Methods and applications for enhancing NK cell survival and anti-tumor activity

Abstract

The present invention provides a method for improving the survival and anti-tumor activity of NK cells and its application. Specifically, an immune cell is provided, and the immune cell expresses a chimeric antigen receptor and a fusion protein, and the fusion protein comprises IL-15Rα and IL-15. The transgenic immune cell can simultaneously express and secrete the chimeric antigen receptor and the fusion protein, enabling the immune cell to target a corresponding antigen and localize to the cell surface expressing the antigen. In addition, the fusion protein further promotes the activation and proliferation of the immune cell, maintains the number and activity of the immune cell in the tumor local microenvironment, enables it to maintain a strong tumor killing activity, and effectively avoids the toxic and side effects caused by systemic high-dose or repeated multiple injections.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to methods for improving the survival and anti-tumor activity of NK cells and their applications. 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 adverse reactions such as cytokine storm, neurotoxicity, and GVHD in clinical applications. Moreover, the therapeutic effect of CAR-T cells on solid tumors is still not ideal, making the clinical application of CAR-T cells still face challenges.

[0003] CAR-NK cells have the advantage of good safety compared with CAR-T cells and generally do not cause side effects such as cytokine storm and GVHD. Moreover, NK cells can directly kill tumor cells without antigen presentation and MHC restriction. CAR-NK cells can recognize and kill tumors through multiple recognition mechanisms such as CAR dependence and NKR dependence, and have a broad anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor therapy and have become a hot spot in the field of cell immunotherapy research and development. However, one of the problems faced in the research and development of CAR-NK cells is that the survival time of NK cells in vivo is relatively short, which affects the exertion of 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 a dimer, they bind to IL-15Rβγc to 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 popular target for drug research and development to enhance the persistence and proliferative activity of lymphocytes in vivo.

[0005] However, the problems of IL-15 in in vivo application are short half-life, limited in vivo drug efficacy, the need to use a large dose, and frequent administration, resulting in various side effects, including hypotension, thrombocytopenia, and elevated AST and ALT, which may cause cancer patients to be unable to tolerate this treatment. In clinical application and drug research and development, it is necessary to maintain the activity of IL-15 to promote lymphocyte proliferation and persistence and promote immune response as much as possible, while reducing the side effects related to IL-15 as much as possible.

[0006] Based on the above research and development status, there is a need to further study safe and effective methods for improving the in vivo persistence of CAR-NK cells. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the present invention provides a genetically modified immune cell, whose proliferation ability, in vivo survival time, and anti-tumor ability are significantly improved compared with natural immune cells, and which has higher safety.

[0008] Therefore, in the first aspect of the present invention, a genetically modified immune cell is provided. According to an embodiment of the present invention, the immune cell expresses a chimeric antigen receptor and a fusion protein, and the fusion protein comprises IL-15Rα and IL-15, namely the so-called super IL-15 hereinafter. The genetically modified immune cell according to the embodiment of the present invention can simultaneously express and secrete the chimeric antigen receptor and the fusion protein. Among them, the chimeric antigen receptor enables the immune cell to target a corresponding antigen and localize to the cell surface expressing the antigen. In addition, the fusion protein further promotes the activation and proliferation of the immune cell, maintains the number and activity of the immune cell in the tumor local microenvironment, enables it to maintain a strong tumor killing activity, and can effectively avoid the toxic and side effects caused by systemic high-dose or repeated multiple injections.

[0009] According to an embodiment of the present invention, the above-mentioned genetically modified immune cell 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 comprises: an extracellular region that can specifically bind to an antigen; a transmembrane region; and an intracellular region that comprises 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 applicable to specifically recognize a variety of antigens.

[0011] According to an embodiment of the present invention, the antigen is a tumor-associated antigen. According to some specific embodiments of the invention, the type of the antigen is not particularly limited.

[0012] According to an embodiment of the present invention, the extracellular region comprises a heavy chain variable region and a light chain variable region of an antibody, and the antibody binds to the antigen. Those skilled in the art can understand that the extracellular region only needs to comprise a binding region that recognizes the antigen, and the extracellular region may comprise at least one of a whole antibody, a Fab antibody, a Fab’ antibody, an F(ab’)2 antibody, an 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 proliferative activity, and have high anti-tumor ability.

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

[0016] According to an embodiment of the present invention, the anti-mesothelin single-chain antibody includes the light-chain variable region of an anti-mesothelin antibody, a linker peptide 1, and the heavy-chain variable region of an anti-mesothelin antibody.

[0017] According to an embodiment of the present invention, the linker peptide 1 has an amino acid sequence represented by (GGGGS)n, where 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 includes the amino acid sequence shown in SEQ ID NO: 14. In some specific embodiments, when the anti-mesothelin single-chain antibody has the above amino acid sequence, the transgenic immune cells can effectively target mesothelin-positive tumors, retain high proliferative 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 selected from the hinge regions of CD8, CD28, and immunoglobulins.

[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: 15.

[0023] According to an embodiment of the present invention, the transmembrane region includes at least one or a fragment selected from CD4, CD8α, CD28, and CD3ζ.

[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: 16.

[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: 17.

[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 can understand 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 all 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: 18.

[0034] According to an embodiment of the present invention, the fusion protein further includes linker peptide 2.

[0035] According to an embodiment of the present invention, the linker peptide 2 has the amino acid sequence shown in SEQ ID NO: 19.

[0036] According to an embodiment of the present invention, the immune cells include at least one of T cells and NK cells. In the present application, the types of the immune stimulatory molecules are not particularly limited, and promoters that can promote the immune function of at least one of T cells and NK cells can all be used. In some specific embodiments of the present application, the immune stimulatory molecule is IL-15.

[0037] According to an embodiment of the present invention, the immune cells are preferably NK cells.

[0038] 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.

[0039] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.

[0040] In a 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 encoding a chimeric antigen receptor; 2) a second nucleic acid molecule encoding a fusion protein including IL-15Rα and IL-15. IL-15 is a pleiotropic cytokine. After IL-15 and IL-15R form a dimer, they bind to IL-15Rc to activate the downstream JAK1 / JAK3 and STAT3 / STAT5 signaling pathways, thereby promoting the proliferation, activation, and effector functions of T cells, B cells, and NK cells. After the isolated nucleic acid according to the embodiment of the present invention is introduced into recipient cells, it can package viruses with a high titer and achieve specific infection of immune cells by the viruses, such as NK cells. After the isolated nucleic acid is introduced into immune cells, the immune cells can simultaneously express and secrete the chimeric antigen receptor and the fusion protein, enabling the immune cells to target the corresponding antigen and localize to the cell surface expressing the antigen. In addition, the fusion protein, such as IL-15Rα and IL-15, further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the tumor local microenvironment, enables them to maintain a strong tumor-killing activity, effectively avoids the toxic and side effects brought by systemic high-dose or repeated injections, and also avoids the toxic and side effects brought by systemic application of high-dose or repeated injections of recombinant IL-15.

[0041] According to an embodiment of the present invention, the above isolated nucleic acid may further include at least one of the following additional technical features:

[0042] According to an embodiment of the present invention, the chimeric antigen receptor is as defined in the first aspect.

[0043] According to an embodiment of the present invention, the second nucleic acid molecule includes the nucleotide sequence encoding IL-15Rα as shown in SEQ ID NO:10 and the nucleotide sequence encoding IL-15 as shown in SEQ ID NO:12.

[0044] According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged to be expressed in immune cells to express the chimeric antigen receptor and the fusion protein, and the fusion protein is in a non-fused form with the chimeric antigen receptor.

[0045] According to an embodiment of the present invention, the isolated nucleic acid further comprises: an internal ribosome entry site sequence, 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 the nucleotide sequence shown in SEQ ID NO: 20.

[0046] 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 arranged between the first nucleic acid molecule and the second nucleic acid molecule, the third nucleic acid molecule encodes a linker peptide 3, and the linker peptide 3 can be cleaved. The linker peptide 3 can separate the first nucleic acid molecule from the second nucleic acid molecule and reduce the functional interference between the two.

[0047] According to an embodiment of the present invention, the linker peptide 3 comprises a 2A peptide or a fragment thereof. Those skilled in the art can understand that the linker peptide 3 is not particularly limited, and any conventional peptide with self-cleavage function can be used.

[0048] According to an embodiment of the present invention, the linker peptide 3 comprises at least one of P2A, T2A, E2A and F2A or a fragment thereof.

[0049] According to an embodiment of the present invention, the linker peptide 3 comprises P2A or a fragment thereof.

[0050] According to an embodiment of the present invention, the linker peptide 3 comprises the amino acid sequence shown in SEQ ID NO: 19.

[0051] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a first promoter, the first promoter is operably linked to the first nucleic acid molecule; and / or a second promoter, the second promoter is operably linked to the second nucleic acid molecule.

[0052] According to an embodiment of the present invention, the first promoter and the second promoter are each independently selected from the U6, H1, CMV, EF-1, LTR or RSV promoter.

[0053] According to an embodiment of the present invention, the isolated nucleic acid further comprises a fourth nucleic acid molecule encoding a signal peptide. According to a specific embodiment of the present invention, the signal peptide encoded by the gene encoding the signal peptide is located at the amino terminus of the chimeric antigen receptor and is the membrane-localized terminal peptide of the chimeric antigen receptor. It helps the chimeric antigen receptor to localize to the endoplasmic reticulum and is hydrolyzed and detached after protein maturation. Therefore, the chimeric antigen receptor on the virus particle does not contain this signal peptide.

[0054] According to an embodiment of the present invention, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.

[0055] According to an embodiment of the present invention, the signal peptide comprises at least one of CSF2R and CD8α or a fragment thereof. Those skilled in the art can understand that the types of the signal peptides are not particularly limited, and conventional signal peptides in the art can be used.

[0056] According to an embodiment of the present invention, the signal peptide comprises CSF2R or a fragment thereof.

[0057] According to an embodiment of the present invention, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 13.

[0058] 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.

[0059] According to an embodiment of the present invention, the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 9.

[0060] According to an embodiment of the present invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 8.

[0061] According to an embodiment of the present invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 2.

[0062] According to an embodiment of the present invention, the isolated nucleic acid has the nucleotide sequence shown in SEQ ID NO: 1.

[0063] 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 isolated nucleic acid described above. When the above-mentioned isolated nucleic acid is ligated to a vector, the isolated nucleic acid can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the isolated nucleic acid, etc., that is, the isolated nucleic acid is operably linked to the control elements. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself.

[0064] According to an embodiment of the present invention, the above-mentioned construct may further include at least one of the following additional technical features:

[0065] 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 high expression efficiency.

[0066] 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 adeno-associated viral vector.

[0067] 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 the constructed vector described above. The recombinant cell according to the embodiment of the present invention can be used to express in vitro and obtain in large quantities the protein encoded by the isolated nucleic acid described above, such as chimeric antigen receptor mesothelin and IL-15Rα and IL-15 fusion protein.

[0068] 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:

[0069] According to an embodiment of the present invention, the recombinant cell includes a eukaryotic cell, preferably a mammalian cell.

[0070] It should be noted that the recombinant cell of the present invention is not particularly limited and may be a prokaryotic cell, a eukaryotic cell, or a phage. Exemplarily, the prokaryotic cell 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, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cell of the present invention is preferably a mammalian cell, including T cells, B cells, NK cells, BHK cells, CHO cells, NSO cells, or COS cells, and does not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0071] In the fifth aspect of the present invention, the present invention provides 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 is capable of simultaneously expressing and secreting a chimeric antigen receptor and a fusion protein. The inventors found in experiments that the fusion protein modification strategy proposed by the present invention enables NK cells or T cells to secrete super IL-15, i.e., the IL-15Rα and IL-15 fusion protein, locally in tumors, significantly improving the in vitro and in vivo proliferation ability of CAR-NK cells or T cells, enhancing the in vivo survival time of NK cells or T cells, and improving the in vivo anti-tumor function of NK cells or T cells. Moreover, this locally continuously and slowly released IL-15 can avoid the toxic and side effects caused by systemic administration of recombinant IL-15 or repeated multiple administrations.

[0072] According to some embodiments of the present invention, the NK cells include at least one selected from peripheral blood NK cells, cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells, and NK-92 cells.

[0073] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.

[0074] In the 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 above-described construct is introduced into a first recipient cell; the first recipient cell into which the construct has been introduced is cultured to obtain the virus. The method according to some preferred embodiments of the present invention can obtain a virus with a high titer.

[0075] According to an embodiment of the present invention, the virus includes a lentivirus.

[0076] According to an embodiment of the present invention, the first recipient cell is 293T.

[0077] In the seventh aspect of the present invention, the present invention provides a virus. According to an embodiment of the present invention, it is obtained by the method for obtaining a virus described above.

[0078] In the eighth aspect of the present invention, the present invention provides a virus. According to an embodiment of the present invention, the virus includes the nucleotide sequence shown in SEQ ID NO:1.

[0079] According to an embodiment of the present invention, the virus includes at least one of a retrovirus, a lentivirus, and an adenovirus.

[0080] According to an embodiment of the present invention, the virus includes a lentivirus.

[0081] In the eighth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the isolated transgenic immune cells, nucleic acids, constructs, recombinant cells, CAR-NK or CAR-T cells, or viruses described above. As mentioned above, the isolated nucleic acids, expression vectors, or cells or viruses carrying the isolated nucleic acids or expression vectors can simultaneously express and secrete chimeric antigen receptors and fusion proteins, enabling immune cells to target corresponding antigens and localize to the cell surface expressing the antigen. In addition, fusion proteins, such as super IL-15, that is, the IL-15Rα and IL-15 fusion protein, further promote the activation and proliferation of immune cells, maintain the number and activity of immune cells in the tumor local microenvironment, keep them with strong tumor-killing activity, effectively avoid the toxic and side effects brought by systemic high-dose or repeated multiple injections, and also avoid the toxic and side effects brought by systemic application of high-dose or repeated multiple injections of recombinant IL-15. Therefore, the pharmaceutical composition containing the above substances also has the above functions, which will not be repeated here.

[0082] According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:

[0083] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, except in the range where any conventional excipients 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 uses are also within the scope contemplated by the present invention.

[0084] For example, the isolated nucleic acids, expression vectors, or cells carrying the isolated nucleic acids or expression vectors of the present invention can be incorporated into drugs suitable for parenteral administration (such as intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (such as injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical drugs are in the form of injection solutions or infusion solutions. The isolated nucleic acids, expression vectors, or cells carrying the isolated nucleic acids or expression vectors can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0085] The effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector according to the present invention may vary with the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). Such factors include, but are not limited to: the 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, depending on the urgency of the treatment situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0086] In the tenth aspect of the present invention, the present invention provides 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 proliferative activity to increase their proliferative activity from low to obtain biological samples that meet expectations.

