Anti-CCR8 monoclonal antibody and application thereof

Abstract

The invention relates to an anti-CCR8 monoclonal antibody and application thereof, in particular to an anti-CCR8 monoclonal antibody which can be combined with CCR8 derived from primates. The invention also relates to a pharmaceutical composition containing the anti-CCR8 monoclonal antibody, and an application of the anti-CCR8 monoclonal antibody and / or the pharmaceutical composition in prevention and / or treatment of tumors or cancers.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an anti-CCR8 monoclonal antibody and an application thereof. Background Art

[0002] Chemokine (CC motif) receptor 8 (CCR8) is a member of the β-chemokine receptor family and a seven-transmembrane G protein-coupled receptor. In humans and mice, CCR8 is found to be mainly expressed on regulatory T cells (Treg) and a small part of Th2, but not on Th1. Through analysis, Treg CCR8 + Upregulation is associated with the occurrence, progression and survival of various tumors.

[0003] CCL1 is one of the main ligands of CCR8. CCL1 is overexpressed in a variety of tumor tissue microenvironments and recruits CCR8 + Treg cells infiltrate into tumor tissues, forming an immunosuppressive microenvironment. By blocking the CCL1-CCR8 action, CCR8 in the tumor environment is eliminated. + Treg, or blocking CCL1-mediated CCR8 + The migration of Treg to tumor tissue can block the immunosuppressive effect caused by Treg, thereby increasing the anti-tumor effect, and is currently a new approach to tumor immunotherapy. Summary of the invention

[0004] The present invention provides an anti-CCR8 antibody, which has high affinity and specificity for human CCR8 protein, can effectively block the binding of CCL1 and CCR8, and inhibit the transduction of downstream signals; in addition, it can also eliminate CCR8 positive cells by mediating antibody-dependent cytotoxicity and complement-dependent cytotoxicity.

[0005] Specifically, on the one hand, the present invention provides a fully human antibody or antigen-binding fragment that specifically binds to human CCR8, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions: H-CDR1, H-CDR2 and H-CDR3, the light chain variable region comprising complementary determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein,

[0006] The H-CDR1 has an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 31;

[0007] The H-CDR2 has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26 or SEQ ID NO:32;

[0008] The H-CDR3 has an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27 or SEQ ID NO: 33;

[0009] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28 or SEQ ID NO:34;

[0010] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29 or SEQ ID NO:35;

[0011] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30 or SEQ ID NO:36.

[0012] The H-CDR1 has an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25 or SEQ ID NO:31.

[0013] SSSAAWS (SEQ ID NO: 1).

[0014] TYWIG (SEQ ID NO: 7).

[0015] SKSVGWH (SEQ ID NO: 13).

[0016] SNSVGWH (SEQ ID NO: 19).

[0017] SKSVGWH (SEQ ID NO: 25).

[0018] SYAIS (SEQ ID NO:31).

[0019] The H-CDR2 has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26 or SEQ ID NO:32.

[0020] RTYYRSKWRSDYAVSVKS(SEQ ID NO:2).

[0021] IINPDDSQARYSPSFQG (SEQ ID NO:8).

[0022] RTYYRSRWNDDYAVSVKS (SEQ ID NO: 14).

[0023] RTYYRSRWNNDYAVSVKS (SEQ ID NO: 20).

[0024] RTYYRSKWNNDYAISVKS (SEQ ID NO: 26).

[0025] IINPSGGNTRYAQKFQG (SEQ ID NO:32).

[0026] The H-CDR3 has an amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27 or SEQ ID NO:33.

[0027] GRWSGFDI (SEQ ID NO: 3).

[0028] PGGP (SEQ ID NO:9).

[0029] GFRYSFDY (SEQ ID NO: 15).

[0030] GFRYSFDY (SEQ ID NO: 21).

[0031] GYNFGFDY (SEQ ID NO: 27).

[0032] DGFGMDSSGYYRLRY (SEQ ID NO:33).

[0033] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28 or SEQ ID NO:34.

[0034] RSSQSLLHSNGYNYLA (SEQ ID NO: 4).

[0035] RSSHSPQFSDGNNYVS(SEQ ID NO:10).

[0036] SGSKSNIGNRTVN (SEQ ID NO: 16).

[0037] SGSSSNIGSRNVN (SEQ ID NO:22).

[0038] SGSSSNIGKNPVS (SEQ ID NO:28).

[0039] RSSQSLVHSDGNTYLS (SEQ ID NO:34).

[0040] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29 or SEQ ID NO:35.

[0041] LGSNRAS (SEQ ID NO: 5).

[0042] RASNRAS (SEQ ID NO: 11).

[0043] GTDQRPS (SEQ ID NO: 17).

[0044] SSNQRPS (SEQ ID NO: 23).

[0045] DNNKRPS (SEQ ID NO: 29).

[0046] KISNRFS (SEQ ID NO: 35).

[0047] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30 or SEQ ID NO:36.

[0048] MQSLQTPPT (SEQ ID NO: 6).

[0049] MQGTHWPWT (SEQ ID NO: 12).

[0050] SSWDDSLKVLL (SEQ ID NO: 18).

[0051] ATWDDSLKVLL (SEQ ID NO: 24).

[0052] ATWDTKGTRLSSVGV (SEQ ID NO:30).

[0053] MQGTRFPHT (SEQ ID NO: 36).

[0054] In some embodiments, the heavy chain variable region comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, a combination selected from the group consisting of:

[0055] (1) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or

[0056] (2) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; or

[0057] (3) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or

[0058] (4) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or

[0059] (5) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; or

[0060] (6) The H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0061] In some embodiments, the fully human antibody comprises at least one of a heavy chain variable region framework region and a light chain variable region framework region, and at least a portion of at least one of the heavy chain variable region framework region and the light chain variable region framework region is derived from at least one of a human antibody or a mutant thereof.

[0062] In some embodiments, the heavy chain variable region of the fully human antibody comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs:59-64.

[0063] QVQLQQSGPGLVRPSQTLSLTCAISGDSISSSSAAWSWIRQSPSGGLEWLGRTYYRSKW RSDYAVSVKSRISVTPDTSKNQFSLQLNSVTPEDTAKYFCARGRWSGFDIWGQGTMVTVSS (SEQ ID NO: 59).

[0064] QVQLVQSGAEVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIINPDDSQ ARYSPSFQGQVTISADKSISTAYVQWSSLKASDSAIYYCARPGGPWGQGTLVTVSS (SEQ ID NO: 60).

[0065] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSKSVGWHWIRQSPSRGLEWLGRTYYRSR WNDDYAVSVKSRITITPDTSKNQFSLQLNSVTPEDTAVYYCARGFRYSFDYWGQGTLVTVS S (SEQ ID NO: 61).

