Fusion protein and use thereof

Abstract

A fusion protein and a use thereof. 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. The fusion protein is used for prevention, treatment, or amelioration of diseases.

Description

Fusion proteins and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to fusion proteins and applications thereof. 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 with 114 amino acids and belongs to the common cytokine receptor gamma 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 + In addition, IL-15 can activate NK cells as well as effector and memory CD8 + 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 cells and NK cells, IL-15 typically forms a complex with the IL-15 receptor α expressed on APCs. The sushi domain (7.5 kDa) of IL-15Rα plays a key role in the formation of the complex between IL-15 and IL-15Rα. Summary of the Invention

[0005] The present invention provides a fusion protein and its application, 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 shown in SEQ ID NO: 79, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 79, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 79; and / or

[0147] The heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 80, or an amino acid sequence having one or more conservative amino acid substitutions compared 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 that has 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. The present invention also provides a polynucleotide encoding the fusion protein or a portion thereof. In some embodiments, the polynucleotide is an isolated polynucleotide.

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

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

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

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

[0165] In some embodiments, the disease includes but is not limited to infection (such as infection caused by bacteria, viruses, fungi or protozoa), autoimmune disease, cancer, 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.

[0166] 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

[0167] FIG1 is a schematic diagram of the structure of fusion protein A.

[0168] Figure 2 shows the experiment of fusion protein A binding to CTLL-2 cells.

[0169] Figure 3 shows the activity experiment of CTLL-2 cells stimulated by fusion protein A.

[0170] FIG4 shows an experiment of fusion protein A binding to HH cells.

[0171] FIG5 is an experiment showing the activation of HH cells by fusion protein A.

[0172] FIG6 is an experiment showing the binding of fusion protein A to CHO-K1-CD122-CD132 cells.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] FIG10 is an experiment showing that fusion protein A inhibits melanoma growth in mice. DETAILED DESCRIPTION

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

[0178] definition

[0179] 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.

[0180] 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.

[0181] 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.

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

[0183] The term "monoclonal antibody" (mAb) refers to a 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.

[0184] 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.

[0185] 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 .

[0186] The variable regions of the antibody light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, 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.

[0187] 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)).

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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).

[0192] "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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] "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.

[0202] As used herein, 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 disclosure. 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.

[0203] 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.

[0204] 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.

[0205] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" 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 of which use 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.

[0206] 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.

[0207] 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).

[0208] 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.

[0209] 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.

[0210] "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.

[0211] “EC 50 "Concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.

[0212] "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.

[0213] 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.

[0214] 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.

[0215] 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.

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

[0217] 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.

[0218] 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.

[0219] "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.

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

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

[0222] 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.

[0223] In some embodiments, the antibodies of the present invention use the "knobs-into-holes" technology (see, for example, John BBRidgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering, 9(7): p.617-21 (1996); Patent US8216805B2). This technology can modify the interface between different chains of an antibody to promote the correct association of each chain of the antibody. Generally, this technology involves introducing "knobs" ("knobs") at the interface of one chain and introducing corresponding "holes" ("holes") at the interface of the other chain to be paired with it, so that the protrusions can be placed in the holes. The protrusions can be constructed by replacing the 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 cavities of the same or similar size to the protuberances are created at the interface of the CH3 domains of the heavy chain constant domains of the other chain to be paired by replacing large amino acid side chains with smaller ones (e.g., amino acid substitutions T366S, L368A, and Y407V (Eu numbering)).

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

[0225] 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.

[0226] 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).

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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).

[0231] In some embodiments, the present invention also provides a fusion protein comprising a PD-L1 binding domain and a domain that stimulates NK and T cell 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.

[0232] 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.

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

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

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

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

[0237] In some embodiments, the IL-15 comprises the amino acid sequence 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.

[0238] 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.

[0239] 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.

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

[0241] In some embodiments, the variant of 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.

[0242] 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.

