CD80 mutant polypeptide and application thereof

Abstract

The invention discloses a CD80 IgV mutant polypeptide. The CD80 IgV mutant polypeptide comprises a CD80 mutant fusion polypeptide compound of the mutant polypeptide and a nucleotide molecule serving as a fusion protein for expressing the mutant fusion polypeptide compound. Also disclosed are methods of inducing or enhancing immunity and methods of treating diseases such as infections and cancer using the mutant polypeptides, fusion polypeptide complexes or nucleotide molecules.

Description

A CD80 mutant polypeptide and its use Technical Field

[0001] The present invention relates to the fields of cancer and immunotherapy, and specifically, to CD80 IgV mutant polypeptides, CD80 ECD mutant polypeptides, CD80 mutant fusion polypeptide complexes comprising the mutant polypeptides, and nucleic acid molecules for expressing the mutant fusion polypeptide complexes as fusion proteins. The present invention also relates to methods for inducing or enhancing immunity, and methods for treating or preventing diseases such as infections and cancer, using the mutant polypeptides, fusion polypeptide complexes, or nucleic acid molecules. Background Art

[0002] CD80 protein is a type of immune-related molecule with stimulatory immune response. It mainly realizes its function by regulating T cell immune response, including participating in immune defense, immune tolerance and immune tissue damage. CD80 mainly exerts immunomodulatory effects to regulate immune response by binding to its ligands CD28, CTLA4 and PDL1. Changing the affinity between CD80 and ligands can regulate CD80-mediated related functions, especially enhancing the body's immune response function and strengthening the control of diseases including tumors and infections.

[0003] As a member of the B7 family of proteins that can costimulate T cell activation, mature CD80 molecules are composed of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain. The extracellular domain (ECD) is the key region for these molecules to bind to their corresponding receptors on T cells. The CD80 extracellular domain contains the immunoglobulin variable-like V (IgV) region and the immunoglobulin constant-like C2 (IgC2) region, and the IgV domain is the key domain directly involved in their receptor binding. The CD80 extracellular domain IgV can bind to CD28 to induce T cell activation, proliferation, and effector function. CTLA4 can also bind to the IgV region of CD80 to participate in immunosuppressive regulation.

[0004] In the immune response regulation, T cell activation is regulated by the binding of co-stimulatory signal receptors such as CD28 and the corresponding ligands CD80 / CD86 on the APC surface. However, CD80 and CD86 are usually not expressed or lowly expressed in the tumor microenvironment, resulting in the inability of T cells to be effectively activated. This is also one of the key mechanisms of tumor immune escape.

[0005] In addition, CTLA4 competes with CD28 for binding to CD80 / CD86, exhibiting higher affinity characteristics, resulting in CD28 being unable to effectively receive the immune stimulation signals mediated by CD80 / CD86, leading to ineffective T cell activation or immunosuppression. Moreover, the binding of PDL1 to CD80 weakens the CD80-mediated immune activation effect and interferes with the normal activation of T cells, thereby suppressing the related immune response.

[0006] Therefore, there is a need in the art for CD80 mutant polypeptides that improve the binding activity of CD80 to its ligand, so that they can effectively stimulate T cells and enhance immune responses, thereby treating or preventing diseases such as infections and tumors caused by suppressed T cell function.

[0007] Summary of the Invention

[0008] The inventors of the present application unexpectedly discovered that the CD80 mutant polypeptide formed by innovatively modifying the CD80 IgV domain can modulate the binding activity of CD80 and its ligand, so that the CD80 mutant polypeptide can effectively stimulate T cells and enhance the immune response, thereby achieving the treatment or prevention of diseases such as infections and tumors caused by the inhibition of T cell function.

[0009] In a first aspect, the present invention provides a CD80 IgV mutant polypeptide, which comprises one or more amino acid mutations selected from the group consisting of T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D relative to the wild-type human CD80 IgV polypeptide sequence SEQ ID NO:45.

[0010] In some embodiments, the CD80 IgV mutant polypeptide comprises a combination of two or more of the amino acid mutations.

[0011] In some embodiments, the combination of two or more of the amino acid mutations is selected from E35N / A71E (SEQ ID NO: 46), E35N / A71N (SEQ ID NO: 47), E35N / A71Q (SEQ ID NO: 48), E35N / A71T (SEQ ID NO: 49), E35S / A71E (SEQ ID NO: 50), E35S / A71N (SEQ ID NO: 51), E35S / A71Q (SEQ ID NO: 52), E35S / A71T (SEQ ID NO: 53), E35Q / A71E (SEQ ID NO: 54), E35Q / A71N (SEQ ID NO: 55), E35Q / A71Q (SEQ ID NO: 56), E35Q / A71T (SEQ ID NO: 57), E35T / A71E (SEQ ID NO: 58), E35T / A71N (SEQ ID NO: 59), E35T / A71T (SEQ ID NO: 60), E35S / A71E (SEQ ID NO: 61), E35S / A71N (SEQ ID NO: 62), E35S / A71Q (SEQ ID NO: 63), E35S / A71T (SEQ ID NO: 64), NO:59), E35T / A71Q (SEQ ID NO:60), E35T / A71T (SEQ ID NO:61), M47A / I69Y / A71E / V83L (SEQ ID NO:62), M47A / I69W / A71E / V83L (SEQ ID NO:63), M47A / I69Y / A71E (SEQ ID NO:64), M47A / I69W / A71E (SEQ ID NO:65), I69Y / A71E / V83L (SEQ ID NO:66), I69W / A71E / V83L (SEQ ID NO:67), I69Y / A71E (SEQ ID NO:68), I69W / A71E(SEQ ID NO:69), H18W / A26M / A71E / L85N(SEQ ID NO:70), H18W / A26M / A71E / L85D(SEQ ID NO:71), H18W / A26C / A71E / L85N(SEQ ID NO:72), H18W / A26C / A71E / L85D(SEQ ID NO:73), A26M / A71E / L85N(SEQ ID NO:74), A26M / A71E / L85D (SEQ ID NO:75), A26C / A71E / L85D (SEQ ID NO:76), A26C / A71E / L85N (SEQ ID NO:77), I69L / A71E / V83L (SEQ ID NO:78), H18W / A26V / E35N / A71E / L85N (SEQ ID NO:79), T13K / M42L / M47H / A71E (SEQ IDNO:80), T13K / M42L / M47Q / A71E (SEQ ID NO:81), T13K / M42L / A71E (SEQ ID NO:82), T13K / M42T / M47H / A71E (SEQ ID NO:83), T13K / M42T / M47T / A71E (SEQ ID NO:84), T13K / M42T / A71E (SEQ ID NO:85), M47H / I69Y / A71E / V83L (SEQ ID NO:86), M47Q / I69Y / A71E / V83L (SEQ ID NO:87) and M47K / I69Y / A71E / V83L (SEQ ID NO:88).

[0012] In a second aspect, the present invention provides a CD80 ECD mutant polypeptide, which comprises one or more amino acid mutations selected from the following: T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N and L85D relative to the wild-type human CD80 ECD polypeptide sequence SEQ ID NO:1.

[0013] In some embodiments, the CD80 ECD mutant polypeptide comprises a combination of two or more of the amino acid mutations.

[0014] In some embodiments, the combination of two or more of the amino acid mutations is selected from E35N / A71E (SEQ ID NO: 2), E35N / A71N (SEQ ID NO: 3), E35N / A71Q (SEQ ID NO: 4), E35N / A71T (SEQ ID NO: 5), E35S / A71E (SEQ ID NO: 6), E35S / A71N (SEQ ID NO: 7), E35S / A71Q (SEQ ID NO: 8), E35S / A71T (SEQ ID NO: 9), E35Q / A71E (SEQ ID NO: 10), E35Q / A71N (SEQ ID NO: 11), E35Q / A71Q (SEQ ID NO: 12), E35Q / A71T (SEQ ID NO: 13), E35T / A71E (SEQ ID NO: 14), E35T / A71N (SEQ ID NO: 15), NO:15), E35T / A71Q (SEQ ID NO:16), E35T / A71T (SEQ ID NO:17), M47A / I69Y / A71E / V83L (SEQ ID NO:18), M47A / I69W / A71E / V83L (SEQ ID NO:19), M47A / I69Y / A71E (SEQ ID NO:20), M47A / I69W / A71E (SEQ ID NO:21), I69Y / A71E / V83L (SEQ ID NO:22), I69W / A71E / V83L (SEQ ID NO:23), I69Y / A71E (SEQ ID NO:24), I69W / A71E(SEQ ID NO:25), H18W / A26M / A71E / L85N(SEQ ID NO:26), H18W / A26M / A71E / L85D(SEQ ID NO:27), H18W / A26C / A71E / L85N(SEQ ID NO:28), H18W / A26C / A71E / L85D(SEQ ID NO:29), A26M / A71E / L85N(SEQ ID NO:30), A26M / A71E / L85D (SEQ ID NO:31), A26C / A71E / L85D (SEQ ID NO:32), A26C / A71E / L85N (SEQ ID NO:33), I69L / A71E / V83L (SEQ ID NO:34), H18W / A26V / E35N / A71E / L85N (SEQ ID NO:35), T13K / M42L / M47H / A71E (SEQ IDNO:36), T13K / M42L / M47Q / A71E (SEQ ID NO:37), T13K / M42L / A71E (SEQ ID NO:38), T13K / M42T / M47H / A71E (SEQ ID NO:39), T13K / M42T / M47T / A71E (SEQ ID NO:40), T13K / M42T / A71E (SEQ ID NO:41), M47H / I69Y / A71E / V83L (SEQ ID NO:42), M47Q / I69Y / A71E / V83L (SEQ ID NO:43) and M47K / I69Y / A71E / V83L (SEQ ID NO:44).

[0015] In a third aspect, the present invention provides a CD80 mutant fusion polypeptide complex comprising the CD80 IgV mutant polypeptide of the first aspect or the CD80 ECD mutant polypeptide of the second aspect and a second domain.

[0016] In some embodiments, the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide is covalently linked to the second domain with or without a linker peptide.

[0017] In some embodiments, the second domain is an antibody or an antigen-binding fragment thereof.

[0018] In some embodiments, the antibody is selected from an immunoglobulin IgG antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody, and / or a bispecific antibody.

[0019] In some embodiments, the immunoglobulin IgG antibody is selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3.

[0020] In some embodiments, the antigen binding fragment is selected from Fab, Fab', Fv, F(ab)2, F(ab')2, scFv, VHH, di-scFv and / or dAb.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets one or more antigens selected from the group consisting of PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36, and / or Tim3.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets PD-L1, PD-1, TIGIT, CTLA4, LAG3, or CD3.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets PD-L1 or PD-1.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets LAG3.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets CD3.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof that targets PD-L1 is Sugemalimab antibody or antigen-binding fragment thereof.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof that targets LAG-3 is Relatlimab antibody or an antigen-binding fragment thereof.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD3 is a CD3B219 antibody or an antigen-binding fragment thereof.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the heavy chain with or without a linker peptide.

[0030] In some embodiments, the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the heavy chain with or without a linker peptide.

[0031] In some embodiments, the heavy chain C-terminus is covalently linked to the N-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the light chain with or without a linker peptide.

[0033] In some embodiments, the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the light chain with or without a linker peptide.

[0034] In some embodiments, the light chain C-terminus is covalently linked to the N-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

[0035] In some embodiments, the CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, and the second polypeptide may be present or absent.

[0036] In some embodiments, the first polypeptide comprises a heavy chain of an antibody or antigen-binding fragment thereof.

[0037] In some embodiments, the first polypeptide is covalently linked from the N-terminus to the C-terminus of the polypeptide by a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, or a heavy chain of an antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the first polypeptide is composed of a heavy chain of an antibody or antigen-binding fragment thereof, a linker peptide, a CD80 IgV mutant polypeptide, or a CD80 ECD mutant polypeptide covalently linked in sequence from the N-terminus to the C-terminus of the polypeptide.

[0039] In some embodiments, the second polypeptide comprises a light chain of an antibody or antigen-binding fragment thereof.

[0040] In some embodiments, the second polypeptide is covalently linked from the N-terminus to the C-terminus of the polypeptide by a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, or a light chain of an antibody or antigen-binding fragment thereof.

[0041] In some embodiments, the second polypeptide is composed of a light chain of an antibody or antigen-binding fragment thereof, a linker peptide, a CD80 IgV mutant polypeptide, or a CD80 ECD mutant polypeptide covalently linked in sequence from the N-terminus to the C-terminus of the polypeptide.

[0042] In some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 97), E35N / A71N (SEQ ID NO: 98), E35N / A71Q (SEQ ID NO: 99), E35N / A71T (SEQ ID NO: 100), E35S / A71E (SEQ ID NO: 101), E35S / A71N (SEQ ID NO: 102), E35S / A71Q (SEQ ID NO: 103), E35S / A71T (SEQ ID NO: 104), E35Q / A71E (SEQ ID NO: 105), E35Q / A71N (SEQ ID NO: 106), E35Q / A71Q (SEQ ID NO: 107), E35Q / A71T (SEQ ID NO: 108), E35T / A71E (SEQ ID NO: 110), E35S / A71N (SEQ ID NO: 111), E35S / A71Q (SEQ ID NO: 112), E35S / A71T (SEQ ID NO: 113), NO:109), E35T / A71N (SEQ ID NO:110), E35T / A71Q (SEQ ID NO:112), E35T / A71T (SEQ ID NO:113), M47A / I69Y / A71E / V83L (SEQ ID NO: 114), M47A / I69W / A71E / V83L (SEQ ID NO: 115), M47A / I69Y / A71E (SEQ ID NO: 116), M47A / I69W / A71E (SEQ ID NO: 117), I69Y / A71E / V83L (SEQ ID NO:118), I69W / A71E / V83L (SEQ ID NO:119), I69Y / A71E (SEQ ID NO:120), I69W / A71E (SEQ ID NO:121), H18W / A26M / A71E / L85N (SEQ ID NO:122),H18W / A26M / A71E / L85D (SEQ ID NO:123), H18W / A26C / A71E / L85N (SEQ ID NO:124), H18W / A26C / A71E / L85D (SEQ ID NO:125), A26M / A71E / L85N (SEQ ID NO:126), A26M / A71E / L85D (SEQ ID NO:127), A26C / A71E / L85D (SEQ ID NO:128), A26C / A71E / L85N (SEQ ID NO:129), I69L / A71E / V83L (SEQ ID NO:130), H18W / A26V / E35N / A71E / L85N (SEQ ID NO:131), T13K / M42L / M47H / A71E (SEQ ID NO:132), T13K / M42L / M47Q / A71E (SEQ ID NO:133), T13K / M42L / A71E (SEQ ID NO:134), T13K / M42T / M47H / A71E (SEQ ID NO:135), T13K / M42T / M47T / A71E (SEQ ID NO:136), T13K / M42T / A71E (SEQ ID NO:136), M47H / I69Y / A71E / V83L (SEQ ID NO:137), M47Q / I69Y / A71E / V83L (SEQ ID NO:138) or M47K / I69Y / A71E / V83L (SEQ ID NO:139).

