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WO2024113649A1 - Method for improving viability and anti-tumor activity of nk cells and use thereof - Google Patents

Method for improving viability and anti-tumor activity of nk cells and use thereof Download PDF

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Publication number
WO2024113649A1
WO2024113649A1 PCT/CN2023/089382 CN2023089382W WO2024113649A1 WO 2024113649 A1 WO2024113649 A1 WO 2024113649A1 CN 2023089382 W CN2023089382 W CN 2023089382W WO 2024113649 A1 WO2024113649 A1 WO 2024113649A1
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Prior art keywords
cells
nucleic acid
cell
car
positive tumor
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French (fr)
Chinese (zh)
Inventor
张彩
胡渊
陈敏华
王烃
伏永玲
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Shanghai NK Cell Tech Co Ltd
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Shanghai NK Cell Tech Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for improving the survival and anti-tumor activity of NK cells and an application thereof.
  • CAR-T chimeric antigen receptor T
  • CAR-NK cells have the advantage of good safety over CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; and NK cells do not require antigen presentation and are not restricted by MHC, and can directly kill tumor cells; CAR-NK cells can identify and kill tumors with a variety of recognition mechanisms such as CAR dependence and NKR dependence, and have a wide anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development. However, one of the difficulties faced in the development of CAR-NK cells is that NK cells have a short survival time in the body, which affects their in vivo effects.
  • IL-15 is a cytokine that can promote the survival, proliferation and function of T cells and NK cells.
  • IL-15 shares the IL-2/15R ⁇ c receptor with IL-2. After IL-15 and IL-15R ⁇ form dimers, they bind to IL-15R ⁇ c and activate the downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways, thereby promoting the proliferation, activation and effector function of NK cells. Therefore, IL-15 has become a hot target for drug development to enhance the persistence and proliferation activity of lymphocytes in the body.
  • IL-15 the problems with the in vivo application of IL-15 are its short half-life and limited in vivo efficacy. It requires the use of larger doses and frequent administration, which can lead to various side effects, including hypotension, thrombocytopenia, and elevated AST and ALT, which may make cancer patients unable to tolerate this treatment. In clinical applications and drug development, the activity of IL-15 in promoting lymphocyte proliferation and persistence and promoting immune response should be maintained as much as possible, while reducing IL15-related side effects as much as possible.
  • the present invention aims to solve one of the technical problems in the related art to at least a certain extent.
  • the present invention proposes a transgenic immune cell, whose proliferation ability and survival time in vivo, as well as its anti-tumor ability are significantly improved compared with natural immune cells, and has higher safety.
  • the present invention proposes a transgenic immune cell.
  • the immune cell expresses a chimeric antigen receptor and a fusion protein, and the fusion protein includes IL-15R ⁇ and IL-15, namely, super IL-15 as referred to below.
  • the transgenic immune cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and a fusion protein.
  • the chimeric antigen receptor can enable the immune cell to target the corresponding antigen and locate on the cell surface expressing the antigen.
  • the fusion protein is further It can promote the activation and proliferation of immune cells in one step, maintain the number and activity of immune cells in the local microenvironment of the tumor, enable them to maintain strong tumor killing activity, and effectively avoid the toxic side effects caused by high-dose or repeated injections throughout the body.
  • the genetically modified immune cells may further include at least one of the following additional technical features:
  • the chimeric antigen receptor includes: an extracellular region, which can specifically bind to an antigen; a transmembrane region; and an intracellular region, which includes an intracellular segment of an immune co-stimulatory molecule and a signal transduction domain; wherein the C-terminus of the extracellular region is connected to the N-terminus of the transmembrane region, and the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region.
  • the type of antigen recognized by the chimeric antigen receptor is not particularly limited, and is suitable for specific recognition of multiple antigens.
  • the antigen is a tumor-associated antigen.
  • the type of the antigen is not particularly limited.
  • the extracellular region includes a heavy chain variable region and a light chain variable region of an antibody, and the antibody binds to the antigen.
  • the extracellular region includes a binding region for recognizing the antigen, and the extracellular region may include at least one of a full antibody, a Fab antibody, a Fab' antibody, a F(ab') 2 antibody, a Fv antibody, a single-chain antibody, and a nanobody.
  • the extracellular region includes a single-chain antibody.
  • the antigen includes at least one selected from mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, GD2, PSCA, IL-13RA2, B7-H3, CD133, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA.
  • the antigen is mesothelin.
  • the transgenic immune cells can effectively target mesothelin-positive tumors, retain high proliferation activity, and have high anti-tumor ability.
  • the extracellular region comprises an anti-mesothelin single-chain antibody.
  • the anti-mesothelin single-chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1, and a heavy chain variable region of an anti-mesothelin antibody.
  • the connecting peptide 1 has an amino acid sequence shown as (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the anti-mesothelin single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the transgenic immune cells can effectively target mesothelin-positive tumors, retain high proliferation activity, and have high anti-tumor ability.
  • the extracellular region further includes a hinge region fragment, and the N-terminus of the hinge region fragment is connected to the C-terminus of the single-chain antibody.
  • the hinge region fragment includes at least one hinge region selected from CD8, CD28 and immunoglobulin.
  • the hinge fragment includes the hinge region of CD8.
  • the hinge fragment comprises the amino acid sequence shown in SEQ ID NO:15.
  • the transmembrane region includes at least one selected from CD4, CD8 ⁇ , CD28 and CD3 ⁇ or a fragment thereof.
  • the transmembrane region includes the CD8 transmembrane region or a fragment thereof.
  • the transmembrane region has an amino acid sequence shown in SEQ ID NO:16.
  • the immune co-stimulatory molecule includes at least one selected from CD28, ICOS, 4-1BB, OX40 and CD27.
  • the intracellular segment of the immune co-stimulatory molecule is the intracellular segment of 4-1BB or CD28 or a fragment thereof.
  • the intracellular segment of the immune co-stimulatory molecule includes the amino acid sequence shown in SEQ ID NO:17.
  • the C-terminus of the intracellular segment of the immune co-stimulatory molecule is connected to the N-terminus of the signal transduction domain.
  • the signal transduction domain includes at least one selected from CD3 ⁇ or Fc ⁇ RI ⁇ or a fragment thereof.
  • hinge region is not particularly limited, and the hinge region, transmembrane region, intracellular segment of the immune co-stimulatory molecule and signal transduction domain available in conventional chimeric antigen receptors in the art can be used.
  • the signal transduction domain includes CD3 ⁇ or a fragment thereof.
  • the signal transduction domain includes the amino acid sequence shown in SEQ ID NO:18.
  • the fusion protein further includes a connecting peptide 2.
  • the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO:19.
  • the immune cell includes at least one of T cells, NKT cells and NK cells.
  • the type of the immunostimulatory molecule is not particularly limited, and a promoting factor that can promote the immune function of at least one of T cells, NKT cells and NK cells can be used.
  • the immunostimulatory molecule is IL-15.
  • the immune cells are preferably NK cells.
  • the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells and ⁇ T cells.
  • the present invention proposes an isolated nucleic acid.
  • the isolated nucleic acid includes: 1) a first nucleic acid molecule, the first nucleic acid molecule encodes a chimeric antigen receptor; 2) a second nucleic acid molecule, the second nucleic acid molecule encodes a fusion protein, and the fusion protein includes IL-15R ⁇ and IL-15.
  • IL-15 is a pleiotropic cytokine.
  • IL-15 and IL-15R form a dimer, they bind to IL-15Rc to activate the downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways, thereby promoting the proliferation, activation and effector functions of T cells, B cells and NK cells.
  • the isolated nucleic acid After the isolated nucleic acid according to the embodiment of the present invention is introduced into the recipient cell, it can package a higher titer of virus and achieve specific infection of immune cells by the virus, such as NK cells.
  • the isolated nucleic acid is introduced into the immune cell, the immune cell can simultaneously express and secrete the chimeric antigen receptor and the fusion protein, so that the immune cell can target the corresponding antigen and locate to the cell surface expressing the antigen.
  • fusion proteins such as IL-15R ⁇ and IL-15 further promote the activation and proliferation of immune cells, maintain immunity
  • the number and activity of cells in the local microenvironment of the tumor enable them to maintain strong tumor-killing activity, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body, and also avoiding the toxic side effects caused by high-dose or repeated injections of recombinant IL-15.
  • the isolated nucleic acid may further include at least one of the following additional technical features:
  • the chimeric antigen receptor is as defined in the first aspect.
  • the second nucleic acid molecule includes a nucleotide sequence encoding the IL-15R ⁇ as shown in SEQ ID NO:10, and a nucleotide sequence encoding the IL-15 as shown in SEQ ID NO:12.
  • the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the fusion protein in an immune cell, and the fusion protein and the chimeric antigen receptor are in a non-fused form.
  • the separated nucleic acid further includes: an internal ribosome entry site sequence, wherein the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO: 20.
  • the isolated nucleic acid further comprises a third nucleic acid molecule, the third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, the third nucleic acid molecule encodes a connecting peptide 3, and the connecting peptide 3 can be cut.
  • the connecting peptide 3 can separate the first nucleic acid molecule from the second nucleic acid molecule to reduce the functional interference between the two.
  • the connecting peptide 3 includes a 2A peptide or a fragment thereof.
  • the connecting peptide 3 is not particularly limited, and any conventional peptide with self-cleavage function can be used.
  • the connecting peptide 3 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.
  • the connecting peptide 3 includes P2A or a fragment thereof.
  • the connecting peptide 3 includes the amino acid sequence shown in SEQ ID NO:19.
  • the isolated nucleic acid further comprises: a first promoter, which is operably linked to the first nucleic acid molecule; and/or a second promoter, which is operably linked to the second nucleic acid molecule.
  • the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter.
  • the isolated nucleic acid further comprises a fourth nucleic acid molecule, and the fourth nucleic acid molecule encodes a signal peptide.
  • the signal peptide expressed by the gene encoding the signal peptide is located at the amino terminus of the chimeric antigen receptor, and is a chimeric antigen receptor membrane localization terminal peptide, which helps the chimeric antigen receptor to be localized to the endoplasmic reticulum, and is hydrolyzed and separated after the protein matures, so the chimeric antigen receptor on the virus particle does not contain the signal peptide.
  • the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.
  • the signal peptide includes at least one selected from CSF2R and CD8 ⁇ or a fragment thereof.
  • the type of the signal peptide is not particularly limited, and any conventional signal peptide in the art can be used.
  • the signal peptide includes CSF2R or a fragment thereof.
  • the signal peptide includes the amino acid sequence shown in SEQ ID NO:13.
  • the first nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3, 4, 5, 6 and 7.
  • the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:9.
  • the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:8.
  • the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:2.
  • the isolated nucleic acid has a nucleotide sequence shown in SEQ ID NO:1.
  • the present invention provides a construct.
  • the construct carries the above-mentioned isolated nucleic acid.
  • the isolated nucleic acid can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the isolated nucleic acid, that is, the isolated nucleic acid is operably connected to the control elements.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself.
  • the above construct may further include at least one of the following additional technical features:
  • the vector of the construct is a non-pathogenic viral vector.
  • the expression vector when it is a viral vector, it has a higher expression efficiency.
  • the viral vector includes at least one selected from a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the isolated nucleic acid or constructed vector described above.
  • the recombinant cell according to an embodiment of the present invention can be used to express in vitro under suitable conditions and obtain a large amount of proteins encoded by the isolated nucleic acid described above, such as chimeric antigen receptor mesothelin and IL-15R ⁇ and IL-15 fusion protein.
  • the above-mentioned recombinant cell may further include at least one of the following additional technical features:
  • the recombinant cell includes a eukaryotic cell, preferably a mammalian cell.
  • the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages.
  • the prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
  • the eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.
  • the recombinant cells of the present invention are preferably mammalian cells, including T cells, B cells, NK cells, NKT cells, macrophages, BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention proposes a CAR-NK or CAR-T or CAR-NKT cell.
  • the CAR-NK cell carries the isolated nucleic acid or construct described above.
  • the CAR-NK cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and a fusion protein.
  • the modification strategy can make NK cells, NKT cells or T cells secrete super IL-15, i.e.
  • IL-15R ⁇ and IL-15 fusion protein locally in the tumor, significantly improving the proliferation ability of CAR-NK cells, NKT cells or T cells in vivo and in vitro, enhancing the survival time of NK cells, NKT cells or T cells in vivo, and improving the anti-tumor function of NK cells, NKT cells or T cells in vivo.
  • this kind of IL-15 that is continuously and slowly released locally can avoid the toxic side effects caused by systemic application of recombinant IL-15 or repeated administration.
  • the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells and ⁇ T cells.
  • the present invention provides a method for obtaining a virus.
  • the construct described above is introduced into a first receptor cell; the first receptor cell into which the construct is introduced is cultured to obtain the virus.
  • the method according to some preferred embodiments of the present invention can obtain a virus with a higher titer.
  • the virus comprises a lentivirus.
  • the first recipient cell is 293T.
  • the present invention provides a virus.
  • the virus is obtained by the above-mentioned method for obtaining a virus.
  • the present invention provides a virus.
  • the virus comprises a nucleotide sequence shown in SEQ ID NO:1.
  • the virus includes at least one of a retrovirus, a lentivirus and an adenovirus.
  • the virus comprises a lentivirus.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition includes: the aforementioned separated transgenic immune cells, nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells or viruses.
  • the separated nucleic acid, expression vector, or cell or virus carrying the separated nucleic acid or expression vector can simultaneously express and secrete chimeric antigen receptors and fusion proteins, so that immune cells can target the corresponding antigens and localize to the cell surface expressing the antigen.
  • fusion proteins such as super IL-15, i.e., IL-15R ⁇ and IL-15 fusion proteins further promote the activation and proliferation of immune cells, maintain the number and activity of immune cells in the local microenvironment of the tumor, and maintain strong tumor killing activity, effectively avoiding the toxic and side effects caused by high-dose or repeated multiple injections of the whole body, and can also avoid the toxic and side effects caused by high-dose or repeated multiple injections of recombinant IL-15. Therefore, the pharmaceutical composition containing the above-mentioned substances also has the above-mentioned functions, which will not be repeated here.
  • the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier, except for any conventional excipients that are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present invention.
  • the isolated nucleic acid, expression vector, or cell carrying the isolated nucleic acid or expression vector of the present invention can be incorporated into a suitable parenteral In medicines for administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicines can be prepared into various forms.
  • liquid, semisolid and solid dosage forms, etc. include but are not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspending agents, tablets, pills, powders, liposomes and suppositories. Typical medicines are in the form of injection solutions or infusion solutions.
  • the isolated nucleic acid, expression vector or cells carrying the isolated nucleic acid or expression vector can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • the effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
  • the present invention proposes a kit.
  • the kit includes: the isolated nucleic acid, construct or virus described above.
  • the isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells or NKT cells, T cells. Therefore, the kit containing the above substances also has the function of promoting the activation or proliferation of NK cells or NKT cells, T cells.
  • the kit can be used for scientific research, such as reversing NK cells or NKT cells, T cells with low proliferation activity, so that their proliferation activity increases from low to obtain biological samples that meet expectations.
  • the present invention provides a method for introducing a virus into an activated immune cell.
  • the activated immune cell is electroporated or transfected with the aforementioned construct or infected with the aforementioned virus.
  • the immune cells include at least one of T cells, NKT cells and NK cells.
  • the immune cells are preferably NK cells.
  • the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells and ⁇ T cells.
  • the present invention proposes a method for obtaining a chimeric antigen receptor and a fusion protein.
  • the method comprises: introducing the construct or virus described above into a second receptor cell; culturing the second receptor cell introduced with the construct or virus to obtain the chimeric antigen receptor and the fusion protein.
  • the construct or virus can simultaneously express the chimeric antigen receptor and the fusion protein under appropriate conditions, and therefore, the method according to the embodiment of the present invention can obtain a large amount of the chimeric antigen receptor and the fusion protein.
  • the introduction into the second recipient cell is performed by electroporation, transfection or infection.
  • electroporation or “transfection” is a method of introducing a viral vector into a recipient cell
  • infection refers to the process in which the virus actively binds to and fuses with the cell membrane and then enters the cell.
  • electroporation refers to a method of introducing a viral packaging vector into a recipient cell by means of electrical stimulation
  • transfection refers to a method of introducing a viral packaging vector into a recipient cell by means of a chemical mediator, such as a liposome.
  • the second receptor cell is at least one of a T cell, a NKT cell and a NK cell.
  • the second receptor cell is a NK cell.
  • the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells and ⁇ T cells.
  • the virus includes at least one selected from retrovirus, lentivirus and adenovirus.
  • the virus comprises a lentivirus.
  • the present invention proposes a method for obtaining a CAR-NK or CAR-NKT cell or CAR-T cell of a chimeric antigen receptor and a fusion protein.
  • it includes: introducing the aforementioned construct or virus into an NK cell or T cell; culturing the NK cell or T cell introduced with the construct or virus to obtain the CAR-NK or CAR-NKT cell or CAR-T cell.
  • a lentiviral expression vector targeting mesothelin and simultaneously expressing super IL-15 i.e., IL-15 and IL-15R ⁇ fusion protein
  • the virus particles are packaged by the lentivirus to infect NK cells or T cells, and high infection efficiency and CAR-positive NK cells or CAR-NKT cells, T cells are obtained.
  • the CAR-NK or CAR-NKT cells, CAR-T cells can not only target and kill mesothelin-positive malignant tumors, but also, because they can continuously secrete super IL-15 locally, they have higher proliferation ability and killing activity than unmodified NK cells or CAR-NKT cells or T cells, especially they can maintain the long-term survival of NK cells or CAR-NKT cells and T cells in the body, so that NK cells or CAR-NKT cells and T cells can maintain high proliferation activity and killing activity, and exert stronger continuous killing tumor ability. More importantly, this locally secreted IL-15 plays an effective biological function in the local tumor, which can effectively avoid the toxic and side effects caused by systemic high-dose or repeated injections of recombinant IL-15.
  • the introduction of NK cells or CAR-NKT cells, T cells is carried out by electroporation, transfection or infection.
  • the present invention proposes the use of the aforementioned isolated nucleic acid, construct, recombinant cell, CAR-NK or CAR-NKT cell, CAR-T cell or virus in the preparation of a pharmaceutical composition, and the pharmaceutical composition is used to treat or prevent tumors.
  • the isolated nucleic acid, construct, or cell carrying the above substance can simultaneously express and secrete chimeric antigen receptors and fusion proteins, such as IL-15R ⁇ and IL-15 fusion protein under appropriate conditions, so that the cell can target the corresponding antigen and locate it to the cell surface expressing the antigen.
  • IL-15R ⁇ and IL-15 fusion protein further promote the activation and proliferation of immune cells, maintain the number and activity of immune cells in the local microenvironment of the tumor, so that it maintains a strong tumor killing activity, effectively avoiding the toxic and side effects caused by high-dose or repeated injections of the whole body, and can also avoid the toxic and side effects caused by high-dose or repeated injections of recombinant IL-15.
  • the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.
  • the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.
  • the present invention proposes the use of the aforementioned isolated nucleic acid, construct or virus in the preparation of a kit for promoting the activation or proliferation of NK cells, NKT cells or T cells.
  • the isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells, NKT cells or T cells, and therefore, the kit containing the above-mentioned substances also has the function of promoting the activation or proliferation of NK cells or T cells.
  • the kit can be used for scientific research, such as reversing NK cells, NKT cells or T cells with low proliferation activity, so that their proliferation activity is increased from low to obtain a biological sample that meets expectations.
  • the present invention provides a method for treating or preventing a tumor, comprising administering to a subject at least one of the aforementioned transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions.
  • a suitable dose of transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or NKT cells or CAR-T cells, viruses or pharmaceutical compositions is administered to a subject to inhibit the proliferation of tumor cells.
  • the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.
  • the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.
  • the present invention proposes a use of the aforementioned transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions for treating or preventing tumors.
  • different treatment methods can be selected for different types of tumors using the above-mentioned products, including one or any combination of transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions, to improve the pertinence and effectiveness of disease treatment.
  • the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.
  • the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.
  • FIG. 1 is a structural schematic diagram of a CAR targeting MSLN and modified with a fusion protein (RIL) according to Example 1 of the present invention, wherein SP represents a nucleotide sequence encoding a signal peptide, ⁇ -MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB co-stimulatory signal domain, CD3Z represents a nucleotide sequence encoding a CD3Z intracellular region, P2A represents a nucleotide sequence encoding a P2A self-cleavage region, IL15R ⁇ represents a nucleotide sequence encoding the full length of IL15R ⁇ , Linker represents a nucleotide sequence encoding a connecting
  • FIG. 2 is a graph showing the results of detecting the secretion level of IL-15 in NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention
  • FIG. 3 is a graph showing the results of detecting STAT5 phosphorylation levels in NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention
  • FIG. 4 is a graph showing the results of in vitro killing ability test of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention
  • Figure 5 is a graph showing the results of in vitro proliferation ability test of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention, wherein the abscissa (Days) represents the number of days, and the ordinate (Cell number) represents the number of cells;
  • FIG. 6 is a flowchart of the operation of using CAR-NK cells expressing RIL to treat pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention
  • FIG. 7 is a graph showing the viability test results of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells in pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention
  • Figure 8 is a graph showing the anti-tumor ability test results of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells according to Example 3 of the present invention on pancreatic cancer Aspc-1 cell-bearing mice, wherein the horizontal axis (Days after NK cell treatment) represents the days after NK cell treatment, and the vertical axis (Tumor) represents the volume of tumor cells.
  • the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • operably linked means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene.
  • Commonly used vectors may be, for example, viral vectors, plasmids, bacteriophages, etc.
  • suitable conditions refer to conditions suitable for the expression of proteins encoded by the nucleic acid separated in the present application. It is easily understood by those skilled in the art that conditions suitable for the expression of proteins encoded by the nucleic acid separated include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of proteins encoded by the nucleic acid separated according to the specific environment of the laboratory.
  • the present application constructs a transgenic immune cell that simultaneously expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the chimeric antigen receptor can target multiple antigens, so that the immune cell can target the corresponding antigen and locate on the surface of the cell expressing the antigen, and the immunostimulatory molecule can further promote the activation and proliferation of the immune cell, such as the IL-15 used in the present application.
  • the proliferation activity and tumor killing ability of the immune cells that simultaneously express the chimeric antigen receptor and IL-15 are significantly improved, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body.
  • amino acid or nucleic acid sequences referred to herein are shown below.
  • the present invention designs a CAR vector (anti-MSLN-CAR-RIL) sequence that targets mesothelin (MSLN) and expresses super IL-15, comprising a signal peptide (SP), an extracellular region that targets and recognizes MSLN (anti-MSLN single-chain antibody, anti-MSLN scFv), a CD8a hinge region (Hinge) and a transmembrane region (TM), a 4-1BB intracellular co-stimulatory signal domain and an intracellular signal transduction molecule CD3 ⁇ , and an IL-15R ⁇ -linker-IL-15 (RIL) gene fragment connected by P2A.
  • the structure of each gene element in the CAR vector is shown in Figure 1. Among them:
  • the signal peptide is CSF2R, and its nucleotide sequence is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the anti-MSLN scFv is shown in SEQ ID NO: 3;
  • the nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO: 4;
  • the nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO: 5;
  • the nucleotide sequence of the 4-1BB co-stimulatory signal domain is shown in SEQ ID NO: 6;
  • the nucleotide sequence of the CD3 ⁇ intracellular region is shown in SEQ ID NO: 7;
  • the nucleotide sequence of the self-cleavage region P2A is shown in SEQ ID NO: 8;
  • the nucleotide sequence of the RIL is shown in SEQ ID NO: 9;
  • the nucleotide sequence of the IL15R ⁇ is shown in SEQ ID NO.10 in the sequence table;
  • the nucleotide sequence of the Linker is shown in SEQ ID NO.11 in the sequence list;
  • the nucleotide sequence of IL15 is shown as SEQ ID NO.12 in the sequence table.
  • the anti-MSLN-CAR fragment was inserted into the lentiviral vector pLent-EF1 ⁇ -P2A-CMV-GP to construct the pLent-anti-MSLN-P2A-CMV-GP vector.
  • the whole gene synthesized IL-15R-IL-15 (RIL) gene fragment was cut through the Not I restriction site. Inserted into pLent-anti-MSLN-P2A-CMV-GP vector. PCR identification and sequencing verified that the sequence was correct, indicating that the pLent-anti-MSLN-P2A-RIL15-CMV-GP vector was successfully constructed.
  • C GFP positive rate detected by flow cytometry
  • N number of cells at the time of infection (approximately 1 ⁇ 10 5 )
  • D dilution factor of viral vector
  • V volume of diluted virus added.
  • NK-92 cells purchased from ATCC
  • NK-92 cells purchased from ATCC
  • ⁇ -MEM medium to resuspend the cells
  • cell density 5 ⁇ 10 5 cells/mL.
  • Example 2 Determination of IL-15 secretion level and cell proliferation ability of CAR-NK cells
  • CAR-NK-92 (hereinafter referred to as CAR-NK) cells obtained in Example 1 were used to measure the IL-15 secretion level and cell proliferation ability
  • NK-92, ⁇ -MSLN-CAR-NK-92 (carrying CAR targeting MSLN) and ⁇ -MSLN-CAR-RIL-NK-92 (carrying CAR targeting MSLN and expressing IL-15 and IL15R ⁇ ) cells were plated and cultured, and the supernatant was collected for 24 hours.
  • the content of IL-15 in the supernatants of different groups was detected by ELISA.
  • the experimental results are shown in Figure 2, where IL-15 was almost undetectable in the supernatants of NK-92 and ⁇ -MSLN-CAR-NK-92 cells.
  • IL-15 was detected in the supernatant of ⁇ -CAR-IL15-NK-92 cells, with a level of 67.48 ⁇ 5.96pg/mL. It shows that the ⁇ -MSLN-CAR-RIL-NK cells designed and modified by the present invention have the ability to secrete IL-15.
  • IL-15 first binds to the IL-15R ⁇ chain to form a dimer, and then IL-15R ⁇ -IL-15 and IL-15R ⁇ c chain bind to activate downstream STAT5 phosphorylation (pSTAT5). After pSTAT5 enters the cell nucleus, it promotes the expression of activation, proliferation and anti-apoptosis related genes. Therefore, the inventors further observed whether the IL-15 secreted by the modified CAR-NK cells of the present invention has biological activity and phosphorylates downstream STAT5.
  • NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells were cultured in serum-free RPMI 1640 medium for 12 hours for starvation treatment.
  • the starvation treatment is to reduce the phosphorylation level of its own STAT5.
  • the cells were collected and the level of pSTAT5 was detected by flow cytometry.
  • the results are shown in Figure 3.
  • the STAT5 phosphorylation levels in the NK-92 cell group and the ⁇ -MSLN-CAR-NK-92 cell group were low, while the STAT5 phosphorylation level in the ⁇ -MSLN-CAR-RIL-NK-92 cell group was significantly higher than that in the control group.
  • the inventors co-incubated the above-mentioned NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells with the pancreatic cancer cell line Aspc-1 for 5 hours and then detected the killing efficiency.
  • the killing efficiency of ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 on Aspc-1 cells was 48.01 ⁇ 2.00% and 58.42% ⁇ 2.46, respectively, when the effect-target ratio was 5:1, which was significantly higher than that of NK-92 cells (35.59 ⁇ 2.46%); and the killing efficiency of ⁇ -MSLN-CAR-RIL-NK-92 cells was also significantly higher than that of the ⁇ -MSLN-CAR-NK-92 group, indicating that the insertion of the RIL fragment can not only promote the secretion of IL-15 by NK cells, but also improve the killing function of ⁇ -MSLN-CAR-NK-92 cells.
  • ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-RIL-NK-92 cells were plated in 96-well plates, and cell counts were performed every 3 days. After culturing for 24 days, cell proliferation curves were drawn. As can be seen from Figure 5, when cultured to the 12th day, the number of ⁇ -MSLN-CAR-RIL-NK-92 cells began to differ from that of the NK-92 and ⁇ -MSLN-CAR-NK-92 groups of cells. By the 21st day, there were significant differences.
  • modified NK cells of the present invention have a stronger proliferation ability, which may be due to the fact that RIL is a superagonist form of IL-15, which can more effectively bind to the IL-15 ⁇ c chain in a trans-binding manner and activate the downstream signaling pathway.
  • Example 3 Detection of the anti-tumor ability of CAR-NK cells in vivo and the survival ability of CAR-NK cells in vivo
  • pancreatic cancer Aspc-1 cell tumor-bearing mouse transplantation model was established to observe the therapeutic effect of CAR-NK-92 cells on pancreatic cancer.
  • the specific experimental procedures are as follows:
  • mice Six-week-old BALB/c-nu nude mice were selected for subcutaneous tumor bearing in the armpit, and the tumor bearing dose was 2 ⁇ 10 6 cells/mouse.
  • the tumor was formed in about 4 days, and cell therapy was started one week later.
  • the tumor volume was measured before treatment, and the mice were randomly divided into PBS group, NK-92 cell treatment group, ⁇ -MSLN-CAR-NK-92 cell treatment group and ⁇ -MSLN-CAR-RIL-NK-92 cell treatment group according to the tumor volume.
  • mice in the treatment group were injected with 1 ⁇ 10 7 effector cells/mouse through the tail vein, and the untreated group was injected with an equal volume of 1 ⁇ PBS, once every week, for a total of 5 treatments, and IL-2 (5 ⁇ 10 4 IU/mouse) was injected through the tail vein every 3 days.
  • IL-2 5 ⁇ 10 4 IU/mouse
  • peripheral blood of mice was collected on the first, third, and seventh days after treatment. After erythrocyte lysis, PerCP/Cyanine5.5anti-human CD56 antibody was labeled, and the proportion of CD56 cell population in peripheral blood lymphocytes, i.e. the proportion of NK-92 cells, was detected by flow cytometry. The tumor volume was measured every 3 days, and the tumor growth curve was drawn.
  • the proportion of NK-92 cells in each group decreased on the third day, the NK-92 group decreased from 27.3% to 8.32%, the ⁇ -MSLN-CAR-NK-92 group decreased from 29.6% to 9.83%, and the ⁇ -MSLN-CAR-RIL-NK-92 decreased from 29.3% to 16.8%.
  • the proportion of cells in the ⁇ -MSLN-CAR-RIL-NK-92 group in vivo is the highest, and the persistence in vivo is more prominent.
  • the proportions of NK-92 cells, ⁇ -MSLN-CAR-NK-92 cells, and ⁇ -MSLN-CAR-RIL-NK-92 cells in vivo were 0.13%, 3.32%, and 14.7%, respectively. It can be seen that compared with the NK-92 group and the ⁇ -MSLN-CAR-NK-92 group, the RIL gene-modified ⁇ -MSLN-CAR-RIL-NK-92 cells have a higher proportion in vivo and stronger persistence in vivo. Therefore, the expression of RIL can improve the survival ability of NK cells in vivo, and the locally secreted super IL-15 (RIL) has a significant effect on maintaining the survival of NK cells in vivo.
  • RIL super IL-15
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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Abstract

