotide encoding the LC having the amino acid sequence of SEQ ID NO: 16. In one embodiment, the polynucleotide encoding the HC of the antibody comprises the polynucleotide sequence of SEQ ID NO: 27, and the polynucleotide encoding the LC comprises the polynucleotide sequence of SEQ ID NO: 28.

[0095] The present invention further provides a mammalian cell transformed with a DNA molecule of the present invention, wherein the transformed mammalian cell is capable of expressing an antibody of the present invention, the antibody comprising two HCs and two LCs.

[0096] The present invention further provides a mammalian cell transformed with a DNA molecule of the present invention, wherein the transformed mammalian cell is capable of expressing an antibody comprising two HCs and two LCs, each HC comprising the amino acid sequence of SEQ ID NO:15 and each LC comprising the amino acid sequence of SEQ ID NO:16.

[0097] The invention also provides a process for producing an antibody of the invention, the antibody comprising two HCs and two LCs, the process comprising culturing a mammalian cell under conditions to express the antibody and recover the expressed antibody. In one embodiment, the mammalian cell is transformed with a DNA molecule of the invention, the transformed mammalian cell being capable of expressing an antibody of the invention comprising two HCs and two LCs. The invention also provides an antibody obtained by this process.

[0098] The invention also provides a process for producing an antibody, the antibody comprising two HCs and two LCs, each HC comprising the amino acid sequence of SEQ ID NO: 15, and each LC comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the process comprises culturing a mammalian cell under conditions to express the antibody and recover the expressed antibody, the mammalian cell being transformed with a DNA molecule of the invention, the transformed mammalian cell being capable of expressing an antibody comprising two HCs and two LCs, each HC comprising the amino acid sequence of SEQ ID NO: 15, and each LC comprising the amino acid sequence of SEQ ID NO: 16. The invention also provides an antibody obtained by this process.

[0099] The present invention provides a DNA molecule comprising a polynucleotide encoding the HC of an antibody, wherein the HC has the amino acid sequence of SEQ ID NO: 19. In one embodiment, the polynucleotide sequence comprises SEQ ID NO:29.

[0100] The present invention provides a DNA molecule comprising a polynucleotide encoding the LC of an antibody, wherein the LC has the amino acid sequence of SEQ ID NO: 20. In one embodiment, the polynucleotide sequence comprises SEQ ID NO: 30.

[0101] The present invention provides a DNA molecule comprising a polynucleotide encoding an HC of an antibody, wherein the HC has the amino acid sequence of SEQ ID NO: 19. In one embodiment, the polynucleotide encoding the HC of the antibody comprises the polynucleotide sequence of SEQ ID NO: 29. The present invention also provides a DNA molecule comprising a polynucleotide encoding an LC of an antibody, wherein the LC has the amino acid sequence of SEQ ID NO: 20. In one embodiment, the polynucleotide encoding the LC comprises the polynucleotide sequence of SEQ ID NO: 30. The present invention also provides a mammalian cell transformed with a DNA molecule of the present invention, wherein the transformed mammalian cell is capable of expressing an antibody comprising two HCs and two LCs, wherein each HC comprises the amino acid sequence of SEQ ID NO: 19 and each LC comprises the amino acid sequence of SEQ ID NO: 20.

[0102] In some embodiments, a nucleic acid sequence encoding a HC described herein can include any one of SEQ ID NOs: 1195, 1197, 1199, 1201, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, or 1230.

[0103] In some embodiments, a nucleic acid sequence encoding an LC described herein may include any one of SEQ ID NOs: 1196, 1198, 1200, 1202, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, or 1229.

[0104] In some embodiments, a nucleic acid sequence encoding an scFv described herein may comprise any one of SEQ ID NOs: 1163-1194.

[0105] The invention also provides a process for producing an antibody, the antibody comprising two HCs and two LCs, each HC comprising the amino acid sequence of SEQ ID NO: 19, and each LC comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the process comprises culturing a mammalian cell under conditions to express the antibody and to recover the expressed antibody, the mammalian cell being transformed with a DNA molecule of the invention. In one embodiment, the transformed mammalian cell is capable of expressing an antibody comprising two HCs and two LCs, each HC comprising the amino acid sequence of SEQ ID NO: 19, and each LC comprising the amino acid sequence of SEQ ID NO: 20. The invention also provides an antibody obtained by this process.

[0106] The invention also provides a process for producing an antibody comprising two HCs and two LCs, the process comprising culturing a mammalian cell as described above under conditions to express the antibody and recover the expressed antibody, wherein (a) both HCs are selected from the set of amino acids 15, 1125, 1127, 1129, 1131, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, or 1160. and (b) both LCs comprise an amino acid sequence of SEQ ID NO: 16, 365, 1126, 1128, 1130, 1132, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, or 1159, or an amino acid sequence that is at least 90% identical to any one of the foregoing LC amino acid sequences.

[0107] In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to human CCR8 at an epitope, said epitope comprising at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises eight or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises nine or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 31. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO: 31 is SEQ ID NO: 82. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody does not inhibit binding of CCL1 to CCR8. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed in human cells, and the peptide comprises the amino acid sequence given by SEQ ID NO: 82. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display.In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human CCR8 at an epitope, the epitope consisting of at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of eight or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of nine or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 31. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO: 31 is SEQ ID NO: 82. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody does not inhibit binding of CCL1 to CCR8. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO:82.In some embodiments, the epitope is determined by an anti-CCR8 antibody or antigen-binding fragment thereof that binds to a T4R mutation in cynomolgus CCR8. In one embodiment, binding to a T4R mutation is measured by a cell-based affinity assay that compares an antibody that binds to cells expressing cynomolgus CCR8 that includes a T4R mutation with an antibody that binds to cells expressing wild-type cynomolgus CCR8 (including a threonine at position 4). In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates reduced binding to CCR8 that includes a T4R mutation. In certain embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates undetectable binding to CCR8 that includes a T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 that includes a T4R mutation comprises the amino acid sequence given by SEQ ID NO: 556. In one embodiment, the anti-CCR8 antibody or fragment thereof is an antibody.

[0108] In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue of SEQ ID NO:82. In one embodiment, the epitope comprises at least two residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least three residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least four residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least five residues of SEQ ID NO:82. In one embodiment, the epitope comprises six or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises seven or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises eight or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises nine or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises ten or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises eleven or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises all 12 amino acid residues of SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody does not inhibit binding of CCL1 to CCR8. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In some embodiments, the epitope is determined by an anti-CCR8 antibody or antigen-binding fragment thereof that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to the T4R mutation is measured by a cell-based affinity assay in which an antibody that binds to cells expressing a cynomolgus CCR8 comprising a T4R mutation is compared to an antibody that binds to cells expressing a wild-type cynomolgus CCR8 (comprising a threonine at position 4). In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates reduced binding to a CCR8 comprising a T4R mutation. In certain embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates undetectable binding to a CCR8 comprising a T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 556. In one embodiment, the anti-CCR8 antibody or fragment thereof is an antibody.

[0109] In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to human CCR8 at an epitope, wherein the epitope consists of at least one residue of SEQ ID NO:82. In one embodiment, the epitope consists of at least two residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least three residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least four residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least five residues of SEQ ID NO:82. In one embodiment, the epitope consists of six or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of seven or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of eight or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of nine or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of ten or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of eleven or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of all 12 amino acid residues of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody does not inhibit binding of CCL1 to CCR8. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by an antibody that binds to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening for antibodies that bind to CCR8 by phage display. In one embodiment, the epitope is determined by measuring binding to a CCR8 peptide expressed on human cells, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. In some embodiments, the epitope is determined by an anti-CCR8 antibody or antigen-binding fragment thereof that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to the T4R mutation is measured by a cell-based affinity assay in which an antibody that binds to cells expressing a cynomolgus CCR8 comprising a T4R mutation is compared to an antibody that binds to cells expressing a wild-type cynomolgus CCR8 (comprising a threonine at position 4). In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates reduced binding to a CCR8 comprising a T4R mutation. In certain embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof binds to the threonine at position 4 when it indicates undetectable binding to a CCR8 comprising a T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 22. In some embodiments, the cynomolgus CCR8 comprises the amino acid sequence given by SEQ ID NO: 556. In one embodiment, the anti-CCR8 antibody or fragment thereof is an antibody.

[0110] In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope comprising amino acids of SEQ ID NO: 83. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope comprising amino acids of SEQ ID NO: 86. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope comprising amino acids of SEQ ID NO: 84. In some embodiments, the epitope is determined by an antibody binding to a peptide of amino acids of SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 86, and / or SEQ ID NO: 84. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope comprising amino acids at positions 13-35 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope comprising amino acids at positions 13, 14, or 15 of SEQ ID NO: 31.

[0111] In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of the amino acids of SEQ ID NO: 83. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of the amino acids of SEQ ID NO: 86. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of the amino acids of SEQ ID NO: 84. In some embodiments, the epitope is determined by an antibody binding to a peptide of the amino acids of SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 86, and / or SEQ ID NO: 84. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of the amino acids of positions 13 to 35 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of the amino acids of positions 13, 14, or 15 of SEQ ID NO: 31.

[0112] In some embodiments, the anti-CCR8 antibody of the invention or a fragment thereof does not bind to an epitope comprising amino acid residues at positions 13-24 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the invention or a fragment thereof does not bind to an epitope comprising amino acid residues at positions 19-30 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the invention or a fragment thereof does not bind to an epitope comprising amino acid residues at positions 25-35 of SEQ ID NO: 31. In some embodiments, the epitope is determined by the antibody binding to a peptide of amino acids SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 86, and / or SEQ ID NO: 84.

[0113] In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of amino acid residues at positions 13 to 24 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of amino acid residues at positions 19 to 30 of SEQ ID NO: 31. In some embodiments, the anti-CCR8 antibody of the present invention or a fragment thereof does not bind to an epitope consisting of amino acid residues at positions 25 to 35 of SEQ ID NO: 31. In some embodiments, the epitope is determined by an antibody binding to a peptide of amino acids of SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 86, and / or SEQ ID NO: 84.

[0114] As used herein, the term "epitope" refers to a site of an antigen that makes contact with a variable region of an antibody. Epitopes may be contiguous or non-contiguous and may be determined by methods known to those of skill in the art, including flow cytometry, hydrogen-deuterium exchange, alanine scanning, and / or x-ray crystallography of antibodies bound to a peptide.

[0115] The epitope can be an epitope that comprises or consists of amino acid residues determined by an antibody that binds to the peptide as described herein. In some such embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 82. In some such embodiments, the peptide comprises the amino acid sequence of residues 1-12 of SEQ ID NO: 31.

[0116] The epitope may be an epitope that comprises or consists of amino acid residues determined by epitope binning, hi some such embodiments, epitope binning is performed using biotinylated N-terminal CCR8 peptides.

[0117] The epitope may be an epitope comprising or consisting of amino acid residues determined by an antibody binding to the CCR8 peptide-Nanobody complex.

[0118] The epitope can be an epitope comprising or consisting of amino acid residues determined by screening for antibodies that bind to CCR8 by phage display.

[0119] The epitope may be an epitope comprising or consisting of threonine at position 4 in the N-terminal region of CCR8, as determined by reduced binding to CCR8 comprising a T4R mutation compared to binding to wild-type CCR8. For example, binding to a T4R mutation may be verified by measuring binding to wild-type cynomolgus CCR8 (comprising the amino acid sequence provided by SEQ ID NO: 22) compared to binding to a cynomolgus CCR8 comprising a T4R mutation (comprising the amino acid sequence provided by SEQ ID NO: 556).

[0120] The epitope may be an epitope comprising or consisting of amino acid residues determined by measuring binding to a CCR8 peptide expressed in a human cell, the peptide comprising the amino acid sequence given by SEQ ID NO: 82. The CCR8 peptide may be fused to a nanobody, or to another protein or Fc, for expression in a human cell.

[0121] The invention further provides antibodies or antigen-binding fragments that bind to an epitope on human CCR8, said epitope comprising or consisting of SEQ ID NO: 82. In some embodiments, the invention also provides antibodies or antigen-binding fragments that (a) bind to an epitope comprising or consisting of SEQ ID NO: 82 on human CCR8, and (b) do not inhibit binding of CCL1 to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0122] In some embodiments, the present invention provides a molecule that competes with the anti-CCR8 antibody or antigen-binding fragment of the present invention for binding to CCR8. Such a competing molecule for binding can be, for example, an antibody, an antibody fragment, or a polypeptide. In some embodiments, the present invention provides a molecule that binds to the same epitope as the anti-CCR8 antibody or antigen-binding fragment of the present invention. In one embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof is an antibody.

[0123] The present invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of an antibody or antigen-binding fragment that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises eight or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope comprises 9 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope comprises all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 31. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO: 31 is SEQ ID NO: 82. In a particular embodiment, the epitope comprises a threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody or antigen-binding fragment does not inhibit binding of CCL1 to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0124] The present invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of an antibody or antigen-binding fragment that binds to human CCR8 at an epitope, wherein the epitope consists of at least one residue at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least two residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least three residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least four residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of at least five residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of six or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of seven or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of eight or more residues at positions 1-12 of SEQ ID NO:31. In one embodiment, the epitope consists of 9 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 10 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of 11 or more residues at positions 1-12 of SEQ ID NO: 31. In one embodiment, the epitope consists of all 12 amino acid residues at positions 1-12 of SEQ ID NO: 31. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 31. The amino acid sequence of amino acid residues 1-12 of SEQ ID NO: 31 is SEQ ID NO: 82. In a particular embodiment, the epitope consists of a threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody or antigen-binding fragment does not inhibit binding of CCL1 to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0125] The invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of an antibody or antigen-binding fragment that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue of SEQ ID NO:82. In one embodiment, the epitope comprises at least two residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least three residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least four residues of SEQ ID NO:82. In one embodiment, the epitope comprises at least five residues of SEQ ID NO:82. In one embodiment, the epitope comprises six or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises seven or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises eight or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises nine or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises ten or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises eleven or more residues of SEQ ID NO:82. In one embodiment, the epitope comprises all 12 amino acid residues of SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope comprises the threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody or antigen-binding fragment does not inhibit binding of CCL1 to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0126] The invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of an antibody or antigen-binding fragment that binds to human CCR8 at an epitope, wherein the epitope consists of at least one residue of SEQ ID NO:82. In one embodiment, the epitope consists of at least two residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least three residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least four residues of SEQ ID NO:82. In one embodiment, the epitope consists of at least five residues of SEQ ID NO:82. In one embodiment, the epitope consists of six or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of seven or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of eight or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of nine or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of ten or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of eleven or more residues of SEQ ID NO:82. In one embodiment, the epitope consists of all 12 amino acid residues of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 82. In a particular embodiment, the epitope consists of the threonine at position 4 of SEQ ID NO: 22. In some such embodiments, the anti-CCR8 antibody or antigen-binding fragment does not inhibit binding of CCL1 to CCR8. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody.

[0127] In some embodiments, the invention provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a molecule that competes for binding to CCR8 with the anti-CCR8 antibody or antigen-binding fragment of the invention. Such a molecule that competes for binding can be, for example, an antibody, an antibody fragment, or a polypeptide. In some embodiments, the invention provides a molecule that binds to the same epitope as the anti-CCR8 antibody of the invention. In one embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof is an antibody.