[0043] Alternatively, in some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 142), E35N / A71N (SEQ ID NO: 143), E35N / A71Q (SEQ ID NO: 144), E35N / A71T (SEQ ID NO: 145), E35S / A71E (SEQ ID NO: 146), E35S / A71N (SEQ ID NO: 147), E35S / A71Q (SEQ ID NO: 148), E35S / A71T (SEQ ID NO: 149), E35Q / A71E (SEQ ID NO: 150), E35Q / A71N (SEQ ID NO: 151), E35Q / A71Q (SEQ ID NO: 152), E35Q / A71T (SEQ ID NO: 153), E35T / A71E (SEQ ID NO: 154), NO:154), E35T / A71N (SEQ ID NO:155), E35T / A71Q (SEQ ID NO:156) or E35T / A71T (SEQ ID NO:157).

[0044] Alternatively, in some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 192), E35N / A71N (SEQ ID NO: 193), E35N / A71Q (SEQ ID NO: 194), or E35N / A71T (SEQ ID NO: 195).

[0045] Alternatively, in some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 198), E35N / A71N (SEQ ID NO: 199), E35N / A71Q (SEQ ID NO: 200), or E35N / A71T (SEQ ID NO: 201).

[0046] Alternatively, in some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 203), E35N / A71N (SEQ ID NO: 204), E35N / A71Q (SEQ ID NO: 205), or E35N / A71T (SEQ ID NO: 206).

[0047] Alternatively, in some embodiments, the first polypeptide is selected from E35N / A71E (SEQ ID NO: 209), E35N / A71N (SEQ ID NO: 210), E35N / A71Q (SEQ ID NO: 211), or E35N / A71T (SEQ ID NO: 212).

[0048] In some embodiments, the second polypeptide is selected from SEQ ID NO: 141, SEQ ID NO: 197, or SEQ ID NO: 208.

[0049] In some embodiments, the CD80 mutant fusion polypeptide complex further comprises a third domain, which is covalently linked to the CD80 IgV mutant polypeptide of the first aspect, the CD80 ECD mutant polypeptide of the second aspect, or the second domain of the third aspect, with or without a linker peptide.

[0050] In some embodiments, the third domain is an antibody or antigen-binding fragment that is the same as or different from the second domain, or the third domain is a functional protein or an active fragment thereof.

[0051] In some embodiments, when the third domain is a functional protein or an active fragment thereof, the functional protein or the active fragment thereof is capable of activating an immune response.

[0052] In some embodiments, the second domain comprises an antibody heavy chain, and the antibody heavy chain of the second domain is connected to the third domain with or without a linker peptide.

[0053] In some embodiments, the heavy chain of the second domain is connected to the N-terminus of the third domain with or without a linker peptide.

[0054] In some embodiments, the heavy chain of the second domain is connected to the C-terminus of the third domain with or without a linker peptide.

[0055] In some embodiments, the second domain comprises an antibody light chain, and the antibody light chain of the second domain is connected to the third domain with or without a linker peptide.

[0056] In some embodiments, the light chain of the second domain is connected to the N-terminus of the third domain with or without a linker peptide.

[0057] In some embodiments, the light chain of the second domain is connected to the C-terminus of the third domain with or without a linker peptide.

[0058] In some embodiments, the third domain is connected to the CD80 IgV mutant polypeptide of the first aspect or the CD80 ECD mutant polypeptide of the second aspect with or without a linker peptide.

[0059] In some embodiments, the CD80 mutant fusion polypeptide complex includes a first polypeptide and a second polypeptide, and the second polypeptide may be present or absent.

[0060] In some embodiments, the first polypeptide comprises a heavy chain second domain.

[0061] In some embodiments, the first polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from N-terminus to C-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0062] In some embodiments, the first polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from C-terminus to N-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0063] In some embodiments, the second polypeptide comprises a light chain second domain.

[0064] In some embodiments, the second polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from N-terminus to C-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0065] In some embodiments, the second polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from C-terminus to N-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0066] In some embodiments, the third domain comprises LAG-3 or an active fragment thereof.

[0067] In some embodiments, the second domain specifically targets PD-1 or PD-L1.

[0068] In some embodiments, the second domain is sugemalimab or a functional fragment thereof.

[0069] In some embodiments, the first polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO:293, SEQ ID NO:295.

[0070] In some embodiments, the second polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO:141.

[0071] In some embodiments, the second domain is an immunoglobulin Fc domain.

[0072] In some embodiments, the Fc domain is selected from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0073] In some embodiments, the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the immunoglobulin Fc domain with or without a linker peptide.

[0074] In some embodiments, the amino acid sequence of the CD80 mutant fusion polypeptide complex is selected from E35N / A71E (SEQ ID NO: 158), E35N / A71N (SEQ ID NO: 159), E35N / A71Q (SEQ ID NO: 160), E35N / A71T (SEQ ID NO: 161), E35S / A71E (SEQ ID NO: 162), E35S / A71N (SEQ ID NO: 163), E35S / A71Q (SEQ ID NO: 164), E35S / A71T (SEQ ID NO: 165), E35Q / A71E (SEQ ID NO: 166), E35Q / A71N (SEQ ID NO: 167), E35Q / A71Q (SEQ ID NO: 168), E35Q / A71T (SEQ ID NO: 169), E35T / A71E (SEQ ID NO: 170), NO:170), E35T / A71N (SEQ ID NO:171), E35T / A71Q (SEQ ID NO:172) or E35T / A71T (SEQ ID NO:173).

[0075] In some embodiments, the C-terminus of the immunoglobulin Fc domain is covalently linked to the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

[0076] In some embodiments, where a linker peptide is present, the linker peptide is selected from one or more of GGGGS (SEQ ID NO:92), GGGGSGGGGS (SEQ ID NO:93), GGGGSGGGGSGGGGS (SEQ ID NO:94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:95), and GGGGSGGGS (SEQ ID NO:96).

[0077] In a fourth aspect, the present invention provides an immunoconjugate comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, or the CD80 mutant fusion polypeptide complex according to the third aspect.

[0078] In a fifth aspect, the present invention provides a nucleotide molecule encoding the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, or the CD80 mutant fusion polypeptide complex according to the third aspect.

[0079] In a sixth aspect, the present invention provides a vector comprising the nucleotide molecule described in the fifth aspect.

[0080] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, the CD80 mutant fusion polypeptide complex according to the third aspect, the nucleotide molecule according to the fifth aspect and / or the vector according to the sixth aspect.

[0081] In an eighth aspect, the present invention provides a composition comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, the CD80 mutant fusion polypeptide complex according to the third aspect, the immunoconjugate according to the fourth aspect, the nucleotide molecule according to the fifth aspect, or the vector according to the sixth aspect, and a pharmaceutically acceptable carrier.

[0082] In a ninth aspect, the present invention provides a method for preparing the CD80 IgV mutant polypeptide of the first aspect, the CD80 ECD mutant polypeptide of the second aspect, or the CD80 mutant fusion polypeptide complex of the third aspect, comprising culturing the host cell of the seventh aspect to express the CD80 IgV mutant polypeptide, CD80 ECD mutant polypeptide and / or CD80 mutant fusion polypeptide complex.

[0083] In the tenth aspect, the present invention provides a method for regulating T cell activity, comprising contacting the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, or the CD80 mutant fusion polypeptide complex according to the third aspect and / or the composition according to the eighth aspect with T cells to stimulate their activation.

[0084] In the eleventh aspect, the present invention provides a method for inhibiting tumor cell growth or proliferation, comprising administering the CD80 IgV mutant polypeptide as described in the first aspect, the CD80 ECD mutant polypeptide as described in the second aspect, the CD80 mutant fusion polypeptide complex as described in the third aspect, the immunoconjugate as described in the fourth aspect, the nucleotide molecule as described in the fifth aspect, the vector as described in the sixth aspect, the host cell as described in the seventh aspect and / or the composition as described in the eighth aspect.

[0085] In the twelfth aspect, the present invention provides use of the CD80 IgV mutant polypeptide as described in the first aspect, the CD80 ECD mutant polypeptide as described in the second aspect, the CD80 mutant fusion polypeptide complex as described in the third aspect, the immunoconjugate as described in the fourth aspect, the nucleotide molecule as described in the fifth aspect, the vector as described in the sixth aspect, the host cell as described in the seventh aspect and / or the composition as described in the eighth aspect in the preparation of a medicament for preventing, ameliorating and / or treating tumors or cancer.

[0086] The following specific embodiments and examples are described in detail. It is to be understood that the present invention is not limited to the specific embodiments described herein and therefore may include many variations and modifications. It will be appreciated by those skilled in the art that such variations and modifications are within the scope and spirit of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figures 1A to 1O show the expression of CD80 mutant fusion polypeptide complexes detected by SDS-PAGE.

[0088] FIG2A-FIG2D exemplarily show the detection of binding of CD80 mutant fusion polypeptide complexes to human PDL1 protein using ELISA.

[0089] FIG3A-FIG3O exemplarily show the detection of binding of CD80 mutant fusion polypeptide complexes to human CTLA4 protein using ELISA.

[0090] FIG4A-FIG4O exemplarily show the detection of binding of CD80 mutant fusion polypeptide complexes to human CD28 protein using ELISA.

[0091] Figures 5A-5L exemplarily show that the CD80 mutant fusion polypeptide complex relieves the PD-1 / PD-L1-mediated T cell activation immunosuppressive response.

[0092] 6A-6D exemplarily show that CD80 mutant fusion polypeptide complexes regulate Jurkat T cell activation.

[0093] 7A-7C exemplarily show that CD80 mutant fusion polypeptide complexes regulate PBMC activation.

[0094] Figures 8A-8C show that the CD80 mutant fusion polypeptide complex promotes the secretion of chemokines by antigen-presenting cells. DETAILED DESCRIPTION

[0095] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of a conflict, the definitions in this specification shall prevail.

[0096] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. Although only certain exemplary materials and methods are described herein, many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure.

[0097] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein are intended to include the plural forms as well. In addition, the open-ended expressions "including" and "comprising" are interpreted as also including structural components or method steps that are not mentioned, but it should be noted that such open-ended expressions also cover situations where the composition consists only of the components and method steps (i.e., they cover situations where the closed-ended expression "consists of...").

[0098] In general, the term "about" is used herein to refer to a numerical value above and below the stated value with a modification of 5%.

[0099] The term "regulating T cell activity" refers to the effect of regulating lymphoid T cells by the CD80 mutant polypeptide and CD80 mutant fusion polypeptide complex disclosed in the present invention, and can be extended to any other related T cell regulators that affect the expression of T cell NFAT transcription factors, the secretion of cytokines such as IL-2, IFN-γ, TNFα and Granzyme B, cell proliferation and cell killing of target cells including tumor cells after treating T cells. The T cells can be expanded to T cell lines and / or primary T cells including but not limited to CD4 T cells and CD4 T cell subsets Th1, Th2, Th9, Th17, TFH and / or Treg cells; CD8 T cells are also included, including but not limited to tumor tissue infiltrating CD8 T cells, effector CD8 T cells and / or immune memory CD8 T cells.

[0100] The term "anti-tumor activity" refers to any biological activity that reduces or prevents the proliferation or viability of tumor cells in vivo and / or in vitro. In certain embodiments, the anti-tumor activity is the anti-tumor effect of the CD80 mutant polypeptides and fusion polypeptide complexes thereof described herein.

[0101] The term "CD80" refers to a polypeptide or fragment thereof that exhibits at least about 85% amino acid identity with the protein encoded by the gene with NCBI accession number Gene ID: 941 and has binding activity to CD28 (protein encoded by the gene with NCBI accession number Gene ID: 940) and / or CTLA4 (protein encoded by the gene with NCBI accession number Gene ID: 1493) and / or PD-L1 (protein encoded by the gene with NCBI accession number Gene ID: 29126). An exemplary human CD80 amino acid sequence is provided below:

[0102] The term "CD80 extracellular region" refers to a polypeptide or fragment thereof having an amino acid sequence with at least about 85% amino acid identity to a domain in the extracellular region of a CD80 protein, and having CD28 and / or CTLA4 binding activity. An exemplary "CD80 extracellular region amino acid sequence" (SEQ ID NO: 1) is provided below. The term "CD80 IgV" refers to a polypeptide or fragment thereof having an amino acid sequence with at least about 85% amino acid identity to an IgV domain in the extracellular region of a CD80 protein, and having CD28 and / or CTLA4 binding activity. An exemplary "CD80 IgV" (SEQ ID NO: 45) is provided below.

[0103] The term "amino acid identity" refers to the percentage identity when amino acid sequences are compared and aligned (with gaps introduced, if necessary) for maximum correspondence. The percent identity value can be determined using sequence comparison software, algorithms, or by visual inspection. For comparison of amino acid identity between two or more or mixed tandem subunits of the second binding domain, the percent identity of each subunit of the second binding domain should be calculated by comparing each subunit separately. Various algorithms and software are known in the art for obtaining amino acid sequence alignments, such as, but not limited to, NCBI BLAST software.

[0104] The term "immunoglobulin" refers to an antibody composed of an antigen-specific binding amino acid segment and a constant region segment. The antigen-specific binding region is the segment that determines the key difference in immunoglobulins and can also be called an antigen-binding domain, or an "epitope" or "antigenic determinant." The antigen-binding domain is generally composed of the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL). However, it does not necessarily include both. The antigen-binding domain of the antibody disclosed in the present invention is not limited to the domain composed of traditional VH and VL, but also includes any other type of antibody, but not limited to, such as recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies, other unconventional antibodies, and combinations thereof. The constant region refers to the common structural region of immunoglobulins and includes the antibody light chain constant region and heavy chain constant region.

[0105] The term "immunoglobulin Fc domain" refers to the Fc fragment of a conventional antibody IgG, which is formed by papain hydrolysis to form an Fc fragment and two identical Fab fragments, comprising the antibody heavy chain CH2, CH3, and hinge region. Conventional Fc fragments bind to Fc fragment receptors to mediate related biological effects, while site-specific mutations can alter their ability to bind to corresponding target receptors, thereby affecting their biological functions. The immunoglobulin Fc domain disclosed herein includes, but is not limited to, conventional Fc fragments and any other forms of Fc mutants. The following provides an exemplary Fc domain of human immunoglobulin IgG1:

[0106] and the Fc domain of human immunoglobulin IgG4:

[0107] The term "linker peptide" means a short peptide chain with conformational flexibility, formed by a combination of amino acid Gly (G) and Ser (S) residues, wherein the ratio of the number of amino acid Gly to the number of amino acid Ser is guaranteed to be ≥1. The linker peptide disclosed in the present invention can be extended to any short peptide with this property. Exemplary linker peptide amino acid sequences are provided below, including but not limited to GGGGS (SEQ ID NO: 92), GGGGSGGGGS (SEQ ID NO: 93), GGGGSGGGGSGGGGS (SEQ ID NO: 94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95) and GGGGSGGGS (SEQ ID NO: 96).

