CD80 variant polypeptides and their applications

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, and more particularly to CD80 IgV mutant polypeptides, CD80 ECD mutant polypeptides, CD80 mutant fusion polypeptide complexes containing the mutant polypeptides, and nucleotide molecules expressing the mutant fusion polypeptide complexes as fusion proteins. The present invention also relates to methods for inducing or enhancing immunity, as well as methods for treating or preventing diseases such as infectious diseases and cancer, using the mutant polypeptides, fusion polypeptide complexes, or nucleic acid molecules. [Background technology]

[0002] CD80 proteins are a type of immune-related molecule that stimulates immune responses, primarily by regulating T-cell immune responses such as immune defense, immune tolerance, and immune tissue damage.

[0003] CD80 primarily exerts its immunomodulatory effects and regulates the immune response by binding to its ligands, CD28, CTLA4, and PDL1. By altering the affinity between CD80 and its ligands, the related functions mediated by CD80 can be modified, particularly enhancing immune response function and strengthening its control over diseases such as cancer and infectious diseases.

[0004] The CD80 molecule is a member of the B7 molecule family and is involved in the co-stimulatory activation of T cells. A mature CD80 molecule consists of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain. Of these, the extracellular domain (ECD) is crucial for binding to the corresponding receptor on T cells. The extracellular domain of CD80 includes an immunoglobulin variant (IgV) domain and a constant-state immunoglobulin (IgC2) domain, with the IgV structural domain being particularly involved in direct receptor binding. The IgV extracellular domain of CD80 binds to CD28 and is involved in the induction of T cell activation, proliferation, and efficacy. CTLA4 can also bind to the IgV domain of CD80 and participate in immunosuppressive regulation.

[0005] In regulating the immune response, T cell activation is controlled by the binding of the costimulatory signaling receptor CD28 to its corresponding ligand 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, which can prevent T cell activation. This is one of the main mechanisms of immune evasion in tumors.

[0006] Furthermore, CTLA4 competitively binds to CD80 / CD86 with CD28, exhibiting higher affinity properties. As a result, CD28 is unable to effectively accept immune agonist signals mediated by CD80 / CD86, leading to ineffective T cell activation or immunosuppression. In addition, by binding PDL1 to CD80, it weakens the CD80-mediated immune activation effect and interferes with the normal activation of T cells, thus suppressing the associated immune response.

[0007] Therefore, in this field, there is a need for CD80 variant polypeptides with improved binding activity to CD80 and its ligands, which can effectively stimulate T cells to enhance the immune response, thereby enabling the treatment or prevention of diseases such as infections and tumors caused by the suppression of T cell function. [Overview of the Initiative]

[0008] The inventors of this invention unexpectedly discovered that CD80 mutant polypeptides, obtained by innovatively modifying the CD80 IgV domain, can modulate the binding activity of CD80 to its ligand. These CD80 mutant polypeptides can effectively stimulate T cells and enhance the immune response. As a result, treatment or prevention of diseases such as infections and tumors caused by suppressed T cell function becomes possible.

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

[0010] VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIV ILAL RPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKAD (Sequence ID 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 include 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), and E35T / A71T. (Sequence ID 61), M47A / I69Y / A71E / V83L (Sequence ID 62), M47A / I69W / A71E / V83L (Sequence ID 63), M47A / I69Y / A71E (Sequence ID 64), M47A / I69W / A71E (Sequence ID 65), I69Y / A71E / V83L (Sequence ID 66), I69W / A71E / V83L (Sequence ID 67), I69Y / A71E (Sequence ID 68), I69W / A71E (Sequence ID 69), H18W / A26M / A71E / L85N (Sequence ID 70), H18W / A26M / A71E / L85D (Sequence ID 71), H18W / A26C / A71E / L85N (Sequence ID 72), H18W / A26C / A71E / L85D (Sequence ID 73), A26M / A71E / L85N (Sequence ID 74), A26M / A71E / L85D (Sequence ID 75), A26C / A71E / L85D (Sequence ID 76), A26C / A71E / L85N (Sequence ID 77), I69L / A71E / V83L (Sequence ID 78), H18W / A26V / E35N / A71E / L85N (Sequence ID 79), T13K / M42L / M47H / A71E (Sequence ID 80), T13K / M42L / M47Q / A71E (Sequence ID 81), T13K / M42L / A71E (Sequence ID 82), T13K / M42T / M47H / A71E (Sequence ID 83), T13K / M42T / M47T / A71E (Sequence ID 84), T13K / M42T / A71E (Sequence ID 85), M47H / I69Y / A71E / V83LThe group is selected from (Sequence ID 86), M47Q / I69Y / A71E / V83L (Sequence ID 87), and M47K / I69Y / A71E / V83L (Sequence ID 88).

[0013] In a second aspect, the present invention provides CD80 ECD mutant polypeptides 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, and L85D relative to the wild-type human CD80 ECD polypeptide sequence SEQ ID NO: 1.

[0014] VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRR IICST SGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN(Sequence ID 1)

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

[0016] In some embodiments, the combination of 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 (Sequence ID 17), M47A / I69Y / A71E / V83L (Sequence ID 18), M47A / I69W / A71E / V83L (Sequence ID 19), M47A / I69Y / A71E (Sequence ID 20), M47A / I69W / A71E (Sequence ID 21), I69Y / A71E / V83L (Sequence ID 22), I69W / A71E / V83L (Sequence ID 23), I69Y / A71E (Sequence ID 24), I69W / A71E (Sequence ID 25), H18W / A26M / A71E / L85N (Sequence ID 26), H18W / A26M / A71E / L85D (Sequence ID 27), H18W / A26C / A71E / L85N (Sequence ID 28), H18W / A26C / A71E / L85D (Sequence ID 29), A26M / A71E / L85N (Sequence ID 30), A26M / A71E / L85D (Sequence ID 31), A26C / A71E / L85D (Sequence ID 32), A26C / A71E / L85N (Sequence ID 33), I69L / A71E / V83L (Sequence ID 34), H18W / A26V / E35N / A71E / L85N (Sequence ID 35), T13K / M42L / M47H / A71E (Sequence ID 36), T13K / M42L / M47Q / A71E (Sequence ID 37), T13K / M42L / A71E (Sequence ID 38), T13K / M42T / M47H / A71E (Sequence ID 39), T13K / M42T / M47T / A71E (Sequence ID 40), T13K / M42T / A71E (Sequence ID 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 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 Sugenmarimab 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 its antigen-binding fragment comprises a heavy chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently bound to the heavy chain, either via or without a linker peptide.

[0032] In some embodiments, the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently bonded to the N-terminus of the heavy chain, either via a linker peptide or without a linker peptide.

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

[0034] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain, and the CD80 IgV mutant polypeptide or CD80 ECD mutant 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 mutant polypeptide or CD80 ECD mutant polypeptide is covalently bonded to the N-terminus of the light chain, either via a linker peptide or without a linker peptide.

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

[0037] In some embodiments, the CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, the second polypeptide being present or absent.

