Genetically modified immune cells and their applications

Abstract

The present invention proposes a transgenic immune cell and its application, wherein the immune cell expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the immunostimulatory molecule includes IL-15. The transgenic immune cell can simultaneously express and secrete the chimeric antigen receptor and the immunostimulatory molecule, so that the immune cell can target the corresponding antigen and localize to the cell surface expressing the antigen. In addition, the immunostimulatory molecule further promotes the activation and proliferation of the immune cell, maintains the number and activity of the immune cell in the local microenvironment of the tumor, and maintains its strong tumor killing activity, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to transgenic immune cells and applications thereof. Background Art

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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 the side effects associated with IL15 as much as possible.

[0006] 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. Summary of the invention

[0007] 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.

[0008] 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 enables the immune cell to target the corresponding antigen and locate to the cell surface expressing the antigen. In addition, the immunostimulatory molecule further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local microenvironment of the tumor, and maintains a strong tumor killing activity, which can effectively avoid the toxic side effects caused by high-dose or repeated injections of the whole body.

[0009] 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:

[0010] 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.

[0011] 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.

[0012] According to an embodiment of the present invention, the extracellular region includes the heavy chain variable region and the light chain variable region of the antibody, and the antibody binds to the antigen. It can be understood by those skilled in the art that the extracellular region only needs to include the binding region that recognizes the antigen, and the extracellular region can include a full antibody, a Fab antibody, a Fab' antibody, a F(ab') 2 At least one of an antibody, a Fv antibody, a single-chain antibody and a nanobody. According to some preferred embodiments of the present invention, the extracellular region comprises a single-chain antibody.

[0013] 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, CD133, CD19, CD20, CD22, CD30, CD33, and BCMA.

[0014] 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.

[0015] According to an embodiment of the present invention, the extracellular region comprises an anti-mesothelin single-chain antibody.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] According to an embodiment of the present invention, the hinge region fragment includes at least one hinge region selected from CD8, CD28 and immunoglobulin.

[0021] According to an embodiment of the present invention, the hinge fragment includes the hinge region of CD8.

[0022] According to an embodiment of the present invention, the hinge fragment includes the amino acid sequence shown in SEQ ID NO:12.

[0023] 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.

[0024] According to an embodiment of the present invention, the transmembrane region includes the CD8 transmembrane region or a fragment thereof.

[0025] According to an embodiment of the present invention, the transmembrane region has the amino acid sequence shown in SEQ ID NO:13.

[0026] According to an embodiment of the present invention, the immune co-stimulatory molecule includes at least one selected from CD28, ICOS, 4-1BB, OX40 and CD27.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] According to an embodiment of the present invention, the signal transduction domain includes CD3ζ or a fragment thereof.

[0033] According to an embodiment of the present invention, the signal transduction domain includes the amino acid sequence shown in SEQ ID NO:15.

[0034] 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.

[0035] According to an embodiment of the present invention, the immune cells are preferably NK cells.

[0036] 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.

[0037] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

[0038] In the second aspect of the present invention, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid includes: 1) a first nucleic acid molecule, the first nucleic acid molecule encodes a chimeric antigen receptor; 2) a second nucleic acid molecule, the second nucleic acid molecule encodes 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 the embodiment of the present invention is introduced into the recipient cell, it can package a higher titer of virus and achieve specific infection of the virus to immune cells, such as NK cells. After the isolated nucleic acid is introduced into the 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 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 they maintain strong tumor killing activity, effectively avoid the toxic and side effects caused by high-dose or repeated injections of the whole body, and avoid the toxic and side effects caused by high-dose or repeated injections of recombinant IL-15.

[0039] According to an embodiment of the present invention, the isolated nucleic acid may further include at least one of the following additional technical features:

[0040] According to an embodiment of the present invention, the chimeric antigen receptor is as defined in the first aspect.

[0041] 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.

[0042] According to an embodiment of the present invention, the isolated 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 IDNO:16.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] According to an embodiment of the present invention, the connecting peptide 2 includes P2A or a fragment thereof.

[0047] According to an embodiment of the present invention, the connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:17.

