WO2025119308A1 - Fusion protein and use thereof - Google Patents

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 protein and its application Technical Field

The present invention belongs to the field of biomedicine, and in particular relates to fusion protein and its application.

Background Art

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

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, enhance the immunity of tumor-specific CD8 ⁺ T cells, and help the immune system eliminate tumor cells.

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, IL-21. IL-15 is secreted by macrophages, dendritic cells and monocytes. IL-15 can stimulate central memory CD8 ⁺ cells to exert immunity, but has no regulatory effect on other T cells. In addition, IL-15 can activate NK cells as well as effector and memory CD8 ⁺ T cells and can rescue T cells from regulatory T cell (Treg)-induced apoptosis. The IL-15 receptor is composed of the following three subunits: IL-15Rα, IL-15Rβ and IL-15Rγ. Before binding to the functional IL-15Rβ and γ units on T cells and NK cells, IL-15 typically forms a complex with the IL-15 receptor α expressed on APCs. The sushi domain (7.5 kDa) of IL-15Rα plays a key role in the complex formation between IL-15 and IL-15Rα.

Summary of the invention

The present invention provides a fusion protein and an application 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.

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

(a) HCDR1 comprising DSWIH; and/or

(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

(c) HCDR3 comprising _RHWPGGX5X6X7 ( _SEQ ID NO:87 ₎ , _X5 is F or L, _X6 is D or L, and _X7 is Y or P.

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

(a) HCDR1, which comprises DSWIH;

(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

(c) HCDR3 comprising _RHWPGGX5X6X7 ( _SEQ ID NO:87 ₎ , _X5 is F or L, _X6 is D or L, and _X7 is Y or P.

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

(a) HCDR1 comprising DSWIH; and/or

(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

(c) HCDR3 comprising _RHWPGGX5X6X7 ( _SEQ ID NO:87 ₎ , _X5 is F or L, _X6 is D or L, _X7 is Y or P; and/or

(d) _LCDR1 comprising _{X8ASQX9IX10X11X12LX13} ( _SEQ ID _NO :88), _X8 is L, Q or R, _X9 is D, T or G, _X10 is _{G or} S, _X11 is K, T or S, _X12 is H, W, F or Y, and _X13 is N or A; and/or

(e) LCDR2 comprising _{X14ASX15LX16X17} ( _SEQ ID NO:89), _X14 is A or G, _X15 is T, N, S or R, _X16 is _Q or K, _{and X17} is S or T; and/or

(f) LCDR3 comprising _{QQX18X19X20TPX21T ( SEQ} ID NO:90 ₎ , _X18 is Y or S, _X19 is Y or F, _X20 is S or T, and _X21 is R or Y.

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

(a) HCDR1, which comprises DSWIH;

(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;

(c) HCDR3 comprising _RHWPGGX5X6X7 (SEQ ID NO: _{87 )} , _X5 is F or L, _X6 is D or L, and _X7 is Y or P;

(d) _LCDR1 comprising _{X8ASQX9IX10X11X12LX13} ( _SEQ ID _NO :88), _X8 is L, Q or R, _X9 is D, T or G, _X10 is _{G or} S, _X11 is K, T or S, _X12 is H, W, F or Y, and _X13 is N or A;

(e) LCDR2 comprising _{X14ASX15LX16X17} (SEQ ID NO:89), _X14 is A or G, _X15 is T, N, S or R, _X16 is _Q or K, _{and X17} is S or T; _and

(f) LCDR3 comprising _{QQX18X19X20TPX21T ( SEQ} ID NO:90 ₎ , _X18 is Y or S, _X19 is Y or F, _X20 is S or T, and _X21 is R or Y.

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

In some embodiments, HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 91-95 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-11 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NOs: 12 or 13 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, the substitution variant is a conservative amino acid substitution variant.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:92, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:93, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:94, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:95, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:13.

In some embodiments, LCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 14-18 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, LCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 19-22 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, LCDR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 23-26 or a variant thereof having a single site substitution, deletion or insertion. In some embodiments, the substitution variant is a conservative amino acid substitution variant.

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

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

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

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

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

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

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

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 NO: 2-11, the HCDR3 shown in SEQ ID NO: 12 or 13, the LCDR1 shown in any one of SEQ ID NO: 14-18, the LCDR2 shown in any one of SEQ ID NO: 19-22, and the LCDR3 shown in any one of SEQ ID NO: 23-26.

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

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

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.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises a HCDR1 shown in any one of SEQ ID NOs: 91-95, a HCDR2 shown in any one of SEQ ID NOs: 2-11, a HCDR3 shown in SEQ ID NOs: 12 or 13, a LCDR1 shown in any one of SEQ ID NOs: 14-18, a LCDR2 shown in any one of SEQ ID NOs: 19-22, and a LCDR3 shown in any one of SEQ ID NOs: 23-26.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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).

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.

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.

