Method for producing γδT cells

Abstract

To provide a method for producing γδT cells from induced pluripotent stem cells.SOLUTION: There is provided a method for producing γδT cells from induced pluripotent stem cells, in which the induced pluripotent stem cells are derived from cells other than αβT cells. The method comprises the following steps of: (1) establishing induced pluripotent stem cells from cells other than αβT cells; and (2) differentiating the induced pluripotent stem cells established in the step (1) into T cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing γδ T cells from induced pluripotent stem cells, γδ T cells differentiated from induced pluripotent stem cells, and cell populations containing said cells.

[0002] BACKGROUND OF THE INVENTION In recent years, immune cell therapy has been attracting attention as a treatment for cancer. Immune cell therapy is a treatment in which immune cells that have been grown and activated outside the patient's body are administered to the patient, allowing the immune cells to attack cancer cells. Immune cell therapy has the advantage of having almost no side effects compared to the three major conventional treatments: surgery, radiation therapy, and chemotherapy. There are various types of immune cell therapy, but one that is attracting attention is treatment using gamma delta T cells, which are responsible for innate immunity and have cytotoxic activity against cancer cells.

[0003] In order to realize γδ T cell therapy, it is desirable to develop a manufacturing method that will enable the efficient production and stable supply of these cells. Although a method for selecting only γδ T cells from a patient's blood (a method for culturing blood cells in a medium containing zoledronic acid and IL-2 (Patent Document 1)) is known, to the inventors' knowledge, no method for producing γδ T cells from stem cells has been reported. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2006 / 006720 Pamphlet Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a method for producing γδ T cells from stem cells. Another objective of the present invention is to provide γδ T cells differentiated from stem cells and a cell population containing said cells. [Means for solving the problem]

[0006] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors discovered that γδ T cells can be efficiently obtained by inducing induced pluripotent stem cells from cells other than αβ T cells and then inducing these cells into T cells. Furthermore, when a chimeric antigen receptor (CAR) gene was introduced into the thus obtained γδ T cells to generate γδ T cells expressing the CAR, the γδ T cells were shown to exhibit high cytotoxicity against cancer cells that were difficult for the γδ T cells to recognize and damage. Based on these findings, the present inventors conducted further research, leading to the completion of the present invention.

[0007] That is, the present invention provides the following. [1] A method for producing γδ T cells from induced pluripotent stem cells, wherein the induced pluripotent stem cells are derived from cells other than αβ T cells. [2] The method described in [1], comprising the following steps: (1) A process for establishing induced pluripotent stem cells from cells other than αβ T cells (2) Differentiating the induced pluripotent stem cells established in step (1) into T cells. [3] The method according to [1] or [2], wherein the cells other than αβ T cells are mononuclear cells other than αβ T cells. [4] The method according to any one of [1] to [3], wherein the cells other than αβT cells are monocytes. [5] A method for treating cells obtained in any one of steps (1) and (2) above, which recognizes and binds to a tumor-specific antigen or a tumor-associated antigen. (i) a nucleic acid encoding an αTCR and a nucleic acid encoding a βTCR; (ii) a nucleic acid encoding a γTCR and a nucleic acid encoding a δTCR, and / or (iii) The method according to any one of [2] to [4], which comprises a step of introducing a nucleic acid encoding a CAR. [6] The method according to [5], wherein the γTCR is Vγ9TCR and the δTCR is Vδ2TCR. [7] The method according to any one of [1] to [6], which comprises a step of introducing a nucleic acid encoding a fusion protein comprising IL-15 and IL-15Rα into cells obtained in any one of steps (1) and (2). [8] A γδ T cell derived from an induced pluripotent stem cell, wherein the induced pluripotent stem cell is derived from a cell other than an αβ T cell. [9] γδT cells produced by the method described in any one of [1] to [7].

[10] The cells described in [8] or [9], wherein the cells other than αβT cells are mononuclear cells other than αβT cells.

[11] The cell according to any one of [8] to

[10] , wherein the cell other than an αβT cell is a monocyte.

[12] The cells described in any one of [8] to

[11] , wherein the γδ T cells express Vγ9 TCR and Vδ2 TCR.

[13] The cells described in any one of [8] to

[12] , wherein the γδ T cells express a CAR.

[14] The cells according to any one of [8] to

[13] , wherein the γδ T cells express a fusion protein comprising IL-15 and IL-15Rα.

[15] A cell population in which at least 90% or more of the total cells are γδ T cells, wherein the γδ T cells are cells differentiated from induced pluripotent stem cells derived from cells other than αβ T cells.