The present invention provides a method for improving viability and anti-tumor activity of NK cells and use thereof. Specifically, provided is an immune cell. The immune cell expresses a chimeric antigen receptor and a fusion protein, and the fusion protein comprises IL-15Rα and IL-15. The transgenic immune cell can express the chimeric antigen receptor and secrete the fusion protein simultaneously, such that the immune cell can target a corresponding antigen and is directed to a cell surface expressing the antigen.

Description

提高NK细胞存活和抗肿瘤活性的方法及其应用Method for improving NK cell survival and anti-tumor activity and its application 技术领域Technical Field

本发明涉及生物技术领域，具体地，本发明涉及提高NK细胞存活和抗肿瘤活性的方法及其应用。The present invention relates to the field of biotechnology, and in particular to a method for improving the survival and anti-tumor activity of NK cells and an application thereof.

背景技术Background technique

近年来，嵌合抗原受体T(chimeric antigen receptor T，CAR-T)细胞在血液恶性肿瘤治疗中取得了令人瞩目的效果。但是CAR-T细胞在临床应用中易产生细胞因子风暴、神经毒性、GVHD等不良反应，而且，CAR-T细胞对实体瘤的治疗效果尚不理想，使得CAR-T细胞的临床应用仍面临挑战。In recent years, chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. However, CAR-T cells are prone to produce adverse reactions such as cytokine storm, neurotoxicity, and GVHD in clinical applications. In addition, the therapeutic effect of CAR-T cells on solid tumors is not ideal, which makes the clinical application of CAR-T cells still face challenges.

CAR-NK细胞较CAR-T细胞具有安全性好的优势，一般不会引起细胞因子风暴和GVHD等副作用；且NK细胞不需要抗原递呈、不受MHC限制，即可发挥直接的杀伤肿瘤细胞的作用；CAR-NK细胞可以以CAR依赖和NKR依赖等多种识别机制识别和杀伤肿瘤，抗瘤谱广。因此，CAR-NK细胞在抗肿瘤治疗中具有广泛的应用前景，已成为细胞免疫治疗研发领域的热点。然而，CAR-NK细胞研发过程中面临的难题之一是NK细胞在体内的存活时间较短，影响了其在体内效应的发挥。CAR-NK cells have the advantage of good safety over CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; and NK cells do not require antigen presentation and are not restricted by MHC, and can directly kill tumor cells; CAR-NK cells can identify and kill tumors with a variety of recognition mechanisms such as CAR dependence and NKR dependence, and have a wide anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development. However, one of the difficulties faced in the development of CAR-NK cells is that NK cells have a short survival time in the body, which affects their in vivo effects.

