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WO2024113777A1 - Transgenic immune cell and use thereof - Google Patents

Transgenic immune cell and use thereof Download PDF

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Publication number
WO2024113777A1
WO2024113777A1 PCT/CN2023/101463 CN2023101463W WO2024113777A1 WO 2024113777 A1 WO2024113777 A1 WO 2024113777A1 CN 2023101463 W CN2023101463 W CN 2023101463W WO 2024113777 A1 WO2024113777 A1 WO 2024113777A1
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Prior art keywords
cells
nucleic acid
cell
car
isolated nucleic
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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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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of biotechnology, and in particular to transgenic immune cells and applications 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 an immunostimulatory molecule, and the immunostimulatory molecule includes IL-15.
  • the transgenic immune cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and an immunostimulatory molecule.
  • the chimeric antigen receptor can enable the immune cell to target the corresponding antigen and locate it on the surface of the cell expressing the antigen.
  • the immunostimulatory molecule further promotes the immune cell
  • the activation and proliferation of immune cells can maintain the number and activity of immune cells in the local microenvironment of the tumor, enabling them to maintain strong tumor-killing activity, and can 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, PSMA, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, 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: 11.
  • 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 includes the amino acid sequence shown in SEQ ID NO:12.
  • 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 the amino acid sequence shown in SEQ ID NO:13.
  • the immune co-stimulatory molecule includes at least one selected from CD28, ICOS, 4-1BB, OX40, CD40, CD40L 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:14.
  • 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:15.
  • the immune cell includes at least one of a T cell and a NK cell.
  • the type of the immunostimulatory molecule is not particularly limited, and any promoting factor that can promote the immune function of at least one of a T cell and a NK cell 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, which encodes a chimeric antigen receptor; 2) a second nucleic acid molecule, which encodes an immunostimulatory molecule, and the immunostimulatory molecule includes IL-15.
  • IL-15 is a pleiotropic cytokine that has the function of activating T cells, B cells and NK cells, and mediating the proliferation and survival of these cells.
  • IL-15 can activate, maintain and amplify CD8 + memory T cells without activating regulatory T lymphocytes.
  • the isolated nucleic acid After the isolated nucleic acid according to an 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 immune cells, the immune cells can simultaneously express and secrete chimeric antigen receptors and immunostimulatory molecules, so that the immune cells can target the corresponding antigens and locate on the surface of cells expressing the antigens.
  • immunostimulatory molecules such as IL-15 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 they maintain strong tumor killing activity, effectively avoid the toxic and side effects caused by high-dose systemic or repeated injections, and avoid the toxic and side effects caused by high-dose systemic 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 first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the immunostimulatory molecule in an immune cell, and the immunostimulatory molecule and the chimeric antigen receptor are in a non-fusion 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:16.
  • 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 2, and the connecting peptide 2 can be cut.
  • the connecting peptide 2 can separate the first nucleic acid molecule from the second nucleic acid molecule to reduce the functional interference between the two.
  • the connecting peptide 2 includes a 2A peptide or a fragment thereof.
  • the connecting peptide 2 is not particularly limited, and any conventional peptide with self-cleavage function can be used.
  • the connecting peptide 2 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.
  • the connecting peptide 2 includes P2A or a fragment thereof.
  • the connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:17.
  • the isolated nucleic acid further includes: a first promoter, which is operably linked to the first nucleic acid molecule; and 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:10.
  • 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 receptors and immunostimulatory molecules, such as IL-15 and mesothelin.
  • 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 pombe, 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, 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 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 chimeric antigen receptors and immunostimulatory molecules.
  • the inventors found in experiments that the IL-15 modification strategy proposed in the present invention can enable NK cells or T cells to secrete IL-15 locally in the tumor, significantly improve the in vivo and in vitro proliferation ability of CAR-NK cells or T cells, enhance the in vivo survival time of NK cells or T cells, and improve the in vivo anti-tumor function of NK cells or T cells. And this kind of IL-15 that is continuously and slowly released locally can avoid the toxic and side effects caused by systemic application of recombinant IL-15 or repeated multiple administrations.
  • the NK cells include peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cells, At least one of iPSC-derived NK cells and NK-92 cells.
  • 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-T cells or viruses.
  • the separated nucleic acid, expression vector, or cells or viruses carrying the separated nucleic acid or expression vector can simultaneously express and secrete chimeric antigen receptors and immunostimulatory molecules, so that immune cells can target corresponding antigens and localize to the cell surface expressing the antigen.
  • immunostimulatory molecules such as IL-15 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 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. 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 nucleic acid of separation of the present invention, expression vector or the cell carrying the nucleic acid of separation or expression vector can be incorporated into the medicine suitable for parenteral administration (for example intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration for example 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 solution (for example, injection solution and infusion solution), dispersant or suspending agent, tablet, pill, powder, liposome and suppository.
  • Typical medicine is injection solution or infusion solution form.
  • the nucleic acid of separation, expression vector or the cell carrying the nucleic acid of separation or expression vector can be used 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 can be administered
  • the preferred effective amount 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 in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.
  • 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 T cells. Therefore, the kit containing the above 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 or 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 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 an immunostimulatory molecule.
  • the method comprises: introducing the aforementioned construct or virus into a second receptor cell; culturing the second receptor cell into which the construct or virus is introduced, so as to obtain the chimeric antigen receptor and the immunostimulatory molecule.
  • the construct or virus can simultaneously express the chimeric antigen receptor and the immunostimulatory molecule 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 immunostimulatory molecule.
  • 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 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-T cell of a chimeric antigen receptor and an immunostimulatory molecule.
  • it includes: introducing the aforementioned construct or virus into a NK cell or T cell; culturing the NK cell or T cell into which the construct or virus is introduced to obtain the CAR-NK or CAR-T cell.
  • a lentiviral expression vector targeting mesothelin and simultaneously expressing IL-15 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 T cells are obtained.
  • the CAR-NK or CAR-T cell can not only target and kill mesothelin-positive malignant tumors, but also, because it can continuously secrete IL-15 locally, it has a higher proliferation ability and killing activity than unmodified NK cells or T cells, especially it can maintain the long-term survival of NK cells or T cells in the body, so that NK cells or T cells can maintain a higher proliferation activity and killing activity, and exert a stronger ability to continuously kill tumors. More importantly, this locally secreted IL-15 exerts effective biological functions locally in the tumor, and can effectively avoid the toxic side effects caused by systemic high-dose or repeated injections of recombinant IL-15.