[16] A pharmaceutical comprising the cell according to any one of [8] to

[14] or the cell population according to

[15] .

[17] The pharmaceutical agent according to

[16] , for use in the prevention or treatment of a tumor.

[18] A cell-killing agent comprising the cells according to any one of [8] to

[14] or the cell population according to

[15] .

[19] The cell according to any one of [8] to

[14] or the cell population according to

[15] for use in the prevention or treatment of a tumor.

[20] Use of the cells according to any one of [8] to

[14] or the cell population according to

[15] in the manufacture of a preventive or therapeutic agent for tumors.

[21] A method for preventing or treating a tumor, comprising administering the cell according to any one of [8] to

[14] or the cell population according to

[15] . [Effects of the Invention]

[0008] The present invention provides a method for producing γδ T cells from induced pluripotent stem cells, γδ T cells differentiated from induced pluripotent stem cells, and cell populations containing said cells. Furthermore, among the γδ T cells produced by the above method, cells expressing a chimeric antigen receptor (CAR) can exhibit high cytotoxic activity in vitro and in vivo, specific to the antigen recognized by the CAR. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the results of staining the acquired cells using an antibody set (Vδ1 Myltenyi FITC, Vδ2 Myltenyi APC, γδ TCR BD BV510, CD3 BioLegend APC / Cy7, and αβ TCR eBioscience FITC). The solid peaks represent the results of the unstained group, and the blank peaks represent the results of staining with each antigen-specific antibody. [Figure 2] Figure 2 shows the results of flow cytometry in which acquired cells were stained with a set of antibodies (V51 Myltenyi FITC, V52 Myltenyi APC, γδTCR BD BV510, CD3 BioLegend APC / Cy7, and αβTCR eBioscience FITC). [Figure 3] 3 shows the results of measuring the cytotoxic activity of the obtained γδ T cells. The vertical axis shows cytotoxic activity (%), and the horizontal axis shows the ratio of the number of mixed γδ T cells to the number of target cells. [Figure 4] Figure 4 shows the results of measuring cell proliferation of iPS cell-derived γδ T cells (iγδ T cells). The vertical axis shows the cell proliferation rate, and the horizontal axis shows the number of days since the start of stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody. [Figure 5]FIG. 5 shows the expression of CD3 and γδTCR molecules on the cell membrane surface of γδT cells (iγ9δ2T cells) differentiated from iPS cells by introducing the Vγ9Vδ2TCR gene. [Figure 6] Figure 6 shows the expression of CD3 and γδTCR molecules on the cell membrane surface of γδT cells (iHγ9δ2T cells) differentiated from iPS cell-derived hematopoietic progenitor cells (HPCs) by introducing the Vγ9Vδ2TCR gene. [Figure 7] Figure 7 shows the results of measuring cell proliferation of iγδ T cells expressing the anti-CD19-CAR gene (iCD19CAR / IL-15γδ T cells). The vertical axis shows the cell number, and the horizontal axis shows the number of days since the start of stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody. [Figure 8] Figure 8 shows the results of measuring cell proliferation of iHγ9δ2 T cells expressing the anti-CD19-CAR gene (iHCD19CAR / IL-15γ9δ2T). The vertical axis shows the cell number, and the horizontal axis shows the number of days since the start of stimulation with anti-CD3 antibody (UCHT1). [Figure 9] Figure 9 shows the results of measuring the cytotoxic activity of iγδ T cells expressing the anti-CD19-CAR gene (iCD19CAR / IL-15γδ T cells). The vertical axis shows the target cell cytotoxicity rate (%), and the horizontal axis shows the ratio of the number of mixed iCD19CAR / IL-15γδ T cells to the number of target cells. [Figure 10] Figure 10 shows the results of measuring the cytotoxic activity of iHγ9δ2 T cells expressing the anti-CD19-CAR gene (iHCD19CAR / IL-15γ9δ2 T cells). The vertical axis shows the target cell cytotoxicity rate (%), and the horizontal axis shows the ratio of the number of mixed iHCD19CAR / IL-15γ9δ2 T cells to the number of target cells. [Figure 11] Figure 11 shows the effect of in vivo administration of iγδ T cells expressing the anti-CD19-CAR gene (iCD19CAR / IL-15γδ T cells) on the survival time of mice bearing human CD19-positive tumors. The vertical axis shows the survival rate of the mice, and the horizontal axis shows the number of days since the day of cancer cell transplantation. [Figure 12]Figure 12 shows the antitumor effect of in vivo administration of iHγ9δ2T cells expressing the anti-CD19-CAