Cell membrane-expressed il-15 fusion protein and use thereof in cell therapy

Abstract

Provided are a cell membrane-expressed IL-15 fusion protein and a use thereof in cell therapy. The provided fusion protein comprises an IL-15Rα signal peptide, IL-15, an IL-15Rα sushi domain, and a transmembrane region; and a cell modified with the fusion protein can achieve efficient expression of IL-15 on the surface of a cell membrane, can effectively avoid side effects caused by the secretion and expression of IL-15, and is safe and controllable. An immune cell modified with a chimeric antigen receptor containing the membrane-expressed IL-15 fusion protein has a stronger tumor killing function, higher IFN-γ secretion, and a greater proliferative capacity; after treatment with lactic acid and TGF-β which are abundant in a tumor microenvironment, the immune cell retain a strong tumor killing function; and the immune cell can activate the anti-tumor function of immune cells other than the immune cell. The constructed immune cell can improve the therapeutic effect against tumors, prolong the duration of disease remission, and improve overall survival in patients.

Description

A cell membrane-expressed IL-15 fusion protein and its application in cell therapy Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cell membrane-expressing IL-15 fusion protein and its applications. Background Technology

[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 and genetically modified to express receptors that recognize tumor antigens, thereby enabling them to identify and attack tumor cells. CAR-T cell therapy has achieved significant breakthroughs in the treatment of hematological malignancies; however, it currently does not benefit most cancer patients due to factors such as the difficulty in target selection, the complex and costly manufacturing process, and the need to inhibit the tumor microenvironment.

[0003] CAR-NK cells are a novel immunotherapy that has emerged in recent years, combining the characteristics of CAR technology and NK cells. Compared to traditional CAR-T cells, CAR-NK cells have several unique advantages: ① Broad-spectrum anti-tumor activity: CAR-NK cells can recognize and attack multiple types of tumors, greatly expanding the treatment scope; ② Lower risk of cytokine release syndrome (CRS): Compared to CAR-T cells, CAR-NK cells typically cause milder 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 for faster treatment. NK cells can also be derived from healthy donors, enabling allogeneic infusion at a low cost. However, CAR-NK cells also have some drawbacks: ① Low persistence and survival: CAR-NK cells typically have a short survival time in vivo, lacking the persistence of CAR-T cells, which may lead to weakened treatment efficacy or an increased risk of tumor recurrence. ② Suppression of the tumor microenvironment: Similar to CAR-T cells, CAR-NK cells are also affected by the tumor microenvironment. The immunosuppressive effect of the tumor microenvironment may inhibit the efficacy of CAR-NK cells.

[0004] γδT cells are a special type of immune cell that possesses both acquired and innate immune response characteristics. CAR-γδT cell therapy is an emerging immunotherapy approach with enhanced tumor cell recognition and elimination capabilities. CAR-γδT cells offer the following advantages: ① Diverse targeting capabilities: γδT cells can recognize a variety of non-specific antigens, such as heat shock proteins, phosphorylated antigens, and some bacterial antigens, thus targeting various types of tumors; ② Innate immune characteristics: γδT cells possess natural killer activity, capable of eliminating tumor cells through direct cytotoxicity and cytokine secretion, independent of specific antigen presentation; ③ Lower risk of cytokine release syndrome: Compared to CAR-T cells, CAR-γδT cells typically have a lower risk of CRS and relatively milder side effects; ④ Rapid expansion and low cost: γδT cells can expand rapidly and respond quickly to infection and tumors in vivo, and can be infused allogeneically, achieving off-the-shelf availability and low cost. Of course, CAR-γδT cells also have some shortcomings: ① Low persistence and survival: CAR-γδT cells usually have low persistence in the body, which may limit the long-term effect of treatment; ② Suppression of the tumor microenvironment: Like CAR-T and CAR-NK cells, CAR-γδT cells are also affected by the tumor microenvironment. The immunosuppressive effect of the tumor microenvironment may inhibit the efficacy of CAR-γδT cells. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

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

[0008] In some embodiments, the amino acid sequence of the IL-15Rα signal peptide is shown in SEQ ID NO:1.

[0009] In some implementations, as long as the IL-15Rα retains the ability to bind with IL-15, it falls within the protection scope of this invention. The IL-15Rα can be the full-length IL-15Rα or a fragment that can bind with IL-15.

[0010] In some implementations, the IL-15Rα is the sushi domain of IL-15Rα.

[0011] In some embodiments, the amino acid sequence of the sushi domain of the IL-15Rα is shown in SEQ ID NO:5.

[0012] In some implementations, the transmembrane region can be any protein that can promote interactions between similar subunits to form dimers or multimers.

[0013] In some embodiments, the transmembrane region is the FcεRIγ transmembrane region.

[0014] In some embodiments, the amino acid sequence of the FcεRIγ transmembrane region is shown in SEQ ID NO:13.

[0015] In some implementations, the fusion protein also includes a safety protection zone or hinge zone.