[0048] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a first promoter, which is operably linked to the first nucleic acid molecule; and a second promoter, which is operably linked to the second nucleic acid molecule.

[0049] 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.

[0050] 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.

[0051] According to an embodiment of the present invention, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.

[0052] 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.

[0053] According to an embodiment of the present invention, the signal peptide includes CSF2R or a fragment thereof.

[0054] According to an embodiment of the present invention, the signal peptide includes the amino acid sequence shown in SEQ ID NO:10.

[0055] 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.

[0056] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:9.

[0057] According to an embodiment of the present invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:8.

[0058] According to an embodiment of the present invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:2.

[0059] According to an embodiment of the present invention, the isolated nucleic acid has the nucleotide sequence shown in SEQ ID NO:1.

[0060] 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.

[0061] According to an embodiment of the present invention, the above construct may further include at least one of the following additional technical features:

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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:

[0066] According to an embodiment of the present invention, the recombinant cell includes a eukaryotic cell, preferably a mammalian cell.

[0067] 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. Exemplarily, 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.

[0068] 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.

[0069] According to some embodiments of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

[0070] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 +T cells and γδ T cells.

[0071] 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.

[0072] According to an embodiment of the present invention, the virus comprises a lentivirus.

[0073] According to an embodiment of the present invention, the first recipient cell is 293T.

[0074] 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.

[0075] 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.

[0076] According to an embodiment of the present invention, the virus includes at least one of a retrovirus, a lentivirus and an adenovirus.

[0077] According to an embodiment of the present invention, the virus comprises a lentivirus.

[0078] In the eighth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned separated transgenic immune cells, nucleic acids, constructs, recombinant cells, CAR-NK or CAR-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.

[0079] 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:

[0080] 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.

[0081] 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.

[0082] The effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0083] 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.

[0084] 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.

[0085] According to an embodiment of the present invention, the immune cells include at least one of T cells and NK cells.

[0086] According to an embodiment of the present invention, the immune cells are preferably NK cells.

[0087] 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.

[0088] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

[0089] 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 construct or virus described above into a second receptor cell; culturing the second receptor cell introduced with the construct or virus to obtain the chimeric antigen receptor and the immunostimulatory molecule. As described 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.

[0090] 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.

[0091] According to an embodiment of the present invention, the second receptor cell is at least one of a T cell and a NK cell.

[0092] According to an embodiment of the present invention, the second receptor cell is a NK cell.

[0093] 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.

[0094] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

[0095] According to an embodiment of the present invention, the virus includes at least one selected from retrovirus, lentivirus and adenovirus.

[0096] According to an embodiment of the present invention, the virus comprises a lentivirus.

[0097] 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.

[0098] According to an embodiment of the present invention, the introducing of NK cells or T cells is performed by electroporation, transfection or infection.

[0099] 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.

[0100] According to an embodiment of the present invention, the tumor includes at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, a CD133-positive tumor, a CD19-positive tumor, a CD20-positive tumor, a CD22-positive tumor, a CD30-positive tumor, a CD33-positive tumor and a BCMA-positive tumor.

[0101] 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.

[0102] 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, and therefore, the kit containing the above-mentioned substances also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells or T cells with low proliferation activity, so that their proliferation activity is increased from low to obtain a biological sample that meets expectations. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] 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, CD8 hinge+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;

[0104] Figure 2 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;

[0105] Figure 3 This is a graph showing the results of detecting the STAT5 phosphorylation level 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;

[0106] Figure 4 This 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;

[0107] Figure 5 This is a graph showing the results of in vitro proliferation ability testing of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells according to Example 2 of the present invention;

[0108] Figure 6 This 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;

[0109] Figure 7 is a graph showing the viability test results of NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 cells in mice bearing pancreatic cancer Aspc-1 cells according to Example 3 of the present invention; and

[0110] Figure 8 This 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. DETAILED DESCRIPTION

[0111] 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, but should not be construed as limiting the present invention.

[0112] 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 of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] The amino acid or nucleic acid sequences referred to herein are shown below.

[0119]

[0120]

[0121]

[0122]

[0123] Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro AlaPhe Leu Leu Ile Pro (SEQ ID NO: 10).

