Anti-PD-L1 antibodies and their applications

Abstract

The present invention provides anti-PD-L1 antibodies and their applications. The antibodies or antigen-binding fragments of the present invention can specifically bind to PD-L1 and block the interaction between PD-1 and PD-L1. The antibodies or antigen-binding fragments of the present invention can contribute to the elimination of tumor cells by the immune system and can also be used in the diagnosis and prognosis of tumors or cancer.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, in particular to anti-PD-L1 antibodies and their applications. [Background technology]

[0002] Programmed death receptor-1 (PD-1) is a type I transmembrane glycoprotein with a molecular weight of approximately 55 kDa. PD-1 is an immunosuppressive receptor expressed on activated T cells, B cells, and myeloid cells, and belongs to the CD28 immunoglobulin superfamily. PD-1 ligand, PD-L1, is a widely distributed type I transmembrane glycoprotein expressed on the surface of antigen-presenting cells such as B cells, T cells, dendritic cells, and macrophages, as well as on tumor tissues.

[0003] The interaction between PD-1 and PD-L1 negatively regulates antigen receptor signaling and attenuates T cell responses. Numerous studies have shown that the interaction between PD-1 and PD-L1 reduces lymphocytes infiltrating tumors, reduces T cell receptor-mediated proliferation, and allows cancer cells to evade the immune system. Blocking the interaction between PD-1 and PD-L1 increases T cell proliferation and cytokine production, and inhibits tumor-specific CD8 + It can improve T cell immunity and contribute to the elimination of tumor cells by the immune system.

[0004] The PD-1 / PD-L1 pathway is a target for antibody therapy in cancer treatment. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2019 / 032431 [Patent Document 2] WO2019 / 018757 [Patent Document 3] WO2015 / 109124 [Patent Document 4] WO2019 / 241353 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides anti-PD-L1 antibodies or antigen-binding fragments that can specifically bind to PD-L1, block the interaction between PD-1 and PD-L1, and contribute to the elimination of tumor cells by the immune system. [Means for solving the problem]

[0007] Some embodiments specifically bind to PD-L1 and (a) an HCDR1 comprising the amino acid sequence represented by X15X16X17X18X19DSWIH, wherein X15 is G, S, or N, X16 is F, W, L, or S, X17 is T, S, A, R, or H, X18 is F, L, T, or P, and X19 is S, K, R, or A; and / or (b) an HCDR2 comprising GWISPYGGSTYYADX20X21X22X23, wherein X20 is S, D, H, G, P or Y, X21 is V, F, L, M or Y, X22 is K, R, G, S, V or H, and X23 is G, H, D, Q, S or A; and / or (c) an anti-PD-L1 antibody or antigen-binding fragment comprising an HCDR3 comprising RHWPGGX24X25X26, where X24 is F or L, X25 is D or L, and X26 is Y or P.

[0008] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1; and (a) HCDR1 comprising the amino acid sequence represented by X15X16X17X18X19DSWIH, wherein X15 is G, S, or N, X16 is F, W, L, or S, X17 is T, S, A, R, or H, X18 is F, L, T, or P, and X19 is S, K, R, or A; (b) an HCDR2 comprising GWISPYGGSTYYADX20X21X22X23, wherein X20 is S, D, H, G, P or Y, X21 is V, F, L, M or Y, X22 is K, R, G, S, V or H, and X23 is G, H, D, Q, S or A; and (c) HCDR3 comprising RHWPGGX24X25X26, wherein X24 is F or L, X25 is D or L, and X26 is Y or P.

[0009] In some embodiments, HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 or a variant thereof with a single-site substitution, deletion or insertion. In some embodiments, HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7 to 16 or a variant thereof with a single-site substitution, deletion or insertion. In some embodiments, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17 or 18 or a variant thereof with a single-site substitution, deletion or insertion. In some embodiments, the substitution variants are conservative amino acid substitution variants.

[0010] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0011] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0012] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:8, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0013] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0014] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:10, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0015] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:11, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0016] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:12, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0017] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:13, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0018] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:14, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0019] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:15, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0020] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0021] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0022] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0023] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0024] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0025] In some embodiments, an antibody that specifically binds to PD-L1 and (a) an HCDR1 comprising the amino acid sequence represented by X15X16X17X18X19DSWIH, wherein X15 is G, S, or N, X16 is F, W, L, or S, X17 is T, S, A, R, or H, X18 is F, L, T, or P, and X19 is S, K, R, or A; and / or (b) an HCDR2 comprising GWISPYGGSTYYADX20X21X22X23, wherein X20 is S, D, H, G, P or Y, X21 is V, F, L, M or Y, X22 is K, R, G, S, V or H, and X23 is G, H, D, Q, S or A; and / or (c) an HCDR3 comprising RHWPGGX24X25X26, wherein X24 is F or L, X25 is D or L, and X26 is Y or P; and / or (d) LCDR1 comprising X1ASQX2IX3X4X5LX6, where X1 is L, Q or R, X2 is D, T or G, X3 is G or S, X4 is K, T or S, X5 is H, W, F or Y, and X6 is N or A; and / or (e) an LCDR2 comprising X7ASX8LX9X10, where X7 is A or G, X8 is T, N, S or R, X9 is Q or K, and X10 is S or T; and / or (f) An antibody or antigen-binding fragment is provided, comprising an LCDR3 comprising QQX11X12X13TPX14T, wherein X11 is Y or S, X12 is Y or F, X13 is S or T, and X14 is R or Y.

[0026] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1; and (a) HCDR1 comprising the amino acid sequence represented by X15X16X17X18X19DSWIH, wherein X15 is G, S, or N, X16 is F, W, L, or S, X17 is T, S, A, R, or H, X18 is F, L, T, or P, and X19 is S, K, R, or A; (b) an HCDR2 comprising GWISPYGGSTYYADX20X21X22X23, wherein X20 is S, D, H, G, P or Y, X21 is V, F, L, M or Y, X22 is K, R, G, S, V or H, and X23 is G, H, D, Q, S or A; (c) an HCDR3 comprising RHWPGGX24X25X26, wherein X24 is F or L, X25 is D or L, and X26 is Y or P; (d) LCDR1 comprising X1ASQX2IX3X4X5LX6, where X1 is L, Q or R, X2 is D, T or G, X3 is G or S, X4 is K, T or S, X5 is H, W, F or Y, and X6 is N or A; (e) LCDR2 comprising X7ASX8LX9X10, where X7 is A or G, X8 is T, N, S or R, X9 is Q or K, and X10 is S or T; and (f) LCDR3 comprising QQX11X12X13TPX14T, wherein X11 is Y or S, X12 is Y or F, X13 is S or T, and X14 is R or Y.

[0027] In some embodiments, LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23 or a variant thereof with a single-site substitution, deletion, or insertion. In some embodiments, LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 24 to 27 or a variant thereof with a single-site substitution, deletion, or insertion. In some embodiments, LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 28 to 31 or a variant thereof with a single-site substitution, deletion, or insertion. In some embodiments, the substitution variants are conservative amino acid substitution variants.

[0028] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:19, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:28.

[0029] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:20, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:25, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:29.

[0030] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:21, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:30.

[0031] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:22, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:27, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:31.

[0032] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:23, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:30.

[0033] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:19, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:31.

