Immune cells induced from iPS cells

Abstract

The present invention addresses the problem of providing an immune cell induced from an iPS cell, which imparts a phenotype that facilitates local migration and infiltration into a solid tumor and proliferation and survival of the immune cell in the solid tumor. [Solution] A cell which is an immune cell induced from an iPS cell and which expresses interleukin 15 and a cell surface molecule that is reactive with a tumor-associated antigen. And a cell which is an immune cell induced from an iPS cell and which expresses a cell surface molecule that is reactive with a tumor-associated antigen and interleukin 12, interleukin 18, or interleukin 21.

Description

Technical Field

[0001] The present invention relates to immune cells induced from iPS cells (induced pluripotent stem cells), wherein the cells express cell surface molecules reactive to tumor-associated antigens and interleukin 15 (IL-15), a method for preparing the cells, a drug containing the cells, a killer of cells expressing tumor-associated antigens containing the cells, and a preventive or therapeutic agent for cancer in mammals, as well as a method for preventing or treating cancer in mammals, wherein the method comprises administering an effective amount of the cells. Background Art

[0002] In recent years, as a new cancer treatment method, cancer immunotherapy research that clears cancer by activating the patient's immune function is being actively carried out. In this cancer immunotherapy, T cells that can specifically recognize and attack cancer are very important. However, the number of T cells that can recognize cancer cells in the patient's body is small. Therefore, cancer immunotherapy using genetically modified T cells is being studied, which express receptors that can specifically recognize cancer antigens or tumor-associated antigens on the cell surface. As such genetically modified T cells, T cells expressing chimeric antigen receptors (CAR) and T cell receptors (TCR) targeting specific cancer antigens or tumor-associated antigens have been reported, and they are respectively referred to as CAR-T cells and TCR-T cells (Patent Documents 1 and 2).

[0003] CAR is a fusion protein that combines the antigen-recognition portion of an antibody that specifically recognizes cancer antigens or tumor-associated antigens with the intracellular domain of a TCR. CAR-T cells can recognize antigens expressed on the cell surface without restriction to human leukocyte antigens (HLA). TCRs are receptors used by T cells to recognize antigens and are composed of dimers of α and β chains or γ and δ chains. TCRs form a complex with CD3 molecules on the T cell surface and activate T cells by recognizing antigen molecules bound to major histocompatibility complex (MHC) molecules.

[0004] In the cancer immunotherapy using the above-mentioned genetically modified T cells, it is difficult to ensure that a sufficient amount of T cells, and T cell exhaustion, such as T cell proliferation ability reduction and immune response to antigens such as target cells reduction, etc. are obstacles to effectively implementing the cancer immunotherapy. In order to overcome this obstacle, the cancer immunotherapy is smoothly implemented, and it is reported that the induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) set up from antigen-specific T cells are propagated and then differentiated into the technology (non-patent literature 1) of cytotoxic T cells. This technology can continuously induce a large amount of functional T cells from iPS cells, is therefore considered to be the important basis for the above-mentioned use of T cells to carry out cancer immunotherapy.

[0005] In cancer immunotherapy using CAR-T cells, significant effects such as complete remission have been achieved for a portion of patients with hematological tumors. However, the above-mentioned use of T cells for cancer immunotherapy may not necessarily achieve satisfactory therapeutic effects for solid tumors (non-patent literature 2). It is reported that in order to enable the above-mentioned genetically modified T cells to exert anti-tumor effects on solid tumors, it is important to enable the T cells that maintain effector properties to migrate and infiltrate into solid tumors and survive for a long time in the tumor, thereby allowing T cells to continue to exert cytotoxic activity (non-patent literature 3).

[0006] As a method for making the above-mentioned genetically modified T cells give full anti-tumor effect to solid tumors, it has been proposed that the factor that improves the T cell function is introduced into the method for the T cell gene.For example, it is disclosed that the cytokine interleukin 7 (IL-7) that plays an important role in T cell survival and proliferation is expressed by gene introduction, and the CAR-T cells of the chemokine CCL19 that plays an important role in the migration of T cells and dendritic cells are effective for solid tumors (patent document 3).In addition, it is reported that interleukin 15 (IL-15) and interleukin 21 (IL-21) CAR-T cells that promote T cell survival, proliferation and cytotoxic activity expression, improve the anti-tumor properties of T cells by gene introduction expression are effective for solid tumors (non-patent literature 4). Prior art literature Patent Literature

[0007] Patent Document 1: International Publication No. 2013 / 070468 Patent Document 2: International Publication No. 2015 / 173112 Patent Document 3: International Publication No. 2017 / 159736 Non-patent literature

[0008] Non-patent literature 1: Minagawa A, et al. Enhancing T cell receptor stability inrejuvenated iPSC-derived T cells improves their use in cancerimmunotherapy. Cell Stem Cell. 2018; 23:850-858. Non-patent literature 2: Hartmann J, et al. Clinical development of CAR T cells-challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med. 2017; 9: 1183-1197. Non-patent literature 3: Chen DS and Mellman I. Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity. 2013; 39: 1-10. Non-patent document 4: Batra SA, et al. Glypican-3-specific CAR T cells coexpressing IL15 and IL21 have superior expansion and antitumor activity against hepatocellular carcinoma. Cancer Immunol Res. 2020; 8: 309-320. Summary of the Invention Problems to be solved by the invention

[0009] Effector cytotoxic T cells can be differentiated and induced from iPS cells. iPS cells are cloned cells that are easy to gene-edit and therefore easy to evaluate for safety. Therefore, in cancer immunotherapy using allogeneic T cells, the use of iPS cells enables off-the-shelf treatment of cells and a stable supply of therapeutic cells. In addition, most cancers are solid tumors, so it is important to give T cells (iPS-T cells) or natural killer (NK) cells (iPS-NK cells) differentiated from iPS cells sufficient anti-tumor effects on solid tumors to improve the treatment results of cancer immunotherapy using these immune cells differentiated from iPS cells.

[0010] To obtain these immune cells with excellent anti-tumor effects against solid tumors, the present invention aims to provide immune cells induced from iPS cells that possess a phenotype that facilitates local migration and infiltration into solid tumors, as well as the proliferation and survival of immune cells within solid tumors. Furthermore, the present invention aims to provide methods for producing these immune cells, pharmaceuticals containing these immune cells, agents that kill cells expressing tumor-associated antigens containing these immune cells, and methods for preventing or treating cancer in mammals, comprising administering an effective amount of these immune cells, and agents for preventing or treating cancer in mammals containing these immune cells. Solutions to Problems

[0011] The present inventors searched for cytokines that improve the function of immune cells induced from iPS cells. They discovered that interleukin-15, or a combination of IL-15 with interleukin-12 (IL-12), interleukin-18 (IL-18), or interleukin-21 (IL-21), can improve the function of these immune cells, leading to the completion of the present invention.

[0012] That is, the present invention provides the following. [1] An immune cell induced from an iPS cell, the cell expressing a cell surface molecule reactive to a tumor-associated antigen and interleukin 15 (IL-15). [2] The cell according to [1], further expressing interleukin 12 (IL-12), interleukin 18 (IL-18) or interleukin 21 (IL-21). 〔3〕 The cell according to [1], wherein the immune cell comprises a nucleic acid encoding the cell surface molecule and a nucleic acid encoding IL-15 introduced from outside the cell. [4] The cell according to [3], further comprising a nucleic acid encoding IL-12, IL-18 or IL-21 introduced from outside the cell. 〔5〕 The cell according to [1], wherein the immune cell comprises an expression vector containing a nucleic acid encoding the cell surface molecule and a nucleic acid encoding IL-15. [6] The cell according to [5], further comprising an expression vector containing a nucleic acid encoding IL-12, IL-18 or IL-21. [7] The cell according to [1], wherein the immune cell is a T cell or a NK cell. 〔8〕 The cell according to [7], wherein the T cell is a CD8 single-positive cytotoxic T cell. 〔9〕 The cell according to [1], wherein the cell surface molecule is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[10] The cell according to [1], wherein the cell surface molecules are chimeric antigen receptor (CAR) and T cell receptor (TCR).

[11] The cell according to [1], wherein the iPS cell is an iPS cell obtained by depleting B cells and T cells from peripheral blood mononuclear cells or reprogramming T cells.

[12] The cell according to [1], wherein the tumor-associated antigen is selected from WT1, GPC3, BCMA, XAGE1, MUC1, MUC5A1, MUC6, EGFRvIII, HER-2 / neu, MAGE-A1, MAGE-A3, telomerase, PRAME, SSX2 / 4, PSCA, CTLA-4, gp100, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53 without gene mutation, p53 with gene mutation, p53 Mutants, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, MelanA / MART1, survivin, Ras, Ras mutants, EGR, bcr-ab1, XBP-1, neoantigens caused by gene mutations, neoantigens caused by splicing abnormalities, HBV, HBs, HPV, EBV, LMP1, EBV, LMP2, EBNA, HPV-E1, HPV-E2, HPV-E6, HPV-E7, HTLV-1Tax and HBZ.

[13] A method, which is the method for preparing cells described in [3], comprising: (1) a step of introducing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells introduced with the nucleic acid obtained in step (1) into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells introduced with the nucleic acid, which were induced to differentiate from iPS cells obtained in step (1), into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells into which the nucleic acid has been introduced and induced to differentiate from the iPS cells obtained in step (1) into mature immune cells; (3) A step of introducing a nucleic acid encoding IL-15 into the mature immune cells obtained in step (1) into which the nucleic acid has been introduced, or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

[14] A method, which is the method for preparing cells described in [4], comprising: (1) a step of introducing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells introduced with the nucleic acid obtained in step (1) into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells introduced with the nucleic acid, which were induced to differentiate from iPS cells obtained in step (1), into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells into which the nucleic acid has been introduced and induced to differentiate from the iPS cells obtained in step (1) into mature immune cells; (3) A step of introducing a nucleic acid encoding IL-15 and a nucleic acid encoding IL-12, IL-18 or IL-21 into the mature immune cells into which the nucleic acid is introduced obtained in step (1), or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

[15] A method, which is the method for preparing cells described in [5], comprising: (1) a step of introducing an expression vector containing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells obtained in step (1) and into which the expression vector has been introduced into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells obtained in step (1) and induced to differentiate from iPS cells and into which the expression vector has been introduced, into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells obtained in step (1) and induced to differentiate from the iPS cells and into which the expression vector has been introduced, into mature immune cells; (3) A step of introducing an expression vector containing a nucleic acid encoding IL-15 into the mature immune cells obtained in step (1) and into which the expression vector has been introduced, or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

[16] A method, which is the method for preparing cells described in [6], comprising: (1) a step of introducing an expression vector containing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells obtained in step (1) and into which the expression vector has been introduced into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells obtained in step (1) and induced to differentiate from iPS cells and into which the expression vector has been introduced, into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells obtained in step (1) and induced to differentiate from the iPS cells and into which the expression vector has been introduced, into mature immune cells; (3) A step of introducing an expression vector comprising a nucleic acid encoding IL-15 and a nucleic acid encoding IL-12, IL-18 or IL-21 into the mature immune cells obtained in step (1) and into which the expression vector has been introduced, or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

[17] A drug comprising the cell according to any one of [1] to

[12] .

[18] The drug according to

[17] is used for the prevention or treatment of cancer.

