Method for linking specific T cells and APC cells

Abstract

The invention discloses a method for linking a specific T cell and an APC cell, and provides a method for promoting binding between the specific T cell and an antigen presenting cell (APC), the method comprises constructing a fusion protein comprising (1) an epitope antigen part capable of being recognized by the specific T cell, and (2) an epitope antigen part capable of being recognized by the specific T cell. And (2) an APC binding domain capable of binding to a specific binding molecule on the surface of an APC cell. The fusion protein is used for mediating stable binding between T cells (including natural T cells or gene-edited T cells) and APC, so that recognition of specific T cells is enhanced in vitro or in vivo, amplification is activated, depletion is reduced or reversed, differentiation of the specific T cells to functional subtypes is promoted, and the killing effect and immune response capacity of the T cells are improved. The invention can be applied to the related fields of tumor immunotherapy, vaccine development, T cell function enhancement and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fusion proteins, and particularly relates to a method for linking specific T cells and APC cells. BACKGROUND

[0002] In the field of tumor immunotherapy and infectious disease prevention and control, specific T cells, as the core effector cells of adaptive immunity, directly determine the efficacy of immune response in terms of quantity and function. However, the recognition efficiency of T cells to tumor antigens or pathogens under natural conditions is extremely low, mainly due to the kinetic defects of the interaction between antigen-presenting cells (APC) and T cells. Although the T cells can be partially activated by the APC loaded with antigen peptides in vitro, there are problems such as low antigen presentation efficiency, insufficient T cell expansion, and short maintenance time of killing activity. In addition, non-specific activation signals easily lead to T cell exhaustion or abnormal functional differentiation, which cannot meet the clinical demand for efficient and persistent immune response.

[0003] Based on the above background, the present application proposes a fusion protein capable of linking specific T cells and antigen-presenting cells, which can promote the combination of antigen-presenting cells containing APC and T cells that specifically recognize antigens, thereby enhancing or assisting the activation, expansion, killing, reducing or reversing exhaustion of specific T cells such as CAR-T cells in any APC-gene edited T cell cultivation system, providing a research tool and new ideas for immune regulation. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a method for linking specific T cells and APC cells, which can promote the combination of T cells targeting DLL3 and APC cells by constructing a fusion protein of surface antigens and APC cell binding domains, realize the specific expansion, reduction or reversal of exhaustion and subtype change of T cells targeting DLL3, and enhance the killing and activation of specific T cells.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The first aspect of the present application provides a fusion protein for linking specific T cells and antigen-presenting cells, which is composed of a surface antigen that can be recognized by T cells, and an APC binding domain that can bind to the surface protein of APC cells.

[0007] Further, the structure of the fusion protein is that the surface antigen and the APC binding domain are sequentially connected, or the APC binding domain and the surface antigen are sequentially connected.

[0008] In the present application, DLL3 is also known as SCDO1. The term encompasses full-length, unprocessed DLL3, as well as any form of DLL3 or portions of the DLL3 protein that result from processing in the cell, or that can bind to a DLL3 antibody. The term encompasses naturally occurring variants of DLL3 (e.g., splice variants or allelic variants). The term encompasses, for example, a DLL3 gene, a DLL3 protein, a human DLL3, and a DLL3 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in the present application, the DLL3 is a human DLL3, Gene ID 10683.

[0009] In the present application, APC (Antigen-presenting cell) is also known as antigen presenting cell, which refers to a kind of immune cells capable of uptaking, processing and presenting antigens to T cells, and is widely distributed in various parts of the body. APCs are mainly divided into professional APCs and non-professional APCs. Professional APCs include monocyte-macrophages, dendritic cells and B lymphocytes, which uptake and process immune antigens by expressing MHC-II molecules and present them to T cells; non-professional APCs include certain endothelial cells and epithelial mesothelial cells, such as fibroblasts, glial cells, thymus, thyroid epithelial cells, vascular endothelial cells and eosinophils.

[0010] The surface antigen includes a tumor-associated antigen that specifically binds to a T cell targeting a tumor antigen.

[0011] Further, the surface antigen binds to a T cell specifically targeting DLL3.

[0012] Further, the surface antigen includes protein fragments of different regions of DLL3, including DLL3-EGF4, DLL3 (277).

[0013] Further, the surface antigen expresses an EGF-like repeat sequence of DLL3.

[0014] Specifically, the DLL3-EGF4 in the present application refers to the 4th part of the EGF domain in the DLL3 sequence; and the DLL3 (277) in the present application refers to the 277 amino acid part of the effective region of the EGF domain in the DLL3 sequence.

[0015] Further, the surface antigen is DLL3-EGF4.

[0016] Further, the amino acid sequence of the DLL3-EGF4 is shown in SEQ ID NO: 10, or has more than 80% identity with SEQ ID NO: 10.

[0017] Further, the surface antigen is DLL3 (277).

[0018] Further, the amino acid sequence of the DLL3 (277) is shown in SEQ ID NO: 11, or has more than 80% identity with SEQ ID NO: 11.

[0019] Further, the fusion protein further comprises a membrane exit signal peptide.

[0020] Further, the membrane exit signal peptide is selected from the membrane exit signal peptide of any one of the following molecules: CD8a, IL-2, CD33, TPA, IgKVIII, GM-CSFRa, CD3zeta, CD28.

[0021] Further, the membrane exit signal peptide is the membrane exit signal peptide of CD8a.

[0022] Further, the amino acid sequence of the membrane exit signal peptide of CD8a is shown in SEQ ID NO: 9.

[0023] The term "identity" also referred to as "homology" refers to an amino acid sequence or a nucleotide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the sequence provided in the present application. To determine sequence identity, sequence alignment can be performed by various means known to one skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software, etc. One skilled in the art is capable of determining appropriate parameters for alignment, including any algorithm needed to achieve optimal alignment over the full length of the sequences being compared. In the present application, antibody sequences that are modified to have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the fusion protein according to the first aspect of the present application are also within the scope of protection of the present application. The term "modification" refers to any form of modification to the amino acid sequence, for example, substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to a change in the amino acid sequence resulting in addition of one or more amino acids compared to the original amino acid sequence.

