CD80 Variant Polypeptide and Its Use

Abstract

The present invention relates to the fields of cancer and immunotherapy, and specifically to CD80 IgV variant polypeptides, CD80 ECD variant polypeptides, CD80 variant fusion polypeptide complexes comprising these variant polypeptides, and nucleic acid molecules that express these variant fusion polypeptide complexes as fusion proteins. The present invention also relates to methods of inducing or enhancing immunity and methods of treating diseases such as infectious diseases and cancer using these variant polypeptides, fusion polypeptide complexes, or nucleic acid molecules.

Description

Technical Field

[0001] The present invention relates to the fields of cancer and immunotherapy. In particular, the present invention relates to CD80 IgV variant polypeptides, CD80 ECD variant polypeptides, CD80 variant fusion polypeptide complexes comprising said variant polypeptides, and nucleotide molecules expressing said variant fusion polypeptide complexes as fusion proteins. The present invention also relates to methods of inducing or enhancing immunity, and methods of treating or preventing diseases such as infectious diseases and cancer, using said variant polypeptides, fusion polypeptide complexes, or nucleic acid molecules.

Background Art

[0002] The CD80 protein is a type of immune-related molecule that stimulates the immune response, and its function is mainly realized by controlling T cell immune responses such as immune defense, immune tolerance, and immune tissue damage.

[0003] CD80 mainly exerts an immunomodulatory effect and regulates the immune response by binding to its ligands CD28, CTLA4, and PDL1. By changing the affinity between CD80 and the ligand, the related functions mediated by CD80 can be adjusted, and in particular, the immune response function can be strengthened, and the control effect against diseases such as cancer and infectious diseases can be strengthened.

[0004] The CD80 molecule is a member of the B7 molecule family and is involved in the co-stimulatory activation of T cells. The mature CD80 molecule is composed of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain. Among them, the extracellular domain (ECD) is an important region in the binding to the corresponding receptor on T cells. The extracellular region of CD80 contains an immunoglobulin variant (IgV) region and an immunoglobulin constant (IgC2) region, and in particular, the IgV structural region is involved in the direct binding to the receptor. The extracellular domain IgV of CD80 binds to CD28 and is involved in the activation, proliferation, and induction of effector functions of T cells. CTLA4 can also bind to the IgV region of CD80 and is involved in immunosuppressive control.

[0005] In the regulation of the immune response, the activation of T cells is regulated by the binding of CD28, a co-stimulatory signal receptor, to the corresponding ligands CD80 / CD86 on the surface of antigen-presenting cells (APCs). However, in the tumor microenvironment, CD80 and CD86 are usually not expressed or are expressed at low levels, so T cells may not be effectively activated. This is one of the main mechanisms of tumor immune escape.

[0006] Furthermore, CTLA4 binds to CD80 / CD86 competitively with CD28 and shows higher affinity characteristics. As a result, CD28 cannot effectively receive the immune agonist signal mediated by CD80 / CD86, and T cells cannot be effectively activated or are immunosuppressed. In addition, by binding to CD80, PDL1 weakens the CD80-mediated immune activation effect and interferes with the normal activation of T cells, so the related immune response is also suppressed.

[0007] Therefore, in the art, there is a need for a CD80 variant polypeptide with improved binding activity of CD80 and its ligand, which can effectively stimulate T cells to enhance the immune response, thereby making it possible to treat or prevent diseases such as infections and tumors caused by the suppression of T cell function.

Summary of the Invention

[0008] The inventor of the present invention unexpectedly discovered that a CD80 variant polypeptide obtained by innovatively modifying the CD80 IgV domain can regulate the binding activity between CD80 and its ligand. This CD80 variant polypeptide can effectively stimulate T cells and enhance the immune response. As a result, it becomes possible to treat or prevent diseases such as infections and tumors caused by the suppression of T cell function.

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

[0010] VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVALARRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKAD (SEQ ID NO: 45).

[0011] In some embodiments, the CD80 IgV mutant polypeptide comprises a combination of two or more of the amino acid mutations.

[0012] In some embodiments, combinations of two or more amino acid mutations are 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 / 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 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 / V83LIt is selected from the group consisting of (SEQ ID NO: 86), M47Q / I69Y / A71E / V83L (SEQ ID NO: 87) and M47K / I69Y / A71E / V83L (SEQ ID NO: 88).

[0013] In a second aspect, the present invention provides a CD80 ECD variant polypeptide comprising one or more amino acid mutations selected from T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, L85D with respect to the wild-type human CD80 ECD polypeptide sequence SEQ ID NO: 1.

[0014] IHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRICTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN (SEQ ID NO: 1).

[0015] In some embodiments, the CD80 ECD variant polypeptide comprises a combination of two or more amino acid mutations.

[0016] In some embodiments, the combination of the two or more amino acid mutations is 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), 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 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 / V83LIt is selected from the group consisting of (SEQ ID NO: 42), M47Q / I69Y / A71E / V83L (SEQ ID NO: 43) and M47K / I69Y / A71E / V83L (SEQ ID NO: 44).

[0017] In a third aspect, the present invention provides a CD80 variant fusion polypeptide complex comprising the CD80 IgV variant polypeptide according to the first aspect or the CD80 ECD variant polypeptide according to the second aspect and a second domain.

[0018] In some embodiments, the CD80 IgV variant polypeptide or the CD80 ECD variant polypeptide is covalently bound to the second domain either via a linker peptide or without a linker peptide.

[0019] In some embodiments, the second domain is an antibody or an antigen-binding fragment thereof.

[0020] In some embodiments, the antibody is selected from the group consisting of immunoglobulin IgG antibodies, recombinant antibodies, chimeric antibodies, heavy chain antibodies, single domain antibodies and / or bispecific antibodies.

[0021] 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.

[0022] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab’, Fv, F(ab)2, F(ab’)2, scFv, VHH, di-scFv and / or dAb.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets one or more antigens selected from 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.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets PD-L1, PD-1, TIGIT, CTLA4, LAG3, or CD3.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets PD-L1 or PD-1.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets LAG3.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof specifically targets CD3.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof that targets PD-L1 is the Sugenamab antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof that targets LAG-3 is the Relatlimab antibody or antigen-binding fragment thereof.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD3 is the CD3B219 antibody or antigen-binding fragment thereof.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain, and the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the heavy chain, either via a linker peptide or directly.

[0032] In some embodiments, the C-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the N-terminus of the heavy chain, either via a linker peptide or directly.

[0033] In some embodiments, the C-terminus of the heavy chain is covalently bound to the N-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide, either via a linker peptide or directly.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the light chain, with or without a linker peptide.

[0035] In some embodiments, the C-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the N-terminus of the light chain, either via a linker peptide or directly.

[0036] In some embodiments, the C-terminus of the light chain is covalently bound to the N-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide, either via a linker peptide or directly.

[0037] In some embodiments, the CD80 variant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, and the second polypeptide may or may not be present.

[0038] In some embodiments, the first polypeptide comprises the heavy chain of an antibody or an antigen-binding fragment thereof.

[0039] In some embodiments, the first polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus of the polypeptide, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, and the heavy chain of an antibody or an antigen-binding fragment thereof.

[0040] In some embodiments, the first polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus of the polypeptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide.

[0041] In some embodiments, the second polypeptide comprises the light chain of an antibody or an antigen-binding fragment thereof.

[0042] In some embodiments, the second polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus of the polypeptide, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, and the light chain of an antibody or an antigen-binding fragment thereof.

[0043] In some embodiments, the second polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus of the polypeptide, the light chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide.

[0044] In some embodiments, the first polypeptide is 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: 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 / A71EIt is selected from the group consisting of 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).

[0045] Alternatively, in some embodiments, 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), 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).

[0046] Alternatively, in some embodiments, the first polypeptide is selected from the group consisting of 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).

[0047] Alternatively, in some embodiments, the first polypeptide is selected from the group consisting of 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).

[0048] Alternatively, in some embodiments, the first polypeptide is selected from the group consisting of 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).

[0049] Alternatively, in some embodiments, the first polypeptide is selected from the group consisting of 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).

[0050] In some embodiments, the second polypeptide is selected from the group consisting of SEQ ID NO: 141, SEQ ID NO: 197, or SEQ ID NO: 208.

[0051] In some embodiments, the CD80 variant fusion polypeptide complex further comprises the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the third domain covalently bound to the second domain according to the third aspect, either via a linker peptide or without a linker peptide.

[0052] In some embodiments, the third domain is the same as or different from the second domain and is an antibody or antigen-binding fragment, or the third domain is a functional protein or an active fragment thereof.

[0053] In some embodiments, 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.

[0054] 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 either via a linker peptide or without a linker peptide.

[0055] In some embodiments, the heavy chain of the second domain is connected to the N-terminus of the third domain, either via a linker peptide or without a linker peptide.

[0056] In some embodiments, the heavy chain of the second domain is connected to the C-terminus of the third domain, either via a linker peptide or without a linker peptide.

[0057] 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, regardless of the presence or absence of a linker peptide.

[0058] In some embodiments, the light chain of the second domain is connected to the N-terminus of the third domain, either via a linker peptide or without a linker peptide.

[0059] In some embodiments, the light chain of the second domain is connected to the C-terminus of the third domain, either via a linker peptide or without a linker peptide.

[0060] In some embodiments, the third domain is connected to the CD80 IgV variant polypeptide according to the first aspect or the CD80 ECD variant polypeptide according to the second aspect, either via a linker peptide or without a linker peptide.

[0061] In some embodiments, the CD80 variant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, and the second polypeptide may or may not be present.

[0062] In some embodiments, the first polypeptide comprises the heavy chain of the second domain.

[0063] In some embodiments, the first polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides therein may be the same or different and may or may not exist independently.

[0064] In some embodiments, the first polypeptide is formed by covalently linking, in order from the C-terminus to the N-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides therein may be the same or different and may or may not exist independently.

[0065] In some embodiments, the second polypeptide comprises the light chain of the second domain.

[0066] In some embodiments, the second polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides therein may be the same or different and may or may not exist independently.

[0067] In some embodiments, the second polypeptide is formed by covalently linking, in order from the C-terminus to the N-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain, wherein the linker peptides therein may be the same or different and may or may not exist independently.

[0068] In some embodiments, the third domain comprises LAG-3 or an active fragment thereof.

[0069] In some embodiments, the second domain specifically targets PD-1 or PD-L1.

[0070] In some embodiments, the second domain is sugemalimab or a functional fragment thereof.

[0071] In some embodiments, the first polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 293 and SEQ ID NO: 295.

[0072] In some embodiments, the second polypeptide is selected from the group consisting of SEQ ID NO: 141.

[0073] In some embodiments, the second domain is an immunoglobulin Fc domain.

[0074] In some embodiments, the Fc domain is selected from the group consisting of 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.

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

[0076] In some embodiments, the amino acid sequence of the CD80 variant 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), E35T / A71E (SEQ ID NO: 170), E35T / A71N (SEQ ID NO: 171), E35T / A71Q (SEQ ID NO: 172), and E35T / A71T (SEQ ID NO: 173).

[0077] In some embodiments, the C-terminus of the immunoglobulin Fc domain is covalently attached to the CD80 IgV variant polypeptide or the CD80 ECD variant polypeptide either via a linker peptide or without a linker peptide.

[0078] In some embodiments, 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), GGGGSGGGSGGGGGS (SEQ ID NO: 94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95), and GGGGSGGGS (SEQ ID NO: 96).