[0034] In some embodiments, a polypeptide that specifically binds to PD-L1 and comprises (a) an HCDR1 having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6, or a variant thereof with a single substitution, deletion, or insertion; and / or (b) an HCDR2 having the amino acid sequence set forth in any one of SEQ ID NOs: 7 to 16, or a variant thereof with a single substitution, deletion, or insertion; and / or (c) an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 17 or 18, or a variant thereof with a single substitution, deletion, or insertion; and / or (d) LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23 or a variant thereof with a substitution, deletion or insertion at a single site, and / or (e) LCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24 to 27 or a variant thereof with a substitution, deletion or insertion at a single site, and / or (f) LCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 28 to 31 or a variant thereof with a substitution, deletion or insertion at a single site.

[0035] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least one, two, three, four, five, or all of the following: HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 16, HCDR3 set forth in SEQ ID NO: 17, LCDR1 set forth in any one of SEQ ID NOs: 19-23, LCDR2 set forth in any one of SEQ ID NOs: 24-27, and LCDR3 set forth in any one of SEQ ID NOs: 28-31.

[0036] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0037] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and the antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 20, an LCDR2 set forth in SEQ ID NO: 25, and an LCDR3 set forth in SEQ ID NO: 29.

[0038] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 21, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 30.

[0039] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 22, an LCDR2 set forth in SEQ ID NO: 27, and an LCDR3 set forth in SEQ ID NO: 31.

[0040] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 23, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 30.

[0041] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and the antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 31.

[0042] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 7, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0043] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 8, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0044] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 9, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0045] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 10, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0046] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1, and comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 11, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28.

[0047] In some embodiments, the heavy chain of the antibody or antigen-binding fragment further comprises FR1, FR2, FR3, and FR4, wherein heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 32, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 32, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 32; and / or

[0048] the heavy chain FR2 of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 33, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 33, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 33; and / or

[0049] the heavy chain FR3 of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 34, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 34, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 34; and / or

[0050] The heavy chain FR4 of the above-described antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:35, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:35, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO:35.

[0051] In some embodiments, heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 32, heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 33, heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 34, and heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 35.

[0052] In some embodiments, the heavy chain variable region (VH) comprises the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 36-50, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 36-50, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 36-50.

[0053] In some embodiments, the light chain of the antibody or antigen-binding fragment further comprises FR1, FR2, FR3, and FR4, wherein light chain FR1 comprises the sequence set forth in any one of SEQ ID NOs: 51-56, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 51-56, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 51-56; and / or

[0054] the light chain FR2 of the antibody or antigen-binding fragment comprises a sequence set forth in any one of SEQ ID NOs: 57 to 60, a sequence having at least 90% identity to a sequence set forth in any one of SEQ ID NOs: 57 to 60, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence set forth in any one of SEQ ID NOs: 57 to 60; and / or

[0055] the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 61 to 64, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 61 to 64, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 61 to 64; and / or

[0056] The light chain FR4 of the above antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 65 to 67, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 65 to 67, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 65 to 67.

[0057] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:51, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:57, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:61, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:65.

[0058] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:52, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:58, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:62, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:66.

[0059] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:53, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:63, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67.

[0060] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:54, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:60, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:64, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67.

[0061] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:55, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:63, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67.

[0062] In some embodiments, the light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:56, the light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:57, the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:61, and the light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67.

[0063] In some embodiments, the light chain variable region (VL) comprises the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In some embodiments, the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 68-73, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 68-73, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 68-73.

[0064] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 36-50, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 68-73.

[0065] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0066] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:69.

[0067] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:70.

[0068] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:71.

[0069] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:72.

[0070] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:73.

[0071] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:37, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0072] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:38, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0073] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:39, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0074] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:40, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0075] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:41, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68.

[0076] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is of one of the following isotypes: IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully humanized antibody.

[0077] In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, compared to the parent Fc region. In some embodiments, the amino acid modifications to the Fc region alter effector function activity compared to the activity of the parent Fc region. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, regulatory activity, or cell binding. In some embodiments, the Fc region amino acid modifications may alter the affinity of the variant Fc region for FcγR (Fcγ receptor) compared to the parent Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A.

[0078] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an scFV, Fab, F(ab)2, or IgG1. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0079] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 74 or 75, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 74 or 75, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 74 or 75; and / or

[0080] The light chain constant region of the above-mentioned antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 76, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 76.

[0081] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 74, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 75, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76.

[0082] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 77-91, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 77-91, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 77-91; and / or

[0083] The light chain of the above antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 92 to 97, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 92 to 97, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 92 to 97.

[0084] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0085] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:93.

[0086] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:94.

[0087] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:95.

[0088] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:96.

[0089] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:97.

[0090] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:78, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0091] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:79, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0092] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:80, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0093] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:81, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0094] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:82, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:92.

[0095] In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).

[0096] In some embodiments, the antibody has two identical heavy chains and two identical light chains, with the Fc regions pairing to form disulfide bonds.

[0097] In one embodiment, the antibody fragment (antigen-binding fragment) is a Fab, Fv, or scFv antibody.

[0098] The present invention further provides a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0099] The present invention further provides a carrier comprising the nucleic acid molecule. In some embodiments, the carrier is an isolated carrier. In some embodiments, the carrier comprising the nucleic acid molecule is a nucleic acid fragment, a plasmid, a phage, or a virus.

[0100] The present invention further provides a host cell comprising the nucleic acid molecule or carrier. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, an HEK cell (e.g., an HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0101] The present invention further provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment and a pharmaceutically acceptable excipient.

[0102] The present invention further provides therapeutic methods and uses. In some embodiments, the present invention provides a method for treating or ameliorating a T cell dysfunction disorder, comprising administering to a patient an effective dose of the above-described antibody or antigen-binding fragment. In some embodiments, the present invention provides the use of the above-described antibody or antigen-binding fragment for treating or ameliorating a T cell dysfunction disorder. In some embodiments, the present invention provides the use of the above-described antibody or antigen-binding fragment in the preparation of a medicament for treating or ameliorating a T cell dysfunction.

[0103] In some embodiments, the T cell dysfunction disorder includes, but is not limited to, an infection caused by a bacterium, virus, fungus, or protozoan, and cancer and tumor, including, but not limited to, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head cancer, neck cancer, and pancreatic cancer.

[0104] The present invention further provides diagnostic methods and uses. In some embodiments, there is provided a method for detecting PD-L1 expression in a sample by contacting the sample with the above-described antibody or antigen-binding fragment, whereby the antibody or antigen-binding fragment binds to PD-L1, and detecting this binding, i.e., the amount of PD-L1 in the sample. In some embodiments, there is provided the use of the above-described antibody or antigen-binding fragment in the preparation of a reagent kit for diagnosing or prognosing cancer or tumors. In some embodiments, there is provided a diagnostic or prognostic reagent kit comprising the above-described antibody or antigen-binding fragment.