[0128] In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the invention bind to CCR8 from a non-human species. In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the invention bind to cynomolgus monkey CCR8. In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the invention bind to mouse CCR8. In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the invention bind to both cynomolgus monkey CCR8 and human CCR8. In certain embodiments, the anti-CCR8 antibodies or antigen-binding fragments of the invention bind to cynomolgus monkey CCR8 and human CCR8 with affinities within 10-fold of each other. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody. [Brief description of the drawings]

[0129] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Antitumor activity of single agent muCLDN18.2 bispecific T cell engager molecule (BiTE®) molecule (□), dual combination of muCLDN18.2 BiTE molecule / anti-CTLA4 (△), triple combination of muCLDN18.2 BiTE molecule / anti-PD-1 / anti-4-1BB (◇), quadruple combination of control BiTE molecule / anti-PD-1 / anti-4-1BB / anti-CTLA4 (◯), or quadruple combination of muCLDN18.2 BiTE molecule / anti-PD-1 / anti-4-1BB / anti-CTLA4 (▽) therapy in the KPC-M5 model. [Figure 2A] Antitumor activity of CCR8 afucosylated mIgG2a antibody in MC38 syngeneic mouse model. Individual tumor growth of isotype control mIgG2a antibody (dashed line) or CCR8 afucosylated mIgG2a antibody (solid line) is shown in Figures 2C and 2B, respectively. Figure 2A shows the mean tumor volume up to day 24 for each group. **** indicates p<0.0001. [Figure 2B]Antitumor activity of CCR8 afucosylated mIgG2a antibody in MC38 syngeneic mouse model. Individual tumor growth of isotype control mIgG2a antibody (dashed line) or CCR8 afucosylated mIgG2a antibody (solid line) is shown in Figures 2C and 2B, respectively. Figure 2A shows the mean tumor volume up to day 24 for each group. **** indicates p<0.0001. [Figure 2C] Antitumor activity of CCR8 afucosylated mIgG2a antibody in MC38 syngeneic mouse model. Individual tumor growth of isotype control mIgG2a antibody (dashed line) or CCR8 afucosylated mIgG2a antibody (solid line) is shown in Figures 2C and 2B, respectively. Figure 2A shows the mean tumor volume up to day 24 for each group. **** indicates p<0.0001. [Diagram 3] Survival rate of mice inoculated with MC38 tumor cells and treated with either isotype control mIgG2a antibody (dashed line) or CCR8 afucosylated mIgG2a antibody (solid line).**** indicates p<0.0001. [Figure 4A] CD8+ / Treg ratios in MC38 tumor-bearing mice treated with either isotype control mIgG2a antibody (○) or CCR8 afucosylated mIgG2a antibody (●). Figures 4A, 4B, 4C, and 4D show the percentage of Foxp3+ Treg, the percentage of CD25+Foxp3+ Treg, CD8 / Treg(Foxp3+), and CD8 / Treg(CD25+Foxp3+), respectively. [Figure 4B] CD8+ / Treg ratios in MC38 tumor-bearing mice treated with either isotype control mIgG2a antibody (○) or CCR8 afucosylated mIgG2a antibody (●). Figures 4A, 4B, 4C, and 4D show the percentage of Foxp3+ Treg, the percentage of CD25+Foxp3+ Treg, CD8 / Treg(Foxp3+), and CD8 / Treg(CD25+Foxp3+), respectively. [Figure 4C]CD8+ / Treg ratios in MC38 tumor-bearing mice treated with either isotype control mIgG2a antibody (○) or CCR8 afucosylated mIgG2a antibody (●). Figures 4A, 4B, 4C, and 4D show the percentage of Foxp3+ Treg, the percentage of CD25+Foxp3+ Treg, CD8 / Treg(Foxp3+), and CD8 / Treg(CD25+Foxp3+), respectively. [Figure 4D] CD8+ / Treg ratios in MC38 tumor-bearing mice treated with either isotype control mIgG2a antibody (○) or CCR8 afucosylated mIgG2a antibody (●). Figures 4A, 4B, 4C, and 4D show the percentage of Foxp3+ Treg, the percentage of CD25+Foxp3+ Treg, CD8 / Treg(Foxp3+), and CD8 / Treg(CD25+Foxp3+), respectively. [Figure 5A] Antitumor activity of CCR8 afucosylated mIgG2a as monotherapy and in combination with TAA-BiTE molecules in a B16F10 syngeneic tumor model expressing tumor-associated antigens (TAA). Individual tumor growth of treatment groups is represented as spider plots (Figures 5A-5D). Animals without measurable tumors, defined as complete responders (CR), were evaluated up to day 48. [Figure 5B] Antitumor activity of CCR8 afucosylated mIgG2a as monotherapy and in combination with TAA-BiTE molecules in a B16F10 syngeneic tumor model expressing tumor-associated antigens (TAA). Individual tumor growth of treatment groups is represented as spider plots (Figures 5A-5D). Animals without measurable tumors, defined as complete responders (CR), were evaluated up to day 48. [Figure 5C] Antitumor activity of CCR8 afucosylated mIgG2a as monotherapy and in combination with TAA-BiTE molecules in a B16F10 syngeneic tumor model expressing tumor-associated antigens (TAA). Individual tumor growth of treatment groups is represented as spider plots (Figures 5A-5D). Animals without measurable tumors, defined as complete responders (CR), were evaluated up to day 48. [Figure 5D]Antitumor activity of CCR8 afucosylated mIgG2a as monotherapy and in combination with TAA-BiTE molecules in a B16F10 syngeneic tumor model expressing tumor-associated antigens (TAA). Individual tumor growth of treatment groups is represented as spider plots (Figures 5A-5D). Animals without measurable tumors, defined as complete responders (CR), were evaluated up to day 48. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0130] Detailed Description The present disclosure provides anti-CCR8 antibodies and methods for producing and using said antibodies. The anti-CCR8 antibodies disclosed herein 1) can bind to human and cynomolgus CCR8 on tumor-resident Treg cells, 2) result in specific depletion of tumor-resident Treg cells, 3) show acceptable pharmacokinetic profiles, and / or 4) show sufficient efficacy for the treatment of cancer. The anti-CCR8 antibodies of the present invention have an improved safety profile compared to other Treg-depleting therapeutic molecules that target other markers that do not specifically deplete tumor-resident Tregs. In addition, treatment with anti-CCR8-depleting antibodies resulted in a significant increase in the CD8+ / Treg ratio in tumors, thereby promoting enhanced anti-tumor immunity.

[0131] The present invention includes anti-CCR8 antibodies that bind to a unique epitope on CCR8, do not inhibit ligand binding to CCR8, and are therefore not neutralizing antibodies. Binding to this unique epitope is believed to contribute to the high affinity and biological activity of the anti-CCR8 antibodies, as well as their acceptable pharmacokinetic profile, compared to antibodies that bind to a different epitope.

[0132] Anti-CCR8 antibodies of the present invention that bind to a unique epitope on CCR8 in the presence of ligand (CCL1) and do not inhibit ligand binding exhibit ADCC activity even at the highest concentration of ligand tested in vitro. In contrast, anti-CCR8 antibodies that bind to a different epitope (and inhibit ligand binding) show reduced ADCC activity in the presence of elevated CCL1 levels. Thus, binding to this unique epitope is believed to contribute to the better efficacy (by ADCC) of the anti-CCR8 antibodies of the present invention, even in the presence of elevated concentrations of ligand. Because CCL1 is highly expressed in tumors such as breast cancer (see, e.g., Kuehnemuth et al., BMC Cancer 18, Article number: 1278 (2018)), anti-CCR8 antibodies that exhibit ADCC activity in the presence of elevated concentrations of ligand are preferred.

[0133] The anti-CCR8 antibodies of the invention are preferably afucosylated and exhibit enhanced ADCC activity.

[0134] Further mechanisms of action envisaged for the anti-CCR8 antibodies or fragments thereof of the present invention to deplete Tregs include antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0135] As used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC comprises a variable region of about 100-120 amino acids that is primarily responsible for recognizing an antigen via the CDRs contained therein. The CDRs are interspersed with more conserved regions termed framework regions ("FRs"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDRs and four FRs, arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. Thus, the functional ability of an antibody to bind a specific antigen is greatly influenced by the amino acid residues within the six CDRs. The amino acid assignments for the CDR domains within the LCVR and HCVR regions of the antibodies of the present invention are based on the well-known Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).It will be understood that other numbering conventions may also be used, such as, for example, Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) and / or North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)). An "anti-CCR8 antibody" is an antibody that binds to CCR8.

[0136] The antibody of the present invention may be IgG1, IgG2, or IgG4. Preferably, the antibody of the present invention is IgG1. IgG1 antibodies are known to induce ADCC. The antibody of the present invention may be a human antibody or a humanized antibody. In the context of monoclonal antibodies, the terms "human" and "humanized" are familiar to the skilled artisan (Weiner LJ, J. Immunother. 2006; 29: 1-9; Mallbris L, et al., J. Clin. Aesthet. Dermatol. 2016; 9: 13-15).

[0137] In addition, the antibodies of the invention are preferably afucosylated. Removal of the core fucose from biantennary complex oligosaccharides attached to Fc significantly increased ADCC effector function without altering antigen binding or CDC effector function. Several methods are known to reduce or eliminate fucosylation of Fc-containing molecules, such as antibodies. These methods include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, mutant CHO line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNA specific for the FUT8 gene, and cell lines co-expressing α-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules may be expressed in non-mammalian cells, such as plant cells, yeast, or prokaryotic cells, e.g., E. coli. Zinc finger nucleases are another known method of generating afucosylated antibodies. See, e.g., Haryadi et al., Bioengineered 4:2, 90-94; March / April 2013; Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Pereira et al. mAbs 2018 Jul; 10(5): 693-711.

[0138] Anti-CCR8 antibodies or fragments thereof are also contemplated in formats including scFv, scFab, Fab, bispecific T cell engager molecules, and bispecific antibodies (which bind to two different epitopes on the same antigen or which bind to two different antigens).

[0139] The scFv or Fab can be converted into an antibody by known methods (see, for example, Reader et al., Molec. Bio. 61, 801-815 (2019)). Constant region sequences are known in the art. Constant region sequences are also exemplified herein, for example, the amino acid sequences of the LC and HC constant regions are given by SEQ ID NO: 1079 and SEQ ID NO: 1080, respectively.

[0140] In certain embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof of the invention is a heterodimeric antibody (used interchangeably herein as "heteroimmunoglobulin" or "heteroIg"), which refers to an antibody comprising two different light chains and two different heavy chains. In some embodiments, the heteroIg comprises two Fab and Fc regions. In some embodiments, the two Fabs are each N-terminal to the Fc region. In some embodiments, the two Fabs are each C-terminal to the Fc region. In some embodiments, at least one Fab is an anti-CCR8 antibody fragment of the invention.

[0141] The heterodimeric antibody may comprise any immunoglobulin constant region. As used herein, the term "constant region" refers to all domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding, but exerts various effector functions. As explained above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) depending on the amino acid sequence of the constant region of their heavy chain. The light chain constant region may be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region, which are found in all five isotypes of antibodies.

[0142] The heavy chain constant region of the heterodimeric antibody can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. In some embodiments, the heterodimeric antibody comprises a heavy chain constant region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin.

[0143] An example of a heterodimeric antibody is Duobody™. Duobodies can be produced by the Duobody™ technology platform (Genmab A / S), as described, for example, in WO 2008 / 119353, WO 2011 / 131746, WO 2011 / 147986, and WO 2013 / 060867, Labrijn AF et al., PNAS, 110(13): 5145-5150 (2013), Gramer et al., mAbs, 5(6): 962-973 (2013), and Labrijn et al., Nature Protocols, 9(10): 2450-2463 (2014). Using this technique, one half of a first monospecific antibody containing two heavy chains and two light chains can be combined with one half of a second monospecific antibody containing two heavy chains and two light chains. The resulting heterodimer contains one heavy chain and one light chain from the first antibody paired with one heavy chain and one light chain from the second antibody. If both monospecific antibodies recognize different epitopes on different antigens, the resulting heterodimer is a multispecific antibody.

[0144] Another exemplary method of generating multispecific antibodies is by the knobs-into-holes technique (Ridgway et al., Protein Eng., 9:617-621 (1996); WO 2006 / 028936). The problem of Ig heavy chain mispairing, which is a major drawback in generating multispecific antibodies, is reduced in this technique by mutating selected amino acids that form the heavy chain interface in IgG. At positions in the heavy chains where two heavy chains directly interact, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (knob) is introduced into the corresponding position of the interacting residue on the other heavy chain. In some examples, the antibodies of the present disclosure have immunoglobulin chains whose heavy chains have been modified by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form multispecific antibodies. Multispecific antibodies can be composed of immunoglobulin chains of the same subclass or different subclasses.

[0145] Yet another method to generate multispecific antibodies is the CrossMab technology. CrossMab is a chimeric antibody composed of two halves of two full-length antibodies. This technology combines two techniques to ensure correct pairing of the chains: (i) knob-into-hole, which favors accurate pairing between the two heavy chains, and (ii) exchange between the heavy and light chains of one of the two Fabs to introduce asymmetry to avoid mispairing of the light chains. See Ridgway et al., Protein Eng., 9:617-621 (1996); Schaefer et al., PNAS, 108:11187-11192 (2011). CrossMab can combine two or more antigen-binding domains to target two or more targets or to introduce bivalency, such as a 2:1 configuration for one target.

[0146] Hetero-Ig molecules may also include the creation of non-canonical disulfide bonds and asymmetric cysteine ​​interfaces, as described in WO 2022 / 040466, which also discloses a set of specific mutations that may be used in the antibodies of the present invention. Both the heavy and light chains may contain complementary amino acid substitutions to facilitate the association of a particular heavy chain with its associated light chain. As used herein, "complementary amino acid substitution" refers to a pairing of a substitution of a positively charged amino acid in one chain with a negatively charged amino acid substitution in the other chain. For example, the heavy chain contains at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain contains at least one amino acid substitution to introduce a charged amino acid, where the charged amino acid introduced into the heavy chain has an opposite charge to the amino acid introduced into the light chain. One or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the first light chain (LC1) and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the corresponding heavy chain (HC1) at the LC1 / HC1 binding interface, while one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the second light chain (LC2) and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the corresponding heavy chain (HC2) at the LC2 / HC2 binding interface. This electrostatic interaction induces LC1 to pair with HC1 and LC2 to pair with HC2 due to the attraction of oppositely charged residues (polarity) at the interface. Heavy / light chain pairs that have the same charged residues (polarity) at the interface (eg, LC1 / HC2 and LC2 / HC1) are repelled, resulting in the inhibition of undesired HC / LC pairing.

[0147] In some embodiments, the hetero-Ig is at least one anti-CCR8 antibody fragment of the present invention. In certain embodiments, the anti-CCR8 antibody fragment is a Fab. In certain embodiments, the anti-CCR8 antibody fragment is a scFab. In certain embodiments, the anti-CCR8 antibody fragment is a scFv. Exemplary anti-CCR8 scFv amino acid sequences include, but are not limited to, any one of SEQ ID NOs: 1093-1124.