IL-15为一种能促进T细胞和NK细胞存活、增殖和功能的细胞因子，IL-15与IL-2共用IL-2/15Rβγc受体，IL-15和IL-15Rα形成二聚体后结合IL-15Rβγc激活下游JAK1/JAK3和STAT3/STAT5信号通路，进而促进NK细胞的增殖、活化和效应功能。因此，IL-15已成为增强淋巴细胞在体内的持久性和增殖活性的药物研发热门靶点。IL-15 is a cytokine that can promote the survival, proliferation and function of T cells and NK cells. IL-15 shares the IL-2/15Rβγc receptor with IL-2. After IL-15 and IL-15Rα form dimers, they bind to IL-15Rβγc and activate the downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways, thereby promoting the proliferation, activation and effector function of NK cells. Therefore, IL-15 has become a hot target for drug development to enhance the persistence and proliferation activity of lymphocytes in the body.

但是，IL-15在体内应用存在的问题是半衰期短、体内药效有限，需要使用较大的剂量，且要频繁施用，导致各种副作用，包括低血压、血小板减少、AST与ALT升高等，这可能导致癌症患者不能耐受这种治疗。临床应用和药物研发中应尽量保持IL-15的促进淋巴细胞增殖与持久性及促进免疫应答的活性，同时尽可能降低IL15相关的副作用。However, the problems with the in vivo application of IL-15 are its short half-life and limited in vivo efficacy. It requires the use of larger doses and frequent administration, which can lead to various side effects, including hypotension, thrombocytopenia, and elevated AST and ALT, which may make cancer patients unable to tolerate this treatment. In clinical applications and drug development, the activity of IL-15 in promoting lymphocyte proliferation and persistence and promoting immune response should be maintained as much as possible, while reducing IL15-related side effects as much as possible.

基于以上研究和开发现状，有待于进一步研究安全有效的提高CAR-NK细胞体内持久性的方法。Based on the above research and development status, further research is needed to find safe and effective methods to improve the persistence of CAR-NK cells in vivo.

发明内容Summary of the invention

本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明提出了一种转基因免疫细胞，其增殖能力和体内存活时间，以及其抗肿瘤能力都较天然的免疫细胞得到显著提高，且具有更高的安全性。The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, the present invention proposes a transgenic immune cell, whose proliferation ability and survival time in vivo, as well as its anti-tumor ability are significantly improved compared with natural immune cells, and has higher safety.

因此，在本发明的第一方面，本发明提出了一种转基因免疫细胞。根据本发明的实施例，所述免疫细胞表达嵌合抗原受体以及融合蛋白，所述融合蛋白包括IL-15Rα和IL-15，即下文所称的超级IL-15。根据本发明实施例的所述转基因免疫细胞能够同时表达和分泌嵌合抗原受体和融合蛋白。其中，所述嵌合抗原受体能够使得免疫细胞靶向对应抗原，定位至表达所述抗原的细胞表面，此外，所述融合蛋白进 一步促进免疫细胞的活化和增殖，维持免疫细胞在肿瘤局部微环境中的数量和活性，使其保持强大的肿瘤杀伤活性，能够有效避免全身高剂量或反复多次注射带来的毒副作用。Therefore, in the first aspect of the present invention, the present invention proposes a transgenic immune cell. According to an embodiment of the present invention, the immune cell expresses a chimeric antigen receptor and a fusion protein, and the fusion protein includes IL-15Rα and IL-15, namely, super IL-15 as referred to below. The transgenic immune cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and a fusion protein. Among them, the chimeric antigen receptor can enable the immune cell to target the corresponding antigen and locate on the cell surface expressing the antigen. In addition, the fusion protein is further It can promote the activation and proliferation of immune cells in one step, maintain the number and activity of immune cells in the local microenvironment of the tumor, enable them to maintain strong tumor killing activity, and effectively avoid the toxic side effects caused by high-dose or repeated injections throughout the body.

根据本发明的实施例，上述转基因免疫细胞还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the genetically modified immune cells may further include at least one of the following additional technical features:

根据本发明的实施例，所述嵌合抗原受体包括：胞外区，所述胞外区能够与抗原特异性结合；跨膜区；以及胞内区，所述胞内区包括免疫共刺激分子胞内段和信号转导结构域；其中，所述胞外区的C端与所述跨膜区的N端相连，所述跨膜区的C端与所述胞内区的N端相连。本申请中，所述嵌合抗原受体识别的抗原种类不受特别限制，适用于特异性识别多种抗原。According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, which can specifically bind to an antigen; a transmembrane region; and an intracellular region, which includes an intracellular segment of an immune co-stimulatory molecule and a signal transduction domain; wherein the C-terminus of the extracellular region is connected to the N-terminus of the transmembrane region, and the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region. In the present application, the type of antigen recognized by the chimeric antigen receptor is not particularly limited, and is suitable for specific recognition of multiple antigens.

根据本发明的实施例，所述抗原是肿瘤相关抗原。根据发明的一些具体实施例，所述抗原的种类不受特别限制。According to an embodiment of the present invention, the antigen is a tumor-associated antigen. According to some specific embodiments of the present invention, the type of the antigen is not particularly limited.

根据本发明的实施例，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体结合所述抗原。本领域技术人员可以理解，所述胞外区包括识别所述抗原的结合区即可，所述胞外区可以包括全抗抗体、Fab抗体、Fab’抗体、F(ab’)2抗体、Fv抗体、单链抗体以及纳米抗体中的至少之一。根据本发明的一些优选的实施例，所述胞外区包括单链抗体。According to an embodiment of the present invention, the extracellular region includes a heavy chain variable region and a light chain variable region of an antibody, and the antibody binds to the antigen. It will be appreciated by those skilled in the art that the extracellular region includes a binding region for recognizing the antigen, and the extracellular region may include at least one of a full antibody, a Fab antibody, a Fab' antibody, a F(ab') 2 antibody, a Fv antibody, a single-chain antibody, and a nanobody. According to some preferred embodiments of the present invention, the extracellular region includes a single-chain antibody.

根据本发明的实施例，所述抗原包括选自间皮素、HER2、EGFR、GPC3、MUC1、CEA、CLDN 18.2、EpCAM、GD2、PSCA、IL-13RA2、B7-H3、CD133、ROR1、CD19、CD20、CD22、CD30、CD33、BCMA中的至少之一。According to an embodiment of the present invention, the antigen includes at least one selected from mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, GD2, PSCA, IL-13RA2, B7-H3, CD133, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA.

根据本发明的实施例，所述抗原为间皮素。根据本发明的一些具体实施例，当所述抗原为间皮素时，所述转基因免疫细胞能够有效靶向间皮素阳性肿瘤，并保留较高的增殖活性，具有较高的抗肿瘤能力。According to an embodiment of the present invention, the antigen is mesothelin. According to some specific embodiments of the present invention, when the antigen is mesothelin, the transgenic immune cells can effectively target mesothelin-positive tumors, retain high proliferation activity, and have high anti-tumor ability.

根据本发明的实施例，所述胞外区包括抗间皮素单链抗体。According to an embodiment of the present invention, the extracellular region comprises an anti-mesothelin single-chain antibody.

根据本发明的实施例，所述抗间皮素单链抗体包括抗间皮素抗体的轻链可变区、连接肽1以及抗间皮素抗体的重链可变区。According to an embodiment of the present invention, the anti-mesothelin single-chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1, and a heavy chain variable region of an anti-mesothelin antibody.

根据本发明的实施例，所述连接肽1具有(GGGGS)n所示的氨基酸序列，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence shown as (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

根据本发明的实施例，抗间皮素单链抗体包括SEQ ID NO:14所示的氨基酸序列。在一些具体的实施例中，当所述抗间皮素单链抗体具有上述氨基酸序列时，所述转基因免疫细胞能够有效靶向间皮素阳性肿瘤，并保留较高的增殖活性，具有较高的抗肿瘤能力。According to an embodiment of the present invention, the anti-mesothelin single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 14. In some specific embodiments, when the anti-mesothelin single-chain antibody has the above amino acid sequence, the transgenic immune cells can effectively target mesothelin-positive tumors, retain high proliferation activity, and have high anti-tumor ability.

根据本发明的实施例，所述胞外区进一步包括铰链区片段，所述铰链区片段的N端与所述单链抗体的C端相连。According to an embodiment of the present invention, the extracellular region further includes a hinge region fragment, and the N-terminus of the hinge region fragment is connected to the C-terminus of the single-chain antibody.

根据本发明的实施例，所述铰链区片段包括选自CD8、CD28和免疫球蛋白的铰链区中的至少之一。According to an embodiment of the present invention, the hinge region fragment includes at least one hinge region selected from CD8, CD28 and immunoglobulin.

根据本发明的实施例，所述铰链片段包括CD8的铰链区。According to an embodiment of the present invention, the hinge fragment includes the hinge region of CD8.

根据本发明的实施例，所述铰链片段包括SEQ ID NO：15所示的氨基酸序列。 According to an embodiment of the present invention, the hinge fragment comprises the amino acid sequence shown in SEQ ID NO:15.

根据本发明的实施例，所述跨膜区包括选自CD4，CD8α，CD28和CD3ζ中的至少之一或其片段。According to an embodiment of the present invention, the transmembrane region includes at least one selected from CD4, CD8α, CD28 and CD3ζ or a fragment thereof.

根据本发明的实施例，所述跨膜区包括CD8跨膜区或其片段。According to an embodiment of the present invention, the transmembrane region includes the CD8 transmembrane region or a fragment thereof.

根据本发明的实施例，所述跨膜区具有SEQ ID NO:16所示的氨基酸序列。According to an embodiment of the present invention, the transmembrane region has an amino acid sequence shown in SEQ ID NO:16.

根据本发明的实施例，所述免疫共刺激分子包括选自CD28、ICOS、4-1BB、OX40和CD27中的至少之一。According to an embodiment of the present invention, the immune co-stimulatory molecule includes at least one selected from CD28, ICOS, 4-1BB, OX40 and CD27.

根据本发明的实施例，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段或其片段。According to an embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule is the intracellular segment of 4-1BB or CD28 or a fragment thereof.

根据本发明的实施例，所述免疫共刺激分子胞内段包括SEQ ID NO:17所示的氨基酸序列。According to an embodiment of the present invention, the intracellular segment of the immune co-stimulatory molecule includes the amino acid sequence shown in SEQ ID NO:17.

根据本发明的实施例，所述免疫共刺激分子胞内段的C端与所述信号转导结构域的N端相连。According to an embodiment of the present invention, the C-terminus of the intracellular segment of the immune co-stimulatory molecule is connected to the N-terminus of the signal transduction domain.

根据本发明的实施例，所述信号转导结构域包括选自CD3ζ或FcεRIγ中的至少之一或其片段。According to an embodiment of the present invention, the signal transduction domain includes at least one selected from CD3ζ or FcεRIγ or a fragment thereof.

本领域技术人员可以理解，所述铰链区、跨膜区、免疫共刺激分子胞内段和信号转导结构域的选择不受特别限制，本领域常规的嵌合抗原受体中可用的铰链区、跨膜区、免疫共刺激分子胞内段和信号转导结构域均可以使用。Those skilled in the art will appreciate that the selection of the hinge region, transmembrane region, intracellular segment of the immune co-stimulatory molecule and signal transduction domain is not particularly limited, and the hinge region, transmembrane region, intracellular segment of the immune co-stimulatory molecule and signal transduction domain available in conventional chimeric antigen receptors in the art can be used.

根据本发明的实施例，所述信号转导结构域包括CD3ζ或其片段。According to an embodiment of the present invention, the signal transduction domain includes CD3ζ or a fragment thereof.

根据本发明的实施例，所述信号转导结构域包括SEQ ID NO:18所示的氨基酸序列。According to an embodiment of the present invention, the signal transduction domain includes the amino acid sequence shown in SEQ ID NO:18.

根据本发明的实施例，所述融合蛋白中进一步包括连接肽2。According to an embodiment of the present invention, the fusion protein further includes a connecting peptide 2.

根据本发明的实施例，所述连接肽2具有SEQ ID NO:19所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO:19.

根据本发明的实施例，所述免疫细胞包括T细胞、NKT细胞和NK细胞中的至少之一。本申请中，所述免疫刺激分子的种类并不受特别限制，能够促进T细胞、NKT细胞和NK细胞中的至少之一的免疫功能的促进因子均可以使用，本申请的一些具体实施例中，所述免疫刺激分子为IL-15。According to an embodiment of the present invention, the immune cell includes at least one of T cells, NKT cells and NK cells. In the present application, the type of the immunostimulatory molecule is not particularly limited, and a promoting factor that can promote the immune function of at least one of T cells, NKT cells and NK cells can be used. In some specific embodiments of the present application, the immunostimulatory molecule is IL-15.

根据本发明的实施例，所述免疫细胞优选为NK细胞。According to an embodiment of the present invention, the immune cells are preferably NK cells.

根据本发明的实施例，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

根据本发明的实施例，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

在本发明的第二方面，本发明提出了一种分离的核酸。根据本发明的实施例，所述分离的核酸包括：1)第一核酸分子，所述第一核酸分子编码嵌合抗原受体；2)第二核酸分子，所述第二核酸分子编码融合蛋白，所述融合蛋白包括IL-15Rα和IL-15。IL-15是一种多效性细胞因子，IL-15和IL-15R形成二聚体后结合IL-15Rc激活下游JAK1/JAK3和STAT3/STAT5信号通路，进而促进T细胞、B细胞和NK细胞的增殖、活化和效应功能。根据本发明实施例的分离的核酸导入受体细胞后能够包装出较高滴度的病毒，并实现病毒对免疫细胞的特异性感染，如NK细胞。将所述分离的核酸导入免疫细胞后，所述免疫细胞能够同时表达和分泌嵌合抗原受体和融合蛋白，使得免疫细胞能够靶向对应抗原，定位至表达所述抗原的细胞表面，此外，融合蛋白，如IL-15Rα和IL-15进一步促进免疫细胞的活化和增殖，维持免疫 细胞在肿瘤局部微环境中的数量和活性，使其保持强大的肿瘤杀伤活性，有效避免全身高剂量或反复多次注射带来的毒副作用，还能避免全身应用高剂量或反复多次注射重组IL-15带来的毒副作用。In the second aspect of the present invention, the present invention proposes an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid includes: 1) a first nucleic acid molecule, the first nucleic acid molecule encodes a chimeric antigen receptor; 2) a second nucleic acid molecule, the second nucleic acid molecule encodes a fusion protein, and the fusion protein includes IL-15Rα and IL-15. IL-15 is a pleiotropic cytokine. After IL-15 and IL-15R form a dimer, they bind to IL-15Rc to activate the downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways, thereby promoting the proliferation, activation and effector functions of T cells, B cells and NK cells. After the isolated nucleic acid according to the embodiment of the present invention is introduced into the recipient cell, it can package a higher titer of virus and achieve specific infection of immune cells by the virus, such as NK cells. After the isolated nucleic acid is introduced into the immune cell, the immune cell can simultaneously express and secrete the chimeric antigen receptor and the fusion protein, so that the immune cell can target the corresponding antigen and locate to the cell surface expressing the antigen. In addition, fusion proteins such as IL-15Rα and IL-15 further promote the activation and proliferation of immune cells, maintain immunity The number and activity of cells in the local microenvironment of the tumor enable them to maintain strong tumor-killing activity, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body, and also avoiding the toxic side effects caused by high-dose or repeated injections of recombinant IL-15.

根据本发明的实施例，上述分离的核酸还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the isolated nucleic acid may further include at least one of the following additional technical features:

根据本发明的实施例，所述嵌合抗原受体是如第一方面所定义的。According to an embodiment of the present invention, the chimeric antigen receptor is as defined in the first aspect.

根据本发明的实施例，所述第二核酸分子中包括如SEQ ID NO:10所示编码所述IL-15Rα的核苷酸序列，和如SEQ ID NO:12所示编码所述IL-15的核苷酸序列。According to an embodiment of the present invention, the second nucleic acid molecule includes a nucleotide sequence encoding the IL-15Rα as shown in SEQ ID NO:10, and a nucleotide sequence encoding the IL-15 as shown in SEQ ID NO:12.

根据本发明的实施例，所述第一核酸分子以及所述第二核酸分子被设置在免疫细胞中表达所述嵌合抗原受体和所述融合蛋白，并且所述融合蛋白与所述嵌合抗原受体呈非融合形式。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the fusion protein in an immune cell, and the fusion protein and the chimeric antigen receptor are in a non-fused form.

根据本发明的实施例，所述分离的核酸进一步包括：内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：20所示的核苷酸序列。According to an embodiment of the present invention, the separated nucleic acid further includes: an internal ribosome entry site sequence, wherein the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO: 20.

根据本发明的实施例，所述分离的核酸进一步包括第三核酸分子，所述第三核酸分子设置在所述第一核酸分子与所述第二核酸分子之间，所述第三核酸分子编码连接肽3，所述连接肽3能够被切割。所述连接肽3能够将所述第一核酸分子与第二核酸分子隔开，减少二者功能干扰。According to an embodiment of the present invention, the isolated nucleic acid further comprises a third nucleic acid molecule, the third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, the third nucleic acid molecule encodes a connecting peptide 3, and the connecting peptide 3 can be cut. The connecting peptide 3 can separate the first nucleic acid molecule from the second nucleic acid molecule to reduce the functional interference between the two.