[0124] Asp Ile Gln Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val LysArg Pro Gly Ala Ser Val Gln ValSer Cys Arg Ala Ser Gly Tyr Ser Ile Asn ThrTyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu GluTrp Met Gly ValIle Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr LeuThr Asn AspThr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser AlaAsp Thr Ala Val Tyr Tyr Cys Ala Arg TrpAla Leu Trp Gly Asp Phe Gly MetAsp ValTrp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly SerGly Gly GlyGly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu SerAla Ser Ile GlyAsp Arg Val Thr Ile Thr Cys ArgAla Ser Glu Gly Ile Tyr His TrpLeuAla Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr Lys Ala SerSer LeuAla Ser GlyAla Pro SerArg Phe Ser Gly Ser Gly Ser Gly ThrAspPhe ThrLeu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln TyrSer Asn Tyr Pro LeuThr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysArg(SEQ ID NO:11)。

[0125] Thr Thr Thr Pro Ala Pro Arg Pro Thr Pro Ala Pro Ala Thr Ala SerGln Pro Leu Ser Leu Arg Pro GluAla Cys Arg Pro AlaAla GlyAla Val HisThrArg Gly LeuAsp PheAla Cys Asp(SEQ ID NO:12)。

[0126] Tyr Isolation Trp Isolation Pro Leu Isolation Gly Thr Cys Gly Val Leu Leu SerLeu Isolation Thr Isolation Tyr Cys(SEQ ID NO:13)。

[0127] Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetArgPro Val Gln Thr Thr Gln Glu GluAsp Gly Cys Ser Cys Arg Phe Pro Glu Glu GluGlu Gly Cys Glu Leu(SEQ ID NO:14)。

[0128] Arg Val Lys Phe SerArg SerAlaAsp Ala Pro Ala Tyr Gln Gln Gly GlnAsnGln Leu TyrAsn Glu Leu AsnLeu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys ArgArg Gly Asp Pro Glu Met Argl Pro Gly Gly LysG Leu TyrAsn Glu Leu Gln Lys Asp Lys MetAla Glu Ala Tyr Ser Glu Ile GlyMetLys Gly Glu ArgArg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrAlaThr Lys Asp Le TyrAsp Met Ala Glu LeQA Pron His NO:15)。

[0129]

[0130] Ala ThrAsn Phe Ser Leu Leu Lys GlnAla GlyAsp Val Glu GluAsn Pro GlyPro (SEQ ID NO: 17).

[0131] 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.

[0132] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0133] Example 1: Preparation of CAR-NK cells

[0134] 1.1 Construction of CAR expression plasmid

[0135] 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 as follows Figure 1 As shown. Among them:

[0136] The full-length gene sequence of the CAR vector composed of the above elements is shown in SEQ ID NO: 1;

[0137] The signal peptide is CSF2R, and its nucleotide sequence is shown in SEQ ID NO: 2;

[0138] The nucleotide sequence of the anti-MSLN scFv is shown in SEQ ID NO: 3;

[0139] The nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO: 4;

[0140] The nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO: 5;

[0141] The nucleotide sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO: 6;

[0142] The nucleotide sequence of the CD3ζ intracellular region is shown in SEQ ID NO: 7;

[0143] The nucleotide sequence of the self-cleavage region P2A is shown in SEQ ID NO: 8;

[0144] The nucleotide sequence of IL-15 is shown in SEQ ID NO:9.

[0145] 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.