[0016] In this invention, the safety protection zone is a region where the safety of the subject is ensured after the gene-edited immune cells are infused into the human body, and where the modified cells need to be controllable. In some embodiments, the safety protection zone includes CD20, iCasp9, tEGFR, and RQR8. In a specific embodiment of this invention, the safety protection zone is selected from CD20. When uncontrollable risks such as abnormal proliferation of the modified cells occur, the clinical drug Rituximab is administered. Rituximab binds to CD20, and the modified cells are cleared by the body's antibody-dependent cell-mediated cytotoxic effect, making it safer. In some embodiments, the amino acid sequence of CD20 is shown in SEQ ID NO:9.

[0017] In some embodiments, the hinge region enables IL-15 to remain outside the plasma membrane and possesses the necessary flexibility to facilitate the binding of membrane-expressed IL-15 to IL-15Rβ and γ receptors, stably delivering IL-15 activation signals. In some embodiments, the hinge region includes a CD28 hinge region, a CD8 hinge region, a CD4 hinge region, a hinge region of immunoglobulin IgG, and a CH2CH3-coupled hinge region of immunoglobulin IgG. In some embodiments, the hinge region is selected from the CD8 hinge region.

[0018] In some embodiments, the amino acid sequence of the hinge region is shown in SEQ ID NO:11.

[0019] In some embodiments, the IL-15Rα signal peptide, IL-15, IL-15Rα, and transmembrane region in the fusion protein are essential for achieving membrane expression. In one optional embodiment, a safety protection zone may be added to ensure the safety of membrane expression of IL-15. In another optional embodiment, a hinge region may be added to achieve better membrane expression of IL-15.

[0020] In some embodiments, the IL-15Rα signal peptide, IL-15, IL-15Rα, and transmembrane region of the fusion protein can be linked sequentially or in other orders. In one embodiment, when a safety protection zone and / or hinge region are added to the fusion protein, they can also be linked sequentially or in other orders. Regardless of the order of linkage, as long as the purpose of IL-15 membrane expression is achieved, it falls within the scope of protection of this invention. In some embodiments, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection zone, and hinge region can be directly linked to each other. In some embodiments, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection zone, and hinge region can be linked to each other through at least one linker, which can be flexible or rigid. In some embodiments, the linker includes glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP, or (GGGGS)n. In some embodiments, the linker is selected from (GGGGS)n.

[0021] In some implementations, the IL-15 and IL-15Rα are connected via a linker (GGGGS)5.

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

[0023] A second aspect of the present invention provides a chimeric antigen receptor that expresses IL-15 on a membrane, the chimeric antigen receptor comprising the fusion protein described in the first aspect of the present invention.

[0024] In this invention, the chimeric antigen receptor expressing IL-15 is a cell membrane that simultaneously expresses IL-15 and a chimeric antigen receptor (CAR). A CAR is a fusion protein comprising an extracellular domain capable of binding antigens and at least one intracellular domain. The CAR is a core component of chimeric antigen receptor immune cells and may include an antigen recognition region (e.g., tumor-specific antigens and / or tumor-associated antigens), a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory domain. The CAR is an engineered receptor that allows for the implantation of any specific receptor onto immune effector cells. Antibodies specifically recognizing tumor antigens can be implanted into immune cells within the CAR. Nucleic acid encoding the CAR can be introduced into immune cells 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] In some embodiments, the chimeric antigen receptor for membrane expression of IL-15 is formed by sequentially linking the fusion protein and CAR as described in the first aspect of the present invention, or by linking them in other orders. Regardless of the order of linking, as long as the purpose of IL-15 membrane expression is achieved, it falls within the protection scope of the present invention.

[0026] In some embodiments, the separate expression can be achieved by adding a self-splitting peptide between membrane-expressed IL-15 and the chimeric antigen receptor.

[0027] In some embodiments, the self-splitting peptide is selected from any one of the following self-splitting peptides: T2A, P2A, E2A, F2A.

[0028] In some embodiments, the self-fracture peptide is T2A.

[0029] In some implementations, the antigen recognition region is selected from antibodies targeting tumor surface antigens.

[0030] In some embodiments, the tumor surface antigen includes 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, and Siglec-6.

[0031] In some implementations, the tumor surface antigen is selected from mesothelin.

[0032] In some embodiments, the heavy chain variable region amino acid sequence of the antibody against tumor surface antigen is shown in SEQ ID NO:17.

[0033] In some embodiments, the amino acid sequence of the light chain variable region of the antibody against tumor surface antigen is shown in SEQ ID NO:18.

[0034] In some embodiments, the transmembrane structural domains include CD8 transmembrane regions, CD8α transmembrane regions, CD28 transmembrane regions, CD4 transmembrane regions, CD28 transmembrane regions, CD3ζ transmembrane regions, CD137 / 4-1BB transmembrane regions, CD134 / OX40 transmembrane regions, ICOS transmembrane regions, CD5 transmembrane regions, CD9 transmembrane regions, CD16 transmembrane regions, CD22 transmembrane regions, CD33 transmembrane regions, CD37 transmembrane regions, CD45 transmembrane regions, CD64 transmembrane regions, CD80 transmembrane regions, CD86 transmembrane regions, CD154 transmembrane regions, TCRα transmembrane regions, and TCRβ transmembrane regions.