[0108] As used herein, the term "CD80 mutant polypeptide" refers to a CD80 ECD mutant polypeptide and / or a CD80 IgV mutant polypeptide. The term "mutant polypeptide fusion polypeptide complex" is also referred to herein as a "mutant polypeptide complex," "polypeptide complex," "fusion polypeptide complex," or "fusion complex." The term "CD80 mutant fusion polypeptide complex" refers to a functional fusion polypeptide complex in which a CD80 ECD mutant or a CD80 IgV mutant is linked to an antibody or an antigen-binding fragment thereof, or an immunoglobulin Fc domain or a functional polypeptide fragment, with or without a linker peptide. The antibodies in the functional fusion polypeptide complex in which the disclosed CD80 mutant is linked to an antibody or an antigen-binding fragment thereof, with or without a linker peptide, include but are not limited to anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-TIGIT antibodies, anti-CTLA4 antibodies, anti-CEA antibodies, anti-BCMA antibodies, anti-LAG3 antibodies, anti-CD3 antibodies, anti-Her2 antibodies, anti-Her3 antibodies, anti-VEGF antibodies, anti-VEGFR antibodies, anti-EGFR antibodies, anti-c-Met antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD38 antibodies, anti-TROP-2 antibodies, anti-CD40 antibodies, anti-4-1BB antibodies, and anti-CD30 antibodies; any form of antibody is linked to the CD80 ECD mutant or CD80 IgV mutant disclosed in the present invention, with or without a linker peptide. Functional fusion proteins covalently linked to IgV mutants or combinations thereof should be considered within the scope of the present invention.

[0109] The term "LAG3" refers to a polypeptide or fragment thereof that has at least about 85% amino acid identity with the protein encoded by the gene with NCBI Accession No. Gene ID: 3902, and has binding activity to the complex protein MHCII (or HLA-DR) and / or FGL1 (protein encoded by the gene with NCBI Accession No. Gene ID: 2267), which is assembled from an α subunit (protein encoded by the gene with NCBI Accession No. Gene ID: 3122) and a β subunit (protein encoded by the gene with NCBI Accession No. Gene ID: 3123, or protein encoded by the gene with NCBI Accession No. Gene ID: 3125), and promotes the activation and maturation of antigen-presenting cells.

[0110] The term "LAG3 active fragment" refers to an active fragment derived from the LAG3 protein that possesses the functional properties of the LAG3 protein. These functional properties include, but are not limited to, binding to MHCII (or HLA-DR) and / or FGL1, and promoting activation and maturation of antigen-presenting cells.

[0111] The activation and maturation of antigen-presenting cells include, but are not limited to, up-regulation of the expression of antigen-presenting cell co-stimulatory receptors, enhanced antigen phagocytosis, enhanced cytokine secretion, and enhanced chemokine secretion.

[0112] The co-stimulatory receptors include but are not limited to ICOSL, CD40L, CD137L, OX40L, CD80, CD83 and CD86.

[0113] The antigen phagocytosis includes but is not limited to the phagocytosis of bacteria, viruses, proteins, polysaccharides and the like.

[0114] The cytokines include but are not limited to IL-1beta, TNFa, IL-6, IL-12 and the like.

[0115] The chemokines include but are not limited to CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, etc.

[0116] The inventors of the present application unexpectedly discovered that the CD80 mutant polypeptide formed by innovatively modifying the CD80 IgV domain can modulate the binding activity of CD80 and its ligand, so that the CD80 mutant polypeptide can effectively stimulate T cells and enhance the immune response, thereby achieving the treatment or prevention of diseases such as infections and tumors caused by the inhibition of T cell function.

[0117] On this basis, the present invention provides a class of CD80 mutant polypeptides including CD80 IgV mutant polypeptides and CD80 ECD mutant polypeptides; CD80 mutant fusion polypeptide complexes; and methods for regulating T cell immune responses and applications thereof in disease treatment.

[0118] In a first aspect, the present invention provides a CD80 IgV mutant polypeptide comprising one or more amino acid substitution mutations at wild-type CD80 IgV, which can be an amino acid substitution mutation of a human CD80 IgV polypeptide (SEQ ID NO: 45), wherein the mutation site is selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85. In some embodiments, the CD80 IgV mutant polypeptide comprises two or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from positions 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on SEQ ID NO: 45; in some embodiments, the CD80 IgV mutant polypeptide comprises three or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from positions 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on SEQ ID NO: 45; in some embodiments, the CD80 IgV mutant polypeptide comprises four or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from positions 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on SEQ ID NO: 45; in some embodiments, the CD80 IgV mutant polypeptide comprises five or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from positions 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on SEQ ID NO: Sites 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on NO:45.

[0119] In some embodiments, the mutation site of the CD80 IgV mutant polypeptide comprises one or more of the mutations T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D at amino acid positions in SEQ ID NO: 45.

[0120] In some preferred embodiments, the mutation site of the CD80 IgV mutant polypeptide comprises a combination of mutations at two or more amino acid sites on SEQ ID NO: 45, and the CD80 IgV mutant polypeptide can be E35N / A71E (SEQ ID NO: 46), E35N / A71N (SEQ ID NO: 47), E35N / A71Q (SEQ ID NO: 48), E35N / A71T (SEQ ID NO: 49), E35S / A71E (SEQ ID NO: 50), E35S / A71N (SEQ ID NO: 51), E35S / A71Q (SEQ ID NO: 52), E35S / A71T (SEQ ID NO: 53), E35Q / A71E (SEQ ID NO: 54), E35Q / A71N (SEQ ID NO: 55), E35Q / A71Q (SEQ ID NO: 56), E35Q / A71T (SEQ ID NO: 57). NO:57), E35T / A71E (SEQ ID NO:58), E35T / A71N (SEQ ID NO:59), E35T / A71Q (SEQ ID NO:60), E35T / A71T (SEQ ID NO:61), M47A / I69Y / A71E / V83L (SEQ ID NO:62), M47A / I69W / A71E / V83L (SEQ ID NO:63), M47A / I69Y / A71E (SEQ ID NO:64), M47A / I69W / A71E (SEQ ID NO:65), I69Y / A71E / V83L (SEQ ID NO:66), I69W / A71E / V83L (SEQ ID NO:67), I69Y / A71E (SEQ ID NO:68), I69W / A71E (SEQ ID NO:69), H18W / A26M / A71E / L85N(SEQ ID NO:70), H18W / A26M / A71E / L85D(SEQ ID NO:71), H18W / A26C / A71E / L85N(SEQ ID NO:72), H18W / A26C / A71E / L85D(SEQ ID NO:73), A26M / A71E / L85N (SEQ ID NO:74), A26M / A71E / L85D (SEQ ID NO:75), A26C / A71E / L85D (SEQ ID NO:76), A26C / A71E / L85N (SEQ ID NO:77), I69L / A71E / V83L(SEQ IDNO:78), H18W / A26V / E35N / A71E / L85N (SEQ ID NO:79), T13K / M42L / M47H / A71E (SEQ ID NO:80), T13K / M42L / M47Q / A71E (SEQ ID NO:81), T13K / M42L / A71E (SEQ ID NO:82), T13K / M42T / M47H / A71E (SEQ ID NO:83), T13K / M42T / M47T / A71E (SEQ ID NO:84), T13K / M42T / A71E (SEQ ID NO:85), M47H / I69Y / A71E / V83L (SEQ ID NO:86), M47Q / I69Y / A71E / V83L(SEQ ID NO:87), M47K / I69Y / A71E / V83L (SEQ ID NO:88).

[0121] In a second aspect, the present invention provides a CD80 ECD mutant polypeptide comprising one or more amino acid substitution mutations on a wild-type CD80 ECD, which can be an amino acid substitution mutation of a human CD80 ECD polypeptide (SEQ ID NO: 1), wherein the mutation site is selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on SEQ ID NO: 1. In some embodiments, the CD80 ECD mutant polypeptide comprises two or more amino acid substitution mutations on SEQ ID NO: 1, and the mutation sites are selected from 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 sites on SEQ ID NO: 1; in some embodiments, the CD80 ECD mutant polypeptide comprises three or more amino acid substitution mutations on SEQ ID NO: 1, and the mutation sites are selected from 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 sites on SEQ ID NO: 1; in some embodiments, the CD80 ECD mutant polypeptide comprises four or more amino acid substitution mutations on SEQ ID NO: 1, and the mutation sites are selected from 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 sites on SEQ ID NO: 1; in some embodiments, the CD80 ECD mutant polypeptide comprises five or more amino acid substitution mutations on SEQ ID NO: 1, and the mutation sites are selected from SEQ ID NO: Sites 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85 on NO:1.

[0122] In some embodiments, the mutation site of the CD80 ECD mutant polypeptide comprises one or more of the mutations T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D at the amino acid positions in SEQ ID NO: 1.

[0123] In some preferred embodiments, the mutation site of the CD80 ECD mutant polypeptide comprises a combination of mutations at two or more amino acid sites on SEQ ID NO: 1, and the CD80 ECD mutant polypeptide can be E35N / A71E (SEQ ID NO: 2), E35N / A71N (SEQ ID NO: 3), E35N / A71Q (SEQ ID NO: 4), E35N / A71T (SEQ ID NO: 5), E35S / A71E (SEQ ID NO: 6), E35S / A71N (SEQ ID NO: 7), E35S / A71Q (SEQ ID NO: 8), E35S / A71T (SEQ ID NO: 9), E35Q / A71E (SEQ ID NO: 10), E35Q / A71N (SEQ ID NO: 11), E35Q / A71Q (SEQ ID NO: 12), E35Q / A71T (SEQ ID NO: 13), NO:13), E35T / A71E (SEQ ID NO:14), E35T / A71N (SEQ ID NO:15), E35T / A71Q (SEQ ID NO:16), E35T / A71T (SEQ ID NO:17), M47A / I69Y / A71E / V83L (SEQ ID NO:18), M47A / I69W / A71E / V83L (SEQ ID NO:19), M47A / I69Y / A71E (SEQ ID NO:20), M47A / I69W / A71E (SEQ ID NO:21), I69Y / A71E / V83L (SEQ ID NO:22), I69W / A71E / V83L (SEQ ID NO:23), I69Y / A71E (SEQ ID NO:24), I69W / A71E (SEQ ID NO:25), H18W / A26M / A71E / L85N(SEQ ID NO:26), H18W / A26M / A71E / L85D(SEQ ID NO:27), H18W / A26C / A71E / L85N(SEQ ID NO:28), H18W / A26C / A71E / L85D(SEQ ID NO:29), A26M / A71E / L85N (SEQ ID NO:30), A26M / A71E / L85D (SEQ ID NO:31), A26C / A71E / L85D (SEQ ID NO:32), A26C / A71E / L85N (SEQ ID NO:33), I69L / A71E / V83L(SEQ ID NO:34), H18W / A26V / E35N / A71E / L85N (SEQ IDNO:35), T13K / M42L / M47H / A71E (SEQ ID NO:36), T13K / M42L / M47Q / A71E (SEQ ID NO:37), T13K / M42L / A71E (SEQ ID NO:38), T13K / M42T / M47H / A71E (SEQ ID NO:39), T13K / M42T / M47T / A71E (SEQ ID NO:40), T13K / M42T / A71E (SEQ ID NO:41), M47H / I69Y / A71E / V83L (SEQ ID NO:42), M47Q / I69Y / A71E / V83L (SEQ ID NO:43), M47K / I69Y / A71E / V83L (SEQ ID NO:44).

[0124] In a third aspect, the present invention provides a fusion polypeptide complex of a CD80 mutant polypeptide, comprising the above-mentioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide, covalently linked to a second domain with or without a linker peptide, wherein the second domain can be an immunoglobulin Fc domain, an antibody or an antigen-binding fragment thereof, or other forms of polypeptide.

[0125] In some embodiments, the fusion polypeptide complex of the CD80 mutant polypeptide comprises the above-mentioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide, covalently linked to an immunoglobulin Fc domain, with or without a linker peptide, wherein the Fc domain includes but is not limited to a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0126] In some embodiments, the CD80 mutant polypeptide is covalently linked to the N-terminus of the immunoglobulin Fc domain with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0127] In some embodiments, the C-terminus of the immunoglobulin Fc domain is covalently linked to a CD80 mutant polypeptide with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide. In some embodiments, the fusion polypeptide complex of the CD80 mutant polypeptide comprises the above-mentioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide, covalently linked to an antibody or its antigen-binding fragment with or without a linker peptide. In some embodiments, the antibody can be selected from immunoglobulin IgG antibodies, recombinant antibodies, chimeric antibodies, heavy chain antibodies, single domain antibodies and / or bispecific antibodies. In some embodiments, the antigen-binding fragment can be selected from Fab, Fab', Fv, F(ab)2, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0128] In some embodiments, the CD80 mutant fusion polypeptide complex comprises the above-mentioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide, covalently linked to an immunoglobulin IgG antibody with or without a linker peptide, wherein the immunoglobulin IgG includes but is not limited to human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b or mouse IgG3.

[0129] In some preferred embodiments, the antibody or antigen-binding fragment in the fusion polypeptide complex of the above-mentioned CD80 mutant polypeptide specifically targets a target selected from but not limited to PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and / or Tim3.

[0130] In a preferred embodiment, the anti-PD-L1 antibody or antigen-binding fragment is Sugemalimab antibody or an antigen-binding fragment thereof.

[0131] In a preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment is Relatlimab antibody or an antigen-binding fragment thereof.

[0132] In a preferred embodiment, the anti-CD3 antibody or antigen-binding fragment is CD3B219 antibody or an antigen-binding fragment thereof.

[0133] Alternatively, in some embodiments, the present invention provides a fusion polypeptide complex of a CD80 mutant polypeptide, wherein the CD80 mutant polypeptide and the antibody or antigen-binding fragment are linked via a linker peptide.

[0134] In one embodiment, the present invention provides a fusion polypeptide complex of a CD80 mutant polypeptide, wherein the CD80 mutant polypeptide and the antibody or antigen-binding fragment are directly linked without a linker peptide.

[0135] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain, and the CD80 mutant polypeptide is covalently linked to the heavy chain with or without a linker peptide.

[0136] In a preferred embodiment, the C-terminus of the CD80 mutant polypeptide is covalently linked to the N-terminus of the heavy chain with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0137] In a preferred embodiment, the C-terminus of the heavy chain is covalently linked to the N-terminus of the CD80 mutant polypeptide with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0138] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 mutant polypeptide is covalently linked to the light chain with or without a linker peptide.

[0139] In a preferred embodiment, the C-terminus of the CD80 mutant polypeptide is covalently linked to the N-terminus of the light chain with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0140] In a preferred embodiment, the C-terminus of the light chain is covalently linked to the N-terminus of the CD80 mutant polypeptide with or without a linker peptide to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0141] In one embodiment, the CD80 mutant polypeptide is covalently linked to the N-terminus of the antibody or antigen-binding fragment thereof with or without a linker peptide.

[0142] In one embodiment, the CD80 mutant polypeptide is covalently linked to the C-terminus of the antibody or antigen-binding fragment thereof with or without a linker peptide.

[0143] In another embodiment, the CD80 mutant fusion polypeptide complex provided by the present invention includes a first polypeptide and a second polypeptide constituting the fusion polypeptide complex. It should be understood that the second polypeptide may not be present in certain specific antibodies.

[0144] In a preferred embodiment, the first polypeptide of the fusion polypeptide complex of the present invention comprises a heavy chain of an antibody or an antigen-binding fragment thereof.

[0145] In a preferred embodiment, the first polypeptide of the fusion polypeptide complex of the present invention is covalently linked from the N-terminus to the C-terminus of the polypeptide by a CD80 mutant polypeptide, a linker peptide, and a heavy chain of an antibody or an antigen-binding fragment thereof.

[0146] In a preferred embodiment, the first polypeptide of the polypeptide complex of the present invention is covalently linked from the N-terminus to the C-terminus of the polypeptide by the heavy chain of an antibody or antigen-binding fragment thereof, a linker peptide, and a CD80 mutant polypeptide.

[0147] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention comprises a light chain of an antibody or an antigen-binding fragment thereof.

[0148] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention is covalently linked from the N-terminus to the C-terminus of the polypeptide by a CD80 mutant polypeptide, a linker peptide, or a light chain of an antibody or antigen-binding fragment thereof.

[0149] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention is covalently linked from the N-terminus to the C-terminus of the polypeptide by a light chain of an antibody or antigen-binding fragment thereof, a linker peptide, and a CD80 mutant polypeptide.

[0150] Alternatively, in some embodiments, the present invention also provides a CD80 mutant fusion polypeptide complex, comprising a CD80 mutant polypeptide, a second domain and a third domain, wherein the CD80 mutant polypeptide and the second domain are the same as described above.

[0151] In some embodiments, the third domain of the present invention is an antibody or antigen-binding fragment that is the same as or different from the second domain, or the third domain is a functional protein capable of immune activation or an active fragment thereof.

[0152] In some embodiments, the second domain of the present invention comprises a heavy chain, and the third domain of the present invention is connected to the heavy chain of the second domain with or without a linker peptide.

[0153] In a specific embodiment, the heavy chain of the second domain of the present invention is connected to the N-terminus of the third domain with or without a linker peptide.

[0154] In a specific embodiment, the heavy chain of the second domain of the present invention is connected to the C-terminus of the third domain with or without a linker peptide.

[0155] Alternatively, in some embodiments, the second domain of the present invention comprises a light chain, and the third domain of the present invention is connected to the light chain of the second domain with or without a linker peptide.

[0156] In a specific embodiment, the light chain of the second domain of the present invention is connected to the N-terminus of the third domain with or without a linker peptide.

[0157] In a specific embodiment, the light chain of the second domain of the present invention is connected to the C-terminus of the third domain with or without a linker peptide.

[0158] In some embodiments, the CD80 mutant fusion polypeptide complex comprising the third domain of the present invention comprises a first polypeptide and a second polypeptide, and the second polypeptide may be present or absent.

[0159] In some embodiments, the first polypeptide comprises a heavy chain second domain.

[0160] In one embodiment, the first polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from N-terminus to C-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0161] In one embodiment, the first polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from C-terminus to N-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

[0162] Alternatively, in some embodiments, the second polypeptide comprises a light chain second domain.

[0163] In some embodiments, the third domain is linked to the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide with or without a linker peptide.

[0164] In one embodiment, the third domain comprises LAG-3 or an active fragment thereof, and the second domain specifically targets PD-1 or PD-L1.

[0165] In one embodiment, the first polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 293, SEQ ID NO: 295; and the second polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 141.

[0166] In some embodiments, the linker peptide is absent.

[0167] In some embodiments, the linker peptide comprises one or more selected from GGGGS (SEQ ID NO:92), GGGGSGGGGS (SEQ ID NO:93), GGGGSGGGGSGGGGS (SEQ ID NO:94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:95), and GGGGSGGGS (SEQ ID NO:96).

[0168] Alternatively, in some embodiments, the CD80 mutant polypeptide can be combined with other forms of polypeptides to form the polypeptide complex using the above-mentioned method.

[0169] In a fourth aspect, the present invention provides an immunoconjugate comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, or the CD80 mutant fusion polypeptide complex according to the third aspect.

[0170] In a fifth aspect, the present invention provides a nucleotide molecule encoding the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, or the CD80 mutant fusion polypeptide complex according to the third aspect.

[0171] In a sixth aspect, the present invention provides a vector comprising the nucleotide molecule described in the fifth aspect.

[0172] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, the CD80 mutant fusion polypeptide complex according to the third aspect, the nucleotide molecule according to the fifth aspect and / or the vector according to the sixth aspect.

[0173] In an eighth aspect, the present invention provides a composition comprising the CD80 IgV mutant polypeptide according to the first aspect, the CD80 ECD mutant polypeptide according to the second aspect, the CD80 mutant fusion polypeptide complex according to the third aspect, the immunoconjugate according to the fourth aspect, the nucleotide molecule according to the fifth aspect, or the vector according to the sixth aspect, and a pharmaceutically acceptable carrier.

[0174] In a ninth aspect, the present invention provides a method for preparing the CD80 IgV mutant polypeptide of the first aspect, the CD80 ECD mutant polypeptide of the second aspect, or the CD80 mutant fusion polypeptide complex of the third aspect, comprising culturing the host cell of the seventh aspect to express the CD80 IgV mutant polypeptide, CD80 ECD mutant polypeptide and / or CD80 mutant fusion polypeptide complex.

[0175] In some embodiments, the present invention provides a method for expressing the polypeptide and polypeptide complex, comprising using the host cell described in the present invention to express the CD80 mutant polypeptide or its fusion polypeptide complex described in the present invention.

[0176] In some embodiments, the present invention provides a method for producing the polypeptide and polypeptide complex, which comprises introducing the polynucleotide encoding the antibody polypeptide complex of the present invention into a host cell, expressing the first polypeptide, including or excluding the second polypeptide, wherein the first polypeptide and the second polypeptide, or the first polypeptide itself can form a stable dimer, and the stable dimer can be maintained by a natural bond or / and a stable dimerization comprising at least one non-natural interchain bond, and the first polypeptide and the second polypeptide assemble into a stable polymer in the host cell, and form the stable complex.

[0177] In certain embodiments, the present invention also provides a method for isolating and purifying the CD80 mutant polypeptide or its fusion polypeptide complex.

[0178] In a tenth aspect, the present invention provides a method for regulating T cell activity, comprising contacting a CD80 IgV mutant polypeptide as described in the first aspect, a CD80 ECD mutant polypeptide as described in the second aspect, or a CD80 mutant fusion polypeptide complex as described in the third aspect, and / or the composition as described in the eighth aspect with a T cell to stimulate activation. The method is not limited to the protocol used in the Examples and can be expanded to other protocols currently published or developed in the future for evaluating T cell activation.

[0179] In the eleventh aspect, the present invention provides a method for inhibiting tumor cell growth or proliferation, comprising administering the CD80 IgV mutant polypeptide as described in the first aspect, the CD80 ECD mutant polypeptide as described in the second aspect, the CD80 mutant fusion polypeptide complex as described in the third aspect, the immunoconjugate as described in the fourth aspect, the nucleotide molecule as described in the fifth aspect, the vector as described in the sixth aspect, the host cell as described in the seventh aspect and / or the composition as described in the eighth aspect.

[0180] In the twelfth aspect, the present invention provides use of the CD80 IgV mutant polypeptide as described in the first aspect, the CD80 ECD mutant polypeptide as described in the second aspect, the CD80 mutant fusion polypeptide complex as described in the third aspect, the immunoconjugate as described in the fourth aspect, the nucleotide molecule as described in the fifth aspect, the vector as described in the sixth aspect, the host cell as described in the seventh aspect and / or the composition as described in the eighth aspect in the preparation of a medicament for preventing, ameliorating and / or treating tumors or cancer.

[0181] The following provides exemplary first polypeptide amino acid sequences of CD80 IgV mutants that are covalently linked to the N-terminus of the heavy chain of sugemalimab antibody via a linker peptide to form a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 97), E35N / A71N (SEQ ID NO: 98), E35N / A71Q (SEQ ID NO: 99), E35N / A71T (SEQ ID NO: 100), E35S / A71E (SEQ ID NO: 101), E35S / A71N (SEQ ID NO: 102), E35S / A71Q (SEQ ID NO: 103), E35S / A71T (SEQ ID NO: 104), E35Q / A71E (SEQ ID NO: 105), E35Q / A71N (SEQ ID NO: 106), E35Q / A71Q (SEQ ID NO: 107), NO:107), E35Q / A71T (SEQ ID NO:108), E35T / A71E (SEQ ID NO:109), E35T / A71N (SEQ ID NO:110), E35T / A71Q (SEQ ID NO:112), E35T / A71T (SEQ ID NO:113), M47A / I69Y / A71E / V83L (SEQ ID NO:114), M47A / I69W / A71E / V83L (SEQ ID NO:115), M47A / I69Y / A71E (SEQ ID NO:116), M47A / I69W / A71E (SEQ ID NO:117), I69Y / A71E / V83L (SEQ ID NO:118), I69W / A71E / V83L (SEQ ID NO:119), I69Y / A71E (SEQ ID NO:120), I69W / A71E (SEQ ID NO:121), H18W / A26M / A71E / L85N (SEQ ID NO:122), H18W / A26M / A71E / L85D (SEQ ID NO:123), H18W / A26C / A71E / L85N (SEQ ID NO:124), H18W / A26C / A71E / L85D (SEQ ID NO:125), A26M / A71E / L85N (SEQ ID NO:126), A26M / A71E / L85D (SEQ ID NO:127), A26C / A71E / L85D (SEQ ID NO:128), A26C / A71E / L85N (SEQ ID NO:129), I69L / A71E / V83L(SEQ IDNO:130), H18W / A26V / E35N / A71E / L85N (SEQ ID NO:131), T13K / M42L / M47H / A71E (SEQ ID NO:132), T13K / M42L / M47Q / A71E (SEQ ID NO:133), T13K / M42L / A71E (SEQ ID NO:134), T13K / M42T / M47H / A71E (SEQ ID NO:135), T13K / M42T / M47T / A71E (SEQ ID NO:136), T13K / M42T / A71E (SEQ ID NO:133) NO:136), M47H / I69Y / A71E / V83L (SEQ ID NO:137), M47Q / I69Y / A71E / V83L (SEQ ID NO: 138), M47K / I69Y / A71E / V83L (SEQ ID NO: 139), and a first polypeptide amino acid sequence (SEQ ID NO: 140) of a non-mutated CD80 IgV and a sugemalimab antibody heavy chain covalently linked to the N-terminus via a linker peptide to form a functional fusion polypeptide complex. The above first polypeptides are respectively assembled with a second polypeptide (SEQ ID NO: 141) of the sugemalimab antibody light chain to form the functional fusion polypeptide complex.

[0182] The following provides exemplary CD80 ECD mutants that are covalently linked to the N-terminus of the heavy chain of the sugemalimab antibody via a linker peptide to form a first polypeptide amino acid sequence of a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 142), E35N / A71N (SEQ ID NO: 143), E35N / A71Q (SEQ ID NO: 144), E35N / A71T (SEQ ID NO: 145), E35S / A71E (SEQ ID NO: 146), E35S / A71N (SEQ ID NO: 147), E35S / A71Q (SEQ ID NO: 148), E35S / A71T (SEQ ID NO: 149), E35Q / A71E (SEQ ID NO: 150), E35Q / A71N (SEQ ID NO: 151), E35Q / A71Q (SEQ ID NO: 152), NO:152), E35Q / A71T (SEQ ID NO:153), E35T / A71E (SEQ ID NO:154), E35T / A71N (SEQ ID NO:155), E35T / A71Q (SEQ ID NO:156), E35T / A71T (SEQ ID NO:157),

[0183] The above-mentioned first polypeptide is assembled with the second polypeptide of the sugemalimab antibody light chain (SEQ ID NO: 141) to form the functional fusion polypeptide complex.

[0184] The following provides exemplary CD80 ECD mutants that are covalently linked to the N-terminus of the Fc domain of human immunoglobulin IgG4 to form functional fusion polypeptide amino acid sequences: E35N / A71E (SEQ ID NO: 158), E35N / A71N (SEQ ID NO: 159), E35N / A71Q (SEQ ID NO: 160), E35N / A71T (SEQ ID NO: 161), E35S / A71E (SEQ ID NO: 162), E35S / A71N (SEQ ID NO: 163), E35S / A71Q (SEQ ID NO: 164), E35S / A71T (SEQ ID NO: 165), E35Q / A71E (SEQ ID NO: 166), E35Q / A71N (SEQ ID NO: 167), E35Q / A71Q (SEQ ID NO: 168), NO: 168), E35Q / A71T (SEQ ID NO: 169), E35T / A71E (SEQ ID NO: 170), E35T / A71N (SEQ ID NO: 171), E35T / A71Q (SEQ ID NO: 172), E35T / A71T (SEQ ID NO: 173), and an unmutated CD80 ECD is covalently linked to the N-terminus of the Fc domain of human immunoglobulin IgG4 to form a functional fusion polypeptide amino acid sequence (SEQ ID NO: 174).

[0185] The following provides exemplary CD80 IgV mutants that are covalently linked to the N-terminus of the Fc domain of human immunoglobulin IgG4 to form functional fusion polypeptide amino acid sequences: E35N / A71E (SEQ ID NO: 175), E35N / A71N (SEQ ID NO: 176), E35N / A71Q (SEQ ID NO: 177), E35N / A71T (SEQ ID NO: 178), E35S / A71E (SEQ ID NO: 179), E35S / A71N (SEQ ID NO: 180), E35S / A71Q (SEQ ID NO: 181), E35S / A71T (SEQ ID NO: 182), E35Q / A71E (SEQ ID NO: 183), E35Q / A71N (SEQ ID NO: 184), E35Q / A71Q (SEQ ID NO: 185), NO: 185), E35Q / A71T (SEQ ID NO: 186), E35T / A71E (SEQ ID NO: 187), E35T / A71N (SEQ ID NO: 188), E35T / A71Q (SEQ ID NO: 189), E35T / A71T (SEQ ID NO: 190), and a functional fusion polypeptide amino acid sequence (SEQ ID NO: 191) formed by covalently linking the unmutated CD80 IgV to the N-terminus of the Fc domain of human immunoglobulin IgG4.

[0186] The following provide exemplary CD80 ECD mutants, each covalently linked to the N-terminus of the heavy chain of the anti-LAG3 antibody Relatlimab via a linker peptide to form a first polypeptide amino acid sequence of a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 192), E35N / A71N (SEQ ID NO: 193), E35N / A71Q (SEQ ID NO: 194), E35N / A71T (SEQ ID NO: 195), and unmutated CD80 ECD (SEQ ID NO: 196). The above first polypeptides are each assembled with the second polypeptide of the light chain of the Relatlimab antibody (SEQ ID NO: 197) to form the functional fusion polypeptide complex.

[0187] The following provide exemplary CD80 IgV mutants, each covalently linked to the N-terminus of the heavy chain of the anti-LAG3 antibody Relatlimab via a linker peptide to form a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 198), E35N / A71N (SEQ ID NO: 199), E35N / A71Q (SEQ ID NO: 200), E35N / A71T (SEQ ID NO: 201), and unmutated CD80 IgV (SEQ ID NO: 202). The above first polypeptides are respectively assembled with the second polypeptide of the light chain of the Relatlimab antibody (SEQ ID NO: 197) to form the functional fusion polypeptide complex.

[0188] The following are exemplary CD80 ECD mutants that are covalently linked to the N-terminus of the heavy chain of the anti-CD3 antibody CD3B219 via a linker peptide to form a first polypeptide amino acid sequence of a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 203), E35N / A71N (SEQ ID NO: 204), E35N / A71Q (SEQ ID NO: 205), E35N / A71T (SEQ ID NO: 206), and unmutated CD80 ECD (SEQ ID NO: 207). The above first polypeptides are respectively assembled with the second polypeptide of the CD3B219 antibody light chain (SEQ ID NO: 208) to form the functional fusion polypeptide complex.

[0189] The following are exemplary CD80 IgV mutants covalently linked to the N-terminus of the heavy chain of the anti-CD3 antibody CD3B219 via a linker peptide to form a first polypeptide amino acid sequence of a functional fusion polypeptide complex: E35N / A71E (SEQ ID NO: 209), E35N / A71N (SEQ ID NO: 210), E35N / A71Q (SEQ ID NO: 211), E35N / A71T (SEQ ID NO: 212), and unmutated CD80 IgV (SEQ ID NO: 213). The above first polypeptides are respectively assembled with the second polypeptide of the CD3B219 antibody light chain (SEQ ID NO: 208) to form the functional fusion polypeptide complex.

[0190] In certain embodiments, the functional antibody Abs-353 is composed of a chimeric antibody Abs-353 first polypeptide (SEQ ID NO: 97) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35N / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-353 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 214) of the first Abs-353 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-353 polypeptide into a host cell for expression.

[0191] In certain embodiments, the functional antibody Abs-354 is composed of a chimeric antibody Abs-354 first polypeptide (SEQ ID NO: 98) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35N / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-354 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 216) of the first Abs-354 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-354 polypeptide into host cells for expression.

[0192] In certain embodiments, the functional antibody Abs-355 is composed of a chimeric antibody Abs-355 first polypeptide (SEQ ID NO: 99) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35N / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-355 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 217) of the first Abs-355 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-355 polypeptide into a host cell for expression.

[0193] In certain embodiments, the functional antibody Abs-356 is composed of a chimeric antibody Abs-356 first polypeptide (SEQ ID NO: 100) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35N / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-356 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 218) of the first Abs-356 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-356 polypeptide into host cells for expression.

[0194] In certain embodiments, the functional antibody Abs-357 is composed of a chimeric antibody Abs-357 first polypeptide (SEQ ID NO: 101) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35S / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-357 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 219) of the first Abs-357 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-357 polypeptide into host cells for expression.

[0195] In certain embodiments, the functional antibody Abs-358 is composed of a chimeric antibody Abs-358 first polypeptide (SEQ ID NO: 102) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35S / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-358 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 220) of the first Abs-358 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-358 polypeptide into host cells for expression.

[0196] In certain embodiments, the functional antibody Abs-359 is composed of a chimeric antibody Abs-first polypeptide (SEQ ID NO: 103) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35S / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-359 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 221) of the Abs-359 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-359 second polypeptide into host cells for expression.

[0197] In certain embodiments, the functional antibody Abs-360 is composed of a chimeric antibody Abs-first polypeptide (SEQ ID NO: 104) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35S / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-360 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 222) of the Abs-360 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-360 second polypeptide into host cells for expression.

[0198] In certain embodiments, the functional antibody Abs-361 is composed of a chimeric antibody Abs-361 first polypeptide (SEQ ID NO: 105) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35Q / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-361 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 223) of the first Abs-361 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-361 polypeptide into host cells for expression.

[0199] In certain embodiments, the functional antibody Abs-362 is composed of a chimeric antibody Abs-362 first polypeptide (SEQ ID NO: 106) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35Q / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-362 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 224) of the first Abs-362 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-362 polypeptide into host cells for expression.

[0200] In certain embodiments, the functional antibody Abs-363 is composed of a chimeric antibody Abs-363 first polypeptide (SEQ ID NO: 107) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35Q / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-363 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 225) of the first Abs-363 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-363 polypeptide into host cells for expression.

[0201] In certain embodiments, the functional antibody Abs-364 is composed of a chimeric antibody Abs-364 first polypeptide (SEQ ID NO: 108) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35Q / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-364 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 226) of the first Abs-364 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-364 polypeptide into host cells for expression.

[0202] In certain embodiments, the functional antibody Abs-365 is composed of a chimeric antibody Abs-365 first polypeptide (SEQ ID NO: 109) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35T / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-365 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 227) of the first Abs-365 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-365 polypeptide into a host cell for expression.

[0203] In certain embodiments, the functional antibody Abs-366 is composed of a chimeric antibody Abs-366 first polypeptide (SEQ ID NO: 110) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35T / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-366 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 228) of the first Abs-366 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-366 polypeptide into host cells for expression.

[0204] In certain embodiments, the functional antibody Abs-367 is composed of a chimeric antibody Ab-367 first polypeptide (SEQ ID NO: 111) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35T / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-367 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 229) of the first Ab-367 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-367 polypeptide into host cells for expression.

[0205] In certain embodiments, the functional antibody Abs-368 is composed of a chimeric antibody Ab-368 first polypeptide (SEQ ID NO: 112) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant E35T / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-368 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 230) of the first Ab-368 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-368 polypeptide into host cells for expression.

[0206] In certain embodiments, the functional antibody Abs-247 is composed of a chimeric antibody Abs-247 first polypeptide (SEQ ID NO: 113) formed by covalently linking the CD80 IgV mutant M47A / I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The functional antibody Abs-247 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 231) of the first Abs-247 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-247 polypeptide into a host cell for expression.

[0207] In certain embodiments, the functional antibody Abs-248 is composed of a chimeric antibody Abs-248 first polypeptide (SEQ ID NO: 114) formed by covalently linking the CD80 IgV mutant M47A / I69W / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The functional antibody Abs-248 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 232) of the first Abs-248 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-248 polypeptide into host cells for expression.

[0208] In certain embodiments, the functional antibody Abs-249 is composed of a chimeric antibody Abs-249 first polypeptide (SEQ ID NO: 115) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by covalently linking the CD80 IgV mutant M47A / I69Y / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker (SEQ ID NO: 95). The functional antibody Abs-249 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 233) of the first Abs-249 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-249 polypeptide into host cells for expression.

[0209] In certain embodiments, the functional antibody Abs-250 is composed of a chimeric antibody Abs-250 first polypeptide (SEQ ID NO: 116) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant M47A / I69W / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker (SEQ ID NO: 95). The assembly into the Abs-250 functional antibody is achieved by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 234) of the first Abs-250 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-250 polypeptide into a host cell for expression.

[0210] In certain embodiments, the functional antibody Abs-251 is composed of a chimeric antibody Abs-251 first polypeptide (SEQ ID NO: 117) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-251 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 235) of the first Abs-251 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-251 polypeptide into host cells for expression.

[0211] In certain embodiments, the functional antibody Abs-252 is composed of a chimeric antibody Abs-252 first polypeptide (SEQ ID NO: 118) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant I69W / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-252 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 236) of the first Abs-252 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-252 polypeptide into host cells for expression.

[0212] In certain embodiments, the functional antibody Abs-253 is composed of a chimeric antibody Abs-253 first polypeptide (SEQ ID NO: 119) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant I69Y / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-253 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 237) of the first Abs-253 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-253 polypeptide into host cells for expression.

[0213] In certain embodiments, the functional antibody Abs-254 is composed of a chimeric antibody Abs-254 first polypeptide (SEQ ID NO: 120) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant I69W / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-254 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 238) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-254 second polypeptide into host cells for expression.

[0214] In certain embodiments, the functional antibody Abs-255 is composed of a chimeric antibody Abs-255 first polypeptide (SEQ ID NO: 121) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant H18W / A26M / A71E / L85N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-255 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 239) of the first Abs-255 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-255 polypeptide into host cells for expression.

[0215] In certain embodiments, the functional antibody Abs-256 is composed of a chimeric antibody Abs-256 first polypeptide (SEQ ID NO: 122) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant H18W / A26M / A71E / L85D to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-256 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 240) of the first Abs-256 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-256 polypeptide into host cells for expression.

[0216] In certain embodiments, the functional antibody Abs-257 is composed of a chimeric antibody Abs-257 first polypeptide (SEQ ID NO: 123) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant H18W / A26C / A71E / L85N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-257 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 241) of the first Abs-257 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-257 polypeptide into host cells for expression.

[0217] In certain embodiments, the functional antibody Abs-258 is composed of a chimeric antibody Abs-258 first polypeptide (SEQ ID NO: 124) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant H18W / A26C / A71E / L85D to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-258 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 242) of the first Abs-258 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-258 polypeptide into host cells for expression.

[0218] In certain embodiments, the functional antibody Abs-259 is composed of a chimeric antibody Abs-259 first polypeptide (SEQ ID NO: 125) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant A26M / A71E / L85N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-259 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 243) of the first Abs-259 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-259 polypeptide into host cells for expression.

[0219] In certain embodiments, the functional antibody Abs-260 is composed of a chimeric antibody Abs-260 first polypeptide (SEQ ID NO: 126) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant A26M / A71E / L85D to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-260 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 244) of the Abs-260 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-260 second polypeptide into host cells for expression.

[0220] In certain embodiments, the functional antibody Abs-261 is composed of a chimeric antibody Abs-261 first polypeptide (SEQ ID NO: 127) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant A26C / A71E / L85D to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-261 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 245) of the first Abs-261 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-261 polypeptide into host cells for expression.

[0221] In certain embodiments, the functional antibody Abs-262 is composed of a chimeric antibody Ab-262 first polypeptide (SEQ ID NO: 128) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant A26C / A71E / L85N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-262 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 246) of the first Ab-262 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-262 polypeptide into host cells for expression.

[0222] In certain embodiments, the functional antibody Abs-263 is composed of a chimeric antibody Ab-263 first polypeptide (SEQ ID NO: 129) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant I69L / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-263 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 247) of the first Ab-263 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-263 polypeptide into host cells for expression.

[0223] In certain embodiments, the functional antibody Abs-264 is composed of a chimeric antibody Ab-264 first polypeptide (SEQ ID NO: 130) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant H18W / A26V / E35N / A71E / L85N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-264 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 248) of the first Ab-264 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-264 polypeptide into host cells for expression.

[0224] In certain embodiments, the functional antibody Abs-265 is composed of a chimeric antibody Ab-265 first polypeptide (SEQ ID NO: 131) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant T13K / M42L / M47H / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-265 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 249) of the first Ab-265 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-265 polypeptide into host cells for expression.

[0225] In certain embodiments, the functional antibody Abs-266 is composed of a chimeric antibody Abs-266 first polypeptide (SEQ ID NO: 132) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant T13K / M42L / M47Q / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-266 is assembled by introducing a vector expressing a polynucleotide sequence of the Abs-268 first polypeptide (SEQ ID NO: 250) and a vector expressing a polynucleotide sequence of the Abs-266 second polypeptide (SEQ ID NO: 215) into a host cell for expression.

[0226] In certain embodiments, the functional antibody Abs-267 is composed of a chimeric antibody Ab-267 first polypeptide (SEQ ID NO: 133) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant T13K / M42L / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-267 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 251) of the first Ab-267 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-267 polypeptide into host cells for expression.

[0227] In certain embodiments, the functional antibody Abs-268 is composed of a chimeric antibody Ab-268 first polypeptide (SEQ ID NO: 134) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant T13K / M42T / M47H / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-268 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 252) of the first Ab-268 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Ab-268 polypeptide into host cells for expression.

[0228] In certain embodiments, the functional antibody Abs-269 is composed of a chimeric antibody Abs-269 first polypeptide (SEQ ID NO: 135) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant T13K / M42T / M47T / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-269 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 253) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-269 second polypeptide into host cells for expression.

[0229] In certain embodiments, the functional antibody Abs-270 is composed of a chimeric antibody Abs-270 first polypeptide (SEQ ID NO: 136) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by covalently linking the CD80 IgV mutant T13K / M42T / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-270 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 254) of the first Abs-270 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-270 polypeptide into host cells for expression.

[0230] In certain embodiments, the functional antibody Abs-271 is composed of a chimeric antibody Abs-271 first polypeptide (SEQ ID NO: 137) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant M47H / I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-271 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 255) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-271 second polypeptide into host cells for expression.

[0231] In certain embodiments, the functional antibody Abs-272 is composed of a chimeric antibody Abs-272 first polypeptide (SEQ ID NO: 138) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant M47Q / I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-272 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 256) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-272 second polypeptide into host cells for expression.

[0232] In certain embodiments, the functional antibody Abs-273 is composed of a chimeric antibody Abs-273 first polypeptide (SEQ ID NO: 139) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 IgV mutant M47K / I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-273 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 257) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-273 second polypeptide into host cells for expression.

[0233] In certain embodiments, the functional antibody Abs-99 is composed of a chimeric antibody Abs-99 first polypeptide (SEQ ID NO: 140) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking a wild-type CD80 IgV to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The assembly into the Abs-functional antibody is achieved by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 258) of the first Abs-99 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-99 polypeptide into a host cell for expression.

[0234] In certain embodiments, the functional antibody Abs-509 is composed of a chimeric antibody Abs-509 first polypeptide (SEQ ID NO: 142) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by covalently linking the CD80 ECD mutant E35N / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-509 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 259) of the first Abs-509 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-509 polypeptide into host cells for expression.

[0235] In certain embodiments, the functional antibody Abs-510 is composed of a chimeric antibody Abs-510 first polypeptide (SEQ ID NO: 143) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 ECD mutant E35N / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker (SEQ ID NO: 95). The functional antibody Abs-510 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 260) of the first Abs-510 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-510 polypeptide into a host cell for expression.

[0236] In certain embodiments, the functional antibody Abs-511 is composed of a chimeric antibody Abs-511 first polypeptide (SEQ ID NO: 144) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 ECD mutant E35N / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-511 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 261) of the first Abs-511 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-511 polypeptide into host cells for expression.

[0237] In certain embodiments, the functional antibody Abs-512 is composed of a chimeric antibody Abs-512 first polypeptide (SEQ ID NO: 145) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the CD80 ECD mutant E35N / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-512 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 262) of the first Abs-512 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-512 polypeptide into host cells for expression.

[0238] In certain embodiments, the functional antibody Abs-98 is composed of a chimeric antibody Abs-98 first polypeptide (SEQ ID NO: 263) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) formed by covalently linking the wild-type CD80 ECD to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-98 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 264) of the first Abs-98 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-98 polypeptide into a host cell for expression.

[0239] In certain embodiments, the functional antibody Abs-480 is composed of a chimeric polypeptide Abs-480 (SEQ ID NO: 158) formed by covalently linking the CD80 ECD mutant E35N / A71E to the N-terminus of IgG4 Fc, and assembly into an Abs-480 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 265) vector expressing the Abs-480 polypeptide into a host cell for expression.