[0105] The present invention provides anti-PD-L1 antibodies or antigen-binding fragments that specifically bind to PD-L1, block the interaction between PD-1 and PD-L1, and contribute to the elimination of tumor cells by the immune system, as well as applications thereof. The antibodies or antigen-binding fragments of the present invention can be used to treat or ameliorate T cell dysfunction disorders, and can also be used in the diagnosis and prognosis of cancer or tumors. [Brief explanation of the drawings]

[0106] [Figure 1A] Blockade of PD-1 / PD-L1 interaction by anti-PD-L1 antibodies is shown in Figures 1A and 1B, where the ordinate represents fluorescence values ​​(in light units) and the abscissa represents concentration. [Figure 1B] Blockade of PD-1 / PD-L1 interaction by anti-PD-L1 antibodies is shown in Figures 1A and 1B, where the ordinate represents fluorescence values ​​(in light units) and the abscissa represents concentration. [Figure 2] Figure 1 shows the binding of anti-PD-L1 antibodies to PD-L1-overexpressing cells, where the ordinate indicates mean fluorescence intensity, the abscissa indicates concentration, and isotype indicates the negative control antibody IgG-isotype. [Figure 3A] The effect of antibodies on tumor volume is shown. [Figure 3B] The effect of antibodies on tumor weight is shown. [Figure 3C] The effect of antibodies on mouse body weight is shown. [Figure 4] Figure 1 shows the binding status of anti-PD-L1 scFv to PD-L1-His-Biotin detected by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0107] term Unless otherwise stated, each of the terms below shall have the meaning set forth below.

[0108] definition It should be noted that the term "one" entity refers to one or more of that entity, e.g., "one antibody" should be understood as one or more antibodies, and therefore the terms "one" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

[0109] As used herein, the terms "comprising" or "including" mean that compositions, methods, etc., include the recited elements, such as components or steps, but do not exclude others. "Consisting essentially of" means that compositions and methods exclude other elements that materially affect the properties of the combination, but do not exclude elements that do not materially affect the composition or method. "Consisting of" means excluding elements not specifically recited.

[0110] The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids, and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of or interchangeably with any one of the above terms. The term "polypeptide" also refers to products modified after expression of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but need not be translated from a designated nucleic acid sequence and can be produced by any method, including chemical synthesis.

[0111] An "amino acid" is an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by a nucleic acid either as such or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (a so-called codon or base triplet). Each amino acid is encoded by at least one codon. The encoding of the same amino acid by different codons is called the "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Naturally occurring 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).

[0112] A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of classes of amino acids containing side chains with similar chemical properties include: 1) aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains such as serine and threonine; 3) amide-containing side chains such as asparagine and glutamine; 4) aromatic side chains such as phenylalanine, tyrosine, and tryptophan; 5) basic side chains such as lysine, arginine, and histidine; and 6) acidic side chains such as aspartic acid and glutamic acid.

[0113] The number of amino acids in the "conservative amino acid substitutions in the frame regions FR1, FR2, FR3, and FR4" is about 1, about 2, about 3, about 4, or about 5. The number of amino acids in the "conservative amino acid substitutions in VL and VH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, or about 15 conservative amino acid substitutions, or a range between any two of these values ​​(including the endpoints), or any value therein. The number of amino acids in a "heavy chain constant region, light chain constant region, heavy chain, or light chain conservative amino acid substitution" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, or about 45 conservative amino acid substitutions, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0114] As used herein, the term "isolated" in reference to a cell, nucleic acid, polypeptide, antibody, etc., refers to a molecule, such as DNA or RNA, that has been isolated from one or more other components of the cell's natural environment. As used herein, the term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" includes nucleic acid fragments that do not occur in the natural state, meaning that they do not occur in the natural state. As used herein, the term "isolated" also refers to a cell or polypeptide that has been isolated from other cellular proteins or tissues. An isolated polypeptide is meant to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc., are generally prepared by at least one purification step. In some embodiments, the purity of an isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0115] The term "recombinant," in reference to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example being one that can be combined to produce a polynucleotide or polypeptide that does not normally occur.

[0116] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0117] In some embodiments, "at least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(inclusive of the endpoints), or any value therein.

[0118] In some embodiments, "at least 90% identity" means about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0119] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity or sequence identity" with another sequence means that, upon sequence alignment, that percentage of bases (or amino acids) are the same in the two sequences being compared. The alignment and percent identity or sequence identity can be determined by visual inspection or by software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, including BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides refer to polynucleotides that have the percentage identity specified above and encode polypeptides that have the same or similar biological activity.

[0120] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented by the characters of a polynucleotide molecule. The characters can be entered into a database in a computer having a central processing unit and used in bioinformatics applications, such as functional genomics and homology searching.

[0121] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE labels), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, carriers, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may also contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Such modifications can occur before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of this disclosure includes the double-stranded form and each of the two known or predicted complementarable single-stranded forms that make up the double-stranded form.

[0122] The term "code," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide, and which, either in its natural state or when manipulated by methods known to those of skill in the art, is capable of producing that polypeptide and / or fragments thereof by transcription and / or translation.

[0123] "Antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be an intact antibody or any antigen-binding fragment thereof, or a single chain thereof. Thus, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the framework region (FR), or any portion thereof, or at least a portion of a binding protein. The CDR regions include the light chain CDR regions (LCDR1-3) and the heavy chain CDR regions (HCDR1-3). Antibodies and antigen-binding fragments can specifically recognize and bind to one or more (e.g., two) antigen polypeptides or polypeptide complexes. An antibody or antigen-binding fragment that specifically recognizes and binds to multiple (e.g., two) antigens may be referred to as a multispecific (e.g., bispecific) antibody or antigen-binding fragment.

[0124] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, and the structural form of an antibody fragment of the present invention can resemble F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. in monospecific antibody fragments. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, enantiomers, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0125] A "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL). In some embodiments, these regions are joined by a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich to increase flexibility or serine- or threonine-rich to increase solubility, and may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are generally known in the art, for example, as described in U.S. Pat. No. 5,892,019.

[0126] The term "antibody" encompasses a wide variety of biochemically distinct polypeptide classes. Those skilled in the art will understand that heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and further include several subclasses (e.g., γ1-γ4). The nature of the chain determines the "type" of an antibody: IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, have been well characterized and their conferred functional specificities are known. All immunoglobulin types are encompassed by the claims disclosed by the present invention. In some embodiments, the immunoglobulin molecule type is IgG. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chains beginning at the mouth of the "Y" and continuing through the variable region to surround the heavy chains.

[0127] Antibodies, antigen-binding fragments or derivatives disclosed by the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2), scFv.

[0128] Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be bound to either a κ or λ light chain. Generally, when immunoglobulins are produced from hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are bound via a covalent bond, and the "tail" portions of the two heavy chains are bound via a covalent disulfide bond or a non-covalent bond. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The κ light chain variable region of an immunoglobulin is Vκ, and the λ light chain variable region of an immunoglobulin is V λ is. The variable regions of the light (VL) and heavy (VH) chains determine antigen recognition and specificity.

[0129] The constant regions of the heavy chains confer important biological properties such as secretion, placental transfer, Fc receptor binding, and complement fixation. By convention, the numbering of the constant regions increases with increasing distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0130] In naturally occurring antibodies, assuming that the antibody exhibits its three-dimensional configuration in an aqueous environment, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous amino acid sequences that specifically bind to antigens, forming the antigen-binding domain. The remaining amino acids in the antigen-binding domain, called "framework" regions, exhibit little inter-molecular variability. Most of the framework regions adopt a β-fold conformation, forming a ring structure to which the CDRs are connected, or in some cases, forming part of the β-fold structure. Thus, the framework regions form a stent, positioning the CDRs in the correct orientation through inter-chain non-covalent interactions. An antigen-binding domain with CDRs at specific positions forms a surface complementary to the antigen epitope, promoting non-covalent binding of the antibody to the antigen epitope. For a given heavy or light chain variable region, one skilled in the art can identify the amino acids comprising the CDR and framework regions by known methods (see Kabat, E. et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0131] Where a term used and / or accepted in the art has more than one definition, the definition of the term used herein includes all of these meanings unless expressly indicated to the contrary. A specific example uses the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), all of which are incorporated herein by reference.