[0148] In some embodiments, the heteroIg comprises an anti-CCR8 antibody fragment of the invention linked to a heteroIg. The anti-CCR8 antibody fragment may be in any format as described herein, including scFv, Fab, or scFab. Such linkage may be via a linker C-terminal or N-terminal to the Fc region, or N-terminal or C-terminal to another binding domain of the heteroIg (e.g., Fab). In some embodiments, the heteroIg comprises at least one binding arm that is a single chain comprising an anti-CCR8 antibody fragment of the invention and further an scFab or scFv.

[0149] The present invention also contemplates T cell engager ("TCE") molecules comprising the anti-CCR8 antibody fragment of the present invention. Such TCE molecules are preferably single chain TCE molecules. Single chain TCE molecules are contemplated having the following orientation: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL). In one embodiment, the TCE molecule further comprises an scFc, having the following orientation: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL)-linker-Fc1 (hinge, CH2, CH3), linker, Fc2 (hinge, CH2, CH3). In some embodiments, the scFv that binds CCR8 is an anti-CCR8 antibody fragment of the present invention.

[0150] The present invention also contemplates TCE molecules having the following orientation from N-terminus to C-terminus: scFv(VH, linker, VL) binding to CCR8-linker-scFv(VH, linker, VL) binding to CD3-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3). In one embodiment, the TCE molecule binds to CCR8 and CD3. The present invention also provides TCE molecules having the following orientation from N-terminus to C-terminus: scFv(VL-linker-VH) binding to CCR8-linker-scFv(VH-linker-VL) binding to CD3-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3). In one embodiment, the TCE molecule binds to CCR8 and CD3. In some embodiments, the scFv binding to CCR8 is an anti-CCR8 antibody fragment of the present invention.

[0151] The present invention contemplates a TCE molecule comprising an orientation, from N-terminus to C-terminus, of an scFab that binds CCR8 (VH, CH1, linker, VL, either Cκ or Cλ), a linker, an scFv that binds CD3 (VH, linker, VL). In some embodiments, the scFab that binds CCR8 is an anti-CCR8 antibody fragment of the invention.

[0152] An scFC is a fusion protein in which a CH2 and CH3 (Fc1) are linked to another CH2 and CH3 (Fc2) via a linker to form a continuous protein chain, the linker being long enough to allow the protein chain to refold on itself.

[0153] A "single-chain antigen-binding fragment" ("scFab") is a fusion protein in which a VH and a CH1 are joined to a VL and a Cκ via a linker to form a contiguous protein chain, which linker is long enough to allow the protein chain to refold upon itself and form a monovalent antigen-binding site. The linker can be, for example, a (G4S)6, (G4S)7, or (G4S)8 linker.

[0154] The scFab, scFv, and / or scFc may also have a cysteine ​​clamp. A "cysteine ​​clamp" refers to the introduction of a cysteine ​​into a polypeptide domain at a specific position, typically by replacing an existing amino acid at that position, which allows the formation of a disulfide bond between the two domains when brought into close proximity with another polypeptide domain that also has a cysteine ​​introduced at that position ("cysteine ​​clamp"). In certain embodiments, the scFC comprises at least one cysteine ​​clamp that creates a disulfide bond across both CH2 domains. In further particular embodiments, the scFC comprises at least two cysteine ​​clamps that create a disulfide bond across both CH2 domains. In other embodiments, the VH and VL domains of the binding construct may comprise cysteine ​​clamps to create disulfide bond formation between the VH and VL domains. These cysteine ​​clamps stabilize the VH and VL domains in the antigen-binding configuration.

[0155] The cysteine ​​clamp may be naturally occurring or may result from a molecule that has been engineered to contain a cysteine. For example, an scFab may have a natural cysteine ​​clamp between the heavy and light chain constant domains. An scFab may also have a natural cysteine ​​clamp between the heavy and light chain constant domains or an engineered cysteine ​​clamp between the cysteine ​​at residue 44 of the heavy chain variable region and the cysteine ​​at residue 100 of the light chain variable region. In addition, an anti-target scFv may also contain a cysteine ​​clamp between the cysteine ​​at residue 44 of the heavy chain variable region and the cysteine ​​at residue 100 of the light chain variable region, whereas an anti-CD3 scFv does not contain an engineered cysteine ​​clamp. An scFC may contain a hinge cysteine ​​clamp, a natural CH2 / CH3 cysteine ​​clamp, and / or an engineered CH2 cysteine ​​clamp (intrachain).

[0156] Antigen-binding fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of antibodies, for example, by pepsin or papain digestion of whole antibodies by conventional methods. As an example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to generate a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic cleavage with papain directly generates two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in Goldenberg, U.S. Pat. No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and by Andrews, SM and Titus, JA in Current Protocols in Immunology (Coligan JE, et al., eds), John Wiley & Sons, New York (2003). pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating the heavy chains to form monovalent light-heavy chain fragments (Fd), further cleaving the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragment binds to the antigen recognized by the intact antibody.

[0157] Antibody fragments may also be any synthetic or genetically engineered protein. For example, antibody fragments include isolated fragments containing the light chain variable region, "Fv" fragments containing the heavy and light chain variable regions, and recombinant single chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker (scFv protein).

[0158] Another form of an antibody fragment is a peptide containing one or more complementarity determining regions (CDRs) of an antibody. CDRs (also called "minimal recognition units" or "hypervariable regions") can be obtained by constructing a polynucleotide encoding the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).

[0159] The antibodies and antigen-binding fragments of the invention bind to human CCR8. Preferably, the antigen-binding fragments of the invention bind to human CCR8 at an epitope that comprises or consists of the amino acid residues of SEQ ID NO: 82. In particular, the antigen-binding fragments of the invention bind to human CCR8 and do not inhibit ligand binding to CCR8.

[0160] Most commonly, T cell engager ("TCE") molecules as described herein comprise a single chain polypeptide capable of binding two different antigens. "TCE molecule" may be used interchangeably with "BiTE molecule." BiTE molecules may comprise scFv or scFab, so long as they are bispecific, meaning they bind to two targets simultaneously (target antigen (here CCR8) and CD3). TCE molecules are antigen-binding molecules. TCE molecules of the present invention may comprise an scFab that binds to a target (e.g., tumor or target antigen; CCR8) and an scFv that binds to CD3. Such molecules may have the following orientation from N-terminus to C-terminus: scFab (VH, CH1, linker, VL, either Cκ or Cλ), linker, scFv (VH, linker, VL). Alternatively, such molecules may have the following orientation from N-terminus to C-terminus: scFab (VL, either Cκ or Cλ, linker, VH, CH1), linker, scFv (VH, linker, VL). In some embodiments, the scFab binds CCR8. In certain embodiments, the TCE molecule comprises Cκ. Such TCE molecules may have the following orientation from N-terminus to C-terminus: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL). In some such embodiments, the scFv or scFab that binds CCR8 is an anti-CCR8 antibody fragment of the invention.

[0161] The TCE molecules of the present invention may also have a half-life extension (HLE) moiety. The HLE moiety can extend the in vivo half-life of the TCE molecules of the present invention. Non-limiting examples of half-life extension moieties include Fc polypeptides, single-chain Fc polypeptides (scFc), albumin, albumin fragments, moieties that bind to albumin or neonatal Fc receptor (FcRn), derivatives of fibronectin modified to bind to albumin or a fragment thereof, peptides that can increase serum half-life, single domain protein fragments, or other polypeptides. In other embodiments, the half-life extension moiety can be a non-polypeptide molecule, such as, for example, polyethylene glycol (PEG). In some embodiments, the HLE is a single-chain Fc (scFc).

[0162] "Nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or modified nucleotides. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "polynucleotide" may be used interchangeably herein to refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. The terms include as equivalents analogs of either RNA or DNA made from nucleotide analogs, as well as modified polynucleotides, such as, but not limited to, methylated and / or end-protected polynucleotides.

[0163] The DNA molecule of the present invention is a DNA molecule comprising a non-naturally occurring polynucleotide sequence encoding a polypeptide having at least one amino acid sequence of the polypeptides (e.g., heavy chain, light chain, variable heavy chain, and variable light chain) of the anti-CCR8 antibody of the present invention.

[0164] The isolated DNA encoding the HCVR region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the HCVR to another DNA molecule encoding a heavy chain constant region. The sequences of the heavy chain constant region genes of other mammals, as well as humans, are known in the art. DNA fragments containing these regions can be obtained, for example, by standard PCR amplification.

[0165] The isolated DNA encoding the LCVR region can be converted into a full-length light chain gene by operably linking the DNA encoding the LCVR to another DNA molecule encoding a light chain constant region. The sequences of the light chain constant region genes of other mammals, as well as humans, are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In some embodiments, the light chain constant region is a kappa constant region.

[0166] The term "encoding" or "encoding" refers to a polynucleotide sequence that codes for one or more amino acids. The term does not require a start or stop codon. The present invention encompasses nucleic acid molecules that code for the polypeptide sequence of an anti-CCR8 antibody.

[0167] The polynucleotide of the present invention can be expressed in a host cell after the sequence is operably linked to an expression control sequence.Expression vectors can typically replicate in the host organism either as episomes or as an integral part of the host chromosomal DNA.Usually, expression vectors contain a selection marker, such as tetracycline, neomycin, and dihydrofolate reductase, so that those cells transformed with the desired DNA sequence can be detected.

[0168] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures. The polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides that are substantially free of contaminating endogenous materials. Cells containing nucleic acids encoding the anti-CCR8 antibody of the present invention also include hybridomas.

[0169] The polynucleotide encoding the amino acid sequence of the anti-CCR8 antibody of the present invention may be of any length suitable for the desired use or function, may include one or more additional sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid, such as a vector. Those skilled in the art will understand that due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by a large number of other nucleic acid sequences. Mutations can also be introduced into a nucleic acid without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues.

[0170] It will be understood that the anti-CCR8 antibody of the present invention may have at least one amino acid substitution, so long as the anti-CCR8 antibody retains the same or better desired binding specificity (e.g., binding to CCR8). Thus, modifications to the anti-CCR8 antibody are encompassed within the scope of the present invention. Such modifications may include amino acid substitutions that may be conservative or non-conservative, without impairing the desired binding ability of the binding construct. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are usually incorporated by chemical peptide synthesis, rather than by synthesis in a living system. These include peptidomimetics and other forms in which amino acid moieties are reversed or inverted. Conservative amino acid substitutions may also include the replacement of natural amino acid residues with standard residues, which have little or no effect on the polarity or charge of the amino acid residue at that position.

[0171] Human CCR8 includes the wild-type human CCR8 sequence, as well as variants and isoforms thereof. The amino acid sequence of human CCR8 comprises the amino acid sequence of SEQ ID NO: 21. The term "variant" as used herein with respect to a nucleic acid sequence means (i) a portion or fragment of a referenced nucleotide sequence, (ii) a complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence that is substantially identical thereto. The term "variant" as used herein with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs from a referenced peptide or polypeptide in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity of the referenced peptide or polypeptide. A variant may also refer to a protein having an amino acid sequence substantially identical to a referenced protein having an amino acid sequence that retains at least one biological activity. The term "isoform" may be used herein to refer to a variant of a polypeptide or protein. Typically, a protein isoform is a member of a series of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. Some protein isoforms exhibit the same or similar biological functions, while other isoforms have unique functions. Isoforms can be generated from alternative splicing of a single gene, use of alternative promoters, or other post-transcriptional modifications.

[0172] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the entire gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or a fragment thereof. In other embodiments, a variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0173] The amino acid sequence of cynomolgus monkey CCR8 comprises the amino acid sequence of SEQ ID NO:22.

[0174] The antibodies of the present invention can be readily produced in mammalian cells, non-limiting examples of which include CHO, NSO, HEK293 or COS cells. Host cells are cultured using techniques well known in the art.

[0175] A vector containing a polynucleotide sequence of interest (e.g., a polynucleotide encoding an antibody polypeptide and an expression control sequence) can be introduced into a host cell by well-known methods that vary depending on the type of cellular host. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0176] The recombinant expression vector of the invention may contain a nucleic acid of the invention in a form suitable for expressing the nucleic acid in a host cell. The recombinant expression vector contains one or more regulatory sequences selected based on the host cell to be used for expression, which regulatory sequences are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287; Maniatis et al., 1987, Science 11:287, which are incorporated by reference in their entireties). 236:1237), as well as those that induce inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and tet- and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems, see ibid. It will be appreciated by those of skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into a host cell to produce a protein or peptide, including a fusion protein or peptide, encoded by a nucleic acid as described herein.

[0177] Typically, expression vectors used in any of the host cells contain sequences for maintaining the plasmid and for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences", typically include, in certain embodiments, one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secreting a polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selection marker element. The leader sequence may comprise the amino acid sequence of SEQ ID NO: 557 (MDMRVPAQLLGLLLLWLRGARC), which is encoded by SEQ ID NO: 558 (atggacatgagagtgcctgcacagctgctgggcctgctgctgctgtggctgagaggcgccagatgc). The leader sequence may comprise the amino acid sequence of SEQ ID NO: 559 (MAWALLLLTLLTQGTGSWA), which is encoded by SEQ ID NO: 560 (atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcc). The present invention contemplates antibody protein sequences that do not include a leader sequence.

[0178] The present invention also contemplates anti-CCR8 antibodies of the invention having clipping of the C-terminal lysine residue of the HC of the antibody. Anti-CCR8 antibodies are contemplated that comprise the HC amino acid sequence of the antibody lacking the C-terminal lysine residue.

[0179] A variety of protein purification methods may be utilized to purify proteins, including but not limited to antibodies, and such methods are known in the art.

[0180] The anti-CCR8 antibodies of the present invention can be biosynthesized, purified, and formulated for administration by well-known methods. For example, suitable host cells, such as HEK293 or CHO, are transiently or stably transfected with an expression system for secreting the antibody, using a predetermined HC:LC vector ratio when two vectors are used, or using a single vector system encoding both the heavy and light chains. Vectors suitable for expressing and secreting antibodies from these commonly used host cells are well known. After the antibody is expressed and secreted, the medium is clarified to remove the cells, and the clarified medium is purified using any of a number of commonly used techniques. For example, the medium may be applied to a Protein A or G column that has been equilibrated with a buffer such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specifically bound components. For example, the bound antibody is eluted by a pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). The antibody fractions are detected, for example, by SDS-PAGE, and then pooled. Further purification is optional depending on the intended use. The antibody may be concentrated and / or sterile filtered using common techniques. Host cell and growth medium components, as well as other substances other than the antibody, such as soluble aggregates and multimers of the antibody, may be effectively reduced or removed by common techniques, including size exclusion chromatography, hydrophobic interaction chromatography, cation exchange chromatography, anion exchange chromatography, affinity chromatography, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is typically greater than 95%. The product may be frozen at -70°C or lyophilized.

[0181] In exemplary embodiments, antibodies of the invention comprise a HC that includes a C-terminal lysine, such as SEQ ID NOs: 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, 544, 554, 1127, 1129, 1131, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, or 1160. In alternative embodiments, antibodies comprise a HC that does not include a C-terminal lysine, such as SEQ ID NOs: 573-592 or SEQ ID NOs: 1238-1254. In addition, the N-terminal glutamine and / or N-terminal glutamic acid of HC may be converted to pyroglutamic acid. Either form is contemplated for the antibody of the present invention.