[0240] In certain embodiments, the functional antibody Abs-481 is composed of a chimeric polypeptide Abs-481 (SEQ ID NO: 159) formed by covalently linking the CD80 ECD mutant E35N / A71N to the N-terminus of IgG4 Fc, and the assembly into the Abs-481 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 266) vector expressing the Abs-481 polypeptide into a host cell for expression.

[0241] In certain embodiments, the functional antibody Abs-482 is composed of a chimeric polypeptide Abs-482 (SEQ ID NO: 160) formed by covalently linking the CD80 ECD mutant E35N / A71Q to the N-terminus of IgG4 Fc, and assembly into an Abs-482 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 267) vector expressing the Abs-482 polypeptide into a host cell for expression.

[0242] In certain embodiments, the functional antibody Abs-484 is composed of a chimeric polypeptide Abs-484 (SEQ ID NO: 161) formed by covalently linking the CD80 ECD mutant E35N / A71T to the N-terminus of IgG4 Fc, and assembly into an Abs-484 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 268) vector expressing the Abs-484 polypeptide into a host cell for expression.

[0243] In certain embodiments, the functional antibody Abs-100 is formed by covalently linking the wild-type CD80 ECD to the N-terminus of IgG4 Fc to form a chimeric polypeptide Abs-100 (SEQ ID NO: 174), and the assembly into the Abs-100 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 269) vector expressing the Abs-100 polypeptide into a host cell for expression.

[0244] In certain embodiments, the functional antibody Abs-427 is composed of a chimeric polypeptide Abs-427 (SEQ ID NO: 175) formed by covalently linking the CD80 IgV mutant E35N / A71E to the N-terminus of IgG4 Fc, and assembly into an Abs-427 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 270) vector expressing the Abs-427 polypeptide into a host cell for expression.

[0245] In certain embodiments, the functional antibody Abs-428 is composed of a chimeric polypeptide Abs-428 (SEQ ID NO: 176) formed by covalently linking the CD80 IgV mutant E35N / A71N to the N-terminus of IgG4 Fc, and assembly into an Abs-428 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 271) vector expressing the Abs-428 polypeptide into a host cell for expression.

[0246] In certain embodiments, the functional antibody Abs-429 is composed of a chimeric polypeptide Abs-429 (SEQ ID NO: 177) formed by covalently linking the CD80 IgV mutant E35N / A71Q to the N-terminus of IgG4 Fc, and assembly into an Abs-429 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 272) vector expressing the Abs-429 polypeptide into a host cell for expression.

[0247] In certain embodiments, the functional antibody Abs-483 is composed of a chimeric polypeptide Abs-483 (SEQ ID NO: 178) formed by covalently linking the CD80 IgV mutant E35N / A71T to the N-terminus of IgG4 Fc, and assembly into an Abs-483 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 273) vector expressing the Abs-483 polypeptide into a host cell for expression.

[0248] In certain embodiments, the functional antibody Abs-504 is composed of a chimeric polypeptide Abs-504 (SEQ ID NO: 191) formed by covalently linking wild-type CD80 IgV to the N-terminus of IgG4 Fc, and assembly into the Abs-504 functional polypeptide complex is achieved by introducing a polynucleotide sequence (SEQ ID NO: 274) vector expressing the Abs-504 polypeptide into a host cell for expression.

[0249] In certain embodiments, the functional antibody Abs-505 is composed of a chimeric antibody Abs-505 first polypeptide (SEQ ID NO: 198) and a Relatlimab light chain second polypeptide (SEQ ID NO: 197) formed by covalently linking the CD80 IgV mutant E35N / A71E to the N-terminus of the Relatlimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-505 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 276) of the Abs-505 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 275) of the Abs-505 second polypeptide into host cells for expression.

[0250] In certain embodiments, the functional antibody Abs-513 is composed of a chimeric antibody Abs-513 first polypeptide (SEQ ID NO: 199) and a Relatlimab light chain second polypeptide (SEQ ID NO: 197) formed by covalently linking the CD80 IgV mutant E35N / A71N to the N-terminus of the Relatlimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-513 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 277) of the first Abs-513 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 275) of the second Abs-513 polypeptide into host cells for expression.

[0251] In certain embodiments, the functional antibody Abs-514 is composed of a chimeric antibody Abs-514 first polypeptide (SEQ ID NO: 200) and a Relatlimab light chain second polypeptide (SEQ ID NO: 197) formed by covalently linking the CD80 IgV mutant E35N / A71Q to the N-terminus of the Relatlimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-514 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 278) of the first Abs-514 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 276) of the second Abs-514 polypeptide into host cells for expression.

[0252] In certain embodiments, the functional antibody Abs-515 is composed of a chimeric antibody Abs-515 first polypeptide (SEQ ID NO: 201) and a Relatlimab light chain second polypeptide (SEQ ID NO: 197) formed by covalently linking the CD80 IgV mutant E35N / A71T to the N-terminus of the Relatlimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-515 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 279) of the first Abs-515 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 276) of the second Abs-515 polypeptide into host cells for expression.

[0253] In certain embodiments, the functional antibody Abs-506 is composed of a chimeric antibody Abs-506 first polypeptide (SEQ ID NO: 202) and a Relatlimab light chain second polypeptide (SEQ ID NO: 197) formed by covalently linking a wild-type CD80 IgV to the N-terminus of the Relatlimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-506 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 280) of the Abs-506 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 276) of the Abs-506 second polypeptide into a host cell for expression.

[0254] In certain embodiments, the functional antibody Abs-507 is composed of a chimeric antibody Abs-506 first polypeptide (SEQ ID NO: 204) and a CD3B219 light chain second polypeptide (SEQ ID NO: 203) formed by covalently linking the CD80 IgV mutant E35N / A71E to the N-terminus of the CD3B219 antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-507 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 282) of the first Abs-507 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the second Abs-507 polypeptide into host cells for expression.

[0255] In certain embodiments, the functional antibody Abs-516 is composed of a chimeric antibody Abs-516 first polypeptide (SEQ ID NO: 205) and a CD3B219 light chain second polypeptide (SEQ ID NO: 203) formed by covalently linking the CD80 IgV mutant E35N / A71N to the N-terminus of the CD3B219 antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-516 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 283) of the first Abs-516 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the second Abs-516 polypeptide into host cells for expression.

[0256] In certain embodiments, the functional antibody Abs-517 is composed of a chimeric antibody Abs-517 first polypeptide (SEQ ID NO: 206) and a CD3B219 light chain second polypeptide (SEQ ID NO: 203) formed by covalently linking the CD80 IgV mutant E35N / A71Q to the N-terminus of the CD3B219 antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-517 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 284) of the first Abs-517 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the second Abs-517 polypeptide into host cells for expression.

[0257] In certain embodiments, the functional antibody Abs-518 is composed of a chimeric antibody Abs-518 first polypeptide (SEQ ID NO: 207) and a CD3B219 light chain second polypeptide (SEQ ID NO: 203) formed by covalently linking the CD80 IgV mutant E35N / A71T to the N-terminus of the CD3B219 antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-518 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 285) of the first Abs-518 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the second Abs-518 polypeptide into host cells for expression.

[0258] In certain embodiments, the functional antibody Abs-508 is composed of a chimeric antibody Abs-508 first polypeptide (SEQ ID NO: 207) and a CD3B219 light chain second polypeptide (SEQ ID NO: 204) formed by covalently linking a wild-type CD80 IgV to the N-terminus of the CD3B219 antibody heavy chain via a linker peptide (SEQ ID NO: 95). The functional antibody Abs-508 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 286) of the Abs-first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the Abs-508 second polypeptide into a host cell for expression.

[0259] In certain embodiments, the functional antibody Abs-16 is composed of a chimeric antibody Abs-16 first polypeptide (SEQ ID NO: 287) formed by the N-terminus of the sugemalimab antibody heavy chain and a sugemalimab light chain second polypeptide (SEQ ID NO: 263), and the assembly into the Abs-16 functional antibody is achieved by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 288) of the Abs-16 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the Abs-16 second polypeptide into host cells for expression.

[0260] In certain embodiments, the functional antibody Abs-58 is composed of a chimeric antibody Abs-58 first polypeptide (SEQ ID NO: 289) formed by the N-terminus of the Relatlimab antibody heavy chain and a Relatlimab light chain second polypeptide (SEQ ID NO: 197), and the assembly into the Abs-58 functional antibody is achieved by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 290) of the Abs-58 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 276) of the Abs-58 second polypeptide into a host cell for expression.

[0261] In certain embodiments, the functional antibody Abs-519 is composed of a chimeric antibody Abs-520 first polypeptide (SEQ ID NO: 291) formed by the N-terminus of the CD3B219 antibody heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 204), and is assembled into the Abs-519 functional antibody by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 292) of the Abs-519 first polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 281) of the Abs-519 second polypeptide into a host cell for expression.

[0262] In certain embodiments, the functional antibody Abs-520 is composed of a chimeric antibody Abs-520 first polypeptide (SEQ ID NO: 293) formed by covalently linking the CD80 IgV mutant E35N / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95), and a sugemalimab light chain second polypeptide (SEQ ID NO: 141) via a linker peptide (SEQ ID NO: 95). The multifunctional antibody Abs-520 is assembled by introducing a vector expressing a polynucleotide sequence (SEQ ID NO: 294) of the first Abs-520 polypeptide and a vector expressing a polynucleotide sequence (SEQ ID NO: 215) of the second Abs-520 polypeptide into a host cell for expression.

[0263] In certain embodiments, the functional antibody Abs-521 is composed of a chimeric antibody Ab-521 first polypeptide (SEQ ID NO: 295) formed by covalently linking the CD80 IgV mutant E35N / A71N to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95), which is then covalently linked to the LAG3 polypeptide at the C-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95), and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The multifunctional antibody Abs-521 is assembled by introducing a vector expressing a polynucleotide sequence of the Ab-520 first polypeptide (SEQ ID NO: 296) and a vector expressing a polynucleotide sequence of the Ab-521 second polypeptide (SEQ ID NO: 215) into a host cell for expression.

[0264] Although various embodiments of the present invention have been described above, it should be understood that these are provided by way of example only and not limitation. Many variations of the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the present invention. Therefore, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

[0265] All documents mentioned in this disclosure are incorporated herein by reference.All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were individually denoted.

[0266] Example

[0267] Example 1. Antibody expression and purification

[0268] In order to further identify the antibodies obtained by screening, it is necessary to express the antibodies in mammalian cells. Therefore, an expression plasmid vector for the heavy chain of the multifunctional / bifunctional antibody and an expression plasmid vector containing the light chain were first constructed. About 24 hours before plasmid transfection, Expi293 cells were passaged to a cell density of approximately 2.4×10 6 cells / ml. When the cell density is 6×10 6 When the cell viability was >95%, 25 μg of the mixed heavy and light chain plasmid vector was taken and transfected into 25 ml of Expi293 cells using Expifectamine 293. The cells were cultured in a shaker at 37°C, 130 rpm, and 8% CO2 for 7 days. The cell culture product was centrifuged and the supernatant was collected. The supernatant was filtered through a 0.45 MCE filter and purified using a Capturem protein A Maxiprep column to collect the target antibody. The antibody was then concentrated by centrifugation using a Vivaspin 20 Centrifugal Concentrator 50K and quantified by the A280 method using a NanoDrop 2000. The antibody purity was determined by SDS-PAGE and SEC-HPLC.

[0269] Example 2. Protein electrophoresis analysis

[0270] The following CD80 proteins selected and purified under reducing conditions (NR) and non-reducing conditions (N) were analyzed by SDS-PAGE. IgV mutant-sugemalimab fusion peptide complexes were analyzed: Abs-99, Abs-0247, Abs-248, Abs-249, Abs-250, Abs-251, Abs-252, Abs-253, Abs-254, Abs-255, Abs-256, Abs-257, Abs-258, Abs-259, Abs-260, Abs-261, Abs-262, Abs-263, Abs-264, Abs-265, Abs-266, Abs-267, Abs-268, Abs-269, Abs-270, Abs-271, Abs-272, Abs-273, Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-358, Abs-359, Abs-360, Abs-361, Abs-362, Abs-363, Abs-364, Abs-365, Abs-366, Abs-367, Abs-368.

[0271] The results of electrophoresis analysis showed that the CD80 IgV mutant-sugemalimab fusion polypeptide complexes Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-360, Abs-362, Abs-364, Abs-367, and Abs-368 antibody assemblies were superior to the wild-type CD80 IgV-sugemalimab fusion polypeptide complex Abs-99, as shown in Figure 1 .

[0272] Example 3. Binding affinity determination

[0273] This example shows the binding of the CD80 mutant fusion polypeptide complex to human PD-L1 protein, human CTLA4 protein and human CD28.

[0274] Specifically, human PD-L1 protein (1 μg / mL in PBS; Acro, PD1-H5258) was incubated in a flat-bottom 96-well Immuno detachable transparent ELISA plate (MaxiSorp) (Thermo Scientific, 446469) at 4°C overnight; then, after washing, 2% BSA (in PBS; VWR Life Science, 0332-1KG) was added to each well and incubated at room temperature for 1 hour. After washing, serial dilutions of the test substance were added to each well and added to the final test concentration and incubated at room temperature for 2 hours. After washing, Goat Anti-Human Lambda-HRP (Southern Biotech, 2060-05) was added to each well and incubated at room temperature for 30 minutes. After washing, the Soluble TMB Kit (Cornwell, CW0050S) was used for detection. The specific operations were performed according to the manufacturer's instructions. The affinity of the candidate multi- / bifunctional fusion proteins to the human PD-L1 protein was detected using EC50 and a binding intensity of 20 nM. The test results are shown in Table 1 and Figure 2.

[0275] Table 1. EC50 and binding strength at 100 nM of CD80 mutant fusion peptide complexes binding to human PDL1 protein

[0276] The results shown in Table 1 and Figure 2 indicate that in the CD80 mutant fusion polypeptide complex binding experiment with human PDL1 antigen, the CD80 mutant fusion polypeptide complexes Abs-427, Abs-428, and Abs-483 had much higher binding activity to the PDL1 antigen than the wild-type CD80 fusion polypeptide complex Ab-504.

[0277] Similarly, human CD28 protein (1 μg / mL in PBS; Acro, CD8-H525a) was incubated in a flat-bottom 96-well Immuno detachable transparent ELISA plate (MaxiSorp) (Thermo Scientific, 446469) at 4°C overnight. The affinity of the test substance to human CD28 protein was detected using a similar method as described above, and the binding strength was expressed as EC50 and 100 nM. The test results are shown in Table 2 and Figure 4.

[0278] Table 2. EC50 and binding strength of CD80 mutant fusion polypeptide complexes binding to human CD28 protein at 100 nM NA: indicates no binding to the target protein

[0279] The results shown in Table 2 and Figure 4 indicate that in the CD80 mutant fusion polypeptide complex binding experiment with human CD28 antigen, the antigen binding activity of the CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-359, Abs-360, Abs-362, Abs-364, Abs-365, Abs-367, and Abs-368 to CD28 was much higher than that of the wild-type CD80 fusion polypeptide complex Ab-99.