[19] A cell-killing agent expressing a tumor-associated antigen, comprising the cell according to any one of [1] to

[12] . 〔20〕 A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the cell according to any one of [1] to

[12] . 〔twenty one〕 A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the drug according to

[17] . 〔twenty two〕 A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the killing agent according to

[19] . 〔twenty three〕 A preventive or therapeutic agent for cancer in a mammal, comprising the cell according to any one of [1] to

[12] . 〔twenty four〕 The cell according to any one of [1] to

[12] , which is used for preventing or treating cancer. 〔25〕 The cell according to any one of [1] to

[12] , which is used for preparing a preventive or therapeutic agent for cancer. Effects of the Invention

[0013] The cells of the present invention are immune cells induced from iPS cells. The cells of the present invention that express cell surface molecules reactive to tumor-associated antigens and IL-15, and the cells that further express IL-12, IL-18, or IL-21, have improved and maintained cytotoxic activity, cell proliferation capacity, and anti-apoptotic capacity against target cells expressing tumor-associated antigens. In addition, the cells of the present invention have enhanced migration ability to solid tumors and enhanced long-term survival ability within solid tumors, thereby being able to inhibit the growth of solid tumors and improve individual survival rates. Drugs containing the cells of the present invention or agents that kill cells expressing tumor-associated antigens are useful for the prevention or treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of an expression vector used to introduce a CAR gene reactive with GPC3 (GPC3-CAR) into iPS cells. Figure 2A Graphs showing the results of flow cytometric analysis of surface markers of T cells differentiated from GPC3-CAR-introduced iPS cells (iPS-CAR-T cells) and fresh PBMCs (peripheral blood mononuclear cells). Figure 2BGraphs showing the results of flow cytometry analysis of CAR expressed in iPS-CAR-T cells. Figure 3 This is a schematic diagram of an expression vector for introducing IL-15, or IL-15 and IL-12, IL-18, or IL-21 genes into GPC3-reactive iPS-CAR-T cells. Figure 4 The graph shows the results of ELISA measurement of the production levels of IL-15, IL-12, IL-18, and IL-21 by GPC3-responsive iPS-CAR-T cells into which cytokine genes were introduced. Figure 5 Graphs showing the results of a study on the effect of cytokine gene introduction on the cell proliferation ability of GPC3-responsive iPS-CAR-T cells stimulated by PHA and PBMC. Figure 6 Graphs showing the cytotoxic activity of cytokine-expressing GPC3-reactive iPS-CAR-T cells. Figure 7A The results are from co-culturing cytokine-expressing GPC3-responsive iPS-CAR-T cells and GPC3-gene-introduced SK-HEP-1 cells, and measuring the cytotoxicity of the SK-HEP-GPC3 cells over time. Figure 7B The graph shows the results of co-culturing cytokine-expressing GPC3-responsive iPS-CAR-T cells and GPC3-gene-introduced SK-HEP-1 cells, and measuring the proliferation of the skHep-GPC3 cells over time. Figure 7C The graph shows the results of co-culturing cytokine-expressing GPC3-responsive iPS-CAR-T cells and GPC3-gene-introduced SK-HEP-1 cells, and measuring the proliferation of the iPS-CAR-T cells over time. Figure 8 The graph shows the results of measuring apoptotic cells by staining GPC3-responsive iPS-CAR-T cells expressing cytokines with Annexin V and PI (propidium iodide) in the presence or absence of SK-HEP-1 cells into which the GPC3 gene was introduced. Figure 9A The graph shows the results of evaluating the migration ability of cytokine-expressing GPC3-responsive iPS-CAR-T cells by a Transwell (registered trademark) assay using culture supernatants of JHH-7 cells and skHep-GPC3 cells. Figure 9BGraphs showing the results of flow cytometry analysis of CXCR3 and CCR5 expression in cytokine-expressing GPC3-responsive iPS-CAR-T cells. Figure 9C Graphs showing the results of evaluating the migration ability of cytokine-expressing GPC3-responsive iPS-CAR-T cells to CXCL9, CXCL10, and CXCL11 by a Transwell (registered trademark) assay. Figure 10A The graph shows the results of measuring the effect of anti-human CXCR3 antibodies on the migration of IL-15- and IL-21-expressing GPC3-reactive iPS-CAR-T cells to the periphery of mouse tumors by in vivo imaging using luciferase luminescence. Figure 10B The graph shows the results of analyzing the effect of anti-human CXCR3 antibodies on the infiltration of GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-21 into mouse tumors. Figure 10C Graphs showing the results of analyzing the proliferative ability of anti-human CXCR3 antibodies on GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-21. Figure 10D Graphs show the results of flow cytometric analysis of CXCR3 expression in GPC3-reactive iPS-CAR-T cells into which IL-15 and IL-21 genes were introduced and infiltrated into mouse tumors. Figure 11A Graphs showing the results of quantitative PCR measurement of CXCR3 expression in cytokine-expressing GPC3-responsive iPS-CAR-T cells. Figure 11B Graphs showing the results of Western blotting analysis of the expression of phospho-STAT1, STAT3, and STAT5 in cytokine-expressing GPC3-responsive iPS-CAR-T cells. Figure 11C Graphs showing the analysis results of the relative expression levels of phospho-STAT1, STAT3, and STAT5 in cytokine-expressing GPC3-responsive iPS-CAR-T cells. Figure 11D A diagram showing the region amplified by ChIP-qPCR in the CXCR3 promoter region. Figure 11E Graphs showing the results of ChIP-qPCR verification of the involvement of STAT1, STAT3, and STAT5 in CXCR3 transcription. Figure 12The graph shows the results of intravenous injection of GPC3-reactive iPS-CAR-T cells into NSG mice that had formed tumors in the human liver cancer-derived cell line SK-HEP-1 by gene introduction of GPC3, and the use of luciferase luminescence in vivo imaging to observe the in vivo dynamics of the iPS-CAR-T cells over time. Figure 13A The graph shows the results of administering cytokine gene-introduced GPC3-responsive iPS-CAR-T cells to NSG mice subcutaneously inoculated with the human liver cancer cell line JHH-7, and measuring the tumor volume in the mice over time. Figure 13B It shows Figure 13A Graph showing the survival rate of mice over time. Figure 14A The graph shows the results of time-dependent measurement of tumor volume in NSG mice inoculated subcutaneously with the human liver cancer-derived cell line SK-HEP-1 into which the GPC3 gene was introduced, after administration of GPC3-reactive iPS-CAR-T cells into the mice. Figure 14B It shows Figure 14A Graph showing the survival rate of mice over time. Figure 15 This graph compares the abundance of GPC3-responsive iPS-CAR-T cells into which cytokine genes have been introduced within tumors. Figure 16 This is a graph of single-cell RNA-seq analysis results drawn by UMAP. Figure 17 This figure shows the clusters plotted on UMAP and the distribution of iPS-CAR-T cells into which cytokine genes have been introduced. Figure 18 It shows Figure 16 A diagram showing the analysis results of the cluster 1 genome. Figure 19 It shows Figure 16 A diagram showing the analysis results of the cluster 4 genome. Figure 20 It shows Figure 17 Shown are diagrams showing the results of genomic analysis of clusters 1 and 4 consisting of iPS-CAR-T cells expressing IL-15 and IL-21. Figure 21 Graph showing that iPS-CAR-T cells expressing IL-15 and IL-21 have a young memory phenotype. Figure 22 This is a graph showing the enhancement of the tumor-infiltrating ability of iPS cell-derived TCR-T cells by IL-15 and IL-21. Figure 23 This is a graph showing the tumor growth inhibitory effect of iPS cell-derived TCR-T cells by IL-15 and IL-21. Figure 24 This is a graph showing the tumor growth inhibitory effect of iPS cell-derived TCR-T cells by IL-15 and IL-21. DETAILED DESCRIPTION

[0015] Immune cells induced from iPS cells The immune cells induced from iPS cells of the present invention are not particularly limited as long as they express cell surface molecules and IL-15 that are reactive to tumor-associated antigens. In one embodiment of the present invention, the immune cells induced from iPS cells express IL-12, IL-18 or IL-21 in addition to cell surface molecules and IL-15 that are reactive to tumor-associated antigens. In one embodiment of the present invention, the immune cells induced from iPS cells can co-express all of IL-12, IL-18 and IL-21, or can express two cytokines selected from IL-12, IL-18 and IL-21. In addition, the immune cells induced from iPS cells of the present invention can also express other cytokines and chemokines and their receptors that regulate cell functions or immune functions such as cytotoxic activity, cell proliferation and cell migration.

[0016] In the present invention, "tumor-associated antigens" refer to antigens that are specifically or non-specifically expressed in tumors, including antigens derived from proteins overexpressed in tumor cells and their variants, antigens derived from tumor viruses, certain differentiated antigens, and novel tumor-associated antigens (neoantigens) generated due to gene mutations and splicing abnormalities. If it is a protein antigen, it can be a peptide (peptide fragment) obtained by fragmenting the antigen. In this specification, tumor-associated antigen is used with the same meaning as tumor antigen. Examples of antigens specifically or nonspecifically expressed in tumors include WT1, GPC3, BCMA, XAGE1, MUC1, MUC5A1, MUC6, EGFRvIII, HER-2 / neu, MAGE-A1, MAGE-A3, telomerase, PRAME, SSX2 / 4, PSCA, CTLA-4, gp100, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53 without gene mutation, p53 with gene mutation, p53 mutant, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, MelanA, / MART1, survivin, Ras, Ras mutant, EGR, bcr-ab1, XBP-1, neoantigens caused by gene mutations, neoantigens caused by splicing abnormalities, HBV, HBs, HPV, EBV, LMP1, EBV, LMP2, EBNA, HPV-E1, HPV-E2, HPV-E6, HPV-E7, HTLV-1Tax and HBZ, but are not limited to these.

[0017] In one embodiment of the present invention, the tumor-associated antigen may be selected from the group consisting of GPC3, WT1, BCMA, XAGE1, LMP2, NY-ESO-1, Epstein-Barr virus antigens and neoantigens and peptide fragments thereof.

[0018] In the present invention, "cell surface molecules reactive to tumor-associated antigens" refers to cell surface molecules that specifically bind to tumor-associated antigens on antigen-presenting cells such as cancer cells. In one embodiment of the present invention, the cell surface molecules are T cell receptors (TCRs) and chimeric antigen receptors (CARs).

[0019] TCR can be a heterodimer composed of an α chain and a β chain, or a heterodimer composed of a γ chain and a δ chain. TCR "is reactive to tumor-associated antigens" means that the immune cell expressing the TCR has a reaction that occurs by selectively binding to an epitope peptide derived from a tumor-associated antigen presented by the major histocompatibility complex (MHC) class I or II on the antigen-presenting cell through the TCR, and refers to the absence of a reaction in the immune cell in which the immune cell binds to an epitope peptide other than the above-mentioned epitope peptide. The immune cell response generated by binding to an epitope peptide derived from a tumor-associated antigen presented by MHC class I or II via TCR can include, for example, cytotoxicity, the production of IFN-γ and granzymes, the expression of T cell activation markers, and the activation of transcription factors such as NF-AT.

