A tigit polypeptide and tigit / ctla4 fusion protein

Abstract

This application provides a TIGIT peptide and a TIGIT / CTLA4 fusion protein. The TIGIT peptide of this application has high affinity, and the TIGIT / CTLA4 fusion protein of this application can simultaneously block the CD226 / CD155 signaling pathway and the CD80 / CD86-CD28 signaling pathway. The peptide and fusion protein have clinical application prospects for treating immune system-related diseases.

Description

A TIGIT polypeptide and a TIGIT / CTLA4 fusion protein

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311873108.7, filed with the State Intellectual Property Office of China on December 29, 2023, entitled “A TIGIT Polypeptide and TIGIT / CTLA4 Fusion Protein,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of recombinant protein technology, and in particular, to a TIGIT and TIGIT / CTLA4 fusion protein. Background Art

[0004] CTLA-4, short for cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is a transmembrane receptor primarily expressed on activated T cells. It is homologous to the T cell surface co-stimulatory protein receptor CD28, and both can bind to CD80 / CD86 (also known as B7-1 and B7-2) on antigen-presenting cells (APCs). CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Studies have shown that CTLA-4 captures B7-1 and B7-2 on the APC membrane and then undergoes endocytosis, preventing them from activating the CD28 signaling pathway. CTLA4 on the cell surface exists as a dimer and is highly endocytic, with only 10% of CTLA4 located on the cell surface. In addition, the ligand CD80 / CD86 is mainly expressed in B cells, DC cells, macrophages and monocytes. CD86 exists in monomeric form while CD80 exists in dimer form. Therefore, CD80 has a stronger affinity than CD86 for both CD28 and CTLA4.

[0005] TIGIT (T cell Ig and ITIM domain) is a member of the poliovirus receptor (PVR) / Nectin family. The transmembrane protein TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a classic immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT) motif. TIGIT is highly expressed in lymphocytes, mainly CD4 + Regulatory T cells and follicular helper T cells, CD8 +Effector T cells and NK cells. Natural TIGIT ligands include CD155, CD112, and CD113, with high affinity for CD155 and low affinity for CD112 and CD113. TIGIT signaling pathways include inhibiting NK cell cytokine production and cytotoxicity, inhibiting T cell activation and proliferation, and enhancing the proliferation and suppressive activity of Treg cells. Furthermore, studies have shown that TIGIT can induce tolerogenic dendritic cells.

[0006] Rheumatoid arthritis (RA) is an autoimmune disease involving multiple genes and multiple immune cells. Specifically, in genetically susceptible individuals, host and environmental factors can lead to the activation of pathogenic T cells and a subsequent series of inflammatory responses. Cytokines are involved in coordinating immune responses and the recruitment and activation of effector cells. While causing inflammation, they can also cause local tissue damage in the patient's joints and involvement of extra-articular organs. Rheumatoid arthritis patients are generally treated with slow-acting antirheumatic drugs (disease-modifying antirheumatic drugs). When patients have an insufficient response to chemical slow-acting antirheumatic drugs, simultaneous treatment with biological slow-acting antirheumatic drugs should be considered. The targets of biological slow-acting antirheumatic drugs are mainly inflammatory factors, CD20-positive B cells, or regulatory T cell co-activation signals. CTLA4 fusion protein can specifically inhibit the CD28-CD80 / CD86 co-activation signaling pathway of T cells, and can also inhibit other CD80 / CD86-positive immune cell groups including activated B cells, macrophages, osteoclasts and endothelial cells. It is a target with great development potential in biological slow-acting anti-rheumatic drugs. Weigong Zhao et al. found in RA model mice that the expression level of TIGIT was negatively correlated with the progression of rheumatoid arthritis. In the synovial fluid of RA-positive T cells, the expression level of TIGIT was significantly lower than that of milder RA-positive cells, while overexpression of TIGIT in CD4-positive cells significantly reduced the cell proliferation level and the secretion of cytokines IFN-γ and IL-17. First, mechanistically, the binding of TIGIT expressed on the surface of T cells to the CD155 receptor on the surface of DC cells will induce the inhibitory signaling pathway of their own T cells, and will also induce the production of immunosuppressive DCs by expressing IL-10. In addition, TIGIT has a higher affinity for CD155 than CD226, thereby disrupting CD226 dimerization and CD226-regulated T cell activation. Therefore, from a mechanistic perspective, TIGIT is also one of the targets of highly promising biological slow-acting anti-rheumatic drugs.

[0007] Type 1 diabetes (T1D) is an autoimmune disease mediated primarily by T cells. Overactivation of the immune system attacks insulin-producing β cells, leading to insufficient insulin secretion. Although the discovery and large-scale production of insulin have made T1D no longer a terminal illness, many patients still experience complications as the disease progresses, including cardiovascular disease, retinopathy, neuropathy, and nephropathy. From the perspective of pathogenesis, the occurrence of T1D is mainly due to CD4 + T and CD8 + T cells involved. CD4 + T cells are involved because their HLA class II haplotype is strongly associated with T1D. In addition to finding CD4 + In addition to direct evidence of T cell pathogenicity, CD4 T cells carrying pancreatic antigens (Ag) have been found in the blood and pancreatic islets of T1D patients. + T cells assist B cells, T cells, and the cytokines they produce to activate macrophages and pancreatic islet-specific CD8 + T cells. Pathogenic CD8 + It will react with various overlapping pancreatic islet antigens (including insulin, IGRP, ZnT8, proinsulin) and some new antigens (such as hybrid peptides and corresponding post-translational modification products), triggering the killing effect on pancreatic beta cells. In addition, other regulatory T cell subsets, such as Tr1, Th3, CD8 + T suppressor cells are also involved in the development of T1D. Fumitaka Haseda et al. found that the expression of CTLA4 in CD4-positive helper T cells of T1D patients was significantly reduced. In the autoimmune-prone non-obese diabetic mouse model that simulates T1D, the expression of CTLA4 was significantly reduced. CTLA4 deficiency can lead to obvious immune system disorders, manifested as CD4 + Overactivation of T cells, CTLA4 for CD4 + T cell co-inhibitory signaling pathway plays an important role in this. Sven Brode et al. found that in the NOD mouse model, CD4 + CD25 + The decrease in the number and functional loss of Tregs also indicates that low CTLA4 expression also affects CD4 + CD25 +The proliferation and function of Treg. For the treatment of T1D, the most direct goal is to ensure the normal function of β cells and improve the immune tolerance and refractoriness of T cells. Similar to the CD28 / CTLA4 / CD80 / CD86 pathway, CD226 can activate CD4 + 、CD8 + Unlike NK cells, TIGIT is the opposite. Therefore, although there is currently little research on the specific mechanisms of CD226 and TIGIT in the pathogenesis of T1D, it can be confirmed that CD226 / TIGIT, which activates and inhibits T cell activation respectively, is crucial for the regulation of T cell immune tolerance.

[0008] The mechanisms of immune system diseases are complex, involving multiple signaling pathways and the activity of multiple immune cells. Conventional single-target drugs have limitations in their treatment, so new dual-target therapeutic drugs still need to be developed. Summary of the Invention

[0009] The present application aims to provide a TIGIT polypeptide variant and its TIGIT / CTLA4 fusion protein. The present application also aims to provide a nucleic acid molecule encoding the polypeptide variant and its fusion protein; an expression vector comprising the nucleic acid molecule; a host cell comprising the expression vector; a method for preparing the polypeptide variant and its fusion protein; a pharmaceutical composition comprising the polypeptide variant and its fusion protein; and medical uses of the polypeptide variant, its fusion protein, or the pharmaceutical composition for preventing or treating immune system-related diseases.

[0010] In order to achieve the above objectives, this application provides the following technical solutions:

[0011] The first aspect of the present application provides a TIGIT polypeptide variant comprising a TIGIT extracellular region or a functional fragment thereof, wherein the variant comprises at least one amino acid mutation relative to the amino acid sequence SEQ ID NO.3, and the mutation position is selected from one or more of the following groups: residues I42, Q56, N70, L73, G74, and Y113.

[0012] The second aspect of the present application provides a fusion protein comprising the TIGIT polypeptide variant.

[0013] The third aspect of the present application provides an expression vector comprising the nucleic acid.

[0014] The fourth aspect of the present application provides a host cell comprising the expression vector or expressing the polypeptide variant or fusion protein.

[0015] The fifth aspect of the present application provides a method for preparing the polypeptide variant or fusion protein, comprising the following steps: synthesizing the polypeptide variant or fusion protein, and / or culturing the host cell under conditions for expressing the polypeptide variant or fusion protein.

[0016] The sixth aspect of the present application provides a pharmaceutical composition comprising the polypeptide variant, the fusion protein and optionally a pharmaceutically acceptable excipient or carrier.

[0017] The seventh aspect of the present application provides a use of the polypeptide variant, fusion protein, or pharmaceutical composition in the preparation of a drug for preventing or treating immune system diseases or tumors.