[0066] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVGWHWIRQSPSRGLEWLGRTYYRSR WNNDYAVSVKSRITITPDTSKNQFSLQLNSVTPEDTAVYYCARGFRYSFDYWGQGTLVTVS S (SEQ ID NO: 62).

[0067] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSKSVGWHWIRQSPSRGLEWLGRTYRSK WNNDYAISVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGYNFGFDYWGQGTLVTVS S (SEQ ID NO: 63).

[0068] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGN TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQ GTLVTVSS (SEQ ID NO: 64).

[0069] In some embodiments, the light chain variable region of the fully human antibody comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs:71-76.

[0070] EIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLAWYLQKPGQSPQLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLQTPPTFGQGTKVEIK (SEQ ID NO: 71).

[0071] EIVLTQSPLSLPVTLGQSASISCRSSHSPQFSDGNNYVSWFQQRPGQAPRLLIYRASNRA SGVPDRFSGSGSGTDFTLSISGVEAEDAGTYYCMQGTHWPWTFGQGTRLEIK (SEQ ID NO: 72).

[0072] LPVLTQPPSASGTPGQRVTISSCSGSKSNIGNRTVNWYQQLPGTAPKLLIHGTDQRPSGVP DRISGSKSGTSASLAISGLQSEDEANYYCSSWDDSLKVLLFGGGTKLTVL (SEQ ID NO: 73).

[0073] NFMLTQPPSASGTPGQRVTISSCSGSSSNIGSRNVNWYQQLPGTAPKLIIHSSNQRPSGVP DRYSGSKSGTSASLAISGLRSDDEAEYFCATWDDSLKVLLFGGTKLTVL (SEQ ID NO: 74).

[0074] QSVVTQPPSVSAAPGQKVTISSCSGSSSNIGKNPVSWYQHLPGAAPKLLIHDNNKRPSGI PDRFSGSKSGTSATLDITGLQTGDEADYYCATWDTKGTRLSSVGVFGGGTKVTVL (SEQ ID NO: 75).

[0075] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIK (SEQ ID NO: 76).

[0076] In some embodiments, the fully human CCR8 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from a combination of the following groups:

[0077] (1) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:59, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:71; or

[0078] (2) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:60, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:72; or

[0079] (3) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73; or

[0080] (4) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:62, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74; or

[0081] (5) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:75; or

[0082] (6) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:64, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:76.

[0083] In some embodiments, the fully human CCR8 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from a combination of the following groups:

[0084] (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:59, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:71; or

[0085] (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72; or

[0086] (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:73; or

[0087] (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74; or

[0088] (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 75; or

[0089] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:76.

[0090] On the other hand, the present invention provides an affinity matured antibody, wherein the affinity matured antibody is prepared by mutation of the fully human CCR8 antibody described in the present invention.

[0091] In some embodiments, the affinity matured antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein,

[0092] The H-CDR1 has an amino acid sequence shown as X1X2X3X4S; wherein X1 is selected from P, S, G, A or R, X2 is selected from R, K, Y or S, X3 is selected from A or T, and X4 is selected from I, Y or V;

[0093] The H-CDR2 has an amino acid sequence shown in IINX5X6X7GX8TRYAQKFQG; wherein X5 is selected from P, I, K or G, X6 is selected from S, W or H, X7 is selected from G or R, and X8 is selected from N, Q, I or K;

[0094] The H-CDR3 has DX9X 10 GMDX 11 X 12 RXGD 13 X 14 The amino acid sequence shown; wherein X9 is selected from G or N, X 10 Selected from F, T or R, X 11 Select from S or K, X 12 Select from R or S, X 13 Select from R or L, X 14 Selected from Y or R;

[0095] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56;

[0096] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57;

[0097] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:58.

[0098] In some embodiments, the affinity matured antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein,

[0099] The H-CDR1 has an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:53;

[0100] The H-CDR2 has an amino acid sequence shown in IINX5X6X7GX8TRYAQKFQG; wherein X5 is selected from P, I, K or G, X6 is selected from S, W or H, X7 is selected from G or R, and X8 is selected from N, Q, I or K;

[0101] The H-CDR3 has DX9X 10 GMDX 11 X 12 RXGD 13 X 14 The amino acid sequence shown; wherein X9 is selected from G or N, X 10 Selected from F, T or R, X 11 Select from S or K, X 12 Select from R or S, X 13 Select from R or L, X 14 Selected from Y or R;

[0102] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56;

[0103] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57;

[0104] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:58.

[0105] In some embodiments, the affinity matured antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein,

[0106] The H-CDR1 has an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:53;

[0107] The H-CDR2 has an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:54;

[0108] The H-CDR3 has an amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51 or SEQ ID NO:55;

[0109] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56;

[0110] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57;

[0111] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:58.

[0112] The H-CDR1 has an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:53.

[0113] SYAIS (SEQ ID NO:31).

[0114] PRAIS (SEQ ID NO: 37).

[0115] SKAYS (SEQ ID NO:40).

[0116] GYTIS (SEQ ID NO: 43).

[0117] AYAVS (SEQ ID NO:49).

[0118] RSAIS (SEQ ID NO:53).

[0119] The H-CDR2 has an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:54.

[0120] IINPSGGNTRYAQKFQG (SEQ ID NO:32).

[0121] IINPSGGQTRYAQKFQG (SEQ ID NO:38).

[0122] IINIWGGNTRYAQKFQG (SEQ ID NO: 41).

[0123] IINKSGGITRYAQKFQG (SEQ ID NO: 44).

[0124] IINPHRGNTRYAQKFQG (SEQ ID NO:50).

[0125] IINGSGGKTRYAQKFQG (SEQ ID NO: 54).

[0126] The H-CDR3 has an amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51 or SEQ ID NO:55.

[0127] DGFGMDSSGYYRLRY (SEQ ID NO:33).

[0128] DGFGMDSRGYYRLRY (SEQ ID NO:39).

[0129] DGTGMDKSGYYRLRY (SEQ ID NO:45).

[0130] DNRGMDSSGYYRLRY (SEQ ID NO:51).

[0131] DGFGMDSSGYYRLLR (SEQ ID NO:55).

[0132] The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56.

[0133] RSSQSLVHSDGNTYLS (SEQ ID NO:34).

[0134] RSSQSLVHSNGNTYLS (SEQ ID NO:42).

[0135] RSSQSLVHSSGNTYLS (SEQ ID NO:46).

[0136] RSSQSLVHSNGNHYLS (SEQ ID NO:52).

[0137] RSSRSLVHSTGNTYLS (SEQ ID NO:56).

[0138] The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57.

[0139] KISNRFS (SEQ ID NO: 35).

[0140] RINNRFS (SEQ ID NO:47).

[0141] KISARFM (SEQ ID NO:57).

[0142] The L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:58.

