Cell membrane expressed il-15 fusion protein and its application in cell therapy

Abstract

The application provides a cell membrane expressed IL-15 fusion protein and application thereof in cell therapy. The fusion protein provided by the application comprises an IL-15R alpha signal peptide, IL-15, an IL-15R alpha sushi domain and a transmembrane region, modified cells of which can realize efficient expression of IL-15 on the cell membrane surface, can effectively avoid side effects caused by IL-15 secretion expression, and are safe and controllable. Chimeric antigen receptor modified immune cells comprising the membrane expressed IL-15 fusion protein of the application have stronger tumor killing function, IFN-gamma secretion and proliferation capacity; after being treated by lactic acid and TGF-beta rich in a tumor microenvironment, the immune cells still have very strong tumor killing function; and can activate the anti-tumor function of immune cells not comprising the application. The immune cells constructed by the application can improve tumor treatment effect, prolong disease remission time and improve patient survival period.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a cell membrane expressing IL-15 fusion protein and application thereof. BACKGROUND

[0002] Chimeric antigen receptor (CAR)-T cell therapy is an innovative immunotherapy approach. CAR-T cells are T cells extracted from a patient's peripheral blood, which are genetically modified to express receptors that can recognize tumor antigens, and thus have the ability to recognize and attack tumor cells. CAR-T cell therapy has made great breakthroughs in the treatment of hematological tumors, but current CAR-T cell therapy cannot benefit most tumor patients, due to difficulties in target selection, complicated and costly production process, and inhibition of tumor microenvironment.

[0003] CAR-NK cells are a new type of immunotherapy that has emerged in recent years, combining the characteristics of CAR technology and NK cells. Compared with traditional CAR-T cells, CAR-NK cells have some unique advantages: ① broad-spectrum anti-tumor activity: CAR-NK cells can recognize and attack multiple types of tumors, greatly improving the treatment range; ② lower risk of cytokine release syndrome (CRS): compared with CAR-T cells, CAR-NK cells usually cause mild CRS, reducing the risk of serious side effects; ③ rapid expansion and low cost: NK cells can be expanded more quickly in vitro, and the relatively short culture time allows the treatment to be carried out more quickly. NK cells can also be derived from healthy donors, enabling allogeneic infusion and low cost. However, CAR-NK cells also have some disadvantages: ① low persistence and survival ability: CAR-NK cells usually have a short survival time in the body, lack the persistence of CAR-T cells, and may lead to a decrease in treatment effect or an increased risk of tumor recurrence. ② inhibition of tumor microenvironment: like CAR-T cells, CAR-NK cells are also affected by the tumor microenvironment, and the immunosuppressive effect of the tumor microenvironment may inhibit the efficacy of CAR-NK cells.

[0004] γδT cells are a special type of immune cells that have both acquired and innate immune response characteristics. CAR-γδT cells are a new type of immune cell therapy that has stronger tumor cell recognition and elimination capabilities. CAR-γδT cells have the following advantages: ① diverse targeting ability: γδT cells can recognize a variety of non-specific antigens, such as heat shock proteins, phosphorylated antigens, and some bacterial antigens, so they can target a variety of different types of tumors; ② natural immune properties: γδT cells have natural killer activity and can eliminate tumor cells through direct cytotoxicity and cytokine secretion, independent of specific antigen presentation; ③ lower risk of cytokine release syndrome: compared with CAR-T cells, CAR-γδT cells generally have a lower risk of CRS and relatively mild side effects; ④ rapid expansion and low cost: γδT cells can be rapidly expanded and respond quickly to infections and tumors in the body, and can be allogeneic transplanted to achieve shelf supply and low cost. Of course, CAR-γδT cells also have some disadvantages: ① low persistence and survival ability: CAR-γδT cells usually have low persistence in the body, which may limit the long-term effectiveness of treatment; ② suppression of tumor microenvironment: like CAR-T and CAR-NK cells, CAR-γδT cells are also affected by the tumor microenvironment, and the immunosuppressive effects of the tumor microenvironment can inhibit the effectiveness of CAR-γδT cells. SUMMARY

[0005] In view of this, in order to make up for the shortcomings of the prior art, the present application is proposed. The purpose of the present application is to provide a fusion protein that can improve the long-term anti-tumor effect of immune cells and is safe and controllable. The membrane expression fusion protein provided by the present application comprises an IL-15Rα signal peptide, IL-15, an IL-15Rα sushi domain, and a transmembrane region. The modified cells can achieve efficient expression of IL-15 on the cell membrane surface, but do not secrete expression, effectively avoiding the side effects of secreted IL-15 expression, and are safe and controllable after adding a safety protection zone. The chimeric antigen receptor comprising the membrane expression IL-15 fusion protein of the present application has stronger tumor killing function, IFN-γ secretion ability and proliferation ability; after treatment with lactic acid and TGF-β rich in the tumor microenvironment, it still has strong tumor killing function; and it can activate the anti-tumor function of immune cells that do not contain the present application. This advantage can improve the tumor treatment effect, prolong the disease remission time, and prolong the survival period of patients in clinical application.

[0006] The above-mentioned purposes of the present application are realized by the following technical solutions:

[0007] The first aspect of the present application provides a cell membrane expressed IL-15 fusion protein, which comprises IL-15, IL-15Rα signal peptide, IL-15Rα and a transmembrane region.

[0008] Further, the amino acid sequence of the IL-15Rα signal peptide is shown as SEQ ID NO: 1.

[0009] Further, the IL-15Rα can be a full-length IL-15Rα.

[0010] Further, the IL-15Rα can also be a fragment capable of binding to IL-15, as long as the binding ability of IL-15Rα to IL-15 is retained.

[0011] Further, the IL-15Rα is a sushi domain of IL-15Rα.

[0012] Further, the amino acid sequence of the sushi domain of IL-15Rα is shown as SEQ ID NO: 5.

[0013] Further, the transmembrane region can be any protein that can promote interaction between homologous subunits to form dimers or multimers.

[0014] Further, the transmembrane region is a FcεRIγ transmembrane region.

[0015] Further, the amino acid sequence of the FcεRIγ transmembrane region is shown as SEQ ID NO: 13.

[0016] Further, the fusion protein further comprises a safety protection region or a hinge region.

[0017] In the present application, the safety protection region is a region that can ensure the safety of the subject after the genetically edited immune cells are infused into the human body, so that the modified cells need to have controllability. Further, the safety protection region comprises CD20, iCasp9, tEGFR, RQR8. In a specific embodiment of the present application, the safety protection region is selected from CD20, and when the modified cells have abnormal proliferation and other uncontrollable risks, the clinical drug Mabthera is given, combined with CD20, to use the antibody-dependent cell-mediated cytotoxic effect of the body to eliminate the modified cells, which is safer. Further, the amino acid sequence of the CD20 is shown as SEQ ID NO: 9.

[0018] Further, the hinge region can keep IL-15 outside of the plasma membrane and has the necessary flexibility to facilitate the binding of membrane-expressed IL-15 to IL-15Rβ and γ receptors, and to stabilize the transmission of IL-15 activation signals. Further, the hinge region comprises a CD28 hinge region, a CD8 hinge region, a CD4 hinge region, a hinge region of an immunoglobulin IgG, a hinge region of an immunoglobulin IgG coupled with a CH2CH3 region. Further, the hinge region is selected from a CD8 hinge region. Further, the amino acid sequence of the hinge region is shown in SEQ ID NO: 11.

[0019] Further, the IL-15Rα signal peptide, IL-15, IL-15Rα, and transmembrane region in the fusion protein are necessary to achieve the purpose of membrane expression. In an alternative, a safety protection region can be added to ensure the safety of membrane-expressed IL-15. In an alternative, a hinge region can be added to achieve better effects of membrane-expressed IL-15.

[0020] Further, the IL-15Rα signal peptide, IL-15, IL-15Rα, and transmembrane region in the fusion protein can be connected in sequence, or connected in other orders. In an embodiment, when a safety protection region and / or a hinge region is added to the fusion protein, it can also be connected in sequence or in other orders. Regardless of the order of connection, as long as the purpose of membrane expression of IL-15 is achieved, it falls within the protection scope of the present application. Further, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection region, and hinge region can be directly connected to each other. Further, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection region, and hinge region can be connected to each other through at least one linker. The linker can be flexible or rigid. Further, the linker comprises a glycine polymer (G)n, a glycine-serine polymer, a glycine-alanine polymer, an alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP, or (GGGGS)n. Further, the linker is selected from (GGGGS)n.

[0021] Further, the IL-15 and IL-15Rα are connected by a linker (GGGGS)5.

[0022] Further, the amino acid sequence of the linker is shown in SEQ ID NO: 7.

[0023] The second aspect of the present application provides a membrane expressing IL-15 chimeric antigen receptor, which comprises the fusion protein of the first aspect of the present application.

[0024] In the present application, the membrane expressing IL-15 chimeric antigen receptor is a chimeric antigen receptor (CAR) and IL-15 which are respectively expressed on the cell membrane. CAR refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor immune cells, which can include an antigen recognition region (e.g., tumor-specific antigen and / or tumor-associated antigen), a transmembrane domain, an intracellular signal domain, a hinge region, and a costimulatory domain. CAR is an engineered receptor that can implant any specific receptor onto an immune effector cell. In CAR, an antibody that specifically recognizes a tumor antigen can be implanted onto an immune cell. A nucleic acid encoding CAR can be introduced into an immune cell using, for example, a retroviral vector. In this way, a large number of cancer-specific immune cells can be generated for adoptive cell therapy.

[0025] Further, the fusion protein of the first aspect of the present application and the CAR can be sequentially connected, or connected in other orders in the membrane expressing IL-15 chimeric antigen receptor. No matter what order the connection is, as long as the purpose of membrane expression of IL-15 is achieved, it falls within the protection scope of the present application.

[0026] Further, the respective expression can be achieved by adding a self-cleavage peptide between the membrane expressing IL-15 and the chimeric antigen receptor.

[0027] Further, the self-cleavage peptide is selected from any one of the following self-cleavage peptides: T2A, P2A, E2A, F2A.

[0028] Further, the self-cleavage peptide is T2A.

[0029] Further, the antigen recognition region is selected from an antibody against a tumor surface antigen.

[0030] Further, the tumor surface antigen comprises one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, FAP, Glypican-3, CEA, CA125, CA199, CA15-3, SCC, NSE, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, GD2, Pro-GRP, CYFRA21-1, TRCP-5b, sB7-H3, DCP, OPN, GP73, CA72-4, MG7-AG, PG, CA50, DC-SIGNR, CHGA, PSA, GPRC5D, Siglec-6.

[0031] Further, the tumor surface antigen is selected from mesothelin.

[0032] Further, the heavy chain variable region amino acid sequence of the antibody against the tumor surface antigen is set forth in SEQ ID NO: 17.

[0033] Further, the light chain variable region amino acid sequence of the antibody against the tumor surface antigen is set forth in SEQ ID NO: 18.

[0034] Further, the transmembrane domain comprises a CD8 transmembrane region, a CD8a transmembrane region, a CD28 transmembrane region, a CD4 transmembrane region, a CD28 transmembrane region, a CD3 zeta transmembrane region, a CD137 / 4-1BB transmembrane region, a CD134 / OX40 transmembrane region, an ICOS transmembrane region, a CD5 transmembrane region, a CD9 transmembrane region, a CD16 transmembrane region, a CD22 transmembrane region, a CD33 transmembrane region, a CD37 transmembrane region, a CD45 transmembrane region, a CD64 transmembrane region, a CD80 transmembrane region, a CD86 transmembrane region, a CD154 transmembrane region, a TCRa transmembrane region, a TCRp transmembrane region.

[0035] Further, the transmembrane domain is selected from a CD28 transmembrane region.

[0036] Further, the amino acid sequence of the transmembrane domain corresponds to the partial amino acid sequence set forth in SEQ ID NO: 19.

[0037] Further, the intracellular signaling domain comprises an intracellular signaling domain of any one of the following molecules: CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, FcR gamma, FcR beta, TCR zeta, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fc epsilon RI, DAP10, DAP12, CD66d.

[0038] Further, the intracellular signaling domain is an intracellular signaling domain of CD3 zeta.

[0039] Further, the amino acid sequence of the intracellular signaling domain is set forth in SEQ ID NO: 20.

[0040] Further, the hinge region comprises a hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3 epsilon, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor.

[0041] Further, the hinge region is selected from a CD28 hinge region.

[0042] Further, the amino acid sequence of the hinge region corresponds to the partial amino acid set forth in SEQ ID NO: 19.

[0043] Further, the co-stimulatory signaling domain comprises the co-stimulatory signaling domain of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8a, CD8b, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226.

[0044] Further, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domain of 4-1BB.

[0045] Further, the amino acid sequence of the co-stimulatory signaling domain is set forth in SEQ ID NO: 21.

[0046] Further, the chimeric antigen receptor further comprises a signal peptide.

[0047] Further, the signal peptide comprises the signal peptide of any one of the following molecules: a chain and b chain of T cell receptor, CD3zeta, CD3epsilon, CD16, CD22, CD33, CD4, CD5, CD8a, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF.

[0048] Further, the signal peptide is selected from the signal peptide of CD8a.

[0049] The third aspect of the present application provides a nucleic acid molecule, which encodes the fusion protein according to the first aspect, or encodes the chimeric antigen receptor according to the second aspect.

[0050] Further, the nucleic acid molecule further comprises a promoter, and / or a restriction site located after the promoter, and / or a kozak sequence located after the restriction site.

[0051] In the present application, nucleic acid molecule refers to DNA molecule and RNA molecule. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is "operably linked" when it is functionally connected to another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the coding sequence.

[0052] Further, the nucleic acid molecule sequence encoding IL-15 expressed by the film corresponds to part or all of SEQ ID NO: 16.

[0053] Further, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of SEQ ID NO: 22.

[0054] The fourth aspect of the present application provides a vector containing the nucleic acid molecule of the third aspect of the present application.

[0055] Further, the type of the vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus and cosmid, which can be changed depending on the cell to be introduced. Virus vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and introduction can be made by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaP04precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors. In some embodiments, viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In addition, in order to evaluate the expression of the protein of interest (e.g., monoclonal antibody), the vector introduced into the cell can also contain either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of the expression cell from the cell population sought to be transfected or infected by the viral vector.

[0056] Further, the vector can be an expression vector, a cloning vector or an integration vector. A typical cloning vector contains a transcription and translation terminator, a start sequence and a promoter that can be used to regulate the expression of the desired nucleic acid sequence. The integration vector contains components that integrate the target sequence into the genome of the cell. These vectors can be used to transform appropriate cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. The sequences usually include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of a polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for insertion of nucleic acid encoding a monoclonal antibody to be expressed and an optional marker element. In a specific embodiment of the present application, the vector used is a lentiviral expression plasmid pCDH vector, a lentiviral packaging plasmid pSPAX2 and a pMD2G vector.

[0057] The fifth aspect of the present application provides an engineered cell and its derivatives, which comprises the nucleic acid molecule of the third aspect of the present application, or comprises the expression vector of the fourth aspect of the present application.

[0058] Further, the derivatives include but are not limited to extracellular vesicles, any derivative produced by the engineered cell of the fifth aspect of the present application falls within the protection scope of the present application.

[0059] In the present application, extracellular vesicles are a group of vesicles with lipid bilayer membrane structure secreted by cells, the contents of extracellular vesicles include miRNA of extracellular vesicles, mRNA of extracellular vesicles or surface proteins of extracellular vesicles, and according to the size of extracellular vesicles, they can be divided into exosomes, microvesicles and apoptotic bodies.

[0060] Further, the extracellular vesicles are selected from exosomes.

[0061] Further, the engineered cell includes eukaryotic cells, prokaryotic cells.

[0062] Further, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells.

[0063] Further, the mammalian cells include immune cells, CHO cells, 293T cells, 293F cells.

[0064] Further, the immune cells include T cells, B cells, NK cells, iNKT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils or any combination thereof.

[0065] Further, the T cells include αβT cells, γδT cells.

[0066] Further, the immune cells are selected from γδT cells, NK cells.

[0067] Further, the γδT cells, NK cells are derived from humans.

[0068] Further, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia.

[0069] The sixth aspect of the present application provides a composition, which comprises the fusion protein of the first aspect of the present application, the chimeric antigen receptor of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the vector of the fourth aspect of the present application, the engineered cell of the fifth aspect of the present application and its derivatives.

[0070] Further, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0071] The seventh aspect of the present application provides a biological preparation comprising the pharmaceutical composition of the sixth aspect of the present application.

[0072] Further, the dosage form of the biological preparation is injection, lyophilized agent, oral agent, cream, gel, drop or patch.

[0073] In some embodiments, the present application does not particularly limit the specific dosage form of the biological preparation, and in some embodiments, the pharmaceutical composition or biological preparation provided by the present application can be formulated for a pharmaceutical composition or biological preparation for topical, intravenous, oral, enteral and / or parenteral administration according to actual needs. In some embodiments, the appropriate dosage of the pharmaceutical composition or biological preparation provided by the present application can be prescribed in various ways according to the formulation method, administration method, age, weight, gender, disease state, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the prescribed dosage effective for the desired treatment or prevention.

[0074] The eighth aspect of the present application provides a kit comprising the fusion protein of the first aspect of the present application, the chimeric antigen receptor of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the vector of the fourth aspect of the present application, the engineered cell and its derivatives of the fifth aspect of the present application, the composition of the sixth aspect of the present application, and the biological preparation of the seventh aspect of the present application.