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

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

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

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

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

[0043] In some embodiments, the second polypeptide is formed by covalently bonding, in order from the N-terminus to the C-terminus, an antibody or its antigen-binding fragment's light chain, a linker peptide, 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 (Sequence ID 112), E35T / A71T (Sequence ID 113), M47A / I69Y / A71E / V83L (Sequence ID 114), M47A / I69W / A71E / V83L (Sequence ID 115), M47A / I69Y / A71E (Sequence ID 116), M47A / I69W / A71E (Sequence ID 117), I69Y / A71E / V83L (Sequence ID 118), I69W / A71E / V83L (Sequence ID 119), I69Y / A71E (Sequence ID 120), I69W / A71E (Sequence ID 121), H18W / A26M / A71E / L85N (Sequence ID 122), H18W / A26M / A71E / L85D (Sequence ID 123), H18W / A26C / A71E / L85N (Sequence ID 124), H18W / A26C / A71E / L85D (Sequence ID 125), A26M / A71E / L85N (Sequence ID 126), A26M / A71E / L85D (Sequence ID 127), A26C / A71E / L85D (Sequence ID 128), A26C / A71E / L85N (Sequence ID 129), I69L / A71E / V83L (Sequence ID 130), H18W / A26V / E35N / A71E / L85N (Sequence ID 131), T13K / M42L / M47H / A71E (Sequence ID 132), T13K / M42L / M47Q / A71E (Sequence ID 133), T13K / M42L / A71E (Sequence ID 134), T13K / M42T / M47H / A71E (Sequence ID 135), T13K / M42T / M47T / A71E (Sequence ID 136), T13K / M42T / A71ESelected from the group consisting of (SEQ ID NO: 136), M47H / I69Y / A71E / V83L (SEQ ID NO: 137), M47Q / I69Y / A71E / V83L (SEQ ID NO: 138), or M47K / I69Y / A71E / V83L (SEQ ID NO: 139).

[0045] Alternatively, in some embodiments, the first polypeptide is 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 Selected from the group consisting of (Sequence ID 156) or E35T / A71T (Sequence ID 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 mutant fusion polypeptide complex also includes a third domain covalently bound to the CD80 IgV mutant polypeptide described in the first embodiment, the CD80 ECD mutant polypeptide described in the second embodiment, or the second domain described in the third embodiment, either via or without a linker peptide.

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

[0053] In some embodiments, if the third domain is a functional protein or an active fragment thereof, the functional protein or its active fragment 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 linked to a third domain, with or without the linker peptide.

[0055] In some embodiments, the heavy chain of the second domain is attached 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 attached 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, with or without a linker peptide.

[0058] In some embodiments, the light chain of the second domain is attached 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 attached 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 linked, with or without the linker peptide, to the CD80 IgV mutant polypeptide described in the first embodiment or the CD80 ECD mutant polypeptide described in the second embodiment.

[0061] In some embodiments, the CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, the second polypeptide being present or absent.

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

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

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

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

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

[0067] In some embodiments, the second polypeptide is formed by covalently bonding, from the C-terminus to the N-terminus, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain, wherein the linker peptides may be the same or different, and may be present independently or absent.

[0068] In some embodiments, the third domain includes 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 includes a sequence selected from the group consisting of SEQ ID NOs: 293 and 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 human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, mouse IgG1 Fc domain, mouse IgG2A Fc domain, mouse IgG2b Fc domain, or mouse IgG3 Fc domain.

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

[0076] In some embodiments, the amino acid sequences of the CD80 mutant fusion polypeptide complex are 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 The group is selected from (Sequence ID 171), E35T / A71Q (Sequence ID 172), and E35T / A71T (Sequence ID 173).

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

[0078] In some embodiments, if 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 mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, or the CD80 mutant fusion polypeptide complex described in the third aspect.

[0080] In a fifth aspect, the present invention provides a nucleotide molecule encoding a CD80 IgV mutant polypeptide as described in the first aspect, a CD80 ECD mutant polypeptide as described in the second aspect, or a CD80 mutant fusion polypeptide complex as described in the third aspect.

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

[0082] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, the CD80 mutant fusion polypeptide complex described in the third aspect, the nucleotide molecule described in the fifth aspect, and / or the vector described in the sixth aspect.

[0083] In the eighth aspect, the present invention provides a composition comprising a CD80 IgV mutant polypeptide as described in the first aspect, a CD80 ECD mutant polypeptide as described in the second aspect, a CD80 mutant fusion polypeptide complex as described in the third aspect, an immune complex as described in the fourth aspect, a nucleotide molecule as described in the fifth aspect, or a vector as described in the sixth aspect, and a pharmaceutically acceptable carrier.

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

[0085] In a tenth aspect, the present invention provides a method for regulating T cell activity, comprising contacting T cells with the CD80 IgV mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, or the CD80 mutant fusion polypeptide complex described in the third aspect, the composition described in the third aspect, and / or the composition described in the eighth aspect to stimulate their activation.

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

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

[0088] The following specific embodiments and examples illustrate 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 therefore many variations and modifications are possible. Those skilled in the art will recognize that such changes and modifications fall within the scope and spirit of the present invention. [Brief explanation of the drawing]

[0089] [Figure 1]Figures 1A-1O show the use of SDS-PAGE to detect the expression of the CD80 mutant fusion polypeptide complex. [Figure 2] Figures 2A–2D show the use of ELISA to detect the binding of CD80 mutant fusion polypeptide complexes to the human PDL1 protein. [Figure 3] Figures 3A-3O show the use of ELISA to detect the binding of CD80 mutant fusion polypeptide complexes to the human CTLA4 protein. [Figure 4] Figures 4A-4O show the use of ELISA to detect the binding of CD80 mutant fusion polypeptide complexes to the human CD28 protein. [Figure 5] Figures 5A–5L exemplify how the CD80 mutant fusion polypeptide complex deactivates the PD-1 / PD-L1-mediated T cell activation immunosuppression response. [Figure 6] Figures 6A–6D exemplify how the CD80 mutant fusion polypeptide complex modulates Jarcat T cell activation. [Figure 7] Figures 7A–7C exemplify how the CD80 mutant fusion polypeptide complex regulates PBMC activation. [Figure 8] Figures 8A-8C show that the CD80 mutant fusion polypeptide complex promotes chemokine secretion from antigen-presenting cells. [Modes for carrying out the invention]

[0090] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, the definitions in this specification shall prevail. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. While only specific exemplary materials and methods are described herein, many similar or equivalent methods and materials may be used in the practice of this disclosure.

[0091] Where used herein, the singular forms "one," "one," and "the foregoing" include the plural unless otherwise clearly indicated by the context. Furthermore, the free expression "contains" and "contains" are interpreted as meaning that they may also include structural components or method steps not described, but it should be noted that this free expression covers only components. The steps of a method consist of situations (i.e., they cover the situations covered by the closed expression "consisting of"). Generally, the term "approximately" is used in this specification to refer to a numerical variation of 5% above or below the stated value.