[0024] Further, the APC binding domain that can bind to an APC cell surface protein comprises an antibody, a ligand protein, or a receptor protein capable of binding to different APC cell surface proteins.

[0025] Further, the APC cell comprises a dendritic cell, a macrophage, a monocyte, a B cell, a CD4 T lymphocyte, a fibroblast, an epithelial cell, an endothelial cell, a glial cell, a Kupffer cell, a hepatic stellate cell, a mast cell, a basophil, an eosinophil, an innate lymphoid cell.

[0026] Further, the APC cell surface proteins include CD40, CD40L, CLEC1A, CLEC2, CLEC4A, CLEC4E, CLEC7A, CLEC9A, CLEC10A, CLEC12A, LILRB1, LILRB2, LILRB3, LILRB3, LILRB4, LILRB5, LFA-1, CD1, CD1c, CD1d, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD17, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD32, CD33, CD35, CD36, CD36L1, CD38, CD39, CD44, CD45RA, CD47, CD49d, CD50, CD54, CD58, CD62L, CD64, CD66b, CD70, CD73, CD80, CD83, CD85j, CD86, CD91, CD103, CD106, CD112, CD115, CD120a, CD120b, CD123, CD134L, CD137L, CD141, CD155, CD163, CD163L1, CD166, CD169, CD172a, CD184, CD204, CD205, CD206, CD207, CD209, CD209L, CD226, CD272, CD276, CD300a, CD300ld, CD301, CD303, CD304, FcR, CR1, CR3, CR4, HLA-DR, CCR2, CCR5, CCR7, CXCR3, CXCR5, CX3CR1, FcyR, FcsRI, MHC-II, ILT7, CLL-1, XCR1, CADM1, XCR1, PD-L1, PD-L2, HLA-G, DEC-205, FOLR2, TREM1, TREM2, uPAR, TIM-3, Siglec-6, FAP, CTLA-4, LAG-3, TIM-3, TIGIT, PD-1, PD-L1, CSF1R, CCR4, GITR, OX40, HVEM, VISTA, S1PR1, Galectin-9, Siglec-10, SLAMF7, TSPAN7, MARCO, SR-A1, B7-H4, TLR2, TLR4, TARM1, MS4A4A.

[0027] Further, the APC binding domain that can bind to an APC cell surface protein comprises CDR1, CDR2 and CDR3 of a light chain or a heavy chain of any one of sCD40L, anti-LILRB4, anti-CLEC7A, or the APC binding domain that can bind to an APC cell surface protein comprises CDR1, CDR2 and CDR3 of a light chain or a heavy chain of any one of sCD40L, anti-LILRB4, anti-CLEC7A with 80% or more sequence identity, which can be obtained according to any numbering scheme.

[0028] Further, the amino acid sequence of the sCD40L is shown in SEQ ID NO: 12, or has 80% or more identity with SEQ ID NO: 12.

[0029] Further, the anti-LILRB4 comprises anti-LILRB4.ATG034, anti-LILRB4.Hz5A7.

[0030] Further, the amino acid sequence of the anti-LILRB4.ATG034 is shown in SEQ ID NO: 13, or has 80% or more identity with SEQ ID NO: 13.

[0031] Further, the amino acid sequence of the anti-LILRB4.Hz5A7 is shown in SEQ ID NO: 14, or has 80% or more identity with SEQ ID NO: 14.

[0032] Further, the anti-CLEC7A comprises anti-CLEC7A.2M24, anti-CLEC7A.15E2.

[0033] Further, the amino acid sequence of the anti-CLEC7A.2M24 is shown in SEQ ID NO: 15, or has 80% or more identity with SEQ ID NO: 15.

[0034] Further, the amino acid sequence of the anti-CLEC7A.15E2 is shown in SEQ ID NO: 16, or has 80% or more identity with SEQ ID NO: 16.

[0035] In the present application, CD40 is also known as p50, Bp50, CDW40, TNFRSF5. The term encompasses full-length, unprocessed CD40, as well as any form of CD40 derived from processing in the cell. The term encompasses naturally occurring variants of CD40, e.g. splice variants or allelic variants. The term encompasses, for example, the CD40 gene, the CD40 protein, 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 the present application, CD40 is human CD40, Gene ID 958.

[0036] In the present application, CLEC7A is also known as CD369, SCARE2, DECTIN-1, CLECSF12. The term encompasses full-length, unprocessed CLEC7A, as well as any form of CLEC7A derived from processing in the cell. The term encompasses naturally occurring variants of CLEC7A, e.g. splice variants or allelic variants. The term encompasses, for example, the CLEC7A gene, the CLEC7A protein, human CLEC7A, and CLEC7A from any other vertebrate source, including mammals such as primates and rodents (e.g. mice and rats). As a preferred embodiment, in the present application, CLEC7A is human CLEC7A, Gene ID 64581.

[0037] In the present application, LILRB4 is also known as CD85k, LIR-5, ILT3, LIR5. The term encompasses full-length, unprocessed LILRB4, as well as any form of LILRB4 derived from processing in the cell. The term encompasses naturally occurring variants of LILRB4, e.g. splice variants or allelic variants. The term encompasses, for example, the LILRB4 gene, the LILRB4 protein, human LILRB4, and LILRB4 from any other vertebrate source, including mammals such as primates and rodents (e.g. mice and rats). As a preferred embodiment, in the present application, LILRB4 is human LILRB4, Gene ID 14727.

[0038] Further, the surface antigen moiety is linked to the APC binding domain via a linker, or directly.

[0039] Further, the linker is a flexible linker or a rigid linker.

[0040] Further, the linker has a length of about 3 to 100 amino acid residues.

[0041] Further, the linker comprises one or more functional tag sequences selected from one or more of FLAG, Myc, HA.

[0042] Further, the linker is a monomeric structure or a multimeric structure.

[0043] Further, the linker is selected from one or more of a polypeptide linker, a polyethylene glycol (PEG) linker, a polyamide linker, a carbohydrate derivative linker, an aliphatic linker, an aromatic linker, a synthetic polymer linker.

[0044] Further, the linker comprises one or more Gly4Ser repeat units, denoted as (Gly-Gly-Gly-Gly-Ser) n wherein n is 1 to 10.