In some embodiments, the heavy 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: 27-41, or an amino acid sequence that is at least 90% identical to 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.

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.

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 NO: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 NO:42-47.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

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 substitution, deletion or insertion. In some embodiments, relative to the parent Fc region activity, the amino acid modification of the Fc region changes the effector function activity. In some embodiments, the variant Fc region may have changed (i.e., increased or reduced) antibody-dependent cellular toxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization or cell binding. In some embodiments, relative to the parent Fc region, the Fc region amino acid modification can change the affinity of the variant Fc region to FcγR (Fcγ receptor). In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A.

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.

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).

In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence that has 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 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 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.

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.

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.

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).

In some embodiments, the heavy chain constant region a and the heavy chain constant region b form a "knobs-into-holes" stable association.

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 in Eu.

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

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.

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.

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.

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 in Eu.

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.

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 has at least 90% identity with the sequence shown in SEQ ID NO:77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:77; and/or

The heavy chain constant region b comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence 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; 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.

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 that is at least 90% identical to the sequence shown in SEQ ID NO:78, or an amino acid sequence that is 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 that is having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:50.

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.

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

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 that has 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

The light chain of the anti-PD-L1 antibody comprises an amino acid sequence shown in any one of SEQ ID NO:66-71, or an amino acid sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: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 NO:66-71.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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

In some embodiments, the heavy chain a comprises the amino acid sequence shown in SEQ ID NO:79, or an amino acid sequence that has 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.

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.

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

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 has at least 90% identity with the sequence shown in SEQ ID NO:81, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:81.

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.

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.

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.

In some embodiments, 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 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.

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.

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.

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.

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.

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

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

In some embodiments, the disease includes but is not limited to infection (such as infection caused by bacteria, viruses, fungi or protozoa), autoimmune diseases, cancer, tumors. In some embodiments, the autoimmune disease includes but is not limited to alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre 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.

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, produce local immune activation, which can not only improve the anti-tumor effect but also increase safety. Compared with the anti-PD-L1 antibody, the fusion protein of the present invention can significantly activate the proliferation activity of CTLL-2 cells, can significantly promote the proliferation of CD8 ⁺ T, NK and NKT cells, and has a better tumor inhibition effect.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

FIG3 is an experiment showing the activity of CTLL-2 cells stimulated by fusion protein A.

FIG. 4 is an experiment showing the binding of fusion protein A to HH cells.

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

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

Figure 7 is an in vitro PBMC activation experiment with fusion protein A; Figure 7a is the total cell number, Figure 7b is the percentage of CD8 ⁺ T cells, Figure 7c is the percentage of NKT cells, and Figure 7d is the percentage of NK cells.

Figure 8 is an experiment of activating PBMC to release cytokines 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.

Figure 9 is an experiment of activating PBMC to release cytokines in liquid phase; Figures 9a-9e 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.

FIG. 10 is an experiment showing that fusion protein A inhibits melanoma growth in mice.

DETAILED DESCRIPTION

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

definition

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.

As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. include the listed elements, such as components or steps, but do not exclude others. "Essentially consisting of" means that the antibody, composition, method, etc. exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically listed.

The term "antibody" as used herein 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.

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 (eg, Fab, Fab' and F(ab') ₂ ).

The term "monoclonal antibody" (mAb) refers to a population of antibody molecules that contain only one molecular species of antibody molecules composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen.

The term "single-chain antibody" (scFv) refers to an antibody formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody through a linker of 15 to 20 amino acids. The linker can be rich in glycine to increase flexibility, 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, and vice versa. Although the protein has been removed of the constant region and introduced into the linker, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, there is a relevant description in U.S. Patent No. 5,892,019.

Those skilled in the art will appreciate that the categories of heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), among which there are some subclasses (e.g., γ1-γ4). The nature of the chain determines the "type" of the antibody, IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been fully characterized and the functional specificity conferred is also known. All immunoglobulin types are within the scope of protection disclosed in the present invention. In one or more embodiments, the type of immunoglobulin molecule is IgG. Two heavy chains and two light chains are connected in a "Y" configuration by disulfide bonds, wherein the light chain starts from the "Y" mouth and continues through the variable region to surround the heavy chain. Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can be combined with a κ or λ light chain. Generally speaking, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light chains and heavy chains are bound by covalent bonds, and the "tail" parts of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of 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 lambda light chain is V _λ .