[0020] CAR is a fusion protein of the antigen recognition portion of an antibody that specifically recognizes a tumor-associated antigen and the intracellular domain of a T cell activation molecule. The antigen recognition portion of the antibody and the intracellular domain of the T cell activation molecule can be bound by a spacer and / or a transmembrane domain. As the extracellular region of the CAR molecule, that is, the antigen recognition portion of the antibody, for example, a single-chain antibody formed by combining the light chain and heavy chain of the variable region of a monoclonal antibody against a tumor-associated antigen in series can be cited. As the intracellular domain of the T cell activation molecule in the CAR molecule, the CD3ζ molecule can be used. In order to fully activate immune cells, costimulatory molecules can also be included in the intracellular domain. As costimulatory molecules, for example, CD27, CD28, 4-IBB, OX40 and ICOS can be cited. The costimulatory molecules can be incorporated into the intracellular domain in combination of two or more. CAR "is reactive to tumor-associated antigens" means that the immune cells expressing CAR specifically bind to tumor-associated antigens expressed on antigen-presenting cells through the antigen recognition portion of the CAR molecule, thereby sending activation signals to the immune cells through the CD3ζ molecule in the intracellular domain of the CAR molecule. Examples of CAR-mediated immune cell responses include inducing apoptosis of antigen-presenting cells by releasing cytotoxic proteins such as perforin and granzymes. Immune cells expressing CAR can recognize antigens expressed on the surface of antigen-presenting cells without restriction using human leukocyte antigens (HLA).

[0021] Preparation of iPS cells In the present invention, iPS cells are prepared by reprogramming mammalian somatic cells. As mammals, humans, monkeys, pigs, dogs, cats, rats, mice, etc. can be mentioned, preferably humans. The somatic cells are not particularly limited, and cells separated from peripheral blood are preferably used. In one embodiment of the present invention, the somatic cells are peripheral blood mononuclear cells or T cells from which B cells and T cells have been removed. Peripheral blood mononuclear cells from which B cells and T cells have been removed can be obtained in the following manner: after monocytes are separated from whole blood using a monocyte separation solution, B cells and T cells are removed using surface antigens expressed by B cells and T cells. As a monocyte separation solution, for example, Lymphoprep (registered trademark) can be mentioned. In order to remove B cells and T cells from monocytes, antibodies against the surface antigens CD19, CD20, CD22 or B cell receptors that B cells have, and the surface antigens CD3, CD4 or CD8 that T cells have can be utilized, for example, using magnetic beads such as flow cytometry or MACS (registered trademark) beads.

[0022] As the collection source of T cells, peripheral blood is preferred because of its low invasiveness, but it is not limited thereto. As other collection sources, cancer tissue or tumor tissue or other tissues, umbilical cord blood, lymph, tissue fluid (interstitial fluid, intercellular fluid and interstitial fluid), body cavity fluid (ascites, pleural effusion, pericardial fluid, cerebrospinal fluid, joint fluid and aqueous humor), nasal discharge, urine, pleural cavity, abdominal cavity, cranial cavity or intraspinal exudate (pleural effusion or ascites etc.) etc. can be enumerated. The cancer tissue or tumor tissue, for example, can be enumerated as being derived from ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphatic leukemia, papillary thyroid carcinoma, colorectal cancer, head and neck cancer, brain tumor, multiple myeloma or B cell non-Hodgkin's lymphoma tissue. In one embodiment of the present invention, the cancer tissue or tumor tissue is hepatocellular carcinoma or hepatoblastoma.

[0023] Methods for producing iPS cells are well known in the art (e.g., see: Takahashi K, Yamanaka S. Cell. 2006; 126: 663-676, Takahashi K, et al. Cell. 2007; 131: 861-872, Nakagawa M, et al. Nat Biotechnol. 2008; 26: 101-106). In one embodiment of the present invention, iPS cells can be induced by introducing cell reprogramming factors into peripheral blood mononuclear cells or T cells from which cells and T cells have been removed. As cell reprogramming factors, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, klf4, klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas and ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 and Glis1 and other genes or gene products can be enumerated. These cell reprogramming factors can be used alone or in combination. In these cell reprogramming factors, from the viewpoint of efficiently setting up iPS cells, preferably Oct3 / 4, Sox2, Klf4 and c-Myc (so-called 4 factors in the mountains) are imported into above-mentioned peripheral blood mononuclear cells or T cells.

[0024] There is no particular limitation on the method for importing cell reprogramming factors into above-mentioned peripheral blood mononuclear cells, monocytes or T cells, methods well known in the art can be used.For example, when the gene encoding the cell reprogramming factors is imported into the peripheral blood mononuclear cells, monocytes or T cells, the gene encoding the cell reprogramming factors (for example, cDNA) is inserted into the expression vector containing the promoter playing a role in the cell, and the expression vector can be imported into cells by infection, lipofection, liposome method, calcium phosphate coprecipitation method, DEAE-dextran method, microinjection or electroporation.When the cell reprogramming factors are in the form of protein, when the protein is imported into peripheral blood mononuclear cells or monocytes or T cells, the method for importing reagents using protein or the method for importing domain fusion proteins using protein, electroporation and microinjection.When the cell reprogramming factors are in the form of messenger RNA (mRNA), when the mRNA is imported into peripheral blood mononuclear cells or monocytes or T cells, the method for importing reagents using mRNA and the method for adding the mRNA into culture medium are used.

[0025] Examples of expression vectors for gene introduction through infection include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, and animal cell expression plasmids. However, from the perspective of being less susceptible to insertional mutations, having high gene introduction efficiency, and having a large number of copies of the introduced gene, it is preferred to use Sendai virus to introduce the gene encoding the cell reprogramming factor into the peripheral blood mononuclear cells or T cells.

[0026] As the promoter used in the expression vector used when the gene encoding the cell reprogramming factor is introduced into the peripheral blood mononuclear cells or T cells, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, ubiquitin promoter, etc. can be enumerated. These promoters can also be substances that can control the expression of genes inserted downstream of the promoter by the presence or absence of drugs such as tetracycline. In addition to the promoter, the expression vector can also contain an enhancer, a polyA tailing signal, a selection marker gene (such as a neomycin resistance gene), an SV40 origin of replication, etc.

[0027] [Introducing cell surface molecules reactive to tumor-associated antigens into iPS cells] In the present invention, for the iPS cells obtained as described above, nucleic acids encoding cell surface molecules reactive to tumor-associated antigens are introduced into iPS cells from outside the cells. The nucleic acids encoding the cell surface molecules can also be introduced into hematopoietic stem cells, immature immune cells or mature immune cells induced by differentiation from iPS cells. The nucleic acids encoding the cell surface molecules are preferably nucleic acids derived from humans. As the nucleic acids encoding cell surface molecules reactive to tumor-associated antigens, for example, nucleic acids encoding TCR and nucleic acids encoding CAR can be cited, which can be nucleic acids of natural origin or artificially synthesized nucleic acids. The sequence information of these nucleic acids can be obtained from well-known documents and databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0028] In the case where the cell surface molecule is TCR, the cDNA encoding TCR alpha chain and beta chain or the cDNA encoding gamma chain and delta chain can be prepared and incorporated into an expression vector. For example, the PCR fragment amplified by these cDNAs as templates can be integrated into a viral vector or a non-viral vector (transposon vector) using the Gibson assembly system. Specifically, in the case of importing TCR, the gene obtained by connecting the cDNA encoding TCR alpha chain and TCR beta chain by the T2A sequence is connected to the downstream of the ubiquitin promoter, and then the marker genes such as EGFR (EGFRt, truncated EGFR) or CD19 of the intracellular domain having the ligand binding site and the intracellular domain removed relative to the IRES (internal ribosome entry site) sequence are connected in its downstream, and the construct is integrated into a viral vector or a non-viral vector. The cDNA encoding TCR alpha chain and the cDNA encoding beta chain can also be respectively incorporated into different expression vectors.

[0029] In one embodiment of the present invention, the preparation of cDNA encoding TCR α chain and β chain respectively can be performed on a single cell using a T cell population that is reactive to tumor-associated antigens and has genetic diversity as a whole as a supply source. After the T cells obtained from the subject are cultured with the target tumor-associated antigen, single cells can be separated from the T cell population that reacts to the tumor-associated antigen used by a cell sorter, etc. using an activation marker. As an activation marker, cell surface CD137 can be cited. As a known method for isolating human T cells, for example, flow cytometry using antibodies against T cell surface markers such as CD3 and CD137 and a cell sorter can be cited. Using the PCR method to clone genes from the obtained single T cells, cDNAs encoding TCR α chain and β chain respectively can be amplified.

[0030] In order to obtain single T cells, CD8 single-positive T cells specific for tumor-associated antigens obtained from peripheral blood, etc. can be combined with MHC Dextramer (registered trademark) that forms a complex with the antigen peptide and single cell sorting using a cell sorter. MHC Dextramer is a compound composed of a dextran polymer backbone formed by combining MHC with a fluorescent pigment molecule. MHC tetramers can be used instead of MHC Dextramers. MHC tetramers refer to tetramers made of a complex of an antigen peptide and an MHC molecule through biotin and avidin. In another embodiment, CD8 single-positive T cells specific for tumor-associated antigens obtained from peripheral blood, etc. can be expanded and cultured in the presence of the antigen, and then single cell sorting of CD3 / CD137 double-positive cells can be performed using a cell sorter. In another embodiment, a cell sorter can be used to single-cell sort the cell population bound to the MHC Dextramer that forms a complex with the antigen peptide from the CD3 / CD137 double-positive cells. RNA is extracted from the resulting single cells, and the cDNA obtained by reverse transcription can be used to isolate TCR gene pairs (TCRα chain gene and TCRβ chain gene) through PCR. The isolated TCR gene pairs can be sequenced to analyze the types of tumor antigen-reactive T cells (TCR repertoire) and their frequency.

[0031] In the case where the cell surface molecule is CAR, a cDNA encoding CAR can be prepared and incorporated into an expression vector. For example, total RNA can be extracted from the lymph nodes of an animal immunized with the desired tumor-associated antigen, and cDNA can be synthesized using this as a template; the light and heavy chains of the variable regions of the monoclonal antibodies against the tumor-associated antigens are amplified separately, combined with flexible joints, and amplified using assembly PCR; by encoding the sequence of the transmembrane domain of the CAR molecule, the sequence encoding the fused light and heavy chains is combined with the sequence encoding the intracellular domain of the CAR molecule to prepare a CAR construct. In order to confirm the expression of the gene introduced into the CAR construct, the EGFR (EGFRt) from which the ligand binding site and the intracellular domain have been removed can be connected by the T2A sequence. The flexible linker that binds the light chain and heavy chain of the variable region of the monoclonal antibody is a linker peptide of about 5 to 20 amino acid residues, which is well known in the art (for example, see Ueda T, et al. Cancer Sci. 2020; 111: 1478-1490. Kawasaki Tomomi et al., SCEJ 72nd Annual Meeting (Kyoto, 2007) H209.).

[0032] The antigen-binding domain incorporated into the CAR can also be prepared by phage display rather than immunizing animals with antigens. For example, antibodies that specifically bind to the desired tumor-associated antigen can be screened from a phage-displayed antibody library expressing a large number of human antibody Fab regions.

[0033] For iPS cells, more than one gene encoding the cell surface molecule can also be introduced. For example, a TCR or CAR reactive to a tumor-associated antigen can be introduced alone, or TCR and CAR can be introduced simultaneously. When TCR and CAR are introduced simultaneously, the expression vectors can be the same or different. When TCR and CAR are introduced simultaneously, TCR and CAR can be reactive to the same tumor-associated antigen or to different tumor-associated antigens. The gene encoding the cell surface molecule can also be introduced into hematopoietic stem cells differentiated from iPS cells or immature immune cells and mature immune cells differentiated from the hematopoietic stem cells. Here, the term "immature immune cells" includes cells at all stages of differentiation from hematopoietic stem cells to mature immune cells, for example, used in the sense of including precursor immune cells and immature immune cells.

