Method for treating tumor and use

Abstract

Provided are a method and use for treating a tumor by using a fusion protein. The method comprises administering to a patient an effective amount of the fusion protein. The fusion protein comprises an anti-PD-L1 antibody or an antigen-binding fragment, IL-15 or a fragment thereof, and IL-15Rα or a sushi domain thereof.

Description

Methods and uses for treating tumors Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method and use for treating tumors. 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 immunoinhibitory receptor expressed on activated T cells, B cells, and myeloid cells, and is a member of the CD28 immunoglobulin superfamily. PD-1's ligand, PD-L1, is also a type I transmembrane glycoprotein with a wide distribution: it is expressed on the surface of antigen-presenting cells such as B cells, T cells, dendritic cells, and macrophages, as well as in tumor tissues.

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

[0004] IL-15 is a 14-15 kDa glycoprotein composed of 114 amino acids and belongs to the shared cytokine receptor γ chain family, which also includes IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 is secreted by macrophages, dendritic cells, and monocytes. IL-15 can stimulate central memory CD8+ cells to exert immunity without regulating other T cells. Furthermore, IL-15 can activate NK cells as well as effector and memory CD8+ T cells and can rescue T cells from regulatory T cell (Treg)-induced apoptosis. The IL-15 receptor is composed of three subunits: IL-15Rα, IL-15Rβ, and IL-15Rγ. Before binding to the functional IL-15Rβ and γ units on T 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 complex formation between IL-15 and IL-15Rα. Summary of the Invention

[0005] In one aspect, the present invention provides a method for treating a tumor, such as a solid tumor, comprising administering an effective amount of a fusion protein to a patient; in another aspect, the present invention provides a use of a fusion protein in the preparation of a medicament for treating a tumor, such as a solid tumor; in another aspect, the present invention provides a use of a fusion protein in treating a tumor, such as a solid tumor, wherein the fusion protein comprises an anti-PD-L1 antibody or antigen-binding fragment, IL-15 or a fragment thereof, and IL-15Rα or a sushi domain thereof.

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

[0007] (a) HCDR1 comprising DSWIH; and / or

[0008] (b) HCDR2 comprising WISPYGGSTYYADX1X2X3X4 (SEQ ID NO: 86), X1 is S, D, H, G, P or Y, X2 is V, F, L, M or Y, X3 is K, R, G, S, V or H, and X4 is G, H, D, Q, S or A; and / or

[0009] (c) HCDR3 comprising RHWPGGX5X6X7 (SEQ ID NO: 87), X5 is F or L, X6 is D or L, and X7 is Y or P.

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

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

[0012] (b) HCDR2 comprising WISPYGGSTYYADX1X2X3X4 (SEQ ID NO: 86), X1 is S, D, H, G, P or Y, X2 is V, F, L, M or Y, X3 is K, R, G, S, V or H, and X4 is G, H, D, Q, S or A; and

[0013] (c) HCDR3 comprising RHWPGGX5X6X7 (SEQ ID NO: 87), X5 is F or L, X6 is D or L, and X7 is Y or P.

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

[0015] (a) HCDR1 comprising DSWIH; and / or

[0016] (b) HCDR2 comprising WISPYGGSTYYADX1X2X3X4 (SEQ ID NO: 86), X1 is S, D, H, G, P or Y, X2 is V, F, L, M or Y, X3 is K, R, G, S, V or H, and X4 is G, H, D, Q, S or A; and / or

[0017] (c) HCDR3 comprising RHWPGGX5X6X7 (SEQ ID NO: 87), X5 is F or L, X6 is D or L, and X7 is Y or P; and / or

[0018] (d) LCDR1, which contains X8ASQX9IX 10 X 11 X 12 LX 13 (SEQ ID NO: 88), X8 is L, Q or R, X9 is D, T or G, X 10 G or S, X 11 K, T, or S, X 12 H, W, F or Y, X 13 is N or A; and / or

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

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

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

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

[0023] (b) HCDR2 comprising WISPYGGSTYYADX1X2X3X4 (SEQ ID NO: 86), X1 is S, D, H, G, P or Y, X2 is V, F, L, M or Y, X3 is K, R, G, S, V or H, and X4 is G, H, D, Q, S or A;

[0024] (c) HCDR3 comprising RHWPGGX5X6X7 (SEQ ID NO: 87), X5 is F or L, X6 is D or L, and X7 is Y or P;

[0025] (d) LCDR1, which contains X8ASQX9IX 10 X 11 X 12 LX 13 (SEQ ID NO: 88), X8 is L, Q or R, X9 is D, T or G, X 10 G or S, X 11 K, T, or S, X 12 H, W, F or Y, X 13 N or A;

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

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

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

[0029] In some embodiments, HCDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 91-95, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, HCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 2-11, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, HCDR3 comprises the amino acid sequence of SEQ ID NOs: 12 or 13, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, the substitution variants are conservative amino acid substitution variants.

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

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

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

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

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

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

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

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

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

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

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

[0041] 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:12.

[0042] 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:3, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0043] 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:4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0044] 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:5, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0045] 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:6, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0046] 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:7, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0047] 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:8, and HCDR3 comprises the amino acid sequence set forth 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 of any one of SEQ ID NOs: 14-18, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, LCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 19-22, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, LCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 23-26, or a variant thereof having a single substitution, deletion, or insertion. In some embodiments, the substitution variants are conservative amino acid substitution variants.

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

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

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

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

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

[0062] In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:14, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:21, and LCDR3 comprises the amino acid sequence set forth 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) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having a single substitution, deletion, or insertion; and / or (b) a HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2-11, or a variant thereof having a single substitution, deletion, or insertion; and / or (c) a HCDR3 comprising the amino acid sequence of SEQ ID NOs: 12 or 13, or a variant thereof having a single substitution, deletion, or insertion; and / or (d) a LCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-18, or a variant thereof having a single substitution, deletion, or insertion; and / or (e) a LCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 19-22, or a variant thereof having a single substitution, deletion, or insertion; and / or (f) a LCDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-26, or a variant thereof having a single 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 the HCDR1 shown in SEQ ID NO: 1, the HCDR2 shown in any one of SEQ ID NOs: 2-11, the HCDR3 shown in SEQ ID NOs: 12 or 13, the LCDR1 shown in any one of SEQ ID NOs: 14-18, the LCDR2 shown in any one of SEQ ID NOs: 19-22, and the 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 a HCDR1 set forth in SEQ ID NO: 1, a HCDR2 set forth in any one of SEQ ID NOs: 2-11, a HCDR3 set forth in SEQ ID NOs: 12 or 13, a LCDR1 set forth in any one of SEQ ID NOs: 14-18, a LCDR2 set forth in any one of SEQ ID NOs: 19-22, and a LCDR3 set forth 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) a HCDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 91-95, or a variant thereof having a single substitution, deletion, or insertion; and / or (b) a HCDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2-11, or a variant thereof having a single substitution, deletion, or insertion; and / or (c) a HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13, or a variant thereof having a single substitution, deletion, or insertion; and / or (d) a LCDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 14-18, or a variant thereof having a single substitution, deletion, or insertion; and / or (e) a LCDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 19-22, or a variant thereof having a single substitution, deletion, or insertion; and / or (f) a LCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs: The amino acid sequence shown in any one of NOs: 23-26 or a variant thereof having 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 the HCDR1 shown in any one of SEQ ID NOs: 91-95, the HCDR2 shown in any one of SEQ ID NOs: 2-11, the HCDR3 shown in SEQ ID NOs: 12 or 13, the LCDR1 shown in any one of SEQ ID NOs: 14-18, the LCDR2 shown in any one of SEQ ID NOs: 19-22, and the 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 a HCDR1 set forth in any one of SEQ ID NOs: 91-95, a HCDR2 set forth in any one of SEQ ID NOs: 2-11, a HCDR3 set forth in SEQ ID NOs: 12 or 13, a LCDR1 set forth in any one of SEQ ID NOs: 14-18, a LCDR2 set forth in any one of SEQ ID NOs: 19-22, and a LCDR3 set forth in any one of SEQ ID NOs: 23-26.

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

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

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

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

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

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

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

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

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

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

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

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

[0081] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 set forth in SEQ ID NO:91, HCDR2 set forth in SEQ ID NO:2, HCDR3 set forth in SEQ ID NO:12, LCDR1 set forth in SEQ ID NO:15, LCDR2 set forth in SEQ ID NO:20, and LCDR3 set forth 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 set forth in SEQ ID NO:91, HCDR2 set forth in SEQ ID NO:2, HCDR3 set forth in SEQ ID NO:12, LCDR1 set forth in SEQ ID NO:17, LCDR2 set forth in SEQ ID NO:22, and LCDR3 set forth in SEQ ID NO:26.

