Fusion protein and application thereof

Abstract

The invention provides a fusion protein and application thereof. The fusion protein comprises an anti-PD-L1 antibody or an antigen binding fragment, IL-15 or a fragment thereof, and IL-15R alpha or a sushi structural domain thereof. The fusion protein can be used for preventing, treating or improving diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a fusion protein and its applications. Background Art

[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 members. As a ligand of PD-1, PD-L1 is also a type I transmembrane glycoprotein, which is widely distributed: expressed on the surface of antigen-presenting cells such as B cells, T cells, dendritic cells, macrophages, and in tumor tissues.

[0003] The interaction between PD-1 and PD-L1 negatively regulates antigen receptor signal transduction and weakens T cell responses. So far, a large number of studies have shown that the interaction between PD-1 and PD-L1 leads to a reduction in lymphocytes infiltrating tumors, a reduction in T cell receptor-mediated proliferation, and immune escape of cancer cells. Blocking the interaction between PD-1 and PD-L1 can increase T cell proliferation and cytokine production, and enhance the immunity of tumor-specific CD8 + T cells, which helps the immune system to eliminate tumor cells.

[0004] IL-15 is a 14-15 kDa glycoprotein with 114 amino acids and belongs to the common cytokine receptor γ chain family, which also includes IL-2, IL-4, IL-7, IL-9, IL-21. IL-15 is secreted by macrophages, dendritic cells, and monocytes. IL-15 can stimulate central memory CD8 + cells to exert immunity, and has no regulatory effect on other T cells. In addition, IL-15 can activate NK cells and effector and memory CD8 + T cells and can rescue T cells from apoptosis induced by regulatory T cells (Tregs). The IL-15 receptor consists of the following three subunits: IL-15Rα, IL-15Rβ, and IL-15Rγ. Before binding to the functional IL-15Rβ and γ units on T cells and NK cells, IL-15 typically forms a complex with the IL-15 receptor α expressed on APCs. The sushi domain (7.5 kDa) of IL-15Rα plays a key role in the formation of the complex between IL-15 and IL-15Rα. Summary of the Invention

[0005] The present invention provides a fusion protein and its applications, and the fusion protein comprises an anti-PD-L1 antibody or antigen-binding fragment, IL-15 or its fragment, and IL-15Rα or its sushi domain.

[0006] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises:

[0007] (a) HCDR1, which comprises DSWIH; and / or

[0008] (b) HCDR2, which comprises WISPYGGSTYYADX 1 X 2 X 3 X 4 (SEQ ID NO:86), where X 1 is S, D, H, G, P or Y, X 2 is V, F, L, M or Y, X 3 is K, R, G, S, V or H, X 4 is G, H, D, Q, S or A; and / or

[0009] (c) HCDR3, which comprises RHWPGGX 5 X 6 X 7 (SEQ ID NO:87), where X 5 is F or L, X 6 is D or L, X 7 is Y or P.

[0010] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises:

[0011] (a) HCDR1, which comprises DSWIH;

[0012] (b) HCDR2, which comprises WISPYGGSTYYADX 1 X 2 X 3 X 4 (SEQ ID NO:86), where X 1 is S, D, H, G, P or Y, X 2 is V, F, L, M or Y, X 3 is K, R, G, S, V or H, X 4 is G, H, D, Q, S or A; and

[0013] (c) HCDR3, which comprises RHWPGGX 5 X 6 X 7 (SEQ ID NO:87), where X 5 is F or L, X 6 is D or L, X 7 is Y or P.

[0014] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises:

[0015] (a) HCDR1, which comprises DSWIH; and / or

[0016] (b) HCDR2, which comprises WISPYGGSTYYADX 1 X 2 X 3 X 4 (SEQ ID NO:86), where X 1 is S, D, H, G, P or Y, and X 2 is V, F, L, M or Y, and X 3 is K, R, G, S, V or H, and X 4 is G, H, D, Q, S or A; and / or

[0017] (c) HCDR3, which comprises RHWPGGX 5 X 6 X 7 (SEQ ID NO:87), where X 5 is F or L, and X 6 is D or L, and X 7 is Y or P; and / or

[0018] (d) LCDR1, which comprises X 8 ASQX 9 IX 10 X 11 X 12 LX 13 (SEQ ID NO:88), where X 8 is L, Q or R, and X 9 is D, T or G, and X 10 is G or S, and X 11 is K, T, or S, and X 12 is H, W, F or Y, and X 13 is N or A; and / or

[0019] (e) LCDR2, which comprises X 14 ASX 15 LX 16 X 17 (SEQ ID NO:89), where X 14 is A or G, and X 15 is T, N, S or R, and X 16 is Q or K, and X 17 is S or T; and / or

[0020] (f) LCDR3, which comprises QQX18 X 19 X 20 TPX 21 T (SEQ ID NO:90), X 18 is Y or S, X 19 is Y or F, X 20 is S or T, X 21 is R or Y.

[0021] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises:

[0022] (a) HCDR1, which comprises DSWIH;

[0023] (b) HCDR2, which comprises WISPYGGSTYYADX 1 X 2 X 3 X 4 (SEQ ID NO:86), X 1 is S, D, H, G, P or Y, X 2 is V, F, L, M or Y, X 3 is K, R, G, S, V or H, X 4 is G, H, D, Q, S or A;

[0024] (c) HCDR3, which comprises RHWPGGX 5 X 6 X 7 (SEQ ID NO:87), X 5 is F or L, X 6 is D or L, X 7 is Y or P;

[0025] (d) LCDR1, which comprises X 8 ASQX 9 IX 10 X 11 X 12 LX 13 (SEQ ID NO:88), X 8 is L, Q or R, X 9 is D, T or G, X 10 is G or S, X 11 is K, T, or S, X 12 is H, W, F or Y, X 13 is N or A;

[0026] (e) LCDR2, which comprises X 14 ASX 15 LX 16 X17 (SEQ ID NO:89), X 14 is A or G, X 15 is T, N, S or R, X 16 is Q or K, X 17 is S or T; and

[0027] (f) LCDR3, which comprises QQX 18 X 19 X 20 TPX 21 T (SEQ ID NO:90), X 18 is Y or S, X 19 is Y or F, X 20 is S or T, X 21 is R or Y.

[0028] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:2-11 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12 or 13 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, the substitution variant is a conservative amino acid substitution variant.

[0029] In some embodiments, HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:91-95 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:2-11 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12 or 13 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, the substitution variant is a conservative amino acid substitution variant.

[0030] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0031] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0032] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0033] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0034] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0035] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0036] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:8, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0037] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0038] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0039] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0040] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:13.

[0041] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0042] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0043] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0044] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0045] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0046] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0047] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:8, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0048] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:91, 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:12.

[0049] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:91, 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:12.

[0050] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:91, 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:12.

[0051] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:92, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0052] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:93, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0053] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:94, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0054] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:95, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0055] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:91, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:13.

[0056] In some embodiments, LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-18 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 19-22 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 23-26 or a variant thereof having a single-site substitution, deletion or insertion. In some embodiments, the substitution variant is a conservative amino acid substitution variant.

[0057] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 23.

[0058] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24.

[0059] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0060] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0061] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0062] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0063] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises: (a) HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof having a single-site substitution, deletion or insertion; and / or (b) HCDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-11 or a variant thereof having a single-site substitution, deletion or insertion; and / or (c) HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 12 or 13 or a variant thereof having a single-site substitution, deletion or insertion; and / or (d) LCDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-18 or a variant thereof having a single-site substitution, deletion or insertion; and / or (e) LCDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 19-22 or a variant thereof having a single-site substitution, deletion or insertion; and / or (f) LCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 23-26 or a variant thereof having a single-site substitution, deletion or insertion.

[0064] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises at least one, two, three, four, five or all of HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in any one of SEQ ID NOs: 2-11, HCDR3 shown in SEQ ID NO: 12 or 13, LCDR1 shown in any one of SEQ ID NOs: 14-18, LCDR2 shown in any one of SEQ ID NOs: 19-22, and LCDR3 shown in any one of SEQ ID NOs: 23-26.

[0065] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in any one of SEQ ID NOs: 2-11, HCDR3 shown in SEQ ID NO: 12 or 13, LCDR1 shown in any one of SEQ ID NOs: 14-18, LCDR2 shown in any one of SEQ ID NOs: 19-22, and LCDR3 shown in any one of SEQ ID NOs: 23-26.

[0066] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment specifically binds to PD-L1 and comprises: (a) HCDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 91-95 or a variant thereof with a single-site substitution, deletion, or insertion; and / or (b) HCDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-11 or a variant thereof with a single-site substitution, deletion, or insertion; and / or (c) HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 12 or 13 or a variant thereof with a single-site substitution, deletion, or insertion; and / or (d) LCDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-18 or a variant thereof with a single-site substitution, deletion, or insertion; and / or (e) LCDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 19-22 or a variant thereof with a single-site substitution, deletion, or insertion; and / or (f) LCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 23-26 or a variant thereof with a single-site substitution, deletion, or insertion.

[0067] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises at least one, two, three, four, five, or all of HCDR1 shown in any one of SEQ ID NOs: 91-95, HCDR2 shown in any one of SEQ ID NOs: 2-11, HCDR3 shown in SEQ ID NO: 12 or 13, LCDR1 shown in any one of SEQ ID NOs: 14-18, LCDR2 shown in any one of SEQ ID NOs: 19-22, and LCDR3 shown in any one of SEQ ID NOs: 23-26.

[0068] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in any one of SEQ ID NOs: 91-95, HCDR2 shown in any one of SEQ ID NOs: 2-11, HCDR3 shown in SEQ ID NO: 12 or 13, LCDR1 shown in any one of SEQ ID NOs: 14-18, LCDR2 shown in any one of SEQ ID NOs: 19-22, and LCDR3 shown in any one of SEQ ID NOs: 23-26.

[0069] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0070] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:24.

[0071] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25.

[0072] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:17, LCDR2 shown in SEQ ID NO:22, and LCDR3 shown in SEQ ID NO:26.

[0073] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25.

[0074] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:26.

[0075] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:3, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0076] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0077] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0078] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:6, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0079] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0080] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0081] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:24.

[0082] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25.

[0083] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:17, LCDR2 shown in SEQ ID NO:22, and LCDR3 shown in SEQ ID NO:26.

[0084] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25.

[0085] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:26.

[0086] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:3, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0087] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0088] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0089] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:6, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0090] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23.

[0091] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0092] In some embodiments, the heavy chain variable region comprises the structure: heavy chain FR1-HCDR1-heavy chain FR2-HCDR2-heavy chain FR3-HCDR3-heavy chain FR4.

[0093] In some embodiments, the light chain variable region comprises the structure: light chain FR1-LCDR1-light chain FR2-LCDR2-light chain FR3-LCDR3-light chain FR4.

[0094] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 27-41.

[0095] In some embodiments, the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-47, or an amino acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 42-47, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 42-47.

[0096] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-41, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-47.

[0097] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0098] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 43.

[0099] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 44.

[0100] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 45.

[0101] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 46.

[0102] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 47.

[0103] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0104] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0105] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0106] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0107] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0108] 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 κ or λ chain constant region. In some embodiments, the antibody or fragment thereof is of one isotype of 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 human antibody.

[0109] In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications relative to the parental Fc region, such as substitutions, deletions, or insertions. In some embodiments, the amino acid modification of the Fc region alters the effector function activity relative to the parental Fc region activity. In some embodiments, the variant Fc region can have altered (i.e., increased or decreased) antibody-dependent cell cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In some embodiments, the amino acid modification of the Fc region can alter the affinity of the variant Fc region for FcγR (Fcγ receptor) relative to the parental Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A.

[0110] 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 a scFv, Fab, F(ab) 2 or IgG. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody. In one embodiment, the antigen-binding fragment is a Fab, Fv, or scFv antibody.

[0111] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0112] In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 90% identity to the sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 48 or 49; and / or

[0113] the light chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 90% identity to the sequence shown in SEQ ID NO: 50, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 50.

[0114] In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 50.

[0115] In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 50.

[0116] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region a (CHa), a heavy chain constant region b (CHb), and a light chain constant region (CL).

[0117] In some embodiments, the heavy chain constant region a and the heavy chain constant region b form a stable association of "Knobs-into-Holes".

[0118] In some embodiments, the heavy chain constant region a and / or the heavy chain constant region b comprises an amino acid mutation selected from Y349C, S354C, T366W, T366S, L368A, and Y407V, wherein the amino acid positions are according to the Eu numbering.

[0119] In some embodiments, the heavy chain constant region a and / or the heavy chain constant region b comprises the following amino acid mutation: K447A, wherein the amino acid position is according to the Eu numbering.

[0120] In some embodiments, the heavy chain constant region a comprises an amino acid mutation selected from S354C and T366W; and / or the heavy chain constant region b comprises an amino acid mutation selected from Y349C, T366S, L368A, and Y407V; wherein the amino acid positions are according to the Eu numbering.

[0121] In some embodiments, the heavy chain constant region a comprises the following amino acid mutations: S354C and T366W; and / or the heavy chain constant region b comprises the following amino acid mutations: Y349C, T366S, L368A, and Y407V; wherein the amino acid positions are according to the Eu numbering.

[0122] In some embodiments, the heavy chain constant region a comprises the following amino acid mutations: S354C and T366W; the heavy chain constant region b comprises the following amino acid mutations: Y349C, T366S, L368A, and Y407V; wherein the amino acid positions are according to the Eu numbering.

[0123] In some embodiments, the heavy chain constant region a comprises the following amino acid mutations: S354C, T366W, and K447A; and / or the heavy chain constant region b comprises the following amino acid mutations: Y349C, T366S, L368A, Y407V, and K447A; wherein the amino acid positions are according to the Eu numbering.

[0124] In some embodiments, the heavy chain constant region a comprises the following amino acid mutations: S354C, T366W, and K447A; the heavy chain constant region b comprises the following amino acid mutations: Y349C, T366S, L368A, Y407V, and K447A; wherein the amino acid positions are according to the Eu numbering.

[0125] In some embodiments, the heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:77; and / or

[0126] the heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:78, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:78; and / or

[0127] the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:50.

[0128] In some embodiments, the heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:77; the heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:78, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:78; the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:50.

[0129] In some embodiments, the heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, the heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50.

[0130] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain and a light chain.

[0131] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 51-65; and / or

[0132] the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 66-71, or an amino acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 66-71, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 66-71.

[0133] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 66.

[0134] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 67.

[0135] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 68.

[0136] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 69.

[0137] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO: 70.

[0138] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:71.

[0139] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:66.

[0140] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:66.

[0141] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:66.

[0142] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:66.

[0143] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:66.

[0144] In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibodies), a polyclonal antibody, or a multispecific antibody or antigen-binding fragment (e.g., a bispecific antibody or antigen-binding fragment).

[0145] In some embodiments, the anti-PD-L1 antibody comprises heavy chain a, heavy chain b, and a light chain.

[0146] In some embodiments, the heavy chain a comprises the amino acid sequence shown in SEQ ID NO:79, or an amino acid sequence having at least 90% identity to the sequence shown in SEQ ID NO:79, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:79; and / or

[0147] The heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 80, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 80; and / or

[0148] The light chain comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 66, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 66.

[0149] In some embodiments, the heavy chain a comprises the amino acid sequence shown in SEQ ID NO: 79, the heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 80, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 66.

[0150] In some embodiments, the IL-15 comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 82, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 82.

[0151] In some embodiments, the IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 81, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 81.

[0152] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment is linked to IL-15 or a fragment thereof and IL-15Rα or its sushi domain via a linker.

[0153] In some embodiments, the C-terminus of one heavy chain of the anti-PD-L1 antibody is linked to IL-15 or a fragment thereof via a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is linked to IL-15Rα or its sushi domain via a linker.

[0154] In some embodiments, the linker is a GS linker. In some embodiments, the linker is independently selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3, or any combination thereof. In some embodiments, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4, 5, or 6, and n is 1, 2, 3, 4, or 5.

[0155] In some embodiments, the fusion protein comprises a first polypeptide, a second polypeptide, and a third polypeptide; wherein

[0156] the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:83, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:83; and / or

[0157] the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:84, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:84; and / or

[0158] the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:66, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:66.

[0159] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66.

[0160] In one embodiment, the fusion protein is an isolated fusion protein. The present invention also provides a polynucleotide encoding the fusion protein or a portion thereof. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0161] The present invention also provides a vector comprising the polynucleotide. In some embodiments, the vector is an isolated vector. In some embodiments, the vector is a nucleic acid fragment, plasmid, phage, or virus.

[0162] The present invention also provides a host cell comprising the polynucleotide or vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a HEK cell (such as HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell or a murine L cell.

[0163] The present invention also provides a pharmaceutical composition comprising the fusion protein described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0164] The present invention also provides methods of treatment and uses. In some embodiments, methods for preventing, treating or ameliorating a disease are provided, the methods comprising administering to a patient an effective amount of the fusion protein or pharmaceutical composition described herein. In some embodiments, the use of the fusion protein or pharmaceutical composition described herein in preventing, treating or ameliorating a disease is provided. In some embodiments, the use of the fusion protein or pharmaceutical composition described herein in the preparation of a medicament for preventing, treating or ameliorating a disease is provided.

[0165] In some embodiments, the disease includes but is not limited to infections (such as infections caused by bacteria, viruses, fungi or protozoa), autoimmune diseases, cancers, tumors. In some embodiments, the autoimmune diseases include but are not limited to alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, Wegener's granulomatosis. In some embodiments, the cancers and tumors include but are 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, pancreatic cancer.

[0166] The anti-PD-L1 antibody of the fusion protein of the present invention is in the form of a complete antibody, which can well relieve the immunosuppression caused by tumor cells. At the same time, the activity of IL-15 is weakened, which can avoid systemic immune activation. The anti-PD-L1 antibody end will enrich IL-15 in the tumor microenvironment, resulting in local immune activation, which can not only improve the anti-tumor effect but also increase safety. Compared with the anti-PD-L1 antibody, the fusion protein of the present invention can significantly activate the proliferation activity of CTLL-2 cells and can significantly promote the expansion of CD8 + T, NK, and NKT cells, and has a better tumor inhibitory effect. Description of the Drawings

[0167] Figure 1 It is a schematic structural diagram of fusion protein A.

[0168] Figure 2 It is an experiment of fusion protein A binding to CTLL-2 cells.

[0169] Figure 3 It is an experiment of fusion protein A stimulating the activity of CTLL-2 cells.

[0170] Figure 4 It is an experiment of fusion protein A binding to HH cells.

[0171] Figure 5 It is an experiment of fusion protein A activating HH cells.

[0172] Figure 6 It is an experiment of fusion protein A binding to CHO-K1-CD122-CD132 cells.

[0173] Figure 7 It is an experiment of fusion protein A activating PBMC in vitro; among them Figure 7 a is the total cell number, Figure 7 b is the proportion of CD8 + T cells, Figure 7 c is the proportion of NKT cells, Figure 7 d is the proportion of NK cells.

[0174] Figure 8 is an experiment of activating PBMC to release cytokines in the solid phase state; among them Figures 8a - 8e The concentrations of IL-2, IFN-γ, IL-6, IL-10, and TNF-α are shown respectively; in the figure, donor 1, donor 2, donor 3, and donor 4 are PBMC cells from different people.

[0175] Figure 9 is an experiment of activating PBMC to release cytokines in the liquid phase state; among them Figures 9a - 9e The concentrations of IL-2, IFN-γ, IL-6, IL-10, and TNF-α are shown respectively; in the figure, donor 1, donor 2, donor 3, and donor 4 are PBMC cells from different people.

[0176] Figure 10 Inhibitory effect of fusion protein A on melanoma growth in mice Detailed implementation mode

[0177] Unless otherwise specified, each of the following terms shall have the meaning set forth below

[0178] Definition

[0179] It should be noted that the term "an" entity refers to one or more of such entities. For example, "an antibody" should be understood as one or more antibodies. Therefore, the terms "an" (or "a"), "one or more" and "at least one" may be used interchangeably herein

[0180] As used herein, the term "comprising" or "including" means that an antibody, composition or method, etc. includes the recited elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that an antibody, composition or method, etc. excludes other elements that have a fundamental impact on the characteristics of the combination, but does not exclude elements that have no essential impact on the antibody, composition or method, etc. "Consisting of" means excluding elements not specifically recited

[0181] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule and the immunologically active portion of the immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an antigen (immunologically reacts therewith). Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single-chain antibodies (scFv), Fab, Fab' and F(ab') 2 fragments, Fv and Fab expression libraries

[0182] The antibodies, antigen-binding units or derivatives disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies (scFv), epitope-binding fragments (e.g., Fab, Fab' and F(ab') 2 )

[0183] The term "monoclonal antibody" (mAb) refers to a group of such antibody molecules: which contain only one molecular species of the antibody molecules composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of monoclonal antibodies are the same in all molecules of the population. MAb contains an antigen-binding site capable of immunologically reacting with a specific epitope of an antigen

[0184] The term "single-chain antibody" (scFv) refers to an antibody formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody through a linker of 15 to 20 amino acids. The linker can be rich in glycine to increase flexibility and rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region of the protein is removed and a linker is introduced, its specificity of the original immunoglobulin is retained. ScFv molecules are generally known in the art and are described, for example, in U.S. Patent 5,892,019.

[0185] Those skilled in the art will understand that the classes of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are also some subclasses (such as γ1-γ4). The nature of this chain determines that the "classes" of antibodies are IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc. have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of protection disclosed in the present invention. In one or more embodiments, the class of the immunoglobulin molecule is IgG. Two heavy chains and two light chains are connected in a "Y" configuration through disulfide bonds, where the light chain starts from the "mouth" of the "Y" and continues through the variable region to surround the heavy chain. The light chain can be divided into kappa (κ) or lambda (λ). Each heavy chain can bind to a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are covalently bound, and the "tail" parts of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. 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 variable region of the immunoglobulin κ light chain is V κ ; the variable region of the immunoglobulin λ light chain is V λ .

[0186] The variable regions of the antibody light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant regions increases as they become more distant from the antigen-binding site or amino terminus of the antibody. The N-terminal part is the variable region, and the C-terminal part is the constant region; for example, the CH3 and CL domains of an IgG1 antibody contain the carboxyl termini of the heavy and light chains, respectively.

[0187] In naturally occurring antibodies, when an antibody assumes its three-dimensional conformation in an aqueous environment, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous amino acid sequences that form the antigen-binding domain and bind specifically to an antigen. The remaining other amino acids in the antigen-binding domain, called the "framework" ("FR") regions, exhibit less intermolecular variability. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loop structures that are connected thereto, or in some cases form part of the β-sheet structure. Thus, the framework regions form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain with CDRs in specific positions forms a surface that is complementary to the epitope on the antigen, and this complementary surface promotes the non-covalent binding of the antibody and its antigen epitope. Typically in an antibody molecule, each heavy chain and light chain has three CDRs, which are respectively called HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In order of position, the heavy chain variable region typically contains VH FR1, HCDR1, VH FR2, HCDR2, VH FR3, HCDR3, and VH FR4, and the light chain variable region contains VL FR1, LCDR1, VL FR2, LCDR2, LFR3, LCDR3, and VL FR4. For a given heavy chain or light chain variable region, those of ordinary skill in the art can identify the amino acids containing the CDRs and the 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), etc.).

[0188] The framework regions and CDR regions of a humanized antibody need not correspond precisely to the parental sequences. For example, the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR or framework residue at that position does not correspond to the donor antibody or consensus framework. Typically, at least 80%, at least 85%, at least 90%, or at least 95% of the residues of the humanized antibody will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a related family of immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either one may be included in the consensus sequence.

[0189] In cases where a term used in the art has two or more definitions that are used and / or accepted, the definition of the term used herein includes all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementary 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. in J. Mol. Biol. 196:901 - 917 (1987), which are hereby incorporated by reference in their entirety.

[0190] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data outside of the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also be numbered using EU or Chothia, AbM, Contact, IMGT, etc. numbering systems.

[0191] The antibodies disclosed by the present invention can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be of chondrichthoid origin (e.g., from sharks).

[0192] "Heavy chain constant region" includes at least one of the CH1 domain, hinge (e.g., upper, middle, and / or lower hinge region) domain, CH2 domain, CH3 domain, or variants or fragments thereof. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another embodiment, a portion of the heavy chain can include a chimeric hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0193] "Light chain constant region" includes a portion of the amino acid sequence from the light chain of an antibody. Preferably, the light chain constant region contains at least one of the constant κ domain or the constant λ domain. "Light chain - heavy chain pair" refers to a collection of a light chain and a heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0194] "Disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of cysteine can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0195] "Chimeric antibody" refers to any antibody whose variable region is obtained or derived from a first species, while its constant region (which can be full-length, partial, or modified) is derived from a second species. In certain embodiments, the variable region is from a non-human source (e.g., mouse or primate), and the constant region is from a human source.

[0196] As used herein, the term "epitope" includes any protein determinant region that can specifically bind to an immunoglobulin or its fragment or a T cell receptor. Epitope determinants are typically composed of the chemically reactive surface groups of a molecule (such as amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.

[0197] As used herein, the terms "specifically bind" or "immunoreact" refer to non-covalent interactions that occur between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents a greater affinity. The immunobinding properties of the selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Thus, both the "association rate constant" (kon) and the "dissociation rate constant" (koff) can be determined by calculating the concentrations and the actual association and dissociation rates (see Malmqvist, M., Nature 361:186-87 (1993)). The koff / kon ratio eliminates all parameters unrelated to affinity and is equal to the equilibrium dissociation constant KD (see Davies et al. (1990) Annual Rev Biochem 59:439-473). Specific binding can be measured by radio-ligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those of skill in the art.

[0198] As used in this invention, the term "isolated" when used in reference to cells, nucleic acids, polypeptides, etc., e.g., "isolated" DNA, RNA, polypeptide, refers to a molecule that is separated from other components in the natural environment of the cell, such as one or more of the other components in DNA or RNA. The term "isolated" as used in this invention 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 techniques, or chemical precursors or other chemicals when chemically synthesized. Additionally, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their natural state and would not exist in their natural state. The term "isolated" is also used in this invention to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, etc. are typically prepared by at least one purification step. In one or more embodiments, the purity of the isolated nucleic acid, polypeptide, 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 end values) or any value therein.

[0199] When applied to a polynucleotide, the term "encoding" refers to a polynucleotide that is said to "encode" a polypeptide, which can produce the polypeptide and / or its fragment upon transcription and / or translation, either in its native state or when manipulated by methods well known to those of skill in the art.

[0200] The term "recombinant" with respect to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that does not exist in nature, and non-limiting examples can be produced by combining polynucleotides or polypeptides that do not normally exist.

[0201] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as α-amino acid, β-amino acid, which can be encoded directly or in the form of a precursor by nucleic acid. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (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 "degeneracy of the genetic code". Amino acids include natural amino acids and non-natural amino acids.

[0202] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be components applicable to the polypeptides of the present disclosure. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation used herein, the left hand direction is the amino terminal direction, and the right hand direction is the carboxyl terminal direction, consistent with standard usage and convention. Conventional (or natural) amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), etc.

[0203] The term "polypeptide" is intended to cover both the singular "polypeptide" and the plural "polypeptides", and refers to a molecule formed by amino acid monomers linearly linked by an amide bond (also known as a peptide bond). The term "polypeptide" refers to any single chain 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 peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the above terms, or used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence, and it can be produced in any manner including chemical synthesis.

[0204] The terms "polynucleotide", "polymer of nucleotides" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide consists of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer having a central processing unit and used in bioinformatics applications such as for functional genomics and homology searching. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of polynucleotides without limitation: genes or gene fragments (such as probes, primers, ESTs or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA, RNA, nucleic acid probes and primers. A polynucleotide can contain modified nucleotides such as methylated nucleotides and nucleotide analogs. If such modification exists, the structural modification of the nucleotides can be carried out before or after the assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. After polymerization, the polynucleotide can be further modified, for example, by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any example of a polynucleotide in this disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0205] 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 when the sequences are aligned, that percentage of the bases (or amino acids) in the two sequences being compared are the same. The alignment and the percentage of identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: Genetic code = 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 are polynucleotides having the specified percentage of identity and encoding polypeptides having the same or similar biological activity.