[0087] In the eleventh aspect of the present invention, the present invention provides a method for introducing a virus into activated immune cells. According to an embodiment of the present invention, the activated immune cells are electroporated or transfected with the construct described above or infected with the virus described above.

[0088] According to an embodiment of the present invention, the immune cells include at least one of T cells and NK cells.

[0089] According to an embodiment of the present invention, the immune cells are preferably NK cells.

[0090] According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells, and NK-92 cells.

[0091] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.

[0092] In a twelfth aspect of the present invention, the present invention provides a method for obtaining a chimeric antigen receptor and a fusion protein. According to an embodiment of the present invention, the method includes: introducing the aforementioned construct or virus into a second receptor cell; culturing the second receptor cell into which the construct or virus has been introduced to obtain the chimeric antigen receptor and the fusion protein. As described above, the construct or virus can express the chimeric antigen receptor and the fusion protein simultaneously under suitable conditions. Therefore, the method according to the embodiment of the present invention can obtain a large amount of the chimeric antigen receptor and the fusion protein.

[0093] According to an embodiment of the present invention, the introduction into the second receptor cell is carried out by means of electroporation, transfection or infection. It should be noted that "electroporation" or "transfection" is a method for introducing a viral vector into a receptor cell, and "infection" refers to the process in which a 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 receptor cell by means of electrical stimulation, and "transfection" refers to a method of introducing a viral packaging vector into a receptor cell by means of a chemical mediator, such as liposome.

[0094] According to an embodiment of the present invention, the second receptor cell is at least one of T cells and NK cells.

[0095] According to an embodiment of the present invention, the second receptor cell is an NK cell.

[0096] According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells, and NK-92 cells.

[0097] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.

[0098] According to an embodiment of the present invention, the virus includes at least one selected from retroviruses, lentiviruses, and adenoviruses.

[0099] According to an embodiment of the present invention, the virus includes a lentivirus.

[0100] In a thirteenth aspect of the present invention, the present invention provides a method for obtaining CAR-NK or CAR-T cells expressing a chimeric antigen receptor and a fusion protein. According to an embodiment of the present invention, the method includes: introducing the aforementioned construct or virus into NK cells or T cells; culturing the NK cells or T cells into which the construct or virus has been introduced to obtain the CAR-NK or CAR-T cells. According to some specific embodiments of the present invention, a lentiviral expression vector targeting mesothelin and simultaneously expressing super IL-15 (i.e., a fusion protein of IL-15 and IL-15Rα) is constructed. The lentivirus is packaged to produce virus particles, which are used to infect NK cells or T cells to obtain NK cells or T cells with high infection efficiency and positive for CAR. These CAR-NK or CAR-T cells can not only target and kill mesothelin-positive malignant tumors, but also, due to their ability to continuously secrete super IL-15 locally, have higher proliferation ability and killing activity than unmodified NK cells or T cells. In particular, they can maintain the long-term survival of NK cells or T cells in vivo, enabling NK cells or T cells to maintain high proliferation vitality and killing activity, and exerting a stronger ability to continuously kill tumors. More importantly, this locally secreted IL-15 exerts effective biological functions at the tumor site, and can effectively avoid the toxic and side effects caused by systemic application of high-dose or repeated injections of recombinant IL-15.

[0101] According to an embodiment of the present invention, the introduction of the NK cells or T cells is carried out by means of electroporation, transfection or infection.

[0102] In a fourteenth aspect of the present invention, the present invention provides 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 for treating or preventing tumors. As described above, the isolated nucleic acid, construct, or cell carrying the above substances can simultaneously express and secrete a chimeric antigen receptor and a fusion protein, such as a fusion protein of IL-15Rα and IL-15, enabling the cell to target the corresponding antigen and localize to the cell surface expressing the antigen. In addition, the fusion protein of IL-15Rα and IL-15 further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local tumor microenvironment, enables them to maintain strong tumor-killing activity, effectively avoids the toxic and side effects caused by systemic high-dose or repeated injections, and can also avoid the toxic and side effects caused by systemic application of high-dose or repeated injections of recombinant IL-15.

[0103] According to an embodiment of the present invention, the tumor is at least one of a mesothelin-positive tumor, a HER2-positive tumor, an EGFR-positive tumor, a GPC3-positive tumor, a MUC1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD2-positive tumor, a PSCA-positive tumor, 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.

[0104] According to an embodiment of the present invention, the mesothelin-positive tumor includes at least one of pancreatic cancer, ovarian cancer, mesothelioma, cholangiocarcinoma, and lung cancer.