[0018] The eighth aspect of the present application provides a method for preventing or treating immune system diseases or tumors, comprising administering a pharmaceutically effective amount of the polypeptide variant, fusion protein, or pharmaceutical composition to a subject.

[0019] The objectives of this application also include providing a TIGIT / CTLA4 fusion protein; providing a nucleic acid molecule encoding the fusion protein; providing an expression vector containing the nucleic acid molecule; providing a host cell containing the expression vector; providing a method for preparing the fusion protein; providing a pharmaceutical composition containing the fusion protein; and providing medical uses of the fusion protein or pharmaceutical composition for preventing or treating immune system-related diseases.

[0020] In order to achieve the above objectives, this application provides the following technical solutions:

[0021] The ninth aspect of the present application provides a fusion protein comprising a first structural region and a second structural region, or comprising a first structural region, a second structural region, and a third structural region; the first structural region is a TIGIT polypeptide or a variant thereof, the second structural region is a CTLA4 polypeptide or a variant thereof, and the third structural region is an Fc polypeptide or a variant thereof; the TIGIT or CTLA4 comprises an extracellular region or a functional fragment thereof.

[0022] The tenth aspect of the present application provides a nucleic acid encoding the fusion protein.

[0023] The eleventh aspect of the present application provides an expression vector comprising the nucleic acid.

[0024] The twelfth aspect of the present application provides a host cell comprising the expression vector or expressing the fusion protein.

[0025] The thirteenth aspect of the present application provides a method for preparing the fusion protein, comprising the following steps: synthesizing the fusion protein, and / or culturing the host cell under conditions for expressing the fusion protein.

[0026] The fourteenth aspect of the present application provides a pharmaceutical composition comprising the fusion protein and optionally a pharmaceutically acceptable excipient or carrier.

[0027] The fifteenth aspect of the present application provides the use of the fusion protein or pharmaceutical composition in the preparation of a drug for preventing or treating immune system diseases or tumors.

[0028] The sixteenth aspect of the present application provides a method for preventing or treating immune system diseases or tumors, comprising administering a pharmaceutically effective amount of the fusion protein or pharmaceutical composition to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is an ELISA test chart showing the affinity of CTLA4 fusion protein for CD86.

[0030] Figure 2 is an ELISA test chart of the affinity of TIGIT fusion protein to CD155.

[0031] Figure 3 is a diagram of the bispecific fusion protein configuration.

[0032] Figure 4 is an ELISA test chart showing the affinity of the bispecific fusion protein for CD86.

[0033] Figure 5 is an ELISA test chart showing the affinity of the bispecific fusion protein for CD155.

[0034] Figure 6 is an ELISA test chart of the affinity of the first-round mutated TIGIT fusion protein to CD155.

[0035] Figure 7 is an ELISA test chart of the affinity of the second-round mutated TIGIT fusion protein to CD155.

[0036] Figure 8 is a graph showing the binding kinetics of TIGIT fusion protein to CD155.

[0037] Figure 9 is a diagram showing the blocking effect of TIGIT fusion protein on the CD226 / CD155 signaling pathway.

[0038] Figure 10 is a diagram showing the blocking effect of the bispecific fusion protein on the CD226 / CD155 signaling pathway.

[0039] Figure 11 is a diagram showing the blocking effect of the bispecific fusion protein on the CD80 / CD86-CD28 signaling pathway.

[0040] Figure 12 shows the bispecific fusion protein inhibiting CD4 + T cell activation assay diagram.

[0041] Figure 13 shows the bispecific fusion protein inhibiting CD8 +T cell activation assay diagram.

[0042] FIG14 shows the fusion protein inhibiting the limb arthritis scores of the collagen-induced mouse arthritis model.

[0043] FIG15 shows the measurement data of bilateral hind limb footpad swelling in a mouse arthritis model induced by collagen, which was inhibited by the fusion protein. DETAILED DESCRIPTION

[0044] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure. The technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, not all. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0045] The present application aims to provide a TIGIT polypeptide variant and its TIGIT / CTLA4 fusion protein. The present application also aims to provide a nucleic acid molecule encoding the polypeptide variant and its fusion protein; an expression vector comprising the nucleic acid molecule; a host cell comprising the expression vector; a method for preparing the polypeptide variant and its fusion protein; a pharmaceutical composition comprising the polypeptide variant and its fusion protein; and medical uses of the polypeptide variant, its fusion protein, or the pharmaceutical composition for preventing or treating immune system-related diseases.

[0046] In order to achieve the above objectives, this application provides the following technical solutions:

[0047] The first aspect of the present application provides a TIGIT polypeptide variant comprising a TIGIT extracellular region or a functional fragment thereof, wherein the variant comprises at least one amino acid mutation relative to the amino acid sequence SEQ ID NO.3, and the mutation position is selected from one or more of the following groups: residues I42, Q56, N70, L73, G74, and Y113.

[0048] In an alternative embodiment, the amino acid mutation is an amino acid substitution.

[0049] In an optional embodiment, the amino acid mutation is selected from one or more of the following groups: I42V, I42L, Q56N, Q56E, N70Q, N70H, L73I, L73V, G74S, Y113W, Y113T, Y113F.

[0050] In an optional embodiment, the TIGIT polypeptide variant comprises the amino acid sequence of SEQ ID NO.21 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence of SEQ ID NO.21.

[0051] The second aspect of the present application provides a fusion protein comprising the TIGIT polypeptide variant.

[0052] In an alternative embodiment, the fusion protein further comprises (a) a CTLA4 polypeptide or a variant thereof, and / or b) an Fc polypeptide or a variant thereof, wherein the CTLA4 polypeptide comprises an extracellular region or a functional fragment thereof.

[0053] In an optional embodiment, the CTLA4 polypeptide variant comprises A30Y and / or L105E amino acid mutations relative to the amino acid sequence of SEQ ID NO.1.

[0054] In an alternative embodiment, the CTLA4 polypeptide or variant thereof comprises an amino acid sequence selected from the group consisting of:

[0055] a) SEQ ID NO. 1;

[0056] b) SEQ ID NO. 9;

[0057] c) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 9.

[0058] In alternative embodiments, the Fc polypeptide is a monomer or dimer of human IgG1, IgG2 or IgG4 Fc polypeptide.

[0059] In alternative embodiments, the Fc polypeptide comprises a hinge region, a CH2 region, and a CH3 region, or a CH2 region and a CH3 region.

[0060] In alternative embodiments, the Fc polypeptide variant comprises amino acid mutations that increase Fc stability or half-life.

[0061] In an alternative embodiment, the Fc polypeptide variant comprises one or more amino acid mutations selected from the following group: M428L, N434S, M252Y, S254T, T256E, IgG4 (S228P).

[0062] In an alternative embodiment, the IgG1 Fc polypeptide variant comprises M428L, N434S or M428L / N434S amino acid mutations.

[0063] In alternative embodiments, the IgG4 Fc polypeptide variant comprises S228P, M428L, N434S, M428L / N434S, M428L / S228P, N434S / S228P, or M428L / N434S / S228P amino acid mutations.

[0064] In an alternative embodiment, the Fc polypeptide or variant thereof comprises an amino acid sequence selected from the group consisting of:

[0065] a) SEQ ID NO. 7;

[0066] b) SEQ ID NO. 8;

[0067] c) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 7 or SEQ ID NO. 8. In an alternative embodiment, the fusion protein comprises a polypeptide monomer or a homologous or heterologous dimer thereof selected from the group consisting of:

[0068] a) AL-Fc-LB;

[0069] b) BL-Fc-LA;

[0070] c) ALBL-Fc;

[0071] d) BLAL-Fc;

[0072] A is a TIGIT polypeptide variant, B is a CTLA4 polypeptide or its variant, and L is absent or a linker.

[0073] In an optional embodiment, the linker is (G4S)n or (SG4)m, wherein n or m is a positive integer selected from 1, 2, 3, 4, 5, 6.

[0074] In an optional embodiment, the fusion protein comprises an amino acid sequence selected from SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 29, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

[0075] In an optional embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO. 27, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

[0076] The second aspect of the present application provides a nucleic acid encoding the polypeptide variant or the fusion protein.

[0077] In an optional embodiment, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, and SEQ ID NO.30.

[0078] The third aspect of the present application provides an expression vector comprising the nucleic acid.

[0079] The fourth aspect of the present application provides a host cell comprising the expression vector or expressing the polypeptide variant or fusion protein.

[0080] The fifth aspect of the present application provides a method for preparing the polypeptide variant or fusion protein, comprising the following steps: synthesizing the polypeptide variant or fusion protein, and / or culturing the host cell under conditions for expressing the polypeptide variant or fusion protein.

[0081] The sixth aspect of the present application provides a pharmaceutical composition comprising the polypeptide variant, the fusion protein and optionally a pharmaceutically acceptable excipient or carrier.