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

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 specifically bind to the antigen, forming the antigen binding domain, assuming that the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, known as the "framework" ("FR") region, show less intermolecular variability. Most of the framework region adopts a β-fold conformation, and the CDR forms a ring structure connected thereto, or in some cases forms part of the β-fold structure. Therefore, the framework region positions the CDR in the correct orientation by forming a scaffold through non-covalent interactions between chains. The antigen binding domain with CDRs in specific positions forms a surface complementary to the epitope on the antigen, which promotes the non-covalent binding of the antibody and its antigenic epitope. Typically, in an antibody molecule, each heavy chain and light chain has three CDRs, which are referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. In order of position, the heavy chain variable region usually includes VH FR1, HCDR1, VH FR2, HCDR2, VH FR3, HCDR3 and VH FR4, and the light chain variable region includes VL FR1, LCDR1, VL FR2, LCDR2, LFR3, LCDR3 and VL FR4. For a given heavy chain or light chain variable region, a person skilled in the art can identify the amino acids comprising the CDR and framework regions by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

The framework region and CDR region of the humanized antibody do not have to correspond exactly to the parent sequence, for example, the donor antibody CDR or the common framework may be mutated by substitution, insertion and/or deletion of at least one amino acid residue, so that the CDR or framework residue at the site does not correspond to the donor antibody or the common framework. Typically, at least 80%, at least 85%, at least 90% or at least 95% of the humanized antibody residues will correspond to those residues of the parental FR and CDR sequences. As used herein, the term "common framework" refers to the framework region in the common immunoglobulin sequence. As used herein, the term "common immunoglobulin sequence" refers to the sequence formed by the most frequently occurring amino acids (or nucleotides) in the 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 common 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 common sequence.

Where a term is used and/or accepted in the art and has two or more definitions, 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-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety.

Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. A person of ordinary skill in the art can apply the "Kabat numbering" system to any variable region sequence without relying on other experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia, AbM, Contact, IMGT and other numbering systems.

The antibodies disclosed in the present invention 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 antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

"Heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., an 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.

The "light chain constant region" includes a portion of the amino acid sequence from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light chains and heavy chains that can form dimers through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

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

"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.

The term "epitope" as used herein includes any protein determining region that can specifically bind to an immunoglobulin or fragment thereof or a T cell receptor. The epitope determining region is usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually has specific three-dimensional structural properties and specific charge properties.

As used herein, the term "specific binding" or "immunoreaction" refers to the non-covalent interaction between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents a greater affinity. The 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 an 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 the "binding rate constant" (kon) and the "dissociation rate constant" (koff) can be determined by calculating the concentration and the actual association and dissociation rates (see Malmqvist, M., Nature 361: 186-87 (1993)). The koff/kon ratio eliminates all affinity-independent parameters 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.

The term "isolated" used in the present invention for cells, nucleic acids, polypeptides, etc., for example, "isolated" DNA, RNA, polypeptides refer to molecules separated from one or more of other components such as DNA or RNA in the natural environment of the cell. The term "isolated" used in the present invention also refers to nucleic acids or peptides that are substantially free of cell material, viral material or cell culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals during chemical synthesis. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in a natural state and will not exist in a natural state. The term "isolated" is also used in the present invention to refer to cells or polypeptides separated from other cell proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, etc. are generally prepared by at least one purification step. In one or more embodiments, the purity of isolated nucleic acids, polypeptides, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including end values) between any two of these values or any value therein.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which, 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.

The term "recombinant" in relation 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 of polynucleotides or polypeptides that do not normally occur.

"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.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, E.S. Golub and D.R. Gren, ed., 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 notation 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.

The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule formed by amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, 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" can be used to replace any of the above terms, or to be used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection/blocking groups, proteolytic cleavage or non-natural amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.

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 the letters of the polynucleotide molecule. The letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as 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 may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modifications to the nucleotides may be performed before or after assembling the polynucleotides. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may 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 a double-stranded form.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" with another sequence means that when the sequences are aligned, that percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and the identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; 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.

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 roughly divided 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, 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 a single replacement of leucine with isoleucine or valine, replacement of aspartate with glutamate, replacement of threonine with serine, or similar replacement of 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 where the replacement 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 determinations 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.

In some embodiments, the amino acid substitution has the following effects: (1) reducing sensitivity to proteolysis, (2) reducing sensitivity to oxidation, (3) changing binding affinity for forming protein complexes, (4) changing binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. Analogs may 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) may 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 change the structural properties of the parent sequence (for example, 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., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

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 of 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.

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 linked by spacer arms of various lengths to reduce possible steric hindrance. The term "agent" or "drug" refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

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

"EC ₅₀ " or concentration for 50% of maximal effect (EC ₅₀ ) refers to the concentration that can cause 50% of the maximal effect.

"Treatment" refers to both therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent, slow down, ameliorate or stop undesirable physiological changes or disorders, 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 extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or disappearance of the disease state (whether partial or complete), extension of the expected survival period compared to not receiving treatment, etc. Patients in need of treatment include patients who already have a disease or disorder, patients who are susceptible to a disease or disorder, or patients in need of prevention of the disease or disorder, and patients who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions disclosed in the present invention for detection, diagnostic procedures and/or treatment.

The term "tumor" means or is intended to describe a physiological state of a mammal, which is 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.