[0035] In some implementations, the transmembrane domain is selected from the CD28 transmembrane region.

[0036] In some embodiments, the amino acid sequence of the transmembrane domain corresponds to a portion of the amino acid sequence shown in SEQ ID NO:19.

[0037] In some embodiments, the intracellular signal transduction domain includes the intracellular signal transduction domain of any of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d.

[0038] In some implementations, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

[0039] In some embodiments, the amino acid sequence of the intracellular signal transduction domain is shown in SEQ ID NO:20.

[0040] In some embodiments, the hinge region includes the hinge region of any of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor.

[0041] In some implementations, the hinge region is selected from the CD28 hinge region.

[0042] In some embodiments, the amino acid sequence of the hinge region corresponds to a portion of the amino acids shown in SEQ ID NO:19.

[0043] In some embodiments, the co-stimulatory signaling domain includes any of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226.

[0044] In some implementations, the costimulatory signaling domain is selected from the costimulatory signaling domains of 4-1BB.

[0045] In some embodiments, the amino acid sequence of the co-stimulatory signaling domain is shown in SEQ ID NO:21.

[0046] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0047] In some embodiments, the signal peptide includes a signal peptide of any of the following molecules: α and β chains of T cell receptors, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8α, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF.

[0048] In some embodiments, the signal peptide is selected from the CD8α signal peptide.

[0049] A third aspect of the present invention provides a nucleic acid molecule that encodes a fusion protein as described in the first aspect, or a chimeric antigen receptor as described in the second aspect.

[0050] In some embodiments, the nucleic acid molecule further includes a promoter, and / or an enzyme cleavage site following the promoter, and / or a kozak sequence following the enzyme cleavage site.

[0051] In this invention, nucleic acid molecules refer to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0052] In some embodiments, the nucleic acid molecule sequence encoding IL-15 expressed by the membrane corresponds to part or all of the sequence shown in SEQ ID NO:16.

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

[0054] A fourth aspect of the present invention provides a carrier containing the nucleic acid molecule described in the third aspect of the present invention.

[0055] In some embodiments, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and granules may be modified depending on the cells to be introduced. Introduction can be performed using standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modalities include, for example, naked DNA, CaPO4 precipitation, DEAE glucan, electroporation, protoplast fusion, liposome transfection, 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, herpesviruses, and lentiviruses. Furthermore, to assess the expression of a target protein (e.g., a monoclonal antibody), the vector introduced into the cells may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a cell population seeking transfection or infection via the viral vector.

[0056] In some embodiments, the vector may be an expression vector, a cloning vector, or an integration vector. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter for regulating the expression of a desired nucleic acid sequence. An integration vector contains components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences typically 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 splicing sites, a leader sequence encoding a polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-linker region for inserting a nucleic acid encoding the monoclonal antibody to be expressed, and optional marker elements. In specific embodiments of the invention, the vectors used are the lentiviral expression plasmid pCDH, the lentiviral packaging plasmid pSPAX2, and the pMD2G vector.

[0057] The fifth aspect of the present invention provides an engineered cell and its derivatives, wherein the engineered cell and its derivatives comprise the nucleic acid molecule described in the third aspect of the present invention, or comprise the expression vector described in the fourth aspect of the present invention.

[0058] In some embodiments, the derivatives include, but are not limited to, extracellular vesicles, and any derivatives produced by the engineered cells described in the fifth aspect of the present invention fall within the scope of protection of the present invention.

[0059] In this invention, extracellular vesicles are a group of vesicles secreted by cells and having a lipid bilayer membrane structure. The contents of extracellular vesicles include extracellular vesicle miRNA, extracellular vesicle mRNA, or extracellular vesicle surface proteins. Based on the size of extracellular vesicles, they can be divided into exosomes, microvesicles, and apoptotic bodies.

[0060] In some implementations, the extracellular vesicles are selected from exosomes.

[0061] In some implementations, the engineered cells include eukaryotic cells and prokaryotic cells.

[0062] In some implementations, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0063] In some embodiments, the mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells.

[0064] In some embodiments, 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] In some implementations, the T cells include αβT cells and γδT cells.

[0066] In some embodiments, the immune cells are selected from γδT cells and NK cells.

[0067] In some implementations, the γδT cells and NK cells are derived from humans.

[0068] In some implementations, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0069] A sixth aspect of the present invention provides a composition comprising the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, and the engineered cell and its derivatives of the fifth aspect of the present invention.

[0070] In some embodiments, the composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0071] A seventh aspect of the present invention provides a biological agent comprising the pharmaceutical composition described in the sixth aspect of the present invention.

[0072] In some embodiments, the dosage form of the biological agent is an injection, a lyophilized preparation, an oral preparation, a cream, a gel, drops, or a patch.

