Fusion protein linked with CD40-expressing cells containing APC and target-specific T cells and application of fusion protein

Abstract

The invention discloses a fusion protein linked with CD40-expressing cells containing APC and target specific T cells and application thereof, CD40L and other elements combined with CD40 are fused with target antigens specifically recognized by molecules in cells such as CAR-T or TCR-T to form a bifunctional protein, the DC maturation of the CD40L and other elements combined with CD40 can be enhanced, and meanwhile, the CD40L and other elements combined with CD40 can be combined with the target antigens specifically recognized by the molecules in the cells such as CAR-T or TCR-T to form the bifunctional protein, so that the CD40L and other elements combined with CD40 can be combined with the target antigens specifically recognized by the molecules in the cells. The target antigen is effectively delivered to the surface of the DC, so that the activation effect of the DC on cells such as CAR-T or TCR-T is enhanced, and particularly, the effect of enhancing the functions such as terminal amplification and reverse depletion of the cells such as CAR-T or TCR-T is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of fusion proteins, specifically relating to a class of fusion proteins that link CD40-expressing cells containing APCs with target-specific T cells and their applications. Background Technology

[0002] In recent years, TNF-R-SF members, including CD40, have been key targets for immunotherapy. CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on immune cells, particularly B cells, dendritic cells, and monocytes. CD40 plays a crucial role in connecting innate and adaptive immunity. CD40L and CD40 antibodies can bind to CD40 on the surface of dendritic cells (DCs), inducing DC maturation, upregulating co-stimulatory molecules (such as CD80 and CD86) and MHC (major histocompatibility complex) molecules, and secreting various cytokines. This helps DCs more effectively activate T cells, thereby inducing a stronger antigen-specific immune response. CD40L is a type II transmembrane protein belonging to the tumor necrosis factor gene superfamily, mainly expressed on activated T cells and platelets. After stimulation by T cell receptors (TCRs) and CD28, CD40L is regulated on the surface of T cells, reaching peak levels after 6 hours and then remaining expressed on their surface for 24 hours.

[0003] A key function of the CD40-CD40L interaction is the activation and "permissioning" of dendritic cells (DCs) to initiate CD8+ effector T cells. This is achieved through the upregulation of cell surface co-stimulatory molecules and MHC molecules, as well as the production of cytokines by DCs, leading to the activation of effective T cells. In the absence of CD40 signaling, "unpermissioned" DC activation results in T cell unresponsiveness or absence and the production of Tregs. Similarly, CD40 binding on B cells leads to their activation, proliferation, and enhanced antigen presentation. Furthermore, CD40-activated macrophages can kill tumor cells and, in some cases, deplete the tumor matrix. Therefore, CD40 on APCs plays a crucial role in inducing an effective immune response.

[0004] Based on the above background, this invention proposes a fusion protein that can link CD40-expressing cells, including APCs, with target-specific T cells. This protein can promote the binding of CD40-expressing cells, including APCs, to T cells that specifically recognize antigens. This enhances or assists the population function and expansion of specific T cells, such as CAR-T cells, in any APC-gene-edited T cell culture system, providing research tools and new ideas for immune regulation. Summary of the Invention

[0005] This invention fuses a CD40 targeting element (containing CD40L and CD40 antibody) with a target antigen that can activate T cells to form a bifunctional protein. This allows the CD40 targeting element to enhance DC maturation while effectively delivering the target antigen to the surface of DCs, thereby enhancing the activation effect of DCs on CAR-T cells. In particular, it has significant effects on enhancing the late-stage expansion of CAR-T cells and reversing their exhaustion.

[0006] To achieve the above objectives, the present invention proposes the following technical solution:

[0007] The first aspect of the present invention provides a fusion protein linking CD40-expressing cells, including APCs, and target-specific T cells, said fusion protein being composed of an antigen unit and a target unit.

[0008] An antigen (Ag) is a substance that can induce antibody production; it is any substance that can trigger an immune response. Foreign molecules can be recognized by immunoglobulins on B cells or processed by antigen-presenting cells and bind to the major histocompatibility complex to form a complex, which then reactivates T cells, triggering a continuous immune response. In this invention, an antigen unit refers to a substance with immunogenicity. In some embodiments, an antigen unit can be full-length or a fragment containing antigenic determinants.

[0009] Furthermore, the antigen unit is derived from single-chain antibodies, single-chain peptide-MHC, bacterial antigens, viral antigens, cancer-associated antigens, cancer-specific antigens, or fragments with antigenic determinants.

[0010] In some embodiments, the adapter, such as the (GGGGS)3 adapter, links VH and VL in the single-chain antibody. In some embodiments, the single-chain antibody is derived from monoclonal Ig produced by myeloma or lymphoma cells. In some embodiments, the antigen unit is telomerase or a functional portion thereof. Telomerase may be hTERT. In some embodiments, the antigen unit is melanoma antigen. Melanoma antigen may be tyrosinase, TRP-1, or TRP-2. In some embodiments, the antigen unit is prostate cancer antigen. Prostate cancer antigen may be PSA. In some embodiments, the antigen unit is cervical cancer antigen. Cervical cancer antigen is selected from E1, E2, E4, E6, E7, L1, and L2. In some embodiments, the antigen unit is hepatocellular carcinoma antigen. Hepatocellular carcinoma antigen may be GPC3. In some embodiments, the antigen unit is derived from bacteria. In some embodiments, the bacterial-derived antigen unit is tuberculosis antigen. In some embodiments, the bacterial-derived antigen unit is brucellosis antigen. In some embodiments, the antigen unit is derived from a virus. In some embodiments, the viral-derived antigen unit is derived from HIV. In some embodiments, the HIV-derived antigenic unit is derived from gp120 or Gag. In some embodiments, the antigenic unit is selected from influenza virus hemagglutinin (HA), nucleoprotein, and M2 antigen; and herpes simplex 2 antigen glycoprotein D.