[0182] Similarly, in exemplary embodiments, the anti-PD-1 antibody comprises a heavy chain that includes a C-terminal lysine, such as, for example, SEQ ID NO: 41. In an alternative embodiment, the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO: 636 that does not include a C-terminal lysine. In other exemplary embodiments, the anti-PD-1 antibody comprises a heavy chain that includes a C-terminal lysine. In an alternative embodiment, the anti-PD-1 antibody comprises a heavy chain that does not include a C-terminal lysine.

[0183] The anti-CCR8 antibody of the present invention, or pharmaceutical composition comprising the same, may be administered by parenteral route, non-limiting examples of which are subcutaneous administration and intravenous administration. Other possible administration routes are intramuscular, intraarterial, intralesional, and peritoneal bolus injection. The anti-CCR8 antibody may also be administered by injection, for example, intravenous or subcutaneous injection. The anti-CCR8 antibody of the present invention may be administered to a patient in a single dose or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. Optionally, the composition further comprises one or more physiologically active agents. The pharmaceutical composition of the present invention may be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and comprises an antibody as disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0184] As used interchangeably herein, "treatment" and / or "treating" and / or "treating" are intended to refer to any process that may slow, hinder, arrest, control, halt, or reverse the progression of the disorders described herein, but does not necessarily indicate the complete elimination of all symptoms of the disorder. Treatment includes administration of the anti-CCR8 antibodies of the present invention to treat a human disease or condition that benefits from the activity of the anti-CCR8 antibodies of the present invention, including (a) inhibiting further progression of the disease; and (b) relieving the disease, i.e., causing regression of the disease or disorder, or alleviating its symptoms or complications.

[0185] A therapeutically effective amount (or dose) of the anti-CCR8 antibody of the present invention can be administered. As used herein, "effective amount" refers to the amount of the anti-CCR8 antibody of the present invention or pharmaceutical composition comprising such an antibody that induces a biological or medical response or induces a desired therapeutic effect in a tissue, system, animal, mammal, or human as determined by a researcher, physician, or other clinician. An effective amount of an antibody can vary depending on factors such as the disease state, age, sex, and weight of an individual, and the ability of the antibody to induce a desired response in an individual. An effective amount is also an amount in which any toxic or harmful effects of the antibody are outweighed by the therapeutically beneficial effects. Such benefits include amelioration of signs or symptoms of cancer. An effective amount of the anti-CCR8 antibody of the present invention can be administered in a single dose or multiple doses. In determining an effective amount for a patient, the attending physician will take into account many factors, including, but not limited to, the patient's size (e.g., weight or mass), body surface area, age, and general health; the particular disease or disorder involved; the extent, or complications, or severity of the disease or disorder; the individual patient's response; the particular compound being administered; the method of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances known to the physician.

[0186] Dosage, frequency of administration, formulation, and effective amounts of PD-1 / PD-L1 pathway antagonists, bispecific T cell engager molecules, and / or immune cell costimulatory receptor agonists can also be determined as described herein.

[0187] Suitable PD-L1 antagonist antibodies for use in the methods of the invention include, but are not limited to, atezolizumab, avelumab, or durvalumab. Examples of suitable PD-1 antagonist antibodies for use in the methods of the invention include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, pidilizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, antibody 20C1.006, zelvalimab, antibody 20A2.003, antibody 22D4.006, or antibody 22D4.017, and any of the PD-1 antagonist antibodies described in WO 2019 / 140196. Such methods include a method of treating cancer in a patient comprising administering to the patient an effective amount of an anti-CCR8 antibody of the invention and a PD-L1 antagonist antibody or a PD-1 antagonist antibody. Also, such methods include a method of treating cancer in a patient comprising administering to the patient an effective amount of a Treg-depleting antibody and one or more of a bispecific T cell engager molecule, an agonist of a T cell costimulatory receptor, and an antagonist of the PD-1 / PD-L1 pathway.

[0188] Bispecific T cell engager molecules are recombinant protein constructs made from two flexibly linked antibody derived binding domains. (商標登録)The bispecific T cell engager molecules may be used interchangeably with "bispecific T cell engager molecule." One binding domain of the bispecific T cell engager is specific for a selected tumor-associated surface antigen on the target cell, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on the T cell. Due to their special design, bispecific T cell engager molecules are uniquely suited to transiently link T cells to target cells and simultaneously potently activate the intrinsic cytolytic capacity of T cells against the target cells (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). "Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies". International Journal of Molecular Sciences. 18 (1): 48 (2016)). Bispecific T cell engager molecules are bispecific, meaning that they bind to two targets (target antigen and CD3) simultaneously. Exemplary scFv sequences that bind to CD3 include I2E and I2C, and are listed in Table 15. Bispecific T cell engager molecules suitable for use in the methods of the present invention include, but are not limited to, the bispecific T cell engager molecules provided in Table 15.

[0189] The CD3 binding domain I2C comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3, VH, VL, and VH-VL amino acid sequences of SEQ ID NOs: 87-95, respectively. The CD3 binding domain I2E comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3, VH, and VL amino acid sequences of SEQ ID NOs: 96-103, respectively. An example of a CD33 T cell engager molecule comprises the CDR, VH / VL domain, and bispecific single chain molecule amino acid sequences of SEQ ID NOs: 104-118. An example of an EGFRvIII T cell engager molecule comprises the CDR, VH / VL domain, and bispecific single chain molecule amino acid sequences of SEQ ID NOs: 119-129. An example of an MSLN T cell engager molecule comprises the CDR, VH / VL domain, and bispecific single chain molecule amino acid sequences of SEQ ID NOs: 130-141. An example of a CDH19 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 142-159. An example of a FLT3 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 160-170. An example of a DLL3 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 171-181. An example of a CD19 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 182-191. An example of a BCMA T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 192-202. An example of a PSMA T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 203 to 240. An example of a CD70 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 241 to 250. An example of a CLDN18.2 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 251 to 266.An example of a MUC17 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 267-302. An example of a CDH3 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 303-313. An example of a CD19 T cell engager molecule comprises the CDRs, VH / VL regions, and bispecific single chain molecule amino acid sequence of SEQ ID NOs: 314-332.

[0190] Methods of the invention include methods of treating cancer in a patient comprising administering an effective amount of an anti-CCR8 antibody and a bispecific T cell engager molecule of the invention. Such methods also include methods of treating cancer in a patient comprising administering to the patient an effective amount of a Treg-depleting antibody and one or more of a bispecific T cell engager molecule, an agonist of a T cell costimulatory receptor, and an antagonist of the PD-1 / PD-L1 pathway.

[0191] An agonist of immune cell costimulatory receptor is a molecule that binds to a costimulatory receptor on an immune cell (e.g., an activated T cell) and promotes the activity of the receptor. Examples of costimulatory receptors include CD2, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB), TNFRSF14 (HVEM), TNFRSF18 (GITR), and ICOS. EXAMPLES

[0192] Working Example Example 1: CCR8 specificity Transfected cells were used to assess the binding specificity of the antibodies using flow cytometry into host human embryonic kidney (HEK) 293T cells. Human CCR8 (SEQ ID NO: 21), human CCR8 with A27G point mutation (SEQ ID NO: 23), mouse CCR8 (SEQ ID NO: 24), rat CCR8 (SEQ ID NO: 25), human CCR4 (SEQ ID NO: 26), or a control expression vector, Gibco, were transfected according to the manufacturer's instructions. (商標)Opti-MEM® medium (Gibco), and 293Fectin (商標) Proteins were expressed in HEK293T cells by transfection using reagents (Invitrogen). A human T-cell lymphoma (HuT78) cell line was also used to measure specificity for endogenously expressed CCR8.

[0193] Transfected HEK293T cells (24 hours after transfection) or HuT78 cells were resuspended in FACS buffer (PBS + 2% fetal bovine serum) and added to 96-well plates. Hybridoma supernatant samples containing control antibodies 433H (BD Biosciences) or L263G8 (BioLegend), Antibody 1 IgG2, or Antibody 2 IgG2 were added to a final concentration of 5.0 μg / ml, cells were resuspended, and incubated for 1 hour at 4° C. Plates were washed twice with FACS buffer, centrifuged to pellet cells, supernatant removed, and resuspended in FACS buffer to remove unbound antibody.

[0194] Alexa Fluor 647 goat anti-human or rat IgG secondary antibody (specific for Fcγ fragments) (Jackson ImmunoResearch) made up in FACS buffer at 5.0 μg / mL was added to each well, cells were resuspended, and incubated for 15 minutes at 4° C. Plates were washed twice with FACS buffer, centrifuged to pellet cells, supernatant removed, and resuspended in FACS buffer to remove unbound secondary antibody. Samples were resuspended in FACS buffer and autosampled using an IntelliCyt® iQue or BD Accuri using an Intellicyt HyperCyt autosampler. (商標) The results were read on either a flow cytometer or a flow cytometer. Data derived from the three cohorts of immunized animals are shown in Table 2.

[0195] [Table 1]

[0196] These data show that hybridoma supernatants containing IgG2 of Antibody 1 or IgG2 of Antibody 2 bound to human CCR8, including human CCR8 with the A27G mutation, rat, and mouse CCR8. None of the antibodies in the supernatants tested bound to human CCR4 or 293T cells transfected with a control expression vector.

[0197] Example 2: Antibody binding to human and cynomolgus monkey regulatory T cells Binding of anti-CCR8 antibodies to endogenous CCR8 expressed by primary human and cynomolgus regulatory T cells (human or cynomolgus T-reg) was assessed by flow cytometry. Freshly isolated human (n=3) and cynomolgus (N=2) peripheral blood mononuclear cells (PBMCs) were incubated with anti-CCR8 hybridoma culture supernatant at a final concentration of 20% in the presence of human Fc Block for 1 h at 4°C. The primary antibodies were washed from the cells and secondary anti-human or anti-rat IgG Fc antibodies and a cocktail of human / cynomolgus cross-reactive anti-CD4 / anti-CD25 / anti-CD127 antibodies were added and incubated for 30 min at 4°C. 200,000 events were collected using a FACS Canto flow cytometer and binding was detected on viable cells gated for CD4+ / CD25+ / CD127-. The percentage of positive cells indicates the percentage of human or cynomolgus Tregs stained by the hybridoma culture supernatant containing the antibody under test. The control antibody, 433H, was purified and used at a concentration of 20 μg / ml. The results are shown in Table 3.

[0198] [Table 2]

[0199] These data demonstrate that hybridoma supernatants containing the antibodies of the invention bind to endogenously expressed human and cynomolgus CCR8 expressed on primary T cells.

[0200] Example 3: Epitope binning To allow epitope mapping of anti-CCR8 antibodies, hybridoma supernatants that bound human CCR8 were tested for binding to five biotinylated N-terminal CCR8 peptides generated from the 1-35 amino acid N-terminal portion of CCR8 (SEQ ID NO:31). Each of the five peptides was 12 amino acids long and overlapped by 6 amino acids. The amino acid sequences of peptide 1, peptide 2, peptide 3, peptide 4, and peptide 5 comprise amino acids 1-12 (SEQ ID NO:82), 7-18 (SEQ ID NO:85), 13-24 (SEQ ID NO:83), 19-30 (SEQ ID NO:86), and 25-35 (SEQ ID NO:84) of SEQ ID NO:31, respectively.

[0201] Biotinylated human CCR8 peptide was captured on streptavidin polystyrene beads (Spherotech) in FACS buffer (PBS + 2% fetal bovine serum) at a final protein concentration of 50-100 ng / mL and incubated at room temperature for 30 min. Beads were washed twice with FACS buffer to remove unbound protein, centrifuged to pellet the beads, resuspended and pooled together in StabilGuard (SurModics). Pooled biotinylated human CCR8 coated beads were added to hybridoma supernatant samples in 96-well plates for a final antibody concentration of 5.0 μg / mL, then incubated at room temperature for 1 h.

[0202] Plates were washed twice with FACS buffer, centrifuged to pellet the beads, the supernatant removed, and resuspended in FACS buffer to remove unbound antibody. Alexa Fluor 488 goat anti-human or rat IgG secondary antibody (specific for Fcγ fragment) (Jackson ImmunoResearch) made up in FACS buffer at 5.0 μg / mL was added to each well, resuspended with the beads, and incubated at room temperature for 15 minutes. Plates were washed twice with FACS buffer, centrifuged to pellet the beads, the supernatant removed, and resuspended in FACS buffer to remove unbound secondary antibody. Samples were then resuspended in FACS buffer and read on either an IntelliCyt® iQue flow cytometer.

[0203] The results are shown in Table 4. Data are presented as the ratio of the geometric mean of binding to beads coated with a specific peptide divided by the geometric mean of binding to beads coated with a negative control peptide (a peptide with an unrelated sequence). Values ​​above 2 indicate binding.

[0204] [Table 3]

[0205] Interestingly, hybridoma supernatants containing Antibody 1 IgG2 bound to the most N-terminal region (1-12), suggesting that Antibody 1 IgG2 binds to a unique epitope on CCR8, which may contribute to the high affinity and biological activity of Antibody 1 IgG2.

[0206] Example 4: Epitope Clustering The extracellular domain of human CCR8 contains three loops and a 35 amino acid N-terminal peptide. For epitope mapping, the N-terminal peptide of human CCR8 (designated as P_1-35 (SEQ ID NO: 31)) was divided into three consecutive segments (designated as P_1-12 (SEQ ID NO: 82), P_13-24 (SEQ ID NO: 83), and P_25-35 (SEQ ID NO: 84)). Two additional overlapping fragments (designated as P_7-18 (SEQ ID NO: 85) and P_19-30 (SEQ ID NO: 86)) were generated to cover adjacent N- or C-terminal regions of the consecutive segments. The full-length N-terminal peptide of human CCR8 and all truncated N-terminal peptides described above were fused to the C-terminal end with a V5 tag via a G4S linker. The V5 tag was further fused to chicken albumin via a G4S linker, followed by a FLAG tag, BAP (biotin acceptor protein) for in vivo biotinylation, and H3G, each fused via an SG linker. All the constructs described above were cloned into the pEFDHFR vector and transiently transfected into HEK293 cells.

[0207] HEK293 cells (1 × 10 8 ) and transfected with 4 ml of OptiMEM (Gibco 31985-047), 100 μl of 293fectin (Invitrogen 12347-019), and 50 μg of DNA encoding either full-length or N-terminally truncated CCR8 constructs according to the manufacturer's protocol. Cells were incubated at 37 °C for 2 h at 8% CO 2 The cells were grown in FreeStyle expression medium at 130 rpm in a humidified incubator at 37 °C for 72 h. The cells were centrifuged at 1,500 rpm for 10 min and the supernatant was collected. 10 ml of each of the transfected cell supernatants or 9 ml of the HEK293 cell supernatant as a negative control were concentrated 20-fold to 500 μL in Amicon Ultra-15 tubes (UFC901008). 18 × 10 CCR8 constructs were added to each of the full-length and truncated N-terminal CCR8 constructs and the HEK293 negative control. 6 Washed streptavidin beads (Streptavidin Microspheres, 6 μm; Polysciences 24172-1) were resuspended in 500 μl of concentrated supernatant and incubated for 1 h with gentle shaking. Beads bound to the respective antigen or negative control were washed and stored overnight at 4°C.