[0073] In some embodiments, the present invention does not impose particular limitations on the specific dosage form of the biological agent. In some embodiments, the pharmaceutical composition or biological agent provided by the present invention can be formulated for topical, intravenous, oral, enteral and / or parenteral administration as needed.

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

[0075] The ninth aspect of the present invention provides a method for preparing the fusion protein described in the first aspect of the present invention, the method comprising culturing the engineered cells and their derivatives described in the fifth aspect of the present invention to enable them to express the fusion protein described in the first aspect of the present invention.

[0076] The ninth aspect of the present invention also provides a method for preparing engineered cells and their derivatives as described in the fifth aspect of the present invention, the method comprising introducing a nucleic acid molecule as described in the third aspect of the present invention or a vector as described in the fourth aspect of the present invention into cells.

[0077] The tenth aspect of this invention provides any of the following applications:

[0078] 1) The use of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in the preparation of diagnostic and / or therapeutic antitumor drugs;

[0079] 2) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in improving the survival ability of immune cells.

[0080] 3) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in inhibiting apoptosis of immune cells;

[0081] 4) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in enhancing the proliferation capacity of immune cells;

[0082] 5) The use of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in the preparation of drugs that enhance the anti-tumor ability of immune cells;

[0083] 6) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in enhancing the cytokine level of immune cells;

[0084] 7) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in the preparation of drugs that alter the tumor microenvironment;

[0085] 8) The application of the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in the in vitro killing of tumor cells;

[0086] 9) The use of the engineered cells and their derivatives as described in the fifth aspect of the present invention in the preparation of the fusion protein described in the first aspect of the present invention;

[0087] 10) The use of the nucleic acid molecule described in the third aspect of the present invention or the carrier described in the fourth aspect of the present invention in the preparation of the engineered cells and their derivatives described in the fifth aspect of the present invention.

[0088] In some implementations, the tumors include, but are not limited to, adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumors, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, and retinoblastoma.

[0089] In some implementations, the tumor is selected from leukemia, ovarian cancer, and lymphoma.

[0090] In some implementations, the tumor is ovarian cancer.

[0091] In some embodiments, the immune cells include T cells, B cells, NK cells, iNKT cells, γδT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof.

[0092] In some embodiments, the immune cells are selected from γδT cells and NK cells.

[0093] In some implementations, the γδT cells and NK cells are derived from humans.

[0094] In some implementations, the cytokines include interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors.

[0095] In some implementations, the cytokines are selected from interleukins and interferons.

[0096] In some implementations, the cytokine is interferon.

[0097] In some implementations, the interferon includes IFN-α, IFN-β, IFN-κ, IFN-γ, and IFN-λ.

[0098] In some implementations, the interferon is selected from IFN-γ.

[0099] The eleventh aspect of this invention provides any of the following methods:

[0100] 1) A method for treating tumors, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0101] 2) A method for enhancing the survival ability of immune cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0102] 3) A method for inhibiting apoptosis of immune cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0103] 4) A method for enhancing the proliferative capacity of immune cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0104] 5) A method for enhancing the anti-tumor ability of immune cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0105] 6) A method for enhancing the cytokine levels of immune cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0106] 7) A method for altering the tumor microenvironment, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0107] 8) A method for killing tumor cells, the method comprising administering the fusion protein of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the engineered cells and their derivatives of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, the biological agent of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention.

[0108] In some implementation schemes, the explanations regarding tumors, immune cells, and cytokines are as described above.

[0109] The advantages and beneficial effects of this invention are as follows: This invention provides a cell membrane-expressed IL-15 fusion protein and its application in cell therapy technology. This invention connects the IL-15Rα signal peptide, IL-15, the IL-15Rα sushi domain, and the transmembrane region in series with a CAR, modifying immune cells and achieving efficient expression of IL-15 on the surface of immune cell membranes. Experiments have demonstrated that the engineered immune cells of the IL-15-IL-15Rα sushi-FcεRI can achieve efficient membrane expression of IL-15, strong tumor cell killing, and potent anti-tumor effects. Furthermore, these engineered immune cells also promote immune cell proliferation, increase IFN-γ expression levels, and alter the tumor microenvironment. Attached Figure Description

[0110] Figure 1 is a schematic diagram of the membrane expression of IL-15;

[0111] Figure 2 shows the results of γδT purity detection in CAR-γδT cells;

[0112] Figure 3 shows the results of CAR expression efficiency detection in CAR-γδT cells;

[0113] Figure 4 shows the results of detecting the expression efficiency of IL-15 (mIL-15) on the surface of CAR-γδT cells;

[0114] Figure 5 shows the results of the detection of the killing effect of CAR-γδT cells on tumor cells;

[0115] Figure 6 shows the results of the detection of the re-killing effect of CAR-γδT cells on tumor cells;

[0116] Figure 7 shows the results of CAR-γδT cell proliferation assay.