[0034] As expression vectors, viral vectors and non-viral vectors can be used. As viral vectors, for example, viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus and Sendai virus, as well as animal cell expression plasmids can be mentioned. Preferably, lentivirus or retrovirus can be used. In the case of retroviral or lentiviral infection, a rotation infection method can be used. As non-viral vectors, transposon vectors such as piggyBac (registered trademark) vectors can be mentioned. In the case of replacing the iPS cell-specific TCR with an extracellularly introduced TCR or CAR, genome editing technology can be used. As genome editing technology, CRISPR / Cas9 method, CRISPR / MAD method and CRISPR / CAS3 method can be mentioned. As a gene introduction method for a non-viral vector or a method for introducing a guide RNA and donor DNA for genome editing, lipofection, liposome method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection and electroporation can be mentioned. TCR and / or CAR gene introduction can be performed on the TCR locus or other loci (such as β2 microglobulin locus). In the case where it is not desired to modify or destroy existing genes including TCRs by the introduced gene, it can also be performed on a safe harbor locus. As such a safe harbor, for example, the AAVS1 (Adeno-associated virus integration site 1) region in the human genome can be cited. As methods for introducing genes targeting such safe harbors, for example, CRISPR / Cas9 and TALEN methods can be cited.

[0035] iPS cells introduced with cell surface molecules reactive to tumor-associated antigens are composed of a large number of iPS cell clones. Therefore, it is also possible to screen for iPS cell clones that have confirmed the genetic introduction of the cell surface molecules or that have maintained certain properties from a collection of iPS cell clones. Examples of methods for screening iPS cell clones include colony picking. There are no particular limitations on colony picking methods, and methods such as using a pipette under a microscope, limiting dilution methods, and methods using fully automated colony selectors can be used. The resulting single iPS cell clone can be cryopreserved after expansion culture. Methods for cryopreservation of cells are well known to those skilled in the art. For example, cultured iPS cell clones can be recovered, washed with a buffer or culture medium, and after counting the number of cells, concentrated by centrifugation, suspended in a freezing medium (e.g., a culture medium containing 10% DMSO), and cryopreserved at low temperatures. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is a temperature suitable for cell storage. Examples include -20°C, -80°C, and -120 to -196°C, preferably below -150°C.

[0036] As a culture medium for culturing iPS cells, there is no particular limitation. A culture medium for culturing animal cells can be used as a basal culture medium, and cytokines for maintaining the undifferentiated ability of iPS cells can be added thereto for preparation. As basal culture media, for example, Iscove's modified Dulbecco's medium (IMDM), culture medium 199, Eagle's minimum essential medium (EMEM), αMEM culture medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F12 culture medium, RPMI 1640 culture medium, Fischer's culture medium, neurobasal medium (Life Technologies), StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply) and mixed culture media thereof can be cited. Serum can be added to the culture medium, or it can be serum-free. As a cytokine, bFGF is preferred, and its concentration in the culture medium is, for example, 1 to 100 μg / mL (preferably 50 μg / mL)

[0037] Differentiation into immune cells from iPS cells into which cell surface molecules reactive to tumor-associated antigens have been introduced In the present invention, as immune cells differentiated from iPS cells, as long as they have immune response capabilities and have the ability to kill cells expressing tumor-associated antigens as targets, there are no particular limitations, and examples include lymphocyte lineage cells such as T cells, B cells, natural killer (NK) cells, and NKT cells. In one embodiment of the present invention, the immune cells are T cells and NK cells. In one embodiment of the present invention, the T cells are mature T cells and CD8 single-positive cytotoxic T cells.

[0038] In one embodiment of the present invention, cell surface molecules iPS cell clones that have been introduced that are reactive to tumor-associated antigens are differentiated into mature T cells as CD8 single-positive T cells via hematopoietic stem cells and immature T cells. The hematopoietic stem cell is a cell that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes and megakaryocytes, and is identified by being double-positive by surface antigens CD34 and CD43. It should be noted that hematopoietic stem cells and hematopoietic progenitor cells (HPCs) are not distinguished from each other and refer to the same cells unless otherwise specified. The immature T cells are cells in all stages of differentiation between hematopoietic stem cells and mature T cells, for example, including precursor T cells and immature T cells. Therefore, the immature T cells are T cells in each stage from the T cell stage in which neither the TCR α chain nor the β chain is expressed to the CD4 / CD8 double-positive cells expressing the TCR α chain and the β chain. In one embodiment of the present invention, mature immune cells induced from iPS cells refer to T cells that express TCR α and β chains and have progressed from CD4 / CD8 double-positive cells to CD8 single-positive cells, preferably CD8 α chain / β chain double-positive.

[0039] Hematopoietic stem cells are preferably prepared by culturing iPS cells in a culture medium supplemented with vitamin C. Vitamin C refers to L-ascorbic acid and its derivatives. Examples of L-ascorbic acid derivatives include ascorbic acid phosphate, ascorbyl glucoside, ethyl ascorbic acid, vitamin C esters, ascorbic acid tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6-palmitate. For example, vitamin C can be contained in the culture medium at a concentration of 5 to 500 μg / mL.

[0040] The culture medium for preparing hematopoietic stem cells is not particularly limited. A culture medium for culturing animal cells can be used as a basal culture medium and prepared by adding vitamin C or the like. Examples of basal culture media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's, Neurobasal Medium (Life Technologies), StemPro34 (Life Technologies), and mixed cultures thereof. Serum can be added to the culture medium, or serum-free medium can be used. As needed, the basal culture medium may contain one or more selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, etc.

[0041] Cytokines selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and FLT3L (Flt3 ligand) can be further added to the culture medium for preparing hematopoietic stem cells. The concentrations of these cytokines in the culture medium are, for example, 1-100 ng / mL for BMP4, 1-100 ng / mL for VEGF, 1-100 ng / mL for bFGF, 10-100 ng / mL for SCF, 1-100 ng / mL for TPO, and 1-100 ng / mL for FLT3L.

[0042] TGFβ inhibitors can be added to the culture medium for hematopoietic stem cells. TGFβ inhibitors are low-molecular-weight inhibitors that interfere with TGFβ family signaling. Examples include SB431542 and SB202190 (e.g., see Lindemann RK, et al. Mol Cancer. 2003; 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, and SD208 (Scios), and LY2109761, LY364947, and LY580276 (Lilly Research Laboratories). The concentration of these inhibitors added to the culture medium is preferably 0.5 to 100 μM.

[0043] iPS cells can be co-cultured with feeder cells such as C3H10T1 / 2 (see, for example, Takayama N, et al. J Exp Med. 2010; 2817-2830) or allogeneic stromal cells (see, for example, Niwa A, et al. J Ce11 Physiol. 2009; 221: 367-377).

[0044] The culture method of iPS cells of hematopoietic stem cells during preparation can be adhesion culture or suspension culture, preferably suspension culture. For example, iPS cells can be cultured to 80% confluence relative to the culture dish used to separate the colonies, dissociate into single cells, and then provide for suspension culture. As a method for isolating iPS cells, for example, a physical separation method using a cell scraper, a dissociation solution using protease activity and collagenase activity (for example, Accutase (registered trademark) and Accumax (registered trademark) etc.) or a separation method using a dissociation solution having collagenase activity can be cited.

[0045] Suspension culture refers to culturing cells in a state where the cells do not adhere to the culture vessel. Suspension culture is not particularly limited and can be carried out in a culture vessel that has not been artificially treated (e.g., coated with an extracellular matrix, etc.) to improve the adhesion to the cells, or using a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or a nonionic surfactant polyol (Pluronic F-127, etc.)). When suspension culture is carried out, it is preferably formed into embryoid bodies (EBs) for cultivation. When the embryoid bodies are suspended and cultured to obtain hematopoietic stem cells, it is preferably dissociated into single cells and then subjected to adhesion culture.

[0046] Hematopoietic stem cells can also be prepared from cyst-like structures (also called iPS-sacs) obtained by culturing iPS cells. Here, "cyst-like structure" refers to a three-dimensional sac-like structure (with a space inside) derived from iPS cells, which is formed by a group of endothelial cells and contains hematopoietic stem cells.

[0047] Hematopoietic stem cells can be cultured in adhesion or suspension, preferably in adhesion culture. In the case of adhesion culture, the culture vessel can be coated for use. For example, as coating agents, matrigel (reference Niwa A, et al. PLoS One. 6 (7): e22261, 2011), collagen, gelatin, laminin, heparan sulfate proteoglycan, fibronectin, Fc-DLL4 or nidogen, and combinations thereof can be cited.

[0048] CD4 / CD8 double-positive T cells are cells that are positive for both surface antigens CD4 and CD8 (CD8 + CD4 + Since T cells can be identified by their surface antigens CD3 and CD45, CD4 / CD8 double-positive T cells can be identified as cells that are positive for CD4, CD8, CD3, and CD45. CD4 / CD8 double-positive T cells can be induced to differentiate into CD4 single-positive cells or CD8 single-positive cells.

[0049] CD4 / CD8 double-positive T cells can be prepared by a method comprising the step of culturing hematopoietic stem cells in a medium supplemented with a p38 inhibitor and / or SDF-1.

[0050] A p38 inhibitor is defined as a substance that inhibits the function of the p38 protein (p38 MAP kinase). p38 inhibitors include, but are not limited to, chemical inhibitors of p38, dominant negative mutants of p38, or nucleic acids encoding the same.

[0051] Chemical inhibitors of p38 include, but are not limited to, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB202190 (4-(4-fluorophenyl)-2)-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol) and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SCl0-469, SCIO-323, SCIO-323, VX-702, or FR167653. These compounds are commercially available, for example, SB203580, SB202190, SC239063, SB220025, and PD169316 are available from Calbiochem, and SC10-469 and SC10-323 are available from Scios, etc. The p38 inhibitor is added to the culture medium at, for example, about 1 μM to about 50 μM.

[0052] Examples of dominant negative mutants of p38 include p38T180A, in which the threonine at position 180 in the DNA binding region of p38 is point mutated to alanine, and p38Y182F, in which the tyrosine at position 182 in human and mouse p38 is point mutated to phenylalanine.

[0053] SDF-1 (stromal cell-derived factor 1) is not limited to SDF-1α or its mature form, but may also be isoforms such as SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, or SDF-1φ, or their mature forms, or mixtures thereof in any ratio. SDF-1α is preferably used. SDF-1 is added to the culture medium, for example, at a concentration of about 10 ng / mL to about 100 ng / mL.

[0054] The culture medium for producing CD4 / CD8 double positive T cells is not particularly limited. It can be prepared by using a culture medium for culturing animal cells as a basal culture medium, and adding a p38 inhibitor and / or SDF-1 and preferably adding vitamin C. As a basal culture medium, for example, Iscove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, αMEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, Ham's F12 culture medium, RPMI1640 culture medium, Fischer's Neurobasal Medium (Life Technologies) and mixed culture medium thereof can be cited. Serum can be added to the culture medium, or it can be serum-free. The basal culture medium may contain, for example, one or more substances selected from albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts and cytokines.

[0055] The culture medium for preparing CD4 / CD8 double-positive T cells may also contain cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT3L, and IL-7. The concentrations of these cytokines are, for example, 10-100 ng / mL for SCF, 10-200 ng / mL for TPO, 1-100 ng / mL for FLT3L, and 1-100 ng / mL for IL-7.