[0079] In a fourth aspect, the present invention provides an immune complex comprising the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect.

[0080] In a fifth aspect, the present invention provides a nucleotide molecule encoding the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect.

[0081] In a sixth aspect, the present invention provides a vector comprising the nucleotide molecule according to the fifth aspect.

[0082] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant 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.

[0083] In an eighth aspect, the present invention provides a composition comprising the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant fusion polypeptide complex according to the third aspect, the immune complex 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.

[0084] In a ninth aspect, the present invention provides a method for preparing the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect, comprising culturing the host cell according to the seventh aspect to express the CD80 IgV variant polypeptide, the CD80 ECD variant polypeptide, and / or the CD80 variant fusion polypeptide complex.

[0085] In a tenth aspect, the present invention provides a method for modulating T cell activity, comprising contacting a T cell with the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, and / or the CD80 variant fusion polypeptide complex according to the third aspect, the composition according to the third aspect, and / or the composition according to the eighth aspect to stimulate its activation.

[0086] In an eleventh aspect, the present invention provides a method for inhibiting the growth or proliferation of tumor cells, comprising administering the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant fusion polypeptide complex according to the third aspect, the immune complex according to the fourth aspect, the nucleotide molecule according to the fifth aspect, the vector according to the sixth aspect, the host cell according to the seventh aspect, and / or the composition according to the eighth aspect.

[0087] In a twelfth aspect, the present invention provides the use of the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant fusion polypeptide complex according to the third aspect, the immune complex according to the fourth aspect, the nucleotide molecule according to the fifth aspect, the vector according to the sixth aspect, the host cell according to the seventh aspect, and / or the composition according to the eighth aspect in the preparation of a medicament for preventing, ameliorating and / or treating a tumor or cancer.

[0088] The following specific embodiments and examples illustrate the embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein, and thus many variations and modifications are possible. Those skilled in the art will recognize that such changes and modifications are within the scope and spirit of the present invention.

Brief Description of the Drawings

[0089]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 8

Modes for Carrying Out the Invention

[0090] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure belongs. In case of conflict, the definitions in this specification shall prevail. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. Although only specific 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 this disclosure.

[0091] As used herein, the singular forms "a", "one", and "the" include the plural forms as well, unless the context clearly dictates otherwise. Further, the free expressions "comprising" and "including" are to be construed to mean that they may include structural components or method steps not described, but it should also be noted that this free expression may only cover components. Method steps are configured according to the situation (i.e., it covers situations covered by the closed formula "consisting of"). Generally, the term "about" is used herein to refer to a numerical change of up to 5% above and below the recited value.

[0092] The term "modulatory effect on T cell activity" refers to the effect of regulating the activity of lymphoid T cells by the CD80 mutant polypeptides and CD80 mutant fusion polypeptide complexes disclosed in the present invention. This can also be extended to the effects of other related T cell regulators that affect the expression of NFAT transcription factors in T cells, the secretion of cytokines such as IL-2, IFN-γ, TNFα, Granzyme B, cell proliferation, and cytotoxic effects on target cells including tumor cells after T cell treatment. The term "T cell" can be extended to T cell lines and / or primary T cells, including but not limited to CD4 T cells and CD4 T cell subsets (such as Th1, Th2, Th9, Th17, TFH, and / or Treg cells). It also includes CD8 T cells, including but not limited to tumor tissue-infiltrating CD8 T cells, effector CD8 T cells, and / or immune memory CD8 T cells.

[0093] The term "anti-tumor activity" means any biological activity that decreases or prevents the growth or survival rate 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 of the present invention and their fusion polypeptide complexes.

[0094] The term "CD80" refers to a polypeptide or a fragment thereof that has at least about 85% amino acid identity with the protein encoded by the gene of NCBI accession number Gene ID: 941 and has the activity of binding to CD28 (the protein encoded by the gene of NCBI accession number Gene ID: 940), and / or CTLA4 (the protein encoded by the gene of NCBI accession number Gene ID: 1493), and / or PD-L1 (the protein encoded by the gene of NCBI accession number Gene ID: 29126). The following provides an exemplary human CD80 amino acid sequence: VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 89).

[0095] The term "CD80 extracellular domain" refers to the amino acid sequence of a domain in the extracellular region of the CD80 protein, which has at least about 85% amino acid identity and refers to a polypeptide or a fragment thereof having CD28 and / or CTLA4 binding activity. The following provides an exemplary "CD80 extracellular domain amino acid sequence" (SEQ ID NO: 1). The term "CD80 IgV" refers to the amino acid sequence of the IgV domain in the extracellular region of the CD80 protein, which has at least about 85% amino acid identity and refers to a polypeptide or a fragment thereof having CD28 and / or CTLA4 binding activity. The following provides an exemplary "CD80 IgV" (SEQ ID NO: 45).

[0096] The term "amino acid identity" refers to the comparison of amino acid sequences to obtain the maximum match, and (gaps are introduced as necessary) alignment. The value of the percent identity can be determined using sequence comparison software, algorithms, or visual inspection. In the case of comparing two or more amino acid identities or mixed tandem amino acid identities in a second domain, the second domain should be as follows. The percent identity between a certain subunit and any subunit of the second domain is calculated by comparing them individually. In this field, various algorithms and software that can be used to obtain an alignment of amino acid sequences are known, including but not limited to NCBI BLAST software.

[0097] The term "immunoglobulin" refers to an antibody consisting of an antigen-specific binding region and an amino acid fragment of a constant region. The antigen-specific binding region is the fragment that determines the important differences in immunoglobulins and is also called the antigen-binding domain, or "epitope" or "antigen determinant". The antigen-binding domain is usually composed of the variable region of the heavy chain (VH) of the antibody and the variable region of the light chain (VL) of the antibody. However, it is not necessarily required to include both. The antigen-binding domain of the antibody disclosed in the present invention is not limited to the domain composed of conventional VH and VL, and also includes the antigen-binding domains of other types of antibodies including recombinant antibodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies, bispecific antibodies, and other non-conventional antibodies and combinations thereof. The constant region refers to the common structural region of immunoglobulins including the constant region of the light chain and the constant region of the heavy chain of the antibody.

[0098] In the present application, the term "immunoglobulin Fc domain" generally refers to one Fc fragment and two identical FAB fragments formed by papain hydrolysis of conventional antibody IgG, and the Fc fragment is referred to as the Fc domain. The Fc domain may include the antibody heavy chain CH2, CH3, and hinge region fragments. The conventional Fc fragment has the function of binding to the Fc fragment receptor and mediating the related biological effects. However, site-specific mutations may change the binding ability to the corresponding target receptor and may affect its biological function. The immunoglobulin Fc domains disclosed in the present application include, but are not limited to, conventional Fc fragments and any other forms of Fc variants. The following are exemplary Fc domains of human immunoglobulin IgG1 (SEQ ID NO:90) and human immunoglobulin IgG4 (SEQ ID NO:91):

[0099] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90); ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 91).

[0100] The term "linker peptide" refers to a short peptide chain with conformational flexibility formed by a combination of Gly (G) and Ser (S) amino acid residues, and it is guaranteed that the ratio of the number of Gly amino acids to the number of Ser amino acids is 1 or more. The linker peptides disclosed in the present invention can be extended to any short peptide having this property. Exemplary amino acid sequences of linker peptides are shown below, but are not limited thereto: 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).

[0101] In the present invention, the term "CD80 variant polypeptide" refers to a CD80 ECD variant polypeptide and / or a CD80 IgV variant polypeptide. The term "fusion polypeptide complex of variant polypeptides" is also referred to as "variant polypeptide complex", "polypeptide complex", "fusion polypeptide complex" or "fusion complex" in the present invention. "CD80 variant fusion polypeptide complex" refers to a functional fusion polypeptide complex in which a CD80 ECD variant or a CD80 IgV variant is bound to an antibody or an antigen-binding fragment thereof, or an immunoglobulin Fc domain, or a functional polypeptide fragment, either via a linker peptide or without a linker peptide. In the functional fusion polypeptide complex in which the CD80 variant disclosed in the present invention is bound to an antibody or an antigen-binding fragment thereof, either via a linker peptide or without a linker peptide, the antibody includes, but is not limited to, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-TIGIT antibody, an anti-CTLA4 antibody, an anti-CEA antibody, an anti-BCMA antibody, an anti-LAG3 antibody, an anti-CD3 antibody, an anti-Her2 antibody, an anti-Her3 antibody, an anti-VEGF antibody, an anti-VEGFR antibody, an anti-EGFR antibody, an anti-c-Met antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD38 antibody, an anti-TROP-2 antibody, an anti-CD40 antibody, an anti-4-1BB antibody, an anti-CD30 antibody, etc. Any form of antibody that is covalently bound to the CD80 ECD variant or CD80 IgV variant disclosed in the present invention, either via a linker peptide or without a linker peptide, alone or in combination, is considered to be within the scope of the present invention.

[0102] The term "LAG3" refers to a peptide or a fragment thereof that has at least about 85% amino acid identity with the protein encoded by the gene with NCBI accession number Gene ID: 3902, and binds to the complex protein MHCII (or HLA-DR) composed of an α subunit (the protein encoded by the gene with NCBI accession number Gene ID: 3122) and a β subunit (the protein encoded by the gene with NCBI accession number Gene ID: 3123 or NCBI accession number Gene ID: 3125) and / or FGL1 (the protein encoded by the gene with NCBI accession number Gene ID: 2267), and promotes the activation and maturation of antigen-presenting cells.

[0103] The term "LAG3 active fragment" refers to an active fragment derived from the LAG3 protein and having the functional characteristics of the LAG3 protein. The functional characteristics of the LAG3 protein include, but are not limited to, binding to MHCII (or HLA-DR) and / or FGL1, and promoting the activation and maturation of antigen-presenting cells.

[0104] Activation and maturation of antigen-presenting cells include, but are not limited to, upregulation of the expression of co-stimulatory receptors on antigen-presenting cells, enhanced phagocytosis of antigens, increased cytokine secretion, and enhanced chemokine secretion.

[0105] The co-stimulatory receptors include, but are not limited to, ICOS-L, CD40L, CD137L, OX40L, CD80, CD83, and CD86.

[0106] Phagocytosis of antigens includes, but is not limited to, phagocytosis of bacteria, viruses, proteins, polysaccharides, etc.

[0107] The cytokines include, but are not limited to, IL-1β, TNFα, IL-6, IL-12, etc.

[0108] The chemokines include, but are not limited to, CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, etc.

[0109] The inventor of the present application unexpectedly discovered that a CD80 variant polypeptide formed by innovatively modifying the CD80 IgV domain can regulate the binding activity between CD80 and its ligand, and as a result, the CD80 variant polypeptide can effectively stimulate T cells and enhance the immune response. As a result, it becomes possible to treat or prevent diseases such as infections and tumors caused by the suppression of T cell function.

[0110] Based on this, the present invention provides a class of CD80 variant polypeptides including CD80 IgV variant polypeptides and CD80 ECD variant polypeptides, as well as methods for regulating T cell immune responses and applications in the treatment of diseases.