[0075] The ninth aspect of the present application provides any one of the following methods:

[0076] 1) A method for preparing the fusion protein of the first aspect of the present application, the method comprising culturing the engineered cell and its derivatives of the fifth aspect of the present application so that it can express the fusion protein of the first aspect of the present application.

[0077] 2) A method for preparing the engineered cell and its derivatives of the fifth aspect of the present application, the method comprising introducing the nucleic acid molecule of the third aspect of the present application or the vector of the fourth aspect of the present application into the cell.

[0078] The tenth aspect of the present application provides any one of the following uses:

[0079] 1) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for preparing a diagnostic and / or therapeutic anti-tumor drug.

[0080] 2) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for improving the survival ability of immune cells.

[0081] 3) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for inhibiting the apoptosis of immune cells.

[0082] 4) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for enhancing the proliferation ability of immune cells.

[0083] 5) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for preparing a drug for improving the anti-tumor ability of immune cells.

[0084] 6) The fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell and its derivatives according to the fifth aspect of the present application, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, and the kit according to the eighth aspect of the present application are used for enhancing the cytokine level of immune cells.

[0085] 7) Use of the fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell according to the fifth aspect of the present application and its derivatives, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, the kit according to the eighth aspect of the present application in the preparation of a medicine for changing tumor microenvironment.

[0086] 8) Use of the fusion protein according to the first aspect of the present application, the chimeric antigen receptor according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the vector according to the fourth aspect of the present application, the engineered cell according to the fifth aspect of the present application and its derivatives, the composition according to the sixth aspect of the present application, the biological agent according to the seventh aspect of the present application, the kit according to the eighth aspect of the present application in killing tumor cells in vitro.

[0087] 9) Use of the engineered cell according to the fifth aspect of the present application and its derivatives in the preparation of the fusion protein according to the first aspect of the present application.

[0088] 10) Use of the nucleic acid molecule according to the third aspect of the present application or the vector according to the fourth aspect of the present application in the preparation of the engineered cell according to the fifth aspect of the present application and its derivatives.

[0089] Further, the tumor includes, but is not limited to, adrenal cortex cancer, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid tumor, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, leukemia, brain low-grade glioma, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, retinoblastoma.

[0090] Further, the tumor is selected from leukemia, ovarian cancer, lymphoma.

[0091] Further, the tumor is ovarian cancer.

[0092] Further, the immune cell includes T cells, B cells, NK cells, iNKT cells, γδ T cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof.

[0093] Further, the immune cells are selected from the group consisting of γδT cells, NK cells.

[0094] Further, the γδT cells, NK cells are derived from human.

[0095] Further, the cytokines include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors.

[0096] Further, the cytokines are selected from the group consisting of interleukins, interferons.

[0097] Further, the cytokines are interferons.

[0098] Further, the interferons include IFN-α, IFN-β, IFN-κ, IFN-γ, IFN-λ.

[0099] Further, the interferons are selected from the group consisting of IFN-γ.

[0100] The present application has the advantages and beneficial effects:

[0101] The present application provides a cell membrane expressed IL-15 fusion protein and its application in cell therapy technology, the present application connects the IL-15Rα signal peptide, IL-15, IL-15Rαsushi domain, transmembrane region in series with CAR, modifies immune cells, realizes the efficient expression of IL-15 on the membrane of immune cells. The present application proves by experiment that the engineered immune cells of IL-15-IL-15Rαsushi-FcεRI can realize efficient membrane expression of IL-15, strong killing of tumor cells and strong anti-tumor effect, and the engineered immune cells also have the functions of promoting immune cell proliferation, improving IFN-γ expression level and changing tumor microenvironment. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 is the structural mode diagram of membrane expressed IL-15;

[0103] Figure 2 is the γδT purity detection result diagram of CAR-γδT cells;

[0104] Figure 3 is the CAR expression efficiency detection result diagram of CAR-γδT cells;

[0105] Figure 4 is the IL-15 (mIL-15) expression efficiency detection result diagram of CAR-γδT cell surface;

[0106] Figure 5 is the tumor cell killing effect detection result diagram of CAR-γδT cells;

[0107] Figure 6 Figure 7 is a graph showing the results of repeated killing of tumor cells by CAR-γδ T cells;

[0108] Figure 7 Figure 8 is a graph showing the results of proliferation of CAR-γδ T cells;

[0109] Figure 8 Figure 9 is a graph showing the results of CD107a expression analysis of CAR-γδ T cells;

[0110] Figure 9 Figure 10 is a graph showing the results of IFN-γ expression analysis of CAR-γδ T cells;

[0111] Figure 10 Figure 11 is a graph showing the change in tumor killing function of CAR-γδ T cells after treatment with lactic acid or TGF-β;

[0112] Figure 11 Figure 12 is a graph showing the effect of CAR-γδ T cells on tumor killing by γδ T cells;

[0113] Figure 12 Figure 13 is a graph showing the effect of CAR-γδ T cells on IFN-γ secretion by γδ T cells;

[0114] Figure 13 Figure 14 is a graph showing the analysis of the clearing effect of rituximab on CAR-γδ T cells;

[0115] Figure 14 Figure 15 is a graph showing the results of killing of tumor cells by CAR-NK cells;

[0116] Figure 15 Figure 16 is a graph showing the results of detection of IL-15 secretion levels by CAR-NK cells. DETAILED DESCRIPTION

[0117] The present application will be further described below in conjunction with specific examples, which are intended to explain the present application, but not to limit the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturers. The reagents, biological materials, etc. used in the following examples, if not otherwise specified, can be obtained from commercial channels.

[0118] Example 1 Construction of IL-15 expressed on the membrane

[0119] The signal peptide of IL-15Rα is directly connected with the human IL-15 coding gene, the sushi region of IL-15Rα is connected with a flexible linker (G4S), the binding region of Rituximab against CD20 is connected with the sushi region of IL-15Rα, the CD8 hinge region is added after the Rituximab binding region, and the intracellular segment of the transmembrane region FcεRIγ (the coding gene is FCER1G) is connected with the CD8 hinge region. The structure of the membrane expressed IL-15 constructed is shown in Figure 1 The sequences of the genes involved in the construction of the membrane expressed IL-15 are shown in Table 1.