  • the introducing of NK cells or T cells is performed by electroporation, transfection or infection.
  • the present invention proposes the use of the aforementioned isolated nucleic acid, construct, recombinant cell, CAR-NK or 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 immunostimulatory molecules under appropriate conditions, so that the cell can target the corresponding antigen and locate to the cell surface expressing the antigen.
  • immunostimulatory molecules such as IL-15 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 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.
  • the tumor includes at least one of 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, PSMA-positive tumor, IL-13RA2-positive tumor, B7-H3-positive tumor, CD133-positive tumor, CD70-positive tumor, C-MET-positive tumor, FAP-positive tumor, TROP-2-positive tumor, ROR1-positive tumor, CD19-positive tumor, CD20-positive tumor, CD22-positive tumor, CD30-positive tumor, CD33-positive tumor and 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 or T cells.
  • the isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells or T cells. Therefore, the kit containing the above 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 proliferation activity NK cells or T cells with low proliferation activity can be increased from a low level to obtain biological samples that meet the expectations.
  • the present invention proposes a method for treating or preventing tumors.
  • the method comprises: administering to the subject at least one of the transgenic immune cells described in the first aspect, the isolated nucleic acid described in the second aspect, the construct described in the third aspect, the recombinant cell described in the fourth aspect, the CAR-NK or CAR-T cell described in the fifth aspect, the virus described in the seventh aspect or the eighth aspect, or the pharmaceutical composition described in the ninth aspect.
  • a suitable dose of transgenic immune cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-T cells, viruses or pharmaceutical compositions are administered to the subject to inhibit the proliferation of tumor cells.
  • the present invention proposes a transgenic immune cell described in the first aspect, an isolated nucleic acid described in the second aspect, a construct described in the third aspect, a recombinant cell described in the fourth aspect, a CAR-NK or CAR-T cell described in the fifth aspect, a virus described in the seventh aspect or the eighth aspect, or a pharmaceutical composition described in the ninth aspect for use in treating or preventing tumors.
  • different treatment methods can be selected for different types of tumors using the above-mentioned products, including one or more combinations of transgenic immune cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-T cells, viruses or pharmaceutical compositions, to improve the pertinence and effectiveness of tumor treatment.
  • Figure 1 is a structural schematic diagram of a CAR targeting MSLN and modified with IL-15 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, CD3 ⁇ represents a nucleotide sequence encoding a CD3 ⁇ intracellular region, P2A represents a nucleotide sequence encoding a P2A self-cleavage region, and IL15 represents a nucleotide sequence encoding a full-length IL15;
  • SP represents a nucleotide sequence encoding a signal peptide
  • 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-IL15-NK-92 cells according to Example 2 of the present invention;
  • FIG. 3 is a graph showing the results of detecting STAT5 phosphorylation levels of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-NK-92 cells of IL-15 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-IL15-NK-92 cells of IL-15 according to Example 2 of the present invention;
  • FIG. 5 is a graph showing the results of in vitro proliferation ability test of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-NK-92 cells according to Example 2 of the present invention
  • FIG. 6 is a flowchart of the operation of using IL-15-expressing CAR-NK cells to treat pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention
  • FIG. 7 is a graph showing the pancreatic NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-NK-92 cells according to Example 3 of the present invention.
  • Figure 1 shows the results of the in vivo viability test of adenocarcinoma Aspc-1 cell-bearing mice;
  • Figure 8 is a graph showing the results of detecting the anti-tumor ability of NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-NK-92 cells according to Example 3 of the present invention on mice bearing pancreatic cancer Aspc-1 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-IL15) sequence that targets mesothelin (MSLN) and expresses 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 and a transmembrane region (TM), a 4-1BB intracellular co-stimulatory signal domain and an intracellular signal transduction molecule CD3 ⁇ , and an IL-15 gene fragment connected by P2A.
  • SP signal peptide
  • TM transmembrane region
  • 4-1BB intracellular co-stimulatory signal domain
  • CD3 ⁇ intracellular signal transduction molecule
  • 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 costimulatory 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 IL-15 is shown in SEQ ID NO: 9.
  • the anti-MSLN-CAR fragment was inserted into the lentiviral vector pLent-EF1 ⁇ -P2A-CMV-GP to construct the pLent-anti-MSLN-CAR-P2A-CMV-GP vector.
  • the IL-15 gene fragment was amplified from the cDNA of human PBMC cells, and the IL-15 gene fragment was inserted into the pLent-anti-MSLN-CAR-P2A-CMV-GP vector through the restriction site Not I. The sequence was verified by PCR identification and sequencing, indicating that the pLent-anti-MSLN-CAR-P2A-IL15-CMV-GP vector was successfully constructed.
  • N number of cells at the time of infection (approximately 1 ⁇ 10 5 )
  • 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 /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-IL15-NK-92 (carrying CAR targeting MSLN and expressing IL-15) 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 After IL-15 binds to the IL-15 receptor, it activates 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, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-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. As shown in Figure 3, the STAT5 phosphorylation level of the NK-92 cell group and the ⁇ -MSLN-CAR-NK-92 cell group was low, while the STAT5 phosphorylation level of the ⁇ -MSLN-CAR-IL15-NK-92 cell group was significantly higher than that of the control group.
  • the inventors co-incubated the above NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL15-NK-92 cells with the pancreatic cancer cell line Aspc-1 for 5 hours and then tested the killing efficiency.
  • ⁇ -MSLN-CAR-NK-92 and The killing efficiency of ⁇ -MSLN-CAR-IL15-NK-92 against Aspc-1 cells was 48.01 ⁇ 2.00% and 48.60 ⁇ 1.78% respectively when the effector-target ratio was 5:1, which was significantly higher than that of NK-92 cells (35.59 ⁇ 2.46%); however, there was no significant difference in the killing efficiency between ⁇ -MSLN-CAR-IL15-NK-92 cells and ⁇ -MSLN-CAR-NK-92 groups.
  • the inventors further verified the pro-survival effect of autocrine IL-15 on NK-92 cells.
  • the same number of cells of the NK-92, ⁇ -MSLN-CAR-NK-92 and ⁇ -MSLN-CAR-IL-15-NK-92 cells were plated in 96-well plates, and the cells were counted every 3 days. After culturing for 24 days, the cell proliferation curve was drawn.
  • Figure 5 when cultured to the 12th day, the number of ⁇ -MSLN-CAR-IL-15-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 very significant differences. It shows that the secreted IL-15 can significantly promote the survival and proliferation ability of CAR-NK cells.
  • Example 3 Detection of the anti-tumor ability of CAR-NK cells expressing IL-15 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-IL15-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, from 27.3% to 8.32% in the NK-92 group, from 29.6% to 9.83% in the ⁇ -MSLN-CAR-NK-92 group, and from 28.7% to 11.762% in the ⁇ -MSLN-CAR-IL15-NK-92 group.
  • the proportions of NK-92 cells, ⁇ -MSLN-CAR-NK-92 cells, and ⁇ -MSLN-CAR-IL15-NK-92 cells in the body were 0.13%, 3.32%, and 9.27%, respectively.
  • the IL-15-modified ⁇ -MSLN-CAR-IL15-NK-92 cells had the highest proportion in the body and more prominent persistence in the body. Therefore, the expression of IL-15 can improve the survival ability of NK cells in vivo, and the locally secreted IL-15 has a significant effect on maintaining the survival of NK cells in vivo.
  • 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 transgenic immune cell and a use thereof. The immune cell expresses a chimeric antigen receptor and an immunostimulatory molecule, and the immunostimulatory molecule comprises IL-15. The transgenic immune cell can express and secrete the chimeric antigen receptor and the immunostimulatory molecule, so that the immune cell can target a corresponding antigen and is positioned to the surface of a cell expressing the antigen.