[0206] Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed within the present disclosure, provided that the amino acid sequence identity is maintained at at least 90%, such as at least 92%, 95%, 98% or 99%. In some embodiments, the variations are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that occur within amino acid families that are related in their side chains. The amino acids encoded by genes are generally grouped into the following categories: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other families of amino acids include (i) serine and threonine of the aliphatic - hydroxy family; (ii) asparagine and glutamine of the amide - containing family; (iii) alanine, valine, leucine and isoleucine of the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine of the aromatic family. In some embodiments, the groups of conservative amino acid substitutions are: valine - leucine - isoleucine, phenylalanine - tyrosine, lysine - arginine, alanine - valine, glutamate - aspartate, and asparagine - glutamine. For example, it can be reasonably predicted that substitution of leucine with isoleucine or valine alone, substitution of aspartate with glutamate, substitution of threonine with serine, or substitution of one amino acid with a structurally related amino acid analog will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within the binding site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Such assays are described in detail herein. Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by one of ordinary skill in the art.

[0207] In some embodiments, the amino acid substitutions have the following effects: (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. The analogs can include various mutant proteins with sequences different from the naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the polypeptide portion outside the domain forming intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural properties of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence, or disrupt other types of secondary structure characteristic of the parental sequence). Examples of the secondary and tertiary structures of artificially identified polypeptides are described in Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (edited by C. Branden and J. Tooze, Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0208] The number of conservative amino acid substitutions in VL, VH can be 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 conservative amino acid substitutions, or a range between any two of these values (including the end values) or any value therein. The number of conservative amino acid substitutions in the heavy chain constant region, the light chain constant region, the heavy chain or the light chain can be 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, about 45 conservative amino acid substitutions, or a range between any two of these values (including the end values) or any value therein.

[0209] As used herein, the terms "label" or "labeled" refer to the incorporation of a detectable label, e.g., by incorporation of a radiolabeled amino acid, or attachment of a polypeptide to a biotinyl moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or an enzyme activity detectable by optical or calorimetric methods). In some cases, the label or labeling may also be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the label is attached via spacer arms of various lengths to reduce possible steric hindrance. The term "agent" or "drug" refers to a compound or composition that, when appropriately administered to a patient, is capable of inducing a desired therapeutic effect.

[0210] "About" refers to the conventional error range of the corresponding value that is readily known to those skilled in the relevant art. In some embodiments, the use of "about" herein refers to the recited value and ranges of ±10%, ±5%, or ±1% thereof.

[0211] "EC 50 ", i.e., the concentration for 50% of maximal effect (EC 50 ), refers to the concentration that elicits 50% of the maximal effect.

[0212] "Treatment" refers to therapeutic treatment and prophylactic or preventive measures that are intended to prevent, slow down, improve, or stop an adverse physiological change or disorder, such as the progression of a disease, and includes but is not limited to the following detectable or non-detectable outcomes: remission of symptoms, reduction in the degree of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement in the disease state, palliation, alleviation, or disappearance (whether partial or complete), and extension of the survival period compared to that expected without treatment. Patients in need of treatment include those who already have a condition or disorder, those who are predisposed to having a condition or disorder, or those who need to prevent the condition or disorder, and who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions disclosed herein for detection, diagnostic procedures, and / or treatment.

[0213] The term "tumor" means or is intended to describe a physiological state in a mammal that is typically characterized by uncontrolled cell growth, including both benign tumors and malignant tumors such as cancer. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include but are not limited to colorectal cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, colon cancer, salivary gland cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, rectal cancer, prostate cancer, vulvar cancer, testicular cancer, squamous cell carcinoma, small cell lung cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma multiforme, mesothelioma, oral epithelioma, choriocarcinoma, and head and neck cancer.

[0214] As used herein, the terms "administer", "administration", and "administering" are used interchangeably and mean the delivery of a substance (e.g., an antibody or a fusion protein) for a therapeutic purpose (e.g., treating a disease associated with PD-L1 or IL-15). The mode of administration can be parenteral, enteral, and topical. Parenteral administration is typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0215] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, such as an antibody or a fusion protein, that is sufficient to reduce or ameliorate a disorder (such as cancer) or the severity and / or duration of one or more symptoms thereof; prevent progression of the disorder; cause regression of the disorder; prevent recurrence, development, onset, or progression of one or more symptoms associated with the disorder; detect the disorder; or enhance or improve the prophylactic or therapeutic effect of another therapy (such as a prophylactic or therapeutic agent). For example, an effective amount of an antibody or a fusion protein can inhibit tumor growth (e.g., inhibit an increase in tumor volume); reduce tumor growth (e.g., reduce tumor volume); reduce the number of cancer cells; and / or alleviate to some extent one or more symptoms associated with cancer. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0216] The term "patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc.

[0217] As used herein, the term "in need" means that a patient has been identified as in need of a particular method or treatment. In some embodiments, the identification can be made by any diagnostic means. In any of the methods and treatments described herein, a patient may be in need.

[0218] The term "drug for treating tumors" as used herein refers to a reagent having functional properties that inhibit the development or progression of tumors in the human body, particularly malignant (cancerous) lesions such as cancer, sarcoma, lymphoma, or leukemia. Inhibiting metastasis is, in many cases, a property of anti-tumor drugs.

[0219] A "pharmaceutical composition" refers to a mixture formed by one or more compounds, their pharmaceutically acceptable salts or prodrugs, and other chemical components, wherein the "other chemical components" refer to pharmaceutically acceptable excipients and / or one or more other therapeutic agents.

[0220] All relevant descriptions of publications mentioned herein are incorporated herein by reference in their entirety.

[0221] Anti-PD-L1 antibodies and fusion proteins

[0222] The present invention provides antibodies or antigen-binding fragments having high affinity for the PD-L1 protein. The antibodies exhibit effective binding activity, 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 are used to treat various types of cancers, tumors, or infections and other related diseases.

[0223] In some embodiments, the antibodies of the present invention utilize the "Knobs-into-Holes" technology (see, for example, John B. B. Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering, 9(7): p. 617-21 (1996); Patent US8216805B2). This technology can engineer the interface between different chains of an antibody to promote the correct association of each chain of the antibody. Generally, this technology involves introducing "knobs" at the interface of one chain and corresponding "holes" at the interface of the other chain to which it is to be paired, such that the knobs can be placed in the holes. The knobs can be constructed by replacing the amino acid side chains at the interface of the CH3 domain of the heavy chain constant domain from one chain with larger side chains (such as the amino acid substitution T366W (Eu numbering)). Compensatory holes of the same or similar size to the knobs are constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired by replacing large amino acid side chains with smaller side chains (such as amino acid substitutions T366S, L368A, and Y407V (Eu numbering)).

[0224] In some embodiments, the constant region of one heavy chain of the antibody comprises the following amino acid mutations: Y349C, T366S, L368A, and Y407V (EU numbering), and the constant region of the other heavy chain of the antibody comprises the following amino acid mutations: S354C and T366W (EU numbering), forming a stable association of "Knobs-into-Holes".

[0225] One of ordinary skill in the art should also understand that the antibody or antigen-binding fragment sequences disclosed in the present invention can be replaced, and the amino acid sequence after replacement is different from the naturally occurring amino acid sequence of the antibody. For example, the amino acid sequence after replacement can be similar to the starting sequence, such as having a certain percentage identity with the starting sequence, such as it can have an identity with the starting sequence of 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.

[0226] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence having one or more modifying groups. For example, the antibody or antigen-binding fragment disclosed in the present invention can comprise a flexible linker sequence, or can be modified to add functional groups (such as PEG, drugs, toxins, or tags).

[0227] The antibodies, antigen-binding fragments, and fusion proteins thereof disclosed in the present invention include modified derivatives, i.e., modifications by covalent attachment of any type of molecule to the antibody or antigen-binding fragment or fusion protein thereof, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment or fusion protein thereof from binding to the epitope. Examples include, but are not limited to, that the antibody or antigen-binding fragment or fusion protein thereof can be glycosylated, acetylated, polyethylene glycolated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytically cleaved, linked to a cell ligand or other protein, etc. Any one of numerous chemical modifications can be carried out by the prior art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0228] In some embodiments, the antibody or antigen-binding fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, bioreactant regulator, pharmaceutical agent, or PEG.

[0229] The antibody or antigen-binding fragment can be detectably labeled by conjugating it to a chemiluminescent compound. Then the presence of the chemiluminescently labeled antibody or antigen-binding fragment is determined by detecting the luminescence that appears during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalates.

[0230] In some embodiments, to facilitate the expression of the antibody in host cells, signal peptide sequences can also be added to the heavy and light chains of the antibody, such as the heavy chain signal peptide: MEFGLSWVFLVAILKGVQC (SEQ ID NO:75), and the light chain signal peptide: MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO:76).

[0231] In some embodiments, the present invention also provides a fusion protein comprising a PD-L1 binding domain and a domain that stimulates NK and T cell activities. In some embodiments, the PD-L1 binding domain is an anti-PD-L1 antibody or antigen-binding fragment. In some embodiments, the domain that stimulates NK and T cell activities comprises IL-15 or its receptor-binding fragment or variant and IL-15Rα or its sushi domain or variant. The sushi domain binds IL-15 with high affinity, and the complex of IL-15 and the sushi domain has high activity for stimulating NK and T cell proliferation.