[0011] In some implementations, the antigen unit is a single-chain peptide-MHC.

[0012] Furthermore, the single-chain peptide-MHC comprises (1) a heavy chain domain; (2) a β2-microglobulin (β2m) domain; and (3) a specific peptide sequence.

[0013] Furthermore, the heavy chain domain includes the heavy chain domain of the MHC molecule.

[0014] Furthermore, the β2-microglobulin (β2m) domain includes the β2-microglobulin (β2m) domain of the MHC molecule.

[0015] In this invention, the term "single-chain peptide-MHC" refers to a single continuous polypeptide chain constructed through genetic engineering. This polypeptide chain comprises at least: (1) a heavy chain domain of an MHC molecule; (2) a β2-microglobulin (β2m) domain; and (3) a specific peptide sequence. The above three components are covalently linked into a single polypeptide chain, enabling the polypeptide chain to stably present the peptide and maintain a conformation similar to that of the native MHC molecule for use in specific antigen presentation and related immune response processes. The MHC molecules include three major classes: class I antigens (HLA-A, B, C, E, F, G), class II antigens (HLA-DP, HLA-DQ, and HLA-DR), and class III antigens. Class I antigens include conventional / classical MHC Ia antigens, HLA-A, HLA-B, and HLA-C, as well as non-classical MHC Ib antigens HLA-E, HLA-F, and HLA-G. MHC class I antigens contain three globular domains (α1, α2, and α3). The MHC I complex also contains β2-microglobulin and a presented peptide bound to a peptide-binding groove, which includes the α1 and α2 domains.

[0016] HLA is an expression product of the human histocompatibility complex (MHC) and is an important antigenic substance constituting transplant rejection. MHC is divided into class I and class II molecules. MHC class I molecules are widely distributed on the surface of almost all types of cells, including all nuclear cells and some plasma membrane cells. MHC class II molecules are mainly expressed on the surface of specialized antigen-presenting cells, such as macrophages, B cells, and some dendritic cells. The process of MHC molecules binding to antigens is highly specific and dynamic. The antigen-binding region of an MHC molecule can adapt to a variety of antigen fragments. This binding leads to the formation of an MHC-antigen complex, which allows T cells to recognize and interact with it.

[0017] In this invention, the term "specific peptide sequence" or immunomodulatory peptide refers to an immunomodulatory peptide obtained from organisms or food proteins through various pathways. In a broad sense, it can refer to all peptide molecules with immunomodulatory activity; in a narrow sense, it refers to small (oligo)peptides with relatively small molecular weights that possess immunomodulatory activity. Immunomodulatory peptides can be classified in various ways, including by biological species (e.g., animals, plants, microorganisms), method of acquisition, location in the organism, type of precursor protein, and immunomodulatory effect (stimulation / inhibition).

[0018] Further, single chain peptide-MHC includes HLA-E, HLA-DR, HLA-PRAME, HLA-NY-ESO-1, HLA-HPV E7, HLA-MAGEA1, HLA-MAGEA4, HLA-Survivin, HLA-WT1, HLA-MUC1, HLA-KRAS, HLA-P53, HLA-EBV, HLA-HBV, HLA-AFP.

[0019] Furthermore, the targeting unit is a CD40 binding domain.

[0020] Furthermore, the CD40 binding domain is derived from the extracellular domain of CD40L, other fragments of CD40L that can bind to CD40, and an antibody against CD40.

[0021] In this invention, CD40 is also known as p50, Bp50, CDW40, and TNFRSF5. This term encompasses full-length, unprocessed CD40, as well as any form of CD40 derived from cell processing. This term encompasses naturally occurring variants of CD40 (e.g., splice variants or allelic variants). This term encompasses, for example, the CD40 gene, human CD40, and CD40 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, CD40 is human CD40, with a gene ID of 958.

[0022] CD40L is a homologous ligand of CD40, also known as CD154 or TNFSF5, and is a 39 kDa type II transmembrane protein.

[0023] In this invention, CD40L is also known as IGM, IMD3, TRAP, gp39, CD154, CD40LG, HIGM1, T-BAM, and TNFSF5. This term encompasses full-length, unprocessed CD40L, as well as any form of CD40L derived from cell-processed sources. This term encompasses naturally occurring variants of CD40L (e.g., splice variants or allelic variants). This term encompasses, for example, the CD40L gene, human CD40L, and CD40L from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, CD40L is human CD40L with gene ID 959.

[0024] Furthermore, the fusion protein also includes a linker.

[0025] In this invention, a linker refers to the site in a fusion protein containing two recombined genes or proteins used for linking. Linkers can be flag, His, HA, Myc, or E-TAG. Linkers can be flexible linkers, rigid linkers, or spliceable linkers. A flexible linker refers to the amino acid spacer between the gene and the tag; it can be GGAGGG, (Gly)n, or a GS combination, i.e., (Gly-Gly-Gly-Gly-Ser)n or (GGGGS)n. A rigid linker can be a linker formed by an α-helix with an (EAAAK)n sequence or a Pro-rich sequence (XP)n linker. Spliceable linkers can be 2A peptides and IRES linking peptides.

[0026] Furthermore, the antigen unit and the target unit may or may not be connected by a connector.

[0027] Furthermore, the fusion protein is obtained by sequentially connecting an antigen unit and a targeting unit.

[0028] Furthermore, the fusion protein is obtained by sequentially connecting an antigen unit, a linker, and a targeting unit.

[0029] Furthermore, the antigen units are derived from AFP, AXL, B4GALNT1, BCMA, CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD99, CD123, CD133, CD174, CD274, CD276, CDH17, CDS, CTAG1B, CEA, CLEC12A, CLDN6, CLDN 18.2, CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, GUCY2C, HER2, and HPV. E7, IL1RAP, IL3RA, IL13Rα2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PROM1, PSCA, PSMA, RO R1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNFRSF8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, ULBP2, HLA-E, HLA-DR, HLA-PRAME, HLA-NY-ESO-1, HLA-HPV E7, HLA-MAGEA1, HLA-MAGEA4, HLA-Survivin, HLA-WT1, HLA-MUC1, HLA-KRAS, HLA-P53, HLA-EBV, HLA-HBV, HLA-AFP.

[0030] Furthermore, the antigen unit is selected from GPC3, HLA-PRAME, HLA-NY-ESO-1, CD19, and MSLN.

[0031] Furthermore, the GPC3 comprises an amino acid sequence such as SEQ ID NO:1 or having at least 80% sequence identity with SEQ ID NO:1.

[0032] Furthermore, the HLA-PRAME contains an amino acid sequence such as SEQ ID NO:2 or having at least 80% sequence identity with SEQ ID NO:2.

[0033] Furthermore, the HLA-NY-ESO-1 contains an amino acid sequence such as SEQ ID NO:3 or having at least 80% sequence identity with SEQ ID NO:3.

[0034] Furthermore, CD19 comprises an amino acid sequence such as SEQ ID NO:4 or having at least 80% sequence identity with SEQ ID NO:4.

[0035] Furthermore, the MSLN contains an amino acid sequence such as SEQ ID NO:5 or having at least 80% sequence identity with SEQ ID NO:5.

[0036] Furthermore, the targeting unit is selected from the CD40L extracellular domain, antibody structures that recognize CD40, or other elements that bind to CD40.

[0037] Furthermore, the CD40L extracellular segment contains an amino acid sequence such as SEQ ID NO:6 or having at least 80% sequence identity with SEQ ID NO:6.

[0038] Furthermore, the antibody structure that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:7 or having at least 80% sequence identity with SEQ ID NO:7.

[0039] Furthermore, the antibody structure that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:8 or having at least 80% sequence identity with SEQ ID NO:8.

[0040] Furthermore, the connector includes a flexible linker, a rigid linker, and a splicable linker.

[0041] Furthermore, the linker is selected from the hinge region of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d.

[0042] Furthermore, the connector is a flexible linker.

[0043] Furthermore, the flexible linker is selected from flag and GS linker.

[0044] Furthermore, the GS linker includes (GGGGX). n The amino acid sequence represented by (G4X) is where n is an integer from 1 to 8 and X is C or S.

[0045] Furthermore, the GS linker is (GGGGS)3 or (G4S)3.

[0046] Furthermore, the flag contains an amino acid sequence such as SEQ ID NO:9 or having at least 80% sequence identity with SEQ ID NO:9.

[0047] Furthermore, (GGGGS)3 comprises an amino acid sequence such as SEQ ID NO:10 or having at least 80% sequence identity with SEQ ID NO:10.

[0048] Furthermore, the fusion protein also includes a signal peptide.

[0049] Furthermore, the signal peptide is selected from SP and SP-HLA.

[0050] Furthermore, the SP contains an amino acid sequence such as SEQ ID NO:11 or having at least 80% sequence identity with SEQ ID NO:11.

[0051] Furthermore, the SP-HLA contains an amino acid sequence such as SEQ ID NO:12 or having at least 80% sequence identity with SEQ ID NO:12.

[0052] In some embodiments, those skilled in the art may change the combination type and sequence of antigen units, target units, linkers and signal peptides according to actual conditions or needs. Regardless of the form of change, as long as the fusion protein has the target T cell corresponding receptor or edited recognition protein provided by the present invention and the CD40 molecule of APC cells or any cells expressing CD40, thereby achieving the linking of the two types of cells and activating downstream CD40 and downstream T cell receptor signals, it falls within the protection scope of the present invention.

[0053] Furthermore, the fusion protein comprises amino acid sequences such as SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20 or having at least 80% sequence identity with SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20.

[0054] The term "identity," also known as "homology," refers to an amino acid or nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared. In this invention, modified antibody sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of the fusion protein described in the first aspect of this invention are also within the scope of protection of this invention. The term "modification" refers to any form of modification to an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in a given amino acid sequence with a different amino acid. The term "deletion" refers to reducing one or more amino acids in a given amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence that result in the addition of one or more amino acids compared to the original amino acid sequence.

[0055] A second aspect of the present invention provides a biomaterial comprising any one of the following: 1) a nucleic acid encoding the fusion protein of the first aspect of the present invention; 2) a recombinant vector comprising the nucleic acid of 1); 3) a recombinant host cell comprising the nucleic acid of 1) and / or the recombinant vector of 2).

[0056] In this invention, the term "nucleic acid" or "nucleic acid molecule" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the fusion protein described in this invention, or a sequence having more than 80% identity with it, is isolated, the fusion protein can be obtained in large quantities using recombinant technology.

[0057] Furthermore, the nucleic acid contains nucleotide sequences encoding antigen units and target units.

[0058] Furthermore, the antigen unit and the target unit are derived from nucleotide sequences encoding single-chain antibodies, single-chain peptide-MHC, bacterial antigens, viral antigens, cancer-associated antigens, cancer-specific antigens, or fragments having antigenic determinants.