[0111] In a first aspect, the present invention includes one or more amino acid site substitution mutations of wild-type CD80 IgV, is obtained by amino acid substitution mutations of a human CD80 IgV polypeptide (SEQ ID NO: 45), and the mutation sites are selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, and 85. In some embodiments, the CD80 IgV variant polypeptide includes two or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83; in some embodiments, the CD80 IgV variant polypeptide includes three or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 on SEQ ID NO: 45; in some embodiments, the CD80 IgV variant polypeptide includes four or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 on SEQ ID NO: 45; in some embodiments, the CD80 IgV variant polypeptide includes five or more amino acid substitution mutations on SEQ ID NO: 45, and the mutation sites are selected from sites 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 on SEQ ID NO: 45.

[0112] In some embodiments, the mutation sites of the CD80 IgV variant polypeptide include 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, L85D of the amino acid sites on SEQ ID NO: 45.

[0113] In some preferred embodiments, the mutated sites of the CD80 IgV variant polypeptide include combinations of mutations at two or more amino acid sites on SEQ ID NO: 45, and the CD80 IgV variant polypeptide is 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 / 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 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 / A71EIt may be (Accession No. 84), T13K / M42T / A71E (Accession No. 85), M47H / I69Y / A71E / V83L (Accession No. 86), M47Q / I69Y / A71E / V83L (Accession No. 87), M47K / I69Y / A71E / V83L (Accession No. 88).

[0114] In a second aspect, the present invention includes one or more amino acid site substitution mutations on wild-type CD80 ECD, is obtained by amino acid substitution mutations of human CD80 ECD polypeptide (Accession No. 1), and the mutation sites are selected from the 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 sites on SEQ ID NO:1. In some embodiments, the CD80 ECD mutant polypeptide includes two or more amino acid substitution mutations on SEQ ID NO:1, and the mutation sites are selected from the 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 sites on SEQ ID NO:1; in some embodiments, the CD80 ECD mutant polypeptide includes three or more amino acid substitution mutations on SEQ ID NO:1, and the mutation sites are selected from the 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 sites on SEQ ID NO:1; in some embodiments, the CD80 ECD mutant polypeptide includes four or more amino acid substitution mutations on SEQ ID NO:1, and the mutation sites are selected from the 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 sites on SEQ ID NO:1; in some embodiments, the CD80 ECD mutant polypeptide includes five or more amino acid substitution mutations on SEQ ID NO:1, and the mutation sites are selected from the 13, 18, 26, 35, 42, 47, 69, 71, 83, 85 sites on SEQ ID NO:1.

[0115] In some embodiments, the mutation sites of the CD80 ECD mutant polypeptide include 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, L85D at the amino acid positions on SEQ ID NO:1.

[0116] In some preferred embodiments, the mutation sites of the CD80 ECD variant polypeptide are combinations of mutations at two or more amino acid positions on SEQ ID NO: 1, and the CD80 ECD variant polypeptide is 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), 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 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 / A71EIt can be (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).

[0117] In a third aspect, the present invention provides a fusion polypeptide complex of a CD80 variant polypeptide, which comprises the CD80 ECD variant polypeptide or the CD80 IgV variant polypeptide and is covalently bound to a second domain with or without a linker peptide, wherein the second domain may be an immunoglobulin Fc domain, an antibody or an antigen-binding fragment thereof, or another form of polypeptide.

[0118] In some embodiments, the fusion polypeptide complex of the CD80 variant polypeptide comprises the CD80 ECD variant polypeptide or the CD80 IgV variant polypeptide and is covalently bound to an immunoglobulin Fc domain with or without a linker peptide. 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.

[0119] In some embodiments, the CD80 variant polypeptide is covalently bound to the N-terminus of the immunoglobulin Fc domain with or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0120] In some embodiments, the C-terminus of the immunoglobulin Fc domain is covalently bound to the CD80 variant polypeptide with or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0121] In some embodiments, the fusion polypeptide complex of the CD80 variant polypeptide comprises the above-described CD80 ECD variant polypeptide or CD80 IgV variant polypeptide covalently bound to an antibody or an antigen-binding fragment thereof, either via a linker peptide or without a linker peptide.

[0122] In some embodiments, the antibody can be selected from the group consisting of 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 the group consisting of Fab, Fab’, Fv, F(ab)2, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

[0123] In some embodiments, the CD80 variant fusion polypeptide complex comprises the above-described CD80 ECD variant polypeptide or CD80 IgV variant polypeptide covalently bound to an immunoglobulin IgG antibody, either via a linker peptide or without a linker peptide, and 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.

[0124] In some preferred embodiments, the specific target of the antibody or antigen-binding fragment in the fusion polypeptide complex of the above-described CD80 variant polypeptide is selected from the group consisting of, 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-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.

[0125] In a preferred embodiment, the anti-PD-L1 antibody or antigen-binding fragment is the Sugemalimab antibody or an antigen-binding fragment thereof.

[0126] In a preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment is the Relatlimab antibody or an antigen-binding fragment thereof.

[0127] In a preferred embodiment, the anti-CD3 antibody or antigen-binding fragment is the CD3B219 antibody or an antigen-binding fragment thereof.

[0128] Alternatively, in some embodiments, the present invention provides a fusion polypeptide complex of a CD80 variant polypeptide, wherein the CD80 variant polypeptide and an antibody or antigen-binding fragment are linked by a linker peptide.

[0129] The present invention provides a fusion polypeptide complex of a CD80 variant polypeptide, wherein the CD80 variant polypeptide and the antibody or antigen-binding fragment are directly linked without a linker peptide.

[0130] In one embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain, and the CD80 variant polypeptide is covalently bound to the heavy chain via a linker peptide or without a linker peptide.

[0131] In a preferred embodiment, the C-terminus of the CD80 variant polypeptide is covalently bound to the N-terminus of the heavy chain via a linker peptide or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0132] In a preferred embodiment, the C-terminus of the heavy chain is covalently bound to the N-terminus of the CD80 variant polypeptide via a linker peptide or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0133] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 variant polypeptide is covalently bound to the light chain either via a linker peptide or without a linker peptide.

[0134] In a preferred embodiment, the C-terminus of the CD80 variant polypeptide is covalently bound to the N-terminus of the light chain either via a linker peptide or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0135] In a preferred embodiment, the C-terminus of the light chain is covalently bound to the N-terminus of the CD80 variant polypeptide either via a linker peptide or without a linker peptide to form a fusion polypeptide complex of the CD80 variant polypeptide.

[0136] In one embodiment, the CD80 variant polypeptide is covalently bound to the N-terminus of the antibody or antigen-binding fragment thereof either via a linker peptide or without a linker peptide.

[0137] In one embodiment, the CD80 variant polypeptide is covalently bound to the C-terminus of the antibody or antigen-binding fragment thereof either via a linker peptide or without a linker peptide.

[0138] In another embodiment, the CD80 variant fusion polypeptide complex provided by the present invention comprises a first polypeptide and a second polypeptide that constitute the fusion polypeptide complex, but it is obvious that the second polypeptide may not exist in some special antibodies.

[0139] In a preferred embodiment, the first polypeptide of the fusion polypeptide complex of the present invention comprises the heavy chain of an antibody or an antigen-binding fragment thereof.

[0140] In a preferred embodiment, the first polypeptide of the fusion polypeptide complex of the present invention is composed of, from the N-terminal side to the C-terminal of the polypeptide, a CD80 variant polypeptide, a linker peptide, and a heavy chain of an antibody or an antigen-binding fragment thereof, which are covalently bonded in this order.

[0141] In a preferred embodiment, the first polypeptide of the polypeptide complex of the present invention is composed of, from the N-terminal side to the C-terminal of the polypeptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a CD80 variant polypeptide, which are covalently bonded in this order.

[0142] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention contains a light chain of an antibody or an antigen-binding fragment thereof.

[0143] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention is composed of, from the N-terminal side to the C-terminal of the polypeptide, a CD80 variant polypeptide, a linker peptide, and a light chain of an antibody or an antigen-binding fragment thereof, which are covalently bonded in this order.

[0144] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention is composed of, from the N-terminal side to the C-terminal of the polypeptide, a light chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a CD80 variant polypeptide, which are covalently bonded in this order.

[0145] Alternatively, in some embodiments, the present invention also provides a CD80 variant fusion polypeptide complex comprising a CD80 variant polypeptide, a second domain, and a third domain, wherein the CD80 variant polypeptide and the second domain are the same as those described above.

[0146] In some embodiments, the third domain of the present invention is the same as or different from the second domain and is an antibody or an antigen-binding fragment thereof, or the third domain is a functional protein capable of immunological activation or an active fragment thereof.

[0147] 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 either via a linker peptide or without a linker peptide.

[0148] In certain embodiments, the heavy chain of the second domain of the present invention is connected to the N-terminus of the third domain either via a linker peptide or without a linker peptide.

[0149] In certain embodiments, the heavy chain of the second domain of the present invention is connected to the C-terminus of the third domain either via a linker peptide or without a linker peptide.

[0150] 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 either via a linker peptide or without a linker peptide.

[0151] In certain embodiments, the light chain of the second domain of the present invention is connected to the N-terminus of the third domain either via a linker peptide or without a linker peptide.

[0152] In certain embodiments, the light chain of the second domain of the present invention is connected to the C-terminus of the third domain either via a linker peptide or without a linker peptide.

[0153] In some embodiments, the CD80 variant fusion polypeptide complex of the present invention comprising the third domain comprises a first polypeptide and a second polypeptide, and the second polypeptide may or may not be present.

[0154] In some embodiments, the first polypeptide comprises the heavy chain of the second domain.

[0155] In one embodiment, the first polypeptide is composed of, from the N-terminus to the C-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently bonded in sequence, and the linker peptides may be the same or different and may or may not exist independently of each other.

[0156] In one embodiment, the first polypeptide is composed of, from the C-terminus to the N-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain covalently bonded in sequence, and the linker peptides may be the same or different and may or may not exist independently of each other.

[0157] Alternatively, in some embodiments, the second polypeptide includes the light chain of the second domain.

[0158] In some embodiments, the third domain is linked to the CD80 IgV variant polypeptide or the CD80 ECD variant polypeptide with or without a linker peptide linker peptide.

[0159] In one embodiment, the third domain includes LAG-3 or an active fragment thereof, and the second domain specifically targets PD-1 or PD-L1.

[0160] In one embodiment, the first polypeptide includes a sequence selected from the group consisting of SEQ ID NO: 293 and SEQ ID NO: 295; the second polypeptide includes a sequence selected from the group consisting of SEQ ID NO: 141.

[0161] In some embodiments, there is no linker peptide.

[0162] In some embodiments, the linker peptide is selected from one or more of GGGGS (SEQ ID NO: 92), GGGGSGGGGS (SEQ ID NO: 93), GGGGSGGGSGGGGGS (SEQ ID NO: 94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95), and GGGGSGGGS (SEQ ID NO: 96).

[0163] Alternatively, in some embodiments, the CD80 variant polypeptide can be combined with other forms of polypeptides to form a polypeptide complex by the above method.

[0164] In a fourth aspect, the present invention provides an immune complex comprising the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect.

[0165] In a fifth aspect, the present invention provides a nucleotide molecule encoding the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect.

[0166] In a sixth aspect, the present invention provides a vector comprising the nucleotide molecule according to the fifth aspect.

[0167] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the variant 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.

[0168] In an eighth aspect, the present invention provides a composition comprising the CD80 IgV variant polypeptide described in the first aspect, the CD80 ECD variant polypeptide described in the second aspect, the CD80 variant fusion polypeptide complex described in the third aspect, the immune complex described in the fourth aspect, the nucleotide molecule described in the fifth aspect, or the vector described in the sixth aspect, and a pharmaceutically acceptable carrier.