[0232] In some embodiments, the fusion protein comprises the anti-PD-L1 antibody or antigen-binding fragment, IL-15 or its receptor-binding fragment or variant, and IL-15Rα or its sushi domain or variant as described herein.

[0233] In some embodiments, the present invention provides a fusion protein comprising an anti-PD-L1 antibody or antigen-binding fragment described herein, IL-15, and an IL-15Rα fusion protein.

[0234] In some embodiments, the present invention provides a fusion protein comprising an anti-PD-L1 antibody or antigen-binding fragment described herein, IL-15, and the sushi domain of IL-15Rα.

[0235] In some embodiments, the present invention provides a fusion protein comprising an anti-PD-L1 antibody or antigen-binding fragment described herein, an IL-15 receptor-binding fragment, and the sushi domain of IL-15Rα.

[0236] In some embodiments, the present invention provides a fusion protein comprising an anti-PD-L1 antibody or antigen-binding fragment described herein, a variant of IL-15 or its receptor-binding fragment, and a variant of IL-15Rα or its sushi domain.

[0237] In some embodiments, the IL-15 comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO:82, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:82.

[0238] In some embodiments, the IL-15Rα sushi domain comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO:81, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:81.

[0239] In some embodiments, the variant of IL-15 or its receptor-binding fragment comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to IL-15 or its receptor-binding fragment.

[0240] In some embodiments, the variant of IL-15 or its receptor-binding fragment has one or more conservative amino acid substitutions compared to IL-15 or its receptor-binding fragment.

[0241] In some embodiments, the variant of the IL-15Rα or its sushi domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the IL-15Rα or its sushi domain.

[0242] In some embodiments, the variant of the IL-15Rα or its sushi domain has one or more conservative amino acid substitutions compared to the IL-15Rα or its sushi domain.

[0243] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment of the fusion protein is linked to IL-15 or its receptor-binding fragment or its variant and the IL-15Rα or its sushi domain or its variant via a linker. In some embodiments, the C-terminus of one heavy chain of the anti-PD-L1 antibody is linked to IL-15 or its receptor-binding fragment or its variant via a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is linked to the IL-15Rα or its sushi domain or its variant via a linker. In some embodiments, the C-terminus of one heavy chain of the anti-PD-L1 antibody is linked to IL-15 via a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is linked to the sushi domain of IL-15Rα via a linker. In some embodiments, the constant region of one heavy chain of the anti-PD-L1 antibody contains the following amino acid mutations: Y349C, T366S, L368A and Y407V (EU numbering), and the constant region of the other heavy chain of the anti-PD-L1 antibody contains the following amino acid mutations: S354C and T366W (EU numbering), forming a stable association of "Knobs-into-Holes", which greatly facilitates the correct assembly of the two heavy chains and minimizes mismatching.

[0244] In some embodiments, the linker comprises glycine and serine ("GS linker"). In some embodiments, the linker is (G m S) n , where each m is independently 1, 2, 3, 4, 5 or 6, and n is 1, 2, 3, 4 or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) 2 . In some embodiments, the linker is (GGGGS) 3 , as shown in SEQ ID NO:85. In some embodiments, the linker is (GGGGS) 4 . In some embodiments, the linker is (GGGGS) 5 .

[0245] In some embodiments, the fusion protein of the present invention comprises a first polypeptide, a second polypeptide, and a third polypeptide; the first polypeptide comprises an anti-PD-L1 heavy chain a, a linker, and IL-15Rα or its sushi domain or a variant thereof, the second polypeptide comprises an anti-PD-L1 heavy chain b, a linker, and IL-15 or its receptor-binding fragment or a variant thereof, and the third polypeptide is an anti-PD-L1 light chain. In some embodiments, the schematic structural diagram of the fusion protein is as shown in Figure 1 shown, consisting of 4 polypeptides, including one first polypeptide, one second polypeptide, and two third polypeptides with the same sequence.

[0246] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:83, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO:83.

[0247] In some embodiments, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:84, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO:84.

[0248] In some embodiments, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:66, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO:66.

[0249] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66.

[0250] In some embodiments, the fusion protein is fusion protein A.

[0251] Methods for preparing antibodies and fusion proteins

[0252] The present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, fusion proteins and their derivatives of 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, light chains or fusion proteins, etc. Methods for preparing antibodies and fusion proteins are well known in the art and are described in the present invention.

[0253] In certain embodiments, the prepared antibodies do not cause harmful immune responses in the animals to be treated (such as humans). In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed by the present invention are modified using techniques recognized in the art to reduce their immunogenicity. For example, the antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parental antibody but have lower immunogenicity in humans. This can be achieved by various methods, including (a) transplanting the entire non-human variable region into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into a human framework and constant region, with or without retaining key framework residues; or (c) transplanting the entire non-human variable region, but "hiding" them by replacing surface residues with human-like portions. Usually, the framework residues in the human framework region will be replaced by the corresponding residues from the CDR donor antibody, such as residues that can improve antigen binding. These framework replacements can be identified by methods well known in the art, such as by modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify abnormal framework residues at specific positions. (See U.S. Patent 5,585,089; the entire content of which is incorporated herein by reference). Various techniques well known in the art can be used to humanize antibodies, such as CDR grafting (WO1991009967; U.S. Patents 5,225,539, 5,530,101, and 5,585,089), reshaping or surface rearrangement (EP592,106; EP519,596), and chain shuffling (U.S. Patent 5,565,332), the entire content of which is incorporated herein by reference.

[0254] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering an antibody to modify T cell epitopes (see, for example, WO2000034317 A2). For example, the heavy chain variable region sequence and the light chain variable region sequence from a starting antibody are analyzed, and a human T cell epitope "map" is generated from each variable region, showing the position of the epitope relative to the complementarity determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides. The genes for the complete heavy and light chains containing the modified variable regions and human constant regions are then cloned into an expression vector, and the plasmid is subsequently transfected into a cell line to produce the complete antibody. The antibodies are then compared in appropriate biochemical and biological assays to identify the optimal antibody.

[0255] The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0256] The preparation of scFv can refer to the techniques for producing single-chain units (U.S. Patent 4,946,778). The single-chain units are formed by amino acid bridging of the heavy and light chain fragments of the Fv region to produce single-chain fusion peptides. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 240:1038-1041 (1988)).

[0257] Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies are described in, for example, U.S. Patents 4,946,778 and 5,258,498. For certain uses, including the use of antibodies in humans and in vitro detection assays, chimeric antibodies, humanized antibodies, or fully human antibodies can be used. Chimeric antibodies are molecules in which different parts of the antibody are derived from different animal species, for example, antibodies having the variable regions of a murine monoclonal antibody and the human immunoglobulin constant regions. Methods for producing chimeric antibodies are known in the art and are described in U.S. Patents 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.

[0258] In addition, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992). In particular, this technique can generate primate antibodies containing simian variable region and human constant region sequences. The entire content of this reference is incorporated herein by reference. In addition, this technique is also mentioned in U.S. Patents 5,658,570, 5,693,780, and 5,756,096, and the entire content of each patent is incorporated herein by reference.

[0259] Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. Also refer to U.S. Patents 4,444,887 and 4,716,111, as well as PCT published texts WO 1998050433, WO 1998024893, WO 1998016654, WO 1996034096, WO 1996033735, and WO1991010741, and the entire content of each patent is incorporated herein by reference.

[0260] In other embodiments, using conventional methods (such as using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains), the DNA encoding the desired monoclonal antibody can be isolated and sequenced. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as Escherichia coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. The isolated DNA (which can be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of the antibody, as described in U.S. Patent 5,658,570, the entire content of which is incorporated herein by reference. This method extracts RNA from the selected cells and converts it into cDNA, which is then amplified by PCR technology using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Patent 5,658,570.

[0261] In addition, using conventional recombinant DNA techniques, one or more CDRs of the antibodies of the present invention can be inserted into framework regions, for example, into human framework regions to construct humanized non-human antibodies. The framework regions can be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human framework regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of a target antigen produced by the combination of a framework region and a CDR. One or more amino acid substitutions can be made within the framework region, and amino acid substitutions that can improve the binding of the antibody to its antigen can be selected. Additionally, substitutions or deletions of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be made by this method, thereby generating antibody molecules lacking one or more interchain disulfide bonds. Other alterations to the polynucleotides within the skill of the art are also encompassed by the present invention.

[0262] Antibodies or fusion proteins can be prepared by using conventional recombinant DNA techniques. Vectors and cell lines for producing antibodies or fusion proteins can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, Reference Module in Life Sciences, 2017, the entire content of which including supplementary content is incorporated herein by reference in its entirety.

[0263] In some embodiments, DNA encoding an antibody or fusion protein can be designed and synthesized according to the amino acid sequences of the antibodies or fusion proteins described herein by conventional methods, inserted into an expression vector, and then transfected into a host cell. The transfected host cell is cultured in a medium to produce a monoclonal antibody or fusion protein. In some embodiments, the expression vector for the antibody or fusion protein includes at least one promoter element, an antibody or fusion protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. High-efficiency transcription can be achieved through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, or other cell promoters such as the actin promoter. Suitable expression vectors can include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, or pCS2, etc. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells, and CHO cells, etc.

[0264] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include selection genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, and hygromycin resistance, etc., to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into a host cell lacking the above genes. After culturing in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.

[0265] In addition, mutations can be introduced into the nucleotide sequence encoding the antibodies or fusion proteins of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. Variants (including derivatives) encode substitutions of fewer than 50 amino acids, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids relative to the original target protein. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the biological activities of the resulting mutants can be screened to identify mutants that retain activity.

[0266] Therapeutic methods

[0267] The present invention also provides treatment methods and uses. In some embodiments, there are provided methods for preventing, treating or ameliorating various types of autoimmune diseases, cancers, tumors or diseases related to infections, etc., said methods comprising administering to a patient an effective amount of an anti-PD-L1 antibody or antigen-binding fragment or fusion protein. In some embodiments, there is provided the use of an anti-PD-L1 antibody or antigen-binding fragment or fusion protein in preventing, treating or ameliorating autoimmune diseases, cancers, tumors or diseases related to infections, etc. In some embodiments, there is provided the use of said anti-PD-L1 antibody or antigen-binding fragment or fusion protein in the preparation of a medicament for preventing, treating or ameliorating autoimmune diseases, cancers, tumors or diseases related to infections, etc.

[0268] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or fusion protein or derivative used, the age and weight of the patient, general health status, gender and diet, as well as the time of administration, excretion frequency, drug combination, and the severity of the particular disease being treated. These factors are judged by medical care personnel within the scope of ordinary skill in the art. The dosage will also 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.

[0269] The methods of administering the antibody or fusion protein or derivative include but are not limited to administration by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral routes. The pharmaceutical composition can be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or skin mucosa (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Thus, the pharmaceutical composition containing the antibody or antigen-binding fragment or fusion protein of the present invention can be administered orally, rectally, parenterally, intravesically (such as intravesical perfusion), intrathecally, vaginally, intraperitoneally, topically (such as by powder, ointment, drops or transdermal patch), orally or by oral or nasal spray.

[0270] The term "parenteral" as used in the present invention refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injections and infusions.

[0271] The administration mode can be systemic administration or local administration. In addition, it may be necessary to introduce the antibody or fusion protein of the present invention into the central nervous system through any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be assisted by connecting an intraventricular catheter to a reservoir (which can be an Ommaya reservoir). Administration can also be through the lungs, for example, by using an inhaler or nebulizer, and using a nebulized preparation.

[0272] The antibody or fusion protein of the present invention can be locally administered to the area in need of treatment; it can be achieved by, but not limited to, the following methods: local infusion during surgery, such as local application in combination with a postoperative wound dressing, by injection, by catheter, by means of a suppository or by means of an implant, and the implant is a porous, non-porous or gel-like material, including a membrane (such as a silicone rubber membrane) or a fiber. Preferably, when administering the protein (including antibody or fusion protein) of the present invention, attention must be paid to using a material that does not absorb the protein.

[0273] In some embodiments, the present invention provides a nucleic acid or polynucleotide encoding an antibody or fusion protein. The nucleic acid or polynucleotide can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to promote the expression of the protein it encodes, and then administering the above nucleic acid or polynucleotide or vector to make it an intracellular part by the following methods, such as by using a retroviral vector (see U.S. Patent 4,980,286), or by direct injection, or by using particle bombardment (such as a gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection reagents, or by administering in connection with a homeobox peptide known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Optionally, the nucleic acid can be introduced into cells and integrated into the host cell DNA by homologous recombination for expression.

[0274] In some embodiments, the dose of the antibody or fusion protein of the present 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. After the initial dose, a second dose or multiple doses of the antibody or antigen-binding fragment or fusion protein can be subsequently administered, and the dose is approximately the same as or less than the initial dose, where the subsequent dose can be separated by at least 1 day to 3 days; or at least one week. A lower starting dose can also be used to increase tolerance, and the dose can be increased subsequently. Modifications such as lipidation can be used to enhance the uptake and tissue penetration ability (such as entering the brain) of the antibody or fusion protein, thereby reducing the dose and frequency of administration of the antibody or fusion protein of the present invention.

[0275] Various known delivery systems can be used to administer the antibodies or fusion proteins or derivatives of the present invention or polynucleotides encoding them, such as encapsulation in 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), construction of nucleic acids as part of a retrovirus or other vector, etc.

[0276] Combination therapy

[0277] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment or fusion protein of the present invention can be combined with other treatment or prevention regimens, including the administration of one or more antibodies or antigen-binding fragments or fusion proteins of the present invention and one or more other therapeutic agents or methods together or in combination. In some embodiments, other treatment regimens include, but are not limited to, radiotherapy, chemotherapy, hormone therapy, etc. For combined treatment, the antibody or fusion protein can be administered simultaneously or separately with other therapeutic agents. When administered separately, the antibody or fusion protein of the present invention can be administered before or after the administration of another other therapeutic agent.

[0278] In some embodiments, the antibody or fusion protein of the present invention is administered in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent that can be administered with the antibody or fusion protein of the present invention includes, but is not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, mithramycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxy The invention relates to steroids and combinations thereof, including but not limited to steroids (e.g., steroids such as betamethasone sodium phosphate, steroids such as chlorambucil, styrax, styraxine ...

[0279] In some embodiments, the anti-PD-L1 antibody or fusion protein of the present invention is administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include immunotherapeutic agents, including but not limited to therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include rituximab, trastuzumab, tositumomab, ibritumomab tiuxetan, alemtuzumab, epratuzumab, bevacizumab, cetuximab, and berentuzumab, etc.

[0280] Pharmaceutical composition

[0281] The present invention also provides a pharmaceutical composition. Such a composition comprises an anti-PD-L1 antibody or antigen-binding fragment or fusion protein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1%-99% of an anti-PD-L1 antibody or antigen-binding fragment or fusion protein. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (such as an immune checkpoint inhibitor).

[0282] In some embodiments, the term "pharmaceutically acceptable" refers to substances approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, particularly in humans. In addition, "pharmaceutically acceptable excipients" generally refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, etc.

[0283] The term "excipient" refers to a diluent, adjuvant, excipient or carrier that can be administered to a patient together with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal or vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions, aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifying agent, or pH buffer. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid, chelating agents, and agents for adjusting tonicity such as dextrose are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment or fusion protein, preferably in purified form, together with a suitable amount of excipients to provide a dosage form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The parent formulation can be encapsulated in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0284] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional steps. Compositions for intravenous administration are usually solutions in sterile, isotonic, aqueous buffers. The composition may also contain solubilizing agents and local anesthetics such as lidocaine to relieve the pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit dosage forms, such as in sealed containers (e.g., ampoules or sachets) indicating the content of the active ingredient in the form of dry lyophilized powder or anhydrous concentrate. In the case of administering the composition by infusion, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, sterile water for injection or saline can be used to mix the active ingredient before administration.

[0285] The antibodies, antigen-binding fragments or fusion proteins of the present invention can be in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0286] Embodiment

[0287] The technical solutions of the present invention are further illustrated by the following specific examples. The specific examples do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0288] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0289] Example 1: Preparation of Anti-PD-L1 Antibody and Fusion Protein A

[0290] 1) Preparation of Anti-PD-L1 Antibody

[0291] The composition and related sequences of the exemplary antibodies are shown in Table 1-10; among them, the heavy chain and light chain compositions of the antibodies are shown in Table 1, the CDR regions of the antibody heavy chain and light chain are shown in Table 2, the composition of the antibody heavy chain CDR region is shown in Tables 3 and 4, and the composition of the antibody light chain CDR region is shown in Table 5.

[0292] The DNA sequences encoding the antibody heavy chain and light chain are respectively cloned into an expression vector, and then the plasmids are respectively extracted. The heavy chain and light chain are transiently transfected into HEK293F cells at a plasmid molar ratio of 1:1. After cell culture and purification, the anti-PD-L1 antibody is obtained, and the sequencing result is the same as the expected sequence.

[0293] Table 1 Heavy Chain and Light Chain Compositions of the Antibody

[0294]

[0295]

[0296] Table 2 CDR regions of antibody heavy and light chains

[0297]

[0298]

[0299] Table 3 Composition of antibody heavy chain CDR regions

[0300]

[0301] Table 4 Composition of antibody heavy chain CDR regions

[0302]

[0303]

[0304] Table 5 Composition of antibody light chain CDR regions

[0305]

[0306] Table 6 Antibody heavy chain variable region

[0307]

[0308]

[0309] Table 7 Antibody light chain variable region

[0310]

[0311] Table 8 Antibody constant region

[0312]

[0313]

[0314] Table 9 Antibody heavy chain sequence

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] Table 10 Antibody Light Chain Sequences

[0321]

[0322]

[0323] 2) Preparation of Fusion Protein A

[0324] The structural schematic diagram of fusion protein A is shown in Figure 1 as follows, which is composed of 4 polypeptides: the first polypeptide (shown as SEQ ID NO: 83), the second polypeptide (shown as SEQ ID NO: 84), and two identical third polypeptides (shown as SEQ ID NO: 66); among them, the first polypeptide contains from the N-terminus to the C-terminus: anti-PD-L1 heavy chain a (shown as SEQ ID NO: 79), linker (shown as SEQ ID NO: 85), and IL-15Rα sushi domain (shown as SEQ ID NO: 81), the second polypeptide contains from the N-terminus to the C-terminus: anti-PD-L1 heavy chain b (shown as SEQ ID NO: 80), linker (shown as SEQ ID NO: 85), and IL-15 (shown as SEQ ID NO: 82), and the third polypeptide is anti-PD-L1 light chain; among them, the nucleic acid sequence of the first polypeptide is shown as SEQ ID NO: 72, the nucleic acid sequence of the second polypeptide is shown as SEQ ID NO: 73, and the nucleic acid sequence of the third polypeptide is shown as SEQ ID NO: 74. The related amino acid sequences of fusion protein A are shown in Table 11, and the related nucleic acid sequences are shown in Table 12.