[0208] To verify expression of full-length and N-terminal truncated CCR8 constructs and binding to streptavidin beads, 2 × 10 5The beads were incubated with 5 μg / mL of anti-FLAG antibody (clone M2, Sigma F3165 / F1804), 5 μg / mL of anti-V5 antibody (clone SV5-Pk1; AbD Serotec, MCA 1360), and PE-labeled anti-mouse Fcγ secondary antibody (Jackson 115-116-071) at a dilution of 1:100. The antigen-bound beads were incubated with three different anti-human CCR8 antibodies. Binding of two of the anti-human CCR8 antibodies (clone L263G8; BioLegend, 360602 and clone 433H; BD 747578; each at 5 μg / ml) was detected by PE-labeled anti-mouse Fcγ secondary antibody (Jackson 115-116-071) at a dilution of 1:100. Binding of anti-human CCR8 antibody (polyclonal; Abcam, ab140796) was detected by a 1:50 dilution of PE-labeled anti-goat Fcγ secondary antibody (Jackson 109-116-098).

[0209] Binding of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules to full-length and truncated N-terminal CCR8 constructs bound to streptavidin beads was measured. Most commonly, T cell engager ("TCE") molecules comprise a single polypeptide chain capable of binding to two different antigens. The term "TCE molecule" refers to a molecule that binds to a "BiTE (商標登録) The terms "bispecific T cell engager" and "bispecific T cell engager" may be used interchangeably with the terms "bispecific T cell engager molecule" or "bispecific T cell engager molecule." The TCE molecules tested included a molecule containing an scFab that binds to CCR8 and an scFv that binds to CD3 (scFab-containing TCE molecule), and a molecule containing an scFv that binds to CCR8 and an scFv that binds to CD3. The TCE molecules tested also contained an scFc at the C-terminus as a half-life extension (HLE) site. The CDRs of the antibody of antibody 1 are identical to the CDRs of TCE1 (the CDR amino acid sequences of TCE1 have SEQ ID NOs: 561-566). The CDRs of the IgG2 of antibody 2 are identical to the CDRs of TCE2 (the CDR amino acid sequences of TCE2 have SEQ ID NOs: 567-572).

[0210] Beads were incubated with 5 μg / mL of each TCE molecule. Binding of these CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules was detected using 2 μg / mL anti-histidine antibody (clone AD1.1.10; AbD Serotec MCA 1396) and a 1:100 dilution of PE-labeled anti-mouse Fcγ secondary antibody (Jackson 115-116-071). All antibodies, CCR8-binding TCE molecules, and scFab-containing CCR8-binding TCE molecules were diluted in PBS containing 2% FBS, and all incubations were performed at 4°C for 45 min (primary antibody) or 30 min (secondary antibody). Washing was performed using PBS containing 2% FBS, and the final suspension buffer before FACS analysis was also PBS containing 2% FBS. Antibody-TCE binding was detected using Intellicyte IQue. Changes in mean fluorescence were analyzed by Intellicyte IQue and FlowJo. Binding to various full-length and N-terminal truncated CCR8 constructs was reflected as a positive signal detected by flow cytometry.

[0211] As shown in Tables 5 and 6, expression and binding of full-length and various N-terminal truncated CCR8 constructs to streptavidin beads was verified by flow cytometry.

[0212] [Table 4]

[0213] The data in Table 5 show that the anti-human CCR8 antibodies bind to the full-length N-terminal peptide P_1-35 of human CCR8, indicating that the anti-human CCR8 antibodies recognized the N-terminal peptide of human CCR8. None of the antibodies bound to streptavidin beads alone or to the HEK293 control. The anti-human CCR8 antibodies (clone L263G8 and clone 433H) showed the same binding pattern, but the polyclonal anti-human CCR8 antibody showed additional binding to the overlapping fragment P_7-18.

[0214] [Table 5]

[0215] The data in Table 6 show that CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules bound to the full-length N-terminal CCR8 peptide P_1-35. TCE2 bound to the truncated N-terminal CCR8 peptide P_13-24. Interestingly, TCE1 bound to the truncated N-terminal CCR8 peptide P_1-12, indicating that TCE1 binds to a unique epitope on CCR8.

[0216] Example 5: Antibody Functional Activity Hybridoma supernatants were tested for inhibition of CLL-1-dependent chemotaxis in HUT78 cells (a human T lymphocyte cell line that endogenously expresses CCR8). Tests were performed in 96-well transwell plates with 5 μm pore size in complete HUT-78 growth medium. Cells were preincubated with purified antibodies for 30 min and transferred to the upper transwell chamber (total volume 50 μl, 50,000 cells per well).

[0217] Recombinant Hu CCL1 (R&D) was prepared at a suboptimal concentration of 100 pM and added to the lower transwell chamber at 100 μl per well. The transwell plate was incubated overnight at 37 °C with 5% CO2. The suboptimal concentration of CCL1 was set based on the cell chemotaxis dose-response curve and determined as IC 50Antibody selection of ≦100 pM was allowed. At the end of the incubation, the upper chamber was removed and 50 μl / well of CellTiterGlo reagent (Promega) was added to the lower chamber containing the migrated cells. After 10 min incubation at room temperature, 100 μl of the mixture from the lower chamber was transferred to a black well clear bottom plate for luminescence readout (Envision plate reader). Percentage of chemotaxis inhibition was calculated using Basal and Max chemotaxis control wells present on each plate. Screener analysis software was used to calculate percent inhibition and IC 50 Values ​​were calculated and the average of three experiments is shown in Table 7.

[0218] [Table 6]

[0219] These data indicate that Antibody 1, which binds a unique epitope, does not inhibit chemotactic activity despite binding to CCR8, and is not a neutralizing antibody. These data indicate that Antibody 1 does not inhibit ligand binding to CCR8. Similar data were observed in experiments testing antibodies in hybridoma supernatants.

[0220] Example 6: Antibody-mediated cytotoxicity assay To determine whether anti-CCR8 antibodies can mediate antibody-mediated cytotoxicity (ADCC), killing assays were developed using HUT78.luc target cells stably transfected with a luciferase reporter gene and expressing endogenous human CCR8. Primary NK cells with VF phenotype from six different donors were used as effector cells (for data in Tables 8a and 8b), primary NK cells with VF phenotype from two different donors were used as effector cells (for data in Table 8c), or primary NK cells with FF phenotype from three different donors were used as effector cells with three individual bleeds for one of them (for data in Table 8d). Negative selection of NK cells was performed by leukopack using the StemCell EasySep Hu NK isolation kit.

[0221] Purified antibodies were tested at a range of concentrations starting from 5 μg / ml (35 nM at 1:10 dilution). Antibodies were incubated overnight with target and effector cells in 384-well plates at 37°C with 5% CO2 in a humidified incubator. The effector to target ratio was 5:1 with 20,000 target cells per well in a total of 50 μl per well. At the end of the incubation, 30 μl of BioGlo Reagent (Tables 8a and 8b) or SteadyGlo Reagent (Table 8c) per well was added, mixed and luminescence was read on an Envision plate reader. Luminescence signal was proportional to the amount of viable target cells. Percentage of ADCC was calculated as (1-(luminescence signal in the presence of Ab / luminescence signal in T+E cells alone))×100. EC was calculated using GraphPad Prism 7. 50 The results are shown in Figures 8a, 8b, 8c, and 8d (ND means not determined).

[0222] [Table 7]

[0223] [Table 8]

[0224] [Table 9]

[0225] [Table 10]

[0226] These data demonstrate that the antibodies of the invention exhibit ADCC-based killing via the CCR8 receptor expressed on the surface of HUT78 cells.

[0227] Example 7: Affinity of anti-CCR8 antibodies Hybridoma supernatants containing Antibody 1 IgG2, Antibody 2 IgG2, or Antibody 4 IgG2 were assessed for their affinity to native cynomolgus monkey CCR8 transiently expressed on 293T cells or native human CCR8 expressed on HUT78 cells by equilibrium exclusion assay (KinExA).

[0228] Cynomolgus CCR8:293 T cells KinExA was performed to determine the K from the concentration of free antibody remaining in solution after equilibrium was established between the antibody and the antigen expressed on the cell surface. d was measured. KinExA provides a sensitive measurement of binding affinity for native CCR8 compared to soluble CCR8. Equilibrium exclusion assay was performed essentially as described in Rathanaswami et al. Anal. Biochem: 373(1): 52-60 (2008).

[0229] Briefly, equilibration sets were set up with each antibody using either human CCR8-expressing HUT78 cells or cynomolgus CCR8-expressing 293T cells. Cells were counted using a hemocytometer. HUT78 cells were titrated and incubated with 0.05% sodium azide in HUT medium (RPMI 1640, 10% FBS, 10 mM HEPES, 2 mM L-Glut, 1 mM Sod.Pyr, 0.1 mM NEAA, 50 μM 2-ME) at two different constant antibody concentrations (one 48 pM, the other 2 nM). For the high [Ab] equilibration set, HUT78 cells were titrated from 62.5 million cells per milliliter 1:2 for 10 points in Eppendorf tubes and equilibrated with 2 nM antibody in a total volume of 400 μl. For the low [Ab] equilibration set, HUT78 cells were titrated 1:2 from 3.89 million cells per milliliter for 10 points in 50-ml Fulcon tubes and equilibrated with 48 pM antibody in a total volume of 15.5 mL.

[0230] Cynomolgus CCR8-expressing 293T cells were titrated and incubated with 0.05% sodium azide in 293T medium (Freestyle-expressing 293T medium with 2% FBS and 50 μg / ml G418) at two different constant antibody concentrations (one at 118 pM and the other at 5 nM). In the high [Ab] equilibration set, 293T cells were titrated 1:3 over 10 points from 25 million cells per milliliter in Eppendorf tubes and equilibrated with 5 nM antibody in a total volume of 200 μl. In the low [Ab] equilibration set, 293T cells were titrated 1:3 over 10 points from 980,000 cells per milliliter in 15 ml Fulcon tubes and equilibrated with 118 pM antibody in a total volume of 10.2 mL.

[0231] For each equilibrium set, reference point controls included samples containing cell medium only and samples without cells. The equilibrium sets were incubated for 24 hours at room temperature with shaking. After 24 hours of incubation, the supernatants were separated from the cell pellets by centrifugation at 500×g for 5 minutes. The supernatants from both the high [Ab] and low [Ab] equilibrium sets were then run on a KinExA 3200 instrument.

[0232] Each equilibration sample set was read in duplicate on the KinExA instrument. For the low [Ab] equilibration samples, 6.8 mL and 4.6 mL of each sample were run in duplicate for human and cynomolgus CCR8 equilibration experiments, respectively. For the high [Ab] equilibration samples, 16 μL and 75 μL of each sample were run in duplicate for human and cynomolgus CCR8 equilibration experiments, respectively.

[0233] PMMA (polymethylmethacrylate particles) beads were coated with goat anti-human Fc Ab or goat anti-hIgG (H+L) Ab, followed by blocking with blocking solution (1x PBS pH 7.4 + 10mg / mL BSA + 0.05% sodium azide). For each equilibration sample, free [Ab] was detected by passing the equilibration sample over the coated beads followed by a quick wash with running buffer (1x PBS pH 7.4 + 1% BSA + 0.05% sodium azide). Secondary detection antibody (Alexa 647, goat anti-huIgG (H+L)) was passed through the flow cell at 680ng / mL and 500μL per run. The KinExA voltage output signal was used in the KinExA software to measure the K d was calculated from the plots at two different initial total [Ab] concentrations by curve fitting using n-curve analysis in KinExA Pro software version 4.3.11 (Sapidyne Instruments Inc.). d The 95% confidence interval was calculated for low K D and high K D The results are shown in Table 9.

[0234] [Table 11]

[0235] Native human CCR8 expressed in HUT78 cells Cells were serially diluted in medium and incubated and equilibrated with the active binding sites of the antibody at a concentration of 48 pM or 2 nM in medium in the presence of 0.05% NaN3. The free mAb remaining in the supernatant was measured as described above. The percentage of free antibody was plotted against cell concentration. N curve analysis was performed using the equilibration to obtain the K d A whole-cell approach was performed to determine optimal values ​​for K and antigen expression levels. The software optimized the K while holding other parameters at their optimal values. d Alternatively, the 95% confidence interval was determined by iteratively changing the optimized value of the antigen expression level.

[0236] The affinity of the test antibodies to endogenous human CCR8 expressed in HUT78 cells is shown in Table 10.

[0237] [Table 12]

[0238] The IgG2 antibody was further engineered to increase its affinity to human and / or cynomolgus CCR8.

[0239] Example 8: T4R mutants of cynomolgus CCR8: CHO cells CHO cells expressing cynomolgus CCR8 (containing threonine at position 4; SEQ ID NO: 22) or cynomolgus CCR8 (T4R; containing arginine at position 4; SEQ ID NO: 556) were incubated with decreasing concentrations of anti-CCR8 antibody (0.005-100 nM, step 1:3, 10 steps) at 4 °C for 30 min. Bound anti-CCR8 antibody molecules were detected by Alexa Fluor 647-conjugated goat anti-human IgG (H+L). Subsequently, cells were stained with Zombie Violet viability dye, fixed with 4% PFA on ice, and detected by fluorescence cytometry. Equilibrium dissociation constants (Kd ) values ​​were calculated. The affinities of the anti-CCR8 antibodies are shown in Table 11. "ND" means not detectable.

[0240] [Table 13]

[0241] These data indicate that binding of antibody 1 to cynomolgus CCR8 was reduced by the T4R mutations, while antibody 2 was unaffected, consistent with the respective epitope binning and clustering determined above. These data indicate that antibodies that bind unique epitopes as described herein bind to CCR8 at the threonine at position 4.

[0242] Example 9: IgG1 afucosylated antibodies Afucosylated anti-CCR8 IgG1 antibodies were generated. Examples of antibody amino acid sequences of afucosylated antibodies are SEQ ID NOs: 346-555, 1125-1160, and 1238-1254. SEQ ID NOs: 573-592 and 1237-1254 correspond to the HC of antibodies that do not contain a C-terminal lysine. Antibodies are designated according to the parent molecule. For example, Antibody 5.1, Antibody 5.2, Antibody 5.3, Antibody 5.4, Antibody 5.5, Antibody 5.6, Antibody 5.7, Antibody 5.8, and Antibody 5.9 all refer to antibodies modified from Antibody 5. "IgG2 of Antibody 1" and "IgG2 of Antibody 2" refer to IgG2 antibodies, while "IgG1 of Antibody 1" and "IgG1 of Antibody 2" refer to IgG1 afucosylated antibodies. Additionally, the IgG1 molecule of Antibody 2 was further modified from the IgG2 antibody of Antibody 2, as set forth in the Sequence Listing (Table 16), to obtain, for example, the IgG1 afucosylated antibodies of Antibody 2.1 and Antibody 2.2. The IgG1 of Antibody 2.2 comprises, for example, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HCVR, LCVR, HC, and LC amino acid sequences as set forth in SEQ ID NOs: 376-385, respectively.

[0243] The modified molecules may exhibit desirable properties, such as, but not limited to, increased affinity for humans and / or cynomolgus monkeys. The modification sites are listed in the sequence listing (Table 19).