[0082] The seventh aspect of the present application provides a use of the polypeptide variant, fusion protein, or pharmaceutical composition in the preparation of a drug for preventing or treating immune system diseases or tumors.

[0083] In an optional embodiment, the immune system disease includes lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes (e.g., type 1 diabetes), myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemorrhagic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatic disease (e.g., rheumatoid arthritis), arthritis, psoriasis, T-cell lymphoma, autoimmune graft-versus-host disease (GVHD), and organ transplant rejection.

[0084] The eighth aspect of the present application provides a method for preventing or treating immune system diseases or tumors, comprising administering a pharmaceutically effective amount of the polypeptide variant, fusion protein, or pharmaceutical composition to a subject.

[0085] In an optional embodiment, the immune system disease includes lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes (e.g., type 1 diabetes), myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemorrhagic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatic disease (e.g., rheumatoid arthritis), arthritis, psoriasis, T-cell lymphoma, autoimmune graft-versus-host disease (GVHD), and organ transplant rejection.

[0086] In an alternative embodiment, the method further comprises administering a second active molecule, including but not limited to an immunosuppressive drug, an immunomodulatory drug, or an anti-inflammatory drug.

[0087] The objectives of this application also include providing a TIGIT / CTLA4 fusion protein; providing a nucleic acid molecule encoding the fusion protein; providing an expression vector containing the nucleic acid molecule; providing a host cell containing the expression vector; providing a method for preparing the fusion protein; providing a pharmaceutical composition containing the fusion protein; and providing medical uses of the fusion protein or pharmaceutical composition for preventing or treating immune system-related diseases.

[0088] In order to achieve the above objectives, this application provides the following technical solutions:

[0089] The ninth aspect of the present application provides a fusion protein comprising a first structural region and a second structural region, or comprising a first structural region, a second structural region, and a third structural region; the first structural region is a TIGIT polypeptide or a variant thereof, the second structural region is a CTLA4 polypeptide or a variant thereof, and the third structural region is an Fc polypeptide or a variant thereof; the TIGIT or CTLA4 comprises an extracellular region or a functional fragment thereof.

[0090] In an optional embodiment, the TIGIT polypeptide variant comprises at least one amino acid mutation relative to the amino acid sequence SEQ ID NO.3, and the mutation position is selected from one or more of the following groups: residues I42, Q56, N70, L73, G74, and Y113.

[0091] In an alternative embodiment, the amino acid mutation is an amino acid substitution.

[0092] In an optional embodiment, the TIGIT polypeptide variant comprises one or more amino acid mutations selected from the following group: I42V, I42L, Q56N, Q56E, N70Q, N70H, L73I, L73V, G74S, Y113W, Y113T, Y113F.

[0093] In an alternative embodiment, the TIGIT polypeptide or variant thereof comprises an amino acid sequence selected from the group consisting of:

[0094] a) SEQ ID NO. 3;

[0095] b) SEQ ID NO. 21;

[0096] c) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 21.

[0097] In an optional embodiment, the CTLA4 polypeptide variant comprises A30Y and / or L105E amino acid mutations relative to the amino acid sequence of SEQ ID NO.1.

[0098] In an alternative embodiment, the CTLA4 polypeptide or variant thereof comprises an amino acid sequence selected from the group consisting of:

[0099] a) SEQ ID NO. 1;

[0100] b) SEQ ID NO. 9;

[0101] c) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 9.

[0102] In alternative embodiments, the Fc polypeptide is a monomer or dimer of human IgG1, IgG2 or IgG4 Fc polypeptide.

[0103] In alternative embodiments, the Fc polypeptide comprises a hinge region, a CH2 region, and a CH3 region, or a CH2 region and a CH3 region.

[0104] In alternative embodiments, the Fc polypeptide variant comprises amino acid mutations that increase Fc stability or half-life.

[0105] In an alternative embodiment, the Fc polypeptide variant comprises one or more amino acid mutations selected from the following group: M428L, N434S, M252Y, S254T, T256E, IgG4 (S228P).

[0106] In an alternative embodiment, the IgG1 Fc polypeptide variant comprises M428L, N434S or M428L / N434S amino acid mutations.

[0107] In alternative embodiments, the IgG4 Fc polypeptide variant comprises S228P, M428L, N434S, M428L / N434S, M428L / S228P, N434S / S228P, or M428L / N434S / S228P amino acid mutations.

[0108] In an alternative embodiment, the Fc polypeptide or variant thereof comprises an amino acid sequence selected from the group consisting of:

[0109] a) SEQ ID NO. 7;

[0110] b) SEQ ID NO. 8;

[0111] c) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 7 or SEQ ID NO. 8. In an alternative embodiment, the fusion protein comprises a polypeptide monomer or a homologous or heterologous dimer thereof selected from the group consisting of:

[0112] a) AL-Fc-LB;

[0113] b) BL-Fc-LA;

[0114] c) ALBL-Fc;

[0115] d) BLAL-Fc;

[0116] A is a TIGIT polypeptide or a variant thereof, B is a CTLA4 polypeptide or a variant thereof, and L is absent or a linker.

[0117] In an optional embodiment, the linker is (G4S)n or (SG4)m, wherein n or m is a positive integer selected from 1, 2, 3, 4, 5, 6.

[0118] In an optional embodiment, the fusion protein comprises an amino acid sequence selected from SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.29 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

[0119] In an optional embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO. 27, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

[0120] The tenth aspect of the present application provides a nucleic acid encoding the fusion protein.

[0121] The eleventh aspect of the present application provides an expression vector comprising the nucleic acid.

[0122] In an optional embodiment, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, and SEQ ID NO.30.

[0123] The twelfth aspect of the present application provides a host cell comprising the expression vector or expressing the fusion protein.

[0124] The thirteenth aspect of the present application provides a method for preparing the fusion protein, comprising the following steps: synthesizing the fusion protein, and / or culturing the host cell under conditions for expressing the fusion protein.

[0125] The fourteenth aspect of the present application provides a pharmaceutical composition comprising the fusion protein and optionally a pharmaceutically acceptable excipient or carrier.

[0126] The fifteenth aspect of the present application provides the use of the fusion protein or pharmaceutical composition in the preparation of a drug for preventing or treating immune system diseases or tumors.

[0127] In an optional embodiment, the immune system disease includes lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes (e.g., type 1 diabetes), myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemorrhagic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatic disease (e.g., rheumatoid arthritis), arthritis, psoriasis, T-cell lymphoma, autoimmune graft-versus-host disease (GVHD), and organ transplant rejection.

[0128] The sixteenth aspect of the present application provides a method for preventing or treating immune system diseases or tumors, comprising administering a pharmaceutically effective amount of the fusion protein or pharmaceutical composition to a subject.

[0129] In an optional embodiment, the immune system disease includes lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes (e.g., type 1 diabetes), myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemorrhagic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatic disease (e.g., rheumatoid arthritis), arthritis, psoriasis, T-cell lymphoma, autoimmune graft-versus-host disease (GVHD), and organ transplant rejection.

[0130] In an alternative embodiment, the method further comprises administering a second active molecule. The second active molecule includes but is not limited to an immunosuppressive drug, an immunomodulatory drug, or an anti-inflammatory drug.

[0131] In an optional embodiment, the above-mentioned immune system disease or tumor is associated with the CTLA-4 and / or TIGIT signaling pathway.

[0132] In an alternative embodiment, the immune system disease is an autoimmune disease.

[0133] The amino acid sequences in this application are arranged sequentially from N-terminus to C-terminus.

[0134] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0135] the term

[0136] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0137] TIGIT

[0138] The term "TIGIT" refers to naturally occurring full-length TIGIT or a fragment thereof. Full-length TIGIT is a cell transmembrane protein composed of an extracellular immunoglobulin variable region (IgV) domain (i.e., an extracellular region), a type 1 transmembrane domain, and an intracellular domain having a classical immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT) motif. The "TIGIT extracellular region or a functional fragment thereof" refers to a portion that can bind to the target antigens CD155, CD112, and / or CD113. In a specific embodiment, the TIGIT extracellular region refers to Met22-Pro141 of NCBI gene accession number NP_776160.2.

[0139] CTLA4

[0140] The term "CTLA4" refers to naturally occurring full-length CTLA4 or a fragment thereof. Full-length CTLA4 is a cell surface protein comprising an N-terminal extracellular region, a transmembrane region, and a C-terminal cytoplasmic region. The "CTLA4 extracellular region or a functional fragment thereof" refers to a portion capable of binding to the target antigens CD80 and / or CD86. In one specific embodiment, the CTLA4 extracellular region refers to Ala37-Phe162 of gene accession number Q6GR94.

[0141] Variants

[0142] The term "variant" refers to a peptide that comprises at least one amino acid substitution, deletion, or insertion relative to a wild-type or naturally occurring peptide, such as conservative variants well known to those skilled in the art.

[0143] Sequence homology

[0144] Methods for determining sequence homology known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). The preferred method for determining homology is to obtain the largest match between the sequences tested. Methods for determining homology are compiled in publicly available computer programs. Preferred computer program methods for determining homology between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine homology.