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

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

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

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

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

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

[0090] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 set forth in SEQ ID NO:91, HCDR2 set forth in SEQ ID NO:7, HCDR3 set forth in SEQ ID NO:12, LCDR1 set forth in SEQ ID NO:14, LCDR2 set forth in SEQ ID NO:19, and LCDR3 set forth 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 that is at least 90% identical to 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 an amino acid sequence as shown in any one of SEQ ID NOs: 42-47, or an amino acid sequence that is at least 90% identical to a sequence as 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 a sequence as 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 kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is of one of the IgG, IgM, IgA, IgE, or IgD isotypes. 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, Fc is a variant Fc region. In some embodiments, relative to the parent Fc region, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions. In some embodiments, relative to the parent Fc region activity, the amino acid modifications in the Fc region change the effector function activity. In some embodiments, the variant Fc region can have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization or cell binding. In some embodiments, relative to the parent Fc region, the Fc region amino acid modifications can change the affinity of the variant Fc region to FcγR (Fcγ receptors). 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 of SEQ ID NO: 48 or 49, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 48 or 49, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of 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 that is at least 90% identical 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 "knobs-into-holes" stable association.

[0118] In some embodiments, the heavy chain constant region a and / or 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 numbered according to Eu.

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

[0120] In some embodiments, 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, Y407V; wherein the amino acid positions are numbered in Eu.

[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 numbered according to Eu.

[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 numbered according to Eu.

[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 numbered according to Eu.

[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 numbered according to Eu.

[0125] In some embodiments, the heavy chain constant region a comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of 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 that is at least 90% identical to 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 that is at least 90% identical to 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.

[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 that is at least 90% identical to 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 that is at least 90% identical to 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 that is at least 90% identical 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.

[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 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 the 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 the sequence as shown in any one of SEQ ID NOs: 51-65; and / or

[0132] The light chain of the anti-PD-L1 antibody comprises an amino acid sequence as 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 a full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).

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

[0146] In some embodiments, the heavy chain a comprises the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 79, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of 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 that is at least 90% identical to 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

[0148] The light chain comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO:66, or an amino acid sequence that has one or more conservative amino acid substitutions compared to 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 of SEQ ID NO:82, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:82, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of 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 that is at least 90% identical 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.

[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 a sushi domain thereof 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 a sushi domain thereof 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 of SEQ ID NO:83, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:83, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:83; and / or

[0157] The second polypeptide comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:84, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of 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 that is at least 90% identical to the sequence shown in SEQ ID NO:66, or an amino acid sequence that has 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.

[0161] 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.

[0162] In some embodiments, a method for preventing, treating, or ameliorating a disease is provided, comprising administering to a patient an effective amount of a fusion protein or pharmaceutical composition as described herein. In some embodiments, use of a fusion protein or pharmaceutical composition as described herein in preventing, treating, or ameliorating a disease is provided. In some embodiments, use of a fusion protein or pharmaceutical composition as described herein in the preparation of a medicament for preventing, treating, or ameliorating a disease is provided.

[0163] In some embodiments, the disease includes but is not limited to infection (such as infection caused by bacteria, viruses, fungi or protozoa), autoimmune diseases, cancer, tumors. In some embodiments, the disease is an advanced solid tumor. In some embodiments, the autoimmune disease includes but is 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.

[0164] In some embodiments, the patient has a histologically or cytologically diagnosed metastatic or locally advanced unresectable solid tumor that is not amenable to local therapy.

[0165] In some embodiments, the patient has a metastatic or locally advanced unresectable solid tumor that is histologically or cytologically diagnosed and is not suitable for local treatment; and the patient no longer benefits from or is not suitable for standard treatment. In some embodiments, the patient is diagnosed with a metastatic or locally advanced unresectable solid tumor that is not suitable for local treatment, has failed standard treatment or is not suitable for standard treatment, and has previously received PD-1 / PD-L1 monoclonal antibody treatment. In some embodiments, the effective amount of the fusion protein is about 0.01 μg / kg to about 2000 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 0.01 μg / kg to about 1000 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 0.1 μg / kg to about 1000 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 1 μg / kg to about 1000 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 10 μg / kg to about 800 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 0.1 μg / kg to about 800 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 0.1 μg / kg to about 200 μg / kg per administration. In some embodiments, the effective amount of the fusion protein is about 0.1 μg / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 160 μg / kg, about 200 μg / kg, about 400 μg / kg, about 600 μg / kg, about 800 μg / kg, about 1000 μg / kg, or about 2000 μg / kg, or a range between any two of these values ​​(including the endpoints) or any value therein, per administration.

[0166] In some embodiments, the effective amount of the fusion protein is about 0.001 mg to about 150 mg per administration. In some embodiments, the effective amount of the fusion protein is about 0.001 mg to about 100 mg per administration. In some embodiments, the effective amount of the fusion protein is about 0.006 mg to about 48 mg per administration. In some embodiments, the effective amount of the fusion protein is about 0.001 mg, about 0.006 mg, about 0.1 mg, about 0.6 mg, about 1.8 mg, about 6 mg, about 12 mg, about 24 mg, about 36 mg, about 48 mg, about 100 mg, or about 150 mg, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein.

[0167] In some embodiments, the fusion protein is administered once every 1-8 weeks. In some embodiments, the fusion protein is administered once every 10 days to 8 weeks. In some embodiments, the fusion protein is administered once every 10 days to 3 weeks. In some embodiments, the fusion protein is administered approximately once every 2 weeks.

[0168] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, with each dose ranging from about 0.01 μg / kg to about 2000 μg / kg.

[0169] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, for example, once every 2 weeks, with each dose ranging from about 0.01 μg / kg to about 2000 μg / kg.

[0170] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, for example, once every 2 weeks, with each dose ranging from about 0.01 μg / kg to about 1000 μg / kg.

[0171] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, for example, once every 2 weeks, with each dose being about 0.1 μg / kg to about 800 μg / kg.

[0172] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, for example, once every 2 weeks, with each dose being about 0.1 μg / kg to about 200 μg / kg.

[0173] In some embodiments, the effective amount of the fusion protein is administered once every 1-8 weeks, for example, once every about 2 weeks, with each dose being about 0.1 μg / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 160 μg / kg, about 200 μg / kg, about 400 μg / kg, about 600 μg / kg, or about 800 μg / kg.

[0174] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose ranging from about 0.01 μg / kg to about 2000 μg / kg.

[0175] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose ranging from about 0.01 μg / kg to about 1000 μg / kg.

[0176] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose ranging from about 0.1 μg / kg to about 800 μg / kg.

[0177] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 10 μg / kg.

[0178] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 30 μg / kg.

[0179] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 100 μg / kg.

[0180] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 200 μg / kg.

[0181] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 400 μg / kg.

[0182] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 600 μg / kg.

[0183] In some embodiments, the effective amount of the fusion protein is administered once every 2 weeks, with each dose being about 800 μg / kg.

[0184] In some embodiments, the fusion protein is administered approximately once every 2 weeks at a dose of about 0.01 μg / kg to about 2000 μg / kg. In some embodiments, the fusion protein is administered approximately once every 2 weeks at a dose of about 0.01 μg / kg to about 1000 μg / kg. In some embodiments, the fusion protein is administered approximately once every 2 weeks at a dose of about 0.1 μg / kg to about 800 μg / kg. In some embodiments, the fusion protein is administered approximately once every 2 weeks at a dose of about 0.1 μg / kg to about 200 μg / kg. In some embodiments, the fusion protein is administered about once every 2 weeks, and the dose of each administration is about 0.1 μg / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 160 μg / kg, about 200 μg / kg, about 400 μg / kg, about 600 μg / kg, about 800 μg / kg, about 1000 μg / kg, or about 2000 μg / kg, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0185] In some embodiments, the fusion protein is administered once every 2 weeks, and each dosage is about 0.001 mg-about 150 mg. In some embodiments, the fusion protein is administered once every 2 weeks, and each dosage is about 0.001 mg-about 100 mg. In some embodiments, the fusion protein is administered once every 2 weeks, and each dosage is about 0.006 mg-about 48 mg. In some embodiments, the fusion protein is administered once every 2 weeks, and each dosage is about 0.001 mg, about 0.006 mg, about 0.1 mg, about 0.6 mg, about 1.8 mg, about 6 mg, about 12 mg, about 24 mg, about 36 mg, about 48 mg, about 100 mg, or about 150 mg, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein.