根据本发明的实施例，所述连接肽3包括2A肽或其片段。本领域技术人员可以理解，所述连接肽3不受特别限制，常规的具有自切割功能的肽均可使用。According to an embodiment of the present invention, the connecting peptide 3 includes a 2A peptide or a fragment thereof. Those skilled in the art will appreciate that the connecting peptide 3 is not particularly limited, and any conventional peptide with self-cleavage function can be used.

根据本发明的实施例，所述连接肽3包括P2A、T2A、E2A和F2A中的至少之一或其片段。According to an embodiment of the present invention, the connecting peptide 3 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

根据本发明的实施例，所述连接肽3包括P2A或其片段。According to an embodiment of the present invention, the connecting peptide 3 includes P2A or a fragment thereof.

根据本发明的实施例，所述连接肽3包括SEQ ID NO:19所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide 3 includes the amino acid sequence shown in SEQ ID NO:19.

根据本发明的实施例，所述分离的核酸进一步包括：第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；和/或第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。According to an embodiment of the present invention, the isolated nucleic acid further comprises: a first promoter, which is operably linked to the first nucleic acid molecule; and/or a second promoter, which is operably linked to the second nucleic acid molecule.

根据本发明的实施例，所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子。According to an embodiment of the present invention, the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter.

根据本发明的实施例，所述分离的核酸还包括第四核酸分子，所述第四核酸分子编码信号肽。根据本发明的具体实施例，所述编码信号肽的基因表达的信号肽位于嵌合抗原受体的氨基末端，是嵌合抗原受体膜定位端肽，在帮助嵌合抗原受体定位到内质网上，蛋白成熟后就水解脱离，因此，病毒颗粒上的嵌合抗原受体并不含有该信号肽。According to an embodiment of the present invention, the isolated nucleic acid further comprises a fourth nucleic acid molecule, and the fourth nucleic acid molecule encodes a signal peptide. According to a specific embodiment of the present invention, the signal peptide expressed by the gene encoding the signal peptide is located at the amino terminus of the chimeric antigen receptor, and is a chimeric antigen receptor membrane localization terminal peptide, which helps the chimeric antigen receptor to be localized to the endoplasmic reticulum, and is hydrolyzed and separated after the protein matures, so the chimeric antigen receptor on the virus particle does not contain the signal peptide.

根据本发明的实施例，所述第四核酸分子与所述第一核酸分子可操作性地相连。According to an embodiment of the present invention, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.

根据本发明的实施例，所述信号肽包括选自CSF2R和CD8α中的至少之一或其片段。本领域技术人员可以理解，所述信号肽的种类不受特别限制，本领域常规信号肽均可以使用。According to an embodiment of the present invention, the signal peptide includes at least one selected from CSF2R and CD8α or a fragment thereof. Those skilled in the art will appreciate that the type of the signal peptide is not particularly limited, and any conventional signal peptide in the art can be used.

根据本发明的实施例，所述信号肽包括CSF2R或其片段。 According to an embodiment of the present invention, the signal peptide includes CSF2R or a fragment thereof.

根据本发明的实施例，所述信号肽包括SEQ ID NO:13所示的氨基酸序列。According to an embodiment of the present invention, the signal peptide includes the amino acid sequence shown in SEQ ID NO:13.

根据本发明的实施例，所述第一核酸分子具有SEQ ID NO:3、4、5、6和7所示的核苷酸序列中的至少之一。According to an embodiment of the present invention, the first nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3, 4, 5, 6 and 7.

根据本发明的实施例，所述第二核酸分子具有SEQ ID NO:9所示的核苷酸序列。According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:9.

根据本发明的实施例，所述第三核酸分子具有SEQ ID NO:8所示的核苷酸序列。According to an embodiment of the present invention, the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:8.

根据本发明的实施例，所述第四核酸分子具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:2.

根据本发明的实施例，所述分离的核酸具有SEQ ID NO:1所示的核苷酸序列。According to an embodiment of the present invention, the isolated nucleic acid has a nucleotide sequence shown in SEQ ID NO:1.

在本发明的第三方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体携带前面所述的分离的核酸。在将上述分离的核酸连接到载体上时，可以将所述分离的核酸与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制所述分离的核酸的翻译和表达等即可，即所述分离的核酸与控制元件进行可操作地连接。当然，这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。In the third aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct carries the above-mentioned isolated nucleic acid. When the above-mentioned isolated nucleic acid is connected to a vector, the isolated nucleic acid can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the isolated nucleic acid, that is, the isolated nucleic acid is operably connected to the control elements. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself.

根据本发明的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above construct may further include at least one of the following additional technical features:

根据本发明的实施例，所述构建体的载体是非致病性病毒载体。根据本发明的一些具体实施例，当所述表达载体为病毒载体时，具有较高的表达效率。According to an embodiment of the present invention, the vector of the construct is a non-pathogenic viral vector. According to some specific embodiments of the present invention, when the expression vector is a viral vector, it has a higher expression efficiency.

根据本发明的实施例，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。According to an embodiment of the present invention, the viral vector includes at least one selected from a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.

在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带前面所述的分离的核酸或构建载体。根据本发明实施例的重组细胞可用于在适合条件下体外表达和大量获得前面所述的分离的核酸所编码的蛋白质，如嵌合抗原受体间皮素和IL-15Rα和IL-15融合蛋白。In a fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the isolated nucleic acid or constructed vector described above. The recombinant cell according to an embodiment of the present invention can be used to express in vitro under suitable conditions and obtain a large amount of proteins encoded by the isolated nucleic acid described above, such as chimeric antigen receptor mesothelin and IL-15Rα and IL-15 fusion protein.

根据本发明的实施例，上述重组细胞还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

根据本发明的实施例，所述重组细胞包括真核细胞，优选为哺乳动物细胞。According to an embodiment of the present invention, the recombinant cell includes a eukaryotic cell, preferably a mammalian cell.

需要注意的是，本发明所述重组细胞不受特别限制，可以为原核细胞、真核细胞或噬菌体。示例性的，所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等；所述真核细胞包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中，本发明所述重组细胞优选为哺乳动物细胞，包括T细胞、B细胞、NK细胞、NKT细胞、巨噬细胞、BHK细胞、CHO细胞、NSO细胞或COS细胞，且不包括动物生殖细胞、受精卵或胚胎干细胞。It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. Exemplary, the prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.; the eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including T cells, B cells, NK cells, NKT cells, macrophages, BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

在本发明的第五方面，本发明提出了一种CAR-NK或CAR-T或CAR-NKT细胞。根据本发明的实施例，所述CAR-NK细胞携带前面所述的分离的核酸或构建体。根据本发明实施例的所述CAR-NK细胞能够同时表达和分泌嵌合抗原受体以及融合蛋白。发明人在实验中发现，本发明所提出的融合蛋白修 饰策略可使NK细胞、NKT细胞或T细胞在肿瘤局部分泌超级IL-15，即IL-15Rα和IL-15融合蛋白，明显提高CAR-NK细胞、NKT细胞或T细胞的体内外增殖能力，增强NK细胞、NKT细胞或T细胞的体内存活时间，提高NK细胞、NKT细胞或T细胞的体内抗肿瘤功能。且这种在局部持续缓慢释放的IL-15可避免全身应用重组IL-15或反复多次给药所带来的毒副作用。In a fifth aspect of the present invention, the present invention proposes a CAR-NK or CAR-T or CAR-NKT cell. According to an embodiment of the present invention, the CAR-NK cell carries the isolated nucleic acid or construct described above. The CAR-NK cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and a fusion protein. The inventors found in experiments that the fusion protein modification proposed by the present invention The modification strategy can make NK cells, NKT cells or T cells secrete super IL-15, i.e. IL-15Rα and IL-15 fusion protein, locally in the tumor, significantly improving the proliferation ability of CAR-NK cells, NKT cells or T cells in vivo and in vitro, enhancing the survival time of NK cells, NKT cells or T cells in vivo, and improving the anti-tumor function of NK cells, NKT cells or T cells in vivo. Moreover, this kind of IL-15 that is continuously and slowly released locally can avoid the toxic side effects caused by systemic application of recombinant IL-15 or repeated administration.

根据本发明的一些实施例，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。According to some embodiments of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

根据本发明的实施例，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

在本发明的第六方面，本发明提出了一种获得病毒的方法。根据本发明的实施例，将前面所述的构建体导入第一受体细胞；将导入构建体的第一受体细胞进行培养，以便获得所述病毒。根据本发明一些优选的实施例的方法能够获得较高滴度的病毒。In a sixth aspect of the present invention, the present invention provides a method for obtaining a virus. According to an embodiment of the present invention, the construct described above is introduced into a first receptor cell; the first receptor cell into which the construct is introduced is cultured to obtain the virus. The method according to some preferred embodiments of the present invention can obtain a virus with a higher titer.

根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the present invention, the virus comprises a lentivirus.

根据本发明的实施例，所述第一受体细胞为293T。According to an embodiment of the present invention, the first recipient cell is 293T.

在本发明的第七方面，本发明提出了一种病毒。根据本发明的实施例，是通过前面所述的获得病毒的方法获得的。In a seventh aspect of the present invention, the present invention provides a virus. According to an embodiment of the present invention, the virus is obtained by the above-mentioned method for obtaining a virus.

在本发明的第八方面，本发明提出了一种病毒。根据本发明的实施例，所述病毒包括具有SEQ ID NO:1所示的核苷酸序列。In an eighth aspect of the present invention, the present invention provides a virus. According to an embodiment of the present invention, the virus comprises a nucleotide sequence shown in SEQ ID NO:1.

根据本发明的实施例，所述病毒包括反转录病毒、慢病毒和腺病毒中的至少之一。According to an embodiment of the present invention, the virus includes at least one of a retrovirus, a lentivirus and an adenovirus.

根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the present invention, the virus comprises a lentivirus.

在本发明的第八方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的分离的转基因免疫细胞、核酸、构建体、重组细胞、CAR-NK或CAR-NKT细胞或CAR-T细胞或者病毒。如前所述，所述分离的核酸、表达载体或者携带所述分离的核酸或表达载体的细胞或病毒能够同时表达和分泌嵌合抗原受体和融合蛋白，使得免疫细胞能够靶向对应抗原，定位至表达所述抗原的细胞表面，此外，融合蛋白，如超级IL-15，即IL-15Rα和IL-15融合蛋白进一步促进免疫细胞的活化和增殖，维持免疫细胞在肿瘤局部微环境中的数量和活性，使其保持强大的肿瘤杀伤活性，有效避免全身高剂量或反复多次注射带来的毒副作用，还能避免全身应用高剂量或反复多次注射重组IL-15带来的毒副作用。因此，包含上述物质的药物组合物同样具备上述功能，此处不再累述。In the eighth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned separated transgenic immune cells, nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells or viruses. As mentioned above, the separated nucleic acid, expression vector, or cell or virus carrying the separated nucleic acid or expression vector can simultaneously express and secrete chimeric antigen receptors and fusion proteins, so that immune cells can target the corresponding antigens and localize to the cell surface expressing the antigen. In addition, fusion proteins such as super IL-15, i.e., IL-15Rα and IL-15 fusion proteins further promote the activation and proliferation of immune cells, maintain the number and activity of immune cells in the local microenvironment of the tumor, and maintain strong tumor killing activity, effectively avoiding the toxic and side effects caused by high-dose or repeated multiple injections of the whole body, and can also avoid the toxic and side effects caused by high-dose or repeated multiple injections of recombinant IL-15. Therefore, the pharmaceutical composition containing the above-mentioned substances also has the above-mentioned functions, which will not be repeated here.

根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:

根据本发明的实施例，所述药物组合物进一步包括药学上可接受的载体，除了任何常规的辅料与本发明的化合物不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, except for any conventional excipients that are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present invention.

例如，本发明的分离的核酸、表达载体或者携带分离的核酸或表达载体的细胞可掺入适用于胃肠外 施用(例如静脉内、皮下、腹膜内、肌肉内)的药物中。这些药物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物为注射溶液或输注溶液形式。所述分离的核酸、表达载体或者携带分离的核酸或表达载体的细胞可通过静脉输注或注射或肌肉内或皮下注射来施用。For example, the isolated nucleic acid, expression vector, or cell carrying the isolated nucleic acid or expression vector of the present invention can be incorporated into a suitable parenteral In medicines for administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicines can be prepared into various forms. For example, liquid, semisolid and solid dosage forms, etc., include but are not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspending agents, tablets, pills, powders, liposomes and suppositories. Typical medicines are in the form of injection solutions or infusion solutions. The isolated nucleic acid, expression vector or cells carrying the isolated nucleic acid or expression vector can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

本发明所述的分离的核酸、表达载体或者携带分离的核酸或表达载体的细胞的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，依据治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

在本发明的第十方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括：前面所述的分离的核酸、构建体或病毒。所述分离的核酸、构建体或者病毒能够显著促进NK细胞或NKT细胞、T细胞的活化或者增殖，因此，包含上述物质的试剂盒同样具备促进NK细胞或NKT细胞、T细胞的活化或者增殖的功能。所述试剂盒可以用于科学研究，如逆转增殖活性低的NK细胞或者NKT细胞、T细胞，使其增殖活性由低升高，以获得符合预期的生物样本。In the tenth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit includes: the isolated nucleic acid, construct or virus described above. The isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells or NKT cells, T cells. Therefore, the kit containing the above substances also has the function of promoting the activation or proliferation of NK cells or NKT cells, T cells. The kit can be used for scientific research, such as reversing NK cells or NKT cells, T cells with low proliferation activity, so that their proliferation activity increases from low to obtain biological samples that meet expectations.

在本发明的第十一方面，本发明提出了一种将病毒导入激活免疫细胞的方法。根据本发明的实施例，利用前面所述的构建体电转或转染所述激活免疫细胞或利用前面所述的病毒感染所述激活免疫细胞。In the eleventh aspect of the present invention, the present invention provides a method for introducing a virus into an activated immune cell. According to an embodiment of the present invention, the activated immune cell is electroporated or transfected with the aforementioned construct or infected with the aforementioned virus.

根据本发明的实施例，所述免疫细胞包括T细胞、NKT细胞和NK细胞中的至少之一。According to an embodiment of the present invention, the immune cells include at least one of T cells, NKT cells and NK cells.

根据本发明的实施例，所述免疫细胞优选为NK细胞。According to an embodiment of the present invention, the immune cells are preferably NK cells.

根据本发明的实施例，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

根据本发明的实施例，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

在本发明的第十二方面，本发明提出了一种获得嵌合抗原受体和融合蛋白的方法。根据本发明的实施例，所述方法包括：将前面所述的构建体或病毒导入第二受体细胞；将导入构建体或病毒的第二受体细胞进行培养，以便获得所述嵌合抗原受体和融合蛋白。如前所述，所述构建体或病毒在合适条件下能够同时表达所述嵌合抗原受体和融合蛋白，因此，根据本发明实施例的方法能够大量获得所述嵌合抗原受体和融合蛋白。In the twelfth aspect of the present invention, the present invention proposes a method for obtaining a chimeric antigen receptor and a fusion protein. According to an embodiment of the present invention, the method comprises: introducing the construct or virus described above into a second receptor cell; culturing the second receptor cell introduced with the construct or virus to obtain the chimeric antigen receptor and the fusion protein. As described above, the construct or virus can simultaneously express the chimeric antigen receptor and the fusion protein under appropriate conditions, and therefore, the method according to the embodiment of the present invention can obtain a large amount of the chimeric antigen receptor and the fusion protein.

根据本发明的实施例，所述导入第二受体细胞是通过电转、转染或感染的方式进行的。需要说明的是，所述“电转”或“转染”是将病毒载体导入受体细胞的方法，所述“感染”是指病毒主动结合和融合细胞膜进而进入细胞的过程。其中，“电转”是指通过电刺激的方式，将病毒包装用载体导入受体细胞的方法，所述“转染”是指通过化学介导物，如脂质体，将病毒包装用载体导入受体细胞的方法。 According to an embodiment of the present invention, the introduction into the second recipient cell is performed by electroporation, transfection or infection. It should be noted that the "electroporation" or "transfection" is a method of introducing a viral vector into a recipient cell, and the "infection" refers to the process in which the virus actively binds to and fuses with the cell membrane and then enters the cell. Among them, "electroporation" refers to a method of introducing a viral packaging vector into a recipient cell by means of electrical stimulation, and the "transfection" refers to a method of introducing a viral packaging vector into a recipient cell by means of a chemical mediator, such as a liposome.

根据本发明的实施例，所述第二受体细胞为T细胞、NKT细胞和NK细胞中的至少之一。According to an embodiment of the present invention, the second receptor cell is at least one of a T cell, a NKT cell and a NK cell.

根据本发明的实施例，所述第二受体细胞为NK细胞。According to an embodiment of the present invention, the second receptor cell is a NK cell.