[0169] In a ninth aspect, the present invention provides a method for preparing the CD80 IgV variant polypeptide described in the first aspect, the CD80 ECD variant polypeptide described in the second aspect, or the CD80 variant fusion polypeptide complex described in the third aspect, which comprises culturing the host cell described in the seventh aspect to express the CD80 IgV variant polypeptide, the CD80 ECD variant polypeptide, and / or the CD80 variant fusion polypeptide complex.

[0170] In some embodiments, the present invention provides a method for expressing the polypeptide and polypeptide complex, the method comprising using the host cell of the present invention to express the CD80 variant polypeptide or its fusion polypeptide complex of the present invention.

[0171] In some embodiments, the present invention provides a method for producing a polypeptide and a polypeptide complex, the method comprising introducing a polynucleotide encoding the antibody polypeptide complex of the present invention into a host cell, expressing a first peptide, and including or not including a second peptide. The first peptide and the second peptide, or the first peptide itself, can form a stable dimer, and the stable dimer is maintained by stable dimerization including natural binding and / or at least one non-natural interchain bond. The first peptide and the second peptide form a stable polymer and a stable complex within the host cell.

[0172] In certain embodiments, the invention also provides a method for isolating and purifying the CD80 variant polypeptide or its fusion polypeptide complex.

[0173] In a tenth aspect, the invention provides a method of modulating T cell activation, comprising contacting a T cell with the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, or the CD80 variant fusion polypeptide complex according to the third aspect and / or the composition according to the eighth aspect to stimulate its activation. This method is not limited to the protocol used in the examples and can be extended to other protocols to be published or developed in the future and can be used to evaluate the activation of T cells.

[0174] In an eleventh aspect, the invention provides a method of inhibiting the growth or proliferation of tumor cells, comprising administering the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant fusion polypeptide complex according to the third aspect, the immune complex according to the fourth aspect, the nucleotide molecule according to the fifth aspect, the vector according to the sixth aspect, the host cell according to the seventh aspect, and / or the composition according to the eighth aspect.

[0175] In a twelfth aspect, the invention provides the use of the CD80 IgV variant polypeptide according to the first aspect, the CD80 ECD variant polypeptide according to the second aspect, the CD80 variant fusion polypeptide complex according to the third aspect, the immune complex according to the fourth aspect, the nucleotide molecule according to the fifth aspect, the vector according to the sixth aspect, the host cell according to the seventh aspect, and / or the composition according to the eighth aspect in the preparation of a medicament for preventing, ameliorating and / or treating a tumor or cancer.

[0176] The following is the first polypeptide amino acid sequence of a functional fusion polypeptide complex formed by CD80 IgV variants covalently bound to the N-terminus of the sugemalimab heavy chain via a linker peptide, respectively: 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: 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), M47K / I69Y / A71E / V83L (SEQ ID NO: 139), and the first polypeptide amino acid sequence (SEQ ID NO: 140) of a functional fusion polypeptide complex formed by covalently linking an unmutated CD80 IgV to the N-terminus of the heavy chain of the sugemalimab antibody via a linker peptide. Each of the above first polypeptides assembles with the second polypeptide (SEQ ID NO: 141) of the sugemalimab antibody light chain to form a functional fusion polypeptide complex.

[0177] The following is provided by way of example the first polypeptide amino acid sequences of functional fusion polypeptide complexes formed by covalently linking CD80 ECD variants to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide: 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), E35T / A71N (SEQ ID NO: 155), E35T / A71Q (SEQ ID NO: 156), E35T / A71T (SEQ ID NO: 157). Each of the above first polypeptides assembles with the second polypeptide (SEQ ID NO: 141) of the sugemalimab antibody light chain to form a functional fusion polypeptide complex.

[0178] The following are the amino acid sequences of functional fusion polypeptides formed by covalently linking CD80 ECD mutants to the N-terminus of the Fc domain of human immunoglobulin IgG4, respectively: 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), E35T / A71N (SEQ ID NO: 171), E35T / A71Q (SEQ ID NO: 172), E35T / A71T (SEQ ID NO: 173), and the amino acid sequence of the functional fusion polypeptide formed by covalently linking the non-mutated CD80 ECD to the N-terminus of the Fc domain of human immunoglobulin IgG4 (SEQ ID NO: 174).

[0179] Provided herein by way of example are CD80 IgV variants and functional fusion polypeptide amino acid sequences formed by covalently linking to the N-terminus of the Fc domain of human immunoglobulin IgG4, respectively: 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), 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 the functional fusion polypeptide amino acid sequence (SEQ ID NO: 191) formed by covalently linking non-mutated CD80 IgV to the N-terminus of the Fc domain of human immunoglobulin IgG4.

[0180] Provided herein by way of example are the first polypeptide amino acid sequences of functional fusion polypeptide complexes formed by covalently linking CD80 ECD variants via a linker peptide to the N-terminus of the heavy chain of the anti-LAG3 antibody Relatlimab, respectively: 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 non-mutated CD80 ECD (SEQ ID NO: 196). The above first polypeptide assembles with the second polypeptide (SEQ ID NO: 197) of the Relatlimab antibody light chain to form a functional fusion polypeptide complex.

[0181] Provided by way of example below are the first polypeptide amino acid sequences of the functional fusion polypeptide complexes formed by covalently binding CD80 IgV variants to the N-terminus of the heavy chain of the anti-LAG3 antibody Relatlimab via a linker peptide: 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 non-mutated CD80 IgV (SEQ ID NO: 202). Each of the above first polypeptides assembles with the second polypeptide (SEQ ID NO: 197) of the Relatlimab antibody light chain to form a functional fusion polypeptide complex.

[0182] Provided by way of example below are the first polypeptide amino acid sequences of the functional fusion polypeptide complexes formed by covalently binding CD80 ECD variants to the N-terminus of the heavy chain of the anti-CD3 antibody CD3B219 via a linker peptide: 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 non-mutated CD80 ECD (SEQ ID NO: 207). Each of the above first polypeptides assembles with the second polypeptide (SEQ ID NO: 208) of the CD3B219 antibody light chain to form the functional fusion polypeptide complex.

[0183] Provided by way of example below are the first polypeptide amino acid sequences of the functional fusion polypeptide complexes formed by covalently binding CD80 IgV variants to the N-terminus of the heavy chain of the anti-CD3 antibody CD3B219 via a linker peptide: 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 non-mutated CD80 IgV (SEQ ID NO: 213). Each of the above first polypeptides assembles with the second polypeptide (SEQ ID NO: 208) of the CD3B219 antibody light chain to form the functional fusion polypeptide complex.

[0184] In some embodiments, the functional antibody Abs-353 consists of a chimeric antibody Abs-353 first polypeptide (SEQ ID NO: 97) formed by covalently linking the CD80 IgV variant E35N / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-353 functional antibody is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 214) that expresses the Abs-353 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-353 second polypeptide.

[0185] In some embodiments, the functional antibody Abs-354 consists of a chimeric antibody Abs-354 first polypeptide (SEQ ID NO: 98) formed by covalently linking the CD80 IgV variant E35N / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalima light chain second polypeptide (SEQ ID NO: 141). The Abs-354 functional antibody is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 216) that expresses the Abs-354 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-354 second polypeptide.

[0186] In some embodiments, the functional antibody Abs-355 consists of a chimeric antibody Abs-355 first polypeptide (SEQ ID NO: 99) formed by covalently linking the CD80 IgV variant E35N / A71Q to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-355 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 217) that expresses the Abs-355 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-355 second polypeptide.

[0187] In some embodiments, the functional antibody Abs-356 consists of a chimeric antibody Abs-356 first polypeptide (SEQ ID NO: 100) formed by covalently linking the CD80 IgV variant E35N / A71T to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-356 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 218) that expresses the Abs-356 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-356 second polypeptide.

[0188] In some embodiments, the functional antibody Abs-357 consists of a chimeric antibody Abs-357 first polypeptide (SEQ ID NO: 101) formed by covalently linking the CD80 IgV variant E35S / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-357 functional antibody is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 219) that expresses the Abs-357 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-357 second polypeptide.

[0189] In some embodiments, the functional antibody Abs-358 consists of a chimeric antibody Abs-358 first polypeptide (SEQ ID NO: 102) formed by covalently linking the CD80 IgV variant E35S / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-358 functional antibody is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 220) that expresses the Abs-358 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-358 second polypeptide.

[0190] In some embodiments, the functional antibody Abs-359 consists of a chimeric antibody Abs-first polypeptide (SEQ ID NO: 103) formed by covalently linking the CD80 IgV variant E35S / A71Q to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and the sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-359 functional antibody is constructed by introducing into a host cell for expression a vector of the polynucleotide sequence (SEQ ID NO: 221) that expresses the Abs-359 first polypeptide and a vector of the polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-359 second polypeptide.

[0191] In some embodiments, the functional antibody Abs-360 consists of a chimeric antibody Abs-first polypeptide (SEQ ID NO: 104) formed by covalently linking the CD80 IgV variant E35S / A71T to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and the sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-360 functional antibody is constructed by introducing into a host cell for expression a vector of the polynucleotide sequence (SEQ ID NO: 222) that expresses the Abs-360 first polypeptide and a vector of the polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-360 second polypeptide.

[0192] In some embodiments, the functional antibody Abs-361 consists of a chimeric antibody Abs-361 first polypeptide (SEQ ID NO: 105) formed by covalently linking the CD80 IgV variant E35Q / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-361 functional antibody is constructed by introducing into a host cell for expression a polynucleotide sequence (SEQ ID NO: 223) vector expressing the Abs-361 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-362 second polypeptide.

[0193] In some embodiments, the functional antibody Abs-362 consists of a chimeric antibody Abs-362 first polypeptide (SEQ ID NO: 106) formed by covalently linking the CD80 IgV variant E35Q / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-362 functional antibody is constructed by introducing into a host cell for expression a polynucleotide sequence (SEQ ID NO: 224) vector expressing the Abs-362 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-362 second polypeptide.

[0194] In some embodiments, the functional antibody Abs-363 consists of a chimeric antibody Abs-363 first polypeptide (SEQ ID NO: 107) formed by covalently binding the CD80 IgV variant E35Q / A71Q to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-363 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 225) that expresses the Abs-363 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-363 second polypeptide for expression.

[0195] In some embodiments, the functional antibody Abs-364 consists of a chimeric antibody Abs-364 first polypeptide (SEQ ID NO: 108) formed by covalently binding the CD80 IgV variant E35Q / A71T to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-364 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 226) that expresses the Abs-364 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-364 second polypeptide for expression.

[0196] In some embodiments, the functional antibody Abs-365 is composed of a chimeric antibody Abs-365 first polypeptide (SEQ ID NO: 109) formed by covalently binding the CD80 IgV variant E35T / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-365 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 227) that expresses the Abs-365 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-365 second polypeptide into a host cell for expression.

[0197] In some embodiments, the functional antibody Abs-366 is composed of a chimeric antibody Abs-366 first polypeptide (SEQ ID NO: 110) formed by covalently binding the CD80 IgV variant E35T / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-366 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 228) that expresses the Abs-366 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-366 second polypeptide into a host cell for expression.

[0198] In some embodiments, the functional antibody Abs-367 consists of a chimeric antibody Abs-367 first polypeptide (SEQ ID NO: 111) formed by covalently binding the CD80 IgV variant E35T / A71Q to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-367 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 229) expressing the Abs-367 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-367 second polypeptide into a host cell for expression.