[0280] Similarly, human CTLA4 protein (1 μg / mL in PBS; Acro, CT4-H52H9) was incubated in a flat-bottom 96-well Immuno detachable transparent ELISA plate (MaxiSorp) (Thermo Scientific, 446469) at 4°C overnight. The affinity of the test substance to the human CTLA4 protein was detected using a similar method as described above, and the binding strength was expressed as EC50 and 20 nM. The test results are shown in Table 3 and Figure 3.

[0281] Table 3. EC50 and binding strength of CD80 mutant fusion polypeptide complexes binding to human CTLA4 protein at 20 nM NA: indicates no binding to the target protein

[0282] The results shown in Table 3 and Figure 3 indicate that in the CD80 mutant fusion polypeptide complex binding experiment with human CTLA4 antigen, the CD80 mutant fusion polypeptide complexes Abs-267, Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-362, and Abs-364 had much higher binding activity to the CTLA4 antigen than the wild-type CD80 fusion polypeptide complex Abs-99.

[0283] Example 4. Determination of the CD80 mutant fusion polypeptide complex to relieve PD-1 / PD-L1-mediated T cell immunosuppression

[0284] This example demonstrates that the CD80 mutant fusion polypeptide complex relieves the PD-1 / PD-L1-mediated T cell inhibitory response.

[0285] Specifically, on the first day, TCR activator PD-L1-CHO cells (BPS bioscience) were digested and collected using 0.05% trypsin, counted, and plated. Fresh culture medium was added to mix the cells and the cell density was adjusted to 3.5*10 5 Cells were added to a sterile 96-well flat-bottom plate (CORNING, 3599) at 100ul / well, for a total of 35,000 cells / well; grown overnight in a 37°C incubator until 80% growth; the next day, the culture medium of the TCR activator PD-L1-CHO cells in the 96-well flat-bottom plate was removed, and 50ul of fresh culture medium containing the corresponding concentration of the test substance (serial dilution) was added to each well. After 30 minutes of treatment, Jurkat-Lucia TM TCR-hPD-1 cells (Invivogen) were counted; fresh culture medium was added to mix the cells and the cell density was adjusted to 4*10 5 cells / ml; add 50ul Jurkat-Lucia TM The final density of TCR-hPD-1 cells was 2*10 4 / well, incubate in a 37°C incubator for 5-6 hours in a 96-well flat-bottom plate. After incubation, observe the cell morphology under a microscope and use QUANTI-Luc TM Chemiluminescence was detected by using Invivogen (rep-qlc1). The specific operation was performed according to the manufacturer's instructions. The activity was expressed as EC50 and the highest induction fold. The test results are shown in Tables 4, 5, Figures 5 and 6.

[0286] Table 4. Results of the assay for the regulation of Jurkat T cell activation by CD80 mutant fusion peptide complexes NT: Not tested, NA: No stimulating effect

[0287] The results in Table 4 and Figure 5 show that in the experiment of regulating Jurkat T cell activation by CD80 mutant fusion polypeptide complexes, the CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-358, Abs-361, and Abs-362 had higher abilities to regulate Jurkat T cell activation than the wild-type CD80 fusion polypeptide complex Ab-99.

[0288] Table 5. Results of assays of CD80 mutant fusion peptide complexes regulating Jurkat T cell activation NA: No exciting effect

[0289] The results in Table 5 and Figure 6 show that in the experiment of regulating Jurkat T cell activation by CD80 mutant fusion polypeptide complexes, the ability of CD80 mutant fusion polypeptide complex Abs-428 to regulate Jurkat T cell activation was higher than that of wild-type CD80 fusion polypeptide complex Abs-504.

[0290] Example 5. Determination of the CD80 mutant fusion polypeptide complex's ability to relieve LAG3-mediated T cell immunosuppression

[0291] This example demonstrates that the CD80 mutant fusion polypeptide complex relieves LAG3-mediated T cell inhibitory responses.

[0292] Specifically, Raji cells (ATCC) were collected, counted, and plated. Fresh culture medium was added to mix the cells and the cell density was adjusted to 3.5*10 5 Cells were added to a sterile 96-well flat-bottom plate (CORNING, 3599) at 100 μl / well for a total of 35,000 cells / well. 50 μl of fresh culture medium containing the corresponding concentration of test substance (serial dilutions) was added to each well. After 30 minutes of treatment, LAG3 / NFAT Reporter-Jurkat cells (BPS bioscience) were collected and counted. Fresh culture medium containing 0.4 ng / ml Staphylococcal enterotoxin E (SEE, Toxintechnology) was added to mix the cells and adjust the cell density to 4*10 5 Add 50ul of LAG3 / NFAT Reporter-Jurkat cells to a final cell density of 2*10 4 / well, incubate in a 37°C incubator for 5-6 hours in a 96-well flat-bottom plate. After incubation, observe the cell morphology under a microscope and use QUANTI-Luc TMChemiluminescence was detected using a chemiluminescence analyzer (BPS bioscience; 78262) according to the manufacturer's instructions.

[0293] Table 6. Results of the assay to relieve LAG3-mediated T cell immunosuppression by CD80 mutant fusion polypeptide complexes

[0294] In Table 6, the experiment of CD80 mutant fusion polypeptide complexes relieving LAG3-mediated T cell immunosuppression showed that the CD80 mutant fusion polypeptide complexes Abs-505, Abs-513, Abs-514, and Abs-515 had a higher ability to relieve LAG3-mediated T cell immunosuppression than the wild-type CD80 fusion polypeptide complex Ab-506.

[0295] Example 6. T cell activation immune response assay

[0296] This example shows that the CD80 mutant fusion polypeptide complex regulates T cell immune response.

[0297] Specifically, collect Jurkat-Lucia TM TCR cells (Invivogen) were counted. Fresh culture medium was added to mix the cells and the cell density was adjusted to 4*10 5 cells / ml. 100ul of fresh culture medium containing the corresponding concentration of test substance (serial dilution) was added to each well and incubated in a 96-well flat-bottom plate in a 37°C incubator for 5-6 hours. After incubation, the cell morphology was observed under a microscope and the QUANTI-Luc TM The chemiluminescence value was detected by using Invivogen (rep-qlc1), and the specific operation was performed according to the manufacturer's instructions.

[0298] Table 7. Results of the assay for T cell activation immune response of CD80 mutant fusion polypeptide complexes

[0299] The results in Table 7 show that in the CD80 mutant fusion polypeptide complex T cell activation immune response assay, the CD80 mutant fusion polypeptide complexes Abs-507, Abs-516, Abs-517, and Abs-518 have higher abilities to activate T cell immune responses than the wild-type CD80 fusion polypeptide complex Ab-508.

[0300] Example 7. Primary PBMC immune response assay

[0301] This example demonstrates the activation of T cell immunity in primary PBMCs by CD80 mutant fusion polypeptide complexes. Specifically, PBMCs were collected, counted, and plated. Fresh culture medium was added to mix the cells and the cell density was adjusted to 1×10 5 10 cells / ml. 50 μl of fresh culture medium containing the corresponding test substance (serial dilutions) was added to each well. After 3 days of incubation, the culture supernatant was collected and IL-2 secretion in the supernatant was measured using an IL-2 ELISA kit (R&D, DY202) according to the manufacturer's instructions.

[0302] Table 8. Results of assays to determine the activation of primary PBMC T cell immune responses by CD80 mutant fusion polypeptide complexes

[0303] The results in Table 8 and Figure 7 indicate that the CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, and Abs-356 have higher abilities to activate T cells in primary PBMCs than the wild-type CD80 fusion polypeptide complex Abs-99.

[0304] Example 8. Determination of Chemokine Secretion by Antigen Presenting Cells

[0305] This example shows that Abs-induced antigen-presenting cells secrete the chemokine CCL4.

[0306] Specifically, antigen presenting THP-1 cells (ATCC) were collected, counted, and plated. Fresh culture medium was added to mix the cells and the cell density was adjusted to 1*10 6 100 μl of cells were plated per well at a concentration of 100 cells / ml. 100 μl of fresh culture medium containing the corresponding test compound, Abs_520 or Abs_521, was added to each well. After 6 hours, the cell culture supernatant was collected and assayed for CCL4 expression using a CCL4 detection kit (PROTEINTECH) according to the manufacturer's instructions. The results showed that Abs_520 and Abs_521 induced chemokine secretion from antigen-presenting cells, as shown in Figure 8.

[0307] The results of the above examples demonstrate that the CD80 mutant polypeptides formed by innovatively modifying the CD80 IgV domain can modulate the binding activity of CD80 to its ligands. Specifically, the CD80 mutant fusion polypeptide complexes disclosed herein have significantly higher antigen binding activities to PDL1, CTLA4, and CD28 than the corresponding wild-type CD80 fusion polypeptide complexes. The CD80 mutant fusion polypeptide complexes disclosed herein can relieve PD-1 / PD-L1-mediated T cell activation immunosuppression, regulate Jurkat T cell activation, regulate PBMC activation, and promote chemokine secretion by antigen-presenting cells. In other words, the CD80 mutant fusion polypeptide complexes disclosed herein can effectively stimulate T cells and enhance immune responses, thereby achieving the treatment or prevention of diseases such as infections and tumors caused by suppressed T cell function.

[0308] Although various embodiments of the present invention have been described above, it should be understood that they are provided by way of example only and not limitation. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications will fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. A CD80 IgV mutant polypeptide comprising one or more amino acid mutations selected from the group consisting of T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D relative to the wild-type human CD80 IgV polypeptide sequence SEQ ID NO:

45.

2. The CD80 IgV mutant polypeptide of claim 1 , wherein the CD80 IgV mutant polypeptide comprises a combination of two or more of the amino acid mutations, optionally wherein the combination of two or more of the amino acid mutations is selected from E35N / A71E (SEQ ID NO: 46), E35N / A71N (SEQ ID NO: 47), E35N / A71Q (SEQ ID NO: 48), E35N / A71T (SEQ ID NO: 49), E35S / A71E (SEQ ID NO: 50), E35S / A71N (SEQ ID NO: 51), E35S / A71Q (SEQ ID NO: 52), E35S / A71T (SEQ ID NO: 53), E35Q / A71E (SEQ ID NO: 54), E35Q / A71N (SEQ ID NO: 55), E35Q / A71Q (SEQ ID NO: 56), E35Q / A71T (SEQ ID NO: 57). NO:57), E35T / A71E (SEQ ID NO:58), E35T / A71N (SEQ ID NO:59), E35T / A71Q (SEQ ID NO:60), E35T / A71T (SEQ ID NO:61), M47A / I69Y / A71E / V83L (SEQ ID NO:62), M47A / I69W / A71E / V83L (SEQ ID NO:63), M47A / I69Y / A71E (SEQ ID NO:64), M47A / I69W / A71E (SEQ ID NO:65), I69Y / A71E / V83L (SEQ ID NO:66), I69W / A71E / V83L (SEQ ID NO:67), I69Y / A71E (SEQ ID NO:68), I69W / A71E (SEQ ID NO:69), H18W / A26M / A71E / L85N(SEQ ID NO:70), H18W / A26M / A71E / L85D(SEQ ID NO:71), H18W / A26C / A71E / L85N(SEQ ID NO:72), H18W / A26C / A71E / L85D(SEQ ID NO:73), A26M / A71E / L85N (SEQ ID NO:74), A26M / A71E / L85D (SEQ ID NO:75), A26C / A71E / L85D (SEQ ID NO:76), A26C / A71E / L85N (SEQ ID NO:77), I69L / A71E / V83L(SEQ IDNO: 78), H18W / A26V / E35N / A71E / L85N (SEQ ID NO: 79), T13K / M42L / M47H / A71E (SEQ ID NO: 80), T13K / M42L / M47Q / A71E (SEQ ID NO: 81), T13K / M42L / A71E (SEQ ID NO: 82), T13K / M42T / M47H / A71E (SEQ ID NO: 83), T13K / M42T / M47T / A71E (SEQ ID NO: 84), T13K / M42T / A71E (SEQ ID NO: 85), M47H / I69Y / A71E / V83L (SEQ ID NO: 86), M47Q / I69Y / A71E / V83L (SEQ ID NO: 87) and M47K / I69Y / A71E / V83L (SEQ ID NO: 88).

3. A CD80 ECD mutant polypeptide comprising one or more amino acid mutations selected from the group consisting of T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D relative to the wild-type human CD80 ECD polypeptide sequence SEQ ID NO:

1.

4. The CD80 ECD mutant polypeptide of claim 3, wherein the CD80 ECD mutant polypeptide comprises a combination of two or more of the amino acid mutations, optionally wherein the combination of two or more of the amino acid mutations is selected from E35N / A71E (SEQ ID NO: 2), E35N / A71N (SEQ ID NO: 3), E35N / A71Q (SEQ ID NO: 4), E35N / A71T (SEQ ID NO: 5), E35S / A71E (SEQ ID NO: 6), E35S / A71N (SEQ ID NO: 7), E35S / A71Q (SEQ ID NO: 8), E35S / A71T (SEQ ID NO: 9), E35Q / A71E (SEQ ID NO: 10), E35Q / A71N (SEQ ID NO: 11), E35Q / A71Q (SEQ ID NO: 12), E35Q / A71T (SEQ ID NO: 13), NO:13), E35T / A71E (SEQ ID NO:14), E35T / A71N (SEQ ID NO:15), E35T / A71Q (SEQ ID NO:16), E35T / A71T (SEQ ID NO:17), M47A / I69Y / A71E / V83L (SEQ ID NO:18), M47A / I69W / A71E / V83L (SEQ ID NO:19), M47A / I69Y / A71E (SEQ ID NO:20), M47A / I69W / A71E (SEQ ID NO:21), I69Y / A71E / V83L (SEQ ID NO:22), I69W / A71E / V83L (SEQ ID NO:23), I69Y / A71E (SEQ ID NO:24), I69W / A71E (SEQ ID NO:25), H18W / A26M / A71E / L85N(SEQ ID NO:26), H18W / A26M / A71E / L85D(SEQ ID NO:27), H18W / A26C / A71E / L85N(SEQ ID NO:28), H18W / A26C / A71E / L85D(SEQ ID NO:29), A26M / A71E / L85N (SEQ ID NO:30), A26M / A71E / L85D (SEQ ID NO:31), A26C / A71E / L85D (SEQ ID NO:32), A26C / A71E / L85N (SEQ ID NO:33), I69L / A71E / V83L(SEQ IDNO: 34), H18W / A26V / E35N / A71E / L85N (SEQ ID NO: 35), T13K / M42L / M47H / A71E (SEQ ID NO: 36), T13K / M42L / M47Q / A71E (SEQ ID NO: 37), T13K / M42L / A71E (SEQ ID NO: 38), T13K / M42T / M47H / A71E (SEQ ID NO: 39), T13K / M42T / M47T / A71E (SEQ ID NO: 40), T13K / M42T / A71E (SEQ ID NO: 41), M47H / I69Y / A71E / V83L (SEQ ID NO: 42), M47Q / I69Y / A71E / V83L (SEQ ID NO: 43) and M47K / I69Y / A71E / V83L (SEQ ID NO: 44).