[0146] 1.2 Lentivirus packaging and virus liquid concentration

[0147] Take 5×10 293T cells in the logarithmic growth phase 6 The cells were inoculated into a 10 cm culture dish and 10 mL of DMEM medium was added. The culture dishes were incubated at 37 °C and 5% CO. 2 Culture overnight in the incubator. When the cell density reaches 80%, replace with 10 mL of fresh DMEM medium for virus packaging, and continue to place the cell culture dish in the incubator for use. Prepare the lentiviral packaging system, add 6 μg of lentiviral packaging auxiliary plasmid psPAX2 and 3 μg of pMD2.G, and 6 μg of the target gene vector plasmid to 250 μL of serum-free DMEM medium to prepare a plasmid mixture, and mix well. Add 15 μL of PEIpro to 235 μL of serum-free DMEM medium and mix well. Add the mixture to the above plasmid mixture at once, mix well, incubate at room temperature for 15 minutes, and add the mixture to the 293T cell culture dish after the incubation. Replace the medium after 24 hours and place the culture dish back to 37°C, 5% CO 2Incubate incubator, collect cell supernatant after 48h, centrifuge at 400×g for 5min, remove cell debris, filter supernatant with 0.45μm filter into 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate virus solution, mix evenly by inverting centrifuge tube, and place in 4℃ refrigerator overnight. Centrifuge at 4000×g for 20min at 4℃, discard supernatant, add appropriate amount of serum-free DMEM to resuspend virus precipitate, transfer into EP tube, and store in -80℃ refrigerator.

[0148] 1.3 Lentiviral titer detection

[0149] Take 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 5 / mL. Take a 24-well plate and add 1mL of cell suspension (1×10 5 / well), set up 3 virus volume gradients. Place at 37°C, 5% CO 2 Incubate overnight in an incubator. 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 medium, and mix well. Replace the 293T cells with fresh DMEM medium, pipette 5μL, 50μL, and 500μL of diluted virus solution into the corresponding wells, mark them, and then return the culture plate to 37°C, 5% CO 2 Incubator. After 24 hours, discard the virus solution in the well plate and add 1 mL of fresh DMEM medium. After 72 hours, 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:

[0150] Titer (TU / mL) = (C×N×D×1000) / V

[0151] Where: C = GFP positive rate detected by flow cytometry

[0152] N = the number of cells at the time of infection (approximately 1×10 5 )

[0153] D = dilution factor of viral vector

[0154] V = volume of diluted virus added.

[0155] 1.4 Lentivirus infection of human NK cells

[0156] NK-92 cells (purchased from ATCC) in the logarithmic growth phase were harvested by centrifugation at 100 × g for 5 min, and an appropriate amount of α-MEM medium was added to resuspend the cells to adjust the cell density to 5 × 10 5 5×10 5NK-92 cells, 1 mL of virus concentrate and protamine (purchased from Solebol, final concentration 8 μg / mL) were mixed evenly and placed at 37°C and 5% CO 2 Culture in an incubator. After 24 hours, observe the cell status, change the medium, transfer the infected cells to an EP tube, centrifuge at 100×g for 5 minutes, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells to a cell culture flask, add 10mL of fresh α-MEM medium and IL-2 (final concentration of 200IU / mL) and continue to culture for 48 hours. Transfer the cells to a flow tube, add 3mL of 1×PBS solution, centrifuge at 100×g for 5 minutes, discard the supernatant, flick the cell pellet, and wash it again with 1×PBS solution. Use a flow cytometer to detect the expression rate of GFP. Continue to expand the culture and adjust the state of the infected NK-92 cells for amplification. The infected NK-92 cells were sorted by flow cytometry for GFP-positive CAR-NK-92 cells for later experiments.

[0157] Example 2: Determination of IL-15 secretion level and cell proliferation ability of CAR-NK cells

[0158] 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

[0159] 2.1ELISA detection of IL-15 secretion level of CAR-NK-92 cells

[0160] 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 cultured and the supernatant was collected after 24 hours. ELISA was used to detect the content of IL-15 in the supernatant of different groups. The experimental results are shown in Figure 2. Figure 2 As shown, IL-15 was hardly detected in the supernatant 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. This indicates that the α-MSLN-CAR-IL15-NK cells designed and modified by the present invention have the ability to secrete IL-15.

[0161] 2.2 Detection of STAT5 phosphorylation level in CAR-NK cells

[0162] 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. The results are as follows. Figure 3 As shown, the STAT5 phosphorylation level in the NK-92 cell group and the α-MSLN-CAR-NK-92 cell group was low, while the STAT5 phosphorylation level in the α-MSLN-CAR-IL15-NK-92 cell group was significantly higher than that in the control group.