[0105] In a fifteenth aspect of the present invention, the present invention provides 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 NK cells or T cells with low proliferative activity to increase their proliferative activity from low to obtain biological samples that meet expectations. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 is a schematic structural diagram of a CAR targeting MSLN and modified with a fusion protein (RIL) according to Embodiment 1 of the present invention. Among them, SP represents a nucleotide sequence encoding a signal peptide, α-MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB co-stimulatory signal domain, CD3Z represents a nucleotide sequence encoding an intracellular region of CD3Z, P2A represents a nucleotide sequence encoding a P2A self-cleavage region, IL15Rα represents a nucleotide sequence encoding the full length of IL15Rα, Linker represents a nucleotide sequence encoding a linker peptide, and IL15 represents a nucleotide sequence encoding the full length of IL15;

[0107] Figure 2 is a diagram showing the detection results of the IL-15 secretion levels of NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells according to Embodiment 2 of the present invention;

[0108] Figure 3It is a graph showing the detection results of STAT5 phosphorylation levels in NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention;

[0109] Figure 4 It is a graph showing the detection results of the in vitro killing ability of NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention;

[0110] Figure 5 It is a graph showing the detection results of the in vitro proliferation ability of NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells according to Example 2 of the present invention, where the abscissa (Days) represents the number of days and the ordinate (Cell number) represents the number of cells;

[0111] Figure 6 It is a flowchart of the operation of treating pancreatic cancer Aspc-1 cell-bearing mice with CAR-NK cells expressing RIL according to Example 3 of the present invention;

[0112] Figure 7 It is a graph showing the detection results of the survival ability of NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells in pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention;

[0113] Figure 8 It is a graph showing the detection results of the anti-tumor ability of NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells against pancreatic cancer Aspc-1 cell-bearing mice according to Example 3 of the present invention, where the abscissa (Days after NK cell treatment) represents the number of days after NK cell treatment and the ordinate (Tumor) represents the volume of tumor cells. Detailed implementation manners

[0114] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings. The same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0115] During the description of the present invention, the relevant terms in this article are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the solution and should not be regarded as a limitation to the protection solution of the present invention.

[0116] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0117] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent described event or condition may or may not occur, and this description includes the situation where the event or condition occurs, and the situation where the event or condition does not occur.

[0118] "Operably linked" in this text means that an exogenous gene is linked to a vector such that control elements in the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of a regulatory system, the expression of the aforementioned isolated nucleic acid can be effectively achieved, and then a large amount of the protein encoded by the isolated nucleic acid can be obtained in vitro.

[0119] In this text, the so-called "suitable conditions" refer to the conditions suitable for the expression of the protein encoded by the isolated nucleic acid described in the present application. It is easily understood by those skilled in the art that the conditions suitable for the expression of the protein encoded by the isolated nucleic acid include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the protein encoded by the isolated nucleic acid according to the specific environment of the laboratory.

[0120] The present application constructs a transgenic immune cell that simultaneously expresses a chimeric antigen receptor and an immune stimulatory molecule. Among them, there can be various antigens targeted by the chimeric antigen receptor, enabling the immune cell to target the corresponding antigen and localize to the cell surface expressing the antigen. The immune stimulatory molecule can further promote the activation and proliferation of the immune cell. For example, IL-15 used in the present application, after experimental verification, the proliferation activity and tumor killing ability of the immune cell that simultaneously expresses the chimeric antigen receptor and IL-15 are significantly improved, effectively avoiding the toxic and side effects brought by systemic high-dose or repeated multiple injections.

[0121] The amino acid or nucleic acid sequences involved in this text are shown as follows.

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro AlaPhe Leu Leu Ile Pro(SEQ ID NO:13)

[0128] Asp Ile Gln Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg(SEQ ID NO:14)

[0129] Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala SerGln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro AlaAla Gly GlyAla Val HisThrArg Gly LeuAsp PheAla Cys Asp(SEQ ID NO:15)

[0130] Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu SerLeu Val Ile Thr Leu Tyr Cys(SEQ ID NO:16)

[0131] Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetArgPro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu GluGlu Gly Gly Cys Glu Leu(SEQ ID NO:17)

[0132] Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg(SEQ ID NO:18)

[0133] Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro(SEQ ID NO:19)

[0134]

[0135] Embodiments of the present invention will be described in more detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0136] It should be noted that in the following embodiments, "plasmid" and "vector" have the same meaning and can be used interchangeably.

[0137] Example 1: Preparation of CAR-NK cells

[0138] 1.1 Construction of CAR expression plasmid

[0139] The present invention designs a CAR vector (anti-MSLN-CAR-RIL) sequence targeting mesothelin (MSLN) and expressing super IL-15, which includes a signal peptide (SP), an extracellular region targeting and recognizing MSLN (anti-MSLN single-chain antibody, anti-MSLN scFv), a CD8a hinge region (Hinge) and transmembrane region (TM), an intracellular co-stimulatory signal domain of 4-1BB and an intracellular signal transduction molecule CD3ζ, and a gene fragment of IL-15Rα-linker-IL-15 (RIL) linked by P2A. The structures of each gene element in the CAR vector are as shown in Figure 1 shown. Among them:

[0140] The full-length gene sequence of the CAR vector composed of each element is as shown in SEQ ID NO: 1;