[0045] In some embodiments, the fusion protein is Linker C, which consists of a membrane export signal peptide as previously described, a DLL3-EGF4 as previously described, and a sCD40L as previously described.

[0046] In some embodiments, the fusion protein is Linker C, which consists of a membrane export signal peptide as previously described, a DLL3-EGF4 as previously described, and a sCD40L as previously described.

[0047] Further, the amino acid sequence of the Linker C is set forth in SEQ ID NO: 3, or has 80% or more identity to SEQ ID NO: 3.

[0048] In some embodiments, the fusion protein is Linker 277, which consists of a membrane export signal peptide as previously described, a DLL3(277) as previously described, and a sCD40L as previously described.

[0049] In some embodiments, the fusion protein is Linker 277, which consists of a membrane export signal peptide as previously described, a DLL3(277) as previously described, and a sCD40L as previously described.

[0050] Further, the amino acid sequence of the Linker 277 is set forth in SEQ ID NO: 4, or has 80% or more identity to SEQ ID NO: 4.

[0051] In some embodiments, the fusion protein is Linker ATG034, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-LILRB4.ATG034 as previously described.

[0052] In some embodiments, the fusion protein is Linker ATG034, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-LILRB4.ATG034 as previously described.

[0053] Further, the amino acid sequence of the Linker ATG034 is set forth in SEQ ID NO: 5, or has 80% or more identity to SEQ ID NO: 5.

[0054] In some embodiments, the fusion protein is Linker Hz5A7, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-LILRB4. Hz5A7 as previously described.

[0055] In some embodiments, the fusion protein is Linker Hz5A7, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-LILRB4. Hz5A7 as previously described.

[0056] Further, the amino acid sequence of the Linker Hz5A7 is set forth in SEQ ID NO: 6, or has 80% or more identity to SEQ ID NO: 6.

[0057] In some embodiments, the fusion protein is Linker 2M24, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-CLEC7A.2M24 as previously described.

[0058] In some embodiments, the fusion protein is Linker 2M24, which consists of a secretion signal peptide as previously described, DLL3-EGF4 as previously described, and anti-CLEC7A.2M24 as previously described.

[0059] Further, the amino acid sequence of the Linker 2M24 is set forth in SEQ ID NO: 7, or has 80% or more identity to SEQ ID NO: 7.

[0060] In some embodiments, the fusion protein is Linker 15E2, which consists of a secretion signal peptide as previously described, a DLL3-EGF4 as previously described, and an anti-CLEC7A.15E2 as previously described, sequentially linked.

[0061] In some embodiments, the fusion protein is Linker 15E2, which consists of a secretion signal peptide as previously described, a DLL3-EGF4 as previously described, and an anti-CLEC7A.15E2 as previously described, sequentially linked.

[0062] Further, the amino acid sequence of the Linker 15E2 is set forth in SEQ ID NO: 8, or has 80% or more identity to SEQ ID NO: 8.

[0063] The second aspect of the present application provides an immunotherapeutic composition comprising: the fusion protein of the first aspect of the present application, which is optionally: (a) combined with a domain that prolongs the half-life in vivo, selected from one or more of an Fc fragment, an albumin binding molecule, a PEG modifier, a high molecular weight peptide segment, an XTEN peptide chain, a PAS sequence, or an analogue thereof; and / or,

[0064] (b) combined with at least one immunomodulatory component, selected from a cytokine, a chemokine, an immune adjuvant, or a functional analogue thereof.

[0065] Further, the cytokine is selected from one or more of an interleukin, an interferon, a tumor necrosis factor, a colony stimulating factor, a transforming growth factor.

[0066] Further, the chemokine is selected from one or more of a CCL family, a CXCL family, an XCL family, a CX3CL family, and a functional analogue thereof.

[0067] Further, the immune adjuvant or immune agonist is selected from one or more of a TLR agonist, a STING agonist, a RIG-I agonist, cGAMP, Poly(I:C), a CpG oligonucleotide, QS-21, MF59, AS03, an aluminum adjuvant.

[0068] The third aspect of the present application provides a nucleic acid molecule comprising a nucleic acid molecule encoding the fusion protein of the first aspect of the present application, which is useful for expressing the fusion protein of the first aspect of the present application in a recombinant host cell.

[0069] In the present application, the term "nucleic acid" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, and / or their analogs, including DNA, RNA and DNA / RNA hybrids, which also includes DNA or RNA analogs such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus, nucleic acids of the present application include DNA, cDNA, mRNA, recombinant nucleic acids, and the like. Once the coding sequence of the fusion protein of the present application or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto is obtained, the fusion protein can be obtained in large quantities using recombinant techniques.

[0070] The recombinant cell of the present application refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising the nucleic acid provided by the present application. The recombinant cell includes any progeny of the parent cell that differs from the parent cell in the degradation that occurs during replication. Preferably, the recombinant cell includes prokaryotic cells, eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia; more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, 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.

[0071] Further, the fusion protein expressed by the nucleic acid molecule can exist in the form of a polypeptide, a protein, a fusion protein or other gene expression products.

[0072] Further, the nucleic acid molecule is selected from DNA, RNA or modified forms thereof.

[0073] Further, the RNA is selected from linear mRNA, circular RNA (circRNA), self-amplifying RNA (saRNA) or modified forms thereof.

[0074] Further, the nucleic acid molecule encoding the fusion protein of the first aspect of the present application includes a sequence encoding Linker C, Linker ATG034, Linker Hz5A7, Linker 2M24, Linker 15E2.

[0075] Further, the nucleic acid sequence encoding Linker C is shown in SEQ ID NO: 17, or has more than 80% identity to SEQ ID NO: 17.

[0076] Further, the nucleic acid sequence encoding Linker 277 is set forth in SEQ ID NO: 18, or has 80% or more identity to SEQ ID NO: 18.

[0077] Further, the nucleic acid sequence encoding Linker ATG034 is set forth in SEQ ID NO: 19, or has 80% or more identity to SEQ ID NO: 19.

[0078] Further, the nucleic acid sequence encoding Linker Hz5A7 is set forth in SEQ ID NO: 20, or has 80% or more identity to SEQ ID NO: 20.

[0079] Further, the nucleic acid sequence encoding Linker 2M24 is set forth in SEQ ID NO: 21, or has 80% or more identity to SEQ ID NO: 21.