[0132] The CDRs defined by Kabat and Chothia contain overlapping or subsets of amino acid residues when compared with each other. Nevertheless, it is within the scope of the present invention to use any definition to refer to the CDRs of an antibody or its variants. The exact residue numbers that comprise a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can generally determine the specific residues contained in a CDR based on the amino acid sequence of the variable region of an antibody.

[0133] Kabat et al. also defined a numbering system that can be applied to any antibody variable region sequence. One skilled in the art can apply this "Kabat numbering" system to any variable region sequence, regardless of other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system described by Kabat et al., US Department of Health and Human Services, in "Sequence of Proteins of Immunological Interest" (1983). Antibodies may also use the EU or Chothia numbering system.

[0134] The antibodies disclosed by the present invention may be derived from any animal, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, camel, llama, horse, or chicken. In other embodiments, the variable region may be derived from a chondricthoid (e.g., shark).

[0135] The heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant regions of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In other embodiments, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In other embodiments, some heavy chains may comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0136] A "light chain constant region" comprises a portion of an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" is an assembly of a light chain and a heavy chain that can form a dimer via disulfide bonding between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0137] The "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the "CH1 domain" comprises the first (most amino-terminal) constant region of an immunoglobulin heavy chain. The CH2 domain is not tightly paired with other domains and inserts two N-linked branched carbohydrate chains between the two CH2 domains in an intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and has been described as comprising approximately 108 residues. The "hinge region" comprises a portion of the heavy chain connecting the CH1 and CH2 domains. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0138] A "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of a cysteine ​​can form a disulfide bond or cross-link with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0139] A "chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial, or modified) are derived from a second species. In some embodiments, the variable regions are non-human (e.g., murine or primate) and the constant regions are human.

[0140] "Specific binding" or "specific for" generally refers to the complementary binding of an antibody or antigen-binding fragment to a specific antigen via its antigen-binding domain and epitope, forming a relatively stable complex. "Specific" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody "A" has a higher relative affinity for the same antigen than antibody "B," antibody "A" is considered to have a higher specificity for this antigen than antibody "B." Specific binding can be expressed by the equilibrium dissociation constant (KD), where a smaller KD indicates tighter binding. Methods for determining whether two molecules are specifically bound include, for example, equilibrium dialysis, surface plasmon resonance, biolayer optical interferometry, and the like, and are well known in the art. An antibody that "specifically binds" to antigen a includes an antibody with an equilibrium dissociation constant KD with antigen a of about 100 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less.

[0141] "Treatment" refers to therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent, alleviate, ameliorate, or halt an undesirable physiological change or disorder, such as the progression of a disease, and includes, but is not limited to, detectable or undetectable results such as alleviation of symptoms, lessening of the extent of the disease, stabilization of the disease state (i.e., not worsening), delaying or alleviating the progression of the disease, improvement, palliative, reduction, or elimination (partial or complete) of the disease state, or an increase in expected survival time if not receiving treatment. Patients in need of treatment include those already suffering from a disease condition or disorder, those susceptible to a disease condition or disorder, or those in need of prevention of the disease or disorder, and those who could or would benefit from administering an antibody or pharmaceutical composition disclosed by the present invention for detection, diagnostic processes, and / or treatment.

[0142] "Patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, and the like.

[0143] "About" refers to the normal error range of the corresponding numerical value, which would be readily known to one of ordinary skill in the art. In some embodiments, "about" as referred to herein refers to the stated numerical value and a range of ±10%, ±5%, or ±1% thereof.

[0144] The half maximum effective concentration (EC 50 ” (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can produce 50% of the maximum effect.

[0145] In the present invention, a "parent Fc region" may be a naturally occurring Fc region. The gene encoding the Fc region may be derived from human, mouse, rabbit, camel, or monkey, with human and mouse being preferred. For example, the parent Fc region is the Fc region of SEQ ID NO: 77 or 78.

[0146] Any relevant discussion of publications mentioned herein is hereby incorporated by reference in its entirety.

[0147] Anti-PD-L1 antibody The present invention provides antibodies or antigen-binding fragments with high affinity for PD-L1 protein. The screened antibodies exhibit effective binding and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints, activate lymphocytes to release cytokines, and be used to treat various types of cancer, tumors, or related diseases, such as infectious diseases.

[0148] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0149] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 93.

[0150] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 94.

[0151] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 95.

[0152] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0153] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 77, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 97.

[0154] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 78, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 78, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0155] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 79, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 79, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0156] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 80, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 80, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0157] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 81, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 81, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0158] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 82, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the heavy chain of the antigen-binding fragment comprises an amino acid sequence other than Fc in SEQ ID NO: 82, and the light chain of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 92.

[0159] In some embodiments, an antibody of the invention comprises two identical heavy chains (or heavy chain fragments) and two identical light chains (or light chain fragments).

[0160] Those skilled in the art should also understand that the sequence of the antibody or antigen-binding fragment disclosed herein may be substituted, and the resulting amino acid sequence may differ from the naturally occurring amino acid sequence of the antibody. For example, the resulting amino acid sequence may be similar to the starting sequence, e.g., have a certain percentage of identity with the starting sequence, e.g., about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0161] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence having one or more modifying groups. For example, the disclosed antibodies or antigen-binding fragments may include a flexible linker sequence or may be modified to add a functional group (e.g., PEG, a drug, a toxin, or a label).

[0162] The antibodies and antigen-binding fragments disclosed herein include modified derivatives, i.e., modified by the covalent attachment of any type of molecule to the antibody or antigen-binding fragment, where the covalent attachment does not prevent the antibody or antigen-binding fragment from binding to its epitope. Examples include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and attachment to cellular ligands or other proteins. Any one of a number of chemical modifications may be performed by conventional techniques, including, but not limited to, specific chemical degradation, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0163] In some embodiments, the antibody or antigen-binding fragment can be conjugated to a therapeutic agent, drug precursor, peptide, protein, enzyme, virus, lipid, biological response modifier, drug, or PEG.

[0164] The antibody or antigen-binding fragment may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody or antigen-binding fragment is then determined by detecting the emission of luminescence during a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridine salts, and oxalate esters.

[0165] Methods for preparing antibodies and polynucleotides encoding the antibodies The present invention further discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof described in the present invention. The polynucleotides disclosed by the present invention can encode heavy chain variable regions, light chain variable regions, Fc regions, partial heavy chain variable regions, partial light chain variable regions, heavy chains, or light chains. Methods for preparing antibodies are known in the art and are described in the present invention.