[0141] The signal peptide is CSF2R, and its nucleotide sequence is as shown in SEQ ID NO: 2;

[0142] The nucleotide sequence of the anti-MSLN scFv is as shown in SEQ ID NO: 3;

[0143] The nucleotide sequence of the CD8 hinge region is as shown in SEQ ID NO: 4;

[0144] The nucleotide sequence of the CD8 transmembrane region is as shown in SEQ ID NO: 5;

[0145] The nucleotide sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO: 6;

[0146] The nucleotide sequence of the CD3ζ intracellular region is as shown in SEQ ID NO: 7;

[0147] The nucleotide sequence of the self-cleaving region P2A is as shown in SEQ ID NO: 8;

[0148] The nucleotide sequence of the RIL is as shown in SEQ ID NO: 9;

[0149] The nucleotide sequence of the IL15Rα is as shown in SEQ ID NO.10 in the sequence listing;

[0150] The nucleotide sequence of the Linker is as shown in SEQ ID NO.11 in the sequence listing;

[0151] The nucleotide sequence of the IL15 is as shown in SEQ ID NO.12 in the sequence listing.

[0152] First, the anti-MSLN-CAR fragment was inserted into the lentiviral vector pLent-EF1α-P2A-CMV-GP to construct the pLent-anti-MSLN-P2A-CMV-GP vector. The full gene-synthesized IL-15R-IL-15 (RIL) gene fragment was inserted into the pLent-anti-MSLN-P2A-CMV-GP vector through the restriction enzyme site Not I. After PCR identification and sequencing verification, the sequence was correct, indicating that the pLent-anti-MSLN-P2A-RIL15-CMV-GP vector was successfully constructed.

[0153] 1.2 Packaging of Lentivirus and Concentration of Viral Solution

[0154] Take 5×10 6 293T cells in the logarithmic growth phase and inoculate them into a 10-cm culture dish. Add 10 mL of DMEM medium and culture overnight in an incubator at 37°C and 5% CO2. When the cell density reaches 80%, replace it with 10 mL of fresh DMEM medium for virus packaging, and continue to place the cell culture dish in the incubator for standby. Prepare the lentiviral packaging system. Add 6 μg of the lentiviral packaging helper plasmid psPAX26 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. Then add the mixture to the above plasmid mixture at one time, mix well, incubate at room temperature for 15 min. After incubation, add the mixture to the 293T cell culture dish. Change the medium after 24 h, put the culture dish back into the incubator at 37°C and 5% CO2, collect the cell supernatant after culturing for 48 h, centrifuge at 400×g for 5 min to remove cell debris, filter the supernatant through a 0.45-μm filter tip into a 50-mL centrifuge tube. Add 5×PEG8000 solution for virus solution concentration, invert the centrifuge tube up and down to mix well, and place it in the refrigerator at 4°C overnight. Centrifuge at 4°C and 4000×g for 20 min, discard the supernatant, resuspend the virus precipitate with an appropriate amount of serum-free DMEM, transfer it into an EP tube, and store it in the refrigerator at -80°C.

[0155] 1.3 Detection of Lentivirus Titer

[0156] Take 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 5 / mL. Take a 24-well plate and add 1 mL of cell suspension (1×10 5 / (Holes), set 3 gradients of virus volume added. Incubate overnight in a 37°C, 5% CO2 incubator. First, perform a 10-fold dilution of the concentrated virus solution: Take a 1 mL Eppendorf tube, pipette 60 μL of the virus concentrated solution into the Eppendorf tube, and dilute it with 540 μL of DMEM medium, mixing well. Replace the medium of 293T cells with fresh DMEM medium, and pipette 5 μL, 50 μL, and 500 μL of the diluted virus solution into the corresponding wells respectively, mark them, and then put the culture plate back into the 37°C, 5% CO2 incubator. After 24 h, aspirate the virus solution in the well plate, and then add 1 mL of fresh DMEM medium. After 72 h, digest and harvest the cells with trypsin, and use a flow cytometer to detect the GFP expression rate of 293T cells. Calculate the virus titer according to the following formula:

[0157] Titer (TU / mL) = (C × N × D × 1000) / V

[0158] Where: C = GFP positive rate detected by flow cytometry

[0159] N = Number of cells at the time of infection (about 1 × 10 5 )

[0160] D = Dilution factor of the virus vector

[0161] V = Volume of the diluted virus added.