根据本发明的实施例，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

根据本发明的实施例，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

根据本发明的实施例，所述病毒包括选自反转录病毒、慢病毒和腺病毒中至少之一。According to an embodiment of the present invention, the virus includes at least one selected from retrovirus, lentivirus and adenovirus.

根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the present invention, the virus comprises a lentivirus.

在本发明的第十三方面，本发明提出了一种获得嵌合抗原受体和融合蛋白的CAR-NK或CAR-NKT细胞、CAR-T细胞的方法。根据本发明的实施例，包括：将前面所述的构建体或病毒导入NK细胞或T细胞；将导入构建体或病毒的NK细胞或T细胞进行培养，以便获得所述CAR-NK或CAR-NKT细胞或CAR-T细胞。根据本发明的一些具体实施例，构建靶向间皮素并同时表达超级IL-15(即IL-15和IL-15Rα融合蛋白)的慢病毒表达载体，经慢病毒包装出病毒颗粒感染NK细胞或者T细胞，获得高感染效率和CAR阳性NK细胞或CAR-NKT细胞、T细胞，该CAR-NK或CAR-NKT细胞、CAR-T细胞不仅能够靶向杀伤间皮素阳性的恶性肿瘤，而且，由于其能够在局部持续分泌超级IL-15，因而具有较未修饰NK细胞或CAR-NKT细胞或T细胞更高的增殖能力和杀伤活性，尤其是能够维持NK细胞或CAR-NKT细胞、T细胞在体内的长期存活，使NK细胞或CAR-NKT细胞、T细胞能够保持较高的增殖活力和杀伤活性，发挥更强的持续杀伤肿瘤的能力。更重要的是，这种局部分泌的IL-15在肿瘤局部发挥有效的生物学功能，能够有效避免全身应用高剂量或反复多次注射重组IL-15带来的毒副作用。In the thirteenth aspect of the present invention, the present invention proposes a method for obtaining a CAR-NK or CAR-NKT cell or CAR-T cell of a chimeric antigen receptor and a fusion protein. According to an embodiment of the present invention, it includes: introducing the aforementioned construct or virus into an NK cell or T cell; culturing the NK cell or T cell introduced with the construct or virus to obtain the CAR-NK or CAR-NKT cell or CAR-T cell. According to some specific embodiments of the present invention, a lentiviral expression vector targeting mesothelin and simultaneously expressing super IL-15 (i.e., IL-15 and IL-15Rα fusion protein) is constructed, and the virus particles are packaged by the lentivirus to infect NK cells or T cells, and high infection efficiency and CAR-positive NK cells or CAR-NKT cells, T cells are obtained. The CAR-NK or CAR-NKT cells, CAR-T cells can not only target and kill mesothelin-positive malignant tumors, but also, because they can continuously secrete super IL-15 locally, they have higher proliferation ability and killing activity than unmodified NK cells or CAR-NKT cells or T cells, especially they can maintain the long-term survival of NK cells or CAR-NKT cells and T cells in the body, so that NK cells or CAR-NKT cells and T cells can maintain high proliferation activity and killing activity, and exert stronger continuous killing tumor ability. More importantly, this locally secreted IL-15 plays an effective biological function in the local tumor, which can effectively avoid the toxic and side effects caused by systemic high-dose or repeated injections of recombinant IL-15.

根据本发明的实施例，所述导入NK细胞或CAR-NKT细胞、T细胞是通过电转、转染或感染的方式进行的。According to an embodiment of the present invention, the introduction of NK cells or CAR-NKT cells, T cells is carried out by electroporation, transfection or infection.

在本发明的第十四方面，本发明提出了前面所述的分离的核酸、构建体、重组细胞、CAR-NK或CAR-NKT细胞、CAR-T细胞或者病毒在制备药物组合物中的用途，所述药物组合物用于治疗或预防肿瘤。如前所述，所述分离的核酸、构建体，或者携带上述物质的细胞在合适的条件下能够同时表达和分泌嵌合抗原受体和融合蛋白，如IL-15Rα和IL-15融合蛋白，使得细胞能够靶向对应抗原，定位至表达所述抗原的细胞表面，此外，IL-15Rα和IL-15融合蛋白进一步促进免疫细胞的活化和增殖，维持免疫细胞在肿瘤局部微环境中的数量和活性，使其保持强大的肿瘤杀伤活性，有效避免全身高剂量或反复多次注射带来的毒副作用，还能避免全身应用高剂量或反复多次注射重组IL-15带来的毒副作用。In the fourteenth aspect of the present invention, the present invention proposes the use of the aforementioned isolated nucleic acid, construct, recombinant cell, CAR-NK or CAR-NKT cell, CAR-T cell or virus in the preparation of a pharmaceutical composition, and the pharmaceutical composition is used to treat or prevent tumors. As mentioned above, the isolated nucleic acid, construct, or cell carrying the above substance can simultaneously express and secrete chimeric antigen receptors and fusion proteins, such as IL-15Rα and IL-15 fusion protein under appropriate conditions, so that the cell can target the corresponding antigen and locate it to the cell surface expressing the antigen. In addition, IL-15Rα and IL-15 fusion protein further promote the activation and proliferation of immune cells, maintain the number and activity of immune cells in the local microenvironment of the tumor, so that it maintains a strong tumor killing activity, effectively avoiding the toxic and side effects caused by high-dose or repeated injections of the whole body, and can also avoid the toxic and side effects caused by high-dose or repeated injections of recombinant IL-15.

根据本发明的实施例，所述肿瘤为间皮素阳性肿瘤、HER2阳性肿瘤、EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤中的至少之一。 According to an embodiment of the present invention, the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.

根据本发明的实施例，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。According to an embodiment of the present invention, the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.

在本发明的第十五方面，本发明提出了前面所述的分离的核酸、构建体或病毒在制备试剂盒中的用途，所述试剂盒用于促进NK细胞、NKT细胞或T细胞活化或者增殖。根据本发明的一些具体实施例，所述分离的核酸、构建体或者病毒能够显著促进NK细胞、NKT细胞或T细胞的活化或者增殖，因此，包含上述物质的试剂盒同样具备促进NK细胞或T细胞的活化或者增殖的功能。所述试剂盒可以用于科学研究，如逆转增殖活性低的NK细胞、NKT细胞或者T细胞，使其增殖活性由低升高，以获得符合预期的生物样本。In the fifteenth aspect of the present invention, the present invention proposes the use of the aforementioned isolated nucleic acid, construct or virus in the preparation of a kit for promoting the activation or proliferation of NK cells, NKT cells or T cells. According to some specific embodiments of the present invention, the isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells, NKT cells or T cells, and therefore, the kit containing the above-mentioned substances also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells, NKT cells or T cells with low proliferation activity, so that their proliferation activity is increased from low to obtain a biological sample that meets expectations.

在本发明的第十六方面，本发明提出了一种治疗或预防肿瘤的方法，包括向受试者使用前面所述的转基因细胞、分离的核酸、构建体、重组细胞、CAR-NK或CAR-NKT细胞或CAR-T细胞、病毒或药物组合物中的至少之一。根据本发明的一些具体实施例，通过向受试者施用合适剂量的转基因细胞、分离的核酸、构建体、重组细胞、CAR-NK或NKT细胞或CAR-T细胞、病毒或药物组合物用以抑制肿瘤细胞的增殖。In the sixteenth aspect of the present invention, the present invention provides a method for treating or preventing a tumor, comprising administering to a subject at least one of the aforementioned transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions. According to some specific embodiments of the present invention, a suitable dose of transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or NKT cells or CAR-T cells, viruses or pharmaceutical compositions is administered to a subject to inhibit the proliferation of tumor cells.

根据本发明的实施例，所述肿瘤为间皮素阳性肿瘤、HER2阳性肿瘤、EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤中的至少之一。According to an embodiment of the present invention, the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.

根据本发明的实施例，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。According to an embodiment of the present invention, the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.

在本发明的第十七方面，本发明提出了一种前述转基因细胞、分离的核酸、构建体、重组细胞、CAR-NK或CAR-NKT细胞或CAR-T细胞、病毒或药物组合物用于治疗或预防肿瘤中的用途。根据本发明的一些具体实施例，利用上述产品针对不同类型的肿瘤，可以选择使用不同的治疗手段，包括转基因细胞、分离的核酸、构建体、重组细胞、CAR-NK或CAR-NKT细胞或CAR-T细胞、病毒或药物组合物中的一种或任意组合，提高疾病治疗的针对性和有效性。In the seventeenth aspect of the present invention, the present invention proposes a use of the aforementioned transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions for treating or preventing tumors. According to some specific embodiments of the present invention, different treatment methods can be selected for different types of tumors using the above-mentioned products, including one or any combination of transgenic cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-NKT cells or CAR-T cells, viruses or pharmaceutical compositions, to improve the pertinence and effectiveness of disease treatment.

根据本发明的实施例，所述肿瘤为间皮素阳性肿瘤、HER2阳性肿瘤、EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤中的至少之一。According to an embodiment of the present invention, the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, an IL-13RA2-positive tumor, a B7-H3-positive tumor, a CD133-positive tumor, a ROR1-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.

根据本发明的实施例，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。 According to an embodiment of the present invention, the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1是根据本发明实施例1的靶向MSLN并用融合蛋白(RIL)修饰的CAR的结构模式图，其中，SP表示编码信号肽的核苷酸序列，α-MSLN-scFv表示编码抗MSLN单链抗体的核苷酸序列，CD8hinge+TM表示编码CD8铰链区和跨膜区的核苷酸序列，4-1BB表示编码4-1BB共刺激信号域的核苷酸序列，CD3Z表示编码CD3Z胞内区的核苷酸序列，P2A表示编码P2A自剪切区的核苷酸序列，IL15Rα表示编码IL15Rα全长的核苷酸序列，Linker表示编码连接肽的核苷酸序列，IL15表示编码IL15全长的核苷酸序列；Figure 1 is a structural schematic diagram of a CAR targeting MSLN and modified with a fusion protein (RIL) according to Example 1 of the present invention, wherein SP represents a nucleotide sequence encoding a signal peptide, α-MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB co-stimulatory signal domain, CD3Z represents a nucleotide sequence encoding a CD3Z intracellular region, P2A represents a nucleotide sequence encoding a P2A self-cleavage region, IL15Rα represents a nucleotide sequence encoding the full length of IL15Rα, Linker represents a nucleotide sequence encoding a connecting peptide, and IL15 represents a nucleotide sequence encoding the full length of IL15;

图2是根据本发明实施例2的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞IL-15的分泌水平检测结果图；2 is a graph showing the results of detecting the secretion level of IL-15 in NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention;

图3是根据本发明实施例2的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞STAT5磷酸化水平检测结果图；3 is a graph showing the results of detecting STAT5 phosphorylation levels in NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention;

图4是根据本发明实施例2的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞体外杀伤能力检测结果图；4 is a graph showing the results of in vitro killing ability test of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention;

图5是根据本发明实施例2的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞体外增殖能力检测结果图，其中，横坐标(Days)表示天数，纵坐标(Cell number)表示细胞数量；Figure 5 is a graph showing the results of in vitro proliferation ability test of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention, wherein the abscissa (Days) represents the number of days, and the ordinate (Cell number) represents the number of cells;

图6是根据本发明实施例3的表达RIL的CAR-NK细胞治疗胰腺癌Aspc-1细胞荷瘤小鼠的操作流程图；6 is a flowchart of the operation of using CAR-NK cells expressing RIL to treat pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention;

图7是根据本发明实施例3的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞在胰腺癌Aspc-1细胞荷瘤小鼠体内的生存能力检测结果图；7 is a graph showing the viability test results of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells in pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention;

图8是根据本发明实施例3的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞对胰腺癌Aspc-1细胞荷瘤小鼠抗肿瘤能力检测结果图，其中，横坐标(Days after NK cell treatment)表示经NK细胞治疗后的天数，纵坐标(Tumor)表示肿瘤细胞的体积。Figure 8 is a graph showing the anti-tumor ability test results of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells according to Example 3 of the present invention on pancreatic cancer Aspc-1 cell-bearing mice, wherein the horizontal axis (Days after NK cell treatment) represents the days after NK cell treatment, and the vertical axis (Tumor) represents the volume of tumor cells.

具体实施方式Detailed ways

下面详细描述本发明的实施例，所述实施例的示例在附图中示出。其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

在对本发明描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本发明保护方案的限制。In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。 In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为病毒载体、质粒、噬菌体等等。根据本发明的一些具体实施例的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的分离的核酸的表达，进而实现所述分离的核酸编码的蛋白质的体外大量获得。Herein, "operably linked" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors may be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the above-mentioned isolated nucleic acid can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the isolated nucleic acid.

本文中，所述的“适合条件”，是指适合本申请所述分离的核酸编码的蛋白表达的条件。本领域技术人员容易理解的是，适合所述分离的核酸编码的蛋白表达的条件包括但不限于合适的转化或转染方式、合适的转化或转染条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述分离的核酸编码的蛋白表达的条件。Herein, the "suitable conditions" refer to conditions suitable for the expression of proteins encoded by the nucleic acid separated in the present application. It is easily understood by those skilled in the art that conditions suitable for the expression of proteins encoded by the nucleic acid separated include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of proteins encoded by the nucleic acid separated according to the specific environment of the laboratory.

本申请构建了一种同时表达嵌合抗原受体和免疫刺激分子的转基因免疫细胞，其中，所述嵌合抗原受体靶向的抗原可以有多种，使得免疫细胞能够靶向对应抗原，定位至表达所述抗原的细胞表面，所述免疫刺激分子能够进一步促进所述免疫细胞的活化、增殖，如本申请中使用的IL-15，经实验验证后，同时表达嵌合抗原受体和IL-15的免疫细胞的增殖活性以及肿瘤杀伤能力均得到显著提高，有效避免全身高剂量或反复多次注射带来的毒副作用。The present application constructs a transgenic immune cell that simultaneously expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the chimeric antigen receptor can target multiple antigens, so that the immune cell can target the corresponding antigen and locate on the surface of the cell expressing the antigen, and the immunostimulatory molecule can further promote the activation and proliferation of the immune cell, such as the IL-15 used in the present application. After experimental verification, the proliferation activity and tumor killing ability of the immune cells that simultaneously express the chimeric antigen receptor and IL-15 are significantly improved, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body.

本文中涉及的氨基酸或核酸序列如下所示。





The amino acid or nucleic acid sequences referred to herein are shown below.





下面将更详细地描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention will be described in more detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

需要说明的是，以下实施中所述的“质粒”与“载体”具有相同的意义，可互换使用。It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

实施例1：CAR-NK细胞的制备Example 1: Preparation of CAR-NK cells

1.1 CAR表达质粒的构建1.1 Construction of CAR expression plasmid

本发明设计靶向间皮素(MSLN)并表达超级IL-15的CAR载体(anti-MSLN-CAR-RIL)序列，包含信号肽(SP)、靶向识别MSLN的胞外区(抗MSLN单链抗体，anti-MSLN scFv)、CD8a铰链区(Hinge)与跨膜区(TM)、4-1BB胞内共刺激信号域与胞内信号转导分子CD3ζ及由P2A连接的IL-15Rα-linker-IL-15(RIL)基因片段。所述CAR载体中的各基因元件结构如图1所示。其中：The present invention designs a CAR vector (anti-MSLN-CAR-RIL) sequence that targets mesothelin (MSLN) and expresses super IL-15, comprising a signal peptide (SP), an extracellular region that targets and recognizes MSLN (anti-MSLN single-chain antibody, anti-MSLN scFv), a CD8a hinge region (Hinge) and a transmembrane region (TM), a 4-1BB intracellular co-stimulatory signal domain and an intracellular signal transduction molecule CD3ζ, and an IL-15Rα-linker-IL-15 (RIL) gene fragment connected by P2A. The structure of each gene element in the CAR vector is shown in Figure 1. Among them:

所述各元件组成的CAR载体的基因全长序列如SEQ ID NO：1所示；The full-length gene sequence of the CAR vector composed of the above elements is shown in SEQ ID NO: 1;

所述信号肽为CSF2R，其核苷酸序列如SEQ ID NO：2所示；The signal peptide is CSF2R, and its nucleotide sequence is shown in SEQ ID NO: 2;

所述anti-MSLN scFv的核苷酸序列如SEQ ID NO：3所示；The nucleotide sequence of the anti-MSLN scFv is shown in SEQ ID NO: 3;

所述CD8铰链区的核苷酸序列如SEQ ID NO：4所示；The nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO: 4;

所述CD8跨膜区的核苷酸序列如SEQ ID NO：5所示；The nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO: 5;

所述4-1BB共刺激信号域的核苷酸序列如SEQ ID NO：6所示；The nucleotide sequence of the 4-1BB co-stimulatory signal domain is shown in SEQ ID NO: 6;

所述CD3ζ胞内区的核苷酸序列如SEQ ID NO：7所示；The nucleotide sequence of the CD3ζ intracellular region is shown in SEQ ID NO: 7;

所述自剪切区P2A的核苷酸序列如SEQ ID NO：8所示；The nucleotide sequence of the self-cleavage region P2A is shown in SEQ ID NO: 8;

所述RIL的核苷酸序列如SEQ ID NO：9所示；The nucleotide sequence of the RIL is shown in SEQ ID NO: 9;

所述IL15Rα的核苷酸序列如序列表中SEQ ID NO.10所示；The nucleotide sequence of the IL15Rα is shown in SEQ ID NO.10 in the sequence table;

所述Linker的核苷酸序列如序列表中SEQ ID NO.11所示；The nucleotide sequence of the Linker is shown in SEQ ID NO.11 in the sequence list;

所述IL15的核苷酸序列如序列表中SEQ ID NO.12所示。The nucleotide sequence of IL15 is shown as SEQ ID NO.12 in the sequence table.