[0166] In some embodiments, the antibodies prepared do not provoke an adverse immune response in the treated animal (e.g., a human). In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified using art-recognized techniques to reduce their immunogenicity. For example, antibodies may be humanized, primatized, deimmunized, or prepared as chimeric antibodies. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, and retain or essentially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved in a variety of ways, including (a) grafting the entire non-human variable region onto human constant regions to produce a chimeric antibody; (b) grafting at least a portion of one or more non-human complementarity-determining regions (CDRs) onto human framework and constant regions, with or without retaining critical framework residues; or (c) grafting the entire non-human variable region but "masking" them by replacing surface residues with human-like moieties. Typically, frame residues in the human frame regions are replaced with corresponding residues from the CDR donor antibody, residues that can improve antigen binding, etc. These frame substitutions can be identified by methods known in the art, for example, by simulating the interactions between CDRs and frame residues to identify frame residues that play an important role in antigen binding, and by sequence alignment to identify unusual frame residues at specific positions (see U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988)), the entire contents of which are incorporated herein by reference).Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400, WO 91 / 09967, U.S. Patents 5,225,539, 5,530,101, and 5,585,089), repair or resurfacing (EP 592,106, EP 519,596, Padlan et al., Molecular Immunology 28(4 / 5):489-498(1991), Studnicka et al., Protein Engineering 7(6):805-814(1994), Roguska et al., Proc. Natl. Sci. USA 91:969-973(1994)), and chain rearrangement (U.S. Patent 5,565,332), the entire contents of which are incorporated herein by reference.

[0167] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering an antibody to modify its T cell epitopes (see, for example, WO / 9852976 A1 and WO / 0034317 A2). For example, the heavy and light chain variable region sequences from a starting antibody are analyzed to generate a human T cell epitope "map" from each variable region, indicating the location of the epitope relative to the complementarity-determining regions (CDRs) and other important residues within the sequence. Analysis of a single T cell epitope from the T cell epitope map identifies selectable amino acid substitutions with a low risk of altering antibody activity. A series of selectable heavy and light chain variable region sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into a series of binding polypeptides. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into an expression vehicle, and the plasmids are then introduced into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibody.

[0168] The binding specificity of the antibodies or antigen-binding fragments disclosed by the present invention can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For the preparation of scFv, reference can be made to techniques for producing single-chain units (U.S. Patent: 4,694,778, Bird, Science 242:423-442 (1988), Huston et al., Proc Natl. Acad. Sci. USA 55:5879-5883 (1988), Ward et al., Nature 334:544-554 (1989), and Nie et al., Antibody Therapeutics 3 (1):18-62 (2020)). Single-chain units are formed by amino acid crosslinking of heavy and light chain fragments of the Fv region to form single-chain fusion peptides. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).

[0169] Examples of techniques that can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993), and Skerra et al., Science 240:1038-1040 (1988). For some uses, including antibody administration in humans and in vitro detection experiments, chimeric, humanized, or fully human antibodies can be used. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies having a murine monoclonal antibody variable region and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference. Further, Newman, Biotechnology 10:1455-1460 (1992) discloses another efficient method for producing recombinant antibodies, which, in particular, can produce primate antibodies containing monkey variable regions and human constant region sequences, the entire contents of which are incorporated herein by reference. Further, this technology is also referenced in U.S. Patents 5,658,570, 5,693,780, and 5,756,096, the entire contents of which are incorporated herein by reference.

[0170] Antibodies can be prepared by a variety of methods known in the art, including phage display techniques utilizing antibody libraries derived from immunoglobulin sequences. See U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, the entire contents of each of which are incorporated herein by reference.

[0171] In some other embodiments, DNA encoding the desired monoclonal antibody can be isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding mouse antibody heavy and light chains). Isolated and subcloned hybridoma cells can be the source of such DNA. Once isolated, the DNA can be placed in an expression vehicle and then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or other non-immunoglobulin-producing myeloma cells. The isolated DNA (which may be synthetic as described herein) can be used to prepare antibody constant and variable region sequences, for example, as described in U.S. Patent 5,658,570, the entire contents of which are incorporated herein by reference. This method involves extracting RNA from selected cells, transforming it into cDNA, and then amplifying it by PCR using Ig-specific primers. Suitable probes for this purpose have been mentioned in U.S. Patent 5,658,570.

[0172] Furthermore, humanized, non-fully human antibodies can be constructed using conventional recombinant DNA techniques by inserting one or more CDRs of the antibody of the present invention into the frame regions, e.g., human frame regions. The frame regions may be naturally occurring or consensus frame regions, but human frame regions are preferred (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human frame regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of a target antigen produced by the combination of the frame regions and CDRs. One or more amino acid substitutions can be made within the frame regions, and amino acid substitutions that can improve antibody binding to the antigen can be selected. Furthermore, antibody molecules lacking one or more interchain disulfide bonds can be produced using this method by substituting or deleting cysteine ​​residues in one or more variable regions involved in interchain disulfide bond formation. Other modifications to polynucleotides within the skill of the art are also encompassed by the present invention.

[0173] Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing carriers and cell lines can be selected, constructed, and cultured using techniques known to those skilled in the art. All of these techniques are described in various laboratory manuals and major publications, such as *Recombiant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, F M Wurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which are incorporated herein by reference.

[0174] In some embodiments, antibody-encoding DNA can be designed and synthesized using conventional methods based on the antibody amino acid sequences described herein, inserted into an expression vehicle, and then transfected into host cells. The transfected host cells can be cultured in culture to produce monoclonal antibodies. In some embodiments, the expression vehicle contains at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and RNA splicing donor and acceptor sites on either side of the insertion sequence. Highly efficient transcription can be achieved using the early and late promoters of SV40, long terminal repeats from retroviruses, such as RSV, HTLV-1, HIV-1, and the early promoter of cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vehicles may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0175] In some embodiments, the inserted gene fragment must contain a screening marker; common screening markers include screening genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, and hygromycin resistance, which facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the gene and cultured in a selective medium; successfully transfected cells grow in large quantities and produce the desired target protein.

[0176] Furthermore, mutations, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions, can be introduced into the nucleotide sequences encoding the antibodies described herein by standard techniques known to those of skill in the art. Variants (including derivatives) encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original heavy and light chain variable regions. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

[0177] Treatment method The present invention further provides therapeutic methods and uses. In some embodiments, the present invention provides a method for treating or ameliorating various types of cancer, tumor, or related diseases, such as infectious diseases, comprising administering to a patient an effective dose of an anti-PD-L1 antibody or antigen-binding fragment. In some embodiments, the present invention provides the use of an anti-PD-L1 antibody or antigen-binding fragment for treating or ameliorating a related disease, such as cancer, tumor, or infectious disease. In some embodiments, the present invention provides the use of the anti-PD-L1 antibody or antigen-binding fragment in the preparation of a medicament for treating or ameliorating a related disease, such as cancer, tumor, or infectious disease. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody or derivative used, the patient's age and weight, general health, sex, diet, and administration time, excretion frequency, drug combination, and the severity of the particular disease being treated. These factors are within the skill of a medical professional to determine these factors. The dosage will further depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0178] Methods of administration of antibodies or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. Pharmaceutical compositions may be administered by any convenient route, such as infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in conjunction with other bioactive agents. Thus, pharmaceutical compositions comprising the antibodies or antigen-binding fragments of the invention can be administered orally, rectally, parenterally, intracisternally, vaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), buccally, or by oral or nasal spray.

[0179] The term "enterogastric" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0180] The administration mode may be systemic or local. Furthermore, the antibody of the present invention may need to be introduced into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection, which may be assisted by connecting an intraventricular catheter to a reservoir (which may be an Ommaya reservoir). Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and by using an atomized formulation.

[0181] The antibodies of the present invention can be administered locally to the area to be treated, including, but not limited to, local infusion during surgery, topical application in combination with a wound dressing after surgery, injection, catheter, suppository, or implant, which may be made of a porous, non-porous, or gel-like material, including a membrane (e.g., a silicone rubber membrane) or fiber. Preferably, when administering a protein (including an antibody) of the present invention, care must be taken to use a material that does not absorb the protein.

[0182] In some embodiments, compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody, which can be formulated as part of a suitable nucleic acid expression vehicle for in vivo administration of the nucleic acid to promote expression of the encoded protein, and then the partial vehicle can be administered intracellularly, for example, by using a retroviral carrier (see U.S. Patent No. 4,980,286), by direct injection, by using biolistic techniques (e.g., gene guns, Biolistic, Dupont), or by administering the nucleic acid coated in a liposome, cell surface receptor, or transfection reagent, or linked to a homeobox peptide known to enter the cell nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid can be introduced intracellularly by homologous recombination and incorporated into host cell DNA for expression.

[0183] In some embodiments, the dose of an antibody of the invention administered to a patient is 0.01 mg / kg to 100 mg / kg of the patient's body weight, or 0.1 mg / kg to 20 mg / kg of the patient's body weight. An initial dose can be followed by a second or multiple doses of the antibody or antigen-binding fragment at approximately the same or lower doses, with subsequent doses spaced at least one to three days apart, or at least one week apart. For example, the dose and frequency of administration of an antibody of the invention can be reduced by improving antibody uptake and tissue penetration (e.g., entry into the brain) through modifications such as liposomalization.

[0184] A variety of known delivery systems can be used to administer the antibodies or derivatives of the invention or polynucleotides encoding them, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), or constructing the nucleic acid as part of a retrovirus or other carrier.

[0185] Combination therapy In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments of the invention can be combined with other therapeutic or prophylactic regimens, including the administration of one or more antibodies or antigen-binding fragments of the invention in combination with or in combination with one or more other therapeutic agents or methods. In some embodiments, the other therapeutic regimens include, but are not limited to, radiation therapy, chemotherapy, hormone therapy, etc. In the case of combination therapy, the antibody can be administered simultaneously with the other therapeutic agent or separately. In the case of separate administration, the antibody of the invention can be administered before or after the administration of the other therapeutic agent.

[0186] In some embodiments, the antibodies of the invention are administered in combination with a chemotherapeutic agent. In some embodiments, chemotherapeutic agents that can be administered with the antibodies of the invention include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouridine, interferon α-2b, glutamic acid, mithramycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate). Antibodies include, but are not limited to, steroids and combinations thereof (e.g., betamethasone sodium phosphate), steroids (e.g., steroids), steroids for treating rheumatoid arthritis (e.g., steroids for rheumatoid arthritis), steroids for ...

[0187] In some embodiments, the anti-PD-L1 antibodies of the invention are administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include immunotherapeutic agents, including, but not limited to, therapeutic antibodies used in the treatment of patients. Some examples of therapeutic antibodies include rituximab, trastuzumab, tositumomab, icomab, alemtuzumab, epratuzumab, bevacizumab, toximab, and belentuzumab.

[0188] Pharmaceutical Composition The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an anti-PD-L1 antibody or antigen-binding fragment and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1% to 90% of the anti-PD-L1 antibody or antigen-binding fragment. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0189] In some embodiments, the term "pharmaceutically acceptable" refers to a substance approved by a government regulatory agency or listed in other generally recognized pharmacopeias for animal, and particularly human, use. Furthermore, a "pharmaceutically acceptable excipient" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid, etc.

[0190] The term "excipient" refers to a diluent, adjuvant, excipient, or carrier with which an active ingredient can be administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, plant, animal, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, nonfat dry milk, glycerin, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol and methyl p-hydroxybenzoate, antioxidants such as ascorbic acid and sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity such as sodium chloride and dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions may also be prepared as suppositories with conventional adhesives and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable drug carriers are described in E.W. Martin's Remington's Pharmaceutical Sciences, incorporated herein by reference. Such compositions contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, combined with appropriate quantities of excipients to provide a dosage form suitable for the patient. The formulation should be adapted to the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0191] In some embodiments, the composition is prepared into a pharmaceutical composition suitable for intravenous injection into humans using conventional procedures. Compositions for intravenous administration are generally solutions in sterile, water-permeable buffer solutions. The composition may further include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Typically, the active ingredients are provided singly or in admixture in unit dosage form, for example, in a hermetically sealed container (e.g., an ampoule or sachet) containing an indication of the amount of active agent, such as a dry, lyophilized powder or water-free concentrate. When administering the composition by injection, the composition may be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline. When administering the composition by injection, an ampoule of sterile water for injection or saline may be used to allow the active ingredients to be mixed prior to administration.

[0192] The compounds of the present invention may be prepared in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Example]

[0193] The technical solutions of the present invention will be further described below through specific examples, but the specific examples do not limit the scope of protection of the present invention. Any insubstantial modifications or adjustments made by those skilled in the art based on the spirit of the present invention still fall within the scope of protection of the present invention.

[0194] Materials, reagents and the like used in the examples described below are all available from commercial sources unless otherwise specified.

[0195] Anti-PD-L1 antibody

[0196] Example 1: Antibody Preparation Method The amino acid and nucleic acid sequences associated with exemplary antibodies are shown in Tables 1-13, with the heavy and light chain structures of the antibodies shown in Table 1. The Fc region of the heavy chain in Table 11 is underlined, and the nucleic acid sequences of the variable regions in Table 13 are underlined.

[0197] The heavy and light chain amino acid sequences of the antibody were optimized according to the codon bias of the host cell to obtain the heavy and light chain DNA sequences. Signal peptides were added to the heavy and light chain sequences, respectively, to facilitate expression in the host cell.

[0198] The optimized and synthesized sequence clones were cloned into a carrier (optional carrier: pCDNA3.1 TM (+), Invitrogen, product number V79020), and then large amounts of plasmid were extracted, and the heavy and light chains were transiently expressed in HEK293F cells transfected at a 1:1 molar ratio of the plasmids. Using antibody L1-R2-4-46 as an example, the heavy chain nucleic acid sequence (SEQ ID NO: 99) and the light chain nucleic acid sequence (SEQ ID NO: 101) were cloned into an expression carrier by enzymatic cleavage, where the heavy chain signal peptide nucleic acid sequence was SEQ ID NO: 102 and the light chain signal peptide nucleic acid sequence was SEQ ID NO: 103. Using antibody L1-R2-4-71 as an example, the heavy chain nucleic acid sequence (SEQ ID NO: 100) and the light chain nucleic acid sequence (SEQ ID NO: 101) were cloned into an expression carrier by enzymatic cleavage, where the heavy chain signal peptide nucleic acid sequence was SEQ ID NO: 102 and the light chain signal peptide nucleic acid sequence was SEQ ID NO: 103. After cell expression, the secreted antibody protein containing two heavy chains and two light chains was purified from the culture medium by immobilized metal affinity chromatography (IMAC) using a Protein A column (GE Healthcare) to achieve an antibody purity of >95%. The sequencing results were identical to the predicted sequences and were used for affinity detection and biological activity identification.