[0092] The term "modulation of T cell activity" refers to the modulating effect of the CD80 mutant polypeptides and CD80 mutant fusion polypeptide complexes disclosed in this invention on the activity of lymphoid T cells. This can be extended to include the effects of other relevant T cell modulators that affect the expression of NFAT transcription factors, the secretion of cytokines such as IL-2, IFN-γ, TNFα, and Granzyme B in T cells after T cell treatment, cell proliferation, and cytotoxic effects on target cells, including tumor cells. The term "T cell" can be extended to T cell lineages 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 but is not limited to CD8 T cells, including tumor tissue-infiltrating CD8 T cells, effector CD8 T cells, and / or immunological memory CD8 T cells.

[0093] The term "antitumor activity" means any biological activity that reduces or prevents the proliferation or survival rate of tumor cells in vivo and / or in vitro. In certain embodiments, antitumor activity is the antitumor effect possessed by the CD80 variant polypeptides and their fusion polypeptide complexes of the present invention.

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

[0095] The term "CD80 extracellular domain" refers to the amino acid sequence of the extracellular domain of the CD80 protein, and refers to a polypeptide or fragment thereof that has at least approximately 85% amino acid identity and CD28 and / or CTLA4 binding activity. An example "CD80 extracellular domain amino acid sequence" (SEQ ID NO: 1) is provided below. The term "CD80 IgV" refers to the amino acid sequence of the IgV domain in the extracellular domain of the CD80 protein, and refers to a polypeptide or fragment thereof that has at least approximately 85% amino acid identity and CD28 and / or CTLA4 binding activity. An example "CD80 IgV" (SEQ ID NO: 45) is provided below.

[0096] The term "amino acid identity" refers to the process of comparing and aligning amino acid sequences (with gaps introduced as needed) to obtain the greatest possible match. The identity percentage can be determined using sequence comparison software, algorithms, or visual inspection. In the case of comparing the identity of two or more amino acids of a second domain or mixed tandem amino acid identity, the second domain must be as follows: The identity percentage between a given subunit and any subunit of the second domain is calculated by comparing them individually. Various algorithms and software are known in this field that can be used to obtain amino acid sequence alignment, including, but not limited to, NCBI BLAST software.

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

[0098] In this 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. Fc domains may include antibody heavy chain CH2, CH3 and hinge region fragments. Conventional Fc fragments have the function of binding to Fc fragment receptors and mediating associated biological effects; however, site-directed mutations can alter their ability to bind to corresponding target receptors, potentially affecting their biological function. The immunoglobulin Fc domains disclosed in this application include, but are not limited to, conventional Fc fragments and any other forms of Fc variants. Provided below are exemplary human immunoglobulin IgG1 Fc domains (SEQ ID NO: 90) and human immunoglobulin IgG4 Fc domains (SEQ ID NO: 91):

[0099] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90); ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(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, where the ratio of Gly amino acids to Ser amino acids is guaranteed to be 1 or greater. The linker peptides disclosed in this invention can be extended to any short peptide having this property. Below are, but are not limited to, the amino acid sequences of exemplary linker peptides GGGGS (SEQ ID NO: 92), GGGGSGGGGS (SEQ ID NO: 93), GGGGSGGGGSGGGGGS (SEQ ID NO: 94), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 95), and GGGGSGGGS (SEQ ID NO: 96).

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

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

[0103] The term "LAG3 active fragment" refers to an active fragment derived from the LAG3 protein that possesses the functional properties of the LAG3 protein. These functional properties include, but are not limited to, binding to MHCII (or HLA-DR) and / or FGL1, and promoting 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 costimulatory receptors on antigen-presenting cells, enhanced antigen phagocytosis, increased cytokine secretion, and enhanced chemokine secretion.

[0105] The aforementioned costimulatory receptors include, but are not limited to, ICOSL, CD40L, CD137L, OX40L, CD80, CD83, and CD86.

[0106] Antigen phagocytosis includes, but is not limited to, the phagocytic action of bacteria, viruses, proteins, and polysaccharides.

[0107] The aforementioned cytokines include, but are not limited to, IL-1beta, TNFa, IL-6, and IL-12.

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

[0109] The inventors of this application unexpectedly discovered that CD80 mutant polypeptides formed by innovatively modifying the CD80 IgV domain can modulate the binding activity of CD80 and its ligand, and as a result, these CD80 mutant polypeptides can effectively stimulate T cells and enhance the immune response. Consequently, 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 mutant polypeptides, including CD80 IgV mutant polypeptides and CD80 ECD mutant polypeptides, as well as methods for modulating T cell immune responses and applications in disease treatment.

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

[0112] In some embodiments, the mutation site of the CD80 IgV mutant polypeptide includes one or more amino acid mutations on SEQ ID NO: 45, such as T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D.

[0113] In some preferred embodiments, the mutation site of the CD80 IgV mutant polypeptide comprises a combination of mutations in two or more amino acid sites on SEQ ID NO: 45, and the CD80 IgV mutant 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 (Sequence ID 58), E35T / A71N (Sequence ID 59), E35T / A71Q (Sequence ID 60), E35T / A71T (Sequence ID 61), M47A / I69Y / A71E / V83L (Sequence ID 62), M47A / I69W / A71E / V83L (Sequence ID 63), M47A / I69Y / A71E (Sequence ID 64), M47A / I69W / A71E (Sequence ID 65), I69Y / A71E / V83L (Sequence ID 66), I69W / A71E / V83L (Sequence ID 67), I69Y / A71E (Sequence ID 68), I69W / A71E (Sequence ID 69), H18W / A26M / A71E / L85N (Sequence ID 70), H18W / A26M / A71E / L85D (Sequence ID 71), H18W / A26C / A71E / L85N (Sequence ID 72), H18W / A26C / A71E / L85D (Sequence ID 73), A26M / A71E / L85N (Sequence ID 74), A26M / A71E / L85D (Sequence ID 75), A26C / A71E / L85D (Sequence ID 76), A26C / A71E / L85N (Sequence ID 77), I69L / A71E / V83L (Sequence ID 78), H18W / A26V / E35N / A71E / L85N (Sequence ID 79), T13K / M42L / M47H / A71E (Sequence ID 80), T13K / M42L / M47Q / A71E (Sequence ID 81), T13K / M42L / A71E (Sequence ID 82), T13K / M42T / M47H / A71E (Sequence ID 83), T13K / M42T / M47T / A71E(Sequence ID 84), T13K / M42T / A71E (Sequence ID 85), M47H / I69Y / A71E / V83L (Sequence ID 86), M47Q / I69Y / A71E / V83L (Sequence ID 87), M47K / I69Y / A71E / V83L (Sequence ID 88).

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

[0115] In some embodiments, the mutation site of the CD80 ECD mutant polypeptide includes one or more amino acid position mutations on SEQ ID NO: 1, such as T13K, A26M, A26C, E35N, E35S, E35Q, E35T, M42L, M47Q, M47H, M47K, M47T, I69Y, I69L, I69W, A71E, A71N, A71Q, A71E, V83L, L85N, and L85D.