[0186] In some embodiments, the fusion proteins provided herein can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Thus, the pharmaceutical composition can be administered subcutaneously, orally, rectally, parenterally, intracerebrally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops or transdermal patch), orally, or by oral or nasal spray.

[0187] In some embodiments, the fusion protein can be formulated into a pharmaceutical composition containing a single dose of the fusion protein as described above, such as a sterile isotonic aqueous solution, and administered to the patient in a form suitable for a selected route of administration, such as parenteral, intramuscular, topical or subcutaneous (sc). In some embodiments, the fusion protein is administered by subcutaneous injection.

[0188] The anti-PD-L1 antibody of the fusion protein of the present invention is in the form of a complete antibody, which can effectively 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 of CTLL-2 cells and significantly promote CD8 + T, NK and NKT cells expand and have better tumor suppression effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0189] FIG1 is a schematic diagram of the structure of fusion protein A.

[0190] Figure 2 shows the experiment of fusion protein A binding to CTLL-2 cells.

[0191] Figure 3 shows the activity experiment of CTLL-2 cells stimulated by fusion protein A.

[0192] FIG4 shows an experiment of fusion protein A binding to HH cells.

[0193] Figure 5 shows the experiment of fusion protein A activating HH cells.

[0194] FIG6 is an experiment showing the binding of fusion protein A to CHO-K1-CD122-CD132 cells.

[0195] Figure 7 shows the in vitro activation of PBMC by fusion protein A; Figure 7a shows the total cell count, and Figure 7b shows the CD8 + Figure 7c shows the percentage of T cells, Figure 7c shows the percentage of NKT cells, and Figure 7d shows the percentage of NK cells.

[0196] Figure 8 shows an experiment of cytokine release from activated PBMC in a solid phase state; Figures 8a-8e respectively show the concentrations of IL-2, IFN-γ, IL-6, IL-10, and TNF-α; in the figure, donor 1, donor 2, donor 3, and donor 4 are PBMC cells from different people.

[0197] Figure 9 shows an experiment of cytokine release from activated PBMC in a liquid phase; Figures 9a-9e show the concentrations of IL-2, IFN-γ, IL-6, IL-10, and TNF-α, respectively; in the figure, donor 1, donor 2, donor 3, and donor 4 are PBMC cells from different people.

[0198] FIG10 is an experiment showing the inhibition of melanoma growth by fusion protein A in mice. BIW indicates twice-weekly administration. DETAILED DESCRIPTION

[0199] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0200] definition

[0201] It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0202] As used herein, the terms "comprising" or "including" mean that the antibody, composition, or method, etc. includes the listed elements, such as components or steps, but does not exclude others. "Essentially consisting of" means that the antibody, composition, or method, etc. excludes other elements that have a fundamental effect on the characteristics of the combination, but does not exclude elements that do not substantially affect the antibody, composition, or method, etc. "Consisting of" means excluding elements not specifically listed.

[0203] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. 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.

[0204] 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).

[0205] The term "monoclonal antibody" (mAb) refers to a population of antibody molecules that contains only one species of antibody molecule 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 identical in all molecules in the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen.

[0206] The term "single-chain antibody" (scFv) refers to an antibody formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody via a linker of 15 to 20 amino acids. The linker can be rich in glycine to increase flexibility, as well as rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has been stripped of the constant region and a linker has been introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent No. 5,892,019.

[0207] Those skilled in the art will appreciate that the classes of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody, 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 herein. In one or more embodiments, the class of immunoglobulin molecule is IgG. Two heavy chains and two light chains are connected in a "Y" configuration by disulfide bonds, wherein the light chains start at the mouth of the "Y" and continue through the variable region to surround the heavy chains. Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can be associated with a κ or λ light chain. Generally speaking, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are bound by covalent bonds, and the "tails" 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 kappa light chain is V κ ; The variable region of the immunoglobulin λ light chain is V λ .

[0208] 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, and complement binding. By convention, the numbering of the constant regions increases as they become more distal to the antibody's antigen-binding site, or amino terminus. The N-terminal portion is the variable region, and the C-terminal portion 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.

[0209] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-continuous amino acid sequences that form the antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain, known as the "framework" ("FR") region, exhibit less intermolecular variability. The framework region largely adopts a β-pleated sheet conformation, with the CDRs forming loops attached thereto, or in some cases forming part of the β-pleated sheet structure. Thus, the framework region positions the CDRs in the correct orientation by forming a scaffold through non-covalent interactions between the chains. The antigen binding domain with specifically positioned CDRs forms a surface that is complementary to the epitope on the antigen, which promotes non-covalent binding between the antibody and its antigenic epitope. Typically, in an antibody molecule, each heavy chain and light chain has three CDRs, known as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. In order of position, the heavy chain variable region generally comprises VH FR1, HCDR1, VH FR2, HCDR2, VH FR3, HCDR3, and VH FR4, and the light chain variable region comprises VL FR1, LCDR1, VL FR2, LCDR2, LFR3, LCDR3, and VL FR4. For a given heavy or light chain variable region, one of ordinary skill in the art can identify the amino acids comprising the CDRs and framework regions by known methods (see Kabat, E., et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0210] The framework and CDR regions of the humanized antibody do not necessarily correspond exactly to the parent sequence. For example, the donor antibody CDR or the consensus framework may be mutated by substitution, insertion and / or deletion of at least one amino acid residue so that the CDR or framework residues at that site do not correspond to the donor antibody or the consensus framework. Typically, at least 80%, at least 85%, at least 90% or at least 95% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In the immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that most frequently occurs at that position in the family. If two amino acids occur equally frequently, either one may be included in the consensus sequence.

[0211] In the case where there are two or more definitions of a term used and / or accepted in the art, the definition of the term used herein includes all of these meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., US Pat. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference in their entirety.

[0212] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence independently of experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., US Pat. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies may also use the EU, Chothia, AbM, Contact, IMGT, or other numbering systems.

[0213] The antibodies disclosed herein can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken. In another embodiment, the variable region can be of condricthoid origin (e.g., from shark).

[0214] "Heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant 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.

[0215] A "light chain constant region" comprises a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a set of light and heavy chains that can form a dimer via a disulfide bond between the light chain CL domain and the heavy chain CH1 domain.

[0216] A "disulfide bond" refers to a covalent bond formed between two sulfur atoms. A thiol group of a 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.

[0217] A "chimeric antibody" refers to any antibody whose variable region is obtained or derived from a first species and whose constant region (which may be complete, 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.

[0218] As used herein, the term "epitope" includes any protein determinant region that is capable of specific binding to an immunoglobulin or fragment thereof or a T-cell receptor. Epitope determinants are typically composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and typically have specific three-dimensional structural properties as well as specific charge properties.

[0219] As used herein, the term "specific binding" or "immunoreaction" refers to the non-covalent interaction that occurs between one or more antigenic determinants of an immunoglobulin molecule and its target antigen. The intensity 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 larger affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Therefore, both "association rate constant" (kon) and "dissociation rate constant" (koff) can be determined by calculating the concentration and actual association and dissociation rates (see Malmqvist, M., Nature 361: 186-87 (1993)). The koff / kon ratio eliminates all parameters not related 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 radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those skilled in the art.

[0220] As used herein with respect to cells, nucleic acids, polypeptides, and the like, the term "isolated" refers to molecules that are separated from one or more of the other components of a cell's natural environment, such as DNA or RNA. The term "isolated" also refers to nucleic acids or peptides that are 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. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their natural state and do not exist in their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that have been 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 (inclusive) between any two of these values, or any value therein.

[0221] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide and that, in its native state or when manipulated by methods well known to those skilled in the art, can produce the polypeptide and / or its fragments via transcription and / or translation.

[0222] The term "recombinant" in reference to a polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not occur in nature, and by way of non-limiting example, can be produced by combination with a polynucleotide or polypeptide that does not normally exist.

[0223] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as α-amino acids and β-amino acids, which can be encoded by nucleic acids directly or in the form of precursors. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids.

[0224] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid and other unconventional amino acids of the twenty conventional amino acids may also be components suitable for use in the polypeptides of the present invention. Examples of unconventional 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 method 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.

[0225] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule formed by amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to the specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or interchangeably with, any of the foregoing terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it need not be translated from a specified nucleic acid sequence and may be produced by any means, including chemical synthesis.

[0226] The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) when the polynucleotide is RNA. A "polynucleotide sequence" can be represented by an alphabetical representation of the polynucleotide molecule. This alphabetical 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 searches. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST 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. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotides can be performed before or after assembly of the polynucleotides. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotides can be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of a polynucleotide disclosed herein includes a double-stranded form and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0227] 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" to another sequence means that when the sequences are aligned, that percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and percent identity or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expectation=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage identities and encode polypeptides having the same or similar biological activity.

[0228] Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the identity of the amino acid sequence remains at least 90%, such as at least 92%, 95%, 98% or 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that occur within a family of related amino acids in their side chains. Genetically encoded amino acids are broadly classified into the following categories: (1) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine, and histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine from the aliphatic-hydroxyl family; (ii) asparagine and glutamine from the amide-containing family; (iii) alanine, valine, leucine, and isoleucine from the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine from the aromatic family. In some embodiments, conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. For example, it is reasonable to predict that replacing leucine with isoleucine or valine alone, replacing aspartate with glutamate, replacing threonine with serine, or similarly replacing an amino acid with a structurally related amino acid 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 measuring the specific activity of the polypeptide derivative. Such assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art.

[0229] In some embodiments, the amino acid substitutions have the following effects: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or improve other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins whose sequences differ from the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain that forms intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991).

[0230] The number of amino acids in the conservative amino acid substitutions of VL and 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 amino acids in the 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.

[0231] As used herein, the term "label" or "labeled" refers to a polypeptide that incorporates a detectable label, for example, by incorporation of a radiolabeled amino acid, or attached to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or an enzymatic activity that can be detected by optical methods or calorimetry). In some cases, the label or tag can also be therapeutic. Various methods of 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 markers (e.g., FITC, rhodamine, lanthanide phosphors), enzyme markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent" or "drug" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0232] "About" refers to the normal error range of the corresponding numerical value that is easily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0233] “EC 50 "Concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.

[0234] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, relief of symptoms, reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or elimination of the disease state (whether partial or complete), prolongation of life expectancy compared to that expected in the absence of treatment, etc. Patients in need of treatment include those already suffering from the disease or disorder, those susceptible to the disease or disorder, or those in need of prevention of the disease or disorder, and those 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.

[0235] The term "tumor" refers to or is intended to describe a physiological state of a mammal, typically characterized by uncontrolled cell growth, including benign 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, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer.

[0236] As used herein, the terms "administer," "administer," and "apply" are used interchangeably and refer to the delivery of a substance (e.g., an antibody or fusion protein) to achieve a therapeutic purpose (e.g., treating a disease associated with PD-L1 or IL-15). 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, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0237] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, such as an antibody or fusion protein, that is sufficient to reduce or improve the severity and / or duration of a condition (e.g., cancer) or one or more of its symptoms; prevent the progression of the condition; cause the condition to subside; prevent the recurrence, development, onset, or progression of one or more symptoms associated with the condition; detect the condition; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). For example, an effective amount of an antibody or 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 one or more symptoms associated with cancer to a certain extent. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0238] 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, cows, etc.

[0239] As used herein, the term "in need thereof" refers to a patient who has been identified as being in need of a particular method or treatment. In some embodiments, identification can be performed by any diagnostic means. In any of the methods and treatments described herein, a patient may be in need thereof.

[0240] As used herein, the term "tumor therapeutic drug" refers to an agent having the functional property of inhibiting the development or progression of a tumor in the human body, especially a malignant (cancerous) lesion such as carcinoma, sarcoma, lymphoma or leukemia. Inhibiting metastasis is a property of anti-tumor drugs in many cases.

[0241] "Pharmaceutical composition" refers to a mixture of one or more compounds, pharmaceutically acceptable salts or prodrugs thereof and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable excipients and / or one or more other therapeutic agents.

[0242] The relevant descriptions of publications mentioned herein are incorporated by reference in their entirety.

[0243] Anti-PD-L1 antibodies and fusion proteins

[0244] The present invention provides antibodies or antigen-binding fragments with high affinity for the PD-L1 protein. These antibodies exhibit effective binding and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints and activate lymphocytes to release cytokines, potentially useful in treating various types of cancer, tumors, infections, and other related diseases.

[0245] In some embodiments, the antibodies of the present invention use the "knobs-into-holes" technology (see, e.g., John BB Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering, 9(7): p.617-21 (1996); Shane Atwell et al., Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol, 270: p.26-35 (1997); Paul Carter, Bispecific human IgG by design, Journal of Immunological Methods, 248, 7-15 (2001); US Patent No. 8216805B2). This technology can reshape the interface between different chains of an antibody to promote the correct association of each chain of the antibody. In general, this technique involves introducing "knobs" ("knobs") at the interface of one chain and corresponding "holes" ("holes") at the interface of the other chain to be paired, so that the protrusions can be placed in the holes. The protrusions can be constructed by replacing amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains, such as the amino acid substitution T366W (Eu numbering). Compensatory holes of the same or similar size as the protrusions 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 the amino acid substitutions T366S, L368A, and Y407V (Eu numbering).

[0246] 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 "knobs-into-holes" stable association.

[0247] It will also be understood by those skilled in the art that the sequences of the antibodies or antigen-binding fragments disclosed herein can be substituted, and the amino acid sequence after substitution is different from the naturally occurring amino acid sequence of the antibody. For example, the substituted amino acid sequence can be similar to the starting sequence, such as having a certain ratio of identity with the starting sequence, such as about 80%, about 85%, about 90%, about 95%, about 98%, about 99% identity with the starting sequence, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0248] In some embodiments, the antibodies or antigen-binding fragments comprise amino acid sequences with one or more modifying groups. For example, the antibodies or antigen-binding fragments disclosed herein may comprise a flexible linker sequence, or may be modified to add functional groups (e.g., PEG, drugs, toxins, or labels).

[0249] The antibodies, antigen-binding fragments, and fusion proteins disclosed herein include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody or antigen-binding fragment or its fusion protein, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment or its fusion protein from binding to the epitope. Examples include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0250] In some embodiments, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0251] The antibody or antigen-binding fragment can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody or antigen-binding fragment can then be determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0252] In some embodiments, to facilitate the expression of the antibody in host cells, a signal peptide sequence can also be added to the heavy chain and light chain of the antibody, for example, the heavy chain signal peptide: MEFGLSWVFLVAILKGVQC (SEQ ID NO: 75), the light chain signal peptide: MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 76).

[0253] 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 activity. 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 activity comprises IL-15 or its receptor binding fragment or variant and IL-15Rα or its sushi domain or variant. The sushi domain binds to IL-15 with high affinity, and the complex of IL-15 and the sushi domain has high activity in stimulating NK and T cell proliferation.

[0254] In some embodiments, the fusion protein comprises an anti-PD-L1 antibody or antigen-binding fragment described herein, IL-15 or its receptor-binding fragment or variant thereof, and IL-15Rα or its sushi domain or variant thereof.

[0255] 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 IL-15Rα.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] In some embodiments, the IL-15 comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:82, or an amino acid sequence having one or more conservative amino acid substitutions compared to SEQ ID NO:82.

[0260] In some embodiments, the IL-15Rαsushi domain comprises the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:81, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:81.

[0261] In some embodiments, the variant of IL-15 or its receptor binding fragment comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to IL-15 or its receptor binding fragment.

[0262] 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.

[0263] In some embodiments, the variant of IL-15Rα or its sushi domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to IL-15Rα or its sushi domain.

[0264] In some embodiments, the variant of IL-15Rα or its sushi domain has one or more conservative amino acid substitutions compared to IL-15Rα or its sushi domain.

[0265] 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 variant and IL-15Rα or its sushi domain or 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 variant 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 or 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 comprises 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 comprises the following amino acid mutations: S354C and T366W (EU numbering), forming a stable "knobs-into-holes" association, greatly promoting the correct assembly of the two heavy chains and minimizing mispairing.

[0266] In some embodiments, the linker comprises glycine and serine. 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. 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.

[0267] 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 variant thereof; the second polypeptide comprises an anti-PD-L1 heavy chain b, a linker, and IL-15 or its receptor-binding fragment or variant thereof; and the third polypeptide is an anti-PD-L1 light chain. In some embodiments, the structural schematic diagram of the fusion protein is shown in Figure 1 and consists of four polypeptides, including a first polypeptide, a second polypeptide, and two third polypeptides with identical sequences.

[0268] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO:83, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:83.

[0269] In some embodiments, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:84, or an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO:84, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:84.

[0270] In some embodiments, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO:66, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:66.

[0271] 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.

[0272] In some embodiments, the fusion protein is fusion protein A.