[0199] [Table 1]

[0200] Table 2

[0201] Table 3

[0202] Table 4

[0203] Table 5

[0204] Table 6

[0205] Table 7

[0206] Table 8A Table 8B

[0207] Table 9

[0208] Table 10

[0209] Table 11A [Table 11B] [Table 11C] [Table 11D] [Table 11E] [Table 11F]

[0210] [Table 12A] [Table 12B]

[0211] [Table 13A] [Table 13B] [Table 13C]

[0212] Example 2 Measurement of dissociation constant The equilibrium dissociation constants (KD) of the above antibodies of the present invention binding to their corresponding antigens were determined using a Biacore system (GE) by kinetic binding assay. According to the manual, 5 μg of antigen protein PD-L1-His was immobilized on a chip, and the affinity was measured using anti-PD-L1 antibodies with a maximum concentration of 100 nM (1:2 dilution, 5 gradients) as the mobile phase. The results were analyzed using Biacore T200 Evaluation Software. The positive control was an ATE antibody (the amino acid sequence of the ATE antibody was identical to that of Atezolizumab expressed by HEK293F cells). Preparation of PD-L1-His antigen: PD-L1 recombinant protein was constructed by adding an 8xHIS tag to the C-terminus of the human PD-L1 extracellular domain (ECD) protein. The ECD region of human PD-L1 was genetically synthesized and subcloned into a mammalian expression vector (e.g., pcDNA3.1(+) expression vector). HEK293F cells were transiently transfected, and the PD-L1-His antigen was purified using nickel-based immobilized metal affinity chromatography (GE Healthcare). The amino acid sequence of the human PD-L1 extracellular domain (ECD) was MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNM TIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (sequence number 98).

[0213] 1) As shown in Table 14, the affinities of the antibodies of the present invention L1-R2-4, L1-R2-18, L1-R2-78 and L1-R2-89 for the PD-L1 antigen are substantially similar compared to the ATE antibody.

[0214] [Table 14]

[0215] 2) As shown in Table 15, the antibodies of the present invention L1-R2-4-36, L1-R2-4-46, L1-R2-4-47, L1-R2-4-55, and L1-R2-4-71 have higher affinity for the PD-L1 antigen compared to the ATE antibodies.

[0216] [Table 15]

[0217] Example 3 Detection of anti-PD-L1 antibody activity Anti-PD-1 / PD-L1 antibodies can block the binding of PD-1 to PD-L1, thereby relieving the inhibitory effect on the downstream NFAT signaling pathway. The Mechanisms of Action (MOA) detection system (PD-1 / PD-L1 Blockade Bioassay, Propagation Model, Catalog J1252) provided by Promega was used to detect fluorescent reporter gene expression, reflecting the activation state of NFAT signaling, according to the procedure described in the manual. The negative control antibody IgG isotype was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (catalog number: HG1K).

[0218] CHO-PD-L1 cells (from the MOA detection system described above) were seeded the day before activity detection. CHO-PD-L1 cells were passaged 1-2 days before seeding, the culture supernatant was discarded, and the cells were washed once with sterile PBS. An appropriate amount of Trypsin (Gibco) was added and digested for 3-5 minutes in a 37°C, 5% CO2 incubator. Fresh medium was added to stop the digestion, and the cells were transferred to a 50 mL centrifuge tube and counted. The required volume of cells was collected and centrifuged at 900 rpm for 5 minutes. DMEM-F12 medium (Gibco) was added, and the cells were diluted to 4 x 10 5 The cells were resuspended at 100 cells / mL. The cells were added to a 96-well white cell culture plate (Corning) at 100 μL / well. The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0219] On the day of detection, Jurkat-PD1 cells (from the MOA detection system described above) were processed: after counting, the required volume of cells was collected and centrifuged at 900 rpm for 5 min. 1.25 × 10 cells were diluted with measurement buffer (1640 medium (Gibco) + 1% FBS). 6The cells were resuspended at 37°C / mL and ready for use. 95μL of the culture supernatant from the CHO-PD-L1 cell culture plate was aspirated and removed per well, and 40μL of test sample (the above anti-PD-L1 antibody, ATE antibody, or negative control HG1K antibody, with a maximum concentration of 20μg / mL, diluted 2-fold in assay buffer for a total of 9 gradients) was added, followed by 40μL of Jurkat-PD1 cells. The cells were gently shaken to mix evenly, and then cultured in an incubator at 37°C, 5% CO2 for 6 hours. Detection: ONE-Glo TM The Luciferase Assay System (purchased from Promega, E6120) was pre-thawed. After 6 hours, the ONE-Glo TM The reagent was added at 50 μL / well, and the plate was left at room temperature for 5 to 10 minutes before reading the value.

[0220] 1) As shown in Figure 1A, the anti-PD-L1 antibodies used in this example (L1-R2-4, L1-R2-6, L1-R2-18, L1-R2-78, L1-R2-85, and L1-R2-89) can all effectively block PD-1 / PD-L1 interaction, with the L1-R2-4 antibody exhibiting stronger blocking activity.

[0221] 2) As shown in Figure 1B, the antibodies L1-R2-4-36, L1-R2-4-46, L1-R2-4-47, L1-R2-4-55, and L1-R2-4-71 of the present invention can all effectively block PD-1 / PD-L1 interaction, and compared to the control antibody ATE, the antibodies (L1-R2-4-36 and L1-R2-4-46) have stronger blocking activity.

[0222] Example 4 Detection of anti-PD-L1 antibody binding ability The binding of the anti-PD-L1 antibodies of the present invention to CHO cells overexpressing human PD-L1 was detected by flow cytometry. Test procedure: CHO-PD-L1 cells (from the MOA detection system described above) were uniformly mixed with different dilutions of antibody (the highest working antibody concentration was 20 nM, diluted at a dilution ratio of 1:2, with an 11-fold gradient) and incubated on ice for 1 hour. The cells were washed twice with PBS, and then a fluorescent secondary antibody, a PE-labeled anti-human Fc antibody (Jackson ImmunoResearch), was added and incubated on ice in the dark for 30 minutes. The cells were washed twice with PBS and resuspended in PBS, and the binding of the antibody to the cells was detected by FACS.

[0223] As shown in Figure 2, the anti-PD-L1 antibodies of the invention (L1-R2-4-36, L1-R2-4-46, L1-R2-4-47, L1-R2-4-55, and L1-R2-4-71) can all bind to human PD-L1 overexpressed in CHO cells.

[0224] Example 5 Antitumor activity Experimental mice: Female C57BL / 6 mice (5-8 weeks old) (Jiangsu Jisui Yaokang Biotechnology Co., Ltd.). Experimental cells: Murine colon cancer cells MC38 in the logarithmic growth phase were harvested, the culture medium was removed, and the cells were washed twice with PBS. 1 × 10 6 The mice were inoculated into the right flank area at an inoculum volume of 100 μL per mouse. 3 In this case, tumor-bearing mice were randomly divided into groups. Criteria for grouping: tumor volume CV value less than 30%. The day of inoculation was defined as D0, and administration began on the day of grouping according to the experimental protocol design. The dosage and administration method are shown in Table 16. After cell inoculation, the effects of tumors on the normal behavior of animals were monitored regularly every week. Specific details: activity of experimental animals, feeding and drinking status, weight gain and loss, eyes, hair coat, and other abnormal conditions. Any clinical symptoms observed during the test were recorded in the raw data. After the start of administration, body weight was measured twice a week. Tumor volume was measured twice a week. Tumor volume calculation method: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2Monitoring ended after 35 days. The relative tumor inhibition rate (TGI%) was calculated 35 days after inoculation. Calculation formula: TGI% = (1 - mean relative tumor volume of treatment group / mean relative tumor volume of vehicle group) × 100%.