[0116] In some preferred embodiments, the mutation site of the CD80 ECD mutant polypeptide is a combination of mutations at two or more amino acid positions on SEQ ID NO: 1, and the CD80 ECD mutant 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 (Sequence ID 15), E35T / A71Q (Sequence ID 16), E35T / A71T (Sequence ID 17), M47A / I69Y / A71E / V83L (Sequence ID 18), M47A / I69W / A71E / V83L (Sequence ID 19), M47A / I69Y / A71E (Sequence ID 20), M47A / I69W / A71E (Sequence ID 21), I69Y / A71E / V83L (Sequence ID 22), I69W / A71E / V83L (Sequence ID 23), I69Y / A71E (Sequence ID 24), I69W / A71E (Sequence ID 25), H18W / A26M / A71E / L85N (Sequence ID 26), H18W / A26M / A71E / L85D (Sequence ID 27), H18W / A26C / A71E / L85N (Sequence ID 28), H18W / A26C / A71E / L85D (Sequence ID 29), A26M / A71E / L85N (Sequence ID 30), A26M / A71E / L85D (Sequence ID 31), A26C / A71E / L85D (Sequence ID 32), A26C / A71E / L85N (Sequence ID 33), I69L / A71E / V83L (Sequence ID 34), H18W / A26V / E35N / A71E / L85N (Sequence ID 35), T13K / M42L / M47H / A71E (Sequence ID 36), T13K / M42L / M47Q / A71E (Sequence ID 37), T13K / M42L / A71E (Sequence ID 38), T13K / M42T / M47H / A71E (Sequence ID 39), T13K / M42T / M47T / A71E(Sequence ID 40), T13K / M42T / A71E (Sequence ID 41), M47H / I69Y / A71E / V83L (Sequence ID 42), M47Q / I69Y / A71E / V83L (Sequence ID 43), M47K / I69Y / A71E / V83L (Sequence ID 44) are possible.

[0117] In a third aspect, the present invention provides a fusion polypeptide complex of CD80 mutant polypeptides, comprising the CD80 ECD mutant polypeptide or the CD80 IgV mutant polypeptide, covalently bound to a second domain, with or without linker peptide, wherein the second domain may be an immunoglobulin Fc domain, an antibody or its antigen-binding fragment, or other forms of polypeptide.

[0118] In some embodiments, the CD80 mutant polypeptide fusion polypeptide complex comprises the CD80 ECD mutant polypeptide or the CD80 IgV mutant polypeptide, 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 mutant polypeptide covalently binds to the N-terminus of the immunoglobulin Fc domain, either via or without a linker peptide, to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0120] In some embodiments, the C-terminus of the immunoglobulin Fc domain covalently binds to the CD80 mutant polypeptide, either via or without a linker peptide, to form a fusion polypeptide complex of the CD80 mutant polypeptide.

[0121] In some embodiments, the CD80 mutant polypeptide fusion polypeptide complex comprises the aforementioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide covalently bound to an antibody or its antigen-binding fragment, with or without the linker peptide.

[0122] In some embodiments, the antibody may 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 may 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 mutant fusion polypeptide complex comprises the above-mentioned CD80 ECD mutant polypeptide or CD80 IgV mutant polypeptide covalently bound to an immunoglobulin IgG antibody, with or without the linker peptide, wherein the immunoglobulin IgG includes, but is not limited to, human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3.

[0124] In some preferred embodiments, the specific target of the antibody or antigen-binding fragment in the fusion polypeptide complex of the above-mentioned 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 a Sugemalimab antibody or its antigen-binding fragment.

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

[0127] In preferred embodiments, the anti-CD3 antibody or antigen-binding fragment is a CD3B219 antibody or its antigen-binding fragment.

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

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

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

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

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

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

[0134] In a preferred embodiment, the C-terminus of the CD80 mutant polypeptide is covalently bonded 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 mutant polypeptide.

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

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

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

[0138] In another embodiment, the CD80 mutant fusion polypeptide complex provided by the present invention includes a first polypeptide and a second polypeptide constituting the fusion polypeptide complex, although it is clear that some specific antibodies may lack a second polypeptide.

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

[0140] In a preferred embodiment, the first polypeptide of the fusion polypeptide complex of the present invention is constructed by covalently bonding a CD80 variant polypeptide, a linker peptide, and the heavy chain of an antibody or its antigen-binding fragment in sequence from the N-terminus to the C-terminus of the polypeptide.

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

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

[0143] In a preferred embodiment, the second polypeptide of the polypeptide complex of the present invention is constructed by covalently bonding a CD80 variant polypeptide, a linker peptide, and the light chain of an antibody or its antigen-binding fragment in order from the N-terminus to the C-terminus of the polypeptide.

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

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

[0146] In some embodiments, the third domain of the present invention is the same as or different from the second domain, an antibody or antigen-binding fragment, or the third domain is an immunoactivated functional protein 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 linked to the heavy chain of the second domain, with or without a linker peptide.

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

[0153] In some embodiments, the CD80 mutant fusion polypeptide complex of the present invention, including a third domain, comprises a first polypeptide and a second polypeptide, the second polypeptide being present or absent.

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

[0155] In one embodiment, the first polypeptide is composed of a CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain, which are covalently bonded in order from the N-terminus to the C-terminus, and the linker peptides may be the same or different, and may be present independently or not.

[0156] In one embodiment, the first polypeptide is composed of a CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain, which are covalently bonded in order from the C-terminus to the N-terminus, and the linker peptides may be the same or different, and may be present independently or not.

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

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

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

[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, the linker peptide is absent.

[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 mutant polypeptide can be combined with other forms of polypeptides to form polypeptide complexes by the method described above.

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

[0165] In a fifth aspect, the present invention provides a nucleotide molecule encoding a CD80 IgV mutant polypeptide as described in the first aspect, a CD80 ECD mutant polypeptide as described in the second aspect, or a CD80 mutant fusion polypeptide complex as described in the third aspect.

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

[0167] In a seventh aspect, the present invention provides a host cell comprising the CD80 IgV mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, the mutant fusion polypeptide complex described in the third aspect, the nucleotide molecule described in the fifth aspect, and / or the vector described in the sixth aspect.

[0168] In the eighth aspect, the present invention provides a composition comprising a CD80 IgV mutant polypeptide as described in the first aspect, a CD80 ECD mutant polypeptide as described in the second aspect, a CD80 mutant fusion polypeptide complex as described in the third aspect, an immune complex as described in the fourth aspect, a nucleotide molecule as described in the fifth aspect, or a vector as 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 mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, or the CD80 mutant fusion polypeptide complex described in the third aspect, comprising culturing the host cells described in the seventh aspect to express the CD80 IgV mutant polypeptide, the CD80 ECD mutant polypeptide, and / or the CD80 mutant fusion polypeptide complex.

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

[0171] In some embodiments, the present invention provides a method for producing polypeptides and polypeptide complexes, 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, the stable dimer being maintained by a stable dimerization including a native linkage and / or at least one non-native interchain linkage. The first peptide and the second peptide form a stable polymer in the host cell, forming a stable complex.