[0120] Table 1 Sequences of genes involved in the construction of the membrane expressed IL-15

[0121]

[0122]

[0123]

[0124]

[0125] Example 2 Construction and function detection of CAR-γδT cells with membrane expressed IL-15

[0126] 1. Experimental materials

[0127] The experimental materials are shown in Table 2:

[0128] Table 2 Experimental materials

[0129]

[0130] 2. Experimental methods

[0131] (1) The coding gene of the membrane expressed IL-15 constructed in the application is connected with a CAR targeting mesothelin antibody (Mesothelin, MSLN) (patent number: CN111548420A) by T2A. The CAR structure includes a human CD8α signal peptide, an anti-mesothelin single-chain antibody, a human CD28 hinge region, a transmembrane region and a cytoplasmic region, and human 4-1BB and CD3ζ cytoplasmic regions. The constructed structure is subcloned into a lentiviral expression plasmid pCDH vector, which is mixed with packaging plasmids pSPAX2 and pMD2G, and then transfected into 293FT cells by the calcium phosphate transfection method to produce lentiviral particles, and the virus is concentrated by PEG8000. The amino acid sequences of the components of the CAR are shown in Table 3.

[0132] Table 3 Amino acid sequences of components of the CAR

[0133]

[0134]

[0135]

[0136] Preparation of γδT cells: take the peripheral blood mononuclear cells (PBMCs) of human, adjust to 1-2x10 6 IU / mL of IL-2, 50 μL / mL of glutamine, 5-10% (volume ratio) of autologous plasma, 5 μM / mL of zoledronic acid in serum-free cell culture solution ALyS505N-0), mix gently, and place in a 5% CO2, 37°C constant temperature incubator for culture.

[0137] Adopt lentivirus transfection to logarithmic growth phase γδT cells (PBMCs cultured for 5-7 days, MOI=10), after virus infection for one day, remove the culture medium containing virus, and continue to culture with complete culture medium without zoledronic acid to the 11th day, that is, the cells can be harvested.

[0138] (2) Detect the purity of γδT cells of CAR-γδT cells. Take the cultured cells, adjust the cell density to 1x10 6 cells / mL, take 100 μL and add into a flow tube, add 5 μL of different fluorescently labeled mouse anti-human CD3, Vγ9 and IL-15 antibodies and fluorescently labeled human Mesothelin (MSLN) protein, incubate at room temperature for 15 min, centrifuge at 1500 rpm for 5 min at room temperature, remove the supernatant, add 2 mL of PBS to wash the cells, centrifuge at 500 rpm for 5 min at room temperature, discard the supernatant, add 200 μL of PBS to suspend the cells, and detect by flow cytometry.

[0139] (3) Detect the in vitro tumor killing ability: remove IL-2 from CAR-γδT cells and γδT cells for 24 h, 48 h and 72 h, respectively; adopt Calcein-AM to label Mesothelin-expressing SKOV3 cells, then act with the cells obtained in Example 2 according to the effector-target ratio of 5:1 for 4 h, detect the release of Calcein after the killing of target cells by a microplate reader, and detect the killing effect of CAR-γδT cells and γδT cells on tumor cells.

[0140] (4) After the cultured CAR-γδT cells and SKOV3 cells are reacted for 24 h at an effector-target ratio of 10:1, the CAR-γδT cells are separated, and secondary killing detection is performed by reacting the separated CAR-γδT cells with Calcein-AM-labeled SKOV3 cells at an effector-target ratio of 5:1 for 4 h to detect the killing effect of the CAR-γδT cells on the tumor cells. Tertiary killing is performed by reacting the separated CAR-γδT cells with SKOV3 cells at an effector-target ratio of 10:1 for 24 h, then separating the CAR-γδT cells, and reacting the separated CAR-γδT cells with Calcein-AM-labeled SKOV3 cells at an effector-target ratio of 5:1 for 4 h to detect the killing effect of the CAR-γδT cells on the tumor cells.

[0141] (5) Detection of in vitro proliferation capacity: detection of the expression level of Ki67. The cultured cells are taken, the cell density is adjusted to 1×10 6 6 6

[0142] (6) Detection of CD107a: the cultured cells and SKOV3 cells are reacted at an effector-target ratio of 10:1 for 4 h, and flow cytometry is used to detect the expression of CD107a of the cultured cells.

[0143] (7) After the CAR-γδT cells and tumor cells SKOV3 cells are co-incubated for 4 h, intracellular staining is used to detect the expression of IFN-γ.

[0144] (8) After the CAR-γδT cells are treated with lactic acid or TGF-β for 24 h, the Calcein-Am release method is used to detect the killing effect of the CAR-γδT cells on the tumor cells.

[0145] (9) After the γδT cells from the same donor source and the CAR-γδT cells are co-incubated at a ratio of 1:1 for 24 h, the γδT cells are reacted with Calcein-AM-labeled tumor cells SKOV3 cells for 4 h to detect the killing effect.

[0146] (10) After the γδT cells from the same donor source and the CAR-γδT cells are co-incubated at a ratio of 1:1 for 24 h, the γδT cells are reacted with Calcein-AM-labeled tumor cells SKOV3 cells for 4 h, and intracellular staining is used to detect the expression of IFN-γ.

[0147] (11) NK cells from the same donor and CAR-γδT cells labeled with Calcein-AM were mixed at a ratio of 10:1 and divided into two groups. One group was given Rituximab, and the other group was not given Rituximab. After incubation for 4 hours, the scavenging effect of Rituximab on the CAR-γδT cells cultured in this invention was detected by the Calcein-Am release assay.