[0273] Methods for preparing antibodies and fusion proteins

[0274] DNA encoding the antibody or fusion protein can be designed and synthesized according to conventional methods based on the antibody or fusion protein amino acid sequence described herein, placed into an expression vector, and then transfected into host cells. The transfected host cells are cultured in culture medium to produce the monoclonal antibody or fusion protein. In some embodiments, the vector expressing the antibody or fusion protein includes at least one promoter element, the 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 either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and the early promoters of cytomegalovirus. Promoters from other cells, such as the actin promoter, can also be used. Suitable expression vectors may 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, mouse L cells and CHO cells, etc.

[0275] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection markers to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.

[0276] In addition, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the antibodies or fusion proteins of the present invention, including but not limited to site-directed mutagenesis and PCR-mediated mutations that result in amino acid substitutions. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original target protein. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutation, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

[0277] Treatment

[0278] The present invention provides treatment methods and uses. In some embodiments, a method for preventing, treating, or ameliorating various types of autoimmune diseases, cancers, tumors, infections, and related diseases is provided, comprising administering to a patient an effective amount of an anti-PD-L1 antibody, antigen-binding fragment, or fusion protein. In some embodiments, the use of an anti-PD-L1 antibody, antigen-binding fragment, or fusion protein for preventing, treating, or ameliorating autoimmune diseases, cancers, tumors, infections, and related diseases is provided. In some embodiments, the use of the anti-PD-L1 antibody, antigen-binding fragment, or fusion protein in the preparation of a medicament for preventing, treating, or ameliorating autoimmune diseases, cancers, tumors, infections, and related diseases is provided.

[0279] 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 patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are judged by a medical caregiver within the scope of those 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.

[0280] The method of administration of antibody or fusion protein or derivative includes but is not limited to intradermal, muscle, abdominal cavity, vein, subcutaneous, nasal cavity, epidural and oral injection.Pharmaceutical composition can be used by any convenient approach, for example, by infusion or push injection, by epithelial or mucocutaneous membrane (for example oral mucosa, rectum and intestinal mucosa etc.) absorption, and can be co-administered with other bioactivators.Therefore, the pharmaceutical composition containing antibody of the present invention or antigen-binding fragment or fusion protein can be orally administered, rectally, parenterally, intravesically (such as intravesical instillation), intracisternal administration, intravaginally, intraperitoneally, externally applied (such as by powder, ointment, drops or transdermal patch), oral administration or by oral or nasal spray administration.

[0281] The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0282] Administration can be systemic or local. In addition, it may be necessary to introduce the antibody or fusion protein of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be assisted by connecting the intraventricular catheter to a reservoir such as a reservoir (which can be an Ommaya reservoir). It can also be administered by pulmonary administration, for example, by using an inhaler or nebulizer, and using atomized preparations.

[0283] The antibodies or fusion proteins of the present invention can be administered topically to the area in need of treatment; this can be achieved by, but is not limited to, local infusion during surgery, for example, in conjunction with a postoperative wound dressing, by injection, by catheter, by suppository, or by implantation of porous, non-porous, or gel-like materials, including membranes (e.g., silicone rubber membranes) or fibers. Preferably, when administering a protein of the present invention (including an antibody or fusion protein), care must be taken to use a material that does not absorb the protein.

[0284] In some embodiments, the present invention provides nucleic acids or polynucleotides comprising antibodies or fusion proteins that can be administered in vivo to promote expression of the encoded protein by constructing them into a suitable nucleic acid expression vector, which can then be administered intracellularly, for example, by using retroviral vectors (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection reagents, or by linking to a homeobox peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

[0285] In some embodiments, the dosage of the antibody or fusion protein of the present invention administered to the patient is once every 1-8 weeks, with each dosage being about 0.01 μg / kg to about 2000 μg / kg of patient body weight. After the initial dose, a second dose or multiple doses of the antibody or antigen-binding fragment or fusion protein may be subsequently administered, with the dosage being roughly the same as or less than the initial dose. A lower initial dose may also be used to increase tolerance, followed by increased dosage administration. The uptake and tissue penetration ability (e.g., entry into the brain) of the antibody or fusion protein can be enhanced by modifications such as lipidation, thereby reducing the dosage and frequency of administration of the antibody or fusion protein of the present invention.

[0286] Various known delivery systems can be used to administer the antibodies or fusion proteins or derivatives of the present invention or polynucleotides encoding the same, 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 the nucleic acid as part of a retroviral or other vector, etc.

[0287] Combination therapy

[0288] In some embodiments, the anti-PD-L1 antibodies, antigen-binding fragments, or fusion proteins of the present invention can be used in conjunction with other treatment or prevention regimens, including administration of one or more antibodies, antigen-binding fragments, or fusion proteins of the present invention and one or more other therapeutic agents or methods. In some embodiments, other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, hormone therapy, and the like. For combination therapy, the antibody or fusion protein can be administered simultaneously or separately with the other therapeutic agent. When administered separately, the antibody or fusion protein of the present invention can be administered before or after administration of the other therapeutic agent.

[0289] In some embodiments, the antibodies or fusion proteins of the present invention are administered in combination with chemotherapeutic agents. In some embodiments, chemotherapeutic agents that can be administered with the antibodies or fusion proteins of the present invention include, but are 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, hydroxybenzoate, benzocaine, benzonatine ...

[0014] The present invention relates to a group of drugs that are administered orally, and includes, but are not limited to, steroids (e.g., steroids such as betamethasone sodium phosphate, steroids such as chlorambucil, methylurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate), hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorethoxyquin, and testolactone), nitrogen mustard derivatives (e.g., melphalan, chlorambucil, diethyltetramine (nitrogen mustard), and thiotepa), steroids and combinations thereof (e.g., betamethasone sodium phosphate), and other compounds (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0290] In some embodiments, an 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 a patient. Some examples of therapeutic antibodies include rituximab, trastuzumab, tositumomab, ibritumomab tiuxetan, alemtuzumab, epratuzumab, bevacizumab, cetuximab, and berentuzumab.

[0291] Pharmaceutical composition

[0292] 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 anticancer agent (e.g., an immune checkpoint inhibitor).

[0293] 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 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.

[0294] The term "excipient" refers to a diluent, adjuvant, vehicle, 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, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid, chelating agents, and agents for regulating tension such as or dextrose are also foreseeable. These compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release preparation etc.Said composition can be formulated into suppository with traditional adhesive and carrier such as triglyceride.Oral preparation can comprise standard carrier, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate etc.Such composition will contain the antibody or antigen-binding fragment or fusion protein of clinical effective dose, preferably in the form after purification, together with the adjuvant of suitable amount, to provide the administration form that is suitable for patient.Said preparation should be applicable to administration mode.Parent preparation can be encapsulated in ampoule bottle, disposable syringe or the multiple dose bottle made of glass or plastic.

[0295] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection in human body according to conventional steps. The composition for intravenous administration is generally a solution in a sterile isotonic aqueous buffer. The composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally speaking, the active ingredient is supplied alone or mixed together in a unit dose form, such as in a sealed container (such as an ampoule or a pouch) that indicates the amount of active agent in the form of a dry lyophilized powder or anhydrous concentrate. In the case of administering the composition by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be used so that the active ingredient can be mixed before administration.

[0296] The antibodies, antigen-binding fragments, or fusion proteins of the present invention may be in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, and tartaric acid, and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0297] Example

[0298] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0299] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0300] Example 1: Preparation of anti-PD-L1 antibody and fusion protein A

[0301] 1) Preparation of anti-PD-L1 antibodies

[0302] The composition and related sequences of the exemplary antibodies are shown in Tables 1-10; the composition of the heavy and light chains of the antibodies are shown in Table 1, the CDR regions of the heavy and light chains of the antibodies are shown in Table 2, the composition of the CDR region of the heavy chain of the antibodies is shown in Tables 3 and 4, and the composition of the CDR region of the light chain of the antibodies is shown in Table 5.

[0303] The DNA sequences encoding the antibody's heavy and light chains were cloned into expression vectors, and the plasmids were extracted. The heavy and light chains were transiently transfected into HEK293F cells at a molar ratio of 1:1. After cell culture and purification, the anti-PD-L1 antibody was obtained, and sequencing results were consistent with the predicted sequence.