[0143] MQGTRFPHT (SEQ ID NO: 36).

[0144] MQGSRFPLT (SEQ ID NO:48).

[0145] MQGTSFPLT (SEQ ID NO: 58).

[0146] In some embodiments, the affinity matured antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprising complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, a combination selected from the following groups:

[0147] (1) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; or

[0148] (2) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:33, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:35 and SEQ ID NO:36, respectively; or

[0149] (3) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively; or

[0150] (4) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:33, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; or

[0151] (5) the H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:51, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:35 and SEQ ID NO:36, respectively; or

[0152] (6) The H-CDR1, H-CDR2 and H-CDR3 have the amino acid sequences shown in SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, respectively; and the L-CDR1, L-CDR2 and L-CDR3 have the amino acid sequences shown in SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, respectively.

[0153] In some embodiments, the affinity matured antibody comprises at least one of a heavy chain variable region framework region and a light chain variable region framework region, and at least a portion of at least one of the heavy chain variable region framework region and the light chain variable region framework region is derived from at least one of a human antibody or a mutant thereof.

[0154] In some embodiments, the heavy chain variable region framework region of the affinity matured antibody comprises: H-FR1, H-FR2, H-FR3 and H-FR4; wherein,

[0155] The H-FR1 has an amino acid sequence shown as QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 83);

[0156] The H-FR2 has an amino acid sequence shown as WVRQAPGQGLEWMG (SEQ ID NO: 84);

[0157] The H-FR3 has the amino acid sequence shown in RVTMTRDTATSTVYMELSSLRSEDTAMYYCAR (SEQ ID NO: 85);

[0158] The H-FR4 has an amino acid sequence shown as WGQGTLVTVSS (SEQ ID NO: 86) or WGQGTSVTVSS (SEQ ID NO: 87).

[0159] In some embodiments, the light chain variable region framework region of the affinity matured antibody comprises: L-FR1, L-FR2, L-FR3 and L-FR4; wherein,

[0160] The L-FR1 has an amino acid sequence shown as EIVMTQTPLSSPVTLGQPASISC (SEQ ID NO: 88);

[0161] The L-FR2 has an amino acid sequence shown as WLHQRPGQPPRPLIY (SEQ ID NO: 89);

[0162] The L-FR3 has an amino acid sequence shown as GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 90);

[0163] The L-FR4 has an amino acid sequence shown as FGQGTKLEIK (SEQ ID NO: 91).

[0164] In some embodiments, the affinity matured antibody heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs:65-70.

[0165] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSPRAISWVRQAPGQGLEWMGIINPSGGQ TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSRGYYRLRYWGQ GTSVTVSS (SEQ ID NO: 65).

[0166] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSKAYSWVRQAPGQGLEWMGIINIWGGN TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQ GTLVTVSS (SEQ ID NO: 66).

[0167] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYTISWVRQAPGQGLEWMGIINKSGGIT RYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGTGMDKSGYYRLRYWGQG TSVTVSS (SEQ ID NO: 67).

[0168] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAVSWVRQAPGQGLEWMGIINPHRGN TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQ GTLVTVSS (SEQ ID NO: 68).

[0169] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGN TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDNRGMDSSGYYRLRYWGQ GTSVTVSS (SEQ ID NO: 69).

[0170] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRSAISWVRQAPGQGLEWMGIINGSGGK TRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLLRWGQ GTSVTVSS (SEQ ID NO: 70).

[0171] In some embodiments, the affinity matured antibody light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs:77-82.

[0172] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIK (SEQ ID NO: 77).

[0173] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLHQRPGQPPRPLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIK (SEQ ID NO: 78).

[0174] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSSGNTYLSWLHQRPGQPPRPLIYRINNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGSRFPLTFGQGTKLEIK (SEQ ID NO: 79).

[0175] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIK (SEQ ID NO: 80).

[0176] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNHYLSWLHQRPGQPPRPLIYKISNRF SGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIK (SEQ ID NO: 81).

[0177] EIVMTQTPLSSPVTLGQPASISCRSSRSLVHSTGNTYLSWLHQRPGQPPRPLIYKISARF MGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTSFPLTFGQGTKLEIK (SEQ ID NO: 82).

[0178] In some embodiments, the affinity matured antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from a combination of the following groups:

[0179] (1) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:65, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:77; or

[0180] (2) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:78; or

[0181] (3) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79; or

[0182] (4) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:68, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:80; or

[0183] (5) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:69, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:81; or

[0184] (6) the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:70, and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:82.

[0185] In some embodiments, the affinity matured antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from a combination of the following groups:

[0186] (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 77; or

[0187] (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 78; or

[0188] (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79; or

[0189] (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 80; or

[0190] (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:81; or

[0191] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:70, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:82.

[0192] In some embodiments, the fully human antibody of the present invention and / or the affinity matured antibody of the present invention comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region comes from at least one of a human antibody or its mutants.

[0193] In some embodiments, the heavy chain constant region and the light chain constant region are both derived from human IgG or a mutant thereof.

[0194] In some embodiments, the heavy chain constant region is from human IgG1 or IgG4 or a mutant thereof.

[0195] In some embodiments, the light chain constant region is derived from a human Kappa or Lambda constant region.

[0196] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:92.

[0197] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:92).

[0198] In some embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:93.

[0199] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:93).

[0200] In some embodiments, the fully human antibodies and / or affinity matured antibodies described herein are monoclonal antibodies.

[0201] In some embodiments, the monoclonal fully human antibody and / or affinity matured antibody of the present invention comprises a heavy chain and a light chain, wherein the heavy chain and the light chain are selected from a combination of the following groups:

[0202] (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:94, and the light chain comprises the amino acid sequence shown in SEQ ID NO:101; or

[0203] (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:95, and the light chain comprises the amino acid sequence shown in SEQ ID NO:102; or

[0204] (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:96, and the light chain comprises the amino acid sequence shown in SEQ ID NO:103; or

[0205] (4) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:97, and the light chain comprises the amino acid sequence shown in SEQ ID NO:104; or

[0206] (5) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:98, and the light chain comprises the amino acid sequence shown in SEQ ID NO:105; or

[0207] (6) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:99, and the light chain comprises the amino acid sequence shown in SEQ ID NO:106; or

[0208] (7) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:100, and the light chain comprises the amino acid sequence shown in SEQ ID NO:107.

[0209] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGNTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:94).

[0210] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:101).

[0211] In the present invention, an antibody comprising the heavy chain shown in SEQ ID NO:94 and the light chain shown in SEQ ID NO:101, numbered: 1H4.

[0212] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSPRAISWVRQAPGQGLEWMGIINPSGGQTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSRGYYRLRYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:95).

[0213] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:102).