[0056] The obtained CD4 / CD8 double-positive T cells can be isolated and used. For isolation, methods well known to those skilled in the art can be used. For example, methods include labeling with antibodies against CD4, CD8, CD3, and / or CD45 and separation using a flow cytometer, or purification using an affinity column immobilized with the desired antigen.

[0057] "CD8 single positive T cells" are mature T cells that are positive for the surface antigen CD8 (CD8 + CD4 - ), also known as cytotoxic T cells. Since T cells can be identified by being positive for the surface antigens CD3 and CD45, CD8 single-positive T cells can be identified as cells that are positive for CD8, CD3, and CD45 but negative for CD4.

[0058] CD8 single-positive T cells can be produced by culturing CD4 / CD8 double-positive T cells in a culture medium supplemented with a corticosteroid. The corticosteroid is preferably a glucocorticoid or a derivative thereof, examples of which include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone dipropionate. Dexamethasone is preferably used as a corticosteroid. Its concentration in the culture medium is, for example, 1 to 100 nM.

[0059] The culture medium for manufacturing CD8 single positive T cells is not particularly limited, and the culture medium for animal cell culture can be used as a basal culture medium, and adrenocortical hormone is added thereto to prepare it. As a basal culture medium, for example, IMDM culture medium, Medium 199 culture medium, EMEM culture medium, αMEM culture medium, DMEM culture medium, Ham's F12 culture medium, RPMI 1640 culture medium, Fischer neurobasal culture medium (Fischer's Neurobasal Medium) (Life Technologies) and its mixed culture medium can be enumerated. Serum can be added to the culture medium, or it can be serum-free. As needed, the basal culture medium can contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts and cytokines.

[0060] The culture medium used to prepare CD8 single-positive T cells may further contain anti-CD3 antibodies, vitamin C, or cytokines. Examples of such cytokines include IL-2, IL-7, IL-15, and IL-21. The anti-CD3 antibody is not particularly limited as long as it specifically recognizes CD3. Examples include antibodies produced by the OKT3 clone. The concentration of the anti-CD3 antibody in the culture medium is, for example, 10 to 1000 ng / mL.

[0061] In one embodiment of the present invention, iPS cell clones into which cell surface molecules reactive to tumor-associated antigens have been introduced are differentiated into mature NK cells via hematopoietic stem cells and immature NK cells. The maturation of human NK cells can be judged using cell surface antigens such as CD7, CD16, CD56, and NKG2A as differentiation markers. The immature NK cells are cells at all stages of differentiation between hematopoietic stem cells and mature NK cells, for example, including precursor NK cells and immature NK cells.

[0062] Mature NK cells can be prepared from embryoid bodies (EBs) made from iPS cell clones. To prepare embryoid bodies, iPS cell clones cultured on a culture dish can be dissociated, transferred to a low-adhesion culture dish, and cultured using a culture medium containing a ROCK inhibitor. As a culture medium, for example, StemFit (registered trademark) AK03N (Ajinomoto) can be cited, which can increase the number of iPS cells in an undifferentiated state. As a ROCK inhibitor, for example, Y-27632 can be cited, but it is not limited to this. Y-27632 can be added to the culture medium at a concentration of 1 to 100 μM, for example. As a method for dissociating iPS cell clones, for example, physical separation methods such as cell scrapers and methods using dissociation solutions with protease activity such as trypsin (for example, TrypLE (registered trademark) select, ThermoFisher) can be cited.

[0063] The resulting embryoid bodies can be cultured in EB culture medium containing cytokines such as BMP4, bFGF, and VEGF. The concentrations of these cytokines in the EB culture medium are, for example, 1 to 100 ng / mL for BMP4, 1 to 100 ng / mL for bFGF, and 1 to 100 ng / mL for VEGF. As an EB culture medium, for example, StemPro (registered trademark)-34 (ThermoFisher) containing 2 mM L-glutamine, 400 μM monothioglycerol, 50 μg / mL ascorbic acid-2-phosphate, insulin, transferrin, and selenium can be used.

[0064] As long as the desired cell state can be obtained, the number of culture days is not particularly limited. After 2 to 6 days from the start of culture, the embryoid bodies can be cultured in EB medium supplemented with a hematopoietic cytokine cocktail containing SCF (10 to 100 ng / mL), FLT3L (1 to 100 ng / mL), IL-3 (1 to 100 ng / mL), and TPO (1 to 100 ng / mL). After 10 to 18 days from the start of culture, the differentiated cells can be transferred to a culture dish coated with FcDLL4 and cultured in EB medium supplemented with a T cell line cytokine cocktail containing FLT3L (1 to 100 ng / mL) and IL-7 (1 to 100 ng / mL). 21 to 36 days after the start of culture, the hematopoietic cells differentiate into CD7 / CD45 double-positive lymphocyte precursor cells. The lymphocyte precursor cells are co-cultured with irradiated allogeneic PBMCs (peripheral blood mononuclear cells) as feeder cells and expanded using PHA (phytohemagglutinin) to obtain NK cells that express cell surface molecules reactive to tumor-associated antigens. The prepared mature NK cells are CD3, CD4, CD5, CD8α, and CD8β negative, and CD56, CD159a, CD161, CD226, CD314, CD336, and CD337 positive.

[0065] [Introduction of cytokine genes into immune cells expressing cell surface molecules reactive to tumor-associated antigens] In the present invention, nucleic acids encoding cytokines are introduced extracellularly into hematopoietic stem cells, immature immune cells, or mature immune cells expressing cell surface molecules reactive to tumor-associated antigens, as described above. The cytokines are IL-15, IL-12, IL-18, and IL-21. The nucleic acids encoding these cytokines are preferably human-derived and can be naturally derived or synthetically synthesized. Sequence information for these nucleic acids can be obtained from publicly available literature and databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0066] In the present invention, the cytokine gene introduced into the hematopoietic stem cells, immature immune cells, or mature immune cells is a nucleic acid encoding IL-15, and in one embodiment, a nucleic acid encoding IL-15 and IL-12, a nucleic acid encoding IL-15 and IL-18, or a nucleic acid encoding IL-15 and IL-21. Cytokine-encoding cDNAs can be prepared and incorporated into expression vectors.

[0067] As expression vectors, viral vectors and non-viral vectors can be used. As viral vectors, for example, viral vectors such as retrovirus, slow virus, adenovirus, adeno-associated virus, herpes virus and Sendai virus, and animal cell expression plasmids can be mentioned. Preferably, retrovirus or slow virus can be used. In the case of retrovirus or slow virus infection, rotation infection method etc. can be used. As non-viral vectors, transposon vector piggyBac (registered trademark) vector etc. can be mentioned. As a gene introduction method as a non-viral vector, lipofection method, liposome method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method and electroporation method can be mentioned.

[0068] In order to prevent any region of the locus group specifically expressed in the hematopoietic stem cells, immature immune cells or mature immune cells, or to prevent the mutation or deletion of existing genes caused by the introduced genes, the above-mentioned cytokine genes can also be carried out on the safe harbor locus. As the safe harbor, for example, the AAVS1 (Adeno-associated virus integration site 1) region in the human genome can be cited. As a method for introducing genes targeting the safe harbor, for example, CRISPR / Cas9 method and TALEN method can be cited.

[0069] When introducing two cytokine genes, they can be incorporated into a single expression vector, or they can be incorporated into separate expression vectors. Specifically, the two nucleic acids encoding the two cytokine genes can be transcribed from a single promoter using an IRES (internal ribosome entry site) or a self-cleaving 2A peptide, or they can be transcribed from separate promoters. When incorporating two cytokine genes into a single expression vector, i.e., nucleic acids encoding IL-15 and IL-12, nucleic acids encoding IL-15 and IL-18, and nucleic acids encoding IL-15 and IL-21, one nucleic acid can be positioned upstream or downstream of the other.

[0070] [Medicament containing the cells of the present invention] The drug containing the cells of the present invention can be used as a preventive or therapeutic agent for mammalian cancer. The drug of the present invention can be prepared by methods commonly used in the field of pharmaceutical technology. The drug of the present invention can contain pharmaceutically acceptable additives. Examples of such additives include cell culture medium, physiological saline, and appropriate buffers (e.g., phosphate buffer).

[0071] The medicament of the present invention can be prepared by suspending the cells of the present invention in physiological saline or a suitable buffer (e.g., phosphate buffer). In order to achieve the desired therapeutic effect, a single dose may contain, for example, 1×10 7 More than 1×10 8 More than or 1×10 9 More than 100 cells. The cell content can be adjusted taking into account the sex, age, weight, condition of the affected area and the condition of the cells of the subject. In order to protect the cells, the drug of the present invention may also contain dimethyl sulfoxide (DMSO) and serum albumin in addition to the cells of the present invention. In addition, in order to prevent bacteria from mixing in, it may contain antibiotics and the like. In addition, it may contain vitamins, cytokines, etc. for promoting the activation and differentiation of cells. In addition, the drug of the present invention may contain other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, analgesics, stabilizers, preservatives, preservatives, physiological saline, etc.).

[0072] The pharmaceutical composition comprising the cells of the present invention can be cryopreserved. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is suitable for the storage of the cells. For example, -20°C, -80°C, and -150°C to -196°C can be mentioned, preferably below -150°C. In the case of cryopreservation, the cells can be stored in appropriate containers such as cryovials and freezing bags.

[0073] The medicament of the present invention can be used to prevent or treat cancer. Examples of cancer include, but are not limited to, ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid carcinoma, colorectal cancer, head and neck cancer, brain tumors, multiple myeloma, and B-cell non-Hodgkin's lymphoma.

[0074] The cells of the present invention can kill cells expressing tumor-associated antigens, and thus can be used as a killing agent for cells expressing tumor-associated antigens. The killing agent can be prepared and used in the same manner as the drug.

[0075] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0076] Statistical Analysis The statistically significant differences mentioned in this specification were analyzed by one-way ANOVA using GraphPad Prism 8 software and Tukey's multiple comparison test ( * P < 0.05; ** P < 0.01;*** P < 0.005; **** P<0.001). Example 1

[0077] Preparation of iPS-T cells introduced with a CAR that reacts with GPC3 use Figure 1 The lentiviral expression vector shown was used to introduce a CAR (GPC3-CAR) that reacts with the tumor-associated antigen GPC3 (Glypican-3) into iPS cells. Clonal cells were obtained from iPS cells confirmed to have been gene introduced and differentiated into T cells. The expression of surface markers TCR α chain and β chain, CD3, CD4, CD5, and CD8 α chain and β chain of the differentiated cells (GPC3 reactive iPS-CAR-T cells) was analyzed by flow cytometry. The results confirmed that GPC3 reactive iPS-CAR-T cells were CD8 α chain / β chain double positive cells ( Figure 2A ). In addition, the results of flow cytometric analysis of CAR confirmed that mature GPC3-reactive iPS-CAR-T cells expressed the EGFR gene linked to the T2A sequence, indicating that CAR ( Figure 2B ). Example 2

[0078] [Introduction of cytokine genes into GPC3-responsive iPS-CAR-T cells] use Figure 3 The retroviral expression vector shown was used to introduce cytokine genes (IL-15, IL-12, IL-18, and IL-21) into the CD8α chain / β chain double-positive GPC3-reactive iPS-CAR-T cells prepared in Example 1. Flow cytometry was used to evaluate the expression of the fluorescent protein mCherry incorporated into the expression vector by binding to the IRES sequence. The results confirmed that cells introduced with either vector construct expressed mCherry to the same extent.