As used herein, the terms "administer", "administering" and "applying" 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). The administration method 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.

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 symptoms thereof; prevent the progression of a condition; cause regression of a condition; prevent the recurrence, development, onset or progression of one or more symptoms associated with a condition; detect a 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.

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.

As used herein, the term "in need" refers to a patient who has been identified as needing 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.

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. Inhibition of metastasis is a property of anti-tumor drugs in many cases.

"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.

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

Anti-PD-L1 antibodies and fusion proteins

The present invention provides antibodies or antigen-binding fragments with high affinity for PD-L1 protein. The antibodies exhibit effective binding activity and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints, activate lymphocytes to release cytokines, and are used to treat various types of cancers, tumors, infections, and other related diseases.

In some embodiments, the antibodies of the present invention use the "knobs-into-holes" technology (see, for example, John B.B. Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering, 9(7): p.617-21 (1996); Patent US8216805B2). This technology can modify the interface between different chains of an antibody to promote the correct association of the chains 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 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 domain of the other chain to be paired with by replacing large amino acid side chains with smaller ones (e.g. the amino acid substitutions T366S, L368A and Y407V (Eu numbering)).

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.

It should also be understood by those skilled in the art that the antibody or antigen-binding fragment sequence disclosed in the present invention can be replaced, and its amino acid sequence after replacement is different from the naturally occurring amino acid sequence of the antibody. For example, the replaced amino acid sequence can be similar to the starting sequence, such as having a certain ratio of identity with the starting sequence, such as it can be about 80%, about 85%, about 90%, about 95%, about 98%, about 99% identical to the starting sequence, or a range (including endpoints) between any two of these values or any value therein.

In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence with one or more modification groups. For example, the antibody or antigen-binding fragment disclosed in the present invention may comprise a flexible linker sequence, or may be modified to add a functional group (e.g., PEG, a drug, a toxin, or a label).

The antibodies, antigen binding fragments and fusion proteins disclosed in the present invention 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. Including but not limited to the following examples, the antibody or antigen binding fragment or its fusion protein can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protection/blocking groups, proteolytically cleaved, attached to cell ligands or other proteins, etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

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

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 is then 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.

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

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 thereof and IL-15Rα or its sushi domain or variant thereof. 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.

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.

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.

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.

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 a sushi domain of IL-15Rα.

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.

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

In some embodiments, the IL-15Rαsushi domain comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with 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.

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.

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.

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.

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.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment of the fusion protein is connected 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 connected 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 connected 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 connected to IL-15 via a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is connected 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 association of "Knobs-into-Holes", which greatly promotes the correct assembly of the two heavy chains and minimizes mispairing.

In some embodiments, the linker comprises glycine and serine ("GS linker"). In some embodiments, the linker is ( _GmS ) _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 .

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

In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence that has at least 90% identity with 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.

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

In some embodiments, the third polypeptide 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.

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.

In some embodiments, the fusion protein is fusion protein A.

Methods for preparing antibodies and fusion proteins

The present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, fusion proteins and derivatives thereof of the present invention. The polynucleotides disclosed in the present invention may 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, etc. Methods for preparing antibodies and fusion proteins are well known in the art and are described in the present invention.

In certain embodiments, the prepared antibodies do not cause harmful immune responses in the animal (e.g., human) to be treated. In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed in the present invention 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, usually murine or primate antibodies, which retain or substantially retain the antigen binding properties of the parent antibody but have low immunogenicity in humans. It can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into the 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 the 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 parts. Usually 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 simulating the interaction of CDR and framework residues to identify framework residues that play an important role in antigen binding and by sequence comparison to identify abnormal framework residues at specific positions. (See U.S. Pat. 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. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP592,106; EP519,596; and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

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 the 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 within the sequence. Analyze individual T cell epitopes from the T cell epitope map to identify selectable amino acid substitutions with a lower risk of changing antibody activity. Design a series of optional heavy chain variable region sequences and light chain variable region sequences containing amino acid substitution combinations, and then incorporate these sequences 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 then transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibodies.

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

The preparation of scFv can be seen in the technology of producing single chain units (U.S. Patent 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 of assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 240:1038-1041 (1988)).

Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include those described in U.S. Pat. Nos. 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. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.

In addition, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10: 1455-1460 (1992), in particular, the technology can produce primate antibodies containing monkey variable region and human constant region sequences, and the entire content of this reference 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.

Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT publications 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.

In other embodiments, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that can specifically bind to genes encoding heavy and light chains of antibodies). Separated and subcloned hybridoma cells can be used as the source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a prokaryotic or eukaryotic host cell such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce other immunoglobulins. Separated DNA (which can be synthetic as described herein) can also be used to prepare sequences of 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. The 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.

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 in 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 technical scope of the art are also covered in the present invention.

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 known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated by reference in their entirety.

In some embodiments, the DNA encoding the antibody or fusion protein can be designed and synthesized according to the antibody or fusion protein amino acid sequence described herein by conventional methods, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium to produce a monoclonal antibody or fusion protein. In some embodiments, the expression antibody or fusion protein vector includes at least one promoter element, an antibody or fusion protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and promoters of other cells such as actin promoters 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.

In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein.

In addition, standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding the antibody or fusion protein 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. Or mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutations, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

Treatment

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

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 medical care personnel included in the scope of ordinary technicians in the field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of preparation, 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.

The method of administration of antibody or fusion protein or derivative includes but is not limited to intradermal, muscle, peritoneal, intravenous, subcutaneous, nasal, epidural and oral administration. The pharmaceutical composition can be applied by any convenient route, such as by infusion or push injection, absorbed by epithelium or skin mucosa (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Therefore, the pharmaceutical composition containing antibody or antigen binding fragment of the present invention or fusion protein can be administered orally, rectally, parenterally, intravesically (such as intravesical instillation), intracisternal administration, intravaginal administration, intraperitoneal administration, external application (such as by powder, ointment, drops or transdermal patch), oral administration or by oral or nasal spray administration.

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

The administration mode can be systemic administration or local administration. In addition, it may be necessary to introduce the antibody or fusion protein of the present invention into the central nervous system 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.

The antibody or fusion protein of the present invention can be applied topically to the area in need of treatment; it can be achieved by, but not limited to, local infusion during surgery, such as local application in conjunction with postoperative wound dressings, by injection, by catheter, by suppository, or by implant, which is a porous, non-porous or gel-like material, including membranes (such as silicone rubber membranes) or fibers. Preferably, when administering the protein of the present invention (including antibodies or fusion proteins), care must be taken to use materials that do not absorb the protein.

In some embodiments, the invention provides nucleic acids or polynucleotides comprising antibodies or fusion proteins, which can be administered in vivo by constructing them as part of an appropriate nucleic acid expression vector to promote the expression of the protein encoded by them, and then administering the nucleic acid or polynucleotide or vector to make it intracellular, 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 agents, or by administering them in conjunction with homeobox peptides 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.

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 the patient's body weight, or 0.1 mg/kg to 20 mg/kg of the patient's body weight. After the initial dose, a second dose or multiple doses of the antibody or antigen-binding fragment or fusion protein may be subsequently administered, and the dosage 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, and subsequent dose administration may be increased. 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.

Various known delivery systems can be used to administer the antibodies or fusion proteins or derivatives of the present invention or the polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as part of a retrovirus or other vector, etc.

Combination therapy

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

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

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

Pharmaceutical composition

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).

In some embodiments, the term "pharmaceutically acceptable" refers to substances for animals, particularly for humans, that are approved by a government regulatory agency or listed in a generally recognized pharmacopoeia. 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.

The term "excipient" refers to a diluent, adjuvant, excipient or carrier that can be applied to a patient together with the active ingredient. This type of pharmaceutical carrier can be a sterile liquid, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, 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 injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, 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 adjusting tension such as or dextrose are also foreseeable. These compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulations, etc. The composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain clinically effective doses of antibodies or antigen binding fragments or fusion proteins, preferably in purified form, together with an appropriate amount of adjuvants, to provide a dosage form suitable for the patient. The preparation should be suitable for administration mode. The parent preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.

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 solubilizer 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, sterile water for injection or saline can be used to mix the active ingredient before administration.

The antibody or antigen-binding fragment or fusion protein of the present invention can be in the form of neutral or salt. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Example

The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

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

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

1) Preparation of anti-PD-L1 antibody

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

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

Table 1 Heavy chain and light chain composition of antibodies

Table 2 Antibody heavy chain and light chain CDR regions

Table 3 Composition of antibody heavy chain CDR region

Table 4 Composition of antibody heavy chain CDR region

Table 5 Composition of antibody light chain CDR region

Table 6 Antibody heavy chain variable region

Table 7 Antibody light chain variable region

Table 8 Antibody constant region

Table 9 Antibody heavy chain sequences

Table 10 Antibody light chain sequences

2) Preparation of fusion protein A

The schematic diagram of the structure of fusion protein A is shown in Figure 1, which consists of four polypeptides: a first polypeptide (as shown in SEQ ID NO:83), a second polypeptide (as shown in SEQ ID NO:84) and two third polypeptides with the same sequence (as shown in SEQ ID NO:66); wherein, the first polypeptide comprises, from N-terminus to C-terminus: anti-PD-L1 heavy chain a (as shown in SEQ ID NO:79), a linker (as shown in SEQ ID NO:85) and an IL-15Rαsushi domain (as shown in SEQ ID NO:86). D NO:81), the second polypeptide comprises from N-terminus to C-terminus: anti-PD-L1 heavy chain b (as shown in SEQ ID NO:80), a linker (as shown in SEQ ID NO:85) and IL-15 (as 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 as shown in SEQ ID NO:72, the nucleic acid sequence of the second polypeptide is as shown in SEQ ID NO:73, and the nucleic acid sequence of the third polypeptide is as 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.

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

Table 11 Fusion protein A related amino acid sequence

Table 12 Fusion protein A related nucleic acid sequences

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

A fluorescence activated cell sorting (FACS)-based assay was used to evaluate the binding of fusion protein A to CTLL-2 cells (mouse cytotoxic T lymphocyte cell line) endogenously expressing IL-15Rα, IL-2Rβ, IL-2Rγ. CTLL-2 cells were resuspended in PBS buffer as a single cell suspension and mixed with 100 μL of fusion protein A samples of different concentrations (starting from 100 nM, 2-fold serial dilutions, with 10 concentrations) in equal volumes (all in 96-well plates), 500,000 cells/well. The mixture was equilibrated at 4°C for 60 minutes (min) and washed with PBS buffer. Then a phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, Cat. No.: 12-4998-82) used as a secondary antibody was added and equilibrated at 4°C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. As shown in Figure 2, FACS binding assays demonstrated that fusion protein A was able to significantly bind to CTLL-2 cells.

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 2mM L-glutamine, 1mM sodium pyruvate, 10% fetal bovine serum (FBS) and 10ng/mL IL-2 at ₃₇ °C and 5% CO2. The cells were suspended and cultured until they reached a cell density of 5×10 ⁵ cells per ml before flask division. For activity analysis, 2-3 days after the last flask division, the cells were washed with RPMI-1640 medium, resuspended with RPMI-1640 medium containing 15% FBS, and the cells were plated in a 96-well white plate at a density of 20,000/well (50μL), and the side wells were filled with PBS. Fusion protein A was diluted as follows: the diluent was RPMI-1640 medium containing 15% FBS, the antibody L1-R2-4-71 and fusion protein A samples started from 200nM, diluted 1:3, and 50μL/well was added to the white plate with cells laid above. The plate was placed in a 37℃, 5% CO ₂ incubator for 24 hours (h). When testing, the CellCounting-Lite 2.0 Luminescent Cell Viability Assay reagent was taken out of the -20℃ refrigerator in advance and equilibrated to room temperature. The cell culture plate was taken out of the incubator, equilibrated at room temperature (25±3℃) for 5 to 10 minutes, and 50μL CellCounting-Lite 2.0 Luminescent Cell Viability Assay reagent (Novozyme, catalog number: DD1101-02) was added to each well, and incubated at room temperature in the dark for 5 to 30 minutes. The relative light units (RLU) were read using the Luminescence detection module on the SpectraMax multi-function microplate reader. Data were analyzed using nonlinear regression using GraphPad PRISM 8 (GraphPad Software, San Diego, CA). As shown in Figure 3, CTLL-2 cell proliferation analysis showed that fusion protein A could significantly activate CTLL-2 cell proliferation activity, with an EC ₅₀ value of 0.417 nM.

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

A fluorescence activated cell sorting (FACS)-based assay was used to evaluate the binding of fusion protein A to HH cells (human cutaneous T lymphocytoma cells; ATCC CRL-2105) endogenously expressing IL-2Rβ, IL-2Rγ. HH cells were resuspended in PBS buffer as 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), 500,000 cells/well. The mixture was equilibrated at 4°C for 60 minutes and washed with PBS buffer. Then, phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen, Cat. No.: 12-4998-82) used as a secondary antibody was added and equilibrated at 4°C in the dark for 30 minutes. The cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. As shown in Figure 4, the FACS binding assay showed that fusion protein A was able to significantly bind to HH cells.

After IL-15 binds to IL-15Rα, it will trans-bind to IL-2Rβ and IL-2Rγ, activate the downstream signaling pathway, and lead to STAT5 phosphorylation. After co-incubation with HH cells with different concentrations of fusion protein A samples, STAT5 phosphorylation was observed to evaluate the ability of fusion protein A to activate HH cell activity. HH cells in the logarithmic phase were collected, washed with PBS, and resuspended in preheated RPMI-1640 medium, 2 million/100μL, and incubated at 37℃ for 30min. During this period, antibody L1-R2-4-71 or fusion protein A samples were diluted with RPMI-1640 medium containing 10% FBS, starting at 200nM, 2-fold dilution, with 11 concentrations, each 100μL. 100μL of the above diluted fusion protein A samples were mixed with an equal volume of cells and incubated at 37℃ for 15min. Then immediately put it on ice, add an equal volume of 4% paraformaldehyde (Paraformaldehyde, FPA) solution (final concentration 2%), fix the cells, and place them on ice for 30 minutes. Then wash with pre-cooled PBS buffer. Discard the supernatant, add 1mL of pre-cooled 90% methanol, place it on ice for 30 minutes, wash it with pre-cooled PBS buffer, resuspend it with PBS buffer, add PE anti-STAT5 Phospho (Tyr694) Antibody (BioLegend, Cat. No.: 936904), incubate at room temperature in the dark for 40 minutes, and then wash it and detect it on the machine (Beckman CytoFlex). The mean fluorescence intensity (Mean Fluorescent Intensity, MFI) value is used 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 level shows that fusion protein A can significantly activate HH cell activity, and the EC ₅₀ value is 1.609nM.