Description

转基因免疫细胞及其应用Genetically modified immune cells and their applications 技术领域Technical Field

本发明涉及生物技术领域，具体地，本发明涉及转基因免疫细胞及其应用。The present invention relates to the field of biotechnology, and in particular to transgenic immune cells and applications 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免疫刺激分子，所述免疫刺激分子包括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免疫刺激分子。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，所述免疫刺激分子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 an immunostimulatory molecule, and the immunostimulatory molecule includes IL-15. The transgenic immune cell according to an embodiment of the present invention can simultaneously express and secrete a chimeric antigen receptor and an immunostimulatory molecule. Among them, the chimeric antigen receptor can enable the immune cell to target the corresponding antigen and locate it on the surface of the cell expressing the antigen. In addition, the immunostimulatory molecule further promotes the immune cell The activation and proliferation of immune cells can maintain the number and activity of immune cells in the local microenvironment of the tumor, enabling them to maintain strong tumor-killing activity, and can 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、PSMA、IL-13RA2、B7-H3、CD133、CD70、C-MET、FAP、TROP-2、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, PSMA, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, 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:11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: 11. 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:12所示的氨基酸序列。 According to an embodiment of the present invention, the hinge fragment includes the amino acid sequence shown in SEQ ID NO:12.

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:13所示的氨基酸序列。According to an embodiment of the present invention, the transmembrane region has the amino acid sequence shown in SEQ ID NO:13.

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、CD40、CD40L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, CD40, CD40L 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:14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:14.

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:15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:15.

根据本发明的实施例，所述免疫细胞包括T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 a T cell and a NK cell. In the present application, the type of the immunostimulatory molecule is not particularly limited, and any promoting factor that can promote the immune function of at least one of a T cell and a NK cell 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免疫刺激分子，所述免疫刺激分子包括IL-15。IL-15是一种多效性细胞因子，具有激活T细胞、B细胞和NK细胞，并可介导这些细胞的增殖和存活的功能。此外，IL-15能激活、维持和扩增CD8+记忆性T细胞，而不激活调节性T淋巴细胞。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免疫刺激分子，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，免疫刺激分子，如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, which encodes a chimeric antigen receptor; 2) a second nucleic acid molecule, which encodes an immunostimulatory molecule, and the immunostimulatory molecule includes IL-15. IL-15 is a pleiotropic cytokine that has the function of activating T cells, B cells and NK cells, and mediating the proliferation and survival of these cells. In addition, IL-15 can activate, maintain and amplify CD8 + memory T cells without activating regulatory T lymphocytes. After the isolated nucleic acid according to an 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 immune cells, the immune cells can simultaneously express and secrete chimeric antigen receptors and immunostimulatory molecules, so that the immune cells can target the corresponding antigens and locate on the surface of cells expressing the antigens. In addition, immunostimulatory molecules such as IL-15 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 they maintain strong tumor killing activity, effectively avoid the toxic and side effects caused by high-dose systemic or repeated injections, and avoid the toxic and side effects caused by high-dose systemic 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免疫刺激分子，并且所述免疫刺激分子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 immunostimulatory molecule in an immune cell, and the immunostimulatory molecule and the chimeric antigen receptor are in a non-fusion 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:16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:16.