[0079] GPC3-responsive iPS-CAR-T cells incorporating cytokine genes were cultured for 48 hours, and the production of various cytokines in the culture supernatant was measured by ELISA. The results are shown in Figure 4. The meanings of the terms used in the figure are as follows. That is, iPS-CART+mock refers to mock (mock) cells; iPS-CART+IL-15 refers to IL-15 gene-introduced cells; iPS-CART+IL-15 / 12 refers to IL-15 and IL-12 gene-introduced cells; iPS-CART+IL-15 / 18 refers to IL-15 and IL-18 gene-introduced cells; and iPS-CART+IL-15 / 21 refers to IL-15 and IL-21 gene-introduced cells. The experiment was performed in three independent cases, and the results were expressed as the mean ± standard error of the cytokine concentration in the cell culture medium.

[0080] The GPC3-responsive iPS-CAR-T cells into which the cytokine gene was introduced showed a significant increase in the production of cytokines corresponding to the introduced cytokine gene. Then, in the absence of CAR-specific stimulation, the GPC3-responsive iPS-CAR-T cells into which the cytokine gene was introduced were stimulated with PHA (phytohemagglutinin) and PBMC (peripheral blood mononuclear cells) to study the relationship between the cytokines produced by the cells and the cell proliferation ability. The results are shown in Figure 5 The meanings of the terms in the figure are as described above, and Figure 4 The same. The experiment was performed in three independent cases, and the results are expressed as the mean ± standard error of the number of cells. As a result, the cell proliferation ability of cells expressing IL-15 and IL-18, as well as cells expressing IL-15 and IL-21, was significantly enhanced. On the other hand, the cell proliferation ability of cells expressing IL-15 and IL-12 was significantly reduced. Almost no effect on cell proliferation ability was observed in cells expressing only IL-15. The above results show that by introducing cytokine genes into GPC3-responsive iPS-CAR-T cells to express cytokines, the cell proliferation ability will be affected. Example 3

[0081] Cytotoxic activity of GPC3-responsive iPS-CAR-T cells expressing cytokines The cytokine-expressing GPC3-responsive iPS-CAR-T cells prepared in Example 2 were used as effector cells (E), and GPC3-positive human hepatoma cell lines JHH-7 and HuH-7, as well as the human hepatoma-derived cell line SK-HEP-1 (skHep-GPC3) into which the GPC3 gene was introduced, and as a control, SK-HEP-1 (skHep-vec) into which GPC3 was not introduced, were used as target cells (T). The cytotoxic activity was studied by a non-radioactive cytotoxicity assay. The results are shown in Figure 6. The meanings of the terms used in the figure are as follows. That is, iPS-CART+mock (mock cells), iPS-CART+IL-15 (IL-15 gene introduced into cells), iPS-CART+IL-15 / 12 (IL-15 and IL-12 gene introduced into cells), iPS-CART+IL-15 / 18 (IL-15 and IL-18 gene introduced into cells) and iPS-CART+IL-15 / 21 (IL-15 and IL-21 gene introduced into cells), and control iPS-T cells (iPS-T). The experiment was carried out in 4 independent cases, and the results are expressed as mean ± standard error.

[0082] GPC3-responsive iPS-CAR-T cells expressing IL-15 / 21 (iPS-CART+IL-15 / 21) showed cytotoxic activity against all target cells used (JHH-7, HuH-7, and skHep-GPC3), and their effect was significantly enhanced compared with mock cells without the introduction of cytokine genes ( Figure 6 ). In addition, among the effector cells studied, iPS-CART+IL-15 / 21 showed the strongest cytotoxic activity. The above-mentioned cells expressing IL-15 and IL-18, as well as IL-15 and IL-12, also confirmed stronger cytotoxic activity than the simulated cells. The above-mentioned cells expressing IL-15 showed cytotoxic activity against HuH-7 and skHep-GPC3. In addition, no nonspecific cytotoxic activity that may be produced when cytokines are expressed was observed. From the above, it can be seen that GPC3-responsive iPS-CAR-T cells can maintain specific cytotoxic activity against GPC3 even if cytokines are expressed. It should be noted that for skHep-vec that does not express GPC3, no cytotoxic activity was observed for GPC3-responsive iPS-CAR-T cells expressing the above-mentioned cytokines.

[0083] The results of co-culture of cytokine-expressing GPC3-responsive iPS-CAR-T cells with skHep-GPC3 cells and measurement of cytotoxicity against skHep-GPC3 cells over time are shown in FIG. Figure 7A The results of measuring the proliferation of skHep-GPC3 cells over time are shown in Figure 7B The results of measuring the proliferation of iPS-CAR-T cells responsive to GPC3 expressing cytokines over time are shown in Figure 7C A real-time cell analyzer, xCELLigence (Agilent), was used for all measurements. Figure 7A "Tumor" in A and B indicates the result when the iPS-CAR-T cells are not present, and "iPS-T" indicates the control iPS-T cells without CAR molecules. Figure 7AIn A, B, and C, the experiments were performed in three independent cases, and the results are expressed as the mean ± standard error.

[0084] The cytotoxic activity of GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21 against skHep-GPC3 cells did not diminish over time for more than 100 hours and remained significantly stronger than that of mock cells ( Figure 7A In contrast, the inhibitory effect of GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21 on skHep-GPC3 cell proliferation lasted for more than 100 hours, and was significantly stronger than that of the mock cells ( Figure 7B ). This effect was also confirmed in GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-18, expressing IL-15 and IL-12, and expressing only IL-15, but it was weaker. In addition, about 70 hours after co-culture, the cytotoxicity was eliminated.

[0085] In co-culture with skHep-GPC3 cells, GPC3-reactive iPS-CAR-T cells expressing IL-15, IL-15 and IL-18, and IL-15 and IL-21 increased in cell number in a time-dependent manner until day 7 of co-culture, whereas GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-12 did not show sustained cell proliferation. This suggests that the cytotoxic activity and cell proliferation inhibitory effects of cytokine-expressing GPC3-reactive iPS-CAR-T cells are not solely dependent on cell number. Example 4

[0086] [Anti-apoptotic activity of cytokine-expressing GPC3-responsive iPS-CAR-T cells] The cytokine-expressing GPC3-responsive iPS-CAR-T cells prepared in Example 2 were cultured in the presence or absence of the human liver cancer-derived cell line SK-HEP-1 (skHep-GPC3) into which the GPC3 gene was introduced, and flow cytometry analysis was performed by Annexin V and PI (propidium iodide) staining to detect apoptotic cells. The results are shown in Figure 8 The experiment was performed in three independent cases, and the results are expressed as mean ± standard error.

[0087] In flow cytometry analysis, late apoptotic cells were stained with both Annexin V and PI. In the absence of antigenic cells skHep-GPC3 cells, apoptosis of GPC3-responsive iPS-CAR-T cells expressing IL-15, expressing IL-15 and IL-18, and expressing IL-15 and IL-21 was significantly less than that of mock cells ( Figure 8In the presence of skHep-GPC3 cells, the number of late apoptotic cells in GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21 was significantly less than that in GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-15 and IL-12 ( Figure 8 These results indicate that cytokine-expressing GPC3-responsive iPS-CAR-T cells, particularly those expressing IL-15 and IL-21, exhibit strong anti-apoptotic activity in tumor tissues, with sustained anti-tumor effects. Example 5

[0088] [In vitro migration ability of cytokine-expressing GPC3-responsive iPS-CAR-T cells] The migration ability of the cytokine-expressing GPC3-responsive iPS-CAR-T cells prepared in Example 2 was evaluated by Transwell (registered trademark) using the culture supernatant of JHH-7 cells and skHep-GPC3 cells cultured for 2 days. The test was performed as follows. That is, the culture supernatant was added to the plate wells of the Transwell, and the plate insert (plate insert) (pore size 5μm polycarbonate membrane) containing the iPS-CAR-T cells was inserted into the wells and cultured for 2 hours, and the number of migrating cells was measured. The results are shown in Figure 9A . The experiment was performed in three independent cases, and the results are expressed as mean ± standard error. When the culture supernatant of JHH-7 cells was used, the migration ability of GPC3-responsive iPS-CAR-T cells expressing cytokines was enhanced, especially the iPS-CAR-T cells expressing IL-15 and IL-21 and IL-15 and IL-18 had significantly enhanced migration ability compared with the mock cells. On the other hand, when the culture supernatant of skHep-GPC3 cells was used, the migration ability of iPS-CAR-T cells expressing IL-15 and IL-12 and IL-15 and IL-21 was significantly enhanced compared with the mock cells.

[0089] CCR5 and CXCR3 are representative chemokine receptors that play an important role in cell migration, especially in the migration of primary CD8-positive T cells to solid tumors. Therefore, the expression of CXCR3 and CCR5 in cytokine-expressing GPC3-responsive iPS-CAR-T cells was analyzed by flow cytometry. The results are shown in Figure 9BThe experiment was performed in 7 independent cases, and the results are expressed as mean ± standard error. According to the analysis results, for chemokine receptor expression, CCR5 in iPS-CAR-T cells expressing IL-15 and IL-12, and CXCR3 in iPS-CAR-T cells expressing IL-15 and IL-18 and IL-15 and IL-21 were significantly increased compared with mock cells.

[0090] As described above, CXCR3 expression is increased in GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-18 as well as IL-15 and IL-21. Therefore, the migration ability of CXCL9, CXCL10, and CXCL11, which are secreted as CXCR3 ligands and enhanced in various tumors, was studied. The migration ability was evaluated by the Transwell (registered trademark) assay as described above. The results are shown in Figure 9C The experiment was performed in three independent cases, and the results are expressed as mean ± standard error. The results showed that the migration ability of GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21 to CXCL9, CXCL10, and CXCL11 was significantly enhanced compared with mock cells and IL-15-expressing GPC3-responsive iPS-CAR-T cells. Example 6

[0091] [In vivo migration ability of GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-21] The results of Example 5 showed that GPC3-reactive iPS-CAR-T cells expressing IL-15 and IL-21 (IL-15 / 21) migrated to solid tumors. Therefore, using tumor-bearing mice, the migration characteristics of GPC3-reactive iPS-CAR-T cells expressing IL-15 / 21 prepared in Example 2 to solid tumors were studied.

[0092] NSG (NOD-SCID IL2Rγc) was purchased from Oriental Bio. null On day 0, human liver cancer cell line JHH-7 (0.5×10 6 On day 14, anti-human CXCR3 antibody or IL-15 / 21 gene-transferred GPC3-reactive iPS-CAR-T cells treated with anti-human IgG1 antibody as a control were intravenously administered (1×10 7On day 16, the iPS-CAR-T cells were observed by in vivo imaging using luciferase luminescence. As a result, the luciferase luminescence around the tumor of mice was significantly reduced when the iPS-CAR-T cells treated with anti-human CXCR3 antibody were administered compared with the cells treated with anti-human IgG1 antibody ( Figure 10A ). Then, the mice were dissected and the number of iPS-CAR-T cells infiltrating the mouse tumor was analyzed by measuring human CD45-positive and GFP-positive cells. As a result, it was confirmed that anti-human CXCR3 antibody treatment significantly reduced the number of cells infiltrating the tumor ( Figure 10B ). The iPS-CAR-T cells were labeled with CytoTell Blue (registered trademark, AAT Bioquest), a cell proliferation monitoring reagent, before administration to mice to confirm that the reduction in the number of tumor-infiltrating cells caused by anti-human CXCR3 antibody treatment was not due to cell proliferation ability. That is, no significant difference in the mean fluorescence intensity of CytoTell Blue, which detects cell division, was confirmed between the anti-human CXCR3 antibody-treated group and the anti-human IgG1 antibody-treated group ( Figure 10C ).