[0117] Figure 8 shows the results of CD107a expression analysis in CAR-γδT cells;

[0118] Figure 9 shows the results of IFN-γ expression analysis in CAR-γδT cells;

[0119] Figure 10 shows the changes in tumor-killing function of CAR-γδT cells after treatment with lactate or TGF-β.

[0120] Figure 11 shows the effect of CAR-γδT cells on the tumor-killing effect of γδT cells.

[0121] Figure 12 shows the effect of CAR-γδT cells on the secretion of IFN-γ by γδT cells;

[0122] Figure 13 is a graph showing the effect of Rituximab on the clearance of CAR-γδT cells;

[0123] Figure 14 shows the results of the detection of the killing effect of CAR-NK cells on tumor cells;

[0124] Figure 15 shows the results of detecting the level of IL-15 secreted by CAR-NK cells. Detailed Implementation

[0125] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, the reagents, biological materials, etc., used in the following embodiments are commercially available.

[0126] Example 1: Construction of membrane-expressed IL-15

[0127] The signal peptide of IL-15Rα was directly linked to the human IL-15 encoding gene. The sushi region of IL-15Rα was linked using a flexible linker (G4S). The CD20 binding region of rituximab was linked to the sushi region of IL-15Rα. A CD8 hinge region was added after the rituximab binding region. The intracellular segment of the transmembrane region FcεRIγ (encoding gene FCER1G) was linked to the CD8 hinge region. The structure of the membrane-expressed IL-15 is shown in Figure 1. The sequences of the genes involved in constructing the membrane-expressed IL-15 are shown in Table 1.

[0128] Table 1. Sequences of the genes involved in constructing membrane-expressed IL-15.

[0129] Example 2: Construction and functional detection of CAR-γδT cells expressing IL-15 on membrane

[0130] 1. Experimental Materials

[0131] The experimental materials are shown in Table 2:

[0132] Table 2 Experimental Materials

[0133] 2. Experimental Methods

[0134] (1) The membrane-expressing IL-15 encoding gene constructed in this invention was linked to a CAR targeting mesothelin (MSLN) using T2A (patent number: CN111548420A). This 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, as well as human 4-1BB and CD3ζ cytoplasmic regions. The constructed structure was subcloned into the lentiviral expression plasmid pCDH, which was then mixed with the packaging plasmids pSPAX2 and pMD2G and transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. The virus was concentrated using PEG8000. The amino acid sequences of each component of the CAR are shown in Table 3.

[0135] Table 3. Amino acid sequences of the components that make up the CAR

[0136] γδT cell preparation: Human peripheral blood mononuclear cells (PBMCs) were collected and adjusted to a size of 1-2 × 10⁻⁶. 6 Complete culture medium (ALyS505N-0 serum-free cell culture medium containing 600 IU / mL IL-2, 50 μL / mL glutamine, 5-10% (v / v) autologous plasma, and 5 μM / mL zoledronic acid) was prepared by gentle shaking and cultured in a 5% CO2 incubator at 37°C.

[0137] γδT cells in logarithmic growth phase (PBMCs cultured for 5-7 days, MOI=10) were transfected with lentivirus. One day after viral infection, the virus-containing culture medium was removed, and the cells were cultured in complete culture medium without zoledronic acid until day 11, at which point the cells could be harvested.

[0138] (2) The purity of γδT cells in CAR-γδT cells was detected. Cultured cells were taken and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL, take 100 μL and add it to a flow cytometer tube, add 5 μL each of different fluorescently labeled mouse anti-human CD3, Vγ9 and IL-15 antibodies and fluorescently labeled human Mesothelin (MSLN) protein, incubate at room temperature in the dark for 15 min, centrifuge at 1500 rpm at room temperature for 5 min, discard the supernatant, add 2 mL PBS to wash the cells, centrifuge at 500 rpm at room temperature for 5 min, discard the supernatant, add 200 μL PBS to suspend the cells, and detect by flow cytometry.

[0139] (3) Detection of tumor killing ability in vitro: CAR-γδT cells and γδT cells were cultured for 24h, 48h and 72h respectively after removing IL-2; SKOV3 cells expressing Mesothelin were labeled with Calcein-AM and then treated with the cells obtained in Example 2 at an effector-target ratio of 5:1 for 4h. The release of Calcein after the target cells were killed was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the killing effect of CAR-γδT cells and γδT cells on tumor cells was detected.

[0140] (4) After treating cultured CAR-γδT cells with SKOV3 cells at a 10:1 effector-to-target ratio for 24 hours, CAR-γδT cells were isolated. For the secondary killing assay, the isolated CAR-γδT cells were treated with Calcein-AM-labeled SKOV3 cells at a 5:1 effector-to-target ratio for 4 hours, and the killing effect of CAR-γδT cells on tumor cells was detected. For the tertiary killing assay, the isolated CAR-γδT cells were treated with SKOV3 cells at a 10:1 effector-to-target ratio for another 24 hours, CAR-γδT cells were isolated again, and then treated with Calcein-AM-labeled SKOV3 cells at a 5:1 effector-to-target ratio for 4 hours, and the killing effect of CAR-γδT cells on tumor cells was detected.