[0059] Furthermore, the targeting unit is a nucleotide sequence encoding a CD40 binding domain.

[0060] Furthermore, the CD40 binding domain includes a nucleotide sequence encoding an extracellular segment of CD40L, other fragments of CD40L that can bind to CD40, an anti-CD40 antibody, or other antibodies that can recognize CD40.

[0061] Furthermore, the nucleic acid also contains a nucleotide sequence encoding a linker.

[0062] Furthermore, the nucleic acid comprises a nucleotide sequence such as SEQ ID NO:21-40 or having at least 80% sequence identity with SEQ ID NO:21-40.

[0063] Furthermore, the recombinant vector includes plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, or other viral vectors.

[0064] The recombinant vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in host cells. The recombinant vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the recombinant vector contains a target gene encoding the fusion protein of this invention, a promoter, a terminator, or optionally a marker gene. The recombinant vector can use known vectors or vectors constructed in-house. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include lactoblastic viral vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc.

[0065] Furthermore, the recombinant vector is a lentiviral vector.

[0066] Furthermore, the recombinant host cell is selected from prokaryotic cells and eukaryotic cells.

[0067] The recombinant host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid provided by this invention. The host cell includes any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof.

[0068] A third aspect of the present invention provides a composition comprising the fusion protein described in the first aspect of the present invention.

[0069] Furthermore, the composition also includes pharmaceutical excipients.

[0070] The compositions described in this invention can be pharmaceutical compositions that can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used, non-toxic, pharmaceutically acceptable carrier, excipient, or formulation. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided that the target tissue can be reached.

[0071] In the context of this invention, pharmaceutical excipients or pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulation materials, or any type of formulation aid. The pharmaceutical excipients may also be binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0072] Furthermore, the composition also includes a detectable marker coupled thereto.

[0073] The detectable markers include at least one of the following: radioactive isotopes, metal nanomaterials, fluorescein, biotin, avidin, biotin / avidin protein complex, biotin / avidin protein complex, chromophores, electron-dense substances, and enzymes.

[0074] The fourth aspect of the present invention provides the use of the fusion protein described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, and / or the composition described in the third aspect of the present invention, the use including any one of the following: 1) use in the preparation of products for treating cancer; 2) use in the preparation of products for treating infectious diseases; 3) use in the preparation of products for treating autoimmune diseases; 4) use in the preparation of products for enhancing or assisting specific T cell function.

[0075] Furthermore, the products include pharmaceuticals, reagent kits, and vaccine formulations.

[0076] Furthermore, the drug and the vaccine formulation also include pharmaceutical excipients.

[0077] Furthermore, the cancers mentioned include solid tumors and hematologic malignancies.

[0078] Furthermore, the cancers include pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, breast cancer, and melanoma.

[0079] Furthermore, the lung cancer in question is non-small cell lung cancer.

[0080] Furthermore, the infectious diseases mentioned include chronic viral diseases and tuberculosis.

[0081] Furthermore, the chronic viral diseases include infectious diseases caused by hepatitis C virus, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, John Cunningham virus, and human papillomavirus.

[0082] Furthermore, the autoimmune diseases include ankylosing spondylitis (AS), psoriasis (PS or PSOR), celiac disease (CEL), systemic lupus erythematosus (SLE), common variant immunodeficiency disease (CVID), inflammatory bowel disease (IBD), ulcerative colitis (UC), type 1 diabetes mellitus (T1D), juvenile idiopathic arthritis (JIA), Crohn's disease (CD), alopecia areata (AA), multiple sclerosis (MS), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), rheumatoid arthritis (RA), Sjögren's syndrome (SJO), systemic sclerosis (SSC), spondyloarthritis (SPA), vitiligo (VIT), asthma, or thyroiditis (AITD, THY, or TH).

[0083] Furthermore, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TCR-T cells, and TruC-T cells.

[0084] The fifth aspect of the present invention provides a method comprising any one of the following: 1) a method for promoting the binding of APCs to specific T cells, the method comprising administering to a subject the fusion protein of the first aspect of the present invention and / or the composition of the fifth aspect of the present invention; 2) a method for enhancing or assisting the function of specific T cells, the method comprising administering to a subject the fusion protein of the first aspect of the present invention and / or the composition of the third aspect of the present invention.

[0085] The sixth aspect of the present invention provides a method for expressing the fusion protein described in the first aspect of the present invention, the method comprising artificial synthesis and genetic engineering techniques.

[0086] In some embodiments, the fusion protein of the present invention is obtained through artificial synthesis. Methods for artificially synthesizing proteins are known in the art, for example, the fusion protein of the present invention is obtained through direct amino acid synthesis. In some embodiments, the fusion protein of the present invention is obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0087] Furthermore, the genetic engineering technology refers to culturing the recombinant host cells described in the second aspect of the present invention or transfecting the recombinant vector described in the second aspect of the present invention into the host cells, so that the recombinant vector described in the second aspect of the present invention is transcribed and the fusion protein described in the first aspect of the present invention is expressed.

[0088] Another aspect of the present invention provides a method for treating cancer, infectious diseases, and autoimmune diseases, the method comprising administering to a subject the fusion protein described in the first aspect of the present invention and / or the composition described in the third aspect of the present invention, simultaneously or prior to administering an effective amount of immune cells containing a chimeric antigen receptor (CAR).

[0089] In some embodiments, the subject includes mammals; in a specific embodiment of the invention, the subject is preferably a human.