[0145] Fusion protein

[0146] The term "fusion protein" refers to a new polypeptide sequence obtained by fusing multiple identical or different polypeptide sequences. The term "fusion" refers to direct connection by a peptide bond or connection with the aid of one or more linkers (peptide linkers). The term "linker (peptide linker)" refers to a short peptide that can connect two polypeptide sequences, generally 1-30 amino acids in length. Preferably, the peptide linker is a flexible peptide linker. Examples of suitable linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker can vary with the type of secondary structural elements that need to be achieved in the linker.

[0147] Multiple

[0148] The term "plurality" means at least two.

[0149] Encoding nucleic acid and expression vector

[0150] The present application also provides nucleic acid molecules encoding the above-mentioned fusion proteins. The nucleic acids herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The sequence of the DNA molecule encoding the fusion protein herein may be obtained using conventional techniques, such as PCR amplification. Furthermore, synthetic methods may be used to synthesize the relevant sequence.

[0151] The present application also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The vectors are conventional expression vectors in the art, meaning expression vectors comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vectors can be viral or plasmids, such as appropriate phages or phagemids. For more technical details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. For many known techniques and protocols for nucleic acid manipulation, see Current Protocols in Molecular Biology, 2nd ed., Ausubel et al. The expression vectors described herein are preferably pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF, or pCHO 1.0.

[0152] In this application, the term "host cell" refers to any host cell commonly used in the art, as long as the vector can stably replicate and the polynucleotide molecules carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and preferably include COS, CHO, NS0, sf9, sf21, DH5α, BL21 (DE3), TG1, BL21 (DE3), 293F or 293E cells.

[0153] Pharmaceutical composition

[0154] The term "pharmaceutical composition" refers to a single or compound pharmaceutical preparation composed of the polypeptide or variants thereof, fusion protein, and a pharmaceutically acceptable excipient or carrier. Such excipients or carriers include those familiar to those skilled in the art. Suitable carriers and excipients include, but are not limited to, human serum albumin, saline, buffer, glucose, water, glycerol, ethanol, glycine, sorbic acid, potassium sorbate, and combinations thereof.

[0155] The pharmaceutical compositions of the present application may be in a variety of dosage forms, including but not limited to liquid solutions or suspensions, injections, injections, and liposomes. The preferred dosage form depends on the mode of administration and the intended use. The pharmaceutical compositions of the present application may be administered to a subject using conventional modes of administration, including but not limited to intravenous, peritoneal, intramuscular, and subcutaneous administration.

[0156] After multiple rounds of protein engineering and screening, this application has obtained a bispecific fusion protein CTLA4-TIGIT that can competitively inhibit the activation of CD28-CD80 / 86 and CD226-CD155 pathways, and exerts a synergistic inhibition of CD4 beyond the use of CTLA4 or TIGIT fusion protein alone. + 、CD8 + The T cell activation effect will have a more effective and broader therapeutic effect in related autoimmune diseases.

[0157] Compared with the related art, the beneficial technical effects of this application include:

[0158] 1. Through extensive research and analysis, this application has identified the key amino acid sites that affect the stability of the "LOCK-and-KEY" of the TIGIT / CD155 interaction interface, and on this basis, through multiple rounds of screening, obtained TIGIT polypeptide variants with significantly improved affinity and slower dissociation rate, achieving unexpected technical effects. This TIGIT polypeptide variant has better biological activity, including stronger CD226 / CD155 signaling pathway blocking ability, and can exert stable immunomodulatory activity at a smaller dose. When used in the clinical treatment of immune system diseases, it can benefit from effectiveness, safety, stability, durability and other aspects.

[0159] 2. The present application provides a new dual-target combination solution, which is the first time to combine the TIGIT extracellular domain protein and the CTLA4 extracellular domain protein to construct a new fusion protein. The TIGIT / CTLA4 fusion protein of the present application can better play the role of inhibiting T cell activation or proliferation, inhibiting the release of inflammatory factors, etc. by doubly blocking the CD226 / CD155 signaling pathway and the CD80 / CD86-CD28 signaling pathway, thereby solving clinical problems that cannot be solved by single drugs or combination drugs. As shown by the exemplary TIGIT / CTLA4 fusion protein, it has signaling pathway inhibition ability and T cell inhibitory activity that are superior to each single-target fusion protein, and the T cell inhibitory activity is better than the combination of TIGIT fusion protein and CTLA4 fusion protein, and has unexpected synergistic or superimposed activity.

[0160] In cell experiments or animal models of immune system-related diseases, such as arthritis models, it has demonstrated in vivo or in vitro effects that are superior to existing drug molecules such as Abatacept and Belatacept.

[0161] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples, for which no detailed conditions are specified, were generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0162] Example 1 Preparation of fusion protein

[0163] By gene synthesis and molecular cloning, the CTLA4 extracellular region (Ala37-Phe162, NCBI gene accession number: Q6GR94; SEQ ID NO.1), the TIGIT extracellular region (Met22-Pro141, NCBI gene accession number: NP_776160.2; SEQ ID NO.3), the CD28 extracellular region (Asn19-Pro152, UniProtKB accession number: P10747; SEQ ID NO.5), and the CD226 extracellular region (Glu19-Asn247, UniProtKB accession number: Q15762.2; SEQ ID NO.6) were linked to the human IgG1 constant region Fc (SEQ ID NO.7) or to the human IgG4 constant region Fc (containing the S228P mutation, SEQ ID NO. NO.8) were linked and cloned into the pcDNA3.4 vector, and HEK-293F cells were transfected and incubated in a cell shaker incubator at 37°C and 8% CO2 for protein expression. After 5 days, the cell supernatant was collected by centrifugation and the target protein was purified by Protein A affinity chromatography to obtain various fusion proteins. Fusion proteins linked to the Fc constant region of human IgG1 were named with the suffix -Fc, and fusion proteins linked to the Fc constant region of human IgG4 were named with the suffix -FcG4. For example, the IgG1 and IgG4 subtype fusion proteins of CTLA4 were named CTLA4-Fc and CTLA4-FcG4, respectively.

[0164] The C-termini of the extracellular region of CD86 (Leu20-Asp238, UniProtKB accession number: P42081; SEQ ID NO.11) and the extracellular region of CD155 (Gly27-Asn343, UniProtKB accession number: P15151; SEQ ID NO.12) were respectively linked to 6×His and cloned into the pcDNA3.4 vector. HEK-293F cells were transfected and the CD86-His and CD155-His proteins were expressed and purified, respectively.

[0165] Example 2 Affinity determination of CTLA4 and TIGIT fusion proteins

[0166] In this example, the affinity of each fusion protein to its corresponding ligand was determined using enzyme-linked immunosorbent assay (ELISA).

[0167] Specifically, CD86-His and CD155-His proteins were diluted to 2 and 5 μg / mL, respectively, with ELISA coating solution, and 100 μL / well were placed in a 4°C wet box to coat the ELISA plate for 16 hours; the ELISA plate was washed three times with PBST to remove unbound antigens, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, and then 2% BSA was prepared with PBS, 200 μL / well, and blocked at room temperature for 2 hours; the ELISA plate was washed three times with PBST to remove excess blocking solution, and the ELISA plate was patted dry on absorbent paper to remove excess The remaining liquid was diluted with 1% BSA in PBST for each test protein. The maximum working concentration of CTLA4 and CD28-related fusion proteins was 3600 nM. These proteins were diluted 11 times in a 5-fold gradient and added to the CD86-His ELISA-coated plate. The maximum working concentration of TIGIT and CD226-related fusion proteins was 5000 nM. These proteins were diluted 11 times in a 2.5-fold gradient and added to the CD155-His ELISA-coated plate at 100 μL / well. The plates were incubated at room temperature for 1 hour. Two replicate wells were prepared for each sample. Unbound or nonspecifically bound samples were washed away. Following the antibody instructions, HRP-labeled anti-human Fc secondary antibody was diluted 1:5000 in antibody diluent and added to each ELISA plate at 100 μL / well. The plates were incubated at room temperature for 1 hour. The plates were washed three times with PBST and patted dry on absorbent paper to remove excess liquid. TMB color development solution (purchased from KPL, cat. no. 5120-0075) was added at 100 μL / well and color was developed to an appropriate depth. 2 M H2SO4 (50 μL / well) was added to stop color development. The absorbance was measured at a wavelength of 450 nm in a multifunctional microplate reader. Data were analyzed using GraphPad Prism10 using the "log (agonist) vs. response--Variable slope (four parameters)" parameter setting.

[0168] The results of the affinity determination of CTLA4-LEAY-Fc, CD28-Fc and CTLA4-Fc for CD86-His are shown in Figure 1. As shown in Figure 1, the affinity of CTLA4-LEAY-Fc for CD86-His is stronger than that of CTLA4-Fc and CD28-Fc, among which the EC values ​​of CTLA4-LEAY-Fc, CTLA4-Fc and CD28-Fc are 50 The values ​​were 0.11 nM, 1.24 nM and 154.35 nM, respectively.