[0141] (5) Assess in vitro proliferation capacity: Detect Ki67 expression levels. Cultured cells were harvested and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL, 100 μL was added to a flow cytometry tube, and after membrane perforation, 5 μL of fluorescently labeled mouse anti-human Ki67 antibody was added. The cells were incubated at room temperature in the dark for 20-30 min, centrifuged at 1500 rpm at room temperature for 5 min, the supernatant was discarded, 2 mL of PBS was added to wash the cells, centrifuged at 500 rpm at room temperature for 5 min, the supernatant was discarded, 200 μL of PBS was added to resuspend the cells, and flow cytometry was used for detection.

[0142] (6) Detection of CD107a: The cultured cells were treated with SKOV3 cells at an effector-target ratio of 10:1 for 4 hours, and the expression of CD107a in the cultured cells was detected by flow cytometry.

[0143] (7) After co-incubating CAR-γδT cells with SKOV3 tumor cells for 4 hours, the expression of IFN-γ was detected by intracellular staining.

[0144] (8) After treating CAR-γδT cells with lactate or TGF-β for 24 h, the killing effect on tumor cells was detected by Calcein-Am release assay.

[0145] (9) After co-incubating γδT cells from the same donor with CAR-γδT cells at a 1:1 ratio for 24 h, the γδT cells were then treated with Calcein-AM labeled SKOV3 tumor cells for 4 h, and the killing effect was detected.

[0146] (10) After co-incubating γδT cells from the same donor with CAR-γδT cells at a 1:1 ratio for 24 h, and then treating γδT cells with Calcein-AM labeled tumor cells SKOV3 cells for 4 h, the expression of IFN-γ was detected by intracellular staining.

[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) The results (Figure 2) 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% in both the control CAR-γδT cells (CAR-γδT) and the 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) The results (Figure 3) 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) The results (Figure 4) 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) The results (Figure 5) show that the mIL-15-CAR-γδT cells constructed in this invention have stronger tumor killing effects than the control CAR-γδT cells and γδT cells after 24h, 48h and 72h of culture without IL-2. This indicates that the CAR-γδT cells constructed in this invention still have the strongest anti-tumor ability under the condition of IL-2 removal.

[0153] (5) The results (Figure 6) show that the secondary and tertiary tumor killing abilities of the mIL-15-CAR-γδT cells constructed in this invention are 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) The results (Figure 7) show that the Ki67 expression of the mIL-15-CAR-γδT cells constructed in this invention is significantly higher than that of the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention have the strongest proliferation ability.

[0155] (7) The results (Figure 8) show that the CD107a expression of the mIL-15-CAR-γδT cells constructed in this invention is significantly higher than that of the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention have stronger killing activity against tumor cells.

[0156] (8) The results (Figure 9) show that the IFN-γ expression of the mIL-15-CAR-γδT cells constructed in this invention is significantly higher than that of the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention have stronger anti-tumor function.

[0157] (9) The results (Figure 10) show that the mIL-15-CAR-γδT cells constructed in this invention still have a significantly higher killing effect on tumor cells than the control group after treatment with lactate or TGF-β, indicating that the mIL-15-CAR-γδT cells constructed in this invention still maintain a strong anti-tumor effect even in the tumor microenvironment rich in lactate and TGF-β.

[0158] (10) The results (Figure 11) showed that the CD107a expression of γδT cells co-incubated with the mIL-15-CAR-γδT cells constructed in this invention was significantly higher than that of the control group, indicating that the mIL-15-CAR-γδT cells constructed in this invention can enhance the killing effect of γδT cells that do not contain this invention on tumor cells.

[0159] (11) The results (Figure 12) show that the IFN-γ expression of γδT cells co-incubated with the mIL-15-CAR-γδT cells constructed in this invention is significantly increased, indicating that the mIL-15-CAR-γδT cells constructed in this invention can promote the secretion of more IFN-γ by γδT cells that do not contain this invention.

[0160] (12) The results (Figure 13) showed that the killing effect of NK cells on the CAR-γδT cells of the present invention and the control was low and there was no statistical difference; however, when NK cells and CAR-γδT cells were interacting, rituximab was added at the same time, and the killing effect of NK cells on the mIL-15-CAR-γδT cells constructed in the present invention was significantly higher than that on the control CAR-γδT cells, indicating that the mIL-15-CAR-γδT cells constructed in the present invention can be cleared by rituximab-dependent NK cells.

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

[0162] 1. Experimental Methods

[0163] (1) Use lentivirus to transfect human NK cells in the logarithmic growth phase (NK cells cultured for 5-7 days, MOI=10). One day after viral infection, remove the culture medium containing the virus, replace it with new NK cell culture medium and continue culturing until day 11, at which point the cells can be harvested.

[0164] (2) NK cells, control CAR-NK cells and the mIL-15-CAR-NK cells constructed in this invention were cultured for 24 h after removing IL-2, and then treated with SKOV3 cells at an effector-target ratio of 2.5:1 for 4 h. The killing effect of NK and CAR-NK cells on tumor cells was then detected.