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

[0173] In a tenth aspect, the present invention provides a method for modulating T cell activity, comprising contacting T cells with the CD80 IgV mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, or the CD80 mutant fusion polypeptide complex described in the third aspect and / or the composition described in the eighth aspect to stimulate their 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 T cell activation.

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

[0175] In a twelfth aspect, the present invention provides the use of the CD80 IgV mutant polypeptide described in the first aspect, the CD80 ECD mutant polypeptide described in the second aspect, the CD80 mutant fusion polypeptide complex described in the third aspect, the immune complex described in the fourth aspect, the nucleotide molecule described in the fifth aspect, the vector described in the sixth aspect, the host cell described in the seventh aspect, and / or the composition described in the eighth aspect in the preparation of agents for preventing, improving and / or treating tumors or cancer.

[0176] The following are exemplary examples of the amino acid sequences of the first polypeptides of functional fusion polypeptide complexes formed by the covalent bonding of CD80 IgV mutants to the N-terminus of the sugemalimab antibody heavy chain via linker peptides: 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 (Sequence ID 121), H18W / A26M / A71E / L85N (Sequence ID 122), H18W / A26M / A71E / L85D (Sequence ID 123), H18W / A26C / A71E / L85N (Sequence ID 124), H18W / A26C / A71E / L85D (Sequence ID 125), A26M / A71E / L85N (Sequence ID 126), A26M / A71E / L85D (Sequence ID 127), A26C / A71E / L85D (Sequence ID 128), A26C / A71E / L85N (Sequence ID 129), I69L / A71E / V83L (Sequence ID 130), H18W / A26V / E35N / A71E / L85N (Sequence ID 131), T13K / M42L / M47H / A71E (Sequence ID 132), T13K / M42L / M47Q / A71E (Sequence ID 133), T13K / M42L / A71EThe amino acid sequences of the first polypeptide of a functional fusion polypeptide complex formed by the covalent bonding of (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 unamutant CD80 IgV to the N-terminus of the heavy chain of the sugemalimab antibody via a linker peptide are shown (SEQ ID NO: 140). Each of the above first polypeptides is assembled with the second polypeptide of the sugemalimab antibody light chain (SEQ ID NO: 141) to form a functional fusion polypeptide complex.

[0177] The following are exemplary examples of functional fusion polypeptide complexes formed by covalent bonding of the CD80 ECD mutant to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide. The first polypeptide amino acid sequences are: 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), and E35Q / A71T. These are (SEQ ID NO: 153), E35T / A71E (SEQ ID NO: 154), E35T / A71N (SEQ ID NO: 155), E35T / A71Q (SEQ ID NO: 156), and E35T / A71T (SEQ ID NO: 157). Each of the above first polypeptides is assembled with the second polypeptide of the sugemalimab antibody light chain (SEQ ID NO: 141) to form a functional fusion polypeptide complex.

[0178] The following are exemplary examples of functional fusion polypeptide amino acid sequences formed by the covalent bonding of CD80 ECD variants to the N-terminus of the Fc domain of human immunoglobulin IgG4: 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 These are (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 (sequence number 174) of a functional fusion polypeptide formed by covalent bonding of a non-mutant CD80 ECD with the N-terminus of the Fc domain of human immunoglobulin IgG4.

[0179] The following are exemplary examples of functional fusion polypeptide amino acid sequences formed by covalent bonding of CD80 IgV mutants to the N-terminus of the Fc domain of human immunoglobulin IgG4: 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 These include (SEQ ID NO: 187), E35T / A71N (SEQ ID NO: 188), E35T / A71Q (SEQ ID NO: 189), E35T / A71T (SEQ ID NO: 190), and the amino acid sequence of a functional fusion polypeptide formed by covalent bonding of non-mutant CD80 IgV and the N-terminus of the Fc domain of human immunoglobulin IgG4 (SEQ ID NO: 191).

[0180] The following are illustrative examples of functional fusion polypeptide complexes formed by the covalent bonding of CD80 ECD mutants to the N-terminus of the anti-LAG3 antibody Relatlimab heavy chain via a linker peptide: the amino acid sequences of the first polypeptide 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 the non-mutant CD80 ECD (SEQ ID NO: 196). The first polypeptide is assembled with the second polypeptide (SEQ ID NO: 197) of the Relatlimab antibody light chain to form a functional fusion polypeptide complex.

[0181] The following are illustrative examples of functional fusion polypeptide complexes formed by the covalent bonding of CD80 IgV mutants to the N-terminus of the anti-LAG3 antibody Relatlimab heavy chain via a linker peptide: the first polypeptide amino acid sequences 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-mutant CD80 IgV (SEQ ID NO: 202). Each of these 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] The following are illustrative examples of functional fusion polypeptide complexes formed by the covalent bonding of CD80 ECD mutants to the N-terminus of the CD3B219 heavy chain via a linker peptide: the amino acid sequences of the first polypeptides are 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-mutant CD80 ECD (SEQ ID NO: 207). Each of these first polypeptides is assembled with the second polypeptide (SEQ ID NO: 208) of the CD3B219 antibody light chain to form the functional fusion polypeptide complex.

[0183] The following are illustrative examples of functional fusion polypeptide complexes formed by the covalent bonding of CD80 IgV mutants to the N-terminus of the CD3B219 heavy chain via a linker peptide: the amino acid sequences of the first polypeptides are 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-mutant CD80 IgV (SEQ ID NO: 213). Each of these first polypeptides is assembled 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) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant E35N / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-353 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 214) vector expressing the Abs-353 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-353 second polypeptide into host cells for expression.

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

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

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

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

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

[0190] In some embodiments, the functional antibody Abs-359 consists of a chimeric antibody Abs-1 polypeptide (SEQ ID NO: 103) and a sugemalimab light chain 2 polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant E35S / A71Q to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-359 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 221) vector expressing the Abs-359 1 polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-359 2 polypeptide into host cells for expression.

[0191] In some embodiments, the functional antibody Abs-360 consists of a chimeric antibody Abs-1 polypeptide (SEQ ID NO: 104) and a sugemalimab light chain 2 polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant E35S / A71T to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-360 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 222) vector expressing the Abs-360 1 polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-360 2 polypeptide into host cells for expression.

[0192] In some embodiments, the functional antibody Abs-361 consists of a chimeric antibody Abs-361 first polypeptide (SEQ ID NO: 105) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant E35Q / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-361 functional antibody is constructed by introducing 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-361 second polypeptide into host cells for expression.