[0304] Table 1 Heavy and light chain composition of antibodies

[0305] Table 2 Antibody heavy chain and light chain CDR regions

[0306] Table 3 Composition of antibody heavy chain CDR region

[0307] Table 4 Composition of antibody heavy chain CDR region

[0308] Table 5 Composition of antibody light chain CDR region

[0309] Table 6 Antibody heavy chain variable region

[0310] Table 7 Antibody light chain variable region

[0311] Table 8 Antibody constant region

[0312] Table 9 Antibody heavy chain sequences

[0313] Table 10 Antibody light chain sequences

[0314] 2) Preparation of fusion protein A

[0315] The structural schematic diagram of fusion protein A is shown in Figure 1 , which consists of four polypeptides: a first polypeptide (shown in SEQ ID NO:83), a second polypeptide (shown in SEQ ID NO:84), and two third polypeptides with identical sequences (shown in SEQ ID NO:66); wherein, the first polypeptide comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain a (shown in SEQ ID NO:79), a linker (shown in SEQ ID NO:85), and an IL-15Rα sushi domain (shown in SEQ ID NO:81); the second polypeptide comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain b (shown in SEQ ID NO:80), a linker (shown in SEQ ID NO:85), and IL-15 (shown in SEQ ID NO:82); and the third polypeptide is an anti-PD-L1 light chain; wherein the nucleic acid sequence of the first polypeptide is shown in SEQ ID NO:72, the nucleic acid sequence of the second polypeptide is shown in SEQ ID NO:73, and the nucleic acid sequence of the third polypeptide is shown in SEQ ID NO:74. The amino acid sequence of fusion protein A is shown in Table 11, and the nucleic acid sequence is shown in Table 12.

[0316] The DNA sequences encoding the first polypeptide, the second polypeptide and the third polypeptide of the fusion protein A were cloned into expression vectors respectively, and then transiently transfected into HEK293F cells. The fusion protein A was obtained through cell culture and purification.

[0317] Table 11 Fusion protein A related amino acid sequence

[0318] Table 12 Fusion protein A related nucleic acid sequences

[0319] Example 2: Determination of binding of fusion protein A to CTLL-2 cells and detection of activity

[0320] A fluorescence-activated cell sorting (FACS)-based assay was used to assess the binding of fusion protein A to CTLL-2 cells (a 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 a single cell suspension and mixed with 100 μL of fusion protein A samples of different concentrations (starting from 100 nM, 2-fold serial dilution, with 10 concentrations) in equal volumes (all in 96-well plates), with 500,000 cells per well. The mixture was equilibrated at 4°C for 60 minutes (min) and washed with PBS buffer. Phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, Cat. No. 12-4998-82) was then added as a secondary antibody and equilibrated at 4°C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. As shown in FIG2 , FACS binding assay demonstrated that fusion protein A was able to significantly bind to CTLL-2 cells.

[0321] The activity of fusion protein A was evaluated in a cell proliferation assay using CTLL-2 cells. 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 at 37°C with 5% CO2. The cells were cultured in suspension until they reached 5 × 10 cells per ml before being split. 5For viability analysis, 2-3 days after the final split, cells were washed with RPMI-1640 medium, resuspended in RPMI-1640 medium supplemented with 15% FBS, and plated at a density of 20,000 cells / well (50 μL) in a 96-well white plate. Side wells were filled with PBS. Fusion protein A was diluted as follows: Antibody L1-R2-4-71 and Fusion Protein A samples were diluted 1:3 starting at 200 nM in RPMI-1640 medium supplemented with 15% FBS, and 50 μL / well was added to the plated white plate. The plate was incubated at 37°C, 5% CO2 in a humidified incubator for 24 hours. Before the assay, the CellCounting-Lite 2.0 Luminescent Cell Viability Assay reagent was removed from the -20°C freezer and equilibrated to room temperature. The cell culture plate was removed from the incubator and equilibrated at room temperature (25±3°C) for 5 to 10 minutes. 50 μL of CellCounting-Lite 2.0 Luminescent Cell Viability Assay reagent (Norvozymes, Cat. No.: DD1101-02) was added to each well and incubated at room temperature in the dark for 5 to 30 minutes. Relative light units (RLU) were read using the Luminescence detection module on the SpectraMax multi-function microplate reader. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. As shown in Figure 3, the CTLL-2 cell proliferation assay showed that fusion protein A could significantly activate the proliferation activity of CTLL-2 cells, and EC 50 The value is 0.417nM.

[0322] Example 3: Determination of binding of fusion protein A to HH cells and detection of its activity

[0323] A fluorescence-activated cell sorting (FACS)-based assay was used 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. HH cells were resuspended in PBS buffer to a single-cell suspension and mixed with 100 μL of different concentrations of antibody L1-R2-4-71 or fusion protein A samples (starting concentration of 100 nM, 2-fold dilution, 11 concentration gradients) in equal volumes (all 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. Phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, catalog number: 12-4998-82) was then added as a secondary antibody and equilibrated at 4°C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using nonlinear regression using GraphPad PRISM 8 (GraphPad Software, San Diego, CA).As shown in Figure 4, FACS binding assays demonstrated that fusion protein A was able to significantly bind to HH cells.

[0324] After IL-15 binds to IL-15Rα, it transactivates and binds to IL-2Rβ and IL-2Rγ, activating downstream signaling pathways and leading to STAT5 phosphorylation. Fusion protein A samples were co-incubated with HH cells at varying concentrations to observe STAT5 phosphorylation and evaluate the ability of fusion protein A to activate HH cell viability. HH cells in the logarithmic phase were harvested, washed with PBS, and resuspended in prewarmed RPMI-1640 medium at 2 million μL / 100 μL. The cells were incubated at 37°C for 30 minutes. During this time, antibody L1-R2-4-71 or fusion protein A samples were diluted in RPMI-1640 medium containing 10% FBS, starting at 200 nM and then diluted two-fold to a total of 11 concentrations, 100 μL each. 100 μL of each diluted fusion protein A sample was mixed with an equal volume of cells and incubated at 37°C for 15 minutes. Then immediately place on ice, add an equal volume of 4% paraformaldehyde (FPA) solution (final concentration 2%), fix the cells, and place on ice for 30 minutes. Then wash with pre-cooled PBS buffer. Discard the supernatant, add 1 mL of pre-cooled 90% methanol, place on ice for 30 minutes, wash with pre-cooled PBS buffer, resuspend with PBS buffer, add PE anti-STAT5 Phospho (Tyr694) Antibody (BioLegend, Product No.: 936904), incubate at room temperature in the dark for 40 minutes, wash and detect on the machine (Beckman CytoFlex), and calculate the mean fluorescence intensity (MFI) value to evaluate the phosphorylation level of STAT5. Using nonlinear regression, GraphPad PRISM 8 (GraphPad Software, San Diego, CA) was used to analyze the data. As shown in Figure 5, the analysis of STAT5 phosphorylation levels showed that fusion protein A can significantly activate HH cell activity, EC 50 The value is 1.609nM.

[0325] Example 4: Determination of binding of fusion protein A to CHO-K1-CD122-CD132 cells

[0326] A fluorescence-activated cell sorting (FACS)-based assay was used to assess the binding of fusion protein A to CHO-K1 cells exogenously expressing IL-2Rβ (CD122) and IL-2Rγ (CD132) (i.e., CHO-K1-CD122-CD132 cells). CHO-K1-CD122-CD132 cells were resuspended in PBS buffer to a single-cell suspension and mixed with 100 μL of fusion protein A or antibody L1-R2-4-71 samples (starting from 100 nM, 2-fold serial dilution) at different concentrations (all in a 96-well plate), with 500,000 cells per well. The mixture was equilibrated at 4°C for 60 minutes and washed with PBS buffer. A phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, Cat. No. 12-4998-82) was then added as a secondary antibody and equilibrated at 4°C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression.As shown in Figure 6, FACS binding assays demonstrated that fusion protein A was able to significantly bind to CHO-K1-CD122-CD132 cells.

[0327] Method for constructing CHO-K1-CD122-CD132 cells: The vector connected to the CD122 gene sequence (NCBI Reference Sequence: NM_000878.5) was linearized and then electroporated into CHO-K1. After culture, the CHO-CD122 stable cell line was screened; based on the CHO-CD122 cell line, the lentivirus containing the CD132 gene sequence (NCBI Reference Sequence: NM_000206.3) was further infected, and the CHO-K1-CD122-CD132 stable cell line was screened after culture.