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2，所述连接肽2能够被切割。所述连接肽2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 2, and the connecting peptide 2 can be cut. The connecting peptide 2 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2包括2A肽或其片段。本领域技术人员可以理解，所述连接肽2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 2 includes a 2A peptide or a fragment thereof. Those skilled in the art will appreciate that the connecting peptide 2 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2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 2 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2包括P2A或其片段。According to an embodiment of the present invention, the connecting peptide 2 includes P2A or a fragment thereof.

根据本发明的实施例，所述连接肽2包括SEQ ID NO:17所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:17.

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；以及第二启动子，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 includes: a first promoter, which is operably linked to the first nucleic acid molecule; and 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:10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:10.

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，如嵌合抗原受体和免疫刺激分子，如IL-15和间皮素。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 receptors and immunostimulatory molecules, such as IL-15 and mesothelin.

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、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 pombe, 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, 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免疫刺激分子。发明人在实验中发现，本发明所提出的IL-15修饰策略可使NK细胞或T细胞在肿瘤局部分泌IL-15，明显提高CAR-NK细胞或T细胞的体内外增殖能力，增强NK细胞或T细胞的体内存活时间，提高NK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 the fifth aspect of the present invention, the present invention proposes a CAR-NK or CAR-T 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 chimeric antigen receptors and immunostimulatory molecules. The inventors found in experiments that the IL-15 modification strategy proposed in the present invention can enable NK cells or T cells to secrete IL-15 locally in the tumor, significantly improve the in vivo and in vitro proliferation ability of CAR-NK cells or T cells, enhance the in vivo survival time of NK cells or T cells, and improve the in vivo anti-tumor function of NK cells or T cells. And this kind of IL-15 that is continuously and slowly released locally can avoid the toxic and side effects caused by systemic application of recombinant IL-15 or repeated multiple administrations.

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 peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cells, At least one of 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.

在本发明的第九方面，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-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免疫刺激分子，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，免疫刺激分子，如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。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 ninth 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-T cells or viruses. As mentioned above, the separated nucleic acid, expression vector, or cells or viruses carrying the separated nucleic acid or expression vector can simultaneously express and secrete chimeric antigen receptors and immunostimulatory molecules, so that immune cells can target corresponding antigens and localize to the cell surface expressing the antigen. In addition, immunostimulatory molecules such as IL-15 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 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. 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 nucleic acid of separation of the present invention, expression vector or the cell carrying the nucleic acid of separation or expression vector can be incorporated into the medicine suitable for parenteral administration (for example 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 solution (for example, injection solution and infusion solution), dispersant or suspending agent, tablet, pill, powder, liposome and suppository. Typical medicine is injection solution or infusion solution form. The nucleic acid of separation, expression vector or the cell carrying the nucleic acid of separation or expression vector can be used 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 can be administered The preferred effective amount 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 in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

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 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 T cells. Therefore, the kit containing the above 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 or 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 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免疫刺激分子的方法。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免疫刺激分子。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免疫刺激分子，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免疫刺激分子。In the twelfth aspect of the present invention, the present invention proposes a method for obtaining a chimeric antigen receptor and an immunostimulatory molecule. According to an embodiment of the present invention, the method comprises: introducing the aforementioned construct or virus into a second receptor cell; culturing the second receptor cell into which the construct or virus is introduced, so as to obtain the chimeric antigen receptor and the immunostimulatory molecule. As mentioned above, the construct or virus can simultaneously express the chimeric antigen receptor and the immunostimulatory molecule 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 immunostimulatory molecule.

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 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免疫刺激分子的CAR-NK或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-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IL-15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T细胞，该CAR-NK或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IL-15，因而具有较未修饰NK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T细胞在体内的长期存活，使NK细胞或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-T cell of a chimeric antigen receptor and an immunostimulatory molecule. According to an embodiment of the present invention, it includes: introducing the aforementioned construct or virus into a NK cell or T cell; culturing the NK cell or T cell into which the construct or virus is introduced to obtain the CAR-NK or CAR-T cell. According to some specific embodiments of the present invention, a lentiviral expression vector targeting mesothelin and simultaneously expressing IL-15 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 T cells are obtained. The CAR-NK or CAR-T cell can not only target and kill mesothelin-positive malignant tumors, but also, because it can continuously secrete IL-15 locally, it has a higher proliferation ability and killing activity than unmodified NK cells or T cells, especially it can maintain the long-term survival of NK cells or T cells in the body, so that NK cells or T cells can maintain a higher proliferation activity and killing activity, and exert a stronger ability to continuously kill tumors. More importantly, this locally secreted IL-15 exerts effective biological functions locally in the tumor, and can effectively avoid the toxic 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 introducing of NK cells or T cells is performed 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-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免疫刺激分子，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，免疫刺激分子，如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 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-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 immunostimulatory molecules under appropriate conditions, so that the cell can target the corresponding antigen and locate to the cell surface expressing the antigen. In addition, immunostimulatory molecules such as IL-15 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 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.