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

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

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

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

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

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

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

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

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

[0202] In some embodiments, the functional antibody Abs-249 consists of a chimeric antibody Abs-249 first polypeptide (SEQ ID NO: 115) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant M47A / I69Y / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-249 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 233) vector expressing the Abs-249 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing 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) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant M47A / I69W / A71E to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-250 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 234) vector expressing the Abs-250 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing 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) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of the CD80 IgV mutant I69Y / A71E / V83L to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-251 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 235) vector expressing the Abs-251 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-251 second polypeptide into host cells for expression.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0219] In some embodiments, the functional antibody Abs-266 consists of a chimeric antibody Abs-266 first polypeptide (SEQ ID NO: 132) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by covalently binding the CD80 IgV mutant T13K / M42L / M47Q / A71E linker peptide (SEQ ID NO: 95) to the N-terminus of the sugemalimab antibody heavy chain. The Abs-266 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-268 first polypeptide into host cells for expression.

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

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

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

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

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

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

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

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

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

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

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

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

[0232] In some embodiments, the functional antibody Abs-98 consists of a chimeric antibody Abs-98 first polypeptide (SEQ ID NO: 263) and a sugemalimab light chain second polypeptide (SEQ ID NO: 141), formed by the covalent bonding of wild-type CD80 ECD to the N-terminus of the sugemalimab antibody heavy chain via a linker peptide (SEQ ID NO: 95). The Abs-98 functional antibody is constructed by introducing 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 into host cells 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 binding the CD80 ECD variant E35N / A71E to the N-terminus of IgG4 Fc. The Abs-480 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 265) vector expressing the Abs-480 polypeptide into host cells 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 binding the CD80 ECD mutant E35N / A71N to the N-terminus of IgG4 Fc. The Abs-481 functional polypeptide complex is constructed by introducing a polynucleotide sequence (SEQ ID NO: 266) vector expressing the Abs-481 polypeptide into host cells for expression.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0253] In some embodiments, the functional antibody Abs-16 consists of a chimeric antibody Abs-16 first polypeptide (SEQ ID NO: 287) formed by the N-terminus of the sugemalimab antibody heavy chain and a sugemalimab light chain second polypeptide (SEQ ID NO: 263). The Abs-16 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 288) vector expressing the Abs-16 first polypeptide and a polynucleotide sequence (SEQ ID NO: 215) vector expressing the Abs-16 second polypeptide into host cells 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 by the N-terminus of the Relatlimab antibody heavy chain and a second polypeptide (SEQ ID NO: 197) formed by the Relatlimab light chain. The Abs-58 functional antibody is constructed by introducing a polynucleotide sequence (SEQ ID NO: 290) vector expressing the Abs-58 first polypeptide and a polynucleotide sequence (SEQ ID NO: 276) vector expressing the Abs-58 second polypeptide into host cells for expression.

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

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

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

[0258] While various embodiments of the present invention have been described above, it should be understood that these are provided only as examples 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 invention. Therefore, the breadth and scope of the invention should not be limited by any of the embodiments described above.

[0259] All documents referenced in this disclosure are incorporated herein by reference. All publications and patent documents cited in this application are incorporated herein by reference for any purpose as if each individual publication or patent document were shown separately.

[0260] Examples Example 1. Antibody expression and purification To further identify the antibodies obtained by screening, it is necessary to express them in mammalian cells. Therefore, we first constructed expression plasmid vectors containing the heavy chain and light chain of multifunctional / bifunctional antibodies. Approximately 24 hours before plasmid transfection, Expi293 cells were transfected to approximately 2.4 10 6 The cells were passaged until a cell density of 6 × 10⁻¹⁶ cells / ml was reached. 6 If the viability exceeded 95% at a cell / ml level, 25 μg of the plasmid vector for discovering the mixed heavy and light chains was collected and 25 ml of Expi293 cells were transfected with Expifectamine 293. After culturing for 7 days at 37°C, 130 rpm, and 8% CO2 with shaking, the cell culture product was centrifuged, the supernatant was collected and filtered through a 0.45 MCE filter, and the target antibody was purified and recovered using a Capturem protein A Maxiprep column, then concentrated by centrifugation using a Vivaspin 20 Centrifugal Concentrator 50K. The antibody purity was measured by A280 measurement using NanoDrop 2000 and 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 (NR) and non-reducing (N) conditions, 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] Electrophoretic analysis revealed 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, and Abs-99 are antibody aggregates. 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 demonstrates 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 at 4 °C in a flat-bottomed 96-well Immuno removable transparent enzyme plate (MaxiSorp) (Thermo Scientific, 446469). 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 procedure was performed using a Soluble TMB Kit (CoWin Biosciences, Inc., CW0050S) according to the manufacturer's instructions. The affinity between candidate multifunctional / bifunctional fusion proteins and human PD-L1 protein is shown by binding strengths at EC50 and 20 nM, and the test results are shown in Table 1 and Figure 2.

[0265] Table 1. Binding strength of CD80 mutant fusion polypeptide complex to human PDL1 protein at EC50 and 100 nM. JPEG0007850830000001.jpg81170

[0266] The results shown in Table 1 and Figure 2 demonstrate that, in binding experiments between CD80 mutant fusion polypeptide complexes and the human PDL1 antigen, the binding activity of CD80 mutant fusion polypeptide complexes Abs-427, Abs-428, and Abs-483 to the PDL1 antigen was significantly higher than that 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-bottomed 96-well Immuno removable transparent microplate (MaxiSorp) (Thermo Scientific, 446469). The affinity of the test substance to human CD28 protein was detected using the same method as described above, and the detection results, expressed as binding strengths of EC50 and 100 nM, are shown in Table 2 and Figure 4.

[0268] Table 2. Binding strength of CD80 mutant fusion polypeptide complex to human CD28 protein at EC50 and 100 nM. JPEG0007850830000002.jpg25586

[0269] NA: Indicates that it does not bind to the target protein.

[0270] The results shown in Table 2 and Figure 4 demonstrate that, in binding experiments between CD80 mutant fusion polypeptide complexes and the human CD28 antigen, the CD28 antigen-binding activity of CD80 mutant fusion polypeptide complexes Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-359, Abs-360, Abs-362, Abs-364, Abs-365, Abs-367, and Abs-368 is far higher than that 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-bottomed 96-well Immuno removable clear enzyme plate (MaxiSorp) (Thermo Scientific, 446469). The affinity of the test substance to human CTLA4 protein was detected using the same method as above, measured by binding strength at EC50 and 20 nM, and the results are shown in Table 3 and Figure 3.

[0272] Table 3. Binding strengths of CD80 mutant fusion polypeptide complex and human CTLA4 protein-binding EC50 and 20 nM. JPEG0007850830000003.jpg25594

[0273] NA: Indicates that it does not bind to the target protein.

[0274] The results shown in Table 3 and Figure 3 demonstrate that, in binding experiments between CD80 mutant fusion polypeptide complexes and the human CTLA4 antigen, the binding activity of CD80 mutant fusion polypeptide complexes Abs-267, Abs-353, Abs-354, Abs-355, Abs-356, Abs-357, Abs-362, and Abs-364 to the CTLA4 antigen was significantly higher than that of the wild-type CD80 fusion polypeptide complex Abs-99.

[0275] Example 4. Measurement of CD80 mutant fusion polypeptide complex that reverses PD-1 / PD-L1-mediated T cell immunosuppression response.