[0328] Example 5: In vitro activation of PBMC by fusion protein A

[0329] This example summarizes the effects of using a fusion protein A sample to induce selective activation and expansion of effector lymphocytes in human peripheral blood. The night before, 500 μL of 200 ng / mL anti-CD3 antibody (Nearshore Bio, GMP-A018) was used to coat a 6-well cell culture plate. The next morning, the supernatant was discarded and PBMCs resuspended in RPMI-1640 medium containing 15% FBS were added to each well at 1×10 6Cells were cultured in a total volume of 3 mL per well, and fusion protein A or antibody L1-R2-4-71 was added to final concentrations of 0.5 nM and 20 nM, respectively. PBMCs were cultured at 37°C, 5% CO2 for 7 days. PBMCs were then transferred to a new 6-well cell culture plate (without the anti-CD3 antibody) and supplemented with fusion protein A or antibody L1-R2-4-71, and cultured for another 5 days. CD8 + T, NK and NKT cells proliferate. + T is CD3CD8 double positive T cells (i.e. CD3 + CD8 + T cells), NK cells are CD16 or CD56 positive cells, and NKT cells are CD3 positive CD4CD8 double negative T cells (i.e. CD3 + CD4 - CD8 - T cells), the antibodies used for detection were 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 shown in Figure 7a-d. Compared with the antibody L1-R2-4-71, fusion protein A can significantly promote the expression of CD8 + T, NK, and NKT cell expansion.

[0330] Example 6: Fusion protein A cytokine release experiment

[0331] This example uses liquid and solid phase incubation systems to evaluate the cytokine release induced by different test articles. The dry pack method, i.e., solid phase incubation system, is to coat a 96-well cell culture plate with 25 μg / mL fusion protein A sample at a volume of 40 μL per well. After drying overnight in a clean bench, 1×10 5 PBMC cells (Leide Biotechnology, Guangzhou) were prepared in a volume of 200 μL. Wet pack method, i.e., liquid phase incubation system, 25 μg / mL fusion protein A sample was added to a 96-well cell culture plate, 100 μL / well, and 1×10 5PBMCs were cultured in a 100 μL volume. Anti-CD3 antibodies (Nearshore Bio, GMP-A018) and TGN1412 (a CD28 agonist antibody; sequence derived from patent US8709414B2) were used as positive control antibodies. After three days of incubation, supernatants were collected and assayed for IL-2, IFN-γ, IL-10, IL-6, and TNF-α (MABTECH, ELISABATIC kit). As shown in Figures 8a-8e and 9a-9e, TGN1412 significantly activated PBMCs in both liquid and solid-phase incubation systems, with release of IL-2, IL-10, IFN-γ, and TNF-α significantly exceeding that of the other test products. Anti-CD3 antibodies, used as control antibodies, also activated PBMCs to varying degrees. Fusion protein A, however, failed to activate PBMCs to release IL-2 in the solid-phase incubation system, but its efficacy in activating other cytokines was comparable to that of antibody L1-R2-4-71.

[0332] Example 7: In vivo anti-tumor efficacy of fusion protein A

[0333] This example describes in vivo experiments evaluating the functional blockade of PD-L1 and the functional activation of IL-15R by fusion protein A. Because PD-L1 monoclonal antibodies also bind to mouse PD-L1 and IL-15 also recognizes mouse receptors, wild-type mice can be used to directly evaluate the efficacy of different test articles in mouse tumor xenograft models.

[0334] 1) Mouse tumor-bearing model was prepared by implanting tumor cells into C57BL / 6 mice. In this assay, the mouse melanoma cell line B16F10 stably expressing human PD-L1 (i.e., B16F10-hPD-L1; Southern Model Animal Center) was used. B16F10-hPD-L1 (1×10 6 ) were injected subcutaneously into 8-week-old C57BL / 6 mice. When the average tumor volume reached 75 mm 3 Around 6 days after tumor implantation, the mice were randomly divided into groups according to the tumor volume, with 10 mice in each group. The 6th day after tumor implantation was the grouping day, which was defined as D0 day. The drug was administered on the grouping day D0 day, twice a week, and the tumor volume was measured twice. IgG1 control (Sino Biologics, HG1K), antibody L1-R2-4-71 and fusion protein A were administered to mice by intravenous injection (iv). The efficacy of different test products was evaluated by evaluating the inhibition of tumor size. The tumor volume inhibition rate (TGI) was calculated as follows:

[0335] TGI = [1-(TVt-TVinitial) / (CVt-CVinitial)] × 100%, where TVt represents the tumor volume of the treatment group at each measurement; TVinitial represents the tumor volume of the treatment group at the time of group dosing; CVt represents the tumor volume of the control group at each measurement; and CVinitial represents the tumor volume of the control group at the time of group dosing. Figure 10 shows that this model is insensitive to PD-L1 monoclonal antibodies. The antibody L1-R2-4-71 has no significant effect, while fusion protein A has a relatively significant tumor inhibitory effect.

[0336] 2) The antitumor activity of Fusion Protein A was verified using wild-type mice with a Balb / c background subcutaneously inoculated with CT26 colon cancer cells overexpressing human PD-L1 (CT26-hPD-L1; Southern Animal Center). Mice were dosed weekly. On day 14 after the start of dosing, the tumor volume inhibition (TGI) in the Fusion Protein A (2 mg / kg) group was 53.81%, a statistically significant difference compared to the PBS control group (P < 0.01).

[0337] Example 8: Clinical trial of fusion protein A

[0338] This study is designed as a multicenter, open-label, dose-escalation and expansion phase I clinical trial. The study mainly evaluates the safety, tolerability, pharmacokinetic characteristics and preliminary efficacy of fusion protein A in patients with advanced solid tumors, and provides a basis for recommended doses in subsequent clinical trials.

[0339] The study was divided into two parts: Part I: A single-agent dose-escalation study. Accelerated titration and a "3+3" dose-escalation strategy were used to explore the safe dose range. The following dose groups were set up: 0.1μg / kg, 1μg / kg, 5μg / kg, 10μg / kg, 20μg / kg, 30μg / kg, 40μg / kg, 80μg / kg, 100μg / kg, 120μg / kg, 160μg / kg, 200μg / kg, 400μg / kg, 600μg / kg, and 800μg / kg (subcutaneous injection, every 2 weeks). The first two groups used an accelerated titration approach, followed by dose escalation according to the standard "3+3" strategy. Part II: A dose-expansion study. Based on the preliminary safety and efficacy data from the dose-escalation phase, appropriate doses and tumor types were selected for expansion studies to further investigate the safety and clinical efficacy of Fusion Protein A and provide a basis for subsequent clinical studies.

[0340] Dosage regimen: Subcutaneous injection, once every 2 weeks (Q2W).

[0341] Clinical efficacy evaluation

[0342] Objective response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were defined according to RECIST 1.1.

[0343] Clinical results: Among patients who have received at least one treatment evaluation to date, one patient with ovarian cancer achieved stable disease in the low-dose 0.1 μg / kg group, one patient with breast cancer achieved stable disease in the 1 μg / kg group, two patients with stable disease in the 5 μg / kg group (thymic carcinoma and lung cancer), and three patients with stable disease in the 10 μg / kg group (one with breast cancer and two with lung cancer). Low-dose fusion protein A has initially shown an inhibitory effect on tumor progression. Due to the short follow-up period, further observation of its anti-tumor effects requires subsequent dose escalation and dose expansion phases.

[0344] Tolerability and Safety: No dose-limiting toxicity (DLT) was observed in the 0.1-5 μg / kg dose group of Fusion Protein A. DLT evaluation is ongoing for the 10 μg / kg dose group, and the maximum tolerated dose (MTD) has not yet been determined. Fusion Protein A has a controllable safety profile and is well tolerated.

[0345] Inclusion criteria

[0346] 1. Ages 18-75, genders are acceptable;

[0347] 2. Depending on the research stage, subjects must meet the following requirements:

[0348] 2.1. Dose escalation phase: Patients with histologically or cytologically diagnosed metastatic or locally advanced unresectable solid tumors that are not suitable for local treatment; and the subjects no longer benefit from standard treatment or are not suitable for standard treatment.

[0349] 2.2. Extension Phase: Subjects are diagnosed with metastatic or locally advanced unresectable solid tumors that are not suitable for local treatment, have failed standard treatment or are not suitable for standard treatment, and have previously received PD-1 / PD-L1 monoclonal antibody treatment.

[0350] 3. There must be at least one evaluable lesion in the dose-escalation phase; in the expansion phase, there must be at least one measurable lesion according to RECIST version 1.1;

[0351] 4. ECOG performance status score is 0 or 1;

[0352] 5. Life expectancy greater than 12 weeks;

[0353] 6. Have sufficient organ and bone marrow reserve function,

[0354] 7. Female subjects of childbearing potential must have a negative serum pregnancy test within 7 days before the first dose and be willing to take effective birth control / contraception methods to prevent pregnancy from the first dose of study treatment to 180 days after the last dose of study treatment; male subjects must agree to take adequate contraceptive measures from the first dose of study treatment to 180 days after the last dose of study treatment.