[0199] In some embodiments, the functional antibody Abs-368 consists of a chimeric antibody Abs-368 first polypeptide (SEQ ID NO: 112) formed by covalently binding the CD80 IgV variant E35T / A71T to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-368 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 230) expressing the Abs-368 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-368 second polypeptide into a host cell for expression.

[0200] In some embodiments, the functional antibody Abs-247 consists of a chimeric antibody Abs-247 first polypeptide (SEQ ID NO: 113) formed by covalently binding the CD80 IgV variant M47A / I69Y / A71E / V83L to the N-terminus of the sugemalimab heavy chain via (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-247 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 231) expressing the Abs-247 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-247 second polypeptide into a host cell for expression.

[0201] In some embodiments, the functional antibody Abs-248 consists of a chimeric antibody Abs-248 first polypeptide (SEQ ID NO: 114) formed by covalently binding the CD80 IgV variant M47A / I69W / A71E / V83L to the N-terminus of the sugemalimab heavy chain via (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-248 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 232) expressing the Abs-248 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-248 second polypeptide into a host cell for expression.

[0202] In some embodiments, the functional antibody Abs-249 consists of a chimeric antibody Abs-249 first polypeptide (SEQ ID NO: 115) formed by covalently binding the CD80 IgV variant M47A / I69Y / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-249 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 233) that expresses the Abs-249 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-249 second polypeptide into host cells for expression.

[0203] In some embodiments, the functional antibody Abs-250 consists of a chimeric antibody Abs-250 first polypeptide (SEQ ID NO: 116) formed by covalently binding the CD80 IgV variant M47A / I69W / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-250 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 234) that expresses the Abs-250 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-250 second polypeptide into host cells for expression.

[0204] In some embodiments, the functional antibody Abs-251 consists of a chimeric antibody Abs-251 first polypeptide (SEQ ID NO: 117) formed by covalently linking the CD80 IgV variant I69Y / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-251 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 235) that expresses the Abs-251 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-251 second polypeptide for expression.

[0205] In some embodiments, the functional antibody Abs-252 consists of a chimeric antibody Abs-252 first polypeptide (SEQ ID NO: 118) formed by covalently linking the CD80 IgV variant I69W / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-252 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 236) that expresses the Abs-252 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-252 second polypeptide for expression.

[0206] In some embodiments, the functional antibody Abs-253 consists of a chimeric antibody Abs-253 first polypeptide (SEQ ID NO: 119) formed by covalently linking the CD80 IgV variant I69Y / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-253 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 237) that expresses the Abs-253 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-253 second polypeptide for expression.

[0207] In some embodiments, the functional antibody Abs-254 consists of a chimeric antibody Abs-254 first polypeptide (SEQ ID NO: 120) formed by covalently linking the CD80 IgV variant I69W / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-254 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 238) that expresses the Abs-254 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-254 second polypeptide for expression.

[0208] In some embodiments, the functional antibody Abs-255 consists of a chimeric antibody Abs-255 first polypeptide (SEQ ID NO: 121) formed by covalently binding a CD80 IgV variant H18W / A26M / A71E / L85N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-255 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 239) that expresses the Abs-255 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-255 second polypeptide into a host cell for expression.

[0209] In some embodiments, the functional antibody Abs-256 consists of a chimeric antibody Abs-256 first polypeptide (SEQ ID NO: 122) formed by covalently binding a CD80 IgV variant H18W / A26M / A71E / L85D to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-256 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 240) that expresses the Abs-256 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-256 second polypeptide into a host cell for expression.

[0210] In some embodiments, the functional antibody Abs-257 consists of a chimeric antibody Abs-257 first polypeptide (SEQ ID NO: 123) formed by covalently binding a CD80 IgV variant H18W / A26C / A71E / L85N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-257 functional antibody is constructed by introducing into a host cell for expression a polynucleotide sequence (SEQ ID NO: 241) vector expressing the Abs-257 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-257 second polypeptide.

[0211] In some embodiments, the functional antibody Abs-258 consists of a chimeric antibody Abs-258 first polypeptide (SEQ ID NO: 124) formed by covalently binding a CD80 IgV variant H18W / A26C / A71E / L85D to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-258 functional antibody is constructed by introducing into a host cell for expression a polynucleotide sequence (SEQ ID NO: 242) vector expressing the Abs-258 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-258 second polypeptide.

[0212] In some embodiments, the functional antibody Abs-259 consists of a chimeric antibody Abs-259 first polypeptide (SEQ ID NO: 125) formed by covalently binding the CD80 IgV variant A26M / A71E / L85N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-259 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 243) that expresses the Abs-259 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-259 second polypeptide for expression.

[0213] In some embodiments, the functional antibody Abs-260 consists of a chimeric antibody Abs-260 first polypeptide (SEQ ID NO: 126) formed by covalently binding the CD80 IgV variant A26M / A71E / L85D to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-260 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 244) that expresses the Abs-260 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-260 second polypeptide for expression.

[0214] In some embodiments, the functional antibody Abs-261 is composed of a chimeric antibody Abs-261 first polypeptide (SEQ ID NO: 127) formed by covalently binding the CD80 IgV variant A26C / A71E / L85D to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-261 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 245) that expresses the Abs-261 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-261 second polypeptide into a host cell for expression.

[0215] In some embodiments, the functional antibody Abs-262 is composed of a chimeric antibody Abs-262 first polypeptide (SEQ ID NO: 128) formed by covalently binding the CD80 IgV variant A26C / A71E / L85N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-262 functional antibody is constructed by introducing a vector of a polynucleotide sequence (SEQ ID NO: 246) that expresses the Abs-262 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-262 second polypeptide into a host cell for expression.

[0216] In some embodiments, the functional antibody Abs-263 consists of a chimeric antibody Abs-263 first polypeptide (SEQ ID NO: 129) formed by covalently linking the CD80 IgV variant I69L / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-263 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 247) that expresses the Abs-263 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-263 second polypeptide for expression.

[0217] In some embodiments, the functional antibody Abs-264 consists of a chimeric antibody Abs-264 first polypeptide (SEQ ID NO: 130) formed by covalently linking the CD80 IgV variant H18W / A26V / E35N / A71E / L85N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-264 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 248) that expresses the Abs-264 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-264 second polypeptide for expression.

[0218] In some embodiments, the functional antibody Abs-265 consists of a chimeric antibody Abs-265 first polypeptide (SEQ ID NO: 131) formed by covalently linking the CD80 IgV variant T13K / M42L / M47H / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-265 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 249) that expresses the Abs-265 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-265 second polypeptide.

[0219] In some embodiments, the functional antibody Abs-266 consists of a chimeric antibody Abs-266 first polypeptide (SEQ ID NO: 132) formed by covalently linking the CD80 IgV variant T13K / M42L / M47Q / A71E via a linker peptide (SEQ ID NO: 95) to the N-terminus of the sugemalimab heavy chain and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-266 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 250) that expresses the Abs-266 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-266 second polypeptide.

[0220] In some embodiments, the functional antibody Abs-267 consists of a chimeric antibody Abs-267 first polypeptide (SEQ ID NO: 133) formed by covalently linking the CD80 IgV variant T13K / M42L / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-267 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 251) that expresses the Abs-267 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-267 second polypeptide for expression.

[0221] In some embodiments, the functional antibody Abs-268 consists of a chimeric antibody Abs-268 first polypeptide (SEQ ID NO: 134) formed by covalently linking the CD80 IgV variant T13K / M42T / M47H / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-268 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 252) that expresses the Abs-268 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-268 second polypeptide for expression.

[0222] In some embodiments, the functional antibody Abs-269 consists of a chimeric antibody Abs-269 first polypeptide (SEQ ID NO: 135) formed by covalently binding the CD80 IgV variant T13K / M42T / M47T / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-269 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 253) that expresses the Abs-269 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-269 second polypeptide for expression.

[0223] In some embodiments, the functional antibody Abs-270 consists of a chimeric antibody Abs-270 first polypeptide (SEQ ID NO: 136) formed by covalently binding the CD80 IgV variant T13K / M42T / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-270 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 254) that expresses the Abs-270 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-270 second polypeptide for expression.

[0224] In some embodiments, the functional antibody Abs-271 consists of a chimeric antibody Abs-271 first polypeptide (SEQ ID NO: 137) formed by covalently binding the CD80 IgV variant M47H / I69Y / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-271 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 255) that expresses the Abs-first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-271 second polypeptide into a host cell for expression.

[0225] In some embodiments, the functional antibody Abs-272 consists of a chimeric antibody Abs-272 first polypeptide (SEQ ID NO: 138) formed by covalently binding the CD80 IgV variant M47Q / I69Y / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-272 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 256) that expresses the Abs-first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-272 second polypeptide into a host cell for expression.

[0226] In some embodiments, the functional antibody Abs-273 is composed of a chimeric antibody Abs-273 first polypeptide (SEQ ID NO: 139) formed by covalently binding the CD80 IgV variant M47K / I69Y / A71E / V83L to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-273 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 257) that expresses the Abs-first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-273 second polypeptide into a host cell for expression.

[0227] In some embodiments, the functional antibody Abs-99 is composed of a chimeric antibody Abs-99 first polypeptide (SEQ ID NO: 140) formed by covalently binding the wild-type CD80 IgV to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-99 functional antibody is constructed by introducing a vector with a polynucleotide sequence (SEQ ID NO: 258) that expresses the Abs-99 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-99 second polypeptide into a host cell for expression.

[0228] In some embodiments, the functional antibody Abs-509 consists of a chimeric antibody Abs-509 first polypeptide (SEQ ID NO: 142) formed by covalently binding the CD80 ECD variant E35N / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-509 functional antibody is obtained by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 259) expressing the Abs-509 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-509 second polypeptide for expression.

[0229] In some embodiments, the functional antibody Abs-510 consists of a chimeric antibody Abs-510 first polypeptide (SEQ ID NO: 143) formed by covalently binding the CD80 ECD variant E35N / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-510 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 260) expressing the Abs-510 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) expressing the Abs-510 second polypeptide for expression.

[0230] In some embodiments, the functional antibody Abs-511 consists of a chimeric antibody Abs-511 first polypeptide (SEQ ID NO: 144) formed by covalently linking the CD80 ECD variant E35N / A71Q to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-511 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 261) that expresses the Abs-511 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-511 second polypeptide for expression.

[0231] In some embodiments, the functional antibody Abs-512 consists of a chimeric antibody Abs-512 first polypeptide (SEQ ID NO: 145) formed by covalently linking the CD80 ECD variant E35N / A71T to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-512 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 262) that expresses the Abs-512 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-512 second polypeptide for expression.

[0232] In some embodiments, the functional antibody Abs-98 consists of a chimeric antibody Abs-98 first polypeptide (SEQ ID NO: 263) formed by covalently linking the wild-type CD80 ECD to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-98 functional antibody is constructed by introducing into a host cell a polynucleotide sequence (SEQ ID NO: 264) vector expressing the Abs-98 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-98 second polypeptide for expression.

[0233] In some embodiments, the functional antibody Abs-480 consists of a chimeric polypeptide Abs-480 (SEQ ID NO: 158) formed by covalently linking the CD80 ECD variant E35N / A71E to the N-terminus of the IgG4 Fc. The Abs-480 functional polypeptide complex is constructed by introducing into a host cell a polynucleotide sequence (SEQ ID NO: 265) vector expressing the Abs-480 polypeptide for expression.