根据本发明的实施例，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、PSMA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、CD70阳性肿瘤、C-MET阳性肿瘤、FAP阳性肿瘤、TROP-2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 includes at least one of 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, PSMA-positive tumor, IL-13RA2-positive tumor, B7-H3-positive tumor, CD133-positive tumor, CD70-positive tumor, C-MET-positive tumor, FAP-positive tumor, TROP-2-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 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 or T cells. Therefore, the kit containing the above 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 proliferation activity NK cells or T cells with low proliferation activity can be increased from a low level to obtain biological samples that meet the 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。根据本发明的实施例，所述方法包括：向受试者施用第一方面所述的转基因免疫细胞、第二方面所述的分离的核酸、第三方面所述的构建体、第四方面所述的重组细胞、第五方面所述的CAR-NK或CAR-T细胞、第七方面或第八方面所述的病毒或第九方面所述的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免疫细胞、分离的核酸、构建体、重组细胞、CAR-NK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 proposes a method for treating or preventing tumors. According to an embodiment of the present invention, the method comprises: administering to the subject at least one of the transgenic immune cells described in the first aspect, the isolated nucleic acid described in the second aspect, the construct described in the third aspect, the recombinant cell described in the fourth aspect, the CAR-NK or CAR-T cell described in the fifth aspect, the virus described in the seventh aspect or the eighth aspect, or the pharmaceutical composition described in the ninth aspect. According to some specific embodiments of the present invention, a suitable dose of transgenic immune cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-T cells, viruses or pharmaceutical compositions are administered to the subject to inhibit the proliferation of tumor cells.

在本发明的第十七方面，本发明提出了一种第一方面所述的转基因免疫细胞、第二方面所述的分离的核酸、第三方面所述的构建体、第四方面所述的重组细胞、第五方面所述的CAR-NK或CAR-T细胞、第七方面或第八方面所述的病毒或第九方面所述的药物组合物在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，包括转基因免疫细胞、分离的核酸、构建体、重组细胞、CAR-NK或CAR-T细胞、病毒或药物组合物中的一种或多种组合，以提高肿瘤治疗的针对性和有效性。In the seventeenth aspect of the present invention, the present invention proposes a transgenic immune cell described in the first aspect, an isolated nucleic acid described in the second aspect, a construct described in the third aspect, a recombinant cell described in the fourth aspect, a CAR-NK or CAR-T cell described in the fifth aspect, a virus described in the seventh aspect or the eighth aspect, or a pharmaceutical composition described in the ninth aspect for use in 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 more combinations of transgenic immune cells, isolated nucleic acids, constructs, recombinant cells, CAR-NK or CAR-T cells, viruses or pharmaceutical compositions, to improve the pertinence and effectiveness of tumor treatment.

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IL-15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，CD3ζ表示编码CD3ζ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，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 IL-15 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, CD3ζ represents a nucleotide sequence encoding a CD3ζ intracellular region, P2A represents a nucleotide sequence encoding a P2A self-cleavage region, and IL15 represents a nucleotide sequence encoding a full-length 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-IL15-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-IL15-NK-92 cells according to Example 2 of the present invention;

图3是根据本发明实施例2的IL-15的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-IL15-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 of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells of IL-15 according to Example 2 of the present invention;

图4是根据本发明实施例2的IL-15的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-IL15-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-IL15-NK-92 cells of IL-15 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-IL15-NK-92细胞体外增殖能力检测结果图；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-IL15-NK-92 cells according to Example 2 of the present invention;

图6是根据本发明实施例3的表达IL-15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 IL-15-expressing CAR-NK cells 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-IL15-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；和FIG. 7 is a graph showing the pancreatic NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells according to Example 3 of the present invention. Figure 1 shows the results of the in vivo viability test of adenocarcinoma Aspc-1 cell-bearing mice; and

图8是根据本发明实施例3的NK-92、α-MSLN-CAR-NK-92和α-MSLN-CAR-IL15-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。Figure 8 is a graph showing the results of detecting the anti-tumor ability of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells according to Example 3 of the present invention on mice bearing pancreatic cancer Aspc-1 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

1.1CAR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IL-15的CAR载体(anti-MSLN-CAR-IL15)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(Hinge)区与跨膜区(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-15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-IL15) sequence that targets mesothelin (MSLN) and expresses 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 and a transmembrane region (TM), a 4-1BB intracellular co-stimulatory signal domain and an intracellular signal transduction molecule CD3ζ, and an IL-15 gene fragment connected by P2A. The structure of each gene element in the CAR vector is shown in Figure 1. Wherein:

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 costimulatory 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IL-15的核苷酸序列如SEQ ID NO：9所示。The nucleotide sequence of the IL-15 is shown in SEQ ID NO: 9.

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-CAR-P2A-CMV-GP载体。从人PBMC细胞cDNA中扩增出IL-15基因片段，通过酶切位点Not I将IL-15基因片段插入到pLent-anti-MSLN-CAR-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-CAR-P2A-IL15-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-CAR-P2A-CMV-GP vector. The IL-15 gene fragment was amplified from the cDNA of human PBMC cells, and the IL-15 gene fragment was inserted into the pLent-anti-MSLN-CAR-P2A-CMV-GP vector through the restriction site Not I. The sequence was verified by PCR identification and sequencing, indicating that the pLent-anti-MSLN-CAR-P2A-IL15-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：Take 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 flow cytometry to detect the GFP expression rate of 293T cells, and convert the virus titer according to the following formula:

滴度(TU/mL)＝(C×N×D×1000)/VTiter (TU/mL) = (C×N×D×1000)/V

其中：C＝流式检测的GFP阳性率Where: C = GFP positive rate detected by flow cytometry

N＝感染时细胞的数目(约为1×105)N = number of cells at the time of infection (approximately 1×10 5 )