[0165] (3) NK cells, control CAR-NK cells and the mIL-15-CAR-NK cells constructed in this invention were cultured for 24 h after removing IL-2, and then the culture supernatant was collected and the concentration of IL-15 was detected by ELISA.

[0166] 2. Experimental Results

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

[0168] (2) The results (Figure 15) show that there is no statistically significant difference in the concentration of IL-15 in the supernatant of the mIL-15-CAR-NK cells constructed in this invention and the control CAR-NK cells and NK cells, indicating that the mIL-15-CAR-NK cells constructed in this invention do not secrete IL-15 into the extracellular space.

[0169] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A cell membrane expressed IL-15 fusion protein, characterized in that, The fusion protein includes IL-15, IL-15Rα signal peptide, IL-15Rα, and a transmembrane region; Preferably, the amino acid sequence of the IL-15Rα signal peptide is shown in SEQ ID NO:1; Preferably, the IL-15Rα is a full-length IL-15Rα or a fragment that can bind to IL-15; Preferably, the IL-15Rα is the sushi domain of IL-15Rα; Preferably, the amino acid sequence of the sushi domain of the IL-15Rα is shown in SEQ ID NO:5; Preferably, the transmembrane region is the FcεRIγ transmembrane region; Preferably, the amino acid sequence of the FcεRIγ transmembrane region is as shown in SEQ ID NO:13; Preferably, the fusion protein further includes a safety protection zone or a hinge region; Preferably, the safety protection zone includes CD20, iCasp9, tEGFR, and RQR8; Preferably, the safety protection zone is selected from CD20; Preferably, the amino acid sequence of CD20 is as shown in SEQ ID NO:9; Preferably, the hinge region includes a CD28 hinge region, a CD8 hinge region, a CD4 hinge region, a hinge region of immunoglobulin antibody IgG, and a CH2CH3 region coupled to the hinge region of immunoglobulin antibody IgG. Preferably, the hinge area is selected from the CD8 hinge area; Preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO:11; Preferably, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection region, or hinge region can be linked in any order in which IL-15 is expressed on any membrane. Preferably, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection zone or hinge region can be connected in sequence; Preferably, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection region, or hinge region can be directly connected to each other; Preferably, the IL-15Rα signal peptide, IL-15, IL-15Rα, transmembrane region, safety protection region or hinge region can be connected to each other through at least one linker; Preferably, the linker comprises glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP, or (GGGGS)n; Preferably, the linker is selected from (GGGGS)n; Preferably, the IL-15 and IL-15Rα are connected via linker (GGGGS) 5; Preferably, the amino acid sequence of the linker is shown in SEQ ID NO:

7.

2. A chimeric antigen receptor expressed by a membrane expressing IL-15, characterized in that, The chimeric antigen receptor comprises the fusion protein of claim 1; Preferably, the membrane-expressing IL-15 chimeric antigen receptor further comprises a self-splitting peptide; Preferably, the self-splitting peptide is selected from any one of the following self-splitting peptides: T2A, P2A, E2A, F2A; Preferably, the self-fracture peptide is T2A; Preferably, the chimeric antigen receptor further comprises an antigen recognition region, a transmembrane domain, an intracellular signal transduction domain, a hinge region, or a co-stimulatory signal domain. Preferably, the antigen recognition region is selected from antibodies targeting tumor surface antigens; Preferably, the tumor surface antigen includes 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, and Siglec-6. Preferably, the tumor surface antigen is selected from mesothelin; Preferably, the amino acid sequence of the heavy chain variable region of the antibody against tumor surface antigen is shown in SEQ ID NO:17; Preferably, the amino acid sequence of the light chain variable region of the antibody against tumor surface antigen is shown in SEQ ID NO:18; Preferably, the transmembrane structural domains include CD8 transmembrane regions, CD8α transmembrane regions, CD28 transmembrane regions, CD4 transmembrane regions, CD28 transmembrane regions, CD3ζ transmembrane regions, CD137 / 4-1BB transmembrane regions, CD134 / OX40 transmembrane regions, ICOS transmembrane regions, CD5 transmembrane regions, CD9 transmembrane regions, CD16 transmembrane regions, CD22 transmembrane regions, CD33 transmembrane regions, CD37 transmembrane regions, CD45 transmembrane regions, CD64 transmembrane regions, CD80 transmembrane regions, CD86 transmembrane regions, CD154 transmembrane regions, TCRα transmembrane regions, and TCRβ transmembrane regions; Preferably, the transmembrane structural domain is selected from the CD28 transmembrane region; Preferably, the amino acid sequence of the transmembrane domain corresponds to a portion of the amino acid sequence shown in SEQ ID NO:19; Preferably, the intracellular signal transduction domain includes the intracellular signal transduction domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d; Preferably, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ; Preferably, the amino acid sequence of the intracellular signal transduction domain is shown in SEQ ID NO:20; Preferably, the hinge region includes the hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Preferably, the hinge area is selected from the CD28 hinge area; Preferably, the amino acid sequence of the hinge region corresponds to a portion of the amino acid sequence shown in SEQ ID NO:19; Preferably, the co-stimulatory signaling domain includes any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226; Preferably, the co-stimulation signal structure domain is selected from the co-stimulation signal structure domain of 4-1BB; Preferably, the amino acid sequence of the co-stimulatory signaling domain is shown in SEQ ID NO:21; Preferably, the chimeric antigen receptor further comprises a signal peptide; Preferably, the signal peptide comprises any one of the following signal peptides: α and β chains of T cell receptors, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8α, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; Preferably, the signal peptide is selected from the CD8α signal peptide.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein as described in claim 1, or the chimeric antigen receptor as described in claim 2; Preferably, the nucleic acid molecule further comprises a promoter, and / or an enzyme cleavage site following the promoter, and / or a kozak sequence following the enzyme cleavage site; Preferably, the nucleic acid sequence encoding IL-15 expressed by the membrane corresponds to part or all of the sequence shown in SEQ ID NO:16; Preferably, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of the sequence shown in SEQ ID NO:

22.

4. A vector, characterized in that, The carrier contains the nucleic acid molecule as described in claim 3.

5. An engineered cell and derivatives thereof, characterized in that, The engineered cells and their derivatives comprise the nucleic acid molecules of claim 3, or the expression vector of claim 4; Preferably, the derivative comprises extracellular vesicles; Preferably, the extracellular vesicles include microvesicles, exosomes, and apoptotic bodies; Preferably, the extracellular vesicles are selected from exosomes; Preferably, the engineered cells include eukaryotic cells and prokaryotic cells; Preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; Preferably, the mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells; Preferably, 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; Preferably, the T cells include αβT cells and γδT cells; Preferably, the immune cells are selected from γδT cells and NK cells; Preferably, the γδT cells and NK cells are derived from humans; Preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

6. A composition comprising the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell of claim 5, and derivatives thereof; Preferably, the composition further includes a pharmaceutically acceptable carrier and / or excipients.

7. A biological agent, characterized in that, The biological agent comprises the pharmaceutical composition of claim 6; Preferably, the dosage form of the biological agent is an injection, a lyophilized agent, an oral preparation, a cream, a gel, drops, or a patch.

8. A kit comprising the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, and the biological agent of claim 7.

9. Any one of the following methods: 1) A process for the preparation of a fusion protein according to claim 1, characterized in that, The preparation method includes culturing the engineered cells and their derivatives as described in claim 5, enabling them to express the fusion protein as described in claim 1; 2) A method for preparing engineered cells and their derivatives as described in claim 5, characterized in that the preparation method includes introducing the nucleic acid molecule as described in claim 3 or the vector as described in claim 4 into the cells.

10. Any of the following applications: 1) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in the preparation of antitumor drugs; 2) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in improving the survival ability of immune cells; 3) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in inhibiting apoptosis of immune cells; 4) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in enhancing the proliferative capacity of immune cells; 5) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in the preparation of drugs that enhance the anti-tumor ability of immune cells; 6) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in enhancing the cytokine levels of immune cells; 7) The use of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in the preparation of drugs that alter the tumor microenvironment; 8) The application of the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8 in the in vitro killing of tumor cells; 9) The use of the engineered cells and their derivatives as described in claim 5 in the preparation of the fusion protein as described in claim 1; 10) The use of the nucleic acid molecule of claim 3 or the carrier of claim 4 in the preparation of the engineered cells and their derivatives of claim 5; Preferably, the tumor includes one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, bile duct carcinoma, colonic 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, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, and retinoblastoma. Preferably, the tumor is selected from leukemia, ovarian cancer, or lymphoma; Preferably, the tumor is ovarian cancer; Preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, γδT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof; Preferably, the immune cells are selected from γδT cells and NK cells; Preferably, the γδT cells and NK cells are derived from humans; Preferably, the cytokines include interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors; Preferably, the cytokines are selected from interleukins and interferons; Preferably, the cytokine is interferon; Preferably, the interferon includes IFN-α, IFN-β, IFN-κ, IFN-γ, and IFN-λ; Preferably, the interferon is selected from IFN-γ.

11. Any of the following methods: 1) A method of treating a tumor, characterized in that, The method includes administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, or the kit of claim 8. 2) A method for improving the survival ability of immune cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 3) A method for inhibiting apoptosis of immune cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 4) A method for enhancing the proliferation capacity of immune cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 5) A method for enhancing the anti-tumor ability of immune cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 6) A method for enhancing the cytokine levels of immune cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 7) A method for altering the tumor microenvironment, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8. 8) A method for killing tumor cells, characterized in that the method comprises administering the fusion protein of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the carrier of claim 4, the engineered cell and its derivatives of claim 5, the composition of claim 6, the biological agent of claim 7, and the kit of claim 8.

Images