首先将anti-MSLN-CAR片段插入慢病毒载体pLent-EF1α-P2A-CMV-GP中构建pLent-anti-MSLN-P2A-CMV-GP载体。将全基因合成IL-15R-IL-15(RIL)基因片段通过酶切位点Not I 插入到pLent-anti-MSLN-P2A-CMV-GP载体中。经PCR鉴定和测序验证序列正确，说明成功构建pLent-anti-MSLN-P2A-RIL15-CMV-GP载体。First, the anti-MSLN-CAR fragment was inserted into the lentiviral vector pLent-EF1α-P2A-CMV-GP to construct the pLent-anti-MSLN-P2A-CMV-GP vector. The whole gene synthesized IL-15R-IL-15 (RIL) gene fragment was cut through the Not I restriction site. Inserted into pLent-anti-MSLN-P2A-CMV-GP vector. PCR identification and sequencing verified that the sequence was correct, indicating that the pLent-anti-MSLN-P2A-RIL15-CMV-GP vector was successfully constructed.

1.2慢病毒的包装及病毒液浓缩1.2 Lentivirus packaging and virus liquid concentration

取处于对数生长期的293T细胞5×106接种于10cm的培养皿中，加入10mL DMEM培养基，于37℃、5％CO2培养箱中培养过夜。待细胞密度达到80％时，更换10mL新鲜的DMEM培养基用于病毒包装，将细胞培养皿继续置于培养箱中备用。配制慢病毒包装体系，分别将慢病毒包装辅助质粒psPAX2 6μg与pMD2.G 3μg，目的基因载体质粒6μg加入至体积为250μL无血清DMEM培养基中配制质粒混合液，混合均匀。将15μL PEIpro加到体积为235μL的无血清DMEM培养基中，混合均匀。将混合液一次性加入到上述质粒混合液中，混匀，室温孵育15min，孵育结束后将混合液加入293T细胞培养皿中。24h后进行换液，将培养皿放回37℃，5％CO2培养箱中，培养48h后收取细胞上清，于400×g离心5min，去除细胞碎片，将上清用0.45μm的滤头过滤至50mL离心管中。加入5×PEG8000溶液进行病毒液浓缩，上下颠倒离心管混合均匀，放于4℃冰箱中过夜。4℃，4000×g离心20min，弃上清，加适量无血清DMEM重悬病毒沉淀，转移入EP管中，放于-80℃冰箱中保存。Take 5×10 6 293T cells in the logarithmic growth phase and inoculate them in a 10 cm culture dish, add 10 mL of DMEM culture medium, and culture overnight in a 37°C, 5% CO 2 incubator. When the cell density reaches 80%, replace with 10 mL of fresh DMEM culture medium for virus packaging, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6 μg of lentiviral packaging auxiliary plasmids psPAX2 and pMD2.G 3 μg, and 6 μg of the target gene vector plasmid to 250 μL of serum-free DMEM culture medium to prepare a plasmid mixture, and mix well. Add 15 μL of PEIpro to 235 μL of serum-free DMEM culture medium and mix well. Add the mixture to the above plasmid mixture at one time, mix well, incubate at room temperature for 15 minutes, and add the mixture to the 293T cell culture dish after the incubation. After 24 hours, change the liquid, put the culture dish back into the 37℃, 5% CO2 incubator, collect the cell supernatant after 48 hours of culture, centrifuge at 400×g for 5 minutes, remove the cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by turning the centrifuge tube upside down, and place it in a 4℃ refrigerator overnight. Centrifuge at 4℃, 4000×g for 20 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, transfer it to an EP tube, and store it in a -80℃ refrigerator.

1.3慢病毒滴度检测1.3 Lentiviral titer detection

取对数生长期的293T细胞，调整浓度为1×105/mL。取24孔板，每孔加1mL细胞悬液(1×105/孔)，设置3个加入病毒体积梯度。置于37℃、5％CO2培养箱过夜培养。先将浓缩病毒液进行10倍稀释：取1mL EP管，吸取60μL病毒浓缩液到EP管中，用540μL DMEM培养基进行稀释，混合均匀。对293T细胞用新鲜DMEM培养基进行换液，分别吸取5μL、50μL与500μL稀释后病毒液加到相应的孔中，做好标记，然后将培养板放回37℃、5％CO2培养箱中。24h后，吸弃孔板中病毒液，再加入1mL新鲜DMEM培养基。72h后，用胰酶消化收获细胞，使用流式仪检测293T细胞GFP表达率，根据下列公式换算病毒滴度：
滴度(TU/mL)＝(C×N×D×1000)/VTake 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 5 /mL. Take a 24-well plate, add 1mL of cell suspension (1×10 5 /well) to each well, and set up 3 gradients of virus volume. Place in a 37°C, 5% CO 2 incubator for overnight culture. First dilute the concentrated virus solution 10 times: take a 1mL EP tube, pipette 60μL of concentrated virus solution into the EP tube, dilute with 540μL DMEM culture medium, and mix evenly. Replace the 293T cells with fresh DMEM culture medium, pipette 5μL, 50μL and 500μL of diluted virus solution into the corresponding wells, mark them, and then put the culture plate back into the 37°C, 5% CO 2 incubator. After 24h, discard the virus solution in the well plate and add 1mL of fresh DMEM culture medium. After 72h, harvest the cells with trypsin digestion, use a flow cytometer to detect the GFP expression rate of 293T cells, and convert the virus titer according to the following formula:
Titer (TU/mL) = (C×N×D×1000)/V

其中：C＝流式检测的GFP阳性率
N＝感染时细胞的数目(约为1×105)
D＝病毒载体的稀释倍数
V＝加入的稀释病毒的体积数。Where: C = GFP positive rate detected by flow cytometry
N = number of cells at the time of infection (approximately 1×10 5 )
D = dilution factor of viral vector
V = volume of diluted virus added.

1.4慢病毒感染人NK细胞1.4 Lentivirus infection of human NK cells

取处于生长对数期的NK-92细胞(购自ATCC)，于100×g离心5min收获细胞，加入适量α-MEM培养基重悬细胞，调整细胞密度为5×105个/mL。在24孔板中分别接入5×105个NK-92细胞，病毒浓缩液1mL与鱼精蛋白(购自索莱宝，终浓度8μg/mL)，混合均匀，置于37℃、5％CO2培养箱中培养。24h后，观察细胞状态，换液，将感染的细胞转移入EP管中，100×g离心5min，加入少量新鲜α-MEM 培养基重悬细胞，将细胞转入细胞培养瓶中，加入10mL新鲜α-MEM培养基和IL-2(终浓度为200IU/mL)继续培养48h。将细胞转移入流式管中，加入3mL 1×PBS溶液，100×g离心5min，弃上清，弹起细胞沉淀，使用1×PBS溶液再次洗涤一遍。使用流式仪检测GFP的表达率。继续扩大培养，调整感染后NK-92细胞的状态进行扩增。将感染后的NK-92细胞通过流式仪分选GFP阳性的CAR-NK-92细胞，用于后期实验。Take NK-92 cells (purchased from ATCC) in the logarithmic growth phase, harvest the cells by centrifugation at 100×g for 5 minutes, add an appropriate amount of α-MEM medium to resuspend the cells, and adjust the cell density to 5×10 5 cells/mL. Inoculate 5×10 5 NK-92 cells in 24-well plates, add 1mL of virus concentrate and protamine (purchased from Solebao, final concentration 8μg/mL), mix evenly, and place in a 37°C, 5% CO 2 incubator for culture. After 24 hours, observe the cell state, change the medium, transfer the infected cells into an EP tube, centrifuge at 100×g for 5 minutes, add a small amount of fresh α-MEM Resuspend the cells in the culture medium, transfer the cells to a cell culture flask, add 10 mL of fresh α-MEM culture medium and IL-2 (final concentration of 200 IU/mL) and continue to culture for 48 hours. Transfer the cells to a flow tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 minutes, discard the supernatant, flick off the cell pellet, and wash again with 1×PBS solution. Use a flow cytometer to detect the expression rate of GFP. Continue to expand the culture and adjust the state of the infected NK-92 cells for amplification. The infected NK-92 cells are sorted by flow cytometry for GFP-positive CAR-NK-92 cells for later experiments.

实施例2：CAR-NK细胞分泌IL-15的水平及细胞增殖能力测定Example 2: Determination of IL-15 secretion level and cell proliferation ability of CAR-NK cells

本实施例以实施例1获得的CAR-NK-92(全文简称为CAR-NK)细胞进行IL-15分泌水平及细胞增殖能力的测定In this example, the CAR-NK-92 (hereinafter referred to as CAR-NK) cells obtained in Example 1 were used to measure the IL-15 secretion level and cell proliferation ability

2.1 ELISA检测CAR-NK-92细胞IL-15的分泌水平2.1 ELISA to detect the secretion level of IL-15 in CAR-NK-92 cells

分别将NK-92、α-MSLN-CAR-NK-92(携带靶向MSLN的CAR)和α-MSLN-CAR-RIL-NK-92(携带靶向MSLN的CAR并表达IL-15和IL15Rα)细胞接板培养，24h收集上清。ELISA检测不同组上清中IL-15的含量。实验结果如图2所示，其中，NK-92与α-MSLN-CAR-NK-92细胞上清中几乎检测不到IL-15。但在α-CAR-IL15-NK-92细胞上清检测出明显的IL-15，水平为67.48±5.96pg/mL。说明本发明设计修饰的α-MSLN-CAR-RIL-NK细胞具有分泌IL-15的能力。NK-92, α-MSLN-CAR-NK-92 (carrying CAR targeting MSLN) and α-MSLN-CAR-RIL-NK-92 (carrying CAR targeting MSLN and expressing IL-15 and IL15Rα) cells were plated and cultured, and the supernatant was collected for 24 hours. The content of IL-15 in the supernatants of different groups was detected by ELISA. The experimental results are shown in Figure 2, where IL-15 was almost undetectable in the supernatants of NK-92 and α-MSLN-CAR-NK-92 cells. However, significant IL-15 was detected in the supernatant of α-CAR-IL15-NK-92 cells, with a level of 67.48±5.96pg/mL. It shows that the α-MSLN-CAR-RIL-NK cells designed and modified by the present invention have the ability to secrete IL-15.

2.2 CAR-NK细胞STAT5磷酸化水平检测2.2 Detection of STAT5 phosphorylation level in CAR-NK cells

IL-15发挥作用首先与IL-15Rα链结合形成二聚体，然后IL-15Rα-IL-15和IL-15Rβγc链结合后，激活下游STAT5磷酸化(pSTAT5)，pSTAT5进入细胞核后促进活化、增殖和抗凋亡相关基因的表达。因此，发明人进一步观察本发明修饰的CAR-NK细胞分泌的IL-15是否具有生物活性，使下游STAT5磷酸化。分别将上述NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞在无血清RPMI 1640培养基中培养12h进行饥饿处理，饥饿处理是为了降低自身STAT5的磷酸化水平。收取细胞，通过流式检测pSTAT5的水平。结果如图3所示，NK-92细胞组和α-MSLN-CAR-NK-92细胞组STAT5磷酸化水平较低，而α-MSLN-CAR-RIL-NK-92细胞组STAT5磷酸化水平明显高于对照组。IL-15 first binds to the IL-15Rα chain to form a dimer, and then IL-15Rα-IL-15 and IL-15Rβγc chain bind to activate downstream STAT5 phosphorylation (pSTAT5). After pSTAT5 enters the cell nucleus, it promotes the expression of activation, proliferation and anti-apoptosis related genes. Therefore, the inventors further observed whether the IL-15 secreted by the modified CAR-NK cells of the present invention has biological activity and phosphorylates downstream STAT5. The above-mentioned NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells were cultured in serum-free RPMI 1640 medium for 12 hours for starvation treatment. The starvation treatment is to reduce the phosphorylation level of its own STAT5. The cells were collected and the level of pSTAT5 was detected by flow cytometry. The results are shown in Figure 3. The STAT5 phosphorylation levels in the NK-92 cell group and the α-MSLN-CAR-NK-92 cell group were low, while the STAT5 phosphorylation level in the α-MSLN-CAR-RIL-NK-92 cell group was significantly higher than that in the control group.

2.3 CAR-NK细胞体外杀伤能力检测2.3 Detection of CAR-NK cell killing ability in vitro

发明人将上述NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞分别与胰腺癌细胞系Aspc-1共孵育5h后检测杀伤效率。从图4中可以看出，α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92在效靶比为5：1时对Aspc-1细胞的杀伤效率分别为48.01±2.00％和58.42％±2.46，明显高于NK-92细胞(35.59±2.46％)；而α-MSLN-CAR-RIL-NK-92细胞的杀伤效率亦明显高于α-MSLN-CAR-NK-92组，说明插入RIL片段不仅能促进NK细胞分泌IL-15，而且能够提高α-MSLN-CAR-NK-92细胞的杀伤功能。The inventors co-incubated the above-mentioned NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells with the pancreatic cancer cell line Aspc-1 for 5 hours and then detected the killing efficiency. As can be seen from Figure 4, the killing efficiency of α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 on Aspc-1 cells was 48.01±2.00% and 58.42%±2.46, respectively, when the effect-target ratio was 5:1, which was significantly higher than that of NK-92 cells (35.59±2.46%); and the killing efficiency of α-MSLN-CAR-RIL-NK-92 cells was also significantly higher than that of the α-MSLN-CAR-NK-92 group, indicating that the insertion of the RIL fragment can not only promote the secretion of IL-15 by NK cells, but also improve the killing function of α-MSLN-CAR-NK-92 cells.

2.4 CAR-NK细胞体外增殖能力检测2.4 Detection of CAR-NK cell proliferation ability in vitro

发明人进一步验证自分泌RIL对NK-92细胞的促存活作用。分别将相同细胞数量的所述NK-92、 α-MSLN-CAR-NK-92和α-MSLN-CAR-RIL-NK-92细胞接板于96孔板中，每3天进行细胞计数，培养24天，绘制细胞增殖曲线。从图5中可以看出，培养至第12天时，α-MSLN-CAR-RIL-NK-92细胞的数量与NK-92、α-MSLN-CAR-NK-92两组细胞相比开始出现差异。至第21天时，开始有显著差异。说明本发明修饰的NK细胞具有更强的增殖能力可能是由于RIL为超激动剂型式的IL-15，它以反式结合方式能够更有效地与IL-15βγc链结合，激活下游信号通路。The inventors further verified the pro-survival effect of autocrine RIL on NK-92 cells. α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 cells were plated in 96-well plates, and cell counts were performed every 3 days. After culturing for 24 days, cell proliferation curves were drawn. As can be seen from Figure 5, when cultured to the 12th day, the number of α-MSLN-CAR-RIL-NK-92 cells began to differ from that of the NK-92 and α-MSLN-CAR-NK-92 groups of cells. By the 21st day, there were significant differences. It is shown that the modified NK cells of the present invention have a stronger proliferation ability, which may be due to the fact that RIL is a superagonist form of IL-15, which can more effectively bind to the IL-15βγc chain in a trans-binding manner and activate the downstream signaling pathway.