[0169] The results of the affinity determination of TIGIT-Fc and CD226-Fc for CD155-His are shown in Figure 2. As shown in Figure 2, the affinity of TIGIT-Fc for CD155-His is stronger than that of CD226-Fc, wherein the EC values ​​of TIGIT-Fc and CD226-Fc are 50 The values ​​were 244 nM and 1465.5 nM, respectively.

[0170] Example 3 Preparation of bispecific fusion protein

[0171] Through gene synthesis and molecular cloning techniques, the extracellular regions of CTLA4 and TIGIT, as well as the human IgG1 constant region Fc, were constructed into multiple bispecific molecules in different structural forms using a (G4S)3 linker. HEK-293F cells were transfected and, referring to the protein expression and purification method in Example 1, the resulting bispecific fusion proteins were named CTLA4-Fc-TIGIT (SEQ ID NO. 13), CTLA4-TIGIT-Fc (SEQ ID NO. 15), TIGIT-CTLA4-Fc (SEQ ID NO. 17), and TIGIT-Fc-CTLA4 (SEQ ID NO. 19). The structural schematics of the bispecific fusion proteins CTLA4-Fc-TIGIT, CTLA4-TIGIT-Fc, TIGIT-CTLA4-Fc, and TIGIT-Fc-CTLA4 are shown in Figure 3.

[0172] The expression levels of the above-mentioned bispecific fusion proteins expressed by HEK-293F and the purity of the samples after one-step Protein A (PrtA) purification and UPLC detection are statistically shown in Table 1. It can be seen that the transient expression level of the bispecific fusion protein TIGIT-CTLA4-Fc and the purity of the samples after one-step PrtA purification are relatively good.

[0173] Table 1 Bispecific fusion protein expression parameters

[0174] Example 4 Affinity determination of bispecific fusion protein

[0175] Referring to the ELISA detection method of Example 2, the affinities of CTLA4-Fc-TIGIT, CTLA4-TIGIT-Fc, TIGIT-CTLA4-Fc and TIGIT-Fc-CTLA4 to CD86-His and CD155-His were measured respectively.

[0176] As shown in Figure 4, among the bispecific fusion proteins, CTLA4-TIGIT-Fc has the best affinity for CD86-His, and its EC 50was 49.78 nM; followed by TIGIT-CTLA4-Fc and TIGIT-Fc-CTLA4, both of which had an EC 50 The affinity of CTLA4-Fc-TIGIT to CD86-His was the weakest, but the affinity of each bispecific fusion protein to CD86-His was significantly weaker than that of the control single molecule CTLA4-Fc, among which the EC 50 The detection efficiency was 2.64 nM, which may be related to the steric hindrance caused by the position of CTLA4 in the bispecific fusion protein and the bridging effect produced by the secondary antibody.

[0177] As shown in Figure 5 , among the bispecific fusion proteins, TIGIT-CTLA4-Fc and TIGIT-Fc-CTLA4 have the best affinity for CD155-His, among which the EC 50 140.85 nM and 134 nM, respectively, which were better than the control single-molecule TIGIT-Fc. 50 The affinity of CTLA4-Fc-TIGIT to CD155-His was 249.4 nM.

[0178] Example 5 Engineering of TIGIT fusion protein

[0179] Based on the three-dimensional structural analysis of the TIGIT / CD155 complex by Katharina F. Stengel (Stengel, et al. Proc. Natl. Acad. Sci. USA 2012, 109, 5399.), we engineered potential amino acid sites that affect the stability of the "LOCK-and-KEY" region of the TIGIT / CD155 interaction interface using the AI ​​(Artificial Intelligence) algorithm of Wecomput's Protein Virtual Affinity Maturation platform.

[0180] Through comprehensive analysis, the following amino acid sites were selected for site-directed mutagenesis in TIGIT-Fc in the first round: I42V, I42L, Q56N, Q56E, N70Q, N70H, L73I, L73V, G74S, Y113W, and Y113T. The TIGIT-Fc mutants obtained through point mutation cloning were transfected into HEK-293F cells using the pcDNA3.4 expression vector, expressed and purified, to obtain the respective TIGIT-Fc mutant proteins. The affinity of each mutant for CD155 was assayed using the ELISA method described in Example 2.

[0181] As shown in Figure 6, among all the mutant proteins of TIGIT-Fc, TIGIT-Fc-Y113W has the highest affinity for CD155-His. That is, after the 113th Tyr (Y) in the extracellular region of TIGIT is mutated to Trp (W) (named TIGIT extracellular region-Y113W, SEQ ID NO.21), the affinity of TIGIT for CD155 can be significantly improved, which is significantly better than TIGIT-Fc.

[0182] Based on the results of the first round of screening mentioned above, the 113th Y in the "KEY" domain of the TIGIT protein was selected for the second round of mutation according to the principle of "similar and close, maximum difference". The specific mutation information is as follows: Y113W, Y113H, Y113E, Y113K, Y113A, and Y113F. The above-mentioned site-directed mutation clones were constructed into the pcDNA3.4 vector and transfected into HEK-293F cells, and the expression and purification were carried out to obtain each mutant protein. The affinity of each mutant for CD155 was detected according to the ELIS method in Example 2.

[0183] As shown in Figure 7, TIGIT-Fc-Y113W exhibits the best affinity for CD155, and its EC 50 reached less than 1 nM; followed by TIGIT-Fc-Y113F, whose EC 50 The EC values ​​of each sample were obtained by four-parameter curve fitting analysis using GraphPad Prism. 50 The values ​​of the upper (Top) and lower (Bottom) platforms are shown in Table 2.

[0184] Table 2 TIGIT mutant proteins binding to CD155 EC 50

[0185] Example 6 Binding Kinetics of TIGIT Fusion Protein

[0186] Based on the above experimental results, TIGIT-Fc-Y113W was selected to further analyze its binding kinetics to CD155 protein using Bacore8K. The specific experimental process is as follows:

[0187] First, CD155-Fc protein was captured using a CM5 chip. The operating parameters were as follows: CD155-Fc concentration was 12 μg / mL, the diluent used was 10 mM sodium acetate at pH 4.0, the contact time was 300 s, and the flow rate was 10 μL / min. Then, the test proteins TIGIT-Fc-Y113W and TIGIT-Fc were diluted with HBS-EP+pH7.4+150 mM NaCl buffer, the maximum concentration was set to 50 nM, and the gradient was diluted 2-fold to 0.1953125, and a zero concentration point was set. The test protein CD226-Fc was set to a maximum concentration of 8000 nM and diluted 2-fold to 31.25 nM and a zero concentration point. 0.2 M NaOH solution was used as the regeneration buffer. Samples were injected on the 8K using the following parameters: association time 240 s, dissociation time 450 s, flow rate 30 μL / min, regeneration contact time 40 s, flow rate 30 μL / min. The collected data were analyzed using Biacore 8K Evaluation Software.

[0188] The binding and dissociation curves of each protein with CD155 are detailed in Figure 8, and the statistics of the binding kinetic parameters are shown in Table 3. As shown in Table 3, by analyzing the hydrophilic-hydrophobic interaction of the key amino acids in the "key-lock" formation between TIGIT and CD155, a mutant TIGIT-Fc-Y113W with stronger affinity for CD155 was screened and successfully obtained. Compared with the wild-type TIGIT-Fc, the affinity (KD) was increased by about 2.5 times, but the dissociation rate of TIGIT-Fc-Y113W was slower, which was about 7.5 times better than TIGIT-Fc and about 135 times better than CD226-Fc, suggesting that TIGIT-Fc-Y113W may have better biological activity.

[0189] Table 3 Kinetic parameters of fusion protein binding to CD155

[0190] Example 7 Blocking Effect of TIGIT Protein Mutants on CD226 / CD155 Signaling Pathway

[0191] CHO cells overexpressing CD155 and TCR Activator (purchased from Nanjing Kebai, catalog number: CBP74073) and Jurkat cells overexpressing the NFAT-Luc fluorescent reporter system (purchased from Jiman Bio, catalog number: GM-C01459) were co-cultured. At the end of the experiment, the fluorescence signal was detected by adding Luciferin substrate to determine the expression level of Luciferase caused by the activation of the NFAT pathway downstream of CD226 / CD155. The specific experimental process is as follows:

[0192] CD155 / TCR Activator / CHO cells in the logarithmic growth phase were collected, centrifuged, washed once with DPBS, and then resuspended to an appropriate concentration in F12 medium containing 1% FBS. 100 μL per well was plated and cultured at 37°C overnight. The next day, the 96-well cell culture plate seeded with CD155 / TCR Activator / CHO cells was removed, the medium therein was discarded, and the fusion protein to be tested prepared in F12 medium containing 1% FBS was added at a concentration of 9000 nM, 50 μL / well, and incubated at room temperature for 30 minutes. Finally, NFAT-Luc Jukrat cells in the logarithmic growth phase were collected, centrifuged and washed once with DPBS, and then resuspended in F12 medium containing 1% FBS to the corresponding concentration (the ratio of CD155 / TCRActivator / CHO and NFAT-Luc Jurkat cells was 1:8), and added to the above 96-well plate at 50 μL / well. After gentle mixing, the cells were placed in a 37°C incubator for incubation. After 6 hours, Bio-Glu (purchased from Promega, product number G7940) reagent was added to the wells at 100 μL / well, and the fluorescence signal was detected by a microplate reader and the data was analyzed.

[0193] As shown in Figure 9 , in the presence of different numbers of effector cells (NFAT-Luc Jurkat), the activity of TIGIT-Fc-Y113W in blocking CD226 / CD155 downstream signaling was significantly better than that of TIGIT-Fc.

[0194] Example 8 Preparation of a Novel Bispecific Fusion Protein

[0195] Referring to the method of Example 3, Ala at position 30 on CTLA4 of CTLA4-Fc-TIGIT, CTLA4-TIGIT-Fc, TIGIT-CTLA4-Fc and TIGIT-Fc-CTLA4 were mutated to Tyr, Leu at position 105 was mutated to Glu, and Tyr at position 113 of TIGIT was mutated to Tr p. At the same time, M428L and N434S mutations were introduced into the Fc region to achieve the effect of enhancing the half-life of the sample in vivo. The new bispecific fusion proteins finally obtained were named CTLA4-Fc-TIGIT-Enhl, CTLA4-TIGIT-Fc-Enhl, TIGIT-CTLA4-Fc-Enhl and TIGIT-Fc-CTLA4-Enhl, respectively. Furthermore, considering that the mechanism of action of this target may require avoiding the biological function effects of the Fc, we simultaneously constructed novel bispecific fusion proteins in which the Fc region of the aforementioned novel bispecific fusion proteins was replaced with the human IgG4 (S228P) Fc region and containing the M428L and N434S mutations. These proteins were designated CTLA4-FcG4-TIGIT-Enhl (SEQ ID NO. 23), CTLA4-TIGIT-FcG4-Enhl (SEQ ID NO. 25), TIGIT-CTLA4-FcG4-Enhl (SEQ ID NO. 27), and TIGIT-FcG4-CTLA4-Enhl (SEQ ID NO. 29), respectively. The expression data of these novel bispecific fusion proteins transiently transfected into HEK-293F cells are shown in Table 4. As shown in Table 4, the transient expression level of TIGIT-CTLA4-Fc-Enhl was the highest, and the relative purity was also high after one-step Protein A purification. The protein expression results of the protein with the Fc region replaced with IgG4 (S228P) Fc were similar, and the expression level of TIGIT-CTLA4-FcG4-Enhl and the protein purity were also high after one-step Protein A purification.

[0196] Table 4 Expression parameters of novel bispecific fusion proteins

[0197] Example 9: Blocking Effect of Bispecific Fusion Protein on Downstream Signaling Pathways

[0198] Based on the expression results of the above-mentioned bispecific fusion proteins, the bispecific fusion protein TIGIT-CTLA4-FcG4-Enhl was selected for further study. Its TIGIT terminal cell functional activity was identified by co-culture of CD155 / TCR Activator / CHO cells and NFAT-Luc Jurkat cells. The specific experimental method is shown in Example 7. As shown in Figure 10, the bispecific fusion protein TIGIT-CTLA4-FcG4-Enhl has a significantly better inhibitory activity on CD226 / CD155 downstream signal transduction than the single fusion proteins TIGIT-Fc and TIGIT-Fc-Y113W. The IC of TIGIT-CTLA4-FcG4-Enhl was analyzed by four-parameter curve fitting. 50 It is 3191nM.

[0199] The cellular functional activity of the CTLA4 end of TIGIT-CTLA4-FcG4-Enhl can be identified by co-culturing Raji cells (purchased from Nanjing Kebai, catalog number: CB P60272) and IL-2-Luc Jurkat cells. Raji cells highly express CD80 and CD86 proteins on their surface, while IL-2-Luc Jurkat cells overexpress CD28 protein on their surface. CD80 / CD86-CD28 is an important co-stimulatory signal during T cell activation. In the co-culture system, the activation level of the CD80 / CD86-CD28 signaling pathway can be determined by the expression level of the downstream IL-2 promoter-activated Luciferase gene. The specific experimental process is as follows:

[0200] First, Raji cells in the logarithmic growth phase were collected, centrifuged, washed once with DPBS, and then resuspended in serum-free RPMI1640 medium. 6,000 cells were plated per well of a 96-well cell culture plate. 40 μL of anti-CD3 antibody (purchased from Invitrogen, Cat. No. 14-0037-82) was then added to a working concentration of 53.33 ng / mL. Eleven concentrations of the test sample were diluted 3.5-fold, with a maximum working concentration of 5,000 nM. The samples were then added to the plate and incubated at room temperature for 30 minutes. IL-2-Luc Jurkat cells in the logarithmic growth phase were collected, centrifuged, washed once with DPBS, and resuspended in serum-free RPMI1640 medium. 120,000 cells were plated per well of the plate. After gentle shaking, the plate was incubated at 37°C. The final volume per well was 120 μL / well. After incubation for 20 hours, 100 μL of Bio-Glu reagent (purchased from Promega, catalog number G7940) was added to detect fluorescence. The experimental results are shown in Figure 11. The bispecific fusion protein TIGIT-CTLA4-FcG4-Enhl has significantly better inhibitory activity on CD80 / CD86-CD28 downstream signaling than the single fusion proteins CTLA4-Fc and CTLA4-Fc-LEAY. The IC values ​​of TIGIT-CTLA4-FcG4-Enhl, CTLA4-Fc, and CTLA4-Fc-LEAY were obtained by four-parameter curve fitting analysis. 50 They are 74.47nM, 1976nM and 1409nM respectively.

[0201] Example 10 Inhibitory Function of Bispecific Fusion Proteins in Mature DC-Mediated T Cell Activation

[0202] In the process of mature DC-induced T cell activation, the activation of co-activation signaling pathways is key to T cell activation. The TIGIT-CTL A4-FcG4-Enhl bispecific fusion protein can competitively bind to CD155 and CD80 / CD86, respectively, thereby blocking the activation of these two co-activation signaling pathways. In this example, the biological function of the fusion protein was tested by measuring the concentration of IL-2 secreted in the cell supernatant when DC cells induced T cell activation. The specific experimental process is as follows:

[0203] Prepare 5 μg / mL anti-human CD3 antibody (purchased from Invitrogen, catalog number 14-0037-82) to coat a 96-well cell culture plate, seal it with sealing film, and place it at 4 degrees overnight; before adding the isolated T cells, discard the liquid in the plate and add 200 μL sterile PBS to wash twice. According to the instructions, use a negative kit (purchased from STEMCELL, catalog number: 17952) to isolate CD4 from fresh PBMC cells.+ CD8 T cells were isolated from fresh PBMC cells using a positive selection kit (purchased from STEMCELL, catalog number: 17853). + T cells were isolated from fresh PBMC cells using CD14 magnetic beads (Miltenyi Biotec, catalog number 130-050-201). + cells; CD14 + The cells were induced with 50 ng / mL human IL-4 (purchased from R&D, product number 204-IL-020 / CF) and 50 ng / mL rhGM-CSF (purchased from R&D, product number 7954-GM-020 / CF) for 7 days to differentiate into DC cells. The medium was changed every three days. On the eighth day, 50 ng / mL rhGM-CSF + 50 ng / mL IL-4 + 100 ng / mL LPS (purchased from Merck, product number L2630-25MG) was used to induce for 1-2 days to obtain mature DC cells. The mature DC cells obtained above were resuspended in RPMI1640 complete medium to 3.33×10 5 50 μL of the protein was added to each well of a 96-well plate coated with CD3 antibody, and the protein dilution to be tested was added. The cells were incubated at room temperature for 30 minutes, and then the CD4 + T or CD8 + T cells were resuspended in RPMI1640 complete medium to a volume of 1 × 10 6Cells were cultured at 37°C for 1 h. The cell culture supernatant was collected after 24 h for IL-2 detection. The experimental process of IL-2 detection is as follows: human IL-2 antibody (purchased from Invitrogen, product number 14-7029-81) was diluted to 1 μg / mL with ELISA coating solution, coated with ELISA plate, 100 μL / well, placed in a wet box, 4 ° C, coated for 16 hours; the ELISA plate was washed three times with PBST to remove unbound antigens, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, and then blocked with 2% BSA prepared in PBS, 200 μL / well, at room temperature for 2 hours; washed once with PBST to wash away excess blocking solution, and patted the ELISA plate dry to remove excess liquid, added cell supernatant, and at the same time, IL-2 protein standard (purchased from R&D, product number 202-IL-050 / CF) was added at a starting concentration of 500 ng / mL and a 3-fold gradient The plate was diluted 15 times in a concentration gradient, 100 μL / well, and incubated at room temperature for 1.5 h; the ELISA plate was washed three times with PBST and patted dry, and the biotinylated IL-2 antibody (purchased from Invitrogen, product number M600B) was diluted to 1 μg / mL with antibody diluent, added to the ELISA plate, 100 μL / well, and incubated at room temperature for 1 h; the ELISA plate was washed three times with PBST and patted dry, and the HRP-SA secondary antibody was diluted 1:5000 with antibody diluent, added to the ELISA plate, 100 μL / well, and incubated at room temperature for 30 min; the plate was washed five times with PBST, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, and TMB color development solution (purchased from KPL, product number: 5120-0075) was added, 100 μL / well, and color was developed to the appropriate depth, and 2M H2SO4, 50 μL / well, was added to stop the color development, and the absorbance A was measured at a wavelength of 450 nm in a multifunctional microplate reader to analyze the data.