5. A CD80 mutant fusion polypeptide complex comprising the CD80 IgV mutant polypeptide of claim 1 or 2 or the CD80 ECD mutant polypeptide of claim 3 or 4 and a second domain, optionally wherein the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide is covalently linked to the second domain with or without a linker peptide.

6. The CD80 mutant fusion polypeptide complex of claim 5, wherein the second domain is an antibody or an antigen-binding fragment thereof, optionally wherein the antigen-binding fragment is selected from Fab, Fab', Fv, F(ab)2, F(ab')2, scFv, VHH, di-scFv and / or dAb.

7. The CD80 mutant fusion polypeptide complex of claim 6, wherein the antibody is selected from an immunoglobulin IgG antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody and / or a bispecific antibody, optionally wherein the immunoglobulin IgG antibody is selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b or mouse IgG3.

8. The CD80 mutant fusion polypeptide complex of claim 6 or 7, wherein the antibody or antigen-binding fragment thereof specifically targets one or more antigens selected from the group consisting of PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2 neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36, and / or Tim3.

9. The CD80 mutant fusion polypeptide complex of claim 8, wherein the antibody or antigen-binding fragment thereof specifically targets PD-L1, PD-1, TIGIT, CTLA4, LAG3 or CD3.

10. The CD80 mutant fusion polypeptide complex according to claim 9, wherein the antibody or antigen-binding fragment thereof targeting PD-L1 is Sugemalimab antibody or antigen-binding fragment thereof; the antibody or antigen-binding fragment thereof targeting LAG-3 is Relatlimab antibody or antigen-binding fragment thereof; and / or the antibody or antigen-binding fragment thereof targeting CD3 is CD3B219 antibody or antigen-binding fragment thereof.

11. The CD80 mutant fusion polypeptide complex of any one of claims 6 to 9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the heavy chain with or without a linker peptide, optionally wherein the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the heavy chain with or without a linker peptide; and / or the C-terminus of the heavy chain is covalently linked to the N-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

12. The CD80 mutant fusion polypeptide complex of any one of claims 6 to 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the light chain with or without a linker peptide, optionally wherein the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the light chain with or without a linker peptide; and / or the C-terminus of the light chain is covalently linked to the N-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

13. The CD80 mutant fusion polypeptide complex of claim 5, wherein the CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, and the second polypeptide may be present or absent, optionally wherein the first polypeptide comprises a heavy chain of an antibody or an antigen-binding fragment thereof, and / or the second polypeptide comprises a light chain of an antibody or an antigen-binding fragment thereof.

14. The CD80 mutant fusion polypeptide complex of claim 13, wherein the first polypeptide is covalently linked, from the N-terminus to the C-terminus, to a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, and a heavy chain of an antibody or an antigen-binding fragment thereof; or the first polypeptide is covalently linked, from the N-terminus to the C-terminus, to a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide.

15. The CD80 mutant fusion polypeptide complex of claim 14, wherein the second polypeptide is covalently linked, from the N-terminus to the C-terminus, to a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, and a light chain of an antibody or an antigen-binding fragment thereof; or the second polypeptide is covalently linked, from the N-terminus to the C-terminus, to a light chain of an antibody or an antigen-binding fragment thereof, a linker peptide, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide.

16. The CD80 mutant fusion polypeptide complex of claim 13 or 14, wherein the first polypeptide is selected from the group consisting of E35N / A71E (SEQ ID NO: 97), E35N / A71N (SEQ ID NO: 98), E35N / A71Q (SEQ ID NO: 99), E35N / A71T (SEQ ID NO: 100), E35S / A71E (SEQ ID NO: 101), E35S / A71N (SEQ ID NO: 102), E35S / A71Q (SEQ ID NO: 103), E35S / A71T (SEQ ID NO: 104), E35Q / A71E (SEQ ID NO: 105), E35Q / A71N (SEQ ID NO: 106), E35Q / A71Q (SEQ ID NO: 107), E35Q / A71T (SEQ ID NO: 108), NO:108), E35T / A71E (SEQ ID NO:109), E35T / A71N (SEQ ID NO:110), E35T / A71Q (SEQ ID NO:112), E35T / A71T (SEQ ID NO:113), M47A / I69Y / A71E / V83L (SEQ ID NO:114), M47A / I69W / A71E / V83L (SEQ ID NO:115), M47A / I69Y / A71E (SEQ ID NO:116), M47A / I69W / A71E (SEQ ID NO:117), I69Y / A71E / V83L (SEQ ID NO:118), I69W / A71E / V83L (SEQ ID NO:119), I69Y / A71E (SEQ ID NO:120), I69W / A71E (SEQ ID NO:121), H18W / A26M / A71E / L85N (SEQ ID NO:122),H18W / A26M / A71E / L85D (SEQ ID NO: 123), H18W / A26C / A71E / L85N (SEQ ID NO: 124), H18W / A26C / A71E / L85D (SEQ ID NO: 125), A26M / A71E / L85N (SEQ ID NO: 126), A26M / A71E / L85D (SEQ ID NO: 127), A26C / A71E / L85D (SEQ ID NO: 128), A26C / A71E / L85N (SEQ ID NO: 129), I69L / A71E / V83L (SEQ ID NO: 130), H18W / A26V / E35N / A71E / L85N (SEQ ID NO: 131), T13K / M42L / M47H / A71E (SEQ ID NO: 132), T13K / M42L / M47Q / A71E (SEQ ID NO: 133), T13K / M42L / A71E (SEQ ID NO: 134), T13K / M42T / M47H / A71E (SEQ ID NO: 135), T13K / M42T / M47T / A71E (SEQ ID NO: 136), T13K / M42T / A71E (SEQ ID NO: 136), M47H / I69Y / A71E / V83L (SEQ ID NO: 137), M47Q / I69Y / A71E / V83L (SEQ ID NO: 138) or M47K / I69Y / A71E / V83L (SEQ ID NO: 139); or, wherein the first polypeptide is selected from the group consisting of E35N / A71E (SEQ ID NO: 142), E35N / A71N (SEQ ID NO: 143), E35N / A71Q (SEQ ID NO: 144), E35N / A71T (SEQ ID NO: 145), E35S / A71E (SEQ ID NO: 146), E35S / A71N (SEQ ID NO: 147), E35S / A71Q (SEQ ID NO: 148), E35S / A71T (SEQ ID NO: 149), E35Q / A71E (SEQ ID NO: 150), E35Q / A71N (SEQ ID NO: 151), E35Q / A71Q (SEQ ID NO: 152), E35S / A71T (SEQ ID NO: 153), E35S / A71E (SEQ ID NO: 154), E35S / A71N (SEQ ID NO: 155), E35S / A71Q (SEQ ID NO: 156), E35S / A71T (SEQ ID NO: 157). NO: 152), E35Q / A71T (SEQ ID NO: 153), E35T / A71E (SEQ ID NO: 154), E35T / A71N (SEQ ID NO: 155), E35T / A71Q (SEQ ID NO: 156), or E35T / A71T (SEQ ID NO: 157); or wherein the first polypeptide is selected from E35N / A71E (SEQ ID NO: 192), E35N / A71N (SEQ ID NO: 193), E35N / A71Q (SEQ ID NO: 194) or E35N / A71T (SEQ ID NO: 195); or wherein the first polypeptide is selected from E35N / A71E (SEQ ID NO: 198), E35N / A71N (SEQ ID NO: 199), E35N / A71Q (SEQ ID NO: 200) or E35N / A71T (SEQ ID NO: 201); or wherein the first polypeptide is selected from E35N / A71E (SEQ ID NO: 203), E35N / A71N (SEQ ID NO: 204), E35N / A71Q (SEQ ID NO: 205) or E35N / A71T (SEQ ID NO: 206); or The first polypeptide is selected from E35N / A71E (SEQ ID NO: 209), E35N / A71N (SEQ ID NO: 210), E35N / A71Q (SEQ ID NO: 211) or E35N / A71T (SEQ ID NO: 212).

17. The CD80 mutant fusion polypeptide complex of claim 15 or 16, wherein the second polypeptide is selected from SEQ ID NO: 141, SEQ ID NO: 197 or SEQ ID NO:

208.

18. The CD80 mutant fusion polypeptide complex according to any one of claims 5 to 17, further comprising a third domain, which is covalently linked to the CD80 IgV mutant polypeptide according to claim 1 or 2, or the CD80 ECD mutant polypeptide according to claim 3 or 4, or the second domain according to any one of claims 5 to 17, with or without a linker peptide.

19. The CD80 mutant fusion polypeptide complex according to claim 18, wherein the third domain is an antibody or antigen-binding fragment that is the same as or different from the second domain, or the third domain is a functional protein or an active fragment thereof, optionally wherein: When the third domain is a functional protein or an active fragment thereof, the functional protein or the active fragment thereof can activate an immune response. Optionally, the third domain is connected to the CD80 IgV mutant polypeptide of claim 1 or 2 or the CD80 ECD mutant polypeptide of claim 3 or 4 with or without a linker peptide.

20. The CD80 mutant fusion polypeptide complex of claim 19, wherein the second domain comprises an antibody heavy chain, and the antibody heavy chain of the second domain is connected to the third domain via or without a linker peptide, optionally, in, The heavy chain of the second domain is connected to the N-terminus of the third domain via or without a linker peptide, and / or the heavy chain of the second domain is connected to the C-terminus of the third domain via or without a linker peptide; and / or The second domain comprises an antibody light chain, and the antibody light chain of the second domain is connected to the third domain with or without a linker peptide, optionally, wherein the light chain of the second domain is connected to the N-terminus of the third domain with or without a linker peptide, and / or the light chain of the second domain is connected to the C-terminus of the third domain with or without a linker peptide.

21. The CD80 mutant fusion polypeptide complex according to any one of claims 18 to 20, wherein: The CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, wherein the second polypeptide may be present or absent. Optionally, wherein the first polypeptide comprises a heavy chain of the second domain, optionally, wherein the first polypeptide is covalently linked from the N-terminus to the C-terminus by a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides may be the same or different and may be independently present or absent; or wherein the first polypeptide is covalently linked from the C-terminus to the N-terminus by a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides may be the same or different and may be independently present or absent, and / or wherein the second polypeptide comprises a light chain of the second domain, optionally, wherein the second polypeptide is covalently linked from the N-terminus to the C-terminus by a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides may be the same or different and may be independently present or absent; or The second polypeptide is composed of a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently linked in sequence from the C-terminus to the N-terminus, wherein the linker peptides may be the same or different and may be independently present or absent.

22. The CD80 mutant fusion polypeptide complex according to any one of claims 18 to 21, wherein: The third domain includes LAG-3 or an active fragment thereof.

23. The CD80 mutant fusion polypeptide complex according to claim 22, wherein: The second domain specifically targets PD-1 or PD-L1, optionally, wherein the second domain is sugemalimab or a functional fragment thereof.

24. The CD80 mutant fusion polypeptide complex according to claim 22, wherein: The first polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 293, SEQ ID NO: 295; or the second polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:

141.

25. The CD80 mutant fusion polypeptide complex of claim 5, wherein the second domain is an immunoglobulin Fc domain, optionally wherein the Fc domain is selected from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain or a mouse IgG3 Fc domain, optionally wherein the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide is covalently linked to the N-terminus of the immunoglobulin Fc domain with or without a linker peptide.

26. The CD80 mutant fusion polypeptide complex of claim 25, wherein the amino acid sequence of the CD80 mutant fusion polypeptide complex is selected from the group consisting of E35N / A71E (SEQ ID NO: 158), E35N / A71N (SEQ ID NO: 159), E35N / A71Q (SEQ ID NO: 160), E35N / A71T (SEQ ID NO: 161), E35S / A71E (SEQ ID NO: 162), E35S / A71N (SEQ ID NO: 163), E35S / A71Q (SEQ ID NO: 164), E35S / A71T (SEQ ID NO: 165), E35Q / A71E (SEQ ID NO: 166), E35Q / A71N (SEQ ID NO: 167), E35Q / A71Q (SEQ ID NO: 168), E35Q / A71T (SEQ ID NO: 169). NO:169), E35T / A71E (SEQ ID NO:170), E35T / A71N (SEQ ID NO:171), E35T / A71Q (SEQ ID NO:172) or E35T / A71T (SEQ ID NO:173).

27. The CD80 mutant fusion polypeptide complex according to claim 25 or 26, wherein the C-terminus of the immunoglobulin Fc domain is covalently linked to the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide with or without a linker peptide.

28. The CD80 mutant fusion polypeptide complex of any one of claims 5-27, wherein when a linker peptide is present, the linker peptide is selected from one or more of GGGGS (SEQ ID NO: 92), GGGGSGGGGS (SEQ ID NO: 93), GGGGSGGGGSGGGGS (SEQ ID NO: 94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95), and GGGGSGGGS (SEQ ID NO: 96).

29. An immunoconjugate comprising the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, or the CD80 mutant fusion polypeptide complex of any one of claims 5-28.

30. A nucleotide molecule encoding the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, or the CD80 mutant fusion polypeptide complex of any one of claims 5-28.

31. A vector comprising the nucleotide molecule of claim 30.

32. A host cell comprising the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, the CD80 mutant fusion polypeptide complex of any one of claims 5-28, the nucleotide molecule of claim 30 and / or the vector of claim 31.

33. A composition comprising the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, the CD80 mutant fusion polypeptide complex of any one of claims 5 to 28, the immunoconjugate of claim 29, the nucleotide molecule of claim 30, or the vector of claim 31, and a pharmaceutically acceptable carrier.

34. A method for preparing the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, or the CD80 mutant fusion polypeptide complex of any one of claims 5-28, comprising culturing the host cell of claim 32 to express the CD80 IgV mutant polypeptide, CD80 ECD mutant polypeptide and / or CD80 mutant fusion polypeptide complex.

35. A method for regulating T cell activity, comprising contacting a CD80 IgV mutant polypeptide according to claim 1 or 2, a CD80 ECD mutant polypeptide according to claim 3 or 4, or a CD80 mutant fusion polypeptide complex according to any one of claims 5 to 28, and / or the composition according to claim 33 with a T cell to stimulate its activation.

36. A method for inhibiting tumor cell growth or proliferation, comprising administering the CD80 IgV mutant polypeptide of claim 1 or 2, the CD80 ECD mutant polypeptide of claim 3 or 4, the CD80 mutant fusion polypeptide complex of any one of claims 5 to 28, the immunoconjugate of claim 29, the nucleotide molecule of claim 30, the vector of claim 31, the host cell of claim 32, and / or the composition of claim 33.

37. Use of the CD80 IgV mutant polypeptide according to claim 1 or 2, the CD80 ECD mutant polypeptide according to claim 3 or 4, the CD80 mutant fusion polypeptide complex according to any one of claims 5 to 28, the immunoconjugate according to claim 29, the nucleotide molecule according to claim 30, the vector according to claim 31, the host cell according to claim 32 and / or the composition according to claim 33 in the preparation of a medicament for preventing, ameliorating and / or treating tumors or cancer.

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