[0090] Furthermore, the prior administration refers to the administration of CAR immune cells at least about 6 months, at least about 9 months, or at least about 1 year before administering the fusion protein described in the first aspect of the present invention and / or the composition described in the third aspect of the present invention to the subject.

[0091] Furthermore, the simultaneous administration includes contacting or simultaneously administering the fusion protein described in the first aspect of the invention and / or the composition described in the third aspect of the invention with CAR immune cells in vitro prior to administration to the subject.

[0092] Furthermore, the cancers mentioned include solid tumors and hematologic malignancies.

[0093] Furthermore, the cancers include pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, breast cancer, and melanoma.

[0094] Furthermore, the lung cancer in question is non-small cell lung cancer.

[0095] Furthermore, the infectious diseases mentioned include chronic viral diseases and tuberculosis.

[0096] Furthermore, the chronic viral diseases include infectious diseases caused by hepatitis C virus, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, John Cunningham virus, and human papillomavirus.

[0097] Furthermore, the autoimmune diseases include ankylosing spondylitis (AS), psoriasis (PS or PSOR), celiac disease (CEL), systemic lupus erythematosus (SLE), common variant immunodeficiency disease (CVID), inflammatory bowel disease (IBD), ulcerative colitis (UC), type 1 diabetes mellitus (T1D), juvenile idiopathic arthritis (JIA), Crohn's disease (CD), alopecia areata (AA), multiple sclerosis (MS), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), rheumatoid arthritis (RA), Sjögren's syndrome (SJO), systemic sclerosis (SSC), spondyloarthritis (SPA), vitiligo (VIT), asthma, or thyroiditis (AITD, THY, or TH).

[0098] Furthermore, the immune cells are selected from T cells, CAR-T cells, STAR-T cells, TCR-T cells, and TruC-T cells.

[0099] Advantages and beneficial effects of the present invention: The present invention fuses CD40L or CD40 antibody with target antigens that are specifically recognized by molecules in cells such as CAR-T cells to form a bifunctional protein. This allows CD40L to enhance DC maturation while effectively delivering the target antigen to the surface of DCs, thereby enhancing the activation effect of DCs on CAR-T cells. In particular, it has significant effects on enhancing the functions of CAR-T cells in the late stage of expansion and reversing exhaustion. Attached Figure Description

[0100] Figure 1 A schematic diagram of a linker design targeting CD40 and T cell recognition receptors.

[0101] Figure 2Flow cytometry results showing the binding of different types of linkers to CD40-mCherry indicator cells and CD40ΔICD-mCherry cells.

[0102] Figure 3 This is a flow cytometry result of the linker binding to different types of effector T cells.

[0103] Figure 4 The results of DC activation of effector cells corresponding to the target CAR / STAR / TRuC / TCR-T in the 24h linker are shown in the figure.

[0104] Figure 5 The results of achieving specific expansion and subtype alteration of effector cells such as CAR / STAR / TRuC / TCR-T using a 72h linker.

[0105] Figure 6 The results of achieving specific amplification and subtype alteration of CAR-T effector cells using a 72-hour antibody linker are shown in the figure.

[0106] Figure 7 Flow cytometry results for identification of M0, M1, and M2 cells and 24-hour CAR-T activation effect.

[0107] Figure 8 A graph showing the differences between DC-activated CAR-T cells implemented by the linker and CAR-T cells activated by other target cells.

[0108] Figure 9 The diagram shows the results of using the DC autocrine linker to activate CAR-T and increase long-acting CAR-T killing. Detailed Implementation

[0109] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0110] Example

[0111] 1.293 Production of anti-CD40 / CD40L-antigen fusion protein (linker) supernatant can activate CD40 downstream signaling in reporter cells: construct CD40-mCherry expression lentiviral system plasmid, prepare lentivirus, and obtain CD40 / NF-κB reporter 293 cells. Plasmids expressing different proteins (protein linking sequence from N-terminus to C-terminus: Antigen-linker(flag / G4S)-CD40L / anti-CD40) were constructed as shown in the list (CD40L-flag-GPC3 SEQ ID NO 13, CD40L-(G4S)3-GPC3 SEQ ID NO 14, CD40L-flag-HLA-PRAME SEQ ID NO 15, CD40L-flag-HLA-NY-ESO-1 SEQ ID NO 16, anti-CD40scFv1-flag-GPC3[GILORALIMAB ABBV-927] SEQ ID NO 19, anti-CD40 scFv2-flag-GPC3[Selicrelumab-Roche] SEQ ID NO 20, CD40L-flag-MSLN, CD40L-flag-CD19 SEQ ID NO 19).

[0112] 17) After transfecting 293 cells using the PEI system, the supernatant of 293 cells was collected 24 hours later to obtain cells containing the corresponding linker. After co-incubation with 293 reporter cells overnight, flow cytometry analysis showed strong red fluorescence expression in the 293 reporter cells. The positive control was CD40L protein (1 μg / ml, Recombinant Human CD40L, novoprotein, Cat. No.: CI56). This indicates that the linker containing both CD40L and anti-CD40 scFv can bind to CD40 on 293 reporter cells and activate downstream signaling. To rule out potential non-specific activation, negative control reporter cells related to CD40ΔICD-mCherry were constructed. The results are as follows. Figure 2 As shown, this indicates that when these negative reporter cells bind to the linker, mCherry expression cannot be initiated.