[0093] The expression of CXCR3 in GPC3-responsive iPS-CAR-T cells infiltrating mouse tumors into which the IL-15 / 21 gene was introduced was analyzed by flow cytometry and compared with that in the cells before administration to mice ( Figure 10D The results showed that CXCR3 expression was enhanced in the iPS-CAR-T cells infiltrating the tumor. The above results indicate that IL-15 / 21-expressing GPC3-reactive iPS-CAR-T cells migrate in a CXCR3-dependent manner within mouse tumors. This suggests that intravenously administered IL-15 / 21-expressing GPC3-reactive iPS-CAR-T cells migrate to solid tumors, accumulate in tumor tissue, and exert anti-tumor effects. Example 7

[0094] [Analysis of the molecular mechanism underlying enhanced CXCR3 expression in GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21] The molecular mechanism of CXCR3 expression regulation migration in GPC3-responsive iPS-CAR-T cells expressing IL-15 and IL-21 (IL-15 / 21) was analyzed. First, the relative expression of CXCR3 expression relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in GPC3-responsive iPS-CAR-T cells expressing IL-15, IL-21 or IL-15 / 21 prepared in Example 2 was evaluated by quantitative PCR. As a result, compared with the CXCR3 expression of cells expressing IL-15 or IL-21, the expression of CXCR3 in cells expressing IL-15 / 21 was significantly enhanced ( Figure 11A In the figure, iPS-CAR-T cells expressing IL-15 are represented as "iCAR-T+IL-15", iPS-CAR-T cells expressing IL-21 are represented as "iCAR-T+IL-21", and iPS-CAR-T cells expressing IL-15 / 21 are represented as "iCAR-T+IL-15 / 21".

[0095] It has been reported that IL-15 and IL-21 can phosphorylate the tyrosine residues of the transcription factors STAT1, STAT3, and STAT5 and activate transcription (O'Shea JJ, et al. Annu Rev Med. 2015; 66: 311-328). Next, the expression of phospho-STAT1, STAT3, and STAT5 in iCAR-T+IL-15, iCAR-T+IL-21, and iCAR-T+IL-15 / 21 cells was analyzed by Western blotting. The following antibodies were used for detection: GAPDH rabbit mAb (Clone: ​​14C10), Phospho-Stat1 (Tyr701) rabbit mAb (Clone: ​​58D6), Phospho-Stat3 (Tyr705) rabbit mAb (Clone: ​​D3A7), Phospho-Stat5 (Tyr694) rabbit mAb (Clone: ​​C11C5), Stat1 rabbit mAb (Clone: ​​D1K9Y), Stat3 rabbit mAb (Clone: ​​D3Z2G), and Stat5 rabbit mAb (Clone: ​​D2O6Y). These antibodies were purchased from Cell Signaling Technology. The relative expression of STAT1, STAT3, and STAT5 relative to GAPDH was evaluated by image analysis using Compass software.

[0096] The results of Western blotting analysis are shown in Figure 11B and Figure 11C .right Figure 11BThe stained images were analyzed and the results showed that the phosphorylated STAT1, STAT3 and STAT5 in iCAR-T+IL-15 / 21 cells were significantly increased compared with other cells ( Figure 11C In particular, the expression of phosphorylated STAT1 in iCAR-T+IL-15 / 21 cells was significantly increased compared to that in either iCAR-T+IL-15 or iCAR-T+IL-21 cells.

[0097] To verify that STAT1, STAT3, and STAT5 are directly involved in CXCR3 transcription, chromatin immunoprecipitation (ChIP)-qPCR was performed on the CXCR3 promoter region using anti-STAT1, anti-STAT3, and anti-STAT5 antibodies. Anti-rabbit IgG (#2729, Cell Signaling Technology) was used as a control antibody. Figure 11D The figure shows three regions amplified by PCR using three different primer pairs in the promoter region of CXCR3. The results of ChIP-qPCR showed that STAT1 significantly bound to the promoter region of CXCR3 ( Figure 11E ). Example 8

[0098] [In vivo dynamics of GPC3-responsive iPS-CAR-T cells transfected with cytokine genes in mice transplanted with the human hepatocellular carcinoma-derived cell line SK-HEP-1] The human hepatocellular carcinoma-derived cell line SK-HEP-1 (2.5×10 6 After tumor nodules were confirmed in mice, GPC3-responsive iPS-CAR-T cells (5×10 6 , refer to Example 2). Afterwards, luciferase luminescence was observed over time by in vivo imaging. The results are shown in Figure 12 . On the 14th day after the administration of the iPS-CAR-T cells that introduced the cytokine gene, no luciferase luminescence was observed around the mouse tumor for the mock cell, IL-15, and iPS-CAR-T cell administration groups expressing IL-15 and IL-12 (IL-15 / 12). On the other hand, in iPS-CAR-T cells expressing IL-15 and IL-18 (IL-15 / 18) and IL-15 and IL-21 (IL-15 / 21), luciferase luminescence was confirmed to be maintained around the tumor. In particular, iPS-CAR-T cells expressing IL-15 / 21 showed that luciferase luminescence was maintained the longest.

[0099] Then, after administering GPC3-responsive iPS-CAR-T cells into which cytokine genes were introduced, immunofluorescence staining of the mouse tumors was performed using anti-CD3 antibodies, anti-Ki67 antibodies, and DAPI. Compared to tumors administered with iPS-CAR-T cells expressing IL-15, IL-15 / 12, and IL-15 / 18, there were more CD3-positive cells in the tumors of mice administered with iPS-CAR-T cells expressing IL-15 / 21. CD3-positive cells are considered to be a marker of iPS-CAR-T cells, indicating that more iPS-CAR-T cells infiltrate into the tumor by expressing IL-15 / 21. That is, this indicates that the tumor-damaging activity of iPS-CAR-T cells expressing IL-15 / 21 is stronger than that of iPS-CAR-T cells expressing other cytokines. It was shown that CD3 and Ki67 double-positive cells were present in tumor tissues administered with iPS-CAR-T cells expressing IL-15 / 21. The presence of CD3 and Ki67 double-positive cells indicated that iPS-CAR-T cells expressing IL-15 / 21 had cell proliferation ability. Example 9

[0100] [Effects of cytokine gene-transferred GPC3-responsive iPS-CAR-T cells on tumor volume and survival in mice transplanted with the human hepatocellular carcinoma cell line JHH-7] On day 0, human hepatoma cell line JHH-7 (0.5×10 6 Then, on days 3 and 10, cytokine gene-introduced GPC3-responsive iPS-CAR-T cells (5×10 6 The tumor volume (V) was calculated based on the length (L, longest dimension) and width (W, shortest dimension) of the tumor using the formula V = LW2 / 2. Figure 13A 8 mice were used for each condition, and the results are shown as mean ± standard error. In addition, the results of survival rate are shown in Figure 13B .

[0101] Regarding tumor volume, iPS-CAR-T cells expressing IL-15 and IL-21 as well as IL-15 and IL-18 potently inhibited tumor progression ( Figure 13A iPS-CAR-T cells expressing IL-15 and IL-21 showed a significant improvement in survival rate, confirming clear anti-tumor activity ( Figure 13B ). Example 10

[0102] Effects of cytokine gene-transfected GPC3-responsive iPS-CAR-T cells on tumor volume and survival in mice transplanted with the human hepatocellular carcinoma cell line SK-HEP-1 On day 0, SK-HEP-1 (2.5×10 6 Then, on days 10 and 17, GPC3-responsive iPS-CAR-T cells (5×10 6 The tumor volume and survival rate of the mice were then observed. The tumor volume was calculated in the same manner as in Example 9. The results of the tumor volume are shown in Figure 14A The results are shown as mean ± standard error (n = 10). In addition, the results of survival rate are shown in Figure 14B (n=12).

[0103] Regarding tumor volume, iPS-CAR-T cells expressing IL-15 and IL-21 as well as IL-15 and IL-18 potently inhibited tumor progression ( Figure 14A ). In particular, iPS-CAR-T cells expressing IL-15 and IL-21 showed extremely strong anti-tumor activity. In addition, iPS-CAR-T cells expressing IL-15 and IL-21 also improved survival rates, showing excellent preventive or therapeutic effects on solid tumors ( Figure 14B ). Example 11

[0104] Comparison of the amount of GPC3-responsive iPS-CAR-T cells introduced with cytokine genes present in tumors IL-15, IL-21, and IL-15 and IL-21 (IL-15 / 21) genes were introduced into iPS-CAR-T cells (iCAR-T) derived from iPS T cells and expressed respectively. GPC3-positive human liver cancer cell line JHH-7 was subcutaneously inoculated into immunodeficient mice (NSG mice). 14 days later, iCAR-T cells with the above-mentioned cytokine gene introduction and iCAR-T-mock cells without the cytokine gene introduction were intravenously administered. On the 14th day after the administration of iCAR-T, the iCAR-T present in the subcutaneous tumors of NSG mice was recovered and analyzed by flow cytometry. Observation of cells expressing the reporter gene mCherry incorporated into the expression vector used for human CD45 and cytokine gene introduction revealed that the levels of iCAR-T+IL-15, iCAR-T+IL-21, and iCAR-T-mock were less than 1%, while the level of iCAR-T+IL-15 / 21 was 2.19%. This indicates that iCAR-T+IL-15 / 21 expressing both IL-15 and IL-21 is more abundant in the tumor than iCAR-T expressing either IL-15 or IL-21 alone. Figure 15 ). Example 12

[0105] Single-cell RNA-seq analysis Single-cell RNA sequencing was performed using the cells analyzed in Example 11. Human CD45 and mCherry positive cells were sorted, and gene analysis was performed on each individual cell using a next generation sequencer, followed by bioinformatics analysis using Seurat (R package). Figure 16 ). Afterwards, the respective analysis data were displayed in the UMAP graph. (It should be noted that the farther apart the clusters displayed on the UMAP are, the more different the gene expression profiles of the cell populations are.) The distribution of the 7 clusters displayed on the UMAP and the individual iCAR-T cells into which the cytokine genes were introduced is shown in FIG. Figure 17. From these classifications, it can be seen that iCAR-T + IL-15 / 21 is present in clusters 1 and 4. On the other hand, iCAR-T + IL-15, iCAR-T + IL-21 and iCAR-T-mock are present in clusters 0, 2 and 5, showing that they have a gene expression graph that is significantly different from that of iCAR-T + IL-15 / 21 cells. In addition, in cluster 3 close to clusters 1 and 4 composed of iCAR-T + IL-15 / 21, due to the presence of T cells (primary CAR-T) derived from peripheral blood, it is shown that the gene expression profile of iCAR-T + IL-15 / 21 is close to that of T cells derived from peripheral blood. So far, iCAR-T has been considered to be a class of NK cells, which are different cells from T cells derived from peripheral blood. According to the results here, it is speculated that due to the expression of IL-15 and IL-21 in iCAR-T, iCAR-T becomes closer to T cells derived from peripheral blood. They concluded that iCAR-T+IL-15 / 21 had a different gene expression profile compared with iCAR-T+IL-15, iCAR-T+IL-21, and iCAR-T-mock, which was almost identical to the gene expression profile of T cells derived from peripheral blood. Example 13