[0163] 2.3 Detection of CAR-NK cell killing ability in vitro

[0164] 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 detected the killing efficiency. Figure 4 It can be seen that the killing efficiency of α-MSLN-CAR-NK-92 and α-MSLN-CAR-IL15-NK-92 on Aspc-1 cells at an effector-target ratio of 5:1 was 48.01±2.00% and 48.60±1.78%, respectively, 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.

[0165] 2.4 Detection of CAR-NK cell proliferation ability in vitro

[0166] The inventors further verified the pro-survival effect of autocrine IL-15 on NK-92 cells. The same number of 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. Figure 5It can be seen that on the 12th day of culture, 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. On the 21st day, there was a significant difference, indicating that the secreted IL-15 can significantly promote the survival and proliferation of CAR-NK cells.

[0167] 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

[0168] 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:

[0169] Six-week-old BALB / c-nu nude mice were selected for subcutaneous tumor implantation in the axilla, with a tumor-bearing dose of 2×10 6 cells / mouse. Tumors 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 effector cells into the tail vein. 7 The untreated group was injected with an equal volume of 1× PBS once every other week for a total of 5 treatments, and IL-2 (5×10 4 IU / piece), for specific experimental settings and operation procedures, 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.

[0170] The results are as follows Figure 7As shown, on the first day of NK cell treatment, the proportion 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 proportion of NK-92 cells in each group was similar. On the third day, the proportion of NK-92 cells, α-MSLN-CAR-NK-92 cells and α-MSLN-CAR-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.

[0171] Tumor growth curves were drawn by measuring tumor size. Figure 8 It can be seen that compared with the control PBS group and the NK-92 cell treatment group, α-MSLN-CAR-NK92 cell and α-MSLN-CAR-IL15-NK-92 cell treatment 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 survival ability of CAR-NK-92 cells in vivo, and improve the anti-tumor effect of CAR-NK-92 cells.

[0172] 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.

[0173] 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.

[0174] 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

1. A virus, It is characterized in that It includes a nucleotide sequence shown in SEQ ID NO:

1.

2. A kit, It is characterized in that include: The virus of claim 1.

3. A method for introducing a virus into activated immune cells, It is characterized in that Infecting the activated immune cells with the virus of claim 1; Wherein, the immune cells include at least one of T cells and NK cells.

4. The method according to claim 3, It is characterized in that The immune cells are NK cells.

5. The method according to claim 4, It is characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent stem cell (iPSC)-derived NK cells and NK-92 cells.

6. The method according to claim 3, It is characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

7. A method for obtaining chimeric antigen receptors and fusion proteins, It is characterized in that include: Introducing the virus of claim 1 into a second recipient cell; The second recipient cells into which the virus has been introduced are cultured to obtain the chimeric antigen receptor and the fusion protein.

8. The method according to claim 7, It is characterized in that The introduction into the second recipient cell is carried out by electroporation, transfection or infection.

9. The method according to claim 7 or 8, It is characterized in that The second receptor cell is at least one of a T cell and a NK cell.

10. The method according to claim 9, It is characterized in that The second receptor cell is a NK cell.

11. The method according to claim 10, It is characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent stem cell (iPSC)-derived NK cells and NK-92 cells.

12. The method according to claim 9, It is characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

13. The method according to claim 7, It is characterized in that The virus includes a lentivirus.

14. A method for obtaining CAR-NK or CAR-T cells expressing a chimeric antigen receptor and a fusion protein, It is characterized in that include: Introducing the virus of claim 1 into NK cells or T cells; The NK cells or T cells introduced with the virus are cultured to obtain the CAR-NK or CAR-T cells.

15. The method according to claim 14, It is characterized in that The introduction of NK cells or T cells is carried out by electroporation, transfection or infection.

16. The method according to claim 15, It is characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent stem cell (iPSC)-derived NK cells and NK-92 cells.

17. The method according to claim 15, It is characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

18. Use of the virus according to claim 1 in preparing a pharmaceutical composition for treating a tumor, wherein the tumor is pancreatic cancer.

19. Use of the virus according to claim 1 in preparing a kit for promoting the activation or proliferation of NK cells or T cells.

20. The use according to claim 19, It is characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent stem cell (iPSC)-derived NK cells and NK-92 cells.

21. The use according to claim 19, It is characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

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