[0214] In the present invention, an antibody comprising the heavy chain shown in SEQ ID NO:95 and the light chain shown in SEQ ID NO:102, numbered: 1H4-1-H4.

[0215] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSKAYSWVRQAPGQGLEWMGIINIWGGNTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:96).

[0216] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLHQRPGQPPRPLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:103).

[0217] In the present invention, an antibody comprising the heavy chain shown in SEQ ID NO:96 and the light chain shown in SEQ ID NO:103, numbered: 1H4-4H1.

[0218] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYTISWVRQAPGQGLEWMGIINKSGGITRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGTGMDKSGYYRLRYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:97).

[0219] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSSGNTYLSWLHQRPGQPPRPLIYRINNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGSRFPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:104).

[0220] In the present invention, an antibody comprising the heavy chain shown in SEQ ID NO:97 and the light chain shown in SEQ ID NO:104, numbered: 1H4-2A5.

[0221] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAVSWVRQAPGQGLEWMGIINPHRGNTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLRYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:98).

[0222] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRPLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:105).

[0223] In the present invention, the antibody comprising the heavy chain shown in SEQ ID NO:98 and the light chain shown in SEQ ID NO:105 is numbered: 1H4-3D2.

[0224] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGNTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDNRGMDSSGYYRLRYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:99).

[0225] EIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNHYLSWLHQRPGQPPRPLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTRFPHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:106).

[0226] In the present invention, an antibody comprising the heavy chain shown in SEQ ID NO:99 and the light chain shown in SEQ ID NO:106, numbered: 1H4-5B4.

[0227] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRSAISWVRQAPGQGLEWMGIINGSGGKTRYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAMYYCARDGFGMDSSGYYRLLRWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:100).

[0228] EIVMTQTPLSSPVTLGQPASISCRSSRSLVHSTGNTYLSWLHQRPGQPPRPLIYKISARFMGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQGTSFPLTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:107).

[0229] In the present invention, the antibody comprising the heavy chain shown in SEQ ID NO: 100 and the light chain shown in SEQ ID NO: 107 is numbered as: 1H4-2D8.

[0230] In some embodiments, the antigen-binding fragment of the present invention is selected from the group consisting of: Fab, Fab', Fv, sdFv and (Fab')2.

[0231] Among them, Fab is composed of a complete light chain and a heavy chain variable region domain (VH) and the first constant domain (CH1) of a heavy chain. Fab can be a product produced by digesting antibodies with papain; Fab' is different from Fab in that the Fab' fragment adds some residues to the carboxyl terminus of the CH1 domain, and these residues contain one or more missing cysteines from the hinge region of the antibody; (Fab')2 is equivalent to two Fab' fragments with disulfide bonds that have divalent antigen-binding activity and can still cross-link antigens, and (Fab')2 can be the product of pepsin cleavage of the antibody; Fv is composed of a tight, non-covalently associated dimer of a heavy chain variable region domain and a light chain variable region domain; sdFv refers to an Fv fragment connected by a disulfide bond.

[0232] In some embodiments, the antibody of the present invention is a natural complete antibody, a single-chain antibody (scFv), a single-chain Fv-Fc antibody (scFv-Fc), a scFv dimer, and a dimer of a scFv-Fc antibody, wherein the dimer includes a homodimer and a heterodimer.

[0233] The single-chain antibody (scFv) as described above comprises a heavy chain variable region (VH) of an amino acid sequence shown in any one of SEQ ID NOs: 59-70 and a light chain variable region (VL) of an amino acid sequence shown in any one of SEQ ID NOs: 71-82, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region through a connecting peptide Linker, or the C-terminus of the variable region is connected to the N-terminus of the heavy chain variable region through a connecting peptide Linker. It should be noted that the "connecting peptide Linker" of the single-chain antibody described in the present invention is used to connect the heavy chain variable region and the light chain variable region of the antibody, which can be a commonly used connecting peptide Linker for preparing single-chain antibodies, or a connecting peptide Linker modified by scientific researchers. An example of the amino acid sequence of the connecting peptide Linker is: (GnS)m, n is a natural number of 3-5, n is preferably 4, m is a natural number of 1-5, and m is preferably 3.

[0234] A specific example of a linker peptide is: GGGGSGGGGSGGGGS (SEQ ID NO: 108).

[0235] Specifically, the single-chain antibody (scFv) described in the present invention comprises a heavy chain variable region, a light chain variable region and a linker: VH-Linker-VL.

[0236] As a specific example, the single-chain antibody (scFv) of the present invention comprises the amino acid sequence shown in SEQ ID NO:109.

[0237] QVQLQQSGPGLVRPSQTLSLTCAISGDSISSSSSAAWSWIRQSPSGGLEWLGRTYYRSKWRSDYAVSVKSRISVTPDTSKNQFSLQLNSVTPEDTAKYFCARGRWSGFDIWGQGTMVTVSSGGGGS GGGGSGGGGSEIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLAWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLQTPPTFGQGTKVEIK(SEQ ID NO:109).

[0238] In the amino acid sequence shown in SEQ ID NO: 109, the bold portion is the connecting peptide Linker.

[0239] The single-chain antibodies of the present invention can be constructed by referring to the specific examples described above.

[0240] The single-chain Fv-Fc antibody (scFv-Fc) as described above comprises scFv and Fc fragment, wherein the Fc fragment refers to an antibody heavy chain constant region fragment comprising at least a hinge region, CH2 and CH3 domains; the scFv is as described above, and the scFv is directly connected to the Fc fragment or connected via a linker (a specific example of a linker is GGGGSGGGGSGGGGS). In some embodiments, the scFv in the present invention is directly connected to the Fc fragment via a hinge region.

[0241] The Fc fragment as described above is derived from, for example, a heavy chain constant region selected from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; particularly selected from, for example, a heavy chain constant region of human IgG1, IgG2, IgG3 and IgG4, more particularly selected from the heavy chain constant region of human IgG1 or IgG4; and, the Fc fragment has one or more amino acid substitutions, deletions or additions (for example, at most 20, at most 15, at most 10, at most 5, at most 3 or at most 1 substitution, deletion or addition) compared to the native sequence from which it is derived.

[0242] Specifically, the Fc in the single-chain Fv-Fc antibody (scFv-Fc) of the present invention has the amino acid sequence shown in SEQ ID NO:110.

[0243] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:110).

[0244] In the single-chain Fv-Fc antibody (scFv-Fc) of the present invention, the scFv can be constructed with reference to the specific examples of the single-chain antibody described above, and the Fc has the amino acid sequence described above.

[0245] Specifically, the single-chain Fv-Fc antibody (scFv-Fc) described in the present invention further comprises a connecting peptide Linker. The connecting peptide Linker can be a commonly used connecting peptide Linker for preparing antibodies, or a connecting peptide Linker modified by scientific researchers. The connecting peptide Linker is used to connect scFv and Fc, wherein the C-terminus of the scFv is connected to the N-terminus of the Fc through the connecting peptide Linker. An example of the amino acid sequence of the connecting peptide Linker is: GGGGSGGGGSGGGGS (SEQ ID NO: 108).