[0276] This example demonstrates how the CD80 mutant fusion polypeptide complex deactivates the PD-1 / PD-L1-mediated T cell inhibitory response.

[0277] Specifically, on day 1, TCR activator PD-L1-CHO cells (BPS bioscience) were digested using 0.05% trypsin, collected, and then plated. Fresh medium was added, the cells were mixed uniformly, and the cell density was set to 3.5*10. 5The cells were adjusted to the desired cell / ml concentration. 100 μL / well of cells were added to a sterile 96-well flat-bottom plate (CORNING, 3599) and cultured overnight in a 37°C incubator until the cell density reached 80%. The following day, the TCR activator PD-L1-CHO cell medium was removed from the 96-well flat-bottom plate. 50 μL of fresh culture medium containing the corresponding concentrations of test material (serial dilutions) was added to each well and treated for 30 minutes. Jurkat-Lucia® TCR-hPD-1 cells (Invivogen) were then harvested and counted. Fresh medium was added, the cells were homogenized, and the cell density was adjusted to 4*10. 5 The solution was adjusted to cells / ml. 50 μl of Jurkat-Lucia™ TCR-hPD-1 cells were added, and the final density was 2*10. 4 Cells were prepared to reach a concentration of / well and incubated in a 96-well flat-bottom plate at 37°C for 5–6 hours. After incubation, cell morphology was observed under a microscope, and chemiluminescence values ​​were detected using QUANTI-Luc® (Invivogen; rep-qlc1). Specific procedures were performed according to the manufacturer's instructions. Activity is indicated by EC50 and maximum induction factor, and the detection results are shown in Tables 4, 5, Figures 5 and 6. Table 4. Measurement results of CD80 mutant fusion polypeptide complexes that regulate Jurkat T cell activation.

[0278] JPEG0007850830000004.jpg255131

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

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

[0281] Table 5. Measurement results for controlling the activation of Jurkat T cells by CD80 mutant fusion polypeptide complexes JPEG0007850830000005.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 a sterile 96-well flat-bottom plate (CORNING, 3599) at 100 μL / well, resulting in a total of 35,000 cells / well. 50 μl of fresh medium containing the corresponding concentration of the test substance (serial dilution) was added to each well, and 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™ (BPS bioscience; 78262). The specific operation was 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 JPEG0007850830000006.jpg61170

[0288] In Table 6, in the experiment where the CD80 mutant fusion polypeptide complex releases the LAG3-mediated T cell immunosuppressive response, it is shown 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™ 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 corresponding concentration of the test substance (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™ (Invivogen; rep-qlc1). The specific operation was carried out according to the manufacturer's instructions.

[0292] Table 7. Measurement results of T cell activation immune response by CD80 mutation fusion polypeptide complex. JPEG0007850830000007.jpg60170

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

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

[0295] This example demonstrates the activation of T cell immunity in primary PBMCs by a CD80 mutant fusion polypeptide complex. Specifically, PBMCs were collected, counted, and plated. Fresh medium was added, the cells were homogenized, and the cell density was increased to 1 × 10⁶. 5 The cells / ml were adjusted. 50 μl of fresh culture medium containing the corresponding concentration of test material (serial dilution) was added to each well, and after incubation for 3 days, the culture supernatant was collected, and the secretion of IL-2 in the supernatant was detected using the IL-2 ELISA detection kit (R&D, DY202), with specific procedures followed according to the manufacturer's instructions.

[0296] Table 8. Measurement results showing that the CD80 mutant fusion polypeptide complex activates the immune response of primary PBMC T cells. JPEG0007850830000008.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 PBMCs than the wild-type CD80 fusion polypeptide complex Abs-99.

[0298] Example 8. Measurement of chemokine secretion by antigen-presenting cells. This example demonstrates that Abs induces the secretion of the chemokine CCL4 from antigen-presenting cells.

[0299] Specifically, antigen-presenting cells (THP-1 cells, or ATCCs) were collected, counted, and then plated. Fresh culture medium was added, the cells were homogenized, and the cell density was increased to 1 × 10⁻⁶. 6 The culture medium was adjusted to cells / ml, and 100 μl of cells were plated into each well. 100 μl of fresh medium containing the corresponding concentration of test substance Abs_520 or Abs_521 was added to each well. After 6 hours, the cell culture supernatant was collected, and the CCL4 expression level was detected using the CCL4 Detection Kit (PROTEINTECH). Specific procedures were performed according to the manufacturer's instructions. The results, as shown in Figure 8, demonstrate that Abs_520 and Abs_521 can induce chemokine secretion from antigen-presenting cells.

[0300] The results of the above examples demonstrate that CD80 mutant polypeptides formed by innovatively modifying the CD80 IgV domain can modulate the binding activity of CD80 to its ligands. In particular, the CD80 mutant fusion polypeptide complexes disclosed herein exhibit significantly higher antigen-binding activity to PDL1, CTLA4, and CD28 than the corresponding wild-type CD80 fusion polypeptide complexes, and the CD80 mutant fusion polypeptide complexes disclosed herein can deactivate the PD-1 / PD-L1-mediated T cell activation immunosuppressive response; modulate PBMC activation and promote chemokine secretion from antigen-presenting cells. In other words, the CD80 mutant fusion polypeptide complexes 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] While various embodiments of the present invention have been described above, it should be understood that these are provided only as examples and are not limiting. Without departing from the spirit and scope of the invention, various modifications and improvements are possible, and these modifications and improvements fall within the scope of the invention as defined in the claims. The scope of the invention is defined by the appended claims and their equivalents.

Claims

1. The wild-type human CD80 IgV polypeptide sequence SEQ ID NO: 45 has only two amino acid mutation combinations selected from the group consisting of E35N / A71E, E35N / A71N, E35N / A71Q, and E35N / A71T. CD80 IgV mutant polypeptide.

2. The wild-type human CD80 ECD polypeptide sequence SEQ ID NO: 1 has only two amino acid mutation combinations selected from the group consisting of E35N / A71E, E35N / A71N, E35N / A71Q, and E35N / A71T. CD80 ECD mutant polypeptide.

3. A CD80 IgV mutant polypeptide according to claim 1 or a CD80 ECD mutant polypeptide according to claim 2, comprising a second domain, wherein the CD80 IgV mutant polypeptide or the CD80 ECD mutant polypeptide is covalently bound to the second domain, either via or without a linker peptide, the second domain being an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment being 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), specifically targeting one or more antigens selected from MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and / or Tim3, CD80 mutant fusion polypeptide complex.

4. The CD80 variant fusion polypeptide complex according to claim 3, wherein the antibody or its antigen-binding fragment specifically targets PD-L1, PD-1, TIGIT, CTLA4, LAG3, or CD3.

5. The antibody or antigen-binding fragment targeting PD-L1 is Sugemalimab antibody or its antigen-binding fragment; the antibody or antigen-binding fragment targeting LAG-3 is Relatlimab antibody or its antigen-binding fragment; and / or the antibody or antigen-binding fragment targeting CD3 is CD3B219 antibody or its antigen-binding fragment. The CD80 mutant fusion polypeptide complex according to claim 4.