[0080] Further, the nucleic acid sequence encoding Linker 15E2 is set forth in SEQ ID NO: 22, or has 80% or more identity to SEQ ID NO: 22.

[0081] The fourth aspect of the present application provides an expression vector comprising the nucleic acid molecule of the third aspect of the present application, the expression vector being used for expressing the fusion protein in a recombinant host cell.

[0082] The vector of the present application refers to an artificial construct which is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. The vector of the present application can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a gene of interest encoding the fusion protein of the first aspect of the present application, a promoter, a terminator, or optionally further comprises a marker gene. The vector can use known vectors or self-constructed vectors. The known vectors include plasmid vectors, lentivirus vectors, adenovirus vectors, AAV virus vectors, herpes virus vectors and other viral vectors. Other viral vectors can include bacteriophage vectors, baculovirus vectors, animal virus vectors, plant virus vectors, can include papovavirus vectors, herpes virus vectors, poxvirus vectors, RNA virus vectors, can include bovine papilloma virus vectors and Epstein-Barr virus vectors, retrovirus vectors, etc.

[0083] Further, the expression vector is selected from one or more of a DNA vector, a viral vector, an mRNA vector.

[0084] The fifth aspect of the present application provides a delivery system, comprising:

[0085] 1) The nucleic acid molecule according to the third aspect of the present invention and / or the linker expressed by the nucleic acid molecule;

[0086] The linker exists in the form of a polypeptide, protein, fusion protein or other gene expression product; and

[0087] 2) A delivery vehicle selected from the group consisting of: liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viruses, virus-like particles, engineered microorganisms or their spores, cells, inorganic nanoparticles, hydrogels, emulsions, polymer micelles, and biomimetic membrane-encapsulated nanoparticles.

[0088] Furthermore, the inorganic nanoparticles include gold nanoparticles, silica nanoparticles and magnetic nanoparticles, the hydrogel includes thermosensitive or pH-responsive hydrogel, the emulsion includes microemulsion and nanoemulsion, and the biomimetic membrane-wrapped nanoparticles includes an envelope structure derived from red blood cell membrane, macrophage membrane or tumor cell membrane.

[0089] The delivery system is used to deliver the nucleic acid molecule, polypeptide, protein, fusion protein or other gene expression product to the target tissue or cell in a mammal to express the fusion protein described in the first aspect of the present invention.

[0090] Furthermore, the nucleic acid molecule is mRNA, and the lipid nanoparticles contain one or more of ionized lipids, helper lipids, cholesterol and PEG-modified lipids.

[0091] Furthermore, the virus is an oncolytic virus, and the oncolytic virus is selected from one or more of adenovirus, adeno-associated virus, herpes simplex virus, varicella-zoster virus, cowpox virus / vaccinia virus, measles virus, Newcastle disease virus, reovirus, polio virus, and Samba virus.

[0092] Furthermore, the cell carrier is selected from T cells, natural killer cells (NK cells), dendritic cells (DC cells), macrophages, monocytes, red blood cells, mesenchymal stem cells, stromal cells and tumor cells.

[0093] The sixth aspect of the present invention provides a use of the fusion protein of the first aspect of the present invention, the immunotherapy composition of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, and / or the delivery system of the fifth aspect of the present invention, wherein the use comprises any one of the following:

[0094] 1) Application in the preparation of products that enhance the specific activation, expansion, and / or reduction or reversal of exhaustion of specific T cells;

[0095] 2) Application in the preparation of products that promote T cell subtype changes;

[0096] 3) use in the manufacture of a product for treating a tumor.

[0097] The term "treatment" or "prevention" as used herein refers to any type of intervention or treatment directed to an individual, or the administration of an active agent to a subject, with the aim of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, occurrence, aggravation or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease.

[0098] Further, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TRuC-T cells, TCR-T cells.

[0099] Further, the specific T cells are T cells targeting DLL3.

[0100] Further, the T cells targeting DLL3 are CAR-T cells specifically recognizing DLL3.

[0101] Further, the CAR-T cells specifically recognizing DLL3 can be constructed by any one of the following CAR structures: DLL3.Juno CAR, DLL3.T3 CAR, or the corresponding single domain antibody sequences thereof; the domain comprises a region having more than 80% amino acid sequence identity with the CDR1, CDR2 and CDR3 regions defined by any one of the above CARs or the single domain antibody sequences thereof; the CDR regions can be determined according to any one of the antibody numbering systems of Kabat, Chothia, IMGT or Aho.

[0102] Further, the promoting T cell subtype change refers to promoting the subtype change of T cells targeting DLL3.

[0103] Further, the subtype change refers to the transition from a terminally differentiated cell subtype to an effector memory T cell subtype.

[0104] Further, the tumor includes a tumor expressing DLL3.

[0105] Further, the tumor includes small cell lung cancer, neuroendocrine tumor, neuroendocrine cancer.

[0106] Further, the product includes a kit, test paper, nucleic acid membrane strip, chip, system or device.

[0107] The seventh aspect of the present application provides a method, which comprises any one of the following:

[0108] 1) a method for promoting the binding of specific T cells and antigen presenting cells in vitro;

[0109] 2) A method for enhancing T cell specific activation and killing capacity in vitro.

[0110] The steps of the method comprise co-culturing the fusion protein of the first aspect of the application with specific T cells and antigen presenting cells.

[0111] Further, the antigen presenting cells are APC cells.

[0112] Further, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TRuC-T cells, TCR-T cells.

[0113] The eighth aspect of the application provides a recombinant host cell comprising the nucleic acid molecule of the third aspect of the application and / or the expression vector of the fourth aspect of the application.

[0114] In the present application, CAR-T cell refers to a T cell modified by genetic engineering technology, which expresses a chimeric antigen receptor (CAR). The receptor usually includes: 1) antigen recognition domain: such as single chain variable fragment (scFv), which is used for specific recognition of target antigen; 2) transmembrane region: connecting extracellular and intracellular structure; 3) intracellular signal transduction region: such as CD3 zeta chain, and may also contain costimulatory signal domain (such as CD28 or 4-1BB), which is used for activating T cell function.