[0234] In some embodiments, the functional antibody Abs-481 consists of a chimeric polypeptide Abs-481 (SEQ ID NO: 159) formed by covalently linking the CD80 ECD variant E35N / A71N to the N-terminus of the IgG4 Fc. The Abs-481 functional polypeptide complex is constructed by introducing into a host cell a polynucleotide sequence (SEQ ID NO: 266) vector expressing the Abs-481 polypeptide for expression.

[0235] In some embodiments, the functional antibody Abs-482 consists of the chimeric polypeptide Abs-482 (SEQ ID NO: 160) formed by covalently binding the CD80 ECD variant E35N / A71Q to the N-terminus of IgG4 Fc. An Abs-482 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 267) vector expressing the Abs-482 polypeptide into a host cell for expression.

[0236] In some embodiments, the functional antibody Abs-484 consists of the chimeric polypeptide Abs-484 (SEQ ID NO: 161) formed by covalently binding the CD80 ECD variant E35N / A71T to the N-terminus of IgG4 Fc. An Abs-484 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 268) vector expressing the Abs-484 polypeptide into a host cell for expression.

[0237] In some embodiments, the functional antibody Abs-100 consists of the chimeric polypeptide Abs-100 (SEQ ID NO: 174) formed by covalently binding the wild-type CD80 ECD to the N-terminus of IgG4 Fc. An Abs-100 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 269) vector expressing the Abs-100 polypeptide into a host cell for expression.

[0238] In some embodiments, the functional antibody Abs-427 consists of the chimeric polypeptide Abs-427 (SEQ ID NO: 175) formed by covalently binding the CD80 IgV variant E35N / A71E to the N-terminus of IgG4 Fc. An Abs-427 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 270) vector expressing the Abs-427 polypeptide into a host cell for expression.

[0239] In some embodiments, the functional antibody Abs-428 consists of the chimeric polypeptide Abs-428 (SEQ ID NO: 176) formed by covalently binding the CD80 IgV variant E35N / A71N to the N-terminus of IgG4 Fc. An Abs-428 functional polypeptide complex is constructed by introducing a vector of the polynucleotide sequence (SEQ ID NO: 271) that expresses the Abs-428 polypeptide into a host cell for expression.

[0240] In some embodiments, the functional antibody Abs-429 consists of the chimeric polypeptide Abs-429 (SEQ ID NO: 177) formed by covalently binding the CD80 IgV variant E35N / A71Q to the N-terminus of IgG4 Fc. An Abs-429 functional polypeptide complex is constructed by introducing a vector of the polynucleotide sequence (SEQ ID NO: 272) that expresses the Abs-429 polypeptide into a host cell for expression.

[0241] In some embodiments, the functional antibody Abs-483 consists of the chimeric polypeptide Abs-483 (SEQ ID NO: 178) formed by covalently binding the CD80 IgV variant E35N / A71T to the N-terminus of IgG4 Fc. An Abs-483 functional polypeptide complex is constructed by introducing a vector of the polynucleotide sequence (SEQ ID NO: 273) that expresses the Abs-483 polypeptide into a host cell for expression.

[0242] In some embodiments, the functional antibody Abs-504 consists of the chimeric polypeptide Abs-504 (SEQ ID NO: 191) formed by covalently binding the wild-type CD80 IgV to the N-terminus of IgG4 Fc. An Abs-504 functional polypeptide complex is constructed by introducing a vector of the polynucleotide sequence (SEQ ID NO: 274) that expresses the Abs-504 polypeptide into a host cell for expression.

[0243] In some embodiments, the functional antibody Abs-505 consists of a chimeric antibody Abs-505 first polypeptide (SEQ ID NO: 198) in which the CD80 IgV variant E35N / A71E is covalently bound via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the Relatlimab heavy chain, and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The Abs-505 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 276) that expresses the Abs-505 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 275) that expresses the Abs-505 second polypeptide for expression.

[0244] In some embodiments, the functional antibody Abs-513 consists of a chimeric antibody Abs-513 first polypeptide (SEQ ID NO: 199) in which the CD80 IgV variant E35N / A71N is covalently bound via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the Relatlimab heavy chain, and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The Abs-513 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 277) that expresses the Abs-513 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 275) that expresses the Abs-513 second polypeptide for expression.

[0245] In some embodiments, the functional antibody Abs-514 consists of a chimeric antibody Abs-514 first polypeptide (SEQ ID NO: 200) in which the CD80 IgV variant E35N / A71Q is covalently bound via a linker peptide (SEQ ID NO: 95) to form the Relatlimab heavy chain N-terminus and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The functional antibody Abs-514 is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 278) that expresses the Abs-514 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 276) that expresses the Abs-514 second polypeptide for expression.

[0246] In some embodiments, the functional antibody Abs-515 consists of a chimeric antibody Abs-515 first polypeptide (SEQ ID NO: 201) in which the CD80 IgV variant E35N / A71T is covalently bound via a linker peptide (SEQ ID NO: 95) to form the Relatlimab heavy chain N-terminus and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The functional antibody Abs-515 is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 279) that expresses the Abs-515 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 276) that expresses the Abs-515 second polypeptide for expression.

[0247] In some embodiments, the functional antibody Abs-506 consists of a chimeric antibody Abs-506 first polypeptide (SEQ ID NO: 202) formed by covalently linking a wild-type CD80 IgV via a linker peptide (SEQ ID NO: 95) to the N-terminus of the Relatlimab heavy chain and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The functional antibody Abs-506 is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 280) that expresses the Abs-506 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 276) that expresses the Abs-506 second polypeptide.

[0248] In some embodiments, the functional antibody Abs-507 consists of a chimeric antibody Abs-506 first polypeptide (SEQ ID NO: 204) formed by covalently linking a CD80 IgV mutant E35N / A71E via a linker peptide (SEQ ID NO: 95) to the N-terminus of the CD3B219 heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 203). The functional antibody Abs-507 is constructed by introducing into a host cell for expression a vector with a polynucleotide sequence (SEQ ID NO: 282) that expresses the Abs-507 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-507 second polypeptide.

[0249] In some embodiments, the functional antibody Abs-516 consists of a chimeric antibody Abs-516 first polypeptide (SEQ ID NO: 205) in which the CD80 IgV variant E35N / A71N is covalently linked via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the CD3B219 antibody heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 203). The Abs-516 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 283) that expresses the Abs-516 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-516 second polypeptide.

[0250] In some embodiments, the functional antibody Abs-517 consists of a chimeric antibody Abs-517 first polypeptide (SEQ ID NO: 206) in which the CD80 IgV variant E35N / A71Q is covalently linked via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the CD3B219 antibody heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 203). The Abs-517 functional antibody is constructed by introducing into a host cell for expression a vector of a polynucleotide sequence (SEQ ID NO: 284) that expresses the Abs-517 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-517 second polypeptide.

[0251] In some embodiments, the functional antibody Abs-518 consists of a chimeric antibody Abs-518 first polypeptide (SEQ ID NO: 207) in which the CD80 IgV variant E35N / A71T is covalently linked via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the CD3B219 antibody heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 203). The Abs-518 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 285) that expresses the Abs-518 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-518 second polypeptide for expression.

[0252] In some embodiments, the functional antibody Abs-508 consists of a chimeric antibody Abs-508 first polypeptide (SEQ ID NO: 207) in which the wild-type CD80 IgV is covalently linked via a linker peptide (SEQ ID NO: 95) to form the N-terminus of the CD3B219 antibody heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 204). The Abs-508 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 286) that expresses the Abs-508 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-508 second polypeptide for expression.

[0253] In some embodiments, the functional antibody Abs-16 consists of a chimeric antibody Abs-16 first polypeptide (SEQ ID NO: 287) that forms the N-terminus of the sugemalimab antibody heavy chain and a sugemalimab light chain second polypeptide (SEQ ID NO: 263). The Abs-16 functional antibody is constructed by introducing into a host cell a vector with a polynucleotide sequence (SEQ ID NO: 288) that expresses the Abs-16 first polypeptide and a vector with a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-16 second polypeptide for expression.

[0254] In some embodiments, the functional antibody Abs-58 consists of a chimeric antibody Abs-58 first polypeptide (SEQ ID NO: 289) formed at the N-terminus of the Relatlimab heavy chain and a Relatlimab light chain second polypeptide (SEQ ID NO: 197). The Abs-58 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 290) that expresses the Abs-58 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 276) that expresses the Abs-58 second polypeptide for expression.

[0255] In some embodiments, the functional antibody Abs-519 consists of a chimeric antibody Abs-520 first polypeptide (SEQ ID NO: 291) formed at the N-terminus of the CD3B219 heavy chain and a CD3B219 light chain second polypeptide (SEQ ID NO: 204). The Abs-519 functional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 292) that expresses the Abs-519 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 281) that expresses the Abs-519 second polypeptide for expression.

[0256] In some embodiments, the functional antibody Abs-520 consists of a chimeric antibody Abs-520 first polypeptide (SEQ ID NO: 293) formed by covalently binding the CD80 IgV variant E35N / A71E to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and covalently binding the C-terminus of the sugemalimab heavy chain to the LAG3 polypeptide via a linker peptide (SEQ ID NO: 95), and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-520 multifunctional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 294) that expresses the Abs-520 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-520 second polypeptide for expression.

[0257] In some embodiments, the functional antibody Abs-521 consists of a chimeric antibody Abs-521 first polypeptide (SEQ ID NO: 295) formed by covalently binding the CD80 IgV variant E35N / A71N to the N-terminus of the sugemalimab heavy chain via a linker peptide (SEQ ID NO: 95) and covalently binding the C-terminus of the sugemalimab heavy chain to the LAG3 polypeptide via a linker peptide (SEQ ID NO: 95), and a sugemalimab light chain second polypeptide (SEQ ID NO: 141). The Abs-521 multifunctional antibody is constructed by introducing into a host cell a vector of a polynucleotide sequence (SEQ ID NO: 296) that expresses the Abs-520 first polypeptide and a vector of a polynucleotide sequence (SEQ ID NO: 215) that expresses the Abs-521 second polypeptide for expression.

[0258] 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 are not limiting. Many modifications to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the present invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

[0259] All documents mentioned in this disclosure are hereby incorporated by reference into this specification. All publications and patent documents cited in this application are hereby incorporated by reference into this specification for all purposes as if each individual publication or patent document were specifically and individually indicated.

[0260] Examples Example 1. Expression and Purification of Antibodies To further identify the antibodies obtained by screening, it is necessary to express the antibodies in mammalian cells. Therefore, an expression plasmid vector expressing the heavy chain of the multifunctional / bifunctional antibody and an expression plasmid vector containing the light chain were first constructed. Approximately 24 hours before plasmid transfection, Expi293 cells were passaged to a cell density of about 2.4 × 10 6 cells / ml. When the cell density was 6 × 10 6 cells / ml and the viability exceeded 95%, 25 μg of the plasmid vector expressing the mixed heavy and light chains was harvested, and 25 ml of Expi293 cells were transfected with Expifectamine 293. After culturing with shaking at 37°C, 130 rpm, and 8% CO2 for 7 days, the cell culture product was centrifuged, the supernatant was collected, filtered through a 0.45 MCE filter, the target antibody was purified and recovered using a Capturem protein A Maxiprep column, and then centrifugally concentrated using a Vivaspin 20 Centrifugal Concentrator 50K. Quantification was performed by measuring A280 using a NanoDrop 2000, and the purity of the antibody was measured by SDS-PAGE and SEC-HPLC.