[0246] Specifically, the single-chain Fv-Fc antibody (scFv-Fc) of the present invention comprises scFv, Fc and Linker: scFv-Linker-Fc.

[0247] As a specific example, the single-chain Fv-Fc antibody (scFv-Fc) of the present invention comprises the amino acid sequence shown in SEQ ID NO:111.

[0248] QVQLQQSGPGLVRPSQTLSLTCAISGDSISSSSSAAWSWIRQSPSGGLEWLGRTYYRSKWRSDYAVSVKSRISVTPDTSKNQFSLQLNSVTPEDTAKYFCARGRWSG FDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLAWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFT LKISRVEAEDVGVYYCMQSLQTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111).

[0249] The single-chain Fv-Fc antibody (scFv-Fc) of the present invention can be constructed with reference to the specific examples described above.

[0250] In some embodiments, the antibodies of the present invention are capable of mediating antibody-dependent cellular cytotoxicity and / or antibody-dependent cellular phagocytosis (ADCC and / or ADCP) and blocking the signaling pathways of CCR8 and CCL1.

[0251] In some embodiments, the fully human antibodies and / or affinity matured antibodies of the present invention are monospecific antibodies, bispecific antibodies or multispecific antibodies.

[0252] In another aspect, the present invention provides a chimeric antigen receptor comprising the fully human antibody and / or affinity matured antibody of the present invention.

[0253] In yet another aspect, the present invention provides a genetically modified cell comprising the chimeric antigen receptor.

[0254] In some embodiments, the genetically modified cells are immune cells, such as T cells or NK cells.

[0255] In yet another aspect, the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises the fully human antibody and / or affinity matured antibody of the present invention.

[0256] In yet another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fully human antibody and / or affinity matured antibody of the present invention.

[0257] In some embodiments, the nucleic acid molecule is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be a cDNA.

[0258] In yet another aspect, the present invention provides an expression vector, wherein the expression vector carries the nucleic acid molecule of the present invention.

[0259] In some embodiments, the expression vector of the present invention is selected from the group consisting of: a plasmid, a cosmid, a virus, a minichromosome, and an artificial chromosome.

[0260] In some embodiments, the expression vector of the present invention is a eukaryotic expression vector.

[0261] In some embodiments, the eukaryotic expression vector of the present invention is selected from the group consisting of: pCRII, pCR3, pcDNA3.4, pBSII, pET 15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual.

[0262] On the other hand, the present invention provides a nucleic acid construct, which comprises the nucleic acid molecule described in the present invention; further, the nucleic acid construct comprises one or more control sequences operably linked to the nucleic acid molecule, wherein the one or more control sequences direct the production of the antibody or antigen-binding fragment thereof described in the present invention in a suitable host cell, and the one or more control sequences are selected from: a promoter, an enhancer, a stop signal, a signal peptide, a leader sequence, a transcription terminator and any group thereof.

[0263] In yet another aspect, the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0264] In some embodiments, the host cell of the present invention is Escherichia coli, yeast or a eukaryotic cell.

[0265] In some embodiments, the host cell of the invention is a eukaryotic host cell.

[0266] In some embodiments, the host cell is a mammalian host cell.

[0267] On the other hand, the present invention provides a pharmaceutical composition, comprising the fully human antibody or antigen-binding fragment described in the present invention, the affinity matured antibody described in the present invention, the nucleic acid molecule described in the present invention, the expression vector described in the present invention, the host cell described in the present invention and a pharmaceutically acceptable carrier.

[0268] On the other hand, the present invention provides use of the fully human antibody or antigen-binding fragment of the present invention, the affinity matured antibody of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug, wherein the drug is used to prevent, alleviate and / or treat CCR8-related diseases or CCR8-CCL1 pathway-related diseases.

[0269] In some embodiments, the CCR8-related disease or CCR8-CCL1 pathway-related disease described in the present invention includes a tumor, and the tumor is selected from breast cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, metastatic brain cancer, metastatic liver cancer, etc.

[0270] In yet another aspect, the present invention provides a method for preventing, alleviating and / or treating CCR8-related diseases or CCR8-CCL1 pathway-related diseases, the method comprising administering to a subject in need thereof the fully human antibody or antigen-binding fragment of the present invention, the affinity matured antibody of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention or the pharmaceutical composition of the present invention.

[0271] In yet another aspect, the present invention provides a kit comprising the fully human antibody or antigen-binding fragment of the present invention and / or the affinity matured antibody of the present invention and instructions for use.

[0272] Definition of terms

[0273] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0274] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0275] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0276] As used herein, "CCR8" and "CC chemokine receptor type 8" and "chemokine receptor 8" refer to any native CCR8 produced by the expression and processing of CCR8 in a cell, or a variant of native CCR8.

[0277] As used herein, "CCL1" and "CC motif chemokine 1" refer to any native CCR1, or a variant of native CCR1, produced by the expression and processing of CCR1 in a cell.

[0278] The term "specifically binds" an antigen or epitope is a term well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" a target if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds with other substances. For example, an antibody that specifically or preferentially binds to a CCR8 epitope is an antibody that binds to the epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other CCR8 epitopes or non-CCR8 epitopes.

[0279] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen binding molecule (e.g., an antibody, an antibody fragment, or a scaffold protein containing an antibody binding region) binds. An epitope generally includes chemically active surface groups of a molecule such as an amino acid, a polypeptide, or a sugar side chain, and has specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be formed by both continuous residues and / or juxtaposed non-continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule.

[0280] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (such as bispecific T cell engagers) and trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0281] The term "monoclonal antibody" refers to an antibody of a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Therefore, a monoclonal antibody sample can bind to the same epitope on the antigen. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method.

[0282] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The term "CDR" means a complementary determining region defined by one skilled in the art by at least one identification method. In some embodiments, CDRs may be defined according to any Chothia numbering scheme, Kabat numbering scheme, a combination of Kabat and Chothia, AbM definition, Contact definition, and / or a combination of Kabat, Chothia, AbM, and / or Contact definitions. The CDRs of the present invention are defined according to the Kabat numbering scheme.

[0283] The term "heavy chain variable region (VH)" refers to a region comprising at least three heavy chain CDRs. In some embodiments, the heavy chain variable region includes three CDRs and at least FR2 and FR3. In some embodiments, the heavy chain variable region comprises at least heavy chain HCDR1, framework (FR) 2, HCDR2, FR3, and HCDR3. In some embodiments, the heavy chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4.

[0284] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0285] The term "heavy chain constant region" refers to a region comprising at least three heavy chain constant regions, CH1, CH2 and CH3. Of course, unless otherwise indicated, non-functional alterations, deletions and alterations within the domain are encompassed within the scope of the term "heavy chain constant region".

[0286] The term "heavy chain" refers to a polypeptide comprising at least one heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0287] The term "light chain variable region (VL)" refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region comprises three CDRs and at least FR2 and FR3. In some embodiments, the light chain variable region comprises at least light chain LCDR1, framework (FR) 2, LCDR2, FR3 and LCDR3. For example, the light chain variable region can comprise light chain CDR1, framework (FR) 2, CDR2, FR3 and CDR3. In some embodiments, the light chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4.

[0288] The term "light chain constant region" refers to a region comprising the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ. Of course, unless otherwise indicated, non-functional alterations, deletions and alterations within the domain are encompassed within the scope of the term "light chain constant region".

[0289] The term "light chain" refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0290] The term "amino acid" refers to a compound containing two functional groups, an amino group and a carboxyl group, such as α-amino acids. Two or more amino acids can form a polypeptide through an amide bond (also called a peptide bond). A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0291] The term "affinity matured" antibody refers to an antibody with one or more amino acid residue changes in one or more CDRs compared to a parent antibody without the alteration, such alterations resulting in improved affinity of the antibody for antigen.

[0292] The term "fully human antibody" means that the antibody gene sequence is entirely derived from human antibody genes, usually obtained by fully human antibody transgenic animals or by constructing a fully human antibody library in vitro through surface display technology or by human single B cell separation technology. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body.

[0293] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins.

[0294] "Antibody-dependent cell-mediated phagocytosis" or "ADCP" is based on the process of endocytosis of target cells (such as tumor cells) by effector cells with phagocytic potential (such as macrophages, monocytes, neutrophils). Antibodies bind to target cells by recognizing their antigens, and then bind phagocytes with their Fc segments to target cells. Once bound to the Fc receptor (FcR) of the phagocyte, the target cell is phagocytosed, and the phagosome fuses with the lysosome and is degraded. This process also causes the effector cells to produce soluble factors that help initiate and drive the immune response.

[0295] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to an antibody (of the appropriate subclass) bound to its cognate antigen.

[0296] The term "regulatory T cells" (also referred to as "Tregs" or "Treg cells" or "suppressor T cells") are T cell subsets that are immunosuppressive and typically inhibit or downregulate the induction and proliferation of effector T cells. Tregs express CD4, FOXP3, and CD25 (IL-2 receptor α chain). Compared to Treg cells in healthy subjects, Treg cells from cancer patients are typically characterized by a unique expression profile of chemokine receptors such as CCR4, CXCR4, and CCR5, which promotes their migration into tumors in response to corresponding chemokine ligands from the tumor microenvironment.

[0297] The term "antibody-drug conjugate (ADC)" refers to the antibody coupled to the effector molecule, preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity, such as one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0298] An "isolated" protein or peptide is one that has been separated from a component of its natural environment. In some aspects, the protein or peptide is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present in an extrachromosomal or chromosomal position different from its natural chromosomal position.

[0299] The term "expression vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0300] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0301] The term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The host cell may include a single cell or a cell population.

[0302] The term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. Typically, two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0303] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 1 9th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0304] The term "subject" generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0305] The term "tumor" generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. In this application, tumors may include solid tumors and / or non-solid tumors (eg, hematological tumors, lymphomas).

[0306] The term "administering" means a method of administering a dose of a compound (e.g., an anti-CCR8 antibody of the present invention) to a subject. In some aspects, the composition used in the methods herein is administered intravenously. The composition used in the methods described herein can be, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, externally, intratumorally, peritoneally, subcutaneously, subconjunctivally, intracapsularly, mucosally, intrapericardially, intraumbilical, intraocularly, orally, externally, topically, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly irrigating target cells, by catheter, by lavage, in the form of an emulsion or a lipid composition. The method of administration can vary according to a variety of factors (e.g., the compound or composition to be administered and the severity of the condition, disease or disorder to be treated).

[0307] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0308] Figure 1 This is a diagram showing the binding results of some positive phages to CHO-CCR8 cells in Example 1;

[0309] Figure 2 This is a graph showing the binding results of the antibody to the CHO-hCCR8 cell line in Example 3;

[0310] Figure 3 This is a graph showing the binding results of the antibody in Example 3 to the 293T-mCCR8 cell line;

[0311] Figure 4 This is a graph showing the binding results of the antibody in Example 3 to the 293T-cynCCR8 cell line;

[0312] Figure 5 This is a diagram showing the binding results of the antibody in Example 3 to the 293T-hCCR4 cell line;

[0313] Figure 6This is a graph showing the binding results of the antibody to the 293T cell line in Example 3;

[0314] Figure 7 This is a graph showing the binding results of the antibody to the CHO cell line in Example 3;

[0315] Figure 8 This is a graph showing the binding ability of the antibody to CHO-hCCR8 in Example 4;

[0316] Fig. 9 This is a graph showing the binding ability of the affinity mature mutant to CHO-hCCR8 in Example 5;

[0317] Fig.10 This is a graph showing the ADCC effect results of the affinity mature mutant in Example 6. DETAILED DESCRIPTION

[0318] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0319] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0320] Example 1 Generation of antibodies that bind to CCR8

[0321] Through affinity panning, specific antibody clones targeting CCR8 were enriched from the fully human phage library. With the help of cell flow cytometry, monoclonal antibody strains that can bind to CCR8 protein at the phage level were screened. The CCR8 protein extracellular region encoding gene was designed using the NCBI database and constructed into a mammalian eukaryotic expression system to express the CCR8 protein extracellular region loop1 and loop2 proteins for phage antibody library screening. Using loop1-Fc and loop2-Fc as panning proteins, the fully human phage library was subjected to multiple rounds of panning to obtain a phage pool enriched with the target antibody clones. Using different antibody libraries, a significant increase in the recovery rate was observed in each panning after 3 rounds of protein panning. The collected phage pool can be used for the next step of monoclonal selection and ELISA / FACS screening.

[0322] In order to obtain specific clones, it is necessary to isolate single clones and package them into monoclonal phages. The initial screening is performed by enzyme-linked immunosorbent assay (ELISA), and the unique antibody sequence contained therein is determined by DNA sequencing. The unique antibody sequence after sequencing is further detected by flow cytometry (FACS). The ELISA results are shown in Table 1-3. Phages 1G3 and 1H4 have strong binding to loop1-Fc and loo2-Fc. Phages 2G12, 5D6, 5G6 and 8H7 have strong binding to loop1-Fc and weak binding to loop2-Fc. The above phages do not bind to Fc. FACS binding is as shown in Table 1-3. Figure 1 As shown, the peaks of phages 1G3, 1H4, 2G12, 5D6, 5G6 and 8H7 shifted to the right compared with the negative control (NC), indicating that the above phages bind to the overexpression cell line CHO-hCCR8. Figure 1 Among them, 1G3, 1H4, 2G12, 5D6, 5G6 and 8H7 are all phage supernatants in scFv form.