[0115] In the present application, TRuC-T cell (TCR Fusion Construct T cell) refers to a T cell expressing a T cell receptor fusion construct (TCR Fusion Construct, TRuC). The TRuC structure directly fuses the antigen recognition domain (such as scFv) to one subunit of the TCR complex (such as CD3 epsilon, CD3 gamma, TCR alpha, etc.), and the fusion protein is embedded in the TCR complex and mediates T cell activation through the endogenous TCR-CD3 signal pathway. TRuC does not rely on the traditional costimulatory elements in the CAR domain, and its activation relies on the signal transduction mechanism of the TCR complex itself.

[0116] In this invention, STAR-T cells (Synthetic TCR And Antigen Receptor T cells) refer to T cells that express a synthetic T cell receptor and antigen receptor fusion construct (Synthetic TCR and Antigen Receptor, STAR). The STAR structure consists of two polypeptide chains: the first chain is composed of the antibody heavy chain variable region (VH) fused to the TCRα chain constant region (including the transmembrane region), and the second chain is composed of the antibody light chain variable region (VL) fused to the TCRβ chain constant region (including the transmembrane region). The two chains are connected by disulfide bonds to form a heterodimer. They can assemble with TCR complex subunits such as CD3ε, CD3γ, CD3δ, and CD3ζ to form a complete TCR-like structure, thereby initiating the TCR signaling activation process.

[0117] The above-mentioned CAR-T, TRuC-T and STAR-T constructs also include all their structural equivalents, functional analogs or derivatives, such as polypeptides of recognition domains, VHHs, ligand forms, and various transmembrane and intracellular signaling modules (such as co-stimulatory structures, scaffold sequences, connecting peptides, etc.), regardless of whether they adopt the specific forms explicitly stated in this specification, they should be deemed to be covered within the scope of protection of the present invention.

[0118] Advantages and beneficial effects of the present invention: The present invention provides a method for linking specific T cells and APC cells. By using an optimized DLL3 fragment fused with the antibody sequence of the APC CD40 / CLEC7A / LILRB4 protein, DLL3CAR-T and DC cells can be connected to achieve specific activation of the expansion and typing changes of target-specific T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 Schematic diagram of the fusion protein structure.

[0120] Figure 2 The figure shows the flow cytometric detection of the binding ability of fusion proteins expressing different DLL3 regions to DLL3 CAR-T cells.

[0121] Figure 3 This is a statistical chart showing the detection results of CD69 and CD137 expression in DLL3 CAR-T cells.

[0122] Figure 4 This is a statistical chart showing the detection results of CD45RA and CCR7 expression after CAR-T cells, DC cells and fusion protein were co-cultured for 72 hours.

[0123] Figure 5Statistical chart of CD69 and CD137 expression, specific activation of CAR-T, expansion of CAR-T and change of CD8+ CAR+ cell memory cell typing after co-culturing of fusion protein targeting different APC surface proteins with CAR-T cells and DC cells.

[0124] Figure 6 Statistical chart of CD69 and CD137 expression, specific activation of CAR-T, expansion of CAR-T and change of CD8+ CAR+ cell memory cell typing after co-culturing of fusion protein of H82 tumor cell line expressing DLL3 or the application with DLL3 CAR-T. DETAILED DESCRIPTION

[0125] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by a person skilled in the art without creative work fall within the protection scope of the application.

[0126] Example 1: Construction of fusion proteins expressing different DLL3 regions and detection of binding ability

[0127] 1. Culture of DLL3-specific recognition T cells: Synthesize and construct pCDH-EF1a lentivirus expression plasmid corresponding to the sequences of DLL3.Juno CAR-T (WO2024044779A2, SEQ ID NO: 1) and DLL3.T3 CAR-T (US20220249563A1, SEQ ID NO: 2), transfect lentivirus system plasmid (pCDH-EF1a lentivirus expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed in a mass ratio of 3:2:1) into logarithmic growth phase adherent 293T cells, harvest cell culture supernatant 48-72 hours after transfection, obtain lentivirus after concentration and filtration, and store at -80°C. Isolate peripheral blood mononuclear cells (PBMCs) from human peripheral blood, separate T cells using human CD3 / 28 magnetic beads, and perform virus transduction within 72 hours of activation. Change the liquid culture 24 hours after transduction to the 14th day, centrifuge and collect the cells, and resuspend them in physiological saline. Use flow cytometry to identify the G4S molecule part of the T cell editing protein to confirm the positive rate of edited T cells. Incubate the supernatant containing the fusion protein with the corresponding edited T cells for 0.5 hours, then wash the cells with PBS and perform flow cytometry staining.

[0128] 2. Construct plasmid containing fusion protein expressing different DLL3 regions: Linker C (SEQ ID NO: 3), Linker 277 (SEQ ID NO: 4), see Table 1 for detailed sequences.

[0129] Wherein, linker C is constructed by linking sCD40L (SEQ ID NO: 12) to the C-terminus of DLL3-EGF4 (SEQ ID NO: 10); linker 277 is constructed by linking sCD40L to the C-terminus of DLL3 (277) (SEQ ID NO: 11), and the detailed sequence is shown in Table 1.

[0130] After the above plasmids are transfected into 293 by PEI system, the 293 cell supernatant after 24 hours is collected to obtain the fusion protein containing corresponding. After the supernatant containing the fusion protein is incubated with the corresponding edited T cells for 0.5 hours, the cells are washed with PBS, and flow cytometry is performed. The specific parameters of flow cytometry detection are as follows: 3x10 5 cells / well are used to detect the number of cells, anti-CD40L (APC), anti-EGFR (PE) (CAR-T marker) are added, and after incubation for 30 minutes, the cells are washed with PBS and then detected by flow cytometry. The results are shown in Figure 2 Figure 6. Different DLL3 fragments have different binding abilities to DLL3 CAR-T. Linker C and Linker 277 show good binding abilities to DLL3.Juno CAR-T and DLL3.T3 CAR-T.