[0225] The results of tumor inhibition are shown in Figures 3A and 3B and Table 17. At day 35 after inoculation, the tumor inhibition rate of the L1-R2-4-71 antibody at a single dose of 5 mg / kg was approximately 70.89%, and tumor weight was reduced by approximately 61.81% compared to the isotype IgG1 group. The antibody of the present invention exhibited significant antitumor activity one week after administration, and tumors in two mice in the antibody of the present invention group (G2 group) completely regressed by day 35. As shown in Figure 3C, there was no significant difference in mouse weight.

[0226] [Table 16]

[0227] [Table 17]

[0228] Example 6 Yeast-mediated display of anti-PD-L1 antibodies in scFv format The nucleic acid sequence encoding the scFv fragment (including the heavy chain variable region, light chain variable region, and heavy-light chain variable region linker (G4S)3) was synthesized and inserted into pYD1 carrier (Addgene) using restriction site endonuclease enzymes. The resulting clones were then electroporated into Saccharomyces cerevisiae hosts (e.g., Invitrogen, EBY100) to obtain positive yeast clones. The resulting positive yeast clones were selected and cultured as monoclonal clones. The heavy and light chain variable regions of the scFv expressed on the surface of the ATE-positive yeast clones were identical to those of atezolizumab. The scFvs expressed on the surface of the other positive yeast clones are listed in Table 18. The amino acid sequence of the linker was GGGGSGGGSGGGGS (SEQ ID NO: 106), and its nucleic acid sequence was GGTGGAGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG (SEQ ID NO: 107). Taking CP11-46 as an example, the nucleic acid sequences of its VH and VL are shown in Table 13. Taking CP11-71 as an example, the nucleic acid sequences of its VH and VL are shown in Table 13.

[0229] [Table 18]

[0230] Example 7 Antigen-binding ability of yeast-displayed anti-PD-L1 scFv The binding ability of the yeast-displayed anti-PD-L1 scFv to the antigen protein PD-L1-His-Biotin was detected by flow cytometry. The yeast cells were incubated with 1 nM PD-L1-His-Biotin antigen protein at room temperature for 1 hour, washed three times with PBS, pH 7.4, and then incubated with Streptavidin-PE fluorescent secondary antibody at room temperature for 30 minutes. After washing three times with PBS, pH 7.4, fresh PBS was added and the cells were ready for flow cytometry.

[0231] Preparation of PD-L1-His-Biotin: Purified PD-L1-His antigen protein was added to 26.6 μL of 10 nM biotin (Sigma-Aldrich, B4501-1G) prepared in DMSO per mg of protein, incubated at room temperature for 2 h, and then dialyzed against PBS, pH 7.4.

[0232] As shown in Figure 4, the binding ability of the anti-PD-L1 scFvs of the invention (CP11-36, CP11-46, CP11-71, CP14-16, and CP14-80) to PD-L1-His-Biotin antigen protein was clearly superior to that of the positive control ATE scFV, and the binding ability of the anti-PD-L1 scFvs of the invention (CP-11-47, CP-11-55, CP21-8, CP21-47, and CP22-34) to PD-L1-His-Biotin antigen protein was substantially the same as that of the positive control ATE scFV.

Claims

1. An antibody or antigen-binding fragment that specifically binds to PD-L1, The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 8, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 11, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 20, an LCDR2 set forth in SEQ ID NO: 25, and an LCDR3 set forth in SEQ ID NO: 29; or the antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 21, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 30; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 22, an LCDR2 set forth in SEQ ID NO: 27, and an LCDR3 set forth in SEQ ID NO: 31; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 23, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 30; or the antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 26, and an LCDR3 set forth in SEQ ID NO: 31; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 9, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 10, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 7, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 12, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 13, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 14, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 15, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO: 28; or The antibody or antigen-binding fragment comprises at least an HCDR1 set forth in SEQ ID NO: 3, an HCDR2 set forth in SEQ ID NO: 16, an HCDR3 set forth in SEQ ID NO: 17, an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 24, and an LCDR3 set forth in SEQ ID NO:

28. An antibody or antigen-binding fragment thereof.

2. the heavy chain of the antibody or antigen-binding fragment further comprises FR1, FR2, FR3, FR4, wherein heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 32, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 32, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 32; and / or the heavy chain FR2 of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 33, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 33, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 33; and / or the heavy chain FR3 of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 34, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 34, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 34; and / or the heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 35, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 35, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 35; 2. The antibody or antigen-binding fragment of claim 1.

3. heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 32, heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 33, heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 34, and heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 35; The antibody or antigen-binding fragment of claim 2.

4. the light chain of the antibody or antigen-binding fragment further comprises FR1, FR2, FR3, FR4, wherein light chain FR1 comprises the sequence set forth in any one of SEQ ID NOs: 51-56, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 51-56, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NOs: 51-56; and / or the light chain FR2 of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 57-60, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 57-60, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 57-60; and / or the light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 61-64, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 61-64, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 61-64; and / or the light chain FR4 of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 65-67, a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 65-67, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 65-67; The antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:51, light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:57, light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:61, and light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:65; or light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 52, light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 58, light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 62, and light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 66; or light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:53, light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59, light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:63, and light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67; or light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:54, light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:60, light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:64, and light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:67; or light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 55, light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 59, light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 63, and light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 67; or a light chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 56, a light chain FR2 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 57, a light chain FR3 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 61, and a light chain FR4 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 67; The antibody or antigen-binding fragment of claim 4.

6. the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 36-45, 47, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 36-45, 47, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 36-45, 47; and / or the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 68-73, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 68-73, or a sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 68-73; The antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 36 to 45, and 47, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 68 to 73; The antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 36-45, 47, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs: 68-73; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 69; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 70; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 71; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 72; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 73; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:37 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:38 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:39 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:40 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:41 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:42 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:43 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:44 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:45 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:47, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:68; The antibody or antigen-binding fragment according to any one of claims 1 to 7.

9. the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 74 or 75, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 74 or 75, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 74 or 75; and / or the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 76, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 76; The antibody or antigen-binding fragment according to any one of claims 1 to 8.

10. the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76; or the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 75, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 76; The antibody or antigen-binding fragment according to any one of claims 1 to 9.

11. the heavy chain of said antibody comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 77 to 86, 88, a sequence having at least 80% identity to the sequence as set forth in any one of SEQ ID NOs: 77 to 86, 88, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence as set forth in any one of SEQ ID NOs: 77 to 86, 88; and / or the light chain of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 92-97, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 92-97, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 92-97; The antibody or antigen-binding fragment according to any one of claims 1 to 10.

12. An antibody or antigen-binding fragment that specifically binds to PD-L1, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 93; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 94; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 95; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 96; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 77 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 97; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 78 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 79 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 80 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 81 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 86 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; or the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 88, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 92; An antibody or antigen-binding fragment thereof.

13. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 12 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising:

14. Encoding the antibody or antigen-binding fragment of any one of claims 1 to 12. A nucleic acid molecule characterized by:

15. 15. The nucleic acid molecule of claim 14, A carrier or host cell characterized in that:

16. A drug for treating or ameliorating a T cell dysfunction disease, comprising the antibody or antigen-binding fragment described in any one of claims 1 to 12.

17. The T cell dysfunction disease includes, but is not limited to, infectious diseases caused by bacteria, viruses, fungi or protozoa, and cancers and tumors selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head cancer, neck cancer, and pancreatic cancer; The drug of claim 16.

18. Comprising the antibody or antigen-binding fragment of any one of claims 1 to 12. A diagnostic or prognostic reagent kit comprising:

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