[0243] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment of the fusion protein is linked to IL-15 or its receptor-binding fragment or 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.

[0244] In some embodiments, the linker comprises glycine and serine ("GS linker"). 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.

[0245] In some embodiments, the fusion protein of the present invention comprises a first polypeptide, a second polypeptide, and a third polypeptide; the first polypeptide comprises an anti-PD-L1 heavy chain a, a linker, and IL-15Rα or its sushi domain or 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.

[0246] 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.

[0247] 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.

[0248] 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.

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

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

[0251] Methods for preparing antibodies and fusion proteins

[0252] The present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, fusion proteins, and derivatives thereof described herein. The polynucleotides disclosed herein can encode a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, a portion of a light chain variable region, a heavy chain, a light chain, or a fusion protein. Methods for preparing antibodies and fusion proteins are well known in the art and are described herein.

[0253] In certain embodiments, the prepared antibodies do not cause a harmful immune response in the animal to be treated (e.g., a human). In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementary determining regions (CDRs) into a human framework and constant region, retaining or not retaining key framework residues; or (c) transplanting the entire non-human variable region, but "hiding" them by replacing surface residues with human-like portions. Typically, the framework residues in the human framework region will be replaced by corresponding residues from the CDR donor antibody, such as residues that can improve antigen binding. These framework replacements can be identified by methods well known in the art, such as by modeling the interactions of CDRs and framework residues to identify framework residues that play an important role in antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See U.S. Patent No. 5,585,089; the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (WO1991009967; U.S. Patents Nos. 5,225,539, 5,530,101 and 5,585,089), repair or resurfacing (EP592,106; EP519,596; and chain rearrangement (U.S. Patent No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0254] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes changing antibodies to modify T cell epitopes (see, for example, WO2000034317 A2). For example, the heavy chain variable region sequence and light chain variable region sequence from the starting antibody are analyzed, and a human T cell epitope "map" from each variable region is generated, showing the position of the epitope relative to the complementary determining region (CDRs) and other key residues in the sequence. A single T cell epitope from the T cell epitope map is analyzed to identify optional amino acid substitutions with a lower risk of changing the antibody activity. A series of optional heavy chain variable region sequences and light chain variable region sequences containing amino acid substitution combinations are designed, and these sequences are subsequently incorporated into a series of binding polypeptides. The genes for the complete heavy and light chains containing the modified variable regions and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibodies.

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

[0256] The preparation of scFv can be seen in the technology for producing single-chain units (U.S. Patent No. 4,946,778). The heavy chain and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit, producing a single-chain fusion peptide. The technology for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 240:1038-1041 (1988)).

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

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

[0259] Antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. Reference may also be made to U.S. Patents 4,444,887 and 4,716,111, and PCT Publication Nos. WO 1998050433, WO 1998024893, WO 1998016654, WO 1996034096, WO 1996033735, and WO 1991010741, the entire contents of each of which are incorporated herein by reference.

[0260] In other embodiments, using conventional methods (such as using oligonucleotide probes that can specifically bind to the genes encoding antibody heavy and light chains), the DNA encoding the desired monoclonal antibody can be isolated and sequenced. Isolation and subcloning of hybridoma cells can be used as the source of this type of DNA. Once isolated, DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. Isolation of DNA (which can be synthetic as described herein) can also be used to prepare the sequence of the constant and variable regions of antibodies, as described in U.S. Patent No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR technology using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Patent No. 5,658,570.

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

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

[0263] In some embodiments, DNA encoding an antibody or fusion protein can be designed and synthesized according to conventional methods based on the amino acid sequence of the antibody or fusion protein described herein, inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in culture medium to produce the monoclonal antibody or fusion protein. In some embodiments, the vector for expressing the antibody or fusion protein comprises at least one promoter element, the antibody or fusion protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, 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, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV-1, and cytomegalovirus. Other cellular promoters, 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.

[0264] 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.

[0265] 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.