[0355] Exclusion criteria

[0356] 1. Patients who have received any other drug clinical trial treatment or participated in medical device clinical research within 4 weeks before the first administration of the study drug;

[0357] 2. Received other tumor treatment within 4 weeks before the first administration of study drug

[0358] 3. Patients who have undergone major organ surgery (excluding puncture biopsy) within 4 weeks before the first use of the study drug or have not yet recovered after surgery, or have suffered significant trauma, or are planning elective surgery during the trial;

[0359] 4. AEs caused by previous anti-tumor treatment have not recovered to CTCAE 5.0 ≤ 1, except for the following: a. Alopecia; b. Pigmentation; c. A distant toxicity caused by chemotherapy or radiotherapy that is judged to be irreversible; d. Hypothyroidism that has been stabilized by hormone replacement therapy;

[0360] 5. Patients with a known history of severe allergies, or subjects known to have had a grade 3 or higher allergic reaction to large molecular protein preparations / monoclonal antibodies;

[0361] 6. Patients with a history of non-infectious pneumonia requiring glucocorticoid treatment within 1 year before enrollment or currently have or have not ruled out interstitial pneumonia;

[0362] 7. Patients who have experienced grade ≥3 irAEs or have previously discontinued immunotherapy due to irAEs of any grade;

[0363] 8. Patients with symptomatic central nervous system metastases

[0364] 9. Severe cardiovascular disease: New York Heart Association (NYHA) class III or higher heart failure, left ventricular ejection fraction (LVEF) <50%, unstable angina, uncontrolled hypertension, a history of myocardial infarction within the past 6 months, or severe arrhythmia requiring medication control

[0365] 10. Patients with uncontrolled pleural effusion, pericardial effusion or ascites or those who need continued drainage;

[0366] 11. Subjects who have had a serious infection as determined by the investigator within 4 weeks before the first dose of the drug or subjects who have an active infection within 2 weeks before the first dose of the drug;

[0367] 12. Known history of human immunodeficiency virus (HIV) infection;

[0368] 13. Untreated active hepatitis B;

[0369] 14. Subjects with active HCV infection (HCV antibody positive and HCV-RNA level above the detection limit);

[0370] 15. Tuberculosis subjects who have not been treated or are currently being treated

[0371] 16. Those with a history of tissue or organ transplant surgery;

[0372] 17. History of autoimmune disease

[0373] 18. Received systemic glucocorticoids or other immunosuppressive treatment within 14 days before the first use of study drug;

[0374] 19. Patients who have received or plan to receive live / attenuated vaccines within 4 weeks before screening during the study period;

[0375] 20. Previous treatment with IL-2 or IL-15 agonists;

[0376] 21. The subject is pregnant or lactating, or is expected to become pregnant or give birth during the planned study period;

[0377] 22. Known history of psychotropic drug abuse or drug use, which is considered to affect compliance with this study;

[0378] 23. Other circumstances that the researcher deems unsuitable for participation in this study.

Claims

1. A method for treating tumors, characterized in that: The method comprises administering to a patient an effective amount of a fusion protein comprising: i. an anti-PD-L1 antibody or antigen-binding fragment thereof; the anti-PD-L1 antibody or antigen-binding fragment comprising a HCDR1 as set forth in any one of SEQ ID NOs: 1 and 91-95, a HCDR2 as set forth in any one of SEQ ID NOs: 2-11, a HCDR3 as set forth in SEQ ID NOs: 12 or 13, a LCDR1 as set forth in any one of SEQ ID NOs: 14-18, a LCDR2 as set forth in any one of SEQ ID NOs: 19-22, and a LCDR3 as set forth in any one of SEQ ID NOs: 23-26; ii. IL-15 or a fragment thereof; and iii. IL-15Rα or its sushi domain, The effective amount of the fusion protein is administered once every 1-8 weeks, with each administration dose being about 0.01 μg / kg to about 2000 μg / kg of patient body weight.

2. The method according to claim 1, wherein 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 method according to claim 1 or 2, wherein: 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 the amino acid sequence shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence that is at least 90% identical to 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; 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 is at least 90% identical to 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 method according to claim 3, wherein 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 method 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.

6. The method according to any one of claims 1 to 5; wherein The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence that is at least 90% identical 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 The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence that is at least 90% identical to 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.

7. The method according to claim 5, wherein 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.

8. The method according to any one of claims 1 to 6, wherein: 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, wherein the amino acid positions are numbered according to Eu.

9. The method according to claim 8, wherein The heavy chain constant region a comprises an amino acid mutation selected from S354C and T366W.

10. The method according to claim 8 or 9, characterized in that The heavy chain constant region b comprises an amino acid mutation selected from Y349C, T366S, L368A, and Y407V.

11. The method according to any one of claims 8 to 10, characterized in that The heavy chain constant region a comprises an amino acid mutation selected from S354C and T366W, and the heavy chain constant region b comprises an amino acid mutation selected from Y349C, T366S, L368A, and Y407V.

12. The method according to claims 1-6, 8-11, characterized in that The heavy chain constant region comprises an amino acid mutation: K447A, wherein the amino acid positions are numbered according to Eu.

13. The method according to any one of claims 8 to 12, wherein: The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 90% identical to 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.

14. The method according to any one of claims 8 to 13, wherein: The heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence that is at least 90% identical to 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.

15. The method according to any one of claims 8 to 14, wherein: The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 90% identical to 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 that is at least 90% identical to 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.

16. The method according to any one of claims 8 to 15, wherein: The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence that is at least 90% identical to 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.

17. The method according to claims 8 to 16, 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.

18. The method according to any one of claims 1 to 7, wherein: 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 that is at least 90% identical to 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 is at least 90% identical to 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.

19. The method according to claim 18, wherein 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.

20. The method according to any one of claims 1-5, 8-17, wherein 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 that is at least 90% identical 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 The heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence that is at least 90% identical to 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 that is at least 90% identical to the sequence shown in SEQ ID NO:66, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

66.

21. The method according to claim 20, wherein 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.

22. The method according to any one of claims 1 to 21, wherein: 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 to the sequence shown in SEQ ID NO:

82.

23. The method according to any one of claims 1 to 22, wherein: The IL-15 comprises the amino acid sequence shown in SEQ ID NO:

82.

24. The method according to any one of claims 1 to 23, wherein: The IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence that is at least 90% identical 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.

25. The method according to any one of claims 1 to 24, wherein The IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO:

81.

26. The method according to any one of claims 1 to 25, wherein: The anti-PD-L1 antibody or antigen-binding fragment is connected to IL-15 or a fragment thereof and IL-15Rα or a sushi domain thereof via a linker.

27. The method according to claim 26, wherein 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 a sushi domain thereof via a linker.

28. The method according to claim 26 or 27, wherein The linker is a GS linker.

29. The method of claim 28, wherein the linker is independently selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof.

30. The method of claim 28, wherein the linker is (G m S) n ,in, Each m is independently 1, 2, 3, 4, 5 or 6, and n is 1, 2, 3, 4 or 5.

31. A method for treating tumors, characterized in that: The method comprises administering to a patient an effective amount of a fusion protein comprising a first polypeptide, a second polypeptide, and a third polypeptide; wherein The first polypeptide comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:83, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:83; and / or The second polypeptide comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:84, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:84; and / or The third polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 66, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO: 66, The effective amount of the fusion protein is administered once every 1-8 weeks, with each administration dose being about 0.01 μg / kg to about 2000 μg / kg of patient body weight.

32. The method of claim 31, 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.

33. The method according to any one of claims 1 to 32, wherein: The effective amount of the fusion protein is about 0.1 μg / kg to about 1000 μg / kg, or about 1 μg / kg to about 1000 μg / kg, or about 10 μg / kg to about 800 μg / kg per administration.

34. The method according to any one of claims 1 to 33, wherein The effective amount of the fusion protein is a dose of about 0.1 μg / kg, or about 1 μg / kg, or about 5 μg / kg, or about 10 μg / kg, or about 20 μg / kg, or about 30 μg / kg, or about 40 μg / kg, or about 80 μg / kg, or about 100 μg / kg, or about 120 μg / kg, or about 160 μg / kg, or about 200 μg / kg, or about 400 μg / kg, or about 600 μg / kg, or about 800 μg / kg per administration.

35. The method according to any one of claims 1 to 34, wherein The fusion protein is administered once every 10 days to 8 weeks.

36. The method according to any one of claims 1 to 34, wherein The fusion protein is administered once every 10 days to 3 weeks.

37. The method according to any one of claims 1 to 34, wherein The fusion protein is administered approximately once every 2 weeks.

38. The method according to any one of claims 1 to 37, wherein The fusion protein is administered by subcutaneous injection.

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