[0261] Example 2. Protein Electrophoresis Analysis The following CD80 IgV mutant-sugemalimab fusion polypeptide complexes, selected and purified under reducing conditions (N-R) and non-reducing conditions (N), were analyzed by SDS-PAGE: 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.

[0262] As a result of the electrophoresis analysis, it is shown 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 are aggregates of antibodies. As shown in Figure 1, antibodies such as -367 and Abs-368 are superior to the wild-type CD80 IgV-sugemalimab fusion polypeptide complex Abs-99.

[0263] Example 3. Measurement of Binding Affinity This example shows the binding of the CD80 mutant fusion polypeptide complex to human PD-L1 protein, human CTLA4 protein, and human CD28.

[0264] Specifically, human PD-L1 protein (1 μg / mL in PBS, Acro Company, PD1-H5258) was prepared in a flat-bottom 96-well Immuno-removable transparent enzyme plate (MaxiSorp) (Thermo Scientific, 446469) at 4 °C. Subsequently, after washing, 2% BSA (in PBS; VWR Life science company, 0332-1KG) was added to each well and incubated at room temperature for 1 hour. 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 (CoWin Biosciences, Inc., CW0050S) was used to perform the operation according to the manufacturer's instructions. The affinity between the candidate multifunctional / bifunctional fusion protein and the human PD-L1 protein is shown by the EC50 and the binding strength at 20 nM, and the test results are shown in Table 1 and Figure 2.

[0265] Table 1. CD80 mutant fusion polypeptide complex and human PDL1 protein binding EC50 and binding strength at 100 nM JPEG2025521557000001.jpg81170

[0266] The results shown in Table 1 and Figure 2 indicate that in the binding experiment of the CD80 mutant fusion polypeptide complex with the human PDL1 antigen, the binding activities of the CD80 mutant fusion polypeptide complexes Abs-427, Abs-428, and Abs-483 to the PDL1 antigen are much higher than those of the wild-type CD80 fusion polypeptide complex Abs-504.

[0267] Similarly, human CD28 protein (1 μg / mL in PBS, Acro, CD8-H525a) was incubated overnight at 4°C in a flat-bottom 96-well Immuno-removable transparent microplate (MaxiSorp) (Thermo Scientific, 446469). The affinity of the test substance for the human CD28 protein was detected by the same method as above, and the detection results expressed as EC50 and binding strength at 100 nM are shown in Table 2 and Figure 4.

[0268] Table 2. Binding EC50 and binding strength at 100 nM of CD80 mutant fusion polypeptide complex to human CD28 protein JPEG2025521557000002.jpg25586

[0269] NA: Indicates no binding to the target protein

[0270] The results shown in Table 2 and Figure 4 indicate that in the binding experiment between the CD80 mutant fusion polypeptide complex and the human CD28 antigen, the binding activities 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, Abs-368 for the CD28 antigen are much higher than the antigen binding activity of the wild-type CD80 fusion polypeptide complex Abs-99.

[0271] Similarly, human CTLA4 protein (1 μg / mL in PBS, Acro Company, CT4-H52H9) was incubated overnight at 4°C in a flat-bottom 96-well Immuno-removable transparent enzyme plate (MaxiSorp) (Thermo Scientific, 446469). Using the same method as above, the affinity of the test substance for the human CTLA4 protein was detected with EC50 and binding strength at 20 nM, and the results are shown in Table 3 and Figure 3.

[0272] Table 3. CD80 variant fusion polypeptide complex and human CTLA4 protein binding EC50 and binding strength at 20 nM. JPEG2025521557000003.jpg25594

[0273] NA: Indicates no binding to the target protein

[0274] The results shown in Table 3 and Figure 3 indicate that in the binding experiment between the CD80 variant fusion polypeptide complex and the human CTLA4 antigen, the binding activities of the CD80 variant fusion polypeptide complexes Abs-267, Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-362, and Abs-364 to the CTLA4 antigen are much higher than those of the wild-type CD80 fusion polypeptide complex Abs-99.

[0275] Example 4. Measurement of CD80 variant fusion polypeptide complex that relieves PD-1 / PD-L1-mediated T cell immunosuppressive response

[0276] This example shows the situation where the CD80 variant fusion polypeptide complex relieves the PD-1 / PD-L1-mediated T cell inhibitory response.

[0277] Specifically, on the first day, TCR activator PD-L1-CHO cells (BPS bioscience) were digested using 0.05% trypsin, and after collection, they were plated. Fresh medium was added, the cells were mixed uniformly, and the cell density was 3.5*10 5Adjusted to cells / ml. The cells were added to a sterile 96-well flat-bottom plate (CORNING, 3599) at 100 μL / well and grown overnight in an incubator at 37 °C until the culture reached 80%. The next day, the medium of TCR activator PD-L1-CHO cells was removed from the 96-well flat-bottom plate. 50 μl of fresh culture medium containing the test substance at the corresponding concentration (serial dilution) was added to each well and treated for 30 minutes, then Jurkat-Lucia™ TCR-hPD-1 cells (Invivogen) were collected and counted. Fresh medium was added, the cells were mixed uniformly, and the cell density was adjusted to 4×10 5 cells / ml. 50 μl of Jurkat-Lucia™ TCR-hPD-1 cells were added to make the final density 2×10 4 / well, and the cells were incubated in a 96-well flat-bottom plate in an incubator at 37 °C for 5 - 6 hours. After incubation, the cell morphology was observed under a microscope, and the chemiluminescence value was detected using QUANTI-Luc™ (Invivogen; rep-qlc1). Specific operations were performed according to the manufacturer's instructions. The activity was indicated by EC50 and the maximum induction multiple, and the detection results are shown in Table 4, Table 5, Figure 5, and Figure 6. Table 4. Measurement results of CD80 mutant fusion polypeptide complexes that control the activation of Jurkat T cells

[0278] JPEG2025521557000004.jpg255131

[0279] NT: Indicates not tested. NA: Indicates no stimulatory effect.

[0280] The results in Table 4 and Figure 5 show that in the experiment where the CD80 mutant fusion polypeptide complex regulates the activation of Jurkat T cells, the ability of the CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-358, Abs-361, Abs-362 to regulate Jurkat T cell activation is higher than that of the wild-type CD80 fusion polypeptide complex Abs-99.

[0281] Table 5. Measurement results of controlling the activation of Jurkat T cells by CD80 mutant fusion polypeptide complex JPEG2025521557000005.jpg55170

[0282] NA: Indicates no stimulatory effect.

[0283] The results in Table 5 and Figure 6 show that in the experiment where the CD80 mutant fusion polypeptide complex regulates the activation of Jurkat T cells, the ability of the CD80 mutant fusion polypeptide complex Abs-428 to regulate Jurkat T cell activation is higher than that of the wild-type CD80 fusion polypeptide complex Abs-504.

[0284] Example 5. Measurement of relieving the LAG3-mediated T cell immunosuppressive response of the CD80 mutant fusion polypeptide complex

[0285] This example shows the situation where the CD80 mutant fusion polypeptide complex relieves the LAG3-mediated T cell inhibitory response.

[0286] Specifically, RajI cells (ATCC) were collected, counted, and seeded onto plates. Fresh medium was added, the cells were mixed uniformly, and the cell density was adjusted to 3.5*10 5 cells / ml. The cells were added to sterile 96-well flat-bottom plates (CORNING, 3599) at 100 μL / well, resulting in a total of 35,000 cells / well. 50 μl of fresh medium containing the test substance at the corresponding concentration (serial dilution) was added to each well. After treatment for 30 minutes, LAG3 / NFAT Reporter-Jurkat cells (BPS bioscience) were collected and counted. Fresh medium containing 0.4 ng / ml of Staphylococcal enterotoxin E (SEE, Toxintechnology) was added to mix the cells uniformly, and the cell density was adjusted to 4*10 5 cells / m. 50 μL of LAG3 / NFAT Reporter - Jurkat cells were added to a final cell density of 2×10 4Added up to the wells and incubated in a 37 °C incubator in a 96-well flat-bottom plate for 5 - 6 hours. After incubation, the cell morphology was observed under a microscope, and the chemiluminescence value was detected using QUANTI-Luc (trademark) (BPS bioscience; 78262). The specific operations were carried out according to the manufacturer's instructions.

[0287] Table 6. Measurement results of the CD80 mutant fusion polypeptide complex releasing the LAG3-mediated T cell immunosuppressive response JPEG2025521557000006.jpg61170

[0288] In Table 6, in the experiment where the CD80 mutant fusion polypeptide complex releases the LAG3-mediated T cell immunosuppressive response, it shows that the abilities of the CD80 mutant fusion polypeptide complexes Abs-505, Abs-513, Abs-514, and Abs-515 to release the LAG3-mediated T cell immunosuppressive response are higher than that of the wild-type CD80 fusion polypeptide complex Abs-506.

[0289] Example 6. T cell activation immune response assay

[0290] This example shows the situation where the CD80 mutant fusion polypeptide complex regulates the T cell immune response.

[0291] Specifically, Jurkat-Lucia (trademark) TCR cells (INVIVoGEN) were collected and counted. Fresh medium was added, the cells were mixed uniformly, and the cell density was adjusted to 4*10 5 cells / ml. 100 μl of fresh medium containing the test substance at the corresponding concentration (serial dilution) was added to each well and incubated in a 37 °C incubator in a 96-well flat-bottom plate for 5 - 6 hours. After incubation, the cell morphology was observed under a microscope, and the chemiluminescence value was detected using QUANTI-Luc (trademark) (Invivogen; rep-qlc1). The specific operations were carried out according to the manufacturer's instructions.

[0292] Table 7. Measurement results of T cell activation immune response by CD80 mutant fusion polypeptide complex JPEG2025521557000007.jpg60170

[0293] The results in Table 7 show that in the measurement test of T cell activation immune response by the CD80 mutant fusion polypeptide complex, the ability of the CD80 mutant fusion polypeptide complexes Abs-507, Abs-516, Abs-517, and Abs-518 to activate the T cell immune response is higher than that of the wild-type CD80 fusion polypeptide complex Abs-508.

[0294] Example 7. Measurement of primary PBMC immune response

[0295] This example shows the situation of activating T cell immunity in primary PBMC by the CD80 mutant fusion polypeptide complex. Specifically, PBMC were collected, counted, and then plated. Fresh medium was added, the cells were mixed evenly, and the cell density was adjusted to 1×10 5 cells / ml. 50 μl of fresh culture medium containing the test substance at the corresponding concentration (serial dilution) was added to each well, and after incubating for 3 days, the culture supernatant was collected, and the secretion of IL-2 in the supernatant was detected using an IL-2 ELISA detection kit (R&D, DY202), and the specific operation was carried out according to the manufacturer's instructions.

[0296] Table 8. Measurement results of CD80 mutant fusion polypeptide complex activating the immune response of primary PBMC T cells JPEG2025521557000008.jpg55170

[0297] The results in Table 8 and Figure 7 show that the CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, and Abs-356 have a higher ability to activate T cells in primary PBMC than the wild-type CD80 fusion polypeptide complex Abs-99.

[0298] Example 8. Measurement of chemokine secretion by antigen-presenting cells This example shows that chemokine CCL4 is secreted from antigen-presenting cells by Abs.