[0131] 3. Effect of fusion protein on T cell activation and subtype conversion: Attachment Solution Kit (Hycells, Shanghai; CAT: DCT01003-A) is used to promote the adhesion of mononuclear cells in PBMC to the culture dish, and then Dendritic Cells Differentiation Kit (Monocyte-Derived) (Hycells, Shanghai; CAT: DCT01003-kit) is used to induce the adherent mononuclear cells into DC. After about 48 hours of adding the maturation medium to the DC, the DC is used. The corresponding DLL3 CAR-T cells are expanded and cultured to the platform stage, and the positive rate is adjusted to about 20%, and then co-incubated with the DC cells at a ratio of 10:1. The test group is added with 293T cell supernatant of different sources. The culture medium is x-vivo culture medium containing 10 ng / ml IL-7 and 5 ng / ml IL-15 mixed with 293 supernatant containing fusion protein or 293 supernatant not containing fusion protein (control) at a ratio of 1:1. After co-incubation for 24 hours, the expression of CD69 and CD137 of DLL3 CAR-T cells is detected to show the activation state. The results are shown in Figure 3As shown, the results show that only the addition of the fusion protein Linker C and Linker 277 combined with DLL3 CAR-T can activate T cells in the presence of DCs.

[0132] After co-incubation of the CAR-T of the above system with the fusion protein for 72 hours, the proliferation of CAR-T cells and the sub-grouping of CD8+ CAR+ cells were detected by CD45RA and CCR7, and the results are shown in Figure 4 As shown, it shows that in the presence of DCs and corresponding fusion proteins, the number of T cells specifically recognizing antigens can be increased, and the number of terminally differentiated cell subtypes is reduced, and converted to effector memory T cell subtypes.

[0133] Example 2 Construction and binding ability detection of fusion proteins against different APC surface proteins

[0134] 1. Construction of plasmids expressing fusion proteins against different APC surface proteins: including two different fusion proteins of DLL3 fragment (as described in Example 1) and anti-LILRB4 and DLL3 fragment and anti-CLEC7A: Linker ATG034 (SEQ ID NO: 5), Linker Hz5A7 (SEQ ID NO: 6), Linker 2M24 (SEQ ID NO: 7), Linker 15E2 (SEQ ID NO: 8), see Table 1 for detailed sequences.

[0135] After transfection into 293 cells by PEI system, the supernatant of 293 cells after 24 hours was collected to obtain the supernatant containing the corresponding fusion protein. Using the protocol described in Example 1, DLL3.T3 CAR-T was co-cultured with DC cells in the presence or absence of the supernatant of the corresponding fusion protein, and it was found that CAR-T could interact with DC under the action of the corresponding fusion protein, achieving specific activation, expansion and CD8+ CAR+ cell memory cell typing change of CAR-T. The results are shown in Figure 5 As shown, GPC3- is used as a control fusion protein for non-DLL3 recognition.

[0136] 2. H82 tumor cell line and fusion protein control: In order to further verify that the fusion protein can enhance the function of CAR-T and change the cell typing by linking APC and CAR-T. The experiment co-cultured the H82 tumor cell line expressing DLL3 with DLL3 CAR-T, and compared with the DC system co-cultured with the fusion protein, it was found that H82 could promote the activation of DLL3 CAR-T, but did not significantly affect the expansion and typing change of CAR-T, indicating that the fusion protein played a role in promoting the expansion and typing change of CAR-T.

[0137] Table 1. Sequence Listing

[0138] The various reagents involved in the technical solutions and experimental processes described in the present application are common reagents or commercial reagents that can be clearly known and easily obtained by those skilled in the art based on their professional knowledge and conventional practice. The description of the reagents in the present application is intended to clearly set forth the material basis involved in the technical solutions, and those skilled in the art can successfully obtain and correctly use these reagents based on their professional accomplishment and industry common sense to achieve the technical purpose of the present application.

[0139] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A fusion protein linking specific T cells and antigen-presenting cells, characterized in that: The fusion protein consists of a surface antigen that can be recognized by T cells and an APC binding domain that can bind to an APC cell surface protein; Preferably, the structure of the fusion protein is that the surface antigen and the APC binding domain are sequentially connected, or the APC binding domain and the surface antigen are sequentially connected; The surface antigens include tumor-associated antigens, which specifically bind to T cells targeting tumor antigens; Preferably, the surface antigen binds to T cells that specifically target DLL3; Preferably, the surface antigen includes protein fragments of different regions of DLL3, and the different regions include DLL3-EGF4, DLL3 (277); Preferably, the surface antigen is DLL3-EGF4; Preferably, the amino acid sequence of the DLL3-EGF4 is as shown in SEQ ID NO: 10, or has more than 80% identity with SEQ ID NO: 10; Preferably, the surface antigen is DLL3 (277); Preferably, the amino acid sequence of DLL3 (277) is as shown in SEQ ID NO: 11, or has more than 80% identity with SEQ ID NO:

11.

2. The fusion protein according to claim 1, characterized in that The fusion protein further includes a membrane export signal peptide; Preferably, the membrane export signal peptide is selected from the membrane export signal peptide of any one of the following molecules: CD8a, IL-2, CD33, TPA, IgKVIII, GM-CSFRα, CD3ζ, CD28; Preferably, the membrane export signal peptide is the membrane export signal peptide of CD8a; Preferably, the amino acid sequence of the CD8a export signal peptide is shown in SEQ ID NO:

9.