[0162] 1.4 Lentivirus infection of human NK cells

[0163] Take NK-92 cells (purchased from ATCC) in the logarithmic growth phase, harvest the cells by centrifugation at 100 × g for 5 min, resuspend the cells with an appropriate amount of α-MEM medium, and adjust the cell density to 5 × 10 5 cells / mL. In a 24-well plate, seed 5 × 10 5 NK-92 cells, 1 mL of the virus concentrated solution, and protamine (purchased from Solarbio, final concentration 8 μg / mL), mix well, and incubate in a 37°C, 5% CO2 incubator. After 24 h, observe the cell status, change the medium, transfer the infected cells into an Eppendorf tube, centrifuge at 100 × g for 5 min, resuspend the cells with a small amount of fresh α-MEM medium, transfer the cells into a cell culture flask, add 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL), and continue to culture for 48 h. Transfer the cells into a flow cytometry tube, add 3 mL of 1×PBS solution, centrifuge at 100 × g for 5 min, discard the supernatant, flick the cell pellet, and wash again with 1×PBS solution. Use a flow cytometer to detect the GFP expression rate. Continue to expand the culture and adjust the status of the infected NK-92 cells for amplification. Sort the GFP-positive CAR-NK-92 cells from the infected NK-92 cells by flow cytometry for later experiments.

[0164] Example 2: Determination of the level of IL-15 secreted by CAR-NK cells and cell proliferation ability

[0165] In this example, the CAR-NK-92 (abbreviated as CAR-NK throughout the text) cells obtained in Example 1 were used to determine the IL-15 secretion level and cell proliferation ability

[0166] 2.1 Detection of the IL-15 secretion level of CAR-NK-92 cells by ELISA

[0167] NK-92, α-MSLN-CAR-NK-92 (CAR carrying the target of MSLN), and α-MSLN-CAR-RIL-NK-92 (CAR carrying the target of MSLN and expressing IL-15 and IL15Rα) cells were respectively seeded and cultured, and the supernatants were collected after 24 h. The content of IL-15 in the supernatants of different groups was detected by ELISA. The experimental results are as Figure 2 shown. Among them, almost no IL-15 was detected in the supernatants of NK-92 and α-MSLN-CAR-NK-92 cells. However, obvious IL-15 was detected in the supernatant of α-CAR-IL15-NK-92 cells, and the level was 67.48 ± 5.96 pg / mL. It shows that the α-MSLN-CAR-RIL-NK cells designed and modified by the present invention have the ability to secrete IL-15

[0168] 2.2 Detection of the STAT5 phosphorylation level of CAR-NK cells

[0169] IL-15 exerts its function by first binding to the α chain of IL-15Rα to form a dimer, and then after the binding of IL-15Rα-IL-15 and the βγc chain of IL-15R, it activates the phosphorylation of downstream STAT5 (pSTAT5). After pSTAT5 enters the nucleus, it promotes the expression of genes related to activation, proliferation, and anti-apoptosis. Therefore, the inventors further observed whether the IL-15 secreted by the CAR-NK cells modified by the present invention has biological activity and phosphorylates downstream STAT5. The above-mentioned NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells were respectively cultured in serum-free RPMI 1640 medium for 12 h for starvation treatment, and the starvation treatment was to reduce the phosphorylation level of their own STAT5. The cells were harvested, and the level of pSTAT5 was detected by flow cytometry. The results are as Figure 3 shown. The STAT5 phosphorylation levels in the NK-92 cell group and the α-MSLN-CAR-NK-92 cell group were relatively low, while the STAT5 phosphorylation level in the α-MSLN-CAR-RIL-NK-92 cell group was significantly higher than that in the control group

[0170] 2.3 Detection of the in vitro killing ability of CAR-NK cells

[0171] The inventors co-incubated the above-mentioned NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells with the pancreatic cancer cell line Aspc-1 for 5 h respectively, and then detected the killing efficiency. As can be seen from Figure 4 it, when the effector-to-target ratio was 5:1, the killing efficiencies of α-MSLN-CAR-NK-92 and α-MSLN-CAR-RIL-NK-92 against Aspc-1 cells were 48.01±2.00% and 58.42%±2.46 respectively, which were significantly higher than that of NK-92 cells (35.59±2.46%); and the killing efficiency of α-MSLN-CAR-RIL-NK-92 cells was also significantly higher than that of the α-MSLN-CAR-NK-92 group, indicating that inserting the RIL fragment can not only promote NK cells to secrete IL-15, but also improve the killing function of α-MSLN-CAR-NK-92 cells.

[0172] 2.4 Detection of the in vitro proliferation ability of CAR-NK cells

[0173] The inventors further verified the pro-survival effect of autocrine RIL on NK-92 cells. The same number of the above-mentioned NK-92, α-MSLN-CAR-NK-92, and α-MSLN-CAR-RIL-NK-92 cells were seeded in 96-well plates respectively, and cell counting was performed every 3 days for 24 days to draw a cell proliferation curve. As can be seen from Figure 5 it, when cultured to the 12th day, the number of α-MSLN-CAR-RIL-NK-92 cells began to show differences compared with the NK-92 and α-MSLN-CAR-NK-92 cell groups. By the 21st day, there were significant differences. It shows that the modified NK cells of the present invention have stronger proliferation ability, probably because RIL is a super-agonist form of IL-15, and it can bind to the IL-15βγc chain more effectively in a trans-binding manner to activate the downstream signaling pathway.