D＝病毒载体的稀释倍数D = dilution factor of viral vector

V＝加入的稀释病毒的体积数。 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 5min, add an appropriate amount of α-MEM medium to resuspend the cells, and adjust the cell density to 5×10 5 /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 culture in a 37°C, 5% CO 2 incubator. After 24h, observe the cell state, change the medium, transfer the infected cells to an EP tube, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells to a cell culture bottle, add 10mL of fresh α-MEM medium and IL-2 (final concentration of 200IU/mL) and continue to culture for 48h. Transfer the cells to a flow tube, add 3mL of 1×PBS solution, centrifuge at 100×g for 5min, discard the supernatant, flick the cell pellet, and wash it again with 1×PBS solution. The expression rate of GFP was detected by flow cytometry. The culture was continued to be expanded, and the state of the infected NK-92 cells was adjusted for amplification. The infected NK-92 cells were 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-IL15-NK-92(携带靶向MSLN的CAR并表达IL-15)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CAR-IL15-NK-92细胞上清检测出明显的IL-15，水平为74.10±5.86pg/mL。说明本发明设计修饰的α-MSLN-CAR-IL15-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-IL15-NK-92 (carrying CAR targeting MSLN and expressing IL-15) 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 CAR-IL15-NK-92 cells, with a level of 74.10±5.86pg/mL. It is shown that the α-MSLN-CAR-IL15-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与IL-15受体结合后，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-IL15-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-IL15-NK-92细胞组STAT5磷酸化水平明显高于对照组。After IL-15 binds to the IL-15 receptor, it activates 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-IL15-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. As shown in Figure 3, the STAT5 phosphorylation level of the NK-92 cell group and the α-MSLN-CAR-NK-92 cell group was low, while the STAT5 phosphorylation level of the α-MSLN-CAR-IL15-NK-92 cell group was significantly higher than that of 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-IL15-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-IL15-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48.60±1.78％，明显高于NK-92细胞(35.59±2.46％)；但α-MSLN-CAR-IL15-NK-92细胞与α-MSLN-CAR-NK-92组的杀伤效率未见明显差异。The inventors co-incubated the above NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells with the pancreatic cancer cell line Aspc-1 for 5 hours and then tested the killing efficiency. As can be seen from Figure 4, α-MSLN-CAR-NK-92 and The killing efficiency of α-MSLN-CAR-IL15-NK-92 against Aspc-1 cells was 48.01±2.00% and 48.60±1.78% respectively when the effector-target ratio was 5:1, which was significantly higher than that of NK-92 cells (35.59±2.46%); however, there was no significant difference in the killing efficiency between α-MSLN-CAR-IL15-NK-92 cells and α-MSLN-CAR-NK-92 groups.

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IL-15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-IL-15-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-IL-15-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，开始有十分显著的差异。说明分泌的IL-15可明显促进CAR-NK细胞的存活和增殖能力。The inventors further verified the pro-survival effect of autocrine IL-15 on NK-92 cells. The same number of cells of the NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL-15-NK-92 cells were plated in 96-well plates, and the cells were counted every 3 days. After culturing for 24 days, the cell proliferation curve was drawn. As can be seen from Figure 5, when cultured to the 12th day, the number of α-MSLN-CAR-IL-15-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 very significant differences. It shows that the secreted IL-15 can significantly promote the survival and proliferation ability of CAR-NK cells.

实施例3：表达IL-15的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 expressing IL-15 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-IL15-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IL-15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-IL15-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 IL-15 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-IL15-NK-92细胞占外周血淋巴细胞的比例分别为27.3％、29.6％和26.8％，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-IL15-NK-92细胞占外周血淋巴细胞比例分别为8.32％、9.83％、和11.5％。可以看出与第一天相比，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-IL15-NK-92从28.7％下降到11.762％。第七天时，NK-92细胞、α-MSLN-CAR-NK-92细胞和α-MSLN-CAR-IL15-NK-92细胞在体内比例分别为0.13％、3.32％、和9.27％。可以看出与NK-92组和α-MSLN-CAR-NK-92组相比，IL-15修饰的α-MSLN-CAR-IL15-NK-92细胞在体内的比例最高，体内持久性更突出。因此，表达IL-15可以提高NK细胞在体内的生存能力，局部分泌的IL-15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 proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells and α-MSLN-CAR-IL15-NK-92 cells in the peripheral blood lymphocytes of mice were 27.3%, 29.6% and 26.8%, respectively, and the proportions of NK-92 cells in each group were similar. On the third day, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells and α-MSLN-CAR-IL15-NK-92 cells in the peripheral blood lymphocytes of mice were 8.32%, 9.83%, and 11.5%, 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, from 27.3% to 8.32% in the NK-92 group, from 29.6% to 9.83% in the α-MSLN-CAR-NK-92 group, and from 28.7% to 11.762% in the α-MSLN-CAR-IL15-NK-92 group. On the seventh day, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells, and α-MSLN-CAR-IL15-NK-92 cells in the body were 0.13%, 3.32%, and 9.27%, respectively. It can be seen that compared with the NK-92 group and the α-MSLN-CAR-NK-92 group, the IL-15-modified α-MSLN-CAR-IL15-NK-92 cells had the highest proportion in the body and more prominent persistence in the body. Therefore, the expression of IL-15 can improve the survival ability of NK cells in vivo, and the locally secreted IL-15 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PBS组、NK-92细胞治疗组相比，α-MSLN-CAR-NK92细胞和α-MSLN-CAR-IL15-NK-92细胞治疗可明显抑制肿瘤生长，且α-MSLN-CAR-IL15-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。IL-15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 PBS group and the NK-92 cell treatment group, α-MSLN-CAR-NK92 cells and α-MSLN-CAR-IL15-NK-92 cells can significantly inhibit tumor growth, and α-MSLN-CAR-IL15-NK-92 cells show better anti-tumor effects than α-MSLN-CAR-NK-92 cells. The above results show that CAR-NK-92 cells targeting mesothelin can inhibit the growth of pancreatic cancer and have a good therapeutic effect. IL-15 gene modification can improve the persistence of CAR-NK-92 cells in vivo, improve the viability of CAR-NK-92 cells in vivo, 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 (89)