3. The fusion protein according to claim 1, characterized in that The APC binding domain that can bind to APC cell surface proteins includes antibodies, ligand proteins or receptor proteins that can bind to different APC cell surface proteins; Preferably, the APC cells include dendritic cells, macrophages, monocytes, B cells, CD4 T lymphocytes, fibroblasts, epithelial cells, endothelial cells, glial cells, Kupffer cells, hepatic stellate cells, mast cells, basophils, eosinophils, and innate lymphoid cells; Preferably, the APC cell surface proteins include CD40, CD40L, CLEC1A, CLEC2, CLEC4A, CLEC4E, CLEC7A, CLEC9A, CLEC10A, CLEC12A, LILRB1, LILRB2, LILRB3, LILRB3, LILRB4, LILRB5, LFA-1, CD1, CD1c, CD1d, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD17, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD32, CD33, CD35, CD36, CD36L1, CD38, CD39, CD44, CD45RA, CD 47. CD49d, CD50, CD54, CD58, CD62L, CD64, CD66b, CD70, CD73, CD80, CD83, CD85j, CD86, CD91, CD103, CD106, CD112, CD115, CD120a, CD120b, CD123, CD134L, CD137L, CD 141. CD155, CD163, CD163L1, CD166, CD169, CD172a, CD184, CD204, CD205, CD206, CD207, CD209, CD209L, CD226, CD272, CD276, CD300a, CD300ld, CD301, CD303, CD304, FcR, CR1, CR3, CR4, HLA-DR, CCR2, CCR5, CCR7, CXCR3, CXCR5, CX3CR1, FcγR, FcεRI, MHC-II, ILT7, CLL-1, XCR1, CADM1, XCR1, PD-L1, PD-L2, HLA-G, DEC-205, FOLR2, TREM1, TREM2, uPAR, TIM-3, Sig lec-6, FAP, CTLA-4, LAG-3, TIM-3, TIGIT, PD-1, PD-L1, CSF1R, CCR4, GITR, OX40, HVEM, VISTA, S1 PR1, Galectin-9, Siglec-10, SLAMF7, TSPAN7, MARCO, SR-A1, B7-H4, TLR2, TLR4, TARM1, MS4A4A; Preferably, the APC cell surface protein is CD40; Preferably, the APC cell surface protein is CLEC7A; Preferably, the APC cell surface protein is LILRB4; Preferably, the APC-binding domain capable of binding to an APC cell surface protein comprises CDR1, CDR2, and CDR3 of the light chain or heavy chain of any one of sCD40L, anti-LILRB4, and anti-CLEC7A antibodies, or the APC-binding domain capable of binding to an APC cell surface protein comprises CDR1, CDR2, and CDR3 of the light chain or heavy chain of any one of sCD40L, anti-LILRB4, and anti-CLEC7A antibodies having a sequence identity of more than 80%, and the CDR1, CDR2, and CDR3 can be obtained according to any numbering scheme; Preferably, the amino acid sequence of sCD40L is as shown in SEQ ID NO: 12, or has more than 80% identity with SEQ ID NO: 12; Preferably, the anti-LILRB4 includes anti-LILRB4.ATG034 and anti-LILRB4.Hz5A7; Preferably, the amino acid sequence of anti-LILRB4.ATG034 is as shown in SEQ ID NO: 13, or has more than 80% identity with SEQ ID NO: 13; Preferably, the amino acid sequence of anti-LILRB4.Hz5A7 is as shown in SEQ ID NO: 14, or has more than 80% identity with SEQ ID NO: 14; Preferably, the anti-CLEC7A includes anti-CLEC7A.2M24 and anti-CLEC7A.15E2; Preferably, the amino acid sequence of anti-CLEC7A.2M24 is as shown in SEQ ID NO: 15, or has more than 80% identity with SEQ ID NO: 15; Preferably, the amino acid sequence of the anti-CLEC7A.15E2 is as shown in SEQ ID NO: 16, or has more than 80% identity with SEQ ID NO:

16.

4. The fusion protein according to claim 1, characterized in that The surface antigen and the APC binding domain are connected via a linker or directly; Preferably, the linker is a flexible linker or a rigid linker; Preferably, the linker has a length of about 3 to 100 amino acid residues; Preferably, the linker comprises one or more functional tag sequences, and the tags are selected from one or more of FLAG, Myc, and HA; Preferably, the linker is a monomeric structure or a polymeric structure; Preferably, the linker is selected from one or more of a polypeptide linker, a polyethylene glycol (PEG) linker, a polyamide linker, a carbohydrate derivative linker, an aliphatic linker, an aromatic linker, and a synthetic polymer linker; Preferably, the linker comprises one or more Gly4Ser repeating units, represented by (Gly-Gly-Gly-Gly-Ser) n , where n is 1 to 10; Preferably, the fusion protein is Linker C, and the Linker C is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the sCD40L according to claim 3; Preferably, the fusion protein is Linker C, which is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the sCD40L according to claim 3 connected in sequence; Preferably, the amino acid sequence of Linker C is as shown in SEQ ID NO: 3, or has more than 80% identity with SEQ ID NO: 3; Preferably, the fusion protein is Linker 277, and the Linker 277 is composed of the membrane export signal peptide according to claim 2, the DLL3 (277) according to claim 2, and the sCD40L according to claim 3; Preferably, the fusion protein is Linker 277, and the Linker 277 is sequentially connected by the membrane export signal peptide of claim 2, the DLL3 (277) of claim 2, and the sCD40L of claim 3; Preferably, the amino acid sequence of Linker 277 is as shown in SEQ ID NO: 4, or has more than 80% identity with SEQ ID NO: 4; Preferably, the fusion protein is Linker ATG034, and the Linker ATG034 is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-LILRB4.ATG034 according to claim 3; Preferably, the fusion protein is Linker ATG034, which is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-LILRB4.ATG034 according to claim 3 connected in sequence; Preferably, the amino acid sequence of Linker ATG034 is as shown in SEQ ID NO: 5, or has more than 80% identity with SEQ ID NO: 5; Preferably, the fusion protein is Linker Hz5A7, and the Linker Hz5A7 is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-LILRB4. Hz5A7 according to claim 3; Preferably, the fusion protein is Linker Hz5A7, and the Linker Hz5A7 is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-LILRB4. Hz5A7 according to claim 3 connected in sequence; Preferably, the amino acid sequence of Linker Hz5A7 is as shown in SEQ ID NO: 6, or has more than 80% identity with SEQ ID NO: 6; Preferably, the fusion protein is Linker 2M24, and the Linker 2M24 is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-CLEC7A.2M24 according to claim 3; Preferably, the fusion protein is Linker 2M24, which is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-CLEC7A.2M24 according to claim 3 connected in sequence; Preferably, the amino acid sequence of Linker 2M24 is as shown in SEQ ID NO: 7, or has more than 80% identity with SEQ ID NO: 7; Preferably, the fusion protein is Linker 15E2, and the Linker 15E2 is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-CLEC7A.15E2 according to claim 3; Preferably, the fusion protein is Linker 15E2, which is composed of the membrane export signal peptide according to claim 2, the DLL3-EGF4 according to claim 2, and the anti-CLEC7A.15E2 according to claim 3 connected in sequence; Preferably, the amino acid sequence of Linker 15E2 is as shown in SEQ ID NO: 8, or has more than 80% identity with SEQ ID NO:

8.