[0325] The DNA sequences encoding the first polypeptide, the second polypeptide, and the third polypeptide of fusion protein A are respectively cloned into an expression vector, and then transiently transfected into HEK293F cells. Fusion protein A is obtained through cell culture and purification.

[0326] Table 11 Related Amino Acid Sequences of Fusion Protein A

[0327]

[0328]

[0329]

[0330] Table 12 Related Nucleic Acid Sequences of Fusion Protein A

[0331]

[0332]

[0333]

[0334] Example 2: Binding determination and activity detection of fusion protein A to CTLL-2 cells

[0335] A fluorescence-activated cell sorting (FACS)-based assay was used to evaluate the binding of fusion protein A to CTLL-2 cells (mouse cytotoxic T lymphocyte cell line) that endogenously express IL-15Rα, IL-2Rβ, and IL-2Rγ. CTLL-2 cells were resuspended in PBS buffer to form a single-cell suspension and mixed with an equal volume (both in 96-well plates) of 100 μL of fusion protein A samples at different concentrations (starting from 100 nM, serially diluted 2-fold, with 10 concentrations), 500,000 cells per well. The mixture was equilibrated at 4 °C for 60 minutes (min) and washed with PBS buffer. Then, phycoerythrin (PE)-conjugated goat anti-human IgGFc antibody (Invitrogen, catalog number: 12-4998-82), used as the secondary antibody, was added and equilibrated at 4 °C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. The data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA). As Figure 2 shown, the FACS binding assay demonstrated that fusion protein A could significantly bind to CTLL-2 cells.

[0336] The activity of fusion protein A was evaluated using CTLL-2 cells in a cell proliferation assay. Under the conditions of 37 °C and 5% CO 2 2, CTLL-2 cells were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum (FBS), and 10 ng / mL IL-2. The cells were cultured in suspension until they reached a cell density of 5 × 10 5 cells per milliliter before subculturing. For the activity assay, 2-3 days after the last subculturing, the cells were washed with RPMI-1640 medium, resuspended in RPMI-1640 medium containing 15% FBS, and seeded at a density of 20,000 cells per well (50 μL) in a 96-well white plate, and the side wells were supplemented with PBS. The fusion protein A was diluted as follows: the diluent was RPMI-1640 medium containing 15% FBS, and the antibody L1-R2-4-71 and fusion protein A samples started from 200 nM and were diluted 1:3, and 50 μL / well was added to the above plate seeded with cells. The plate was placed at 37 °C and 5% CO 2Cultivate for 24 hours (h) in an incubator. Before detection, take out the CellCounting-Lite2.0 Luminescent Cell Viability Assay reagent from the -20°C refrigerator and equilibrate it to room temperature. Take out the cell culture plate from the incubator and equilibrate it at room temperature (25 ± 3°C) for 5 - 10 min. Add 50 μL of the CellCounting-Lite2.0 Luminescent Cell Viability Assay reagent (Novoprotein, catalog number: DD1101-02) to each well and incubate in the dark at room temperature for 5 - 30 min. Use the Luminescence detection module on the SpectraMax multi-functional microplate reader to read the relative light units (RLU). Use non-linear regression and analyze the data using GraphPad PRISM 8

[0337] (GraphPad Software, San Diego, CA). As Figure 3 shown, the CTLL-2 cell proliferation assay demonstrated that the fusion protein A could significantly activate the proliferation activity of CTLL-2 cells, and the EC 50 value was 0.417 nM.

[0338] Example 3: Binding determination and activity detection of fusion protein A to HH cells

[0339] Use a fluorescence-activated cell sorting (FACS)-based assay to evaluate the binding of fusion protein A to HH cells (human cutaneous T-lymphoma cells; ATCC CRL-2105) that endogenously express IL-2Rβ and IL-2Rγ. Resuspend HH cells in PBS buffer to form a single-cell suspension and mix it in equal volume with 100 μL of antibody L1-R2-4-71 or fusion protein A samples at different concentrations (starting concentration of 100 nM, 2-fold dilution, 11 concentration gradients) (both in 96-well plates), with 500,000 cells per well. Equilibrate the mixture at 4°C for 60 minutes and wash it with PBS buffer. Then add a phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, catalog number: 12-4998-82) used as the secondary antibody and equilibrate it in the dark at 4°C for 30 minutes. Wash the cells again with PBS buffer and analyze by flow cytometry. Use non-linear regression and analyze the data using GraphPad PRISM 8 (GraphPad Software, San Diego, CA). As Figure 4 shown, the FACS binding assay demonstrated that the fusion protein A could significantly bind to HH cells.

[0340] After IL-15 binds to IL-15Rα, it acts in trans to bind to IL-2Rβ and IL-2Rγ, activating the downstream signaling pathway and leading to STAT5 phosphorylation. Different concentrations of fusion protein A samples were co-incubated with HH cells, and the phosphorylation of STAT5 was observed to evaluate the ability of fusion protein A to activate HH cell activity. HH cells in the logarithmic phase were collected, washed with PBS, resuspended in pre-warmed RPMI-1640 medium at a density of 2 million cells per 100 μL, and incubated at 37°C for 30 min. During this period, antibody L1-R2-4-71 or fusion protein A samples were diluted with RPMI-1640 medium containing 10% FBS starting from 200 nM and serially diluted 2-fold to obtain 11 concentrations, each at 100 μL. 100 μL of the above-diluted fusion protein A samples were mixed with an equal volume of cells and incubated at 37°C for 15 min. Then, they were immediately placed on ice, and an equal volume of 4% paraformaldehyde (FPA) solution (final concentration 2%) was added to fix the cells, which were then placed on ice for 30 min. Subsequently, they were washed with pre-cooled PBS buffer. The supernatant was discarded, 1 mL of pre-cooled 90% methanol was added, and after placing on ice for 30 min, they were washed with pre-cooled PBS buffer, resuspended in PBS buffer, and PE anti-STAT5 Phospho(Tyr694) Antibody (BioLegend, catalog number: 936904) was added, followed by incubation in the dark at room temperature for 40 min. After washing, they were analyzed on a Beckman CytoFlex, and the mean fluorescent intensity (MFI) value was statistically analyzed to evaluate the phosphorylation level of STAT5. Nonlinear regression was used to analyze the data using GraphPad PRISM 8 (GraphPad Software, San Diego, CA). As Figure 5 shown, the analysis of STAT5 phosphorylation level demonstrated that fusion protein A could significantly activate HH cell activity, and the EC 50 value was 1.609 nM.

[0341] Example 4: Determination of the binding of fusion protein A to CHO-K1-CD122-CD132 cells

[0342] A fluorescence-activated cell sorting (FACS)-based assay was used to evaluate the binding of fusion protein A to CHO-K1 cells (i.e., CHO-K1-CD122-CD132 cells) that exogenously expressed IL-2Rβ (CD122) and IL-2Rγ (CD132). The CHO-K1-CD122-CD132 cells were resuspended in PBS buffer to form a single-cell suspension and mixed with 100 μL of fusion protein A or antibody L1-R2-4-71 samples at different concentrations (starting from 100 nM, serially diluted 2-fold) in equal volumes (both in 96-well plates), with 500,000 cells per well. The mixture was equilibrated at 4 °C for 60 minutes and washed with PBS buffer. Then, phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, catalog number: 12-4998-82), used as the secondary antibody, was added and equilibrated at 4 °C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. The data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA). As Figure 6 shown, the FACS binding assay demonstrated that fusion protein A could significantly bind to CHO-K1-CD122-CD132 cells.

[0343] Method for constructing CHO-K1-CD122-CD132 cells: The vector ligated with the CD122 gene sequence (NCBI Reference Sequence: NM_000878.5) was linearized and then electrotransfected into CHO-K1. After culture, the CHO-CD122 stable cell line was obtained by screening; on the basis of the CHO-CD122 cell line, it was further infected with lentivirus containing the CD132 gene sequence (NCBI Reference Sequence: NM_000206.3), and the CHO-K1-CD122-CD132 stable cell line was obtained by screening after culture.

[0344] Example 5: Experiment on the in vitro activation of PBMC by fusion protein A

[0345] This example outlines the effect of inducing the selective activation and expansion of effector lymphocytes in human peripheral blood using fusion protein A samples. Six-well cell culture plates were coated with 500 μL of 200 ng / mL anti-CD3 antibody (Novoprotein, GMP-A018) one night in advance; the next morning, after discarding the supernatant, PBMC resuspended in RPMI-1640 medium containing 15% FBS was added, 1×10 6 cells per well, with a total volume of 3 mL per well, and fusion protein A or antibody L1-R2-4-71 was added respectively, with final concentrations of 0.5 nM and 20 nM. At 37 °C, 5% CO 2Cultivate for 7 days, transfer PBMC to a new 6-well cell culture plate (remove anti-CD3 antibody), supplement with fusion protein A or antibody L1-R2-4-71, and cultivate for another 5 days; detect CD8 by flow cytometry + proliferation of T, NK, and NKT cells. Among them, CD8 + T is CD3+CD8+ double-positive T cells (i.e., CD3 + CD8 + T cells), NK cells are CD16+ or CD56+ cells, and NKT cells are CD3+CD4-CD8- double-negative T cells (i.e., CD3 + CD4 - CD8 - T cells). The sources of the antibodies used for detection are as follows: CD3 antibody (elabscience, catalog number: FW2689), CD4 antibody (elabscience, catalog number: FW0218), CD8 antibody (elabscience, catalog number: FW0931), CD16 antibody (BioLegend, catalog number: 302038), CD56 antibody (BioLegend, catalog number: 302630). The results are as Figure 7 shown in a-d. Compared with antibody L1-R2-4-71, fusion protein A can significantly promote the expansion of CD8 + T, NK, and NKT cells.