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免疫刺激分子，所述免疫刺激分子包括IL-15。A transgenic immune cell, characterized in that the immune cell expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the immunostimulatory molecule includes 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、PSMA、IL-13RA2、B7-H3、CD133、CD70、C-MET、FAP、TROP-2、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, PSMA, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, 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:11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:11. 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12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:12所示的氨基酸序列。 The genetically modified immune cell according to claim 12, characterized in that the hinge fragment comprises the amino acid sequence shown in SEQ ID NO:12. 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:13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:13. 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、CD40、CD40L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, CD40, CD40L 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:14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:14. 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:15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:15. 根据权利要求1所述的转基因免疫细胞，其特征在于，所述免疫细胞包括T细胞和NK细胞中的至少之一。The genetically modified immune cell according to claim 1, characterized in that the immune cell comprises at least one of a T cell and a NK cell. 根据权利要求26所述的转基因免疫细胞，其特征在于，所述免疫细胞为NK细胞。The genetically modified immune cell according to claim 26, characterized in that the immune cell is a NK cell. 根据权利要求27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 27 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26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 26, 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免疫刺激分子，所述免疫刺激分子包括IL-15。A second nucleic acid molecule, wherein the second nucleic acid molecule encodes an immunostimulatory molecule, wherein the immunostimulatory molecule includes IL-15. 根据权利要求30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2～26任一项中所定义的。 The isolated nucleic acid according to claim 30, characterized in that the chimeric antigen receptor is as defined in any one of claims 2 to 26. 根据权利要求30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免疫刺激分子，并且所述免疫刺激分子与所述嵌合抗原受体呈非融合形式。The isolated nucleic acid according to claim 30 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 immunostimulatory molecule in an immune cell, and the immunostimulatory molecule and the chimeric antigen receptor are in a non-fused form. 根据权利要求30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 30, 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:16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:16. 根据权利要求30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 30, 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2，所述连接肽2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 2, and the connecting peptide 2 can be cut in the immune cell. 根据权利要求34所述的分离的核酸，其特征在于，所述连接肽2包括2A肽或其片段。The isolated nucleic acid according to claim 34, characterized in that the connecting peptide 2 comprises a 2A peptide or a fragment thereof. 根据权利要求35所述的分离的核酸，其特征在于，所述连接肽2包括P2A、T2A、E2A和F2A中的至少之一或其片段。The isolated nucleic acid according to claim 35 is characterized in that the connecting peptide 2 comprises at least one of P2A, T2A, E2A and F2A or a fragment thereof. 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2包括P2A或其片段。The isolated nucleic acid according to claim 36, characterized in that the connecting peptide 2 comprises P2A 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2包括SEQ ID NO:17所示的氨基酸序列。The isolated nucleic acid according to claim 37 is characterized in that the connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:17. 根据权利要求30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 30, 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；以及a first promoter operably linked to the first nucleic acid molecule; and 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39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 39, 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30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 30, 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41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 41, wherein the fourth nucleic acid molecule is operably linked to the first 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 41 is 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43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 43, wherein the signal peptide comprises CSF2R or a fragment thereof. 根据权利要求44所述的分离的核酸，其特征在于，所述信号肽包括SEQ ID NO:10所示的氨基酸序列。The isolated nucleic acid according to claim 44 is characterized in that the signal peptide comprises the amino acid sequence shown in SEQ ID NO:10. 根据权利要求30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 claim 30 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30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 claim 30 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34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 claim 34 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41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 claim 41 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30～49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 30 to 49. 根据权利要求50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 50 is characterized in that the vector of the construct is a non-pathogenic viral vector. 根据权利要求51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 51 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30～49任一项所述的分离的核酸或权利要求50或51所述的构建体。A recombinant cell, characterized in that it carries the isolated nucleic acid according to any one of claims 30 to 49 or the construct according to claim 50 or 51. 根据权利要求53所述的重组细胞，其特征在于，所述重组细胞包括真核细胞。The recombinant cell of claim 53, wherein the recombinant cell comprises a eukaryotic cell. 根据权利要求54所述的重组细胞，其特征在于，所述重组细胞为哺乳动物细胞。The recombinant cell according to claim 54, characterized in that the recombinant cell is a mammalian cell. 一种CAR-NK或CAR-T细胞，其特征在于，携带权利要求30～49任一项所述的分离的核酸或权利要求50或51所述的构建体。A CAR-NK or CAR-T cell, characterized in that it carries the isolated nucleic acid according to any one of claims 30 to 49 or the construct according to claim 50 or 51. 