[0148] 3. Experimental Results

[0149] (1) Results Figure 2 The results show that the proportion of γδT cells in the CAR-γδT cells (mIL-15-CAR-γδT) targeting Mesothelin constructed in this invention is higher than 90% compared with the control CAR-γδT cells (CAR-γδT) and control γδT cells (γδT), and there is no significant difference. This indicates that the CAR-γδT cells constructed in this invention do not affect the purity of γδT cells and have a very high purity.

[0150] (2) Results Figure 3 The results show that the CAR expression ratio of the mIL-15-CAR-γδT cells constructed in this invention is slightly higher than that of the control CAR-γδT cells, indicating that the CAR-γδT cells constructed in this invention have better CAR expression.

[0151] (3) Results Figure 4 The results show that the mIL-15-CAR-γδT cell membrane constructed in this invention expresses IL-15, while the control group does not express it, indicating that this invention can achieve membrane expression of IL-15.

[0152] (4) Results Figure 5 The results showed that the mIL-15-CAR-γδT cells constructed in this invention had stronger tumor-killing effects than the control CAR-γδT cells and γδT cells after 24h, 48h and 72h of culture with IL-2 removed, indicating that the CAR-γδT cells constructed in this invention still had the strongest anti-tumor ability under the condition of IL-2 removal.

[0153] (5) Results Figure 6 The results showed that the secondary and tertiary tumor-killing abilities of the mIL-15-CAR-γδT cells constructed in this invention were significantly higher than those of the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention have the strongest multiple anti-tumor function.

[0154] (6) Results Figure 7 The results showed that the Ki67 expression in the mIL-15-CAR-γδT cells constructed in this invention was significantly higher than that in the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention had the strongest proliferative capacity.

[0155] (7) ResultsFigure 8 ) shows that the expression of CD 107a of the mIL-15-CAR-gammadelta T cells constructed by the application is significantly higher than that of the control group, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application have stronger killing activity on tumor cells.

[0156] (8) Results Figure 9 ) shows that the expression of IFN-g of the mIL-15-CAR-gammadelta T cells constructed by the application is significantly higher than that of the control group, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application have stronger anti-tumor function.

[0157] (9) Results Figure 10 ) shows that the killing effect of the mIL-15-CAR-gammadelta T cells constructed by the application on tumor cells is still significantly higher than that of the control group after being treated with lactic acid or TGF-b, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application still have strong anti-tumor effect even in the tumor microenvironment rich in lactic acid and TGF-b.

[0158] (10) Results Figure 11 ) shows that the expression of CD 107a of the gammadelta T cells co-incubated with the mIL-15-CAR-gammadelta T cells constructed by the application is significantly higher than that of the control group, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application can improve the killing effect of the gammadelta T cells not containing the application on tumor cells.

[0159] (11) Results Figure 12 ) shows that the expression of IFN-g of the gammadelta T cells co-incubated with the mIL-15-CAR-gammadelta T cells constructed by the application is significantly increased, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application can promote the gammadelta T cells not containing the application to secrete more IFN-g.

[0160] (12) Results Figure 13 ) shows that the killing effect of NK cells on the CAR-gammadelta T cells of the application and the control group is low and has no statistical difference; but when the NK cells and the CAR-gammadelta T cells are added with Mabthera at the same time, the killing effect of the NK cells on the mIL-15-CAR-gammadelta T cells constructed by the application is significantly higher than that of the control group CAR-gammadelta T cells, indicating that the mIL-15-CAR-gammadelta T cells constructed by the application can be cleared by the NK cells dependent on Mabthera.

[0161] Example 3 Construction and function detection of CAR-NK cells expressing IL-15 on membrane

[0162] 1. Experimental method

[0163] (1) Adopt lentivirus transfection to human NK cells in logarithmic growth phase (NK cells cultured for 5-7 days, MOI=10), one day after viral infection, remove the culture medium containing virus and replace it with new NK cell culture medium for continuous culture to the 11th day, at which time the cells can be harvested.

[0164] (2) Remove IL-2 from NK cells, control CAR-NK cells and mIL-15-CAR-NK cells constructed according to the application respectively for 24 h, then act with SKOV3 cells according to the effector-target ratio of 2.5:1 for 4 h, and detect the killing effect of NK and CAR-NK cells on tumor cells.

[0165] (3) Remove IL-2 from NK cells, control CAR-NK cells and mIL-15-CAR-NK cells constructed according to the application respectively for 24 h, then collect the culture supernatant and detect the concentration of IL-15 by Elisa.

[0166] 2、Experimental results

[0167] (1) The results ( Figure 14 ) show that the tumor killing effect of mIL-15-CAR-NK cells constructed according to the application is significantly stronger than that of control CAR-NK cells and NK cells, indicating that the mIL-15-CAR-NK cells constructed according to the application have the strongest anti-tumor ability.

[0168] (2) The results ( Figure 15 ) show that there is no statistical difference in the concentration of IL-15 in the supernatant of mIL-15-CAR-NK cells constructed according to the application and control CAR-NK cells and NK cells, indicating that the mIL-15-CAR-NK cells constructed according to the application do not secrete IL-15 to the outside of the cells.

[0169] The above examples are 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, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A cell membrane expressed IL-15 fusion protein, characterized in that, The fusion protein sequentially connects, from the amino terminus to the carboxyl terminus, an IL-15Rα signal peptide, IL-15, a connecting region, a sushi domain of IL-15Rα, a Mab Meroit binding region, a CD8 hinge region, and a FcεRIγ transmembrane region; The amino acid sequence of the IL-15Rα signal peptide is shown as SEQ ID NO: 1; The amino acid sequence of the IL-15 is shown as SEQ ID NO: 3; The amino acid sequence of the connecting region is shown as SEQ ID NO: 7; The amino acid sequence of the sushi domain of IL-15Rα is shown as SEQ ID NO: 5; The amino acid sequence of the Mab Meroit binding region is shown as SEQ ID NO:

9.

2. The cell membrane expressed IL-15 fusion protein of claim 1, wherein, The amino acid sequence of the FcεRIγ transmembrane region is shown as SEQ ID NO:

13.