[0174] Example 3: Detection of the in vivo anti-tumor ability of CAR-NK cells and the in vivo survival ability of CAR-NK cells

[0175] In this example, a xenograft model of nude mice bearing pancreatic cancer Aspc-1 cells was established to observe the therapeutic effect of CAR-NK-92 cells on pancreatic cancer. The specific experimental operations are as follows:

[0176] Six-week-old BALB / c-nu nude mice were selected for subcutaneous tumor implantation under the armpit, and the tumor implantation dose was 2×10 6Cells / mouse. Tumors formed in about 4 days, and cell therapy began after one week. Before treatment, the tumor volume was measured, and the mice were randomly divided into a PBS group, an NK-92 cell therapy group, an α-MSLN-CAR-NK-92 cell therapy group, and an α-MSLN-CAR-RIL-NK-92 cell therapy group according to the tumor volume. The effector cells (1×10 7 cells / mouse) were injected into the caudal vein of the mice in the treatment groups, and the same volume of 1×PBS was injected into the untreated group. The injection was performed once every other week for a total of 5 times, and IL-2 (5×10 4 IU / mouse) was injected into the caudal vein every 3 days. The specific experimental settings and operation procedures are referred to Figure 6 . To study the pro-survival effect of RIL on CAR-NK-92 cells, peripheral blood of the mice was collected on the first, third, and seventh days after treatment. After red blood cell lysis, the PerCP / Cyanine5.5 anti-human CD56 antibody was labeled, and the proportion of the CD56 cell population in peripheral blood lymphocytes, that is, 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 plotted.

[0177] The results are as Figure 7As shown, on the first day of NK cell treatment, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells, and α-MSLN-CAR-RIL-NK-92 cells in the peripheral blood lymphocytes of mice were 27.3%, 29.6%, and 29.3% respectively, and the proportions of NK-92 cells in each group were similar in vivo. On the third day, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells, and α-MSLN-CAR-RIL-NK-92 cells in the peripheral blood lymphocytes of mice were 8.32%, 9.83%, and 16.8% respectively. It can be seen that compared with the first day, the proportions of NK-92 cells in each group decreased on the third day. The NK-92 group decreased from 27.3% to 8.32%, the α-MSLN-CAR-NK-92 group decreased from 29.6% to 9.83%, and the α-MSLN-CAR-RIL-NK-92 group decreased from 29.3% to 16.8%. However, it can be seen that the proportion of α-MSLN-CAR-RIL-NK-92 cells in vivo was the highest, and the persistence in vivo was more prominent. On the seventh day, the proportions of NK-92 cells, α-MSLN-CAR-NK-92 cells, and α-MSLN-CAR-RIL-NK-92 cells in vivo were 0.13%, 3.32%, and 14.7% respectively. It can be seen that compared with the NK-92 group and the α-MSLN-CAR-NK-92 group, the α-MSLN-CAR-RIL-NK-92 cells modified with the RIL gene had a higher proportion in vivo and stronger persistence in vivo. Therefore, expressing RIL can improve the survival ability of NK cells in vivo, and locally secreted super IL-15 (RIL) has an obvious effect on maintaining the survival of NK cells in vivo.

[0178] The tumor growth curve was plotted by measuring the tumor size. From Figure 8 It can be seen that compared with the control group (PBS group) and the NK-92 cell treatment group, the treatment with α-MSLN-CAR-NK92 cells and α-MSLN-CAR-RIL-NK-92 cells could significantly inhibit tumor growth, and the α-MSLN-CAR-RIL-NK-92 cells showed better anti-tumor effects than the α-MSLN-CAR-NK-92 cells. The above results indicate that CAR-NK-92 cells targeting mesothelin can inhibit the growth of pancreatic cancer and have good therapeutic effects. The modification of the RIL gene can improve the persistence of CAR-NK-92 cells in vivo, enhance the survival ability of CAR-NK-92 cells in vivo, and improve the anti-tumor effect of CAR-NK-92 cells.

[0179] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0180] In the description of this specification, the description with reference to terms such as "an embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection 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, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0181] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

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

1.

2. A kit, characterized in that, Comprising: The virus according to claim 1.

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

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

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

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

7. A method for obtaining a chimeric antigen receptor and a fusion protein, characterized in that, Comprising: Introducing the virus according to claim 1 into a second recipient cell; Culturing the second recipient cell into which the virus has been introduced to obtain the chimeric antigen receptor and the fusion protein.

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

9. The method according to claim 7, characterized in that, The second recipient cell is at least one of T cells and NK cells.

10. The method according to claim 9, characterized in that, The second recipient cell is NK cells.

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

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

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

14. A method for obtaining CAR-NK or CAR-T cells expressing a chimeric antigen receptor and a fusion protein, characterized in that, Comprising: Introducing the virus according to claim 1 into NK cells or T cells; Culturing the NK cells or T cells into which the virus has been introduced to obtain the CAR-NK or CAR-T cells.

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

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

17. The method according to claim 15, 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 the preparation of a pharmaceutical composition for treating a tumor, wherein the tumor is pancreatic cancer.

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

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

21. The use according to claim 19, wherein The T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.

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