[0244] The examples described herein demonstrate in vivo studies showing the activity of afucosylated anti-CCR8 antibodies, such as ADCC activity (Example 16), anti-tumor activity (Example 11) and increased survival (Example 12).

[0245] Example 10: Treg depletion combination therapy The efficacy of administration of bispecific T cell engager molecules, PD-1 antagonist antibodies, 4-1BB agonist antibodies, and Treg-depleting antibodies was measured. Mice genetically modified to express humanized CD3ε molecules on the surface of T cells were treated with 10 10 cells of KPC-M5 cancer cells in 50 μl of PBS mixed with 50 μl of Matrigel. 5 The KPC-M5 syngeneic tumor cell line was injected subcutaneously with an inoculum consisting of 1000 cells. 3 Once tumor volume reached 100 μg / kg, mice were injected with one or more of the bispecific T cell engager molecules (bispecific molecules that bind CD3 and a target antigen), PD-1 antagonist antibodies, 4-1BB agonist antibodies, and Treg-depleting antibodies, and tumor volumes were measured over time. Bispecific T cell engager molecules were administered at doses ranging from 15 to 5,000 μg / kg, depending on the bispecific T cell engager molecule administered.

[0246] Anti-Mouse CLDN18.2 BiTE (商標登録)The molecules were injected intravenously into tumor-bearing mice at a dose of 150 μg / kg once a week. Mice were co-injected intravenously every 3 days with an anti-mouse PD-1 mIgG1 antagonist antibody at a dose of 100 μg per mouse, an agonist antibody against the 4-1BB costimulatory receptor (anti-mouse 41BB rIgG1 (clone LOB12.3, BioXcell) at a dose of 150 μg per mouse, and / or a Treg-depleting antibody (mIgG1) at a dose of 300 μg per mouse. Tumor volumes were measured on days 7, 10, 14, 17, and 20 after injection.

[0247] The data in Figure 1 show that minimal activity was observed with the combination of 4-1BB agonist + anti-PD-1 or anti-CTLA4 with the CLDN18.2 BiTE® molecule alone, whereas the quadruple combination of CLDN18.2 BiTE® molecule + 4-1BB agonist + anti-PD-1 + anti-CTLA4 significantly increased CD4 + The results show that the antitumor effect was robust, similar to that observed with T cell depletion. Notably, this antitumor effect was driven by the expression of intratumoral CD8 + This was associated with a clear increase in the T cell:Treg ratio. Taken together, these data support the notion that CD8 + This study demonstrated the selective activity and dependency of the bispecific T cell engager molecule on T cells, and demonstrated that the antitumor effect mediated by the bispecific T cell engager molecule was associated with CD4 + These results suggest that T cells play a context-dependent inhibitory role.The results also suggest that Tregs may play a major role in suppressing the activity of bispecific T cell engager + anti-4-1BB + anti-PD-1 combination immunotherapy.

[0248] Example 11: CCR8-depleting antibodies are effective in the MC38 in vivo tumor model The antitumor activity of afucosylated anti-CCR8 mIgG2a in the MC38 syngeneic tumor model was measured. MC38 tumor cells were injected subcutaneously into the right flank of female hCD3eKI animals on day 0 of the study. On day 10, tumors grew to 99.93 mm 3Animals were assigned to different treatment groups (n=10 / group) based on mean tumor volume of 1000 mg / kg. On days 11, 14, 17, and 20 (Q3D×4) of the study, animals were administered 10 mg / kg intraperitoneally of either control isotype mIgG2a or anti-CCR8 afucosylated mIgG2a. The anti-CCR8 afucosylated mIgG2a antibodies comprise the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3, LCVR, HCVR, LC, and HC amino acid sequences of SEQ ID NOs: 637-646, respectively.

[0249] Tumor volumes were measured twice weekly. Statistical analysis evaluating the effect of anti-CCR8 antibody compared to isotype control on tumor size over time of treatment was performed using a linear mixed effects (LME) model with Dunnett's post-hoc analysis. **** indicates p<0.0001.

[0250] Individual tumor growth of the treatment groups is represented as spider plots in Figures 2C and 2B. Figure 2A shows the mean tumor volume + / - SEM for each group by the final time point (day 24). Mice treated with anti-CCR8 afucosylated mIgG2a antibody showed a statistically reduced tumor volume by day 24 compared to animals treated with isotype control. As shown in Figure 2B, there was one complete responder. This complete responder animal was evaluated until day 48, at which point there was no measurable tumor. Animals without measurable tumors, defined as complete responders (CR), were evaluated until day 48. These data show that MC38 tumor-bearing animals showed a significant reduction in tumor volume (66.44% TGI, ****p<0.0001) when treated with anti-CCR8 afucosylated mIgG2a antibody compared to isotype control.

[0251] Example 12: CCR8-depleting antibody therapy extends survival in vitro MC38 tumor cells were injected subcutaneously into the right flank of female hCD3eKI animals on day 0 of the study. On day 10, tumors grew to a size of 99.93 mm 3Animals were assigned to different treatment groups (n=10 / group) based on a mean tumor volume of 800 mm. On days 11, 14, 17, and 20 (Q3D×4) of the study, animals were administered 10 mg / kg intraperitoneally of either isotype control mIgG2a or anti-CCR8 afucosylated mIgG2a antibody. All animals were treated with 10 mg / kg of either isotype control mIgG2a or anti-CCR8 afucosylated mIgG2a antibody. 3 or according to IACUC standards for animal welfare. Statistical analysis was performed using the log-rank (Mantel-Cox) test comparing anti-CCR8 afucosylated mIgG2a antibody (treatment group 2) with the isotype control mIgG2a (control group 1). **** indicates p<0.0001.

[0252] Survival data are shown in Figure 3. The median survival time of animals treated with isotype control mIgG2a was 24 days, whereas the median survival time of animals treated with anti-CCR8 afucosylated mIgG2a antibody was 27 days (***p<0.0001). These data show that MC38 tumor-bearing animals had increased survival time when treated with anti-CCR8 afucosylated mIgG2a antibody compared to animals treated with isotype control antibody.

[0253] Example 13: Depletion of Tregs with CCR8 mIgG2a antibodies leads to enhanced CD8+ / Treg ratios in tumors On day 11 of the study, MC38 tumor-bearing animals were treated with a single dose of 10 mg / kg intraperitoneally of either control isotype mIgG2a or anti-CCR8 afucosylated mIgG2a. PD assessment was performed 48 hours after treatment (day 13). Tumor weights were collected during harvest in the different groups and used for normalization to determine absolute cell numbers in the tumor. Single cell suspensions of tumors, draining lymph nodes (DLN), and spleens were prepared for flow cytometric analysis of T cell percentages and phenotypes.

[0254] Total T cells were gated using TCRβ+Thy1.2+ staining within the viable / CD45+ fraction. The percentage and absolute number of Treg cells were assessed within the CD4+ T cell compartment using both Foxp3+ and CD25+Foxp3+ gating, as shown in Figure 4A. CD8+ T cells were gated on total T cells, and the CD8 / Treg ratio in the tumor was calculated as shown in Figure 4B. Each point represents data from an individual mouse. Statistical analysis was performed using unpaired T-test (two-tailed) to compare treatment and control groups (*p<0.05, **p<0.01).

[0255] These data show that the proportion of Tregs was decreased after a single dose of anti-CCR8 afucosylated antibody when assessed using both Foxp3+ and CD25+Foxp3+ gating schemes (Figures 4A and 4B). Importantly, treatment with anti-CCR8 depleting antibody led to a significant increase in the CD8+ / Treg ratio in tumors (Figures 4C and 4D), thereby promoting enhanced anti-tumor immunity.

[0256] Example 14: CCR8-binding scFvs screened by phage display A preferred class of amino acid substitution mutations of the CCR8 binding molecules described herein involves the substitution of one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they are generated. One method for generating such substitution variants involves affinity maturation using phage display. Briefly, several CDR sides (e.g., 6-7 sides) were mutated to generate all possible amino acid substitutions on each side. The antibody variants thus generated were displayed in a monovalent format from filamentous phage particles, for example as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants were then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidates for CDR sides to modify, alanine scanning mutagenesis was performed to identify CDR residues that contribute significantly to antigen binding.

[0257] Once such mutants are generated, the panel of mutants is screened as described herein, and antibodies with superior properties in one or more relevant assays are selected for further development. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991), and Marks et al., J. Mol.Biol., 222: 581-597 (1991).

[0258] Anti-CCR8 scFvs that bind the 1-12 amino acid epitope (amino acid sequence given by SEQ ID NO:82) were generated and screened for epitope binding by phage display essentially as described above. The heavy and light chain amino acid sequences of scFvs that bind to CCR8 in the 1-12 amino acid epitope cluster are shown in Table 12.

[0259] [Table 14]

[0260] Anti-CCR8 scFv MPK20299-A4 was further modified and converted to an afucosylated anti-CCR8 antibody to generate additional anti-CCR8 antibodies that bind 1-12.

[0261] Example 15: Affinity of CCR8-binding antibodies to CCR8 peptide-nanobody complexes Binding affinity (K of Fab of Antibody 1 and CCR8-binding monoclonal antibodies (mAbs) of the present invention to CCR8 1-12 epitopes (SEQ ID NO: 82)-Nanobody (Nb) fusion protein D Equilibrium dissociation constant) and rate constant (k a Binding rate constant, k d Dissociation rate constants (dissociation rate constants) were measured using an OCTET® Biolayer Interferometry system (Sartorius AG, Göttingen, Germany). CCR8-nanobody fusion proteins were expressed in human cells. For Fab binding, biotinylated CCR8 peptide-Nb fusions were captured on streptavidin SAX biosensors at loading concentrations of 2–4 nm and then incubated with a dilution series of soluble Fab (up to 100 nM, 6 points, 1:3 serial dilutions) for 300 s followed by incubation in buffer for 500 s for dissociation. For mAb binding, mAbs were captured on anti-huIgG Fc-uptake biosensors at loading concentrations of 1–2 nm and then incubated with a dilution series of non-biotinylated CCR8 peptide-Nb fusions (up to 100 nM, 6 points, 1:3 serial dilutions) for 300 s followed by incubation in buffer for 500 s for dissociation.

[0262] The OCTET® system uses a mechanism called biolayer interferometry to acquire data over time (seconds); once proteins bind to the biosensor chip, the instrument measures the sensitive binding signal in nm. All fiber optic tips were discarded after a single use; they were not regenerated. OCTET® buffer baseline, dissociation steps, and protein dilutions were performed in OCTET® buffer (10 mM TRIS pH 7.5, 150 mM NaCl, 1 mM CaCl2, 0.13% (v / v) Triton X-100, and 0.10 mg / mL BSA).

[0263] Raw data were processed by the GeneData Screener v18 SPR package using the same data processing as the OCTET® instrument data analysis software (subtracting the average of two reference wells per column, fitting the Y-axis to baseline, fitting inter-step correction to dissociation, and Savitzky-Golay filtering). a ; Unit is M -1 sec -1 ) and dissociation rate constant (k d ; Unit is sec -1 Each Fab or mAb interaction was grouped into its own sensorgram and globally fitted with a 1:1 binding model to determine the equilibrium dissociation constant (K D ;The unit is nanomolar (nM) = 1 x 10 -9 mol / L) to k d / k a Calculated as a ratio.

[0264] The results are shown in Figures 13a and 13b. The errors in the 1:1 model fit to the processed data were reported as standard errors (i.e., k a The error is the standard error of the binding rate constant measurement, k d The error is the standard error of the measured dissociation rate constant. D ) is the relationship between two measured variables and their standard errors (k a , Δka , k d , Δk d ) is calculated from the statistical error propagation as defined above.

[0265] [Table 15]

[0266] [Table 16]

[0267] These data demonstrate that the CCR8-binding antibodies of the present invention bind with high affinity to an N-terminal peptide of human CCR8 containing amino acids 1 to 12 (SEQ ID NO: 82) and amino acids 1 to 25 (residues 1 to 25 of SEQ ID NO: 31) expressed in human cells.

[0268] Example 16: ADCC in the presence or absence of ligand To measure ADCC by anti-CCR8 antibodies that either inhibit or do not inhibit ligand binding, flow cytometry was used to measure live and dead cells in the presence of various concentrations of ligand and anti-CCR8 antibody. In the experiment ("Test A"), 100 pM of an afucosylated anti-CCR8 antibody of the invention that binds to a unique epitope and does not inhibit ligand binding (antibodies containing the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1-6, respectively; "non-inhibitory mAbs") or three anti-CCR8 antibodies that inhibit ligand binding ("inhibitory mAbs") were incubated with HUT78 cells expressing CCR8, the NK cell line NK92MI expressing CD16 (effector cells), and increasing concentrations of CCL1 (ligand) ranging from 0.128 pM to 50 nM. In another experiment ("Test B"), a similar procedure was followed as described above, except that increasing concentrations of CCL1 were first added to HUT78 cells for 30 min, followed by addition of 100 pM of antibody and effector cells. IC 50 The values ​​of the minimum mortality and the maximum mortality are reported in Table 14. Following the procedures essentially as described, the following data was obtained.

[0269] [Table 17]

[0270] These data show that the anti-CCR8 antibodies of the present invention that bind to unique epitopes and do not inhibit ligand binding were highly potent in both Test A and Test B in the presence of ligand, and also showed the highest minimum killing rates, measuring ADCC capacity, at high concentrations of CCL1.

[0271] Example 17: Anti-CCR8 molecules and BiTEs in vivo (商標登録) Molecular Combinations CCR8-depleted murine surrogate antibodies were evaluated in combination with surrogate TAA-BiTE molecules for their ability to enhance anti-tumor activity in the B16F10 tumor model. The B16F10 tumor model was chosen for this combination efficacy study because it is refractory to checkpoint inhibitors (anti-PD1 and anti-CTLA4) and therefore can be used to evaluate meaningful differences from the combination therapy of BiTE molecules and anti-CCR8 mAbs in this example.

[0272] B16F10 tumor cells were engineered to express a BiTE molecule tumor-associated antigen (TAA) and injected into an immunocompetent humanized CD3e KI line that allows evaluation of TAA-BiTE molecules with an I2C anti-CD3 scFv that recognizes human CD3e. B16F10-TAA tumor-bearing animals were treated with either a CCR8-depleted mIgG2a antibody, a TAA-BiTE molecule alone, or a combination of a CCR8-depleted mIgG2a antibody and a TAA-BiTE molecule.

[0273] B16F10-TAA expressing tumor cells were injected subcutaneously into an immunocompetent mouse model expressing a humanized CD3e chain (huCD3e KI) on day 0. On day 12, tumors grew to a size of 108.37 mm 3Animals were assigned to different treatment groups (n=10 / group) based on mean tumor volume of 1000–10 ...

[0274] Tumor volumes were measured twice weekly. Individual tumor growth for treatment groups is depicted as spider plots in Figure 5. Animals without measurable tumors, defined as complete responders (CR), were evaluated through day 48.