实施例3：CAR-NK细胞体内抗肿瘤能力及CAR-NK细胞在体内的存活能力检测Example 3: Detection of the anti-tumor ability of CAR-NK cells in vivo and the survival ability of CAR-NK cells in vivo

本实施例建立了胰腺癌Aspc-1细胞荷瘤小鼠移植模型以观察CAR-NK-92细胞对胰腺癌的治疗作用。具体的实验操作如下：In this example, a pancreatic cancer Aspc-1 cell tumor-bearing mouse transplantation model was established to observe the therapeutic effect of CAR-NK-92 cells on pancreatic cancer. The specific experimental procedures are as follows:

选择6周龄BALB/c-nu裸鼠进行腋下皮下荷瘤，荷瘤剂量为2×106细胞/只。在4天左右成瘤，一周后开始进行细胞治疗。治疗前测量肿瘤体积大小，根据肿瘤体积大小随机分为PBS组、NK-92细胞治疗组、α-MSLN-CAR-NK-92细胞治疗组和α-MSLN-CAR-RIL-NK-92细胞治疗组。给治疗组小鼠尾静脉注射效应细胞1×107个/只，未治疗组注射等体积的1×PBS，每隔一周注射一次，共治疗5次，并且每隔3天尾静脉注射IL-2(5×104IU/只)，具体的实验设置及操作流程参考图6。为了研究RIL对CAR-NK-92细胞的促存活作用，在治疗后第一、三、七天采集小鼠外周血，红细胞裂解后标记PerCP/Cyanine5.5anti-human CD56抗体，流式检测外周血淋巴细胞中CD56细胞群的比例，即NK-92细胞的比例。每隔3天测量肿瘤体积大小，绘制肿瘤生长曲线。Six-week-old BALB/c-nu nude mice were selected for subcutaneous tumor bearing in the armpit, and the tumor bearing dose was 2×10 6 cells/mouse. The tumor was formed in about 4 days, and cell therapy was started one week later. The tumor volume was measured before treatment, and the mice were randomly divided into PBS group, NK-92 cell treatment group, α-MSLN-CAR-NK-92 cell treatment group and α-MSLN-CAR-RIL-NK-92 cell treatment group according to the tumor volume. The mice in the treatment group were injected with 1×10 7 effector cells/mouse through the tail vein, and the untreated group was injected with an equal volume of 1×PBS, once every week, for a total of 5 treatments, and IL-2 (5×10 4 IU/mouse) was injected through the tail vein every 3 days. For specific experimental settings and operation procedures, please refer to Figure 6. In order to study the effect of RIL on the survival of CAR-NK-92 cells, peripheral blood of mice was collected on the first, third, and seventh days after treatment. After erythrocyte lysis, PerCP/Cyanine5.5anti-human CD56 antibody was labeled, and the proportion of CD56 cell population in peripheral blood lymphocytes, i.e. the proportion of NK-92 cells, was detected by flow cytometry. The tumor volume was measured every 3 days, and the tumor growth curve was drawn.

结果如图7所示，在NK细胞治疗一天时，小鼠体内NK-92细胞、α-MSLN-CAR-NK-92细胞和α-MSLN-CAR-RIL-NK-92细胞占外周血淋巴细胞比例分别为27.3％、29.6％和29.3％，各组NK-92细胞在体内比例相近。第三天时，小鼠体内NK-92细胞、α-MSLN-CAR-NK-92细胞和α-MSLN-CAR-RIL-NK-92细胞占外周血淋巴细胞比例分别为8.32％、9.83％、和16.8％。可以看出与第一天相比，在第三天时各组NK-92细胞比例都有所下降，NK-92组从27.3％下降到8.32％，α-MSLN-CAR-NK-92组从29.6％下降到9.83％，α-MSLN-CAR-RIL-NK-92从29.3％下降到16.8％。但可以看出α-MSLN-CAR-RIL-NK-92组细胞在体内的比例最高，体内持久性更突出。第七天时，NK-92细胞、α-MSLN-CAR-NK-92细胞和α-MSLN-CAR-RIL-NK-92细胞在体内比例分别为0.13％、3.32％、和14.7％。可以看出与NK-92组和α-MSLN-CAR-NK-92组相比，RIL基因修饰的α-MSLN-CAR-RIL-NK-92细胞在体内的比例更高，体内持久能力更强。因此，表达RIL可以提高NK细胞在体内的生存能力，局部分泌的超级IL-15(RIL)具有明显的维持NK细胞体内存活的效果。The results are shown in Figure 7. On the first day of NK cell treatment, the proportion of NK-92 cells, α-MSLN-CAR-NK-92 cells and α-MSLN-CAR-RIL-NK-92 cells in the peripheral blood lymphocytes of mice was 27.3%, 29.6% and 29.3%, respectively, and the proportion of NK-92 cells in each group was similar. On the third day, the proportion of NK-92 cells, α-MSLN-CAR-NK-92 cells and α-MSLN-CAR-RIL-NK-92 cells in the peripheral blood lymphocytes of mice was 8.32%, 9.83%, and 16.8%, respectively. It can be seen that compared with the first day, the proportion of NK-92 cells in each group decreased on the third day, the NK-92 group decreased from 27.3% to 8.32%, the α-MSLN-CAR-NK-92 group decreased from 29.6% to 9.83%, and the α-MSLN-CAR-RIL-NK-92 decreased from 29.3% to 16.8%. However, it can be seen that the proportion of cells in the α-MSLN-CAR-RIL-NK-92 group in vivo is the highest, and the persistence in vivo is more prominent. On the seventh day, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells, and α-MSLN-CAR-RIL-NK-92 cells in vivo were 0.13%, 3.32%, and 14.7%, respectively. It can be seen that compared with the NK-92 group and the α-MSLN-CAR-NK-92 group, the RIL gene-modified α-MSLN-CAR-RIL-NK-92 cells have a higher proportion in vivo and stronger persistence in vivo. Therefore, the expression of RIL can improve the survival ability of NK cells in vivo, and the locally secreted super IL-15 (RIL) has a significant effect on maintaining the survival of NK cells in vivo.

通过测量肿瘤大小绘制肿瘤生长曲线，由图8可以看出，与对照组PBS组、NK-92细胞治疗组相比，α-MSLN-CAR-NK92细胞和α-MSLN-CAR-RIL-NK-92细胞治疗可明显抑制肿瘤生长，且α-MSLN-CAR-RIL-NK-92细胞显示出较α-MSLN-CAR-NK-92细胞更好的抗肿瘤作用。以上结果说明靶 向间皮素的CAR-NK-92细胞可以抑制胰腺癌生长，具有良好的治疗效果。RIL基因修饰可以提高CAR-NK-92细胞在体内的持久性，提高CAR-NK-92细胞在体内的生存能力，提高CAR-NK-92细胞的抗肿瘤效果。.By measuring the tumor size and drawing the tumor growth curve, it can be seen from Figure 8 that compared with the control group PBS group and the NK-92 cell treatment group, α-MSLN-CAR-NK92 cell and α-MSLN-CAR-RIL-NK-92 cell treatment can significantly inhibit tumor growth, and α-MSLN-CAR-RIL-NK-92 cells show better anti-tumor effect than α-MSLN-CAR-NK-92 cells. The above results show that target CAR-NK-92 cells expressing mesothelin can inhibit the growth of pancreatic cancer and have a good therapeutic effect. RIL gene modification can improve the persistence of CAR-NK-92 cells in the body, improve the survival ability of CAR-NK-92 cells in the body, and improve the anti-tumor effect of CAR-NK-92 cells. .

此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。 Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (93)