根据权利要求56所述的CAR-NK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 or CAR-T cell according to claim 56, 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56所述的CAR-NK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 or CAR-T cell according to claim 56, 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50或51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 claim 50 or 51 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59所述的方法，其特征在于，所述病毒包括慢病毒。The method of claim 59, wherein the virus comprises a lentivirus. 根据权利要求59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 59 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59～61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 59 to 61. 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1～29任一项所述的转基因免疫细胞、权利要求30～49任一项所述的分离的核酸、权利要求50或51所述的构建体、权利要求53～55任一项所述的重组细胞、权利要求56～58任一项所述的CAR-NK或CAR-T细胞或者权利要求62～63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 29, the isolated nucleic acid according to any one of claims 30 to 49, the construct according to claim 50 or 51, the recombinant cell according to any one of claims 53 to 55, the CAR-NK or CAR-T cell according to any one of claims 56 to 58, or the virus according to any one of claims 62 to 63. 一种试剂盒，其特征在于，包括：权利要求30～49任一项所述的分离的核酸、权利要求50或51 所述的构建体或权利要求62～63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 30 to 49, the isolated nucleic acid according to claim 50 or 51 The construct or the virus according to any one of claims 62 to 63. 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50或51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62～63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 claim 50 or 51, or the activated immune cell is infected with the virus described in any one of claims 62 to 63. 根据权利要求66所述的方法，其特征在于，所述免疫细胞包括T细胞和NK细胞中的至少之一。The method according to claim 66, characterized in that the immune cells include at least one of T cells and NK cells. 根据权利要求67所述的方法，其特征在于，所述免疫细胞为NK细胞。The method according to claim 67, characterized in that the immune cells are 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67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 67, 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免疫刺激分子的方法，其特征在于，包括：A method for obtaining a chimeric antigen receptor and an immunostimulatory molecule, comprising: 将权利要求50或51所述的构建体或权利要求62～63任一项所述的病毒导入第二受体细胞；introducing the construct of claim 50 or 51 or the virus of any one of claims 62 to 63 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免疫刺激分子。The second recipient cell into which the expression vector or virus has been introduced is cultured to obtain the chimeric antigen receptor and immunostimulatory molecule. 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71所述的方法，其特征在于，所述第二受体细胞为T细胞和NK细胞中的至少之一。The method according to claim 71 is characterized in that the second receptor cell is at least one of a T 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74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 74 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免疫刺激分子的CAR-NK或CAR-T细胞的方法，其特征在于，包括：A method for obtaining CAR-NK or CAR-T cells expressing a chimeric antigen receptor and an immunostimulatory molecule, comprising: 将权利要求50或51所述的构建体或权利要求62～63任一项所述的病毒导入NK细胞或T细胞；Introducing the construct of claim 50 or 51 or the virus of any one of claims 62 to 63 into NK cells or T cells; 将导入所述构建体或病毒的NK细胞或T细胞进行培养，以便获得所述CAR-NK或CAR-T细胞。The NK cells or T cells introduced with the construct or virus are cultured to obtain the CAR-NK 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 or T cells is carried out by electroporation, transfection or infection. 根据权利要求78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 78 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78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 78, characterized in that the T cells include CD4 + T cells, CD8 + T cells and γδ T cells. cell. 权利要求1～29任一项所述的转基因免疫细胞、权利要求30～49任一项所述的分离的核酸、权利要求50或51所述的构建体、权利要求53～55任一项所述的重组细胞、权利要求56～58任一项所述的CAR-NK或CAR-T细胞或权利要求62～63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 29, the isolated nucleic acid of any one of claims 30 to 49, the construct of claim 50 or 51, the recombinant cell of any one of claims 53 to 55, the CAR-NK or CAR-T cell of any one of claims 56 to 58, or the virus of any one of claims 62 to 63 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、PSMA阳性肿瘤、IL-13RA2阳性肿瘤、B7-H3阳性肿瘤、CD133阳性肿瘤、CD70阳性肿瘤、C-MET阳性肿瘤、FAP阳性肿瘤、TROP-2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。The use according to claim 82 is characterized in that the tumor includes at least one of 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, PSMA-positive tumor, IL-13RA2-positive tumor, B7-H3-positive tumor, CD133-positive tumor, CD70-positive tumor, C-MET-positive tumor, FAP-positive tumor, TROP-2-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30～49任一项所述的分离的核酸、权利要求50或51所述的构建体或权利要求62～63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细胞或T细胞活化或者增殖。Use of the isolated nucleic acid according to any one of claims 30 to 49, the construct according to claim 50 or 51, or the virus according to any one of claims 62 to 63 in the preparation of a kit for promoting NK cell or T cell activation or proliferation. 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1～29任一项所述的转基因免疫细胞、权利要求30～49任一项所述的分离的核酸、权利要求50或51所述的构建体、权利要求53～55任一项所述的重组细胞、权利要求56～58任一项所述的CAR-NK或CAR-T细胞、权利要求62～63任一项所述的病毒或权利要求64所述的药物组合物中的至少之一。A method for treating or preventing a tumor, characterized in that it comprises administering to a subject at least one of the transgenic immune cells of any one of claims 1 to 29, the isolated nucleic acid of any one of claims 30 to 49, the construct of claim 50 or 51, the recombinant cells of any one of claims 53 to 55, the CAR-NK or CAR-T cells of any one of claims 56 to 58, the virus of any one of claims 62 to 63, or the pharmaceutical composition of claim 64. 权利要求1～29任一项所述的转基因免疫细胞、权利要求30～49任一项所述的分离的核酸、权利要求50或51所述的构建体、权利要求53～55任一项所述的重组细胞、权利要求56～58任一项所述的CAR-NK或CAR-T细胞、权利要求62～63任一项所述的病毒或权利要求64所述的药物组合物在治疗或预防肿瘤中的用途。 Use of the transgenic immune cell of any one of claims 1 to 29, the isolated nucleic acid of any one of claims 30 to 49, the construct of claim 50 or 51, the recombinant cell of any one of claims 53 to 55, the CAR-NK or CAR-T cell of any one of claims 56 to 58, the virus of any one of claims 62 to 63, or the pharmaceutical composition of claim 64 in the treatment or prevention of tumors.
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