[0204] The IL-2 inhibition rate was calculated as follows:

[0205] IL-2 inhibition rate (%) = (1-A 样品 / A 阴性对照 )×100%.

[0206] As shown in Figure 12, TIGIT-CTLA4-FcG4-Enhl can dose-dependently inhibit DC cell-induced CD4 + When T cells secrete IL-2 and the concentration range in this experiment is above 4.89 nM, the inhibitory activity of TIGIT-CTLA4-FcG4-Enhl is significantly better than that of TIGIT and CTLA4 single fusion proteins at equimolar concentrations, as well as the combination of the two single fusion proteins.

[0207] As shown in Figure 13, TIGIT-CTLA4-FcG4-Enhl can dose-dependently inhibit DC cells from inducing CD8 + When T cells secrete IL-2 and the concentration range in this experiment is above 1.39 nM, the inhibitory activity of TIGIT-CTLA4-FcG4-Enhl is significantly better than that of equimolar concentrations of TIGIT and CTLA4 single fusion proteins, as well as the combination of two single fusion proteins (7.8 nM is the maximum inhibitory activity concentration of CTLA4-Fc).

[0208] The above experimental results show that the bispecific fusion protein TIGIT-CTLA4-FcG4-Enhl can inhibit CD4 + and CD8 + There is an additive effect when T cells are activated. Specifically, when the concentration of the fusion protein is higher than 4.89nM, the inhibitory activity exceeds that of TIGIT and CTLA4 single fusion proteins at the same concentration. In addition, it was found that targeting TIGIT has an inhibitory effect on CD4 + The T cell response was significantly stronger than that of CD8 + For T cells, this may be due to the differences in the two co-activation signals CD155 / CD226 and CD80 / CD86 / CD28 among T cell subsets, which seems to explain the superposition effect of TIGIT and CTLA4 bispecific fusion proteins.

[0209] Example 11 In vivo efficacy study of fusion protein in collagen-induced arthritis model

[0210] Collagen-induced arthritis (CIA) is an experimental autoimmune disease that can be induced by immunizing susceptible rodent strains (rats and mice) with type II collagen. Immunized animals can develop an immune-mediated polyarthritis. Polyarthritis and human rheumatoid arthritis have the same clinical, histological, and immunological characteristics. In this example, 6-8 week old DBA / 1 male mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were used for modeling. On Day 0 (Day 0), an 8 mg / mL collagen solution (dissolved in 0.1 M acetic acid) was emulsified with an equal volume of complete Freund's adjuvant (purchased from Sigma) using a high-speed homogenizer. The collagen emulsion was injected subcutaneously at a single point at the base of the tail of the modeling group mice. Three weeks later, on Day 21 (Day 21), the same amount of collagen emulsion as on Day 1 was injected again at the base of the tail. Seven days after the second immunization (Day 28), the modeling mice were divided equally into five groups based on arthritis scores, including a model control group (administered with a vehicle) and four drug-administered groups, each with seven mice. Seven mice not immunized with collagen emulsion were also selected and given a vehicle as a normal control group. Drug administration began on the day of grouping and was intraperitoneally injected three times a week. The dose of TIGIT-Fc, TIGIT-Fc-Y113W, and the control drug Abatacept (sequence shown below) was 5 mg / kg, and the dose of TIGIT-CTLA4-FcG4-Enhl (hereinafter referred to as TIGIT-CTLA4-Fc-Enhl) was 7 mg / kg. From the second immunization onward, the arthritis scores of the limbs of each group were observed twice weekly until the end of the study. The scoring criteria were as follows: 0, no redness or swelling; 1, redness or swelling of the first or second interphalangeal joints or mild redness or swelling of the midfoot (tarsal bones) or ankle joint; 2, redness or swelling of the third or fourth interphalangeal joints or mild redness or swelling from the ankle joint to the midfoot (tarsal bones); 3, moderate redness or swelling from the ankle joint to the metatarsal joint; and 4, severe redness or swelling from the toes or fingers to the ankle joint or wrist joint. Before modeling on day 0, the footpad thickness of each animal's hind paw was measured using a screw micrometer (purchased from Mitutoyo). Starting from day 21, the footpad thickness was measured twice weekly, and the pre- and post-immunization footpad thicknesses were compared. The arthritis score and footpad thickness measurement data were recorded and analyzed. The results are shown in Figures 14 and 15. Compared with the normal control group, the animals in the model control group and each drug treatment group developed obvious arthritis symptoms, proving that the model was successful. Compared with the model control group, the arthritis symptoms of each animal were significantly alleviated after drug treatment. Among them, after treatment with TIGIT-Fc-Y113W and TIGIT-CTLA4-Fc-Enhl, the arthritis symptoms of the animal's limbs and the thickness of the bilateral hind limb pads recovered most significantly, which was better than the TIGIT-Fc treatment group and the control drug Abatacept treatment group.

[0211] This application relates to the sequence:

[0212] SEQ ID NO.1: CTLA4 extracellular region (Ala37-Phe162) amino acid sequence

[0213] SEQ ID NO.2: CTLA4 extracellular region (Ala37-Phe162) nucleic acid sequence

[0214] SEQ ID NO.3: TIGIT extracellular region (Met22-Pro141) amino acid sequence

[0215] The mutation positions are in italic bold and underlined: I42, Q56, N70, L73, G74, Y113.

[0216] SEQ ID NO.4: TIGIT extracellular region (Met22-Pro141) nucleic acid sequence

[0217] SEQ ID NO.5: Amino acid sequence of the extracellular region of CD28 (Asn19-Pro152)

[0218] SEQ ID NO.6: Amino acid sequence of the extracellular region of CD226 (Glu19-Asn247)

[0219] SEQ ID NO.7: IgG1-Fc amino acid sequence

[0220] SEQ ID NO.8: IgG4-Fc (S228P) amino acid sequence

[0221] SEQ ID NO.9: CTLA4-LEAY amino acid sequence

[0222] SEQ ID NO.10: CTLA4-LEAY nucleic acid sequence

[0223] SEQ ID NO.11: Amino acid sequence of the extracellular region of CD86 (Leu20-Asp238)

[0224] SEQ ID NO.12: Amino acid sequence of CD155 extracellular region (Gly27-Asn343)

[0225] SEQ ID NO.13: CTLA4-Fc-TIGIT amino acid sequence

[0226] SEQ ID NO.14: CTLA4-Fc-TIGIT nucleic acid sequence

[0227] SEQ ID NO.15: CTLA4-TIGIT-Fc amino acid sequence

[0228] SEQ ID NO.16: CTLA4-TIGIT-Fc nucleic acid sequence:

[0229] SEQ ID NO.17: TIGIT-CTLA4-Fc amino acid sequence

[0230] SEQ ID NO.18: TIGIT-CTLA4-Fc nucleic acid sequence

[0231] SEQ ID NO.19: TIGIT-Fc-CTLA4 amino acid sequence

[0232] SEQ ID NO.20: TIGIT-Fc-CTLA4 nucleic acid sequence

[0233] SEQ ID NO.21: TIGIT extracellular region-Y113W amino acid sequence

[0234] SEQ ID NO.22: TIGIT extracellular region-Y113W nucleic acid sequence

[0235] SEQ ID NO.23: CTLA4-FcG4-TIGIT-Enhl amino acid sequence

[0236] SEQ ID NO.24: CTLA4-FcG4-TIGIT-Enhl nucleic acid sequence

[0237] SEQ ID NO.25: CTLA4-TIGIT-FcG4-Enhl amino acid sequence

[0238] SEQ ID NO.26: CTLA4-TIGIT-FcG4-Enhl nucleic acid sequence

[0239] SEQ ID NO.27: TIGIT-CTLA4-FcG4-Enhl amino acid sequence

[0240] SEQ ID NO.28: TIGIT-CTLA4-FcG4-Enhl nucleic acid sequence

[0241] SEQ ID NO.29: TIGIT-FcG4-CTLA4-Enhl amino acid sequence

[0242] SEQ ID NO.30: TIGIT-FcG4-CTLA4-Enhl nucleic acid sequence

[0243] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and that such equivalents also fall within the scope of the claims appended hereto. Industrial Applicability

[0244] After multiple rounds of protein engineering and screening, this application has obtained a bispecific fusion protein CTLA4-TIGIT that can competitively inhibit the activation of CD28-CD80 / 86 and CD226-CD155 pathways, and exerts a synergistic inhibition of CD4 beyond the use of CTLA4 or TIGIT fusion protein alone. + 、CD8 + The T cell activation effect will have a more effective and broader therapeutic effect in related autoimmune diseases.