3. The cell membrane expressed IL-15 fusion protein of claim 1, wherein, The amino acid sequence of the CD8 hinge region is shown as SEQ ID NO:

11.

4. A chimeric antigen receptor expressed by a membrane that expresses IL-15, characterized in that, The chimeric antigen receptor comprises the fusion protein of any one of claims 1-3.

5. The membrane chimeric antigen receptor expressing IL-15 of claim 4, wherein, The membrane expressing IL-15 chimeric antigen receptor further comprises a self-cleaving peptide.

6. The membrane expressing IL-15 chimeric antigen receptor of claim 5, wherein, The self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A.

7. The membrane expressing IL-15 chimeric antigen receptor of claim 6, wherein, The self-cleaving peptide is T2A.

8. The membrane expressing IL-15 chimeric antigen receptor of claim 4, wherein, The chimeric antigen receptor further comprises an antigen recognition region, a transmembrane domain, an intracellular signaling domain, a hinge region, or a costimulatory signaling domain.

9. The membrane chimeric antigen receptor expressing IL-15 of claim 8, wherein, The antigen recognition region is selected from an antibody against a tumor surface antigen.

10. The membrane chimeric antigen receptor expressing IL-15 of claim 9, wherein, The tumor surface antigen comprises one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, FAP, Glypican-3, CEA, CA125, CA199, CA15-3, SCC, NSE, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, GD2, Pro-GRP, CYFRA21-1, TRCP-5b, sB7-H3, DCP, OPN, GP73, CA72-4, MG7-AG, PG, CA 50, DC-SIGNR, CHGA, PSA, GPRC5D, Siglec-6.

11. The membrane chimeric antigen receptor expressing IL-15 of claim 10, wherein, The tumor surface antigen is selected from mesothelin.

12. The membrane chimeric antigen receptor expressing IL-15 of claim 9, wherein, The amino acid sequence of the heavy chain variable region of the antibody against the tumor surface antigen is shown as SEQ ID NO: 17; and the amino acid sequence of the light chain variable region of the antibody against the tumor surface antigen is shown as SEQ ID NO:

18.

13. The membrane chimeric antigen receptor expressing IL-15 of claim 8, wherein, The transmembrane domain comprises a CD8 transmembrane region, a CD8a transmembrane region, a CD28 transmembrane region, a CD4 transmembrane region, a CD3 zeta transmembrane region, a CD137 / 4-1BB transmembrane region, a CD134 / OX40 transmembrane region, an ICOS transmembrane region, a CD5 transmembrane region, a CD9 transmembrane region, a CD16 transmembrane region, a CD22 transmembrane region, a CD33 transmembrane region, a CD37 transmembrane region, a CD45 transmembrane region, a CD64 transmembrane region, a CD80 transmembrane region, a CD86 transmembrane region, a CD154 transmembrane region, a TCRa transmembrane region, a TCRp transmembrane region.

14. The membrane chimeric antigen receptor expressing IL-15 of claim 13, wherein, The transmembrane domain is selected from a CD28 transmembrane region.

15. The membrane chimeric antigen receptor expressing IL-15 of claim 8, wherein, The intracellular signaling domain comprises an intracellular signaling domain of any one of the following molecules: CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, FcR gamma, FcR beta, TCR zeta, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fc epsilon RI, DAP10, DAP12, CD66d.

16. The membrane expressing IL-15 chimeric antigen receptor of claim 15, wherein, The intracellular signaling domain is an intracellular signaling domain of CD3 zeta.

17. The membrane chimeric antigen receptor expressing IL-15 of claim 16, wherein, The intracellular signaling domain has an amino acid sequence as set forth in SEQ ID NO:

20.

18. The membrane chimeric antigen receptor expressing IL-15 of claim 8, wherein, The hinge region comprises a hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3 epsilon, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor.

19. The membrane chimeric antigen receptor expressing IL-15 of claim 18, wherein, The hinge region is selected from a CD28 hinge region.

20. The membrane chimeric antigen receptor expressing IL-15 of claim 8, wherein, The costimulatory signaling domain comprises a costimulatory signaling domain of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8a, CD8b, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226.

21. The membrane expressing IL-15 chimeric antigen receptor of claim 20, wherein, The costimulatory signaling domain is selected from a costimulatory signaling domain of 4-1BB.

22. The membrane chimeric antigen receptor expressing IL-15 of claim 21, wherein, The costimulatory signaling domain has an amino acid sequence as set forth in SEQ ID NO:

21.

23. The membrane chimeric antigen receptor expressing IL-15 of claim 4, wherein, The chimeric antigen receptor further comprises a signal peptide.

24. The membrane expressing IL-15 chimeric antigen receptor of claim 23, wherein, The signal peptide comprises a signal peptide of any one of the following molecules: CD3 zeta, CD3 epsilon, CD16, CD22, CD33, CD4, CD5, CD8a, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF.

25. The membrane expressing IL-15 chimeric antigen receptor of claim 24, wherein, The signal peptide is selected from a signal peptide of CD8a.

26. A nucleic acid molecule, wherein, The nucleic acid molecule encodes the fusion protein of any one of claims 1-3, or encodes the chimeric antigen receptor of any one of claims 4-25.

27. The nucleic acid molecule of claim 26, wherein The nucleic acid molecule further comprises a promoter, and / or a restriction site located after the promoter, and / or a kozak sequence located after the restriction site.

28. The nucleic acid molecule of claim 26, wherein The nucleic acid molecule sequence encoding the IL-15 fusion protein expressed by the membrane is shown as SEQ ID NO:

16.

29. A vector, comprising: The vector contains the nucleic acid molecule according to any one of claims 26-28.

30. An engineered cell, comprising: The engineered cell comprises the nucleic acid molecule according to any one of claims 26-28, or comprises the expression vector according to claim 29.

31. The engineered cell of claim 30, wherein, The engineered cell comprises a eukaryotic cell, a prokaryotic cell.

32. The engineered cell of claim 31, wherein, The eukaryotic cell comprises a mammalian cell, an insect cell, a yeast cell.