[0299] Specifically, antigen-presenting cells THP-1 cells (ATCC) were collected, counted, and then plated. Fresh medium was added, the cells were uniformly mixed, and the cell density was adjusted to 1×10 6 cells / ml, and 100 μl of the cells were plated in each well. A test substance Abs_520 or Abs_521 at the corresponding concentration in 100 μl of fresh medium was added to each well, and after 6 hours, the cell culture supernatant was collected, and the expression level of CCL4 was detected using a CCL4 detection kit (PROTEINTECH). The specific operation was performed according to the manufacturer's instructions. The results show that, as shown in Figure 8, Abs_520 and Abs_521 can induce chemokine secretion from antigen-presenting cells.

[0300] The results of the above example prove that the CD80 mutant polypeptide formed by innovatively modifying the CD80 IgV domain can regulate the binding activity between CD80 and its ligand. In particular, the CD80 mutant fusion polypeptide complex disclosed herein has significantly higher antigen-binding activity against PDL1, CTLA4, and CD28 than the corresponding wild-type CD80 fusion polypeptide complex, and the CD80 mutant fusion polypeptide complex disclosed herein can relieve the PD-1 / PD-L1-mediated T cell activation immunosuppressive response. Activation; It regulated the activation of PBMCs and promoted chemokine secretion from antigen-presenting cells. That is, the CD80 mutant fusion polypeptide complex disclosed herein 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 suppression of T cell function.

[0301] 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 are not limiting. Without departing from the spirit and scope of the present invention, various changes and improvements are possible to the present invention, and these changes and improvements are included in the scope of the present invention as recited in the claims. The scope of the present invention 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 with respect to the wild-type human CD80 IgV polypeptide sequence of SEQ ID NO:

45. The CD80 IgV mutant polypeptide.

2. The CD80 IgV variant polypeptide comprises a combination of two or more of the amino acid mutations, and optionally, the combination of the two or more amino acid mutations is 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 / 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 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),Selected from the group consisting of M47Q / I69Y / A71E / V83L (SEQ ID NO: 87) and M47K / I69Y / A71E / V83L (SEQ ID NO: 88), The CD80 IgV mutant polypeptide according to Claim 1.

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 with respect to the wild-type human CD80 ECD polypeptide sequence of SEQ ID NO:

1. The CD80 ECD mutant polypeptide.

4. The CD80 ECD variant polypeptide comprises a combination of two or more of the amino acid mutations, and optionally, the combination of the two or more amino acid mutations is 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), 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 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),Selected from the group consisting of M47Q / I69Y / A71E / V83L (SEQ ID NO: 43) and M47K / I69Y / A71E / V83L (SEQ ID NO: 44), The CD80 ECD mutant polypeptide according to Claim 3.

5. A CD80 mutant fusion polypeptide complex comprising the CD80 IgV mutant polypeptide according to Claim 1 or 2 or the CD80 ECD mutant polypeptide according to Claim 3 or 4 and a second domain, optionally, the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide is covalently bound to the second domain via a linker peptide or without a linker peptide. The CD80 mutant fusion polypeptide complex.

6. The CD80 mutant fusion polypeptide complex according to Claim 5, wherein the second domain is an antibody or an antigen-binding fragment thereof, optionally, 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 according to Claim 6, wherein the antibody is selected from the group consisting of immunoglobulin IgG antibodies, recombinant antibodies, chimeric antibodies, heavy chain antibodies, single domain antibodies, and / or bispecific antibodies, optionally, the immunoglobulin IgG antibody is selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, and mouse IgG3. The CD80 mutant fusion polypeptide complex according to Claim 6.

8. The antibody or antigen-binding fragment thereof specifically targets one or more antigens selected from 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. The CD80 variant fusion polypeptide complex according to claim 6 or 7.

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

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

11. The antibody or antigen-binding fragment thereof contains a heavy chain, and the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the heavy chain either via a linker peptide or without a linker peptide. Optionally, the C-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the N-terminus of the heavy chain either via a linker peptide or without a linker peptide; and / or the C-terminus of the heavy chain is covalently bound to the N-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide either via a linker peptide or without a linker peptide. The CD80 variant fusion polypeptide complex according to any one of claims 6-9.

12. The antibody or antigen-binding fragment thereof comprises a light chain, and the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide is covalently bound to the light chain either via a linker peptide or without a linker peptide, and optionally is covalently bound to the N-terminus of the light chain either via a C-terminal linker peptide of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide or without such a linker peptide; and / or the C-terminus of the light chain is covalently bound to the N-terminus of the CD80 IgV variant polypeptide or CD80 ECD variant polypeptide either via a linker peptide or without a linker peptide. The CD80 variant fusion polypeptide complex according to any one of claims 6-10.

13. The CD80 variant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, the second polypeptide may or may not be present, and optionally, 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. The CD80 variant fusion polypeptide complex according to claim 5.

14. The first polypeptide is formed by covalently binding, in order from the N-terminus to the C-terminus of the polypeptide, a CD80 IgV variant polypeptide or CD80 ECD variant polypeptide, a linker peptide, and a heavy chain of an antibody or an antigen-binding fragment thereof, or the first polypeptide is formed by covalently binding, in order from the N-terminus to the C-terminus of the polypeptide, a heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a CD80 IgV variant polypeptide or CD80 ECD variant polypeptide. The CD80 variant fusion polypeptide complex according to claim 13.

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

16. The first polypeptide is 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: 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), M47K / I69Y / A71E / V83L (SEQ ID NO: 139), selected from, or, 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), E35T / A71N (SEQ ID NO: 155), E35T / A71Q (SEQ ID NO: 156), E35T / A71T (SEQ ID NO: 157); or The first polypeptide is selected from E35N / A71E (SEQ ID NO: 192), E35N / A71N (SEQ ID NO: 193), E35N / A71Q (SEQ ID NO: 194), E35N / A71T (SEQ ID NO: 195); or The first polypeptide is selected from E35N / A71E (SEQ ID NO: 198), E35N / A71N (SEQ ID NO: 199), E35N / A71Q (SEQ ID NO: 200), E35N / A71T (SEQ ID NO: 201); or The first polypeptide is selected from E35N / A71E (SEQ ID NO: 203), E35N / A71N (SEQ ID NO: 204), E35N / A71Q (SEQ ID NO: 205), 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), E35N / A71T (SEQ ID NO: 212), The CD80 variant fusion polypeptide complex according to claim 13 or 14.

17. The CD80 variant fusion polypeptide complex according to claim 15 or 16, wherein the second polypeptide is selected from the group consisting of SEQ ID NO: 141, SEQ ID NO: 197, and SEQ ID NO:

208.

18. The CD80 variant fusion polypeptide complex further comprises a third domain covalently bound to the CD80 IgV variant polypeptide according to claim 1 or 2, or the CD80 ECD variant polypeptide according to claim 3 or 4, or the second domain according to any one of claims 5 - 17, either via a linker peptide or without a linker peptide. The CD80 variant fusion polypeptide complex according to any one of claims 5-17.

19. 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, when the third domain is a functional protein or an active fragment thereof, the functional protein or its active fragment can activate an immune response. Optionally, the third domain is connected to the CD80 IgV variant polypeptide according to claim 1 or 2 or the CD80 ECD variant polypeptide according to claim 3 or 4 either via a linker peptide or without a linker peptide. The CD80 variant fusion polypeptide complex according to claim 18.

20. The second domain contains an antibody heavy chain, and the antibody heavy chain of the second domain is connected to the third domain either via a linker peptide or without a linker peptide. Optionally, the heavy chain of the second domain is connected to the N-terminus of the third domain either via a linker peptide or without a linker peptide, and / or the heavy chain of the second domain is connected to the C-terminus of the third domain either via a linker peptide or without a linker peptide; and / or The second domain contains an antibody light chain, and the antibody light chain of the second domain is connected to the third domain either via a linker peptide or without a linker peptide. Optionally, the light chain of the second domain is connected to the N-terminus of the third domain either via a linker peptide or without a linker peptide, and / or the light chain of the second domain is connected to the C-terminus of the third domain either via a linker peptide or without a linker peptide. The CD80 variant fusion polypeptide complex according to claim 19.

21. The CD80 variant fusion polypeptide complex includes a first polypeptide and a second polypeptide, and the second polypeptide may or may not be present. Optionally, The first polypeptide contains the heavy chain of the second domain. Optionally, The first polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain. The linker peptides may be the same or different and may or may not exist independently; or The first polypeptide is formed by covalently linking, in order from the C-terminus to the N-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain. The linker peptides may be the same or different and may or may not exist independently; and / or The second polypeptide comprises the light chain of a second domain, optionally The second polypeptide is formed by covalently linking, in order from the N-terminus to the C-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain. The linker peptides may be the same or different and may or may not exist independently; or The second polypeptide is formed by covalently linking, in order from the C-terminus to the N-terminus, a CD80 IgV variant polypeptide or a CD80 ECD variant polypeptide, a linker peptide, the heavy chain of an antibody or an antigen-binding fragment thereof, a linker peptide, and a third domain. The linker peptides may be the same or different and may or may not exist independently, The CD80 variant fusion polypeptide complex according to any one of claims 18-20. **Claim 22** The CD80 variant fusion polypeptide complex according to any one of claims 18-21, wherein the third domain comprises LAG-3 or an active fragment thereof. **Claim 23** The second domain specifically targets PD-1 or PD-L1, and optionally, the second domain is sugemalimab or a functional fragment thereof, The CD80 variant fusion polypeptide complex according to claim 22. **Claim 24** The CD80 variant fusion polypeptide complex according to claim 22, wherein the first polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 293 and SEQ ID NO: 295, or the second polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:

141.

25. The second domain is an immunoglobulin Fc domain. Optionally, the Fc domain is selected from the group consisting of 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, and a mouse IgG3 Fc domain. Optionally, the CD80 IgV variant polypeptide or the CD80 ECD variant polypeptide is covalently bound to the N-terminus of the immunoglobulin Fc domain with or without a linker peptide. The CD80 variant fusion polypeptide complex according to claim 5.

26. The amino acid sequence of the CD80 variant 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), E35T / A71E (SEQ ID NO: 170), E35T / A71N (SEQ ID NO: 171), E35T / A71Q (SEQ ID NO: 172), and E35T / A71T (SEQ ID NO: 173). The CD80 variant fusion polypeptide complex according to claim 25.

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

28. 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). The CD80 variant fusion polypeptide complex according to any one of claims 5-27.

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

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

31. A vector comprising the nucleotide molecule according to claim 30.

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

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

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

35. Contacting the CD80 IgV variant polypeptide according to claim 1 or 2, the CD80 ECD variant polypeptide according to claim 3 or 4, or the CD80 variant fusion polypeptide complex according to any one of claims 5-28 and / or the composition according to claim 33 with T cells to stimulate their activation, A method for regulating T cell activity.

36. Administering the CD80 IgV variant polypeptide according to claim 1 or 2, the CD80 ECD variant polypeptide according to claim 3 or 4, the CD80 variant fusion polypeptide complex according to any one of claims 5-28, the immune complex 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, A method for inhibiting the growth or proliferation of tumor cells.

37. Use in the preparation of a medicament for preventing, ameliorating and / or treating tumors or cancers of the CD80 IgV variant polypeptide according to claim 1 or 2, the CD80 ECD variant polypeptide according to claim 3 or 4, the CD80 variant fusion polypeptide complex according to any one of claims 5-28, the immune complex 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.

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