[0266] Treatment

[0267] The present invention also 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.

[0268] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or fusion protein or derivative used, the 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.

[0269] The method of administration of antibody or fusion protein or derivative includes but is not limited to by intradermal, muscle, abdominal cavity, vein, subcutaneous, nasal cavity, epidural and oral administration.The pharmaceutical composition can be used by any convenient approach, for example, by infusion or push injection, by epithelial or mucocutaneous (such as 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.

[0270] The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] In some embodiments, the dosage of the antibody or fusion protein of the present invention administered to the patient is 0.01 mg / kg to 100 mg / kg of patient body weight, or 0.1 mg / kg to 20 mg / 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, the dosage of which is approximately the same as or less than the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; or at least one week. A lower initial dose may also be used to increase tolerance, followed by increased dosing. The uptake and tissue penetration ability (e.g., 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.

[0275] 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.

[0276] Combination therapy

[0277] 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.

[0278] 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).

[0279] 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 patients. Some examples of therapeutic antibodies include rituximab, trastuzumab, tositumomab, ibritumomab tiuxetan, alemtuzumab, epratuzumab, bevacizumab, cetuximab, and berentuzumab, among others.

[0280] Pharmaceutical composition

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

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

[0283] The term "excipient" refers to a diluent, adjuvant, 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.

[0284] 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) representing the active ingredient content 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, the sterile water for injection or saline can be used to mix the active ingredient before administration.

[0285] The antibodies, antigen-binding fragments, or fusion proteins of the present invention 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.

[0286] Example

[0287] 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.

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

[0289] Example 1: Preparation of anti-PD-L1 antibody and fusion protein A

[0290] 1) Preparation of anti-PD-L1 antibodies

[0291] 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.

[0292] 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.

[0293] Table 1 Heavy and light chain composition of antibodies

[0294] Table 2 Antibody heavy chain and light chain CDR regions

[0295] Table 3 Composition of antibody heavy chain CDR region

[0296] Table 4 Composition of antibody heavy chain CDR region

[0297] Table 5 Composition of antibody light chain CDR region

[0298] Table 6 Antibody heavy chain variable region

[0299] Table 7 Antibody light chain variable region

[0300] Table 8 Antibody constant regions

[0301] Table 9 Antibody heavy chain sequences

[0302] Table 10 Antibody light chain sequences

[0303] 2) Preparation of fusion protein A

[0304] 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.

[0305] 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.

[0306] Table 11 Fusion protein A related amino acid sequence

[0307] Table 12 Fusion protein A related nucleic acid sequences

[0308] Example 2: Determination of binding of fusion protein A to CTLL-2 cells and detection of activity

[0309] 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.

[0310] 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 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.

[0311] Example 3: Determination of binding of fusion protein A to HH cells and detection of its activity

[0312] 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.

[0313] 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.

[0314] Example 4: Determination of binding of fusion protein A to CHO-K1-CD122-CD132 cells

[0315] A fluorescence-activated cell sorting (FACS)-based assay was used to assess the binding of fusion protein A to CHO-K1 cells (i.e., CHO-K1-CD122-CD132 cells) that exogenously express IL-2Rβ (CD122) and IL-2Rγ (CD132). 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, 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 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.

[0316] 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.

[0317] Example 5: In vitro activation of PBMC by fusion protein A

[0318] 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.

[0319] Example 6: Fusion protein A cytokine release experiment

[0320] 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.

[0321] Example 7: In vivo anti-tumor efficacy of fusion protein A

[0322] 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.

[0323] The mouse tumor-bearing model was prepared by implanting tumor cells into C57BL / 6 mice. In this assay, the murine melanoma cell line B16F10 stably expressing human PD-L1 (i.e., B16F10-hPD-L1; Southern 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 started on D0 day, and the drug was administered twice a week. 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. 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:

[0324] 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.

Claims

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

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

23.

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

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

42.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region.