一种转基因免疫细胞，其特征在于，所述免疫细胞表达嵌合抗原受体以及融合蛋白，所述融合蛋白包括IL-15Rα和IL-15。A transgenic immune cell, characterized in that the immune cell expresses a chimeric antigen receptor and a fusion protein, wherein the fusion protein includes IL-15Rα and IL-15. 根据权利要求1所述的转基因免疫细胞，其特征在于，所述嵌合抗原受体包括：The genetically modified immune cell according to claim 1, characterized in that the chimeric antigen receptor comprises: 胞外区，所述胞外区能够与抗原特异性结合；An extracellular region, which is capable of specifically binding to an antigen; 跨膜区；以及transmembrane region; and 胞内区，所述胞内区包括免疫共刺激分子胞内段和信号转导结构域；The intracellular region includes the intracellular segment of the immune co-stimulatory molecule and the signal transduction domain; 其中，所述胞外区的C端与所述跨膜区的N端相连，所述跨膜区的C端与所述胞内区的N端相连。The C-terminus of the extracellular region is connected to the N-terminus of the transmembrane region, and the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述抗原是肿瘤相关抗原。The transgenic immune cell according to claim 2, characterized in that the antigen is a tumor-associated antigen. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述胞外区包括抗体的重链可变区和轻链可变区，所述抗体结合所述抗原。The transgenic immune cell according to claim 2, characterized in that the extracellular region includes a heavy chain variable region and a light chain variable region of an antibody, and the antibody binds to the antigen. 根据权利要求4所述的转基因免疫细胞，其特征在于，所述抗体为单链抗体。The genetically modified immune cell according to claim 4, characterized in that the antibody is a single-chain antibody. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述抗原包括选自间皮素、HER2、EGFR、GPC3、MUC1、CEA、CLDN 18.2、EpCAM、GD2、PSCA、IL-13RA2、B7-H3、CD133、ROR1、CD19、CD20、CD22、CD30、CD33、BCMA中的至少之一。The transgenic immune cells according to claim 2 are characterized in that the antigens include at least one selected from mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, GD2, PSCA, IL-13RA2, B7-H3, CD133, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA. 根据权利要求6所述的转基因免疫细胞，其特征在于，所述抗原为间皮素。The genetically modified immune cell according to claim 6, characterized in that the antigen is mesothelin. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述胞外区包括抗间皮素单链抗体。The transgenic immune cell according to claim 2, characterized in that the extracellular region comprises an anti-mesothelin single-chain antibody. 根据权利要求8所述的转基因免疫细胞，其特征在于，所述抗间皮素单链抗体包括抗间皮素抗体的轻链可变区、连接肽1以及抗间皮素抗体的重链可变区。The transgenic immune cell according to claim 8, characterized in that the anti-mesothelin single-chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1, and a heavy chain variable region of an anti-mesothelin antibody. 根据权利要求9所述的转基因免疫细胞，其特征在于，所述连接肽1具有(GGGGS)n所示的氨基酸序列，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。The genetically modified immune cell according to claim 9, characterized in that the connecting peptide 1 has an amino acid sequence shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 根据权利要求8所述的转基因免疫细胞，其特征在于，抗间皮素单链抗体包括SEQ ID NO:14所示的氨基酸序列。The transgenic immune cell according to claim 8 is characterized in that the anti-mesothelin single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:14. 根据权利要求8所述的转基因免疫细胞，其特征在于，所述胞外区进一步包括铰链区片段，所述铰链区片段的N端与所述单链抗体的C端相连。The transgenic immune cell according to claim 8, characterized in that the extracellular region further includes a hinge region fragment, and the N-terminus of the hinge region fragment is connected to the C-terminus of the single-chain antibody. 根据权利要求12所述的转基因免疫细胞，其特征在于，所述铰链区片段包括选自CD8、CD28和免疫球蛋白的铰链区中的至少之一。The transgenic immune cell according to claim 12, characterized in that the hinge region fragment comprises at least one hinge region selected from CD8, CD28 and immunoglobulin. 根据权利要求13所述的转基因免疫细胞，其特征在于，所述铰链片段包括CD8的铰链区。The transgenic immune cell according to claim 13, characterized in that the hinge fragment comprises the hinge region of CD8. 根据权利要求14所述的转基因免疫细胞，其特征在于，所述铰链片段包括SEQ ID NO：15所示的氨基酸序列。 The transgenic immune cell according to claim 14, characterized in that the hinge fragment comprises the amino acid sequence shown in SEQ ID NO: 15. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述跨膜区包括选自CD4，CD8α，CD28和CD3ζ中的至少之一或其片段。The transgenic immune cell according to claim 2, characterized in that the transmembrane region includes at least one or a fragment thereof selected from CD4, CD8α, CD28 and CD3ζ. 根据权利要求16所述的转基因免疫细胞，其特征在于，所述跨膜区包括CD8跨膜区或其片段。The transgenic immune cell according to claim 16, characterized in that the transmembrane region comprises a CD8 transmembrane region or a fragment thereof. 根据权利要求17所述的转基因免疫细胞，其特征在于，所述跨膜区具有SEQ ID NO:16所示的氨基酸序列。The transgenic immune cell according to claim 17 is characterized in that the transmembrane region has the amino acid sequence shown in SEQ ID NO:16. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述免疫共刺激分子包括选自CD28、ICOS、4-1BB、OX40和CD27中的至少之一。The transgenic immune cell according to claim 2, characterized in that the immune co-stimulatory molecule includes at least one selected from CD28, ICOS, 4-1BB, OX40 and CD27. 根据权利要求19所述的转基因免疫细胞，其特征在于，所述免疫共刺激分子胞内段是4-1BB或CD28的胞内段或其片段。The transgenic immune cell according to claim 19, characterized in that the intracellular segment of the immune co-stimulatory molecule is the intracellular segment of 4-1BB or CD28 or a fragment thereof. 根据权利要求20所述的转基因免疫细胞，其特征在于，所述免疫共刺激分子胞内段包括SEQ ID NO:17所示的氨基酸序列。The transgenic immune cell according to claim 20 is characterized in that the intracellular segment of the immune co-stimulatory molecule includes the amino acid sequence shown in SEQ ID NO:17. 根据权利要求2所述的转基因免疫细胞，其特征在于，所述免疫共刺激分子胞内段的C端与所述信号转导结构域的N端相连。The transgenic immune cell according to claim 2, characterized in that the C-terminus of the intracellular segment of the immune co-stimulatory molecule is connected to the N-terminus of the signal transduction domain. 根据权利要求22所述的转基因免疫细胞，其特征在于，所述信号转导结构域包括选自CD3ζ或FcεRIγ中的至少之一或其片段。The transgenic immune cell according to claim 22, characterized in that the signal transduction domain includes at least one or a fragment thereof selected from CD3ζ or FcεRIγ. 根据权利要求23所述的转基因免疫细胞，其特征在于，所述信号转导结构域包括CD3ζ或其片段。The transgenic immune cell according to claim 23, characterized in that the signal transduction domain comprises CD3ζ or a fragment thereof. 根据权利要求24所述的转基因免疫细胞，其特征在于，所述信号转导结构域包括SEQ ID NO:18所示的氨基酸序列。The transgenic immune cell according to claim 24 is characterized in that the signal transduction domain includes the amino acid sequence shown in SEQ ID NO:18. 根据权利要求1所述的转基因免疫细胞，其特征在于，所述融合蛋白中进一步包括连接肽2。The transgenic immune cell according to claim 1, characterized in that the fusion protein further comprises a connecting peptide 2. 根据权利要求26所述的转基因免疫细胞，其特征在于，所述连接肽2具有SEQ ID NO:19所示的氨基酸序列。The transgenic immune cell according to claim 26 is characterized in that the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO:19. 根据权利要求1所述的转基因免疫细胞，其特征在于，所述免疫细胞包括T细胞、NKT细胞和NK细胞中的至少之一，优选为NK细胞。The genetically modified immune cell according to claim 1, characterized in that the immune cell comprises at least one of a T cell, a NKT cell and a NK cell, preferably a NK cell. 根据权利要求28所述的转基因免疫细胞，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The transgenic immune cell according to claim 28, characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求28所述的转基因免疫细胞，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The genetically modified immune cell according to claim 28, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδT cells. 一种分离的核酸，其特征在于，所述分离的核酸包括：An isolated nucleic acid, characterized in that the isolated nucleic acid comprises: 第一核酸分子，所述第一核酸分子编码嵌合抗原受体；以及a first nucleic acid molecule encoding a chimeric antigen receptor; and 第二核酸分子，所述第二核酸分子编码融合蛋白，所述融合蛋白包括IL-15Rα和IL-15。 The second nucleic acid molecule encodes a fusion protein comprising IL-15Rα and IL-15. 根据权利要求31所述的分离的核酸，其特征在于，所述嵌合抗原受体是如权利要求2～25任一项中所定义的。The isolated nucleic acid according to claim 31, characterized in that the chimeric antigen receptor is as defined in any one of claims 2 to 25. 根据权利要求31所述的分离的核酸，其特征在于，所述第二核酸分子中包括如SEQ ID NO:10所示编码所述IL-15Rα的核苷酸序列，和如SEQ ID NO:12所示编码所述IL-15的核苷酸序列。The isolated nucleic acid according to claim 31 is characterized in that the second nucleic acid molecule includes a nucleotide sequence encoding the IL-15Rα as shown in SEQ ID NO:10, and a nucleotide sequence encoding the IL-15 as shown in SEQ ID NO:12. 根据权利要求31所述的分离的核酸，其特征在于，所述第一核酸分子以及所述第二核酸分子被设置在免疫细胞中表达所述嵌合抗原受体和所述融合蛋白，并且所述融合蛋白与所述嵌合抗原受体呈非融合形式。The isolated nucleic acid according to claim 31 is characterized in that the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the fusion protein in an immune cell, and the fusion protein is in a non-fused form with the chimeric antigen receptor. 根据权利要求31所述的分离的核酸，其特征在于，进一步包括：The isolated nucleic acid according to claim 31, further comprising: 内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：20所示的核苷酸序列。An internal ribosome entry site sequence, wherein the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO: 20. 根据权利要求34所述的分离的核酸，其特征在于，进一步包括：The isolated nucleic acid according to claim 34, further comprising: 第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码连接肽3，所述连接肽3能够在所述免疫细胞中被切割。The third nucleic acid molecule is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a connecting peptide 3, and the connecting peptide 3 can be cut in the immune cell. 根据权利要求36所述的分离的核酸，其特征在于，所述连接肽3包括2A肽或其片段。The isolated nucleic acid according to claim 36, characterized in that the connecting peptide 3 comprises a 2A peptide or a fragment thereof. 根据权利要求37所述的分离的核酸，其特征在于，所述连接肽3包括P2A、T2A、E2A和F2A中的至少之一或其片段。The isolated nucleic acid according to claim 37 is characterized in that the connecting peptide 3 comprises at least one of P2A, T2A, E2A and F2A or a fragment thereof. 根据权利要求38所述的分离的核酸，其特征在于，所述连接肽3包括P2A或其片段。The isolated nucleic acid according to claim 38, characterized in that the connecting peptide 3 comprises P2A or a fragment thereof. 根据权利要求39所述的分离的核酸，其特征在于，所述连接肽3包括SEQ ID NO:19所示的氨基酸序列。The isolated nucleic acid according to claim 39 is characterized in that the connecting peptide 3 includes the amino acid sequence shown in SEQ ID NO:19. 根据权利要求31所述的分离的核酸，其特征在于，进一步包括：The isolated nucleic acid according to claim 31, further comprising: 第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；和/或a first promoter, the first promoter being operably linked to the first nucleic acid molecule; and/or 第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。A second promoter is operably linked to the second nucleic acid molecule. 根据权利要求41所述的分离的核酸，其特征在于，所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子。The isolated nucleic acid according to claim 41 is characterized in that the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter. 根据权利要求31所述的分离的核酸，其特征在于，进一步包括：The isolated nucleic acid according to claim 31, further comprising: 第四核酸分子，所述第四核酸分子编码信号肽。A fourth nucleic acid molecule, wherein the fourth nucleic acid molecule encodes a signal peptide. 根据权利要求43所述的分离的核酸，其特征在于，所述第四核酸分子与所述第一核酸分子可操作性地相连。The isolated nucleic acid of claim 43, wherein the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule. 根据权利要求43所述的分离的核酸，其特征在于，所述信号肽包括选自CSF2R和CD8α中的至少之一或其片段。The isolated nucleic acid according to claim 43, characterized in that the signal peptide comprises at least one selected from CSF2R and CD8α or a fragment thereof. 根据权利要求45所述的分离的核酸，其特征在于，所述信号肽包括CSF2R或其片段。 The isolated nucleic acid of claim 45, wherein the signal peptide comprises CSF2R or a fragment thereof. 根据权利要求46所述的分离的核酸，其特征在于，所述信号肽包括SEQ ID NO:13所示的氨基酸序列。The isolated nucleic acid according to claim 46 is characterized in that the signal peptide comprises the amino acid sequence shown in SEQ ID NO:13. 根据权利要求31～47任一项所述的分离的核酸，其特征在于，所述第一核酸分子具有SEQ ID NO:3、4、5、6和7所示的核苷酸序列中的至少之一。The isolated nucleic acid according to any one of claims 31 to 47 is characterized in that the first nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3, 4, 5, 6 and 7. 根据权利要求31～47任一项所述的分离的核酸，其特征在于，所述第二核酸分子具有SEQ ID NO:9所示的核苷酸序列。The isolated nucleic acid according to any one of claims 31 to 47 is characterized in that the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:9. 根据权利要求31～47任一项所述的分离的核酸，其特征在于，所述第三核酸分子具有SEQ ID NO:8所示的核苷酸序列。The isolated nucleic acid according to any one of claims 31 to 47 is characterized in that the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:8. 根据权利要求31～47任一项所述的分离的核酸，其特征在于，所述第四核酸分子具有SEQ ID NO:2所示的核苷酸序列。The isolated nucleic acid according to any one of claims 31 to 47 is characterized in that the fourth nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:2. 一种构建体，其特征在于，所述构建体携带权利要求31～51任一项所述的分离的核酸。A construct, characterized in that it carries the isolated nucleic acid according to any one of claims 31 to 51. 根据权利要求52所述的构建体，其特征在于，所述构建体的载体是非致病性病毒载体。The construct according to claim 52 is characterized in that the vector of the construct is a non-pathogenic viral vector. 根据权利要求53所述的构建体，其特征在于，所述病毒载体包括选自反转录病毒载体、慢病毒载体和腺病毒相关病毒载体的至少之一。The construct according to claim 53 is characterized in that the viral vector comprises at least one selected from a retroviral vector, a lentiviral vector and an adenovirus-associated viral vector. 一种重组细胞，其特征在于，携带权利要求31～51任一项所述的分离的核酸或权利要求52～54任一项所述的构建体。A recombinant cell, characterized in that it carries the isolated nucleic acid according to any one of claims 31 to 51 or the construct according to any one of claims 52 to 54. 根据权利要求55所述的重组细胞，其特征在于，所述重组细胞包括真核细胞，优选为哺乳动物细胞。The recombinant cell according to claim 55, characterized in that the recombinant cell comprises a eukaryotic cell, preferably a mammalian cell. 一种CAR-NK、CAR-NKT或CAR-T细胞，其特征在于，携带权利要求31～51任一项所述的分离的核酸或权利要求52～54任一项所述的构建体。A CAR-NK, CAR-NKT or CAR-T cell, characterized in that it carries the isolated nucleic acid of any one of claims 31 to 51 or the construct of any one of claims 52 to 54. 根据权利要求57所述的CAR-NK、CAR-NKT或CAR-T细胞，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The CAR-NK, CAR-NKT or CAR-T cell according to claim 57, characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求57所述的CAR-NK、CAR-NKT或CAR-T细胞，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The CAR-NK, CAR-NKT or CAR-T cell according to claim 57, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδT cells. 一种获得病毒的方法，其特征在于，将权利要求52～54任一项所述的构建体导入第一受体细胞；将导入所述的构建体的第一受体细胞进行培养，以便获得所述病毒。A method for obtaining a virus, characterized in that the construct described in any one of claims 52 to 54 is introduced into a first recipient cell; and the first recipient cell introduced with the construct is cultured to obtain the virus. 根据权利要求60所述的方法，其特征在于，所述病毒包括慢病毒。The method of claim 60, wherein the virus comprises a lentivirus. 根据权利要求60所述的方法，其特征在于，所述第一受体细胞为293T。The method according to claim 60 is characterized in that the first recipient cell is 293T. 一种病毒，其特征在于，是通过权利要求60～62任一项所述的方法获得的。A virus, characterized in that it is obtained by the method according to any one of claims 60 to 62. 一种病毒，其特征在于，包括具有SEQ ID NO:1所示的核苷酸序列。 A virus, characterized in that it comprises a nucleotide sequence shown in SEQ ID NO:1. 一种药物组合物，其特征在于，包括有效量的权利要求1～30任一项所述的转基因免疫细胞、权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体、权利要求55～56任一项所述的重组细胞、权利要求57～59任一项所述的CAR-NK或CAR-T细胞或者权利要求63～64任一项所述的病毒。A pharmaceutical composition, characterized in that it comprises an effective amount of the transgenic immune cell according to any one of claims 1 to 30, the isolated nucleic acid according to any one of claims 31 to 51, the construct according to any one of claims 52 to 54, the recombinant cell according to any one of claims 55 to 56, the CAR-NK or CAR-T cell according to any one of claims 57 to 59, or the virus according to any one of claims 63 to 64. 一种试剂盒，其特征在于，包括：权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体或权利要求63～64任一项所述的病毒。A kit, characterized in that it comprises: the isolated nucleic acid according to any one of claims 31 to 51, the construct according to any one of claims 52 to 54, or the virus according to any one of claims 63 to 64. 一种将病毒导入激活免疫细胞的方法，其特征在于，利用权利要求52～54任一项所述的构建体电转或转染所述激活免疫细胞或利用权利要求63～64任一项所述的病毒感染所述激活免疫细胞。A method for introducing a virus into an activated immune cell, characterized in that the activated immune cell is electroporated or transfected with the construct described in any one of claims 52 to 54, or the activated immune cell is infected with the virus described in any one of claims 63 to 64. 根据权利要求67所述的方法，其特征在于，所述免疫细胞包括T细胞、NKT细胞和NK细胞中的至少之一，优选为NK细胞。The method according to claim 67 is characterized in that the immune cells include at least one of T cells, NKT cells and NK cells, preferably NK cells. 根据权利要求68所述的方法，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The method according to claim 68 is characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求68所述的方法，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The method according to claim 68, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδ T cells. 一种获得嵌合抗原受体和融合蛋白的方法，其特征在于，包括：A method for obtaining chimeric antigen receptors and fusion proteins, characterized by comprising: 将权利要求52～54任一项所述的构建体或权利要求63～64任一项所述的病毒导入第二受体细胞；introducing the construct of any one of claims 52 to 54 or the virus of any one of claims 63 to 64 into a second recipient cell; 将导入表达载体或病毒的第二受体细胞进行培养，以便获得所述嵌合抗原受体和融合蛋白。The second recipient cell into which the expression vector or virus has been introduced is cultured to obtain the chimeric antigen receptor and fusion protein. 根据权利要求71所述的方法，其特征在于，所述导入第二受体细胞是通过电转、转染或感染的方式进行的。The method according to claim 71 is characterized in that the introduction into the second recipient cell is carried out by electroporation, transfection or infection. 根据权利要求72所述的方法，其特征在于，所述第二受体细胞为T细胞、NKT细胞和NK细胞中的至少之一。The method according to claim 72 is characterized in that the second receptor cell is at least one of a T cell, a NKT cell and a NK cell. 根据权利要求73所述的方法，其特征在于，所述第二受体细胞为NK细胞。The method according to claim 73 is characterized in that the second receptor cell is a NK cell. 根据权利要求73所述的方法，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The method according to claim 73 is characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求73所述的方法，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The method according to claim 73 is characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδT cells. 根据权利要求71所述的方法，其特征在于，所述病毒包括慢病毒。The method of claim 71, wherein the virus comprises a lentivirus. 一种获得表达嵌合抗原受体和融合蛋白的CAR-NK或CAR-NKT细胞或CAR-T细胞的方法，A method for obtaining CAR-NK or CAR-NKT cells or CAR-T cells expressing a chimeric antigen receptor and a fusion protein, 其特征在于，包括：The invention is characterized by comprising: 将权利要求52～54任一项所述的构建体或权利要求63～64任一项所述的病毒导入NK细胞、NKT细胞或T细胞； Introducing the construct according to any one of claims 52 to 54 or the virus according to any one of claims 63 to 64 into NK cells, NKT cells or T cells; 将导入所述构建体或病毒的NK细胞、NKT细胞或T细胞进行培养，以便获得所述CAR-NK、CAR-NKT细胞或CAR-T细胞。The NK cells, NKT cells or T cells introduced with the construct or virus are cultured to obtain the CAR-NK, CAR-NKT cells or CAR-T cells. 根据权利要求78所述的方法，其特征在于，所述导入NK细胞、NKT细胞或T细胞是通过电转、转染或感染的方式进行的。The method according to claim 78 is characterized in that the introduction of NK cells, NKT cells or T cells is carried out by electroporation, transfection or infection. 根据权利要求79所述的方法，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The method according to claim 79 is characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求79所述的方法，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The method according to claim 79, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδ T cells. 权利要求1～30任一项所述的转基因免疫细胞、权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体、权利要求55～56任一项所述的重组细胞、权利要求57～59任一项所述的CAR-NK或CAR-NKT细胞或CAR-T细胞或权利要求63～64任一项所述的病毒在制备药物组合物中的用途，所述药物组合物用于治疗或预防肿瘤。Use of the transgenic immune cell of any one of claims 1 to 30, the isolated nucleic acid of any one of claims 31 to 51, the construct of any one of claims 52 to 54, the recombinant cell of any one of claims 55 to 56, the CAR-NK or CAR-NKT cell or CAR-T cell of any one of claims 57 to 59, or the virus of any one of claims 63 to 64 in the preparation of a pharmaceutical composition for treating or preventing a tumor. 根据权利要求82所述的用途，其特征在于，所述肿瘤包括间皮素阳性肿瘤、HER2阳性肿瘤、EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤。The use according to claim 82 is characterized in that the tumor includes mesothelin-positive tumor, HER2-positive tumor, EGFR-positive tumor, GPC3-positive tumor, MUC1-positive tumor, CEA-positive tumor, CLDN 18.2-positive tumor, EpCAM-positive tumor, GD2-positive tumor, PSCA-positive tumor, IL-13RA2-positive tumor, B7-H3-positive tumor, CD133-positive tumor, ROR1-positive tumor, CD19-positive tumor, CD20-positive tumor, CD22-positive tumor, CD30-positive tumor, CD33-positive tumor and BCMA-positive tumor. 根据权利要求83所述的用途，其特征在于，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。The use according to claim 83 is characterized in that the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer. 权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体或权利要求63～64任一项所述的病毒在制备试剂盒中的用途，所述试剂盒用于促进NK细胞、NKT细胞或T细胞活化或者增殖。Use of the isolated nucleic acid of any one of claims 31 to 51, the construct of any one of claims 52 to 54, or the virus of any one of claims 63 to 64 in the preparation of a kit for promoting the activation or proliferation of NK cells, NKT cells or T cells. 根据权利要求85所述的用途，其特征在于，所述NK细胞包括选自外周血NK细胞、脐带血NK细胞、诱导多能细胞(iPSC)衍生NK细胞和NK-92细胞中的至少之一。The use according to claim 85 is characterized in that the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells. 根据权利要求85所述的用途，其特征在于，所述T细胞包括CD4+T细胞、CD8+T细胞和γδT细胞。The use according to claim 85, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδT cells. 一种治疗或预防肿瘤的方法，其特征在于，包括向受试者施用权利要求1～30任一项所述的转基因免疫细胞、权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体、权利要求55～56任一项所述的重组细胞、权利要求57～59任一项所述的CAR-NK、CAR-NKT或CAR-T细胞、权利要求63～64任一项所述的病毒或权利要求65所述的药物组合物中的至少之一。A method for treating or preventing tumors, characterized in that it comprises administering to a subject at least one of the transgenic immune cells of any one of claims 1 to 30, the isolated nucleic acid of any one of claims 31 to 51, the construct of any one of claims 52 to 54, the recombinant cells of any one of claims 55 to 56, the CAR-NK, CAR-NKT or CAR-T cells of any one of claims 57 to 59, the virus of any one of claims 63 to 64, or the pharmaceutical composition of claim 65. 根据权利要求88所述的方法，其特征在于，所述肿瘤包括间皮素阳性肿瘤、HER2阳性肿瘤、 EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤。The method according to claim 88, characterized in that the tumor comprises a mesothelin-positive tumor, a HER2-positive tumor, EGFR-positive tumors, GPC3-positive tumors, MUC1-positive tumors, CEA-positive tumors, CLDN 18.2-positive tumors, EpCAM-positive tumors, GD2-positive tumors, PSCA-positive tumors, IL-13RA2-positive tumors, B7-H3-positive tumors, CD133-positive tumors, ROR1-positive tumors, CD19-positive tumors, CD20-positive tumors, CD22-positive tumors, CD30-positive tumors, CD33-positive tumors, and BCMA-positive tumors. 根据权利要求89所述的方法，其特征在于，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。The method according to claim 89, characterized in that the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer. 权利要求1～30任一项所述的转基因免疫细胞、权利要求31～51任一项所述的分离的核酸、权利要求52～54任一项所述的构建体、权利要求55～56任一项所述的重组细胞、权利要求57～59任一项所述的CAR-NK或CAR-T细胞、权利要求63～64任一项所述的病毒或权利要求65所述的药物组合物用于治疗或预防肿瘤中的用途。Use of the transgenic immune cell of any one of claims 1 to 30, the isolated nucleic acid of any one of claims 31 to 51, the construct of any one of claims 52 to 54, the recombinant cell of any one of claims 55 to 56, the CAR-NK or CAR-T cell of any one of claims 57 to 59, the virus of any one of claims 63 to 64, or the pharmaceutical composition of claim 65 for treating or preventing tumors. 根据权利要求91所述的用途，其特征在于，所述肿瘤包括间皮素阳性肿瘤、HER2阳性肿瘤、EGFR阳性肿瘤、GPC3阳性肿瘤、MUC1阳性肿瘤、CEA阳性肿瘤、CLDN 18.2阳性肿瘤、EpCAM阳性肿瘤、GD2阳性肿瘤、PSCA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、ROR1阳性肿瘤、CD19阳性肿瘤、CD20阳性肿瘤、CD22阳性肿瘤、CD30阳性肿瘤、CD33阳性肿瘤和BCMA阳性肿瘤。The use according to claim 91 is characterized in that the tumor includes mesothelin-positive tumor, HER2-positive tumor, EGFR-positive tumor, GPC3-positive tumor, MUC1-positive tumor, CEA-positive tumor, CLDN 18.2-positive tumor, EpCAM-positive tumor, GD2-positive tumor, PSCA-positive tumor, IL-13RA2-positive tumor, B7-H3-positive tumor, CD133-positive tumor, ROR1-positive tumor, CD19-positive tumor, CD20-positive tumor, CD22-positive tumor, CD30-positive tumor, CD33-positive tumor and BCMA-positive tumor. 根据权利要求92所述的用途，其特征在于，所述间皮素阳性肿瘤包括胰腺癌、卵巢癌、间皮瘤、胆管癌和肺癌中的至少之一。 The use according to claim 92 is characterized in that the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, bile duct cancer and lung cancer.
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