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

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

Construction method of CHO-K1-CD122-CD132 cells: linearize the vector connected with the CD122 gene sequence (NCBI Reference Sequence: NM_000878.5) and electroporate CHO-K1, and screen and cultivate to obtain CHO-CD122 stable cell line; based on the CHO-CD122 cell line, further infect with a lentivirus containing the CD132 gene sequence (NCBI Reference Sequence: NM_000206.3), and cultivate and screen to obtain CHO-K1-CD122-CD132 stable cell line.

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

This example summarizes the effect of using fusion protein A samples to induce selective activation and expansion of effector lymphocytes in human peripheral blood. 6-well cell culture plates were coated with 500 μL, 200 ng/mL anti-CD3 antibody (Nearshore Bio, GMP-A018) the night before; the next morning, the supernatant was discarded and PBMCs resuspended in RPMI-1640 medium containing 15% FBS were added, 1×10 ⁶ cells per well, and the total volume per well was 3 mL, and fusion protein A or antibody L1-R2-4-71 was added, respectively, with final concentrations of 0.5 nM and 20 nM, respectively. After 7 days of culture at 37°C and 5% CO ₂ , PBMCs were transferred to a new 6-well cell culture plate (anti-CD3 antibody removed), supplemented with fusion protein A or antibody L1-R2-4-71, and cultured for another 5 days; CD8 ⁺ T, NK and NKT cell proliferation was detected by flow cytometry. Among them, CD8 ⁺ T is CD3CD8 double positive T cell (i.e. CD3 ⁺ CD8 ⁺ T cell), NK cell is CD16 or CD56 positive cell, NKT cell is CD3 positive CD4CD8 double negative T cell (i.e. CD3 ⁺ CD4 ^- CD8 ^- T cell), and the sources of antibodies used for detection are as follows: CD3 antibody (elabscience, catalog number: FW2689), CD4 antibody (elabscience, catalog number: FW0218), CD8 antibody (elabscience, catalog number: FW0931), CD16 antibody (BioLegend, catalog number: 302038), CD56 antibody (BioLegend, catalog number: 302630). As shown in Figure 7a-d, fusion protein A can significantly promote the expansion of CD8 ⁺ T, NK and NKT cells compared with antibody L1-R2-4-71.

Example 6: Fusion protein A cytokine release experiment

This example uses liquid and solid phase incubation systems to evaluate the cytokine release caused by different test products. Dry pack method, i.e. solid phase incubation system, 25 μg/mL fusion protein A sample is coated on a 96-well cell culture plate, 40 μL volume/well, and 1×10 ⁵ PBMC cells (Leide Biology, Guangzhou) are added to each well after drying overnight in a clean bench, with a volume of 200 μL. Wet pack method, i.e. liquid phase incubation system, 25 μg/mL fusion protein A sample is added to a 96-well cell culture plate, 100 μL/well, and 1×10 ⁵ PBMC cells are added to each well, with a volume of 100 μL. At the same time, anti-CD3 antibody (Near Shore Bio, GMP-A018) and TGN1412 antibody (cd28 agonist antibody; sequence derived from patent US8709414B2) are selected as positive control antibodies. After three days of incubation, the supernatant was collected to detect IL-2, IFN-γ, IL-10, IL-6 and TNF-α (MABTECH, ELISABATIC kit). As shown in Figures 8a-8e and 9a-9e, TGN1412 antibody can very obviously activate PBMC in both liquid and solid phase incubation systems, as shown by the release of IL-2, IL-10, IFN-γ and TNF-α much higher than other test products. Anti-CD3 antibody, as a control antibody, can also activate PBMC to varying degrees. Fusion protein A cannot activate PBMC to release IL-2 in the solid phase incubation system, but its effect on the release of other cytokines is comparable to that of antibody L1-R2-4-71.

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

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

The mouse tumor-bearing model was prepared by implanting tumor cells into C57BL/6 mice. The mouse melanoma cell line B16F10 (i.e., B16F10-hPD-L1; Southern Model Animal Center) that stably expresses human PD-L1 was used in this assay. B16F10-hPD-L1 (1×10 ⁶ ) was subcutaneously injected into 8-week-old C57BL/6 mice. When the average tumor volume reached about 75 mm ³ , 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 day of grouping, and the day of grouping was defined as D0 day. The drug was administered on the day of grouping D0, twice a week, and the tumor volume was measured twice. IgG1 control (Sino Biological, HG1K), antibody L1-R2-4-71 and fusion protein A were administered to 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:

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 when the drugs were administered in groups; CVt represents the tumor volume of the control group at each measurement; CVinitial represents the tumor volume of the control group when the drugs were administered in groups. Figure 10 shows that the model is insensitive to PD-L1 monoclonal antibody, the antibody L1-R2-4-71 has no obvious efficacy, and the fusion protein A has a relatively obvious tumor inhibition effect.

Claims (38)

A fusion protein comprising: i. an anti-PD-L1 antibody or antigen-binding fragment; the anti-PD-L1 antibody or antigen-binding fragment comprises a HCDR1 as shown in any one of SEQ ID NOs: 1 and 91-95, a HCDR2 as shown in any one of SEQ ID NOs: 2-11, a HCDR3 as shown in SEQ ID NOs: 12 or 13, a LCDR1 as shown in any one of SEQ ID NOs: 14-18, a LCDR2 as shown in any one of SEQ ID NOs: 19-22, and a 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. The fusion protein of claim 1, wherein the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO: 20 and LCDR3 shown in SEQ ID NO: 24; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 21 and LCDR3 shown in SEQ ID NO: 25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 17, LCDR2 shown in SEQ ID NO: 22 and LCDR3 shown in SEQ ID NO: 26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 21 and LCDR3 shown in SEQ ID NO: 25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 21 and LCDR3 shown in SEQ ID NO: 26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 3, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19 and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 4, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19 and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 5, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19 and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19 and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 19 and LCDR3 shown in SEQ ID NO: 23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:20 and LCDR3 shown in SEQ ID NO:24; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:21 and LCDR3 shown in SEQ ID NO:25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:17, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21 and LCDR3 shown in SEQ ID NO:25; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:21 and LCDR3 shown in SEQ ID NO:26; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:3, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:5, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:6, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23; or The anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:91, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:19 and LCDR3 shown in SEQ ID NO:23. 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 that is at least 90% identical to a sequence as shown in any one of SEQ ID NOs: 27-41, or an amino acid sequence that has 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 shown in any one of SEQ ID NO:42-47, or an amino acid sequence that has at least 90% identity with the sequence shown in any one of SEQ ID NO:42-47, 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 NO:42-47. The fusion protein of claim 3, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-41, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-47; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:44; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:45; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42. 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. The fusion protein as described in claim 5 is 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 that has at least 90% identity with the sequence shown in SEQ ID NO:48 or 49, or an amino acid sequence that has 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. The fusion protein of claim 5, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 50; or The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:50. 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. The fusion protein according to claim 8, wherein the heavy chain constant region a comprises an amino acid mutation selected from S354C and T366W. 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. 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. The fusion protein according to any one of claims 1-6 and 8-11, characterized in that the heavy chain constant region comprises an amino acid mutation: K447A, wherein the amino acid position is Eu numbered. 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. 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. 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. 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. 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. 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 that has at least 90% identity with the sequence as shown in any one of SEQ ID NOs:51-65, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence as shown in any one of SEQ ID NOs:51-65; and/or The light chain comprises an amino acid sequence shown in any one of SEQ ID NO:66-71, or an amino acid sequence that has at least 90% identity with the sequence shown in any one of SEQ ID NO: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 NO:66-71. The fusion protein of claim 18, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:69; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:70; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain comprises the amino acid sequence shown in SEQ ID NO:71; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain comprises the amino acid sequence shown in SEQ ID NO:66. 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 that has at least 90% identity with the sequence shown in SEQ ID NO:79, or an amino acid sequence that has 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. The fusion protein as described in claim 20 is 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. The fusion protein as described in any one of claims 1-21 is characterized in that the IL-15 comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO:82, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:82. 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. 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. The fusion protein according to any one of claims 1 to 24, wherein the IL-15Rα or its sushi domain comprises the amino acid sequence shown in SEQ ID NO:81. 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 through a linker. The fusion protein of claim 26, wherein the C-terminus of one heavy chain of the anti-PD-L1 antibody is connected to IL-15 or a fragment thereof through a linker, and the C-terminus of the other heavy chain of the anti-PD-L1 antibody is connected to IL-15Rα or its sushi domain through a linker. The fusion protein according to claim 26 or 27, wherein the linker is a GS linker. 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. The fusion protein according to claim 28, wherein 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. 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 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. A 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, 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. A biological material for (1) A polynucleotide, characterized in that it encodes the fusion protein according to any one of claims 1 to 32 or a portion thereof; (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. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 32; or further comprising a pharmaceutically acceptable excipient. 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 according to any one of claims 1 to 32 or the pharmaceutical composition according to claim 34. The method of claim 35, wherein the disease is an infection, an autoimmune disease, a cancer or a tumor. 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 the preparation of a medicament for preventing, treating or ameliorating a disease. The use according to claim 37, wherein the disease is infection, autoimmune disease, cancer or tumor.