[0275] As shown in Figure 5, monotherapy with CCR8 mIgG2a (group 3; Figure 5C) was ineffective in this anti-CTLA4 refractory cold tumor model. Monotherapy with TAA-BiTE (group 2; Figure 5B) resulted in delayed tumor growth and one tumor-free / complete responder (CR) at the end of the study. Interestingly, the combination of CCR8 mIgG2a and TAA-BiTE (group 4; Figure 5D) resulted in seven CRs, indicating a significant benefit of combining BiTE molecules with CCR8-depleting mAbs to enhance anti-tumor immunity.

[0276] array IgG2 HCDR1 of Antibody 1 (SEQ ID NO: 1) NARMG IgG2 HCDR2 of Antibody 1 (SEQ ID NO:2) RIKSKTEGGTRDYAAPVKG IgG2 HCDR3 of Antibody 1 (SEQ ID NO:3) YSGV Antibody 1 IgG2 LCDR1 (SEQ ID NO: 4) KSSQSVLYSSNNKNYLA Antibody 1 IgG2 LCDR2 (SEQ ID NO:5) WASTRES Antibody 1 IgG2 LCDR3 (SEQ ID NO:6) QQYYSIPIT IgG2 HCDR1 of Antibody 2 (SEQ ID NO:7) NYGMH IgG2 HCDR2 of Antibody 2 (SEQ ID NO:8) VISYDGSNKFYADSVKG IgG2 HCDR3 of Antibody 2 (SEQ ID NO: 9) AGGIGRFDY Antibody 2 IgG2 LCDR1 (SEQ ID NO: 10) KYSQSLLHSDGKTYLF Antibody 2 IgG2 LCDR2 (SEQ ID NO: 11) EVSNRFS Antibody 2 IgG2 LCDR3 (SEQ ID NO: 12) MQTLKLPLT IgG2 HCVR of Antibody 1 (SEQ ID NO: 13) [ka] IgG2 LCVR of Antibody 1 (SEQ ID NO: 14) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYHQKPGQSPKLLISWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYSIPITFGGGTKVEIKR Antibody 1 IgG2 HC (SEQ ID NO: 15) [ka] Antibody 1 IgG2 LC (SEQ ID NO: 16) [ka] IgG2 HCVR of Antibody 2 (SEQ ID NO: 17) [ka] IgG2 LCVR of Antibody 2 (SEQ ID NO: 18) DFVMTQTPLSLSVTPGQPASISCKYSQSLLHSDGKTYLFWYLQKPGQPPHLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCMQTLKLPLTFGGGGTKVEIN Antibody 2 IgG2 HC (SEQ ID NO: 19) [ka] Antibody 2 IgG2 LC (SEQ ID NO: 20) [ka] hCCR8 (SEQ ID NO: 21) [ka] Cynomolgus CCR8 (SEQ ID NO: 22) [ka] Human CCR8[A27G] (SEQ ID NO: 23) [ka] mCCR8 (SEQ ID NO: 24) [ka] Rat CCR8 (SEQ ID NO: 25) [ka] hCCR4 (SEQ ID NO: 26) [ka] Antibody 1 IgG2 HC DNA (SEQ ID NO:27) [ka] Antibody 1 IgG2 LC DNA (SEQ ID NO:28) [ka] Antibody 2 IgG2 HC DNA (SEQ ID NO:29) [ka] Antibody 2 IgG2 LC DNA (SEQ ID NO:30) [ka] Human CCR8 1-35 (SEQ ID NO: 31) MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK Zelvalimuab LCDR1 (SEQ ID NO: 32) RASQGISNWLA Zelvalimuab LCDR2 (SEQ ID NO: 33) AASSLQS Zelvalimuab LCDR3 (SEQ ID NO: 34) QQAESFPHT Zelvalimb HCDR1 (SEQ ID NO: 35) SYDMS Zelvalimb HCDR2 (SEQ ID NO:36) LISGGGSQTYYAESVKG Zelvalimb HCDR3 (SEQ ID NO:37) PSGHYFYAMDV Zelvalimb VL (SEQ ID NO: 38) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAESFPHTFGGGTKVEIK Zelvalimb VH (SEQ ID NO: 39) [ka] Zelvalimb LC (SEQ ID NO: 40) [ka] Zelvalimb HC (SEQ ID NO: 41) [ka] Antibody 20A2.003 LCDR1 (SEQ ID NO: 42) SGDKLGDKYAS Antibody 20A2.003 LCDR2 (SEQ ID NO: 43) QDRKRPS Antibody 20A2.003 LCDR3 (SEQ ID NO: 44) QAFESSTEV Antibody 20A2.003 HCDR1 (SEQ ID NO: 45) NYGMH Antibody 20A2.003 HCDR2 (SEQ ID NO: 46) LIWYDASKKYYAESVKG Antibody 20A2.003 HCDR3 (SEQ ID NO: 47) DPSSLTGSTGYYGMDV Antibody 20A2.003 VL (SEQ ID NO: 48) SYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDRKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAFESSTEVFGGGTKLTVL Antibody 20A2.003 VH (SEQ ID NO: 49) [ka] Antibody 20A2.003 LC (SEQ ID NO:50) [ka] Antibody 20A2.003 HC (SEQ ID NO:51) [ka] Antibody 22D4.006 LCDR1 (SEQ ID NO:52) SGDALPKKYAY Antibody 22D4.006 LCDR2 (SEQ ID NO:53) EDAKRPS Antibody 22D4.006 LCDR3 (SEQ ID NO:54) YSTDASGNHRV Antibody 22D4.006 HCDR1 (SEQ ID NO:55) DYSMS Antibody 22D4.006 HCDR2 (SEQ ID NO:56) GINWNGGRTYADAVKG Antibody 22D4.006 HCDR3 (SEQ ID NO:57) EFNNFESNWFDP Antibody 22D4.006 VL (SEQ ID NO:58) SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKPGQAPVLVISEDAKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDASGNHRVFGGGTKLTVL Antibody 22D4.006 VH (SEQ ID NO:59) [ka] Antibody 22D4.006 LC (SEQ ID NO: 60) [ka] Antibody 22D4.006 HC (SEQ ID NO: 61) [ka] Antibody 22D4.017 LCDR1 (SEQ ID NO: 62) SGDALPKKYAY Antibody 22D4.017 LCDR2 (SEQ ID NO: 63) EDAKRPS Antibody 22D4.017 LCDR3 (SEQ ID NO: 64) YSTDASGNHRV Antibody 22D4.017 HCDR1 (SEQ ID NO: 65) DYSMS Antibody 22D4.017 HCDR2 (SEQ ID NO: 66) GINWNAGRTYADAVKG Antibody 22D4.017 HCDR3 (SEQ ID NO: 67) EFNNFESNWFDP Antibody 22D4.017 VL (SEQ ID NO: 68) SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKPGQAPVLVISEDAKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDASGNHRVFGGGTKLTVL Antibody 22D4.017 VH (SEQ ID NO: 69) [ka] Antibody 22D4.017 LC (SEQ ID NO: 70) [ka] Antibody 22D4.017 HC (SEQ ID NO: 71) [ka] Antibody 20C1.006 LCDR1 (SEQ ID NO: 72) RASQGISNWLA Antibody 20C1.006 LCDR2 (SEQ ID NO: 73) AASSLQS Antibody 20C1.006 LCDR3 (SEQ ID NO: 74) QQAESFPHT Antibody 20C1.006 HCDR1 (SEQ ID NO: 75) SYDMS Antibody 20C1.006 HCDR2 (SEQ ID NO: 76) LISGGGSNTYYAESVKG Antibody 20C1.006 HCDR3 (SEQ ID NO:77) PSGHYFYAMDV Antibody 20C1.006 VL (SEQ ID NO: 78) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAESFPHTFGGGTKVEIK Antibody 20C1.006 VH (SEQ ID NO:79) [ka] Antibody 20C1.006 LC (SEQ ID NO: 80) [ka] Antibody 20C1.006 HC (SEQ ID NO: 81) [ka] CCR8 P_1-12 peptide (SEQ ID NO: 82) MDYTLDLSVTTV CCR8 P_13-24 peptide (SEQ ID NO: 83) TDYYYPDIFSSP CCR8 P_25-35 peptide (SEQ ID NO: 84) CDAELIQTNGK CCR8 P_7-18 peptide (SEQ ID NO: 85) LSVTTVTDYYYP CCR8 P_19-30 peptide (SEQ ID NO: 86) DIFSSPCDAELI

[0277] [Table 18]

[0278] [Table 19]

[0279] [Table 20]

[0280] [Table 21]

[0281] [Table 22]

[0282]

Table 23

[0283]

Table 24

[0284]

Table 25

[0285]

Table 26

[0286]

Table 27

[0287]

Table 28

[0288]

Table 29

[0289]

Table 30

[0290]

Table 31

[0291]

Table 32

[0292]

Table 33

[0293]

Table 34

[0294]

Table 35

[0295]

Table 36

[0296]

Table 37

[0297]

Table 38

[0298]

Table 39

[0299]

Table 40

[0300]

Table 41

[0301]

Table 42

[0302]

Table 43

[0303]

Table 44

[0304]

Table 45

[0305]

Table 46

[0306]

Table 47

[0307]

Table 48

[0308]

Table 49

[0309]

Table 50

[0310]

Table 51

[0311]

Table 52

[0312]

Table 53

[0313]

Table 54

[0314]

Table 55

[0315]

Table 56

[0316]

Table 57

[0317]

Table 58

[0318]

Table 59

[0319]

Table 60

[0320]

Table 61

[0321]

Table 62

[0322]

Table 63

[0323]

Table 64

[0324]

Table 65

[0325]

Table 66

[0326]

Table 67

[0327]

Table 68

[0328]

Table 69

[0329]

Table 70

[0330]

Table 71

[0331]

Table 72

[0332]

Table 73

[0333]

Table 74

[0334] [Table 75]

[0335] [Table 76]

[0336] [Table 77]

[0337] [Table 78]

[0338] [Table 79]

[0339] [Table 80]

[0340] [Table 81]

[0341] Cynomolgus monkey native to Mauritius T4R CCR8 (SEQ ID NO: 556) [ka] Leader sequence (SEQ ID NO:557) MDMRVPAQLLGLLLLWLRGARC DNA encoding the leader sequence of SEQ ID NO:557 (SEQ ID NO:558) atggacatgagagtgcctgcacagctgctgggcctgctgctgctgtggctgagaggcgccagatgc Leader sequence (SEQ ID NO:559) MAWALLLLTLLTQGTGSWA DNA encoding the leader sequence of SEQ ID NO:559 (SEQ ID NO:560) atggcctgggctctgctgctcctcaccctcctcactcagggcacagggtcctgggcc TCE1 CCR8 HCDR1 (SEQ ID NO:561) NARMG TCE1 CCR8 HCDR2 (SEQ ID NO:562) RIKSKTEGGTRDYAAPVKG TCE1 CCR8 HCDR3 (SEQ ID NO:563) YSGV TCE1 CCR8 LCDR1 (SEQ ID NO: 564) KSSQSVLYSSNNKNYLA TCE1 CCR8 LCDR2 (SEQ ID NO: 565) WASTRES TCE1 CCR8 LCDR3 (SEQ ID NO: 566) QQYYSIPIT TCE2 CCR8 HCDR1 (SEQ ID NO:567) NYGMH TCE2 CCR8 HCDR2 (SEQ ID NO:568) VISYDGSNKFYADSVKG TCE2 CCR8 HCDR3 (SEQ ID NO:569) AGGIGRFDY TCE2 CCR8 LCDR1 (SEQ ID NO: 570) KYSQSLLHSDGKTYLF TCE2 CCR8 LCDR2 (SEQ ID NO: 571) EVSNRFS TCE2 CCR8 LCDR3 (SEQ ID NO: 572) MQTLKLPLT

[0342] [Table 82]

[0343] [Table 83]

[0344]

Table 84

[0345]

Table 85

[0346]

Table 86

[0347]

Table 87

[0348]

Table 88

[0349]

Table 89

[0350]

Table 90

[0351]

Table 91

[0352]

Table 92

[0353]

Table 93

[0354]

Table 94

[0355]

Table 95

[0356]

Table 96

[0357]

Table 97

[0358]

Table 98

[0359]

Table 99

[0360]

Table 100

[0361]

Table 101

[0362]

Table 102

[0363]

Table 103

[0364]

Table 104

[0365]

Table 105

[0366]

Table 106

[0367]

Table 107

[0368]

Table 108

[0369]

Table 109

[0370]

Table 110

[0371]

Table 111

[0372]

Table 112

[0373]

Table 113

[0374]

Table 114

[0375]

Table 115

[0376]

Table 116

[0377]

Table 117

[0378]

Table 118

[0379]

Table 119

[0380]

Table 120

[0381]

Table 121

[0382]

Table 122

[0383]

Table 123

[0384]

Table 124

[0385] [Table 125]

[0386] [Table 126]

[0387] [Table 127]

[0388] Zelvalimuab HC (SEQ ID NO: 636) without the C-terminal lysine [ka]

[0389] [Table 128]

[0390] [Table 129]

[0391] [Table 130]

[0392] [Table 131]

[0393] [Table 132]

[0394] [Table 133]

[0395]

Table 134

[0396]

Table 135

[0397]

Table 136

[0398]

Table 137

[0399]

Table 138

[0400]

Table 139

[0401]

Table 140

[0402]

Table 141

[0403]

Table 142

[0404]

Table 143

[0405]

Table 144

[0406]

Table 145

[0407]

Table 146

[0408]

Table 147

[0409]

Table 148

[0410]

Table 149

[0411]

Table 150

[0412]

Table 151

[0413]

Table 152

[0414]

Table 153

[0415]

Table 154

[0416]

Table 155

[0417]

Table 156

[0418]

Table 157

[0419]

Table 158

[0420]

Table 159

[0421]

Table 160

[0422]

Table 161

[0423]

Table 162

[0424]

Table 163

[0425]

Table 164

[0426]

Table 165

[0427]

Table 166

[0428]

Table 167

[0429]

Table 168

[0430]

Table 169

[0431]

Table 170

[0432]

Table 171

[0433]

Table 172

[0434]

Table 173

[0435]

Table 174

[0436]

Table 175

[0437]

Table 176

[0438]

Table 177

[0439]

Table 178

[0440]

Table 179

[0441]

Table 180

[0442]

Table 181

[0443]

Table 182

[0444]

Table 183

[0445]

Table 184

[0446]

Table 185

[0447]

Table 186

[0448]

Table 187

[0449]

Table 188

[0450]

Table 189

[0451]

Table 190

[0452]

Table 191

[0453]

Table 192

[0454]

Table 193

[0455]

Table 194

[0456]

Table 195

[0457]

Table 196

[0458]

Table 197

[0459]

Table 198

[0460]

Table 199

[0461]

Table 200

[0462]

Table 201

[0463]

Table 202

[0464]

Table 203

[0465]

Table 204

[0466]

Table 205

[0467]

Table 206

[0468]

Table 207

[0469]

Table 208

[0470]

Table 209

[0471]

Table 210

[0472]

Table 211

[0473]

Table 212

[0474]

Table 213

[0475]

Table 214

[0476]

Table 215

[0477]

Table 216

[0478]

Table 217

[0479]

Table 218

[0480]

Table 219

[0481]

Table 220

[0482]

Table 221

[0483]

Table 222

[0484]

Table 223

[0485]

Table 224

[0486]

Table 225

[0487]

Table 226

[0488]

Table 227

[0489]

Table 228

[0490]

Table 229

[0491]

Table 230

[0492]

Table 231

[0493]

Table 232

[0494]

Table 233

[0495]

Table 234

[0496]

Table 235

[0497]

Table 236

[0498]

Table 237

[0499]

Table 238

[0500]

Table 239

[0501]

Table 240

[0502]

Table 241

[0503]

Table 242

[0504]

Table 243

Claims

1. An antibody that binds to human C-C chemokine receptor type 8 (CCR8), comprising: (a) the amino acid sequence of heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1; (b) the amino acid sequence of HCDR2 of SEQ ID NO: 2; (d) KSSQSVLYSSNNX １ NYLA (SEQ ID NO: 1235) (wherein X １ is K or R) and the amino acid sequence of light chain complementarity determining region (LCDR) 1 (c) the amino acid sequence of HCDR3 of SEQ ID NO: 3; and an antibody.