6. The fusion protein according to claim 5, characterized in that The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 48 or 49; and / or The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

50.

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

50.

8. The fusion protein according to any one of claims 1 to 6, characterized in that The anti-PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region a, a heavy chain constant region b and a light chain constant region; the heavy chain constant region a and / or the heavy chain constant region b comprises an amino acid mutation selected from Y349C, S354C, T366W, T366S, L368A and Y407V; wherein the amino acid positions are Eu numbered.

9. The fusion protein according to claim 8, characterized in that The heavy chain constant region a comprises an amino acid mutation selected from S354C and T366W.

10. The fusion protein 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 fusion protein 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 fusion protein according to any one of claims 1 to 6 and 8 to 11, characterized in that: The heavy chain constant region comprises an amino acid mutation: K447A, wherein the amino acid positions are Eu numbered.

13. The fusion protein according to any one of claims 8 to 12, characterized in that The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:77, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

77.

14. The fusion protein according to any one of claims 8 to 13, characterized in that The heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:78, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

78.

15. The fusion protein according to any one of claims 8 to 14, characterized in that The heavy chain constant region a comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence 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, and 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 fusion protein according to any one of claims 8 to 15, characterized in that The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

50.

17. The fusion protein according to any one of 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 fusion protein according to any one of claims 1 to 7, characterized in that The anti-PD-L1 antibody comprises a heavy chain and a light chain; wherein The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having at least 90% identity with a sequence as shown in any one of SEQ ID NOs: 51-65, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 51-65; and / or The light chain comprises an amino acid sequence as shown in any one of SEQ ID NOs:66-71, or an amino acid sequence that has at least 90% identity with the sequence shown in any one of SEQ ID NOs:66-71, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs:66-71.

19. The fusion protein according to claim 18, characterized in that The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:69; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:70; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:71; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

66.

20. The fusion protein according to any one of claims 1 to 5 and 8 to 17, characterized in that: The anti-PD-L1 antibody comprises a heavy chain a, a heavy chain b and a light chain; wherein The heavy chain a comprises the amino acid sequence shown in SEQ ID NO: 79, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 79, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 79; and / or The heavy chain b comprises the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:80, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:80; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence that has at least 90% identity with 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 fusion protein according to claim 20, characterized in that The heavy chain a comprises the amino acid sequence shown in SEQ ID NO:79, the heavy chain b comprises the amino acid sequence shown in SEQ ID NO:80, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

66.

22. The fusion protein according to any one of claims 1 to 21, characterized in that The IL-15 comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:82, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO:

82.

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

82.

24. The fusion protein according to any one of claims 1 to 23, characterized in that The IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:81, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

81.

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

81.

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

27. The fusion protein according to claim 26, characterized in that 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 fusion protein according to claim 26 or 27, characterized in that The linker is a GS linker.

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

66.

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

66.

33. A biomaterial comprising (1) A polynucleotide characterized in that: It encodes the fusion protein or a portion thereof according to any one of claims 1 to 32; (2) A vector, characterized in that it contains a polynucleotide encoding the fusion protein according to any one of claims 1 to 32 or a portion thereof; or (3) A cell, characterized in that it contains a polynucleotide encoding the fusion protein according to any one of claims 1 to 32 or a portion thereof.

34. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 32; or further comprising a pharmaceutically acceptable excipient.

35. A method for preventing, treating or ameliorating a disease, characterized in that: The method comprises administering to a patient an effective amount of the fusion protein of any one of claims 1 to 32 or the pharmaceutical composition of claim 34.

36. The method of claim 35, wherein the disease is an infection, an autoimmune disease, a cancer or a tumor.

37. Use of the fusion protein according to any one of claims 1 to 32 or the pharmaceutical composition according to claim 34 in preventing, treating or ameliorating a disease or in preparing a medicament for preventing, treating or ameliorating a disease.

38. The use according to claim 37, wherein the disease is infection, autoimmune disease, cancer or tumor.

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