[0113] 2.293 The linker supernatant can bind to effector T cells corresponding to the target: pCDH-EF1α lentiviral expression plasmid was constructed by synthesizing the sequences corresponding to GPC3 CAR-T (SEQ ID NO 41), CD19 CAR-T (SEQ ID NO 44), PR20 CAR-T (SEQ ID NO 45), and NY-ESO-1TCR-T (SEQ ID NO 46). The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered, and the lentivirus was stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and the cells were cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify the G4S or EGFRt molecule fraction in T cell editing proteins to confirm the CAR-T / TCR-T positivity rate. The supernatant containing the linker was co-incubated with the corresponding edited T cells for 1 hour, followed by washing with PBS and flow cytometry staining. Specific flow cytometry parameters: 3 × 10⁻⁶ cells / mL. 5 Cell counts were determined per well. Anti-flag (PE), anti-CD40L, and anti-G4S (CAR) were added, and after incubation for 30 minutes, the cells were washed with PBS and analyzed by flow cytometry. GPC3 CAR-T specifically recognized the GPC3 molecule linked to G4S / flag, CD40L, and anti-CD40 scfv.

[0114] CD19 CAR-T recognizes the CD19 molecule; PR20 CAR-T recognizes HLA-A*0201 and presents the molecular structure of PRAME (ALYVDSLFFL); NY-ESO-1 TCR-T recognizes HLA-A*0201 and presents the molecular structure of NY-ESO-1 (SLLMWITQC). Results are as follows... Figure 3 As shown, the dual-positive cell population consists of editing T cells and linker-binding cells.

[0115] 3. In the DC-T system, the linker successfully activated and promoted the expansion of target CAR / STAR / TRUC / T-T cells: The Attachment Solution Kit (Hycells, Shanghai; CAT:DCT01003-A) was used to promote the adhesion of monocytes from PBMCs to culture dishes. Then, the Dendritic Cells Differentiation Kit (Monocyte-Derived) (Hycells, Shanghai; CAT:DCT01003-kit) was used to induce the adherent monocytes into DCs. DCs were used approximately 48 hours after being added to maturation medium. The corresponding GPC3 CAR / STAR / TRUC-T cells were expanded to the plateau phase, and after adjusting the positive rate to approximately 20%, they were co-incubated with DCs at a ratio of 10:1. Experimental groups were set up by adding supernatant from 293T cells of different sources. The culture media were all x-vivo medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15, mixed 1:1 with linker 293 supernatant (or without linker 293 supernatant). After co-incubation for 24 hours, the activation status of CPC3 CAR-T (SEQ ID NO 41), STAR-T (SEQ ID NO 42), and TruC-T (SEQ ID NO 43) cells was detected. Following the same protocol, the NY-ESO-1TCR-T (SEQ ID NO 46) group was set up to examine the effect of CD40L-flag-HLA-NY-ESO-1 on TCR-T. The results are as follows: Figure 4 As shown, only the addition of the corresponding CAR / STAR / TRuC / TCR-T antigen can activate T cells by DCs, and only the supernatant containing the linker cannot directly activate T cells.

[0116] After 72 hours of co-incubation, the positivity rate and cell population of CAR / STAR / TRuC / TCR-T cells were detected using CD45RA and CCR7, obtaining the fold increase of the corresponding gene-edited T cell population. Subtype detection was performed within the CD8+ cell population using CCR7 and CD45RA, with results as follows: Figure 5 As shown, the presence of DCs and corresponding connectors can reduce the number of terminally differentiated T cell subtypes that specifically recognize antigens, and convert them into effector memory T cell subtypes.

[0117] When using a linker with anti-CD40 scfv1 / scfv2, such as Figure 6 As shown, after 72 hours, the total number of CAR-T cells increased, and the number of terminally differentiated cell subtypes decreased.

[0118] 4. Function of the linker in co-incubation of different macrophages with GPC3 CAR-T (SEQ ID NO 41) cells: CD14-positive cells from PBMCs were sorted using a CD14 sorting kit and then cultured as follows: 1) M0 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 8 days; 2) M1 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 6 days, followed by stimulation with 100 ng / mL LPS and 50 ng / mL IFN-γ for 48 hours to induce differentiation; 3) M2 cells were cultured in 1640 medium containing 100 ng / mL M-CSF for 6 days, followed by stimulation with 50 ng / mL IL-4 and 50 ng / mL IL-13 for 48 hours to induce differentiation. The corresponding macrophage phenotypes were detected using CD80 and CD206. Different types of macrophages were incubated with CD40L-GPC3 (Figure 40G3), control CD40L-MSLN (Figure 40MN), blank control 293 supernatant (Figure 293T), and 1 μg / ml CD40L positive control (Figure 293T40L) for 24 hours. The activation of CD69 and CD137 in T cells was then measured. The results showed that only CAR-T cells were activated under the action of CD40L-GPC3 during co-incubation, and M0, M1, and M2 cells could all activate GPC3 CAR-T cells. Figure 7 As shown, after 72 hours of co-incubation, the number and type of CAR-T cells were detected. It was found that M0, M1, and M2 cells could all increase CAR-T amplification and reduce the proportion of terminally differentiated cells.

[0119] 5. The linker can preserve the ability of DCs to act on CAR-T cells, rather than simply providing target cells for killing: To verify that the linker can preserve the effect of DCs on GPC3 CAR-T (SEQ ID NO 41) cells, the project designed co-incubation of GPC3-expressing Huh7 tumor cells, 293CD40-mCherry reporter cells, and DCs with GPC CAR-T cells. The linker CD40L-GPC3 was added to the co-incubation system of 293CD40-mCherry reporter cells and DCs, while GPC3 CAR-T cells cultured alone with CD40L-GPC3 were used as a control group. The culture medium was x-vivo medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15, mixed 1:1 with 293 linker supernatant (or without 293 linker supernatant). After 72 hours, CD8 CAR-T cells were collected for mRNA sequencing. The results are as follows: Figure 8As shown, sequencing results indicated a significant difference in CAR-T activity between the DC linker-treated group and ordinary 293T cells containing only target cells or linker-connected cells (PCA grouping). Differentially expressed genes were also observed, primarily enriched in T cell pathways such as cytokines, chemokines, and JAK-STAT activation. This demonstrates that the linker can enhance CAR-T cell activation through linking with DC cells and CAR-T cells, rather than simply stimulating CAR-T cells through cellular protein expression.

[0120] 6. Linkers and DCs can reactivate CAR-T cells during the killing process: 1) GPC3CAR-T cells prepared according to the above protocol were repeatedly co-incubated with GPC3-expressing target cells (Huh-7 cells) until the killing ratio of target cells remained unchanged after 24 hours of co-incubation. Depleted GPC3 CAR-T cells (SEQ ID NO 41) were then sorted. These depleted CAR-T cells were co-incubated for 3 days with supernatants containing control supernatant, CD40L-GPC3 linkers, linkers from different target sites, and DC cells. GPC3 CAR-T cells were then co-incubated with Huh-7 target cells at a ratio of 1:10, and their long-term killing effect was detected in RTCA. The results are as follows: Figure 9 As shown, in the group co-incubated with DC cells containing the CD40L-GPC3 linker (40G3), the long-term killing effect of CAR-T cells significantly increased in the later stages. 2) CHO cells overexpressing GPC3 were co-incubated with GPC3 CAR-T at a ratio of 30:1. The absolute count of CHO-GPC3 was performed daily, and the GPC3 positivity rate was detected by flow cytometry. The percentage of CD3+CAR+ positive cells was calculated by flow cytometry. DC cells were infected with adenovirus overexpressing the CD40L-flag-GPC3 linker. On day 5 of co-incubation, the DC cells were added to the co-incubation system (40G3). The untreated group (T), the DC-added group (DCT), and the DC-added group (40MN) with adenovirus expressing MSLN were set as negative controls. The results are as follows. Figure 9 As shown, CHO-GPC3 levels decreased after the addition of DCs expressing the CD40L-flag-GPC3 linker (40G3), and the proportion of CAR-T cells in this group increased after the addition of DCs, indicating that DCs expressing the corresponding linker can activate and expand GPC3 CAR-T cells in the killing process.

[0121] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A fusion protein linking APCs or other CD40-expressing cells to target-specific T cells, characterized in that, The fusion protein is composed of an antigen unit and a target unit; Preferably, the antigen unit is derived from a single-chain antibody, a single-chain peptide-MHC, a bacterial antigen, a viral antigen, a cancer-associated antigen, a cancer-specific antigen, or a fragment having an antigenic determinant. Preferably, the single-chain peptide-MHC comprises (1) a heavy chain domain; (2) a β2-microglobulin (β2m) domain; and (3) a specific peptide sequence. Preferably, the heavy chain domain includes the heavy chain domain of an MHC molecule; Preferably, the β2-microglobulin (β2m) domain includes the β2-microglobulin (β2m) domain of the MHC molecule; Preferably, the targeting unit is a CD40 binding domain; Preferably, the CD40 binding domain is derived from the extracellular domain of CD40L, other fragments of CD40L that can bind to CD40, or an antibody against CD40; Preferably, the fusion protein further includes a linker; Preferably, the antigen unit and the target unit may be connected by a connector or not; Preferably, the fusion protein is formed by sequentially connecting an antigen unit and a targeting unit in series; Preferably, the fusion protein is formed by connecting an antigen unit, a linker, and a targeting unit in series.

2. The fusion protein according to claim 1, characterized in that, The antigen units are derived from AFP, AXL, B4GALNT1, BCMA, CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD99, CD123, CD133, CD174, CD274, CD276, CDH17, CDS, CTAG1B, CEA, CLEC12A, CLDN6, CLDN 18.2, CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, GUCY2C, HER2, and HPV. E7, IL1RAP, IL3RA, IL13Rα2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PROM1, PSCA, PSMA, RO R1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNFRSF8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, ULBP2, HLA-E, HLA-DR, HLA-PRAME, HLA-NY-ESO-1, HLA-HPV E7, HLA-MAGEA1, HLA-MAGEA4, HLA-Survivin, HLA-WT1, HLA-MUC1, HLA-KRAS, HLA-P53, HLA-EBV, HLA-HBV, HLA-AFP; Preferably, the antigen unit is selected from GPC3, HLA-PRAME, HLA-NY-ESO-1, CD19, and MSLN; Preferably, the GPC3 comprises an amino acid sequence such as SEQ ID NO:1 or having at least 80% sequence identity with SEQ ID NO:1; Preferably, the HLA-PRAME comprises an amino acid sequence such as SEQ ID NO:2 or having at least 80% sequence identity with SEQ ID NO:2; Preferably, the HLA-NY-ESO-1 comprises an amino acid sequence such as SEQ ID NO:3 or having at least 80% sequence identity with SEQ ID NO:3; Preferably, CD19 comprises an amino acid sequence such as SEQ ID NO:4 or having at least 80% sequence identity with SEQ ID NO:4; Preferably, the MSLN comprises an amino acid sequence such as SEQ ID NO:5 or having at least 80% sequence identity with SEQ ID NO:5; Preferably, the targeting unit is selected from the CD40L extracellular domain, an antibody structure that recognizes CD40, or other elements that bind to CD40; Preferably, the CD40L extracellular segment comprises an amino acid sequence such as SEQ ID NO:6 or having at least 80% sequence identity with SEQ ID NO:6; Preferably, the antibody structure that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:7 or having at least 80% sequence identity with SEQ ID NO:7; Preferably, the antibody structure that recognizes CD40 comprises an amino acid sequence such as SEQ ID NO:8 or having at least 80% sequence identity with SEQ ID NO:8; Preferably, the connector includes a flexible linker, a rigid linker, and a splicable linker; Preferably, the linker is selected from the hinge region of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d; Preferably, the connector is a flexible linker; Preferably, the flexible linker is selected from flag and GS linker; Preferably, the GS linker comprises (GGGGX). n Or (G4X) represents an amino acid sequence, where n is an integer from 1 to 8, and X is C or S; Preferably, the GS linker is (GGGGS)3 or (G4S)3; Preferably, the flag contains an amino acid sequence such as SEQ ID NO:9 or having at least 80% sequence identity with SEQ ID NO:9; Preferably, (GGGGS)3 comprises an amino acid sequence such as SEQ ID NO:10 or having at least 80% sequence identity with SEQ ID NO:10; Preferably, the fusion protein further includes a signal peptide; Preferably, the signal peptide is selected from SP and SP-HLA; Preferably, the SP comprises an amino acid sequence such as SEQ ID NO:11 or having at least 80% sequence identity with SEQ ID NO:11; Preferably, the SP-HLA contains an amino acid sequence such as SEQ ID NO:12 or having at least 80% sequence identity with SEQ ID NO:12; Preferably, the fusion protein comprises amino acid sequences such as SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20 or having at least 80% sequence identity with SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20.