2. The antibody according to claim 1, comprising the amino acid sequence of LCDR1 of SEQ ID NO:

4.

3. An antibody according to claim 1 or 2, comprising the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 13 and a light chain variable region (LCVR) comprising the following amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNNX １ NYLAWY X ２ QKPGQ X ３ PKLLISWASTRESGVPDRFSGSGSGSTDFTLTINS LQAE DVA VYYCQ QYY SIPITFGGG TKVEIKR (SEQ ID NO: 1236) (wherein X １ is K or R, X ２ is H or Q, and / or X ３ is S or P)

4. The antibody according to claim 1, comprising the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 13 and the amino acid sequence of the light chain variable region (LCVR) of SEQ ID NO: 14 or SEQ ID NO:

363.

5. The antibody according to claim 1, comprising the amino acid sequence of the heavy chain (HC) of SEQ ID NO: 15 or SEQ ID NO: 573 and the amino acid sequence of the light chain (LC) of SEQ ID NO: 16 or SEQ ID NO:

365.

6. An antibody that binds to human CCR8, comprising: (a) the amino acid sequence of HCDR1 of SEQ ID NO: 839; (b) the amino acid sequence of HCDR2 of SEQ ID NO: 840; (c) the amino acid sequence of HCDR3 of SEQ ID NO: 841; (d) the amino acid sequence of LCDR1 of SEQ ID NO: 842; (e) the amino acid sequence of LCDR2 of SEQ ID NO: 843; and (f) the amino acid sequence of LCDR3 of SEQ ID NO:

844.

7. The antibody according to claim 6, comprising the amino acid sequence of HCVR of SEQ ID NO: 1017 and the amino acid sequence of LCVR of SEQ ID NO: 1018.

8. The antibody according to claim 6 or 7, comprising the amino acid sequence of HC of SEQ ID NO: 1125 or SEQ ID NO: 1237 and the amino acid sequence of LC of SEQ ID NO: 1126.

9. A nucleic acid sequence encoding the antibody according to claim 1.

10. A nucleic acid sequence encoding the heavy chain amino acid sequence of SEQ ID NO: 15, 573, 1125 or 1237.

11. A nucleic acid sequence encoding the light chain amino acid sequence of SEQ ID NO: 16, 365 or 1126.

12. A vector comprising the nucleic acid sequence according to any one of claims 9 to 11.

13. A mammalian cell comprising the nucleic acid sequence according to any one of claims 9 to 11.

14. A pharmaceutical composition comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition for treating cancer in a patient, comprising an effective amount of the antibody according to claim 1.

16. The pharmaceutical composition according to claim 15, wherein the cancer is a solid tumor.

17. The pharmaceutical composition according to claim 15, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.

18. The pharmaceutical composition according to any one of claims 15 to 17, further comprising an effective amount of a PD-1 antagonist antibody.

19. The pharmaceutical composition according to claim 18, wherein the PD-1 antagonist antibody is a monoclonal antibody.

20. The pharmaceutical composition according to claim 18, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, or zelboraf.

21. The antibody according to claim 1, which is afucosylated.

22. A pharmaceutical composition for use in therapy, comprising the antibody according to claim 1.

23. A pharmaceutical composition for use in the treatment of cancer, comprising the antibody according to claim 1.

24. The pharmaceutical composition according to claim 23, wherein the cancer is a solid tumor.

25. The pharmaceutical composition according to claim 23 or 24, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.

26. The pharmaceutical composition according to claim 24, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer.

27. A pharmaceutical composition for treating cancer in a patient, comprising an effective amount of a Treg depletion antibody, and one or more of a bispecific T cell engager molecule, an agonist of a T cell co-stimulatory receptor, and an antagonist of the PD-1 / PD-L1 pathway.

28. The pharmaceutical composition according to claim 27, comprising an effective amount of a Treg depletion antibody and a bispecific T cell engager molecule.

29. The pharmaceutical composition according to claim 28, further comprising an antagonist of the PD-1 / PD-L1 pathway.

30. The pharmaceutical composition according to claim 28 or 29, further comprising an agonist of a T cell co-stimulatory receptor.

31. The pharmaceutical composition according to claim 27, wherein the Treg depletion antibody is an anti-CCR8 antibody.

32. The pharmaceutical composition according to claim 27, wherein the Treg-depleting antibody is the anti-CCR8 antibody according to claim 1.

33. The pharmaceutical composition according to claim 27, wherein the Treg-depleting antibody is an anti-CTLA-4 antibody.

34. The pharmaceutical composition according to any one of claims 27 or 29 to 33, wherein the antagonist of the PD-1 / PD-L1 pathway is a PD-1 antagonist antibody.

35. The pharmaceutical composition according to claim 34, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, or zelboraf.

36. The pharmaceutical composition according to claim 27, wherein the agonist of the immune cell costimulatory receptor is a 4-1BB agonist antibody.

37. A pharmaceutical composition for treating a patient's cancer, comprising an effective amount of an antibody or an antigen-binding fragment thereof that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue of SEQ ID NO:

82.

38. The pharmaceutical composition according to claim 37, wherein the epitope comprises at least two residues of SEQ ID NO:

82.

39. The pharmaceutical composition according to claim 37 or 38, wherein the epitope comprises at least three residues of SEQ ID NO:

82.

40. The pharmaceutical composition according to claim 37 or 38, wherein the epitope comprises at least four residues of SEQ ID NO:

82.

41. The pharmaceutical composition according to claim 37 or 38, wherein the epitope comprises threonine at position 4 of SEQ ID NO:

82.

42. The pharmaceutical composition according to claim 37 or 38, wherein the antibody does not inhibit ligand binding to CCR8.

43. A pharmaceutical composition for treating a patient's cancer, comprising an effective amount of an antibody or an antigen-binding fragment thereof that binds to human CCR8 at an epitope, wherein the epitope consists of threonine at position 4 of SEQ ID NO:

82.

44. The pharmaceutical composition according to claim 37 or 38, wherein the epitope is determined by an anti-CCR8 antibody or an antigen-binding fragment thereof that binds to the T4R mutation in cynomolgus CCR8.

45. The pharmaceutical composition according to claim 44, wherein the epitope is determined by a cell-based affinity assay, and an antibody that binds to cells expressing cynomolgus CCR8 containing the T4R mutation is compared with an antibody that binds to cells expressing wild-type cynomolgus CCR8.

46. The pharmaceutical composition according to claim 37 or 38, wherein the epitope is determined by epitope binning that binds to a CCR8 peptide-nanobody complex and / or screening for binding to CCR8 by phage display.

47. The pharmaceutical composition according to claim 43, wherein the antibody does not inhibit ligand binding to CCR8.

48. An anti-CCR8 antibody that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue of SEQ ID NO:

82.

49. The anti-CCR8 antibody according to claim 48, wherein the epitope comprises at least two residues of SEQ ID NO:

82.

50. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least three residues of SEQ ID NO:

82.

51. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least four residues of SEQ ID NO:

82.

52. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least five residues of SEQ ID NO:

82.

53. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least six residues of SEQ ID NO:

82.

54. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least seven residues of SEQ ID NO:

82.

55. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least eight residues of SEQ ID NO:

82.

56. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least nine residues of SEQ ID NO:

82.

57. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least ten residues of SEQ ID NO:

82.

58. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises at least eleven residues of SEQ ID NO:

82.

59. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises twelve residues of SEQ ID NO:

82.

60. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope comprises threonine at position 4 of SEQ ID NO:

82.

61. The anti-CCR8 antibody according to claim 48 or 49, wherein the anti-CCR8 antibody does not inhibit the binding of CCL1 to CCR8.

62. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope is determined by epitope binning.

63. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope is determined by binding to a CCR8 peptide-nanobody complex.

64. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope is determined by screening for an antibody that binds to CCR8 by phage display.

65. The anti-CCR8 antibody according to claim 48 or 49, wherein the epitope is determined by an anti-CCR8 antibody or an antigen-binding fragment thereof that binds to the T4R mutation in cynomolgus CCR8.

66. The anti-CCR8 antibody according to claim 65, wherein the epitope is determined by a cell-based affinity assay, and an antibody that binds to a cell expressing cynomolgus CCR8 containing the T4R mutation is compared with an antibody that binds to a cell expressing wild-type cynomolgus CCR8.

67. The anti-CCR8 antibody according to claim 65, wherein the antibody or antigen-binding fragment indicates that binding to CCR8 containing the T4R mutation is reduced or undetectable.

68. The anti-CCR8 antibody according to claim 48 or 49, wherein the antibody or its antigen-binding fragment does not inhibit ligand binding to CCR8.

69. The anti-CCR8 antibody according to claim 68, wherein the ligand is CCL1.

70. The antibody according to claim 8, comprising the HC amino acid sequence of SEQ ID NO: 1125.

71. The antibody according to claim 8, comprising the HC amino acid sequence of SEQ ID NO: 1237.

72. (a) an HCDR1 amino acid sequence comprising SEQ ID NO: 839, (b) an HCDR2 amino acid sequence comprising SEQ ID NO: 840, (c) an HCDR3 amino acid sequence comprising SEQ ID NO: 841, (d) an LCDR1 amino acid sequence comprising SEQ ID NO: 842, (e) an LCDR2 amino acid sequence comprising SEQ ID NO: 843, and (f) an LCDR3 amino acid sequence comprising SEQ ID NO: 844 A single-chain variable fragment (scFv), Fab, or single-chain Fab (scFab) that binds to human C-C chemokine receptor type 8 (CCR8).

73. A heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1017, and an amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVL YSSNNX １ NYLAWY X ２ QKPGQ X ３ PKLLISWAS TRE SGV PDRFSGSGSGSTD FT LTINS LQAE DVA VYYCQ QYY SIPITFGGGG TKVEIKR (SEQ ID NO: 1236) [where X １ is R, X ２ is H, X ３ is S or P] A light-chain variable region (LCVR) comprising The scFv, Fab, or scFab according to claim 72, comprising **Claim 74** An LCVR comprising an amino acid sequence containing SEQ ID NO: 1018, and An HCVR comprising an amino acid sequence containing SEQ ID NO: 1017 The scFv, Fab, or scFab according to claim 72, comprising **Claim 75** (a) A heavy chain complementarity determining region (HCDR) 1 amino acid sequence consisting of SEQ ID NO: 839, (b) An HCDR2 amino acid sequence consisting of SEQ ID NO: 840, (c) An HCDR3 amino acid sequence consisting of SEQ ID NO: 841, (d) A light chain complementarity determining region (LCDR) 1 amino acid sequence consisting of SEQ ID NO: 842, (e) An LCDR2 amino acid sequence consisting of SEQ ID NO: 843, and (f) An LCDR3 amino acid sequence consisting of SEQ ID NO: 844 An antibody that binds to human C-C chemokine receptor type 8 (CCR8), comprising **Claim 76** A heavy chain (HC) amino acid sequence consisting of SEQ ID NO: 1237, and A light chain (LC) amino acid sequence consisting of SEQ ID NO: 1126 The antibody according to claim 75, comprising **Claim 77** An HCVR amino acid sequence consisting of SEQ ID NO: 1017, and An LCVR amino acid sequence consisting of SEQ ID NO: 1018 The antibody according to claim 75, comprising **Claim 78** An antibody that binds to human C-C chemokine receptor type 8 (CCR8), wherein the antibody A nucleic acid sequence encoding an HC comprising an HCDR1 amino acid sequence containing SEQ ID NO: 839, an HCDR2 amino acid sequence containing SEQ ID NO: 840, and an HCDR3 amino acid sequence containing SEQ ID NO: 841, and A nucleic acid sequence encoding an LC comprising an LCDR1 amino acid sequence containing SEQ ID NO: 842, an LCDR2 amino acid sequence containing SEQ ID NO: 843, and an LCDR3 amino acid sequence containing SEQ ID NO: 844 Culturing mammalian cells containing a vector comprising the nucleic acid sequences under conditions in which the antibody is expressed, and Recovering the expressed antibody An antibody obtained by a method comprising **Claim 79** A nucleic acid sequence encoding an HCVR amino acid sequence that is at least 90% identical to SEQ ID NO: 1017, and A nucleic acid sequence encoding an LCVR amino acid sequence that is at least 90% identical to SEQ ID NO: 1018 Culturing mammalian cells containing a vector comprising the nucleic acid sequences under conditions in which the antibody is expressed, and Recovering the expressed antibody The antibody according to claim 78, obtained by a method comprising **Claim 80** A nucleic acid sequence encoding an HCVR amino acid sequence containing SEQ ID NO: 1017, and A nucleic acid sequence encoding an LCVR amino acid sequence containing SEQ ID NO: 1018 Culturing mammalian cells containing a vector containing the nucleic acid sequence under conditions in which the antibody is expressed; Recovering the expressed antibody; The antibody according to claim 78, obtained by a method comprising:

81. A nucleic acid sequence encoding an HC amino acid sequence that is at least 90% identical to SEQ ID NO: 1125 or SEQ ID NO: 1237, and A nucleic acid sequence encoding an LC amino acid sequence that is at least 90% identical to SEQ ID NO: 1126 Culturing mammalian cells containing a vector containing the nucleic acid sequence under conditions in which the antibody is expressed; Recovering the expressed antibody; The antibody according to claim 78, obtained by a method comprising:

82. A nucleic acid sequence encoding an HC amino acid sequence containing SEQ ID NO: 1125 or SEQ ID NO: 1237, and A nucleic acid sequence encoding an LC amino acid sequence containing SEQ ID NO: 1126 Culturing mammalian cells containing a vector containing the nucleic acid sequence under conditions in which the antibody is expressed; Recovering the expressed antibody; The antibody according to claim 78, obtained by a method comprising:

83. An antibody that binds to human C-C chemokine receptor type 8 (CCR8), comprising A vector containing a nucleic acid sequence encoding an HCDR1 amino acid sequence containing SEQ ID NO: 839, an HCDR2 amino acid sequence containing SEQ ID NO: 840, and an HCDR3 amino acid sequence containing SEQ ID NO: 841, and A vector containing a nucleic acid sequence encoding an LCDR1 amino acid sequence containing SEQ ID NO: 842, an LCDR2 amino acid sequence containing SEQ ID NO: 843, and an LCDR3 amino acid sequence containing SEQ ID NO: 844 Culturing mammalian cells containing the vector under conditions in which the antibody is expressed; Recovering the expressed antibody; An antibody obtained by a method comprising:

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