6. The antibody or its antigen-binding fragment comprises a heavy chain, the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently bound to the heavy chain, either via or without a linker peptide, and optionally, the C-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently bound to the N-terminus of the heavy chain, either via 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 mutant polypeptide or CD80 ECD mutant polypeptide, either via or without a linker peptide. The CD80 mutant fusion polypeptide complex according to claim 3.

7. The antibody or its antigen-binding fragment comprises a light chain, the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide is covalently bound to the light chain, either via or without a linker peptide, and optionally covalently bound to the N-terminus of the light chain, either via or without a C-terminal linker peptide of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide; and / or the C-terminus of the light chain is covalently bound to the N-terminus of the CD80 IgV mutant polypeptide or CD80 ECD mutant polypeptide, either via or without a linker peptide. The CD80 mutant fusion polypeptide complex according to claim 3.

8. The CD80 mutant 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 the heavy chain of an antibody or its antigen-binding fragment, and / or the second polypeptide comprises the light chain of an antibody or its antigen-binding fragment. The CD80 mutant fusion polypeptide complex according to claim 3.

9. The first polypeptide is formed by sequentially covalently bonding a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, and the heavy chain of an antibody or its antigen-binding fragment from the N-terminus to the C-terminus of the polypeptide, or the first polypeptide is formed by sequentially covalently bonding a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide from the N-terminus to the C-terminus of the polypeptide. The CD80 mutant fusion polypeptide complex according to claim 8.

10. The second polypeptide is formed by sequentially covalently bonding a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, and the light chain of an antibody or its antigen-binding fragment from the N-terminus to the C-terminus of the polypeptide; or the second polypeptide is formed by sequentially covalently bonding a light chain of an antibody or its antigen-binding fragment, a linker peptide, and a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide from the N-terminus to the C-terminus of the polypeptide. The CD80 mutant fusion polypeptide complex according to claim 9.

11. The first polypeptide is selected from E35N / A71E (SEQ ID NO: 97), E35N / A71N (SEQ ID NO: 98), E35N / A71Q (SEQ ID NO: 99), E35N / A71T (SEQ ID NO: 100), 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), 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), and / or The second polypeptide is selected from the group consisting of SEQ ID NO: 141, SEQ ID NO: 197, and SEQ ID NO:

208. The CD80 mutant fusion polypeptide complex according to claim 9.

12. The CD80 mutant fusion polypeptide complex also includes a third domain covalently bonded to the second domain, either via or without a linker peptide. The CD80 mutant fusion polypeptide complex according to claim 3.

13. The third domain is either the same as or different from the second domain, an antibody or antigen-binding fragment, or the third domain is a functional protein or an active fragment thereof, and optionally the third domain is a functional protein or an active fragment thereof, the functional protein or its active fragment can activate an immune response, and optionally the third domain is linked, with or without linker peptide, to the CD80 IgV mutant polypeptide described in claim 1 or the CD80 ECD mutant polypeptide described in claim 2. The CD80 mutant fusion polypeptide complex according to claim 12.

14. The second domain comprises an antibody heavy chain, the antibody heavy chain of the second domain is linked to the third domain with or without a linker peptide, optionally, the heavy chain of the second domain is linked to the N-terminus of the third domain with or without a linker peptide, and / or the heavy chain of the second domain is linked to the C-terminus of the third domain with or without a linker peptide; and / or The second domain comprises an antibody light chain, the antibody light chain of the second domain is linked to the third domain via or without a linker peptide, optionally, the light chain of the second domain is linked to the N-terminus of the third domain via or without a linker peptide, and / or the light chain of the second domain is linked to the C-terminus of the third domain via or without a linker peptide. The CD80 mutant fusion polypeptide complex according to claim 13.

15. The CD80 mutant fusion polypeptide complex comprises a first polypeptide and a second polypeptide, the second polypeptide may or may not be present, optionally. The first polypeptide comprises a heavy chain of a second domain, optionally, The first polypeptide is formed by covalently bonding, from the N-terminus to the C-terminus, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain in that order, wherein the linker peptides may be the same or different, and may be present independently or not; or The first polypeptide is formed by covalently bonding, from the C-terminus to the N-terminus, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain in that order, wherein the linker peptides may be the same or different, and may be present independently or not; and / or The second polypeptide comprises a light chain of the second domain, optionally, The second polypeptide is formed by covalently bonding, in order from the N-terminus to the C-terminus, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain, wherein the linker peptides may be the same or different, and may be present independently or absent; The second polypeptide is formed by covalently bonding, in order from the C-terminus to the N-terminus, a CD80 IgV mutant polypeptide or a CD80 ECD mutant polypeptide, a linker peptide, a heavy chain of an antibody or its antigen-binding fragment, a linker peptide, and a third domain. The linker peptides may be the same or different, and may be present independently or absent. The CD80 mutant fusion polypeptide complex according to claim 12.

16. The CD80 mutant fusion polypeptide complex according to claim 12, wherein the third domain comprises LAG-3 or an active fragment thereof, and / or the second domain specifically targets PD-1 or PD-L1, and optionally the second domain is sugemalimab or a functional fragment thereof.

17. The CD80 mutant fusion polypeptide complex according to claim 16, 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.

18. The second domain is an immunoglobulin Fc domain, which is optionally selected from the group consisting of human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, mouse IgG1 Fc domain, mouse IgG2a Fc domain, mouse IgG2b Fc domain, and mouse IgG3 Fc domain, and optionally the CD80 IgV mutant polypeptide or / or CD80 ECD mutant polypeptide is covalently bound to the N-terminus or C-terminus of the immunoglobulin Fc domain, with or without the linker peptide. The CD80 mutant fusion polypeptide complex according to claim 3.

19. The amino acid sequence of the CD80 mutant fusion polypeptide complex is selected from the group consisting of E35N / A71E (SEQ ID NO: 158), E35N / A71N (SEQ ID NO: 159), E35N / A71Q (SEQ ID NO: 160), and E35N / A71T (SEQ ID NO: 161). The CD80 mutant fusion polypeptide complex according to claim 18.

20. If 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 mutant fusion polypeptide complex according to claim 3.

21. An immune complex comprising the CD80 IgV mutant polypeptide described in claim 1, or the CD80 ECD mutant polypeptide described in claim 2.

22. A nucleotide molecule encoding the CD80 IgV mutant polypeptide described in claim 1, or the CD80 ECD mutant polypeptide described in claim 2.

23. A composition comprising the CD80 IgV mutant polypeptide described in claim 1, or the CD80 ECD mutant polypeptide described in claim 2.

24. A CD80 IgV mutant polypeptide according to claim 1, or a CD80 ECD mutant polypeptide according to claim 2, comprising A drug used to regulate T cell activity.

25. A CD80 IgV mutant polypeptide according to claim 1, or a CD80 ECD mutant polypeptide according to claim 2, comprising A drug used to inhibit the growth or proliferation of tumor cells.

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