[0106] [Genomic Analysis of Clusters 1 and 4 Composed of iCAR-T+IL-15 / 21] Analysis of the gene sets of clusters 1 and 4 composed of iCAR-T+IL-15 / 21 showed that the expression of chemokine-related genes, cytotoxic molecules and cell cycle-related genes was enhanced ( Figure 18 and Figure 19 ). Pathway analysis was performed using genes that were significantly expressed in each cluster. As a result, it was found that the pathways of chemokine-related genes and cell cycle-related genes in clusters 1 and 4 composed of iCAR-T+IL-15 / 21 were enhanced. Similarly, it was found that the pathways of immune-related genes and cytokine-related genes in iCAR-T+IL-15 / 21 were enhanced ( Figure 20 Although the data are not shown, this tendency is also present in T cells derived from peripheral blood. Example 14

[0107] [Properties of iCAR-T+IL-15 / 21] The memory phenotype of each iCAR-T was observed using the cells analyzed in Example 11. It is believed that the memory phenotype changes in the order of immature, stem cell memory, central memory, and effector memory. It is known that in cell therapy, the more immature-like young T cells there are, the higher the therapeutic effect. It is known that the expression of CD62L, CCR7, and CD45RO of iCAR-T+IL-15 / 21 is enhanced. T cells expressing these cell surface markers are generally central memory-like T cells. Although not shown in the data, iCAR-T that does not produce IL-15 and IL-21 or expresses either IL-15 and IL-21, based on the expressed cell surface markers, shows a phenotype that is considered to be effector memory to terminally differentiated T cells. In conclusion, iCAR-T+IL-15 / 21 is shown to have a young memory phenotype ( Figure 21 ). Example 15

[0108] [Enhancement of tumor-infiltrating ability of iPS cell-derived TCR-T cells by IL-15 and IL-21] Colorectal cancer cell sphere culture cells (2×10 5 NSG mice (PDSX mice) were intravenously injected with 10 7 The following cells were used: cloned T cells (TI-CTLs) of tumor-infiltrating T cells isolated when colorectal cancer cell sphere culture cells were established, T cells (iTCR-Ts) differentiated and induced by iPS cells (FF-I01s04 cell line provided by the iPS Cell Research Institute of Kyoto University) into which TCRs obtained from the cloned T cells that can recognize colorectal cancer cell sphere culture cells were introduced, and iTCR-Ts (IL-15-21iTCR-Ts) in which IL-15 and IL-21 genes were introduced into the iTCR-Ts to continuously produce IL-15 and IL-21. Tumors were recovered after 14 days. After immunohistochemical staining of tumor tissue sections with anti-human CD8 antibodies, TI-CTLs, iTCR-Ts, and IL-15-21iTCR-Ts infiltrating the tumor were analyzed. The infiltrating T cells accumulated around stromal cells and epithelial cells in the tumor tissue. The results were converted into the ratio of human CD8 cells to the total number of cells and presented in a graph ( Figure 22 ). TI-CTLs showed good tumor tissue infiltration, while iTCR-Ts showed lower infiltration ability than TI-CTLs. On the other hand, IL-15-21iTCR-Ts showed equal or higher tumor infiltration ability than TI-CTLs due to the production of IL-15 and IL-21 ( Figure 22This indicates that T cells derived from iPS cells into which TCRs have been introduced exhibit significantly enhanced tumor-infiltrating ability due to the introduction of IL-15 and IL-21, and that the degree of infiltration is higher than that of T cells that have infiltrated tumors. Example 16

[0109] [Tumor growth inhibitory effects of iPS cell-derived TCR-T cells induced by IL-15 and IL-21 (I)] Colorectal cancer cell spheres (1×10 5 NSG mice (PDSX mice) were intravenously injected with 6×10 6 The following cells were administered 3 times: cloned T cells (TI-CTLs) of tumor-infiltrating T cells isolated when colorectal cancer cell sphere culture cells were established, iTCR-Ts (IL-15-21iTCR-Ts) that were differentiated and induced by iPS cells (FF-I01s04 cell line provided by the iPS Cell Research Institute of Kyoto University) that were differentiated and induced by TCRs obtained from the cloned T cells and capable of recognizing colorectal cancer cell sphere culture cells, and phosphate-buffered saline (PBS), and tumor proliferation was observed over time by IVISspectrumCT. Compared with the TI-CTLs administration group, the IL-15-21iTCR-Ts administration group showed a stronger tumor proliferation inhibitory effect ( Figure 23 This indicates that T cells derived from TCR-introduced iPS cells showed significant suppression of tumor growth due to the introduction of IL-15 and IL-21, and that the degree of suppression was higher than that of T cells infiltrating the tumor. Example 17

[0110] [Tumor growth inhibitory effects of iPS cell-derived TCR-T cells induced by IL-15 and IL-21 (II)] Colorectal cancer cell spheres (1×10 5 NSG mice (PDSX mice) were intravenously injected with 1.2×10 7The following cells were injected 3 times: T cells (iTCR-Ts) differentiated and induced by iPS cells (FF-I01s04 cell line provided by the iPS Cell Research Institute of Kyoto University) that were obtained by introducing cloned T cells from tumor-infiltrating T cells isolated during the establishment of colorectal cancer cell sphere culture cells and capable of recognizing colorectal cancer cell sphere culture cells, iTCR-Ts (IL-15-21iTCR-Ts) that were able to continuously produce IL-15 and IL-21 by introducing IL-15 and IL-21 genes into the iTCR-Ts, or PBS, and tumor proliferation was observed over time by IVISspectrumCT. Compared with the iTCR-Ts administration group, the IL-15-21iTCR-Ts administration group showed a stronger tumor proliferation inhibition effect ( Figure 24 This indicates that T cells derived from iPS cells into which TCRs have been introduced exhibit significant tumor growth inhibition due to the introduction of IL-15 and IL-21, and that the extent of inhibition is greater than that of T cells derived from iPS cells that do not produce IL-15 and IL-21.

Claims

An immune cell induced from an iPS cell, the cell expressing a cell surface molecule reactive to a tumor-associated antigen and interleukin 15 (IL-15). 2 . The cell according to claim 1 , further expressing interleukin 12 (IL-12), interleukin 18 (IL-18) or interleukin 21 (IL-21). 3 . The cell according to claim 1 , wherein the immune cell comprises a nucleic acid encoding the cell surface molecule and a nucleic acid encoding IL-15 introduced from outside the cell. The cell according to claim 3 , further comprising a nucleic acid encoding IL-12, IL-18 or IL-21 introduced from outside the cell.

5. The cell of claim 1, wherein the immune cell comprises an expression vector comprising a nucleic acid encoding the cell surface molecule and a nucleic acid encoding IL-15.

6. The cell of claim 5, further comprising an expression vector comprising a nucleic acid encoding IL-12, IL-18 or IL-21. The cell according to claim 1 , wherein the immune cell is a T cell or a NK cell.

8. The cell according to claim 7, wherein the T cell is a CD8 single-positive cytotoxic T cell.

9. The cell of claim 1, wherein the cell surface molecule is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

10. The cell of claim 1, wherein the cell surface molecules are a chimeric antigen receptor (CAR) and a T cell receptor (TCR). The cell according to claim 1 , wherein the iPS cell is an iPS cell obtained by depleting B cells and T cells from peripheral blood mononuclear cells or reprogramming T cells.

12. The cell according to claim 1, wherein the tumor-associated antigen is selected from WT1, GPC3, BCMA, XAGE1, MUC1, MUC5A1, MUC6, EGFRvIII, HER-2 / neu, MAGE-A1, MAGE-A3, telomerase, PRAME, SSX2 / 4, PSCA, CTLA-4, gp100, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53 without gene mutation, p53 with gene mutation, p 53 mutants, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, MelanA / MART1, survivin, Ras, Ras mutants, EGR, bcr-ab1, XBP-1, neoantigens caused by gene mutations, neoantigens caused by splicing abnormalities, HBV, HBs, HPV, EBV, LMP1, EBV, LMP2, EBNA, HPV-E1, HPV-E2, HPV-E6, HPV-E7, HTLV-1Tax and HBZ.

13. A method, which is the method for preparing the cell according to claim 3, comprising: (1) a step of introducing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells introduced with the nucleic acid obtained in step (1) into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells into which the nucleic acid has been introduced and induced to differentiate from the iPS cells obtained in step (1) into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells into which the nucleic acid has been introduced and induced to differentiate from the iPS cells obtained in step (1) into mature immune cells; (3) A step of introducing a nucleic acid encoding IL-15 into the mature immune cells obtained in step (1) into which the nucleic acid has been introduced, or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

14. A method, which is the method for preparing the cell according to claim 4, comprising: (1) a step of introducing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells introduced with the nucleic acid obtained in step (1) into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells introduced with the nucleic acid, which were induced to differentiate from iPS cells obtained in step (1), into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells into which the nucleic acid has been introduced and induced to differentiate from the iPS cells obtained in step (1) into mature immune cells; (3) A step of introducing a nucleic acid encoding IL-15 and a nucleic acid encoding IL-12, IL-18 or IL-21 into the mature immune cells into which the nucleic acid is introduced obtained in step (1), or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

15. A method, which is the method for preparing the cell according to claim 5, comprising: (1) a step of introducing an expression vector containing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells introduced with the expression vector obtained in step (1) into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells introduced with the expression vector, which were induced to differentiate from the iPS cells obtained in step (1), into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells introduced with the expression vector and induced to differentiate from the iPS cells obtained in step (1) into mature immune cells; (3) A step of introducing an expression vector containing a nucleic acid encoding IL-15 into the mature immune cells introduced with the expression vector obtained in step (1), or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

16. A method, which is the method for preparing the cell according to claim 6, comprising: (1) a step of introducing an expression vector containing a nucleic acid encoding the cell surface molecule into iPS cells or hematopoietic stem cells, immature immune cells, or mature immune cells induced to differentiate from iPS cells; (2) Including the following steps (2-1), (2-2) or (2-3): (2-1) a step of differentiating the iPS cells obtained in step (1) and into which the expression vector has been introduced into hematopoietic stem cells, immature immune cells, or mature immune cells; (2-2) a step of differentiating the hematopoietic stem cells obtained in step (1) and induced to differentiate from iPS cells and into which the expression vector has been introduced, into immature immune cells or mature immune cells; (2-3) a step of differentiating the immature immune cells obtained in step (1) and induced to differentiate from the iPS cells and into which the expression vector has been introduced, into mature immune cells; (3) A step of introducing an expression vector comprising a nucleic acid encoding IL-15 and a nucleic acid encoding IL-12, IL-18 or IL-21 into the mature immune cells obtained in step (1) and into which the expression vector has been introduced, or the hematopoietic stem cells, immature immune cells or mature immune cells obtained in step (2-1), (2-2) or (2-3).

17. A medicament comprising the cell according to any one of claims 1 to 12. The medicament according to claim 17, which is used for the prevention or treatment of cancer.

19. An agent for killing cells expressing tumor-associated antigens, comprising the cells according to any one of claims 1 to 12.

20. A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the cell according to any one of claims 1 to 12.

21. A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the medicament according to claim 17.

22. A method for preventing or treating cancer in a mammal, comprising administering to the mammal an effective amount of the killing agent according to claim 19.

23. A preventive or therapeutic agent for cancer in a mammal, comprising the cell according to any one of claims 1 to 12.

24. The cell according to any one of claims 1 to 12, for use in the prevention or treatment of cancer.

25. The cell according to any one of claims 1 to 12, which is used for preparing a preventive or therapeutic agent for cancer.

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