3. A biomaterial comprising any one of the following: 1) A nucleic acid encoding the fusion protein according to any one of claims 1-2; 2) A recombinant vector comprising the nucleic acid described in 1); 3) A recombinant host cell comprising the nucleic acid described in 1) and / or the recombinant vector described in 2).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid contains a nucleotide sequence encoding an antigen unit and a target unit; Preferably, the antigen unit and the targeting unit are derived from nucleotide sequences encoding single-chain antibodies, single-chain peptide-MHC, bacterial antigens, viral antigens, cancer-associated antigens, cancer-specific antigens, or fragments having antigenic determinants. Preferably, the targeting unit is a nucleotide sequence encoding a CD40 binding domain; Preferably, the CD40 binding domain comprises a nucleotide sequence encoding an extracellular segment of CD40L, other fragments of CD40L that can bind to CD40, an anti-CD40 antibody, or other antibodies that can recognize CD40; Preferably, the nucleic acid further comprises a nucleotide sequence encoding a linker; Preferably, the nucleic acid comprises a nucleotide sequence such as SEQ ID NO:21-40 or having at least 80% sequence identity with SEQ ID NO:21-40.

5. The biomaterial according to claim 3, characterized in that, The recombinant vector includes plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, or other viral vectors; Preferably, the recombinant vector is a lentiviral vector; Preferably, the recombinant host cell is selected from prokaryotic cells or eukaryotic cells.

6. A composition, characterized in that, The composition comprises the fusion protein according to any one of claims 1-2; Preferably, the composition further includes pharmaceutical excipients; Preferably, the composition further includes a detectable marker coupled thereto.

7. The use of the fusion protein according to any one of claims 1-2, the biomaterial according to any one of claims 3-5, and / or the composition according to claim 6, characterized in that, The application includes any of the following: 1) Application in the preparation of cancer treatment products; 2) Application in the preparation of products for treating infectious diseases; 3) Application in the preparation of products for treating autoimmune diseases; 4) Application in the preparation of products that enhance or assist specific T cell function.

8. The application according to claim 7, characterized in that, The products include pharmaceuticals, reagent kits, and vaccine formulations; Preferably, the drug and the vaccine formulation further include pharmaceutical excipients; Preferably, the cancer includes solid tumors and hematologic malignancies; Preferably, the cancers include pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, breast cancer, and melanoma. Preferably, the lung cancer is non-small cell lung cancer; Preferably, the infectious disease includes chronic viral diseases and tuberculosis; Preferably, the chronic viral diseases include infectious diseases caused by hepatitis C virus, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, John Cunningham virus, and human papillomavirus; Preferably, the autoimmune diseases include ankylosing spondylitis, psoriasis, celiac disease, systemic lupus erythematosus, common variant immunodeficiency disease, inflammatory bowel disease, ulcerative colitis, type I diabetes, juvenile idiopathic arthritis, Crohn's disease, alopecia areata, multiple sclerosis, primary biliary cirrhosis, primary sclerosing cholangitis, rheumatoid arthritis, Sjögren's syndrome, systemic sclerosis, spondyloarthritis, vitiligo, asthma, or thyroiditis; Preferably, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TCR-T cells, and TruC-T cells.

9. A method comprising any one of the following: 1) A method for promoting the binding of APCs or other CD40-expressing cells to specific T cells for non-therapeutic purposes, the method comprising: Administer to a subject the fusion protein according to any one of claims 1-2 and / or the composition according to claim 6; 2) A method for enhancing or assisting specific T cell function for non-therapeutic purposes, the method comprising: administering to a subject the fusion protein of any one of claims 1-2 and / or the composition of claim 6.

10. A method for expressing the fusion protein according to any one of claims 1-2, characterized in that, The method includes artificial synthesis and genetic engineering techniques; Preferably, the genetic engineering technology refers to culturing the recombinant host cell as described in any one of claims 3-5 or transfecting the recombinant vector as described in any one of claims 3-5 into the host cell, so that it transcribes the nucleotide sequence as described in any one of claims 3-5 and expresses the fusion protein as described in any one of claims 1-2.

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