[0245] Through extensive research and analysis, this application has identified the key amino acid sites that affect the stability of the "LOCK-and-KEY" of the TIGIT / CD155 interaction interface, and on this basis, through multiple rounds of screening, a TIGIT polypeptide variant with significantly improved affinity and slower dissociation rate has been obtained, achieving unexpected technical effects. This TIGIT polypeptide variant has better biological activity, including stronger CD226 / CD155 signaling pathway blocking ability, and can exert stable immunomodulatory activity at a smaller dose. When used in the clinical treatment of immune system diseases, it can benefit from effectiveness, safety, stability, durability and other aspects.

[0246] This application provides a new dual-target combination solution, which is the first time to combine the TIGIT extracellular domain protein and the CTLA4 extracellular domain protein to construct a new fusion protein. The TIGIT / CTLA4 fusion protein of this application can better play the role of inhibiting T cell activation or proliferation, inhibiting the release of inflammatory factors, etc. by doubly blocking the CD226 / CD155 signaling pathway and the CD80 / CD86-CD28 signaling pathway, thereby solving clinical problems that cannot be solved by single drugs or combination drugs. As shown by the exemplary TIGIT / CTLA4 fusion protein, it has signaling pathway inhibition ability and T cell inhibitory activity that are superior to each single-target fusion protein, and the T cell inhibitory activity is better than the combination of TIGIT fusion protein and CTLA4 fusion protein, and has unexpected synergistic or superimposed activity.

Claims

1. A TIGIT polypeptide variant comprising the extracellular region of TIGIT or a functional fragment thereof, characterized in that, The variant relative to the amino acid sequence SEQ ID NO.3 contains at least one amino acid mutation, and the mutation positions are selected from one or more of the following groups: residues I42, Q56, N70, L73, G74, Y113.

2. The TIGIT polypeptide variant according to claim 1, wherein The amino acid mutation is an amino acid substitution.

3. The TIGIT polypeptide variant according to claim 1 or 2, characterized in that, The amino acid mutation is selected from one or more of the following groups: I42V, I42L, Q56N, Q56E, N70Q, N70H, L73I, L73V, G74S, Y113W, Y113T, Y113F.

4. The TIGIT polypeptide variant according to any one of claims 1-3, characterized in that Comprising the amino acid sequence SEQ ID NO.21 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence SEQ ID NO.

21.

5. A fusion protein comprising a TIGIT polypeptide variant according to any one of claims 1-4.

6. The fusion protein according to claim 5, wherein, Further comprising (a) a CTLA4 polypeptide or a variant thereof, and / or b) an Fc polypeptide or a variant thereof, wherein the CTLA4 polypeptide comprises an extracellular region or a functional fragment thereof.

7. The fusion protein according to claim 6, wherein The CTLA4 polypeptide variant relative to the amino acid sequence SEQ ID NO.1 contains A30Y and / or L105E amino acid mutations.

8. The fusion protein according to claim 6 or 7, characterized in that, The CTLA4 polypeptide or a variant thereof comprises an amino acid sequence selected from the following: a) SEQ ID NO.1; b) SEQ ID NO.9; c) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO.1 or SEQ ID NO.

9.

9. The fusion protein according to claim 6, wherein The Fc polypeptide is a monomer or dimer of a human IgG1, IgG2, or IgG4 Fc polypeptide.

10. The fusion protein according to claim 6, wherein, The Fc polypeptide comprises a hinge region, a CH2 region, and a CH3 region or a CH2 region and a CH3 region; preferably, the Fc polypeptide variant contains amino acid mutations that can improve the stability or half-life of the Fc; preferably, comprising one or more amino acid mutations selected from the following groups: M428L, N434S, M252Y, S254T, T256E, IgG4(S228P); more preferably, the IgG1 Fc polypeptide variant contains M428L, N434S or M428L / N434S, M252Y, S254T, T256E or M252Y\S254T\T256E amino acid mutations; the IgG4 Fc polypeptide variant contains S228P, M428L, N434S, M428L / N434S, M428L / S228P, N434S / S228P, M428L / N434S / S228P, M252Y / S228P, S254T / S228P, T256E / S228P or M252Y\S254T\T256E / S228P amino acid mutations.

11. The fusion protein according to claim 6 or 10, wherein The Fc polypeptide or a variant thereof comprises an amino acid sequence selected from the following: a) SEQ ID NO.7; b) SEQ ID NO.8; c) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO.7 or SEQ ID NO.

8.

12. The fusion protein according to claim 6, wherein The fusion protein comprises a polypeptide monomer selected from the following or a homologous or heterologous dimer thereof: a) A-L-Fc-L-B; b) B-L-Fc-L-A; c) A-L-B-L-Fc; d) B-L-A-L-Fc; e) Fc-L-A-L-B; f) Fc-L-B-L-A; Wherein A is a TIGIT polypeptide variant, B is a CTLA4 polypeptide or a variant thereof, and L is absent or is a linker; preferably, the linker is (G4S)n or (SG4)m, where n or m is a positive integer selected from 0, 1, 2, 3, 4, 5, 6.

13. The fusion protein according to claim 5, wherein The fusion protein comprises an amino acid sequence selected from SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.29 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology therewith.

14. The fusion protein according to claim 5, wherein The fusion protein comprises the amino acid sequence of SEQ ID NO.27 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 99% sequence homology therewith.

15. A nucleic acid, characterized in that, Encoding the polypeptide variant according to any one of claims 1-4 or the fusion protein according to any one of claims 5-14.

16. An expression vector, characterized in that, Comprising the nucleic acid according to claim 15.

17. A host cell, characterized in that, Comprising the expression vector according to claim 16 or expressing the polypeptide variant according to any one of claims 1-4 or the fusion protein according to any one of claims 5-14.

18. A method for preparing a polypeptide variant according to any one of claims 1-4 or a fusion protein according to any one of claims 5-14, characterized in that, Comprising the following steps: Synthesizing the polypeptide variant according to any one of claims 1-4 or the fusion protein according to any one of claims 5-14, and / or culturing the host cell according to claim 17 under conditions for expressing the polypeptide variant according to any one of claims 1-4 or the fusion protein according to any one of claims 5-14.

19. A pharmaceutical composition, characterized in that, Comprising the polypeptide variant according to any one of claims 1-4, the fusion protein according to any one of claims 5-14 and optionally a pharmaceutically acceptable excipient or carrier.

20. Use of the polypeptide variant according to any one of claims 1-4, the fusion protein according to any one of claims 5-14, or the pharmaceutical composition according to claim 19 in the preparation of a drug for preventing or treating immune system diseases or tumors; preferably, the immune system diseases include lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes, myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatism, arthritis, psoriasis, T cell lymphoma, autoimmune graft-versus-host disease (GVHD), organ transplant rejection.

21. The method according to claim 20, wherein The diabetes is type I diabetes.

22. The method according to claim 20, wherein The rheumatism or arthritis is rheumatoid arthritis.

23. A method for preventing or treating an immune system disease or a tumor, comprising administering to a subject a pharmaceutically effective amount of the polypeptide variant according to any one of claims 1-4, the fusion protein according to any one of claims 5-14, or the pharmaceutical composition according to claim 19.

24. The method according to claim 23, wherein The immune system diseases include lupus erythematosus, lupus nephritis, Hashimoto's thyroiditis, diabetes, myasthenia gravis, pemphigus, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, chronic active hepatitis, ulcerative colitis, rheumatism, arthritis, psoriasis, T-cell lymphoma, and autoimmune graft-versus-host disease (GVHD).

25. The method according to claim 24, wherein The diabetes is type I diabetes.

26. The method according to claim 24, wherein The rheumatism or arthritis is rheumatoid arthritis.

27. The method according to any one of claims 23-26, characterized in that, The method further comprises administering a second active molecule, and the second active molecule includes an immunosuppressive drug, an immunomodulatory drug, or an anti-inflammatory drug.

28. A fusion protein, characterized in that, It comprises a first structural region and a second structural region, or comprises a first structural region, a second structural region, and a third structural region; the first structural region is a TIGIT polypeptide or a variant thereof, the second structural region is a CTLA4 polypeptide or a variant thereof, and the third structural region is an Fc polypeptide or a variant thereof; the TIGIT or CTLA4 comprises an extracellular region or a functional fragment thereof.

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