5. An immunotherapy composition comprising: The fusion protein according to any one of claims 1 to 4, wherein the fusion protein is optionally: (a) combined with a domain that prolongs the in vivo half-life, wherein the domain that prolongs the in vivo half-life is selected from one or more of an Fc fragment, an albumin binding molecule, a PEG modification, a high molecular weight peptide segment, an XTEN peptide chain, a PAS sequence, or an analog thereof; and / or, (b) in combination with at least one immunomodulatory component selected from cytokines, chemokines, immune adjuvants or functional analogs thereof; Preferably, the cytokine is selected from one or more of interleukin, interferon, tumor necrosis factor, colony stimulating factor, and transforming growth factor; Preferably, the chemokine is selected from one or more of the CCL family, CXCL family, XCL family, CX3CL family and functional analogs thereof; Preferably, the immune adjuvant or immune agonist is selected from one or more of: TLR agonist, STING agonist, RIG-I agonist, cGAMP, Poly (I: C), CpG oligonucleotide, QS-21, MF59, AS03, and aluminum adjuvant.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 4, and the nucleic acid molecule can be used to express the fusion protein according to any one of claims 1 to 4 in a recombinant host cell; Preferably, the fusion protein expressed by the nucleic acid molecule may exist in the form of a polypeptide, protein, fusion protein or other gene expression product; Preferably, the nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 4 comprises sequences encoding Linker C, Linker ATG034, Linker Hz5A7, Linker 2M24, and Linker 15E2; Preferably, the nucleic acid sequence encoding Linker C is as shown in SEQ ID NO: 17, or has more than 80% identity with SEQ ID NO: 17; Preferably, the nucleic acid sequence encoding Linker 277 is as shown in SEQ ID NO: 18, or has more than 80% identity with SEQ ID NO: 18; Preferably, the nucleic acid sequence encoding Linker ATG034 is as shown in SEQ ID NO: 19, or has more than 80% identity with SEQ ID NO: 19; Preferably, the nucleic acid sequence encoding Linker Hz5A7 is as shown in SEQ ID NO: 20, or has more than 80% identity with SEQ ID NO: 20; Preferably, the nucleic acid sequence encoding Linker 2M24 is as shown in SEQ ID NO: 21, or has more than 80% identity with SEQ ID NO: 21; Preferably, the nucleic acid sequence encoding Linker 15E2 is as shown in SEQ ID NO: 22, or has more than 80% identity with SEQ ID NO:

22.

7. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule according to claim 6, and the expression vector is used to express the fusion protein in a recombinant host cell; Preferably, the expression vector is selected from one or more of a DNA vector, a viral vector, and an mRNA vector.

8. A delivery system comprising: 1) The nucleic acid molecule, polypeptide, protein, fusion protein or other gene expression product of claim 6; as well as 2) a delivery vehicle selected from the group consisting of: liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viruses, virus-like particles, engineered microorganisms or their spores, cells, inorganic nanoparticles, hydrogels, emulsions, polymer micelles, and biomimetic membrane-encapsulated nanoparticles; The delivery system is used to deliver the nucleic acid molecule, polypeptide, protein, fusion protein or other gene expression product to a target tissue or cell in a mammal to express the fusion protein according to any one of claims 1 to 4; Preferably, the nucleic acid molecule is mRNA, and the lipid nanoparticles comprise one or more of ionized lipids, helper lipids, cholesterol, and PEG-modified lipids; Preferably, the virus is an oncolytic virus, and the oncolytic virus is selected from one or more of adenovirus, adeno-associated virus, herpes simplex virus, varicella-zoster virus, vaccinia virus / vaccinia virus, measles virus, Newcastle disease virus, reovirus, poliovirus, and Samba virus; Preferably, the cell carrier is selected from T cells, natural killer cells, dendritic cells, macrophages, monocytes, red blood cells, mesenchymal stem cells, stromal cells and tumor cells.

9. Use of the fusion protein according to any one of claims 1 to 4, the immunotherapy composition according to claim 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 and / or the delivery system according to claim 8, characterized in that: The application includes any of the following: 1) Application in the preparation of products that enhance the specific activation, expansion, and / or reduction, or reversal of exhaustion of specific T cells; 2) Application in the preparation of products that promote T cell subtype changes; 3) Application in the preparation of tumor treatment products; Preferably, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TRuC-T cells, and TCR-T cells; Preferably, the specific T cells are T cells targeting DLL3; Preferably, the T cells targeting DLL3 are CAR-T cells that specifically recognize DLL3; Preferably, the DLL3-specifically recognized CAR-T cell can be constructed using any of the following CAR structures: DLL3.JunoCAR (SEQ ID NO: 1), DLL3.T3 CAR (SEQ ID NO: 2), or their corresponding single-domain antibody sequences; the structural domain comprises a region having more than 80% amino acid sequence identity with the CDR1, CDR2, and CDR3 regions defined by any of the above-mentioned CARs or their single-domain antibody sequences; the CDR regions can be identified according to any of the antibody numbering systems of Kabat, Chothia, IMGT, or Aho; Preferably, the promoting the change of T cell subtype refers to promoting the change of T cell subtype targeting DLL3; Preferably, the subtype change refers to the transition from a terminally differentiated cell subtype to an effector memory T cell subtype; Preferably, the tumor comprises a tumor expressing DLL3; Preferably, the tumor includes small cell lung cancer, neuroendocrine tumor, neuroendocrine carcinoma; Preferably, the product includes a kit, a test paper, a nucleic acid membrane strip, a chip, a system or a device.

10. A method, characterized in that The method includes any of the following: 1) A method for promoting the binding of specific T cells and antigen-presenting cells in vitro; 2) A method for enhancing T cell-specific activation and killing ability in vitro; The method comprises the steps of: co-culturing the fusion protein according to any one of claims 1 to 4 with specific T cells and antigen-presenting cells; Preferably, the antigen presenting cells are APC cells; Preferably, the specific T cells are selected from T cells, CAR-T cells, STAR-T cells, TRuC-T cells, and TCR-T cells.

11. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule of claim 6 and / or the expression vector of claim 7.

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