33. The engineered cell of claim 32, wherein, The mammalian cell comprises an immune cell, a CHO cell, a 293T cell, a 293F cell.

34. The engineered cell of claim 33, wherein, The immune cell comprises a T cell, a B cell, a NK cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, a neutrophil, or any combination thereof.

35. The engineered cell of claim 33, wherein, The immune cell comprises an iNKT cell, a CTL cell.

36. The engineered cell of claim 33, wherein, The immune cell comprises an NK92 cell.

37. The engineered cell of claim 34, wherein, The T cell comprises an αβ T cell, a γδ T cell.

38. The engineered cell of claim 34, wherein, The immune cell is selected from an NK cell.

39. The engineered cell of any of claims 37-38, wherein, The γδ T cell, the NK cell is derived from a human.

40. A composition comprising the fusion protein according to any one of claims 1-3, the chimeric antigen receptor according to any one of claims 4-25, the nucleic acid molecule according to any one of claims 26-28, the vector according to claim 29, the engineered cell according to any one of claims 30-39.

41. The composition of claim 40, wherein, The composition further comprises a pharmaceutically acceptable carrier and / or excipient.

42. A biological agent, characterized in that, The biological preparation comprises the composition according to any one of claims 40-41.

43. The biological preparation of claim 42, wherein, The dosage form of the biological preparation is an injection, a lyophilized agent, a cream, a gel, a drop, or a patch.

44. A kit comprising the fusion protein according to any one of claims 1-3, the chimeric antigen receptor according to any one of claims 4-25, the nucleic acid molecule according to any one of claims 26-28, the vector according to claim 29, the engineered cell according to any one of claims 30-39, the composition according to any one of claims 40-41, the biological preparation according to any one of claims 42-43.

45. Any one of the following methods: 1) A process for the preparation of a fusion protein according to any one of claims 1 to 3, characterized in that, The preparation method comprises culturing the engineered cell according to any one of claims 30-39, so that it can express the fusion protein according to any one of claims 1-3. 2) A preparation method of the engineered cell according to any one of claims 30-39, wherein the preparation method comprises introducing the nucleic acid molecule according to any one of claims 26-28 or the vector according to claim 29 into the cell.

46. Any one of the following uses: 1) Use of the fusion protein according to any one of claims 1-3, the chimeric antigen receptor according to any one of claims 4-25, the nucleic acid molecule according to any one of claims 26-28, the vector according to claim 29, the engineered cell according to any one of claims 30-39, the composition according to any one of claims 40-41, the biological preparation according to any one of claims 42-43, or the kit according to claim 44 in the preparation of an anti-tumor drug. 2) Use of the fusion protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-25, the nucleic acid molecule of any one of claims 26-28, the vector of claim 29, the engineered cell of any one of claims 30-39, the composition of any one of claims 40-41, the biological agent of any one of claims 42-43, or the kit of claim 44 in the preparation of a medicament for increasing the survival ability of immune cells; 3) Use of the fusion protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-25, the nucleic acid molecule of any one of claims 26-28, the vector of claim 29, the engineered cell of any one of claims 30-39, the composition of any one of claims 40-41, the biological agent of any one of claims 42-43, or the kit of claim 44 in the preparation of a medicament for inhibiting the apoptosis of immune cells; 4) Use of the fusion protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-25, the nucleic acid molecule of any one of claims 26-28, the vector of claim 29, the engineered cell of any one of claims 30-39, the composition of any one of claims 40-41, the biological agent of any one of claims 42-43, or the kit of claim 44 in the preparation of a medicament for enhancing the proliferation ability of immune cells; 5) Use of the fusion protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-25, the nucleic acid molecule of any one of claims 26-28, the vector of claim 29, the engineered cell of any one of claims 30-39, the composition of any one of claims 40-41, the biological agent of any one of claims 42-43, or the kit of claim 44 in the preparation of a medicament for increasing the anti-tumor ability of immune cells; 8) Use of the fusion protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-25, the nucleic acid molecule of any one of claims 26-28, the vector of claim 29, the engineered cell of any one of claims 30-39, the composition of any one of claims 40-41, the biological agent of any one of claims 42-43, or the kit of claim 44 in killing tumor cells in vitro; 9) Use of the engineered cell of any one of claims 30-39 in the preparation of the fusion protein of any one of claims 1-3; 10) Use of the nucleic acid molecule of any one of claims 26-28 or the vector of claim 29 in the preparation of the engineered cell of any one of claims 30-39.

47. The use according to claim 46, wherein The tumor comprises one or more of adrenal cortical carcinoma, bladder urothelial carcinoma, breast carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, leukemia, brain low-grade glioma, hepatocellular carcinoma, mesothelioma, ovarian carcinoma, pancreatic carcinoma, pheochromocytoma and paraganglioma, prostate carcinoma, rectal carcinoma, sarcoma, stomach carcinoma, testicular germ cell tumor, thyroid carcinoma, thymus carcinoma, endometrial carcinoma, uterine sarcoma, myeloma, lymphoma, lung carcinoma, sarcoma, anal carcinoma, melanoma, retinoblastoma.

48. The use of claim 46, wherein, The tumor comprises uveal melanoma.

49. The use of claim 47, wherein, The tumor is selected from leukemia, ovarian cancer, lymphoma.

50. The use of claim 49, wherein, The tumor is ovarian cancer.

51. The use of claim 46, wherein, The immune cells comprise T cells, B cells, NK cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof.

52. The use of claim 46, wherein, The immune cells comprise iNKT cells, CTL cells.

53. The use of claim 46, wherein, The immune cells comprise NK92 cells.

54. The use of claim 51, wherein, The T cells comprise γδ T cells.

55. The use of claim 46, wherein, The immune cells are selected from γδ T cells, NK cells.

56. The use of claim 55, wherein, The γδ T cells, NK cells are derived from humans.

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