[0346] Example 6: Cytokine Release Experiment of Fusion Protein A

[0347] In this example, a liquid and solid-phase incubation system was used to evaluate the cytokine release induced by different test articles. The dry-pack method, i.e., the solid-phase incubation system, coated a 96-well cell culture plate with a 25 μg / mL fusion protein A sample, 40 μL per well, and dried it overnight in a laminar flow hood. Then, 1×10 5 PBMC cells (Red Biotechnology, Guangzhou) were added to each well, with a volume of 200 μL. The wet-pack method, i.e., the liquid-phase incubation system, added a 25 μg / mL fusion protein A sample to a 96-well cell culture plate, 100 μL per well, and 1×10 5 PBMC cells were added to each well, with a volume of 100 μL. At the same time, anti-CD3 antibody (Novus Biologicals, GMP-A018) and TGN1412 antibody (a CD28 agonist antibody; the sequence is derived from patent US8709414B2) were used as positive control antibodies. After incubating for three days, the supernatants were collected to detect IL-2, IFN-γ, IL-10, IL-6, and TNF-α (MABTECH, ELISABATIC kit). Figures 8a - 8e and Figures 9a - 9eAs shown, the TGN1412 antibody can significantly activate PBMCs in both liquid-phase and solid-phase incubation systems, as manifested by far higher release levels of IL-2, IL-10, IFN-γ, and TNF-α than those of other test articles. The anti-CD3 antibody, used as a control antibody, can also activate PBMCs to varying degrees. The fusion protein A cannot activate PBMCs to release IL-2 in the solid-phase incubation system, and its effects on the release of other cytokines are comparable to those of antibody L1-R2-4-71.

[0348] Example 7: Anti-tumor Efficacy of Fusion Protein A in Vivo

[0349] This example describes an in vivo experiment to evaluate the functional blockade of PD-L1 and the functional activation of IL-15R by fusion protein A. Since the PD-L1 monoclonal antibody also binds to murine PD-L1 and IL-15 can also recognize the murine receptor, wild-type mice can be used to directly evaluate the efficacy of different test articles in a murine tumor xenograft model in vivo.

[0350] A murine tumor-bearing model was prepared by implanting tumor cells into C57BL / 6 mice. The murine melanoma cell line B16F10 stably expressing human PD-L1 (i.e., B16F10-hPD-L1; Southern Model Animal Center) was used in this assay. B16F10-hPD-L1 (1×10 6 ) was subcutaneously injected into 8-week-old C57BL / 6 mice. When the average tumor volume reached approximately 75 mm 3 , the mice were randomly grouped according to tumor volume, with 10 mice in each group. The sixth day after tumor implantation was the grouping day, which was defined as D0 day, and drug administration started on D0 day. The drugs were administered twice a week, and the tumor volume was measured twice. IgG1 control (Sino Biological, HG1K), antibody L1-R2-4-71, and fusion protein A were administered to the mice via intravenous injection. The efficacy of different test articles was evaluated by assessing the inhibition of tumor size. The tumor volume inhibition rate (TGI) was calculated as follows:

[0351] TGI = [1 - (TVt - TVinitial) / (CVt - CVinitial)] × 100%, where TVt represents the tumor volume at each measurement in the treatment group; TVinitial represents the tumor volume of the treatment group at the time of grouping and drug administration; CVt represents the tumor volume at each measurement in the control group; CVinitial represents the tumor volume of the control group at the time of grouping and drug administration. Figure 10 It can be seen that this model is insensitive to the PD-L1 monoclonal antibody, antibody L1-R2-4-71 has no obvious drug effect, while fusion protein A has a relatively obvious tumor inhibitory effect.

Claims

1. A fusion protein comprising: i. an anti-PD-L1 antibody or antigen-binding fragment; the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 as shown in any one of SEQ ID NOs: 1 and 91-95, HCDR2 as shown in any one of SEQ ID NOs: 2-11, HCDR3 as shown in SEQ ID NOs: 12 or 13, LCDR1 as shown in any one of SEQ ID NOs: 14-18, LCDR2 as shown in any one of SEQ ID NOs: 19-22, and LCDR3 as shown in any one of SEQ ID NOs: 23-26; ii. IL-15 or a fragment thereof; and iii. IL-15Rα or its sushi domain.

2. The fusion protein according to claim 1, characterized in that The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 24; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22, and LCDR3 shown in SEQ ID NO: 26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 21, and LCDR3 shown in SEQ ID NO: 26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 3, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 4, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 5, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:24; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:17, LCDR2 shown in SEQ ID NO:22, and LCDR3 shown in SEQ ID NO:26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:21, and LCDR3 shown in SEQ ID NO:26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:3, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:6, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:

23.

3. The fusion protein according to claim 1 or 2, characterized in that The anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; wherein The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence having at least 90% identity with a sequence as shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 27-41; and / or The light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs:42-47, or an amino acid sequence that has at least 90% identity with the sequence shown in any one of SEQ ID NOs:42-47, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs:42-47.

4. The fusion protein according to claim 3, characterized in that The heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-41, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-47; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

42.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region; wherein The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 48 or 49; and / or The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

50.

6. The fusion protein according to claim 5, characterized in that The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:48, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50; or The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

50.

7. The fusion protein according to any one of claims 1 to 4, characterized in that The anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region a, a heavy chain constant region b and a light chain constant region; the heavy chain constant region a and / or the heavy chain constant region b comprises an amino acid mutation selected from Y349C, S354C, T366W, T366S, L368A and Y407V; or The heavy chain constant region a comprises an amino acid mutation selected from S354C, T366W; and / or The heavy chain constant region b comprises an amino acid mutation selected from Y349C, T366S, L368A, and Y407V; The amino acid positions are Eu numbered.

8. The fusion protein according to claims 1-5 and 7, characterized in that: The heavy chain constant region comprises an amino acid mutation: K447A, wherein the amino acid positions are Eu numbered.

9. The fusion protein according to claim 7 or 8, characterized in that The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 77; and / or The heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO: 78, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 78, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 78; and / or The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

50.

10. The fusion protein according to claim 9, characterized in that The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, the heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

50.

11. The fusion protein according to any one of claims 1 to 6, characterized in that The anti-PD-L1 antibody comprises a heavy chain and a light chain; wherein The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having at least 90% identity with a sequence as shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 51-65; and / or The light chain comprises an amino acid sequence as shown in any one of SEQ ID NOs:66-71, or an amino acid sequence that has at least 90% identity with the sequence shown in any one of SEQ ID NOs:66-71, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs:66-71.

12. The fusion protein according to claim 11, characterized in that The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:69; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:70; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:71; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

66.

13. The fusion protein according to any one of claims 1 to 4 and 7 to 10, characterized in that: The anti-PD-L1 antibody comprises a heavy chain a, a heavy chain b and a light chain; wherein The heavy chain a comprises the amino acid sequence shown in SEQ ID NO: 79, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 79, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 79; and / or The heavy chain b comprises the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:80, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:80; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:66, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

66.

14. The fusion protein according to claim 13, characterized in that The heavy chain a comprises the amino acid sequence shown in SEQ ID NO:79, the heavy chain b comprises the amino acid sequence shown in SEQ ID NO:80, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

66.

15. The fusion protein according to any one of claims 1 to 14, characterized in that The IL-15 comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:82, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO:

82.

16. The fusion protein according to any one of claims 1 to 15, characterized in that The IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:81, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

81.

17. The fusion protein according to any one of claims 1 to 16, characterized in that The anti-PD-L1 antibody or antigen-binding fragment is connected to IL-15 or a fragment thereof and IL-15Rα or its sushi domain via a linker; alternatively, the C-terminus of one heavy chain of the anti-PD-L1 antibody is connected to IL-15 or a fragment thereof via a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is connected to IL-15Rα or its sushi domain via a linker.

18. The fusion protein according to claim 17, characterized in that The linker is a GS linker; or, the linker is independently selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof; or, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4, 5 or 6, and n is 1, 2, 3, 4 or 5.

19. A fusion protein, characterized in that The fusion protein comprises a first polypeptide, a second polypeptide and a third polypeptide; wherein The first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:83, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:83; and / or The second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:84, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:84; and / or The third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:66, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

66.

20. A fusion protein comprising a first polypeptide, a second polypeptide and a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:

66.

21. A biomaterial comprising (1) A polynucleotide characterized in that: It encodes the fusion protein or a portion thereof according to any one of claims 1 to 20; (2) A vector, characterized in that it contains a polynucleotide encoding the fusion protein according to any one of claims 1 to 20 or a portion thereof; or (3) A cell, characterized in that it contains a polynucleotide encoding the fusion protein according to any one of claims 1 to 20 or a portion thereof.

22. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 20; or further comprising a pharmaceutically acceptable excipient.

23. Use of the fusion protein according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 22 in preventing, treating or ameliorating a disease or in preparing a medicament for preventing, treating or ameliorating a disease; or, the disease is an infection, an autoimmune disease, cancer or a tumor.

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