[0323] Table 1 Results of ELISA assay for binding of some positive phages to loop1-Fc

[0324] serial number 1G3-scFv 1H4-scFv 2G12-scFv 5D6-scFv 5G6-scFv 8H7-scFv OD450 2.575 2.766 1.808 1.121 1.287 1.912

[0325] Table 2 Results of ELISA assay for binding of some positive phages to loop2-Fc

[0326] serial number 1G3-scFv 1H4-scFv 2G12-scFv 5D6-scFv 5G6-scFv 8H7-scFv OD450 2.387 2.364 0.334 0.378 0.334 0.741

[0327] Table 3 Results of ELISA assay of some positive phages binding to Fc

[0328] serial number 1G3-scFv 1H4-scFv 2G12-scFv 5D6-scFv 5G6-scFv 8H7-scFv OD450 0.09 0.117 0.108 0.074 0.068 0.104

[0329] Example 2 Expression, purification and identification of antibodies

[0330] A series of antibody expression vectors were constructed by molecular cloning and recombinant expression was performed in the 293F expression system. The nucleotide sequences encoding a series of monoclonal antibody light and heavy chains were obtained by extracting plasmids from panning phage solutions. The obtained scFv sequences were double-digested and inserted into the same restriction sites of the eukaryotic expression vector to construct a series of antibody expression vectors. Then, a series of verified correct expression vectors were extracted using the Invitrogen plasmid extraction kit, and purified and recovered after linearization with restriction endonucleases and stored at -20°C.

[0331] After the 293F host cells were revived with Opim medium, when the cell density was about 3*10 6 When the concentration of 1g / mL was 2.57kcal / mL, the cells were collected and transfected with PEI reagent. On the second day of culture, feed was added and glucose was added to the culture concentration of 8g / L. On the sixth day of culture, the cell culture fluid was collected.

[0332] A series of antibodies were tested at the translation level. The collected cell culture fluid was purified using a Protein A chromatography column, and the absorption peak was collected for mass spectrometry detection. The mass spectrometry detection showed that the molecular weight of a series of chimeric antibody scFv-Fc was about 100KD, which was consistent with the theoretical molecular weight and was in the form of a dimer. At the same time, the collected samples were reduced and non-reduced and then tested by 10% SDS-PAGE electrophoresis. The reduced SDS-PAGE electrophoresis pattern showed about 50KD, and the non-reduced SDS-PAGE electrophoresis pattern showed a single band at about 100KD. The size of the electrophoresis band was consistent with the theory. The purified samples were dialyzed overnight at 4°C using 0.02M PBS buffer at pH 7.4.

[0333] Example 3 Using multiple cell lines to identify antibody specificity and species cross-reactivity by FACS

[0334] The obtained antibody (prepared in Example 2) was reacted with the overexpressed human CCR8 cell line CHO-CCR8, the mouse CCR8 cell line 293T-CCR8-M and the monkey CCR8 cell line 293T-CCR8-Cyn to analyze whether the antibody can bind to CCR8 antigens of different species. At the same time, the overexpressed human CCR4 cell line 293-CCR4 and CHO, 293T cell lines were reacted to analyze whether the obtained antibody specifically binds to CCR8 and has any non-specific binding with other cell lines that do not express CCR8. In this example, the cells overexpressing human CCR8 were digested with trypsin, counted and resuspended to a cell density of 1E7 / ml. 100 μl / well cell suspension was added to a U-bottom 96-well plate, centrifuged at 300g for 5 minutes, and the supernatant was discarded. The obtained anti-CCR8 antibody was used as the primary antibody, and the Shionogi antibody 10A11 (whole antibody) was used as the positive reference at a concentration of 10ug / mL. The cells were resuspended with the diluted primary antibody, 100μl / well, and incubated at 4°C for 1 hour. Wash twice with PBS, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 100μl / well of the secondary antibody diluted 1:500 with FACS buffer to resuspend the cells, and then incubate at 4°C for 40 minutes. Wash twice with PBS, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 200μl / well DPBS to resuspend and analyze on the FACS machine.

[0335] The results are as follows Figure 2 As shown in the figure, the peak of 1H4 is significantly shifted to the right compared with the NC control, indicating that 1H4 has strong binding to the overexpression cell line CHO-hCCR8, while 1G3, 2G12, 5D6, 5G6 and 8H7 have weak binding. Figure 3 and Figure 4 As shown in the figure, 5D6, 5G6 and 8H7 have a certain binding to the mouse CCR8 overexpressing cell line 293T-mCCR8, while 1G3, 1H4 and 2G12 do not bind to the cell line 293T-mCCR8; in addition, 1H4, 5D6, 5G6 and 8H7 can bind to the monkey CCR8 overexpressing cell line 293-cynCCR8, while 1G3 and 2G12 do not. The specific binding results are shown in the figure. Figure 5 , Figure 6 and Figure 7 As shown, 1H4, 1G3 and 2G12 do not bind to 293T-hCCR4, CHO, and 293T cell lines, while 5D6, 5G6 and 8H7 have some binding to 293T-hCCR4 and 293T cell lines. In addition, the positive antibody has weak binding to 293T-hCCR4 and 293T cell lines. In summary, 1H4 has better specificity than the positive antibody. Among them, the positive control 10A11 contains the heavy chain shown in SEQ ID NO: 112 and the light chain shown in SEQ ID NO: 113.

[0336] EVQLVESGGALVKPGGSLRLSCAASGFTFSTYALYWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRARFYYSDYGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:112).

[0337] DIVMTQSPDSLAVSLGERATINCRSSKSLLHSNGNTYLYWYQQKPGQPPKLLIYRMSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCMQHLEYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:113).

[0338] Example 4 Binding ability of anti-CCR8 antibody 1H4 to high-expression cell line CHO-CCR8

[0339] This example uses the FACS binding method to analyze the half effective concentration (EC50) of antibody molecules, providing important information for the research and development process. In this implementation, the anti-CCR8 antibody prepared by Example 2 starts from 100ug / mL and is diluted 10 times in sequence, for a total of 8 concentrations. The binding ability of the antibody 1H4-(scFv-Fc) to the high-expression cell line CHO-CCR8 is tested. The steps are the same as Example 3. Affinity refers to the strength of binding of a single molecule to its ligand. The binding ability to positive cell lines can usually be detected by FACS to evaluate the strength of the interaction between the two molecules and rank them. The smaller the EC50 value, the greater the affinity of the antibody for its target. Figure 8 As shown, the EC50 of the binding ability of the obtained CCR8 antibody 1H4-(scFv-Fc) to the overexpression cell line CHO-CCR8 was greater than 10 nM.

[0340] Example 5 Anti-CCR8 Antibody Affinity Maturation

[0341] In this example, affinity maturation of antibody 1H4 was performed in order to improve its ability to bind to the target. By designing NNK degenerate primers, four mutation saturation libraries for the first round of affinity maturation were constructed: H1H2 (VH-CDR1+VH-CDR2), H3 (VH-CDR3), L1L2 (VL-CDR1+VL-CDR2), and L3 (VL-CDR3). Subsequently, the phage libraries after three rounds of screening with these four libraries were used as templates to construct the second round of affinity maturation libraries H1H2H3, H1H2L1L2, H1H2L3, H3L1L2, H3L3, and L1L2L3. Two to three rounds of affinity maturation were performed in this mode. Preliminary screening was performed using ELISA and FACS, and the sequences after affinity maturation were constructed into mammalian expression vectors for protein expression and purification. The purified mutants were then identified using FACS. The results are shown in Figure 2. Fig. 9 As shown in Table 4 , the affinity-matured mutants had stronger binding activity than the parent strain and the overexpression cell line CHO-hCCR8, and their binding activity was comparable to that of the positive 10A11.

[0342] Table 4 Binding ability of 1H4 affinity mature mutant antibody to CHO-hCCR8

[0343]

[0344] Example 6 Mutants have ADCC reporter gene biological activity

[0345] Digest and collect CHO-hCCR8 cells overexpressing human CCR8, and adjust the cell density to 4*10 5cells / mL, added to a white ViewPlate 96-well plate, 12.5μL / well. The antibody to be tested (prepared in Example 5) was diluted with 1640 medium, the positive antibody was 10A11, the starting concentration was 100μg / mL, 5-fold gradient dilution, a total of 7 concentrations. 25μl / well of the diluted antibody was added to a white ViewPlate 96-well plate and incubated at 37°C, 5% CO2 for 1h. Jurkat-NFAT-Luc2 / CD16 cells were collected and the cell density was adjusted to 4*10 5 cells / mL, added to a white ViewPlate 96-well plate, 12.5μL / well. The 96-well plate was placed in a 37°C incubator for overnight incubation. 6, 30μL / well Luciferase substrate was added, and RLU was read by an ELISA reader. The data were analyzed using GraphPadPrism. The results are shown in Fig.10 As shown, the affinity mature mutant 1H4-1-H4 has a stronger ADCC effect with an EC50 of 0.08 nM, which is comparable to that of the positive antibody 10A11.

[0346] In the description of this specification, the description with reference to the terms "some embodiments", "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0347] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A fully human antibody or antigen-binding fragment that specifically binds to human CCR8, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementary determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein: The H-CDR1 has an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 31; The H-CDR2 has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26 or SEQ ID NO:32; The H-CDR3 has an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27 or SEQ ID NO: 33; The L-CDR1 has an amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28 or SEQ ID NO:34; The L-CDR2 has an amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 35; The L-CDR3 has an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30 or SEQ ID NO:

36.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody comprises at least one of a heavy chain variable region framework region and a light chain variable region framework region, and at least a portion of at least one of the heavy chain variable region framework region and the light chain variable region framework region is derived from at least one of a human antibody or a mutant thereof.

3. The antibody or antibody fragment according to claim 1 or 2, characterized in that The heavy chain variable region of the antibody comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 59-64.

4. The antibody or antibody fragment according to any one of claims 1 to 3, characterized in that The light chain variable region of the antibody comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 71-76.

5. An affinity matured antibody, characterized in that: The affinity matured antibody is prepared by mutation of the CCR8 antibody according to any one of claims 1 to 4.

6. The affinity matured antibody according to claim 5, characterized in that The affinity matured antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementary determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein: The H-CDR1 has an amino acid sequence shown as X1X2X3X4S; wherein X1 is selected from P, S, G, A or R, X2 is selected from R, K, Y or S, X3 is selected from A or T, and X4 is selected from I, Y or V; The H-CDR2 has an amino acid sequence shown in IINX5X6X7GX8TRYAQKFQG; wherein X5 is selected from P, I, K or G, X6 is selected from S, W or H, X7 is selected from G or R, and X8 is selected from N, Q, I or K; The H-CDR3 has DX9X 10 GMDX 11 X 12 RXGD 13 X 14 The amino acid sequence shown; wherein X9 is selected from G or N, X 10 Selected from F, T or R, X 11 Select from S or K, X 12 Select from R or S, X 13 Select from R or L, X 14 Selected from Y or R; The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56; The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57; and the L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:

58.

7. The affinity matured antibody according to claim 5 or 6, characterized in that: The affinity matured antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions: H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises complementary determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein: The H-CDR1 has an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:53; The H-CDR2 has an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:54; The H-CDR3 has an amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51 or SEQ ID NO:55; The L-CDR1 has an amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:56; The L-CDR2 has an amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:57; and the L-CDR3 has an amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48 or SEQ ID NO:

58.

8. The affinity matured antibody according to any one of claims 5 to 7, characterized in that: The affinity matured antibody comprises at least one of a heavy chain variable region framework region and a light chain variable region framework region, and at least a portion of at least one of the heavy chain variable region framework region and the light chain variable region framework region is derived from at least one of a human antibody or a mutant thereof.

9. The affinity matured antibody according to any one of claims 5 to 8, characterized in that: The affinity matured antibody heavy chain variable region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 65-70.

10. The affinity matured antibody according to any one of claims 5 to 9, characterized in that: The affinity matured antibody light chain variable region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 77-82.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 4 or the affinity matured antibody according to any one of claims 5 to 10, characterized in that: The antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region comes from at least one of a human antibody or a mutant thereof.

12. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1-4 or the affinity matured antibody of any one of claims 5-11.

13. An expression vector, characterized in that: The expression vector carries the nucleic acid molecule of claim 12.

14. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 12 or the expression vector of claim 13.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment of any one of claims 1-4, the affinity matured antibody of any one of claims 5-11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the host cell of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the antibody or antigen-binding fragment of any one of claims 1 to 4, the affinity matured antibody of any one of claims 5 to 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the host cell of claim 14 or the pharmaceutical composition of claim 15 in the preparation of a drug for preventing, alleviating and / or treating CCR8-related diseases.

17. The use according to claim 16, characterized in that The CCR8-related diseases include tumors, which are selected from breast cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, metastatic brain cancer, metastatic liver cancer, etc.

18. A kit, characterized in that: The kit comprises the antibody or antigen-binding fragment according to any one of claims 1 to 4 and / or the affinity-matured antibody according to any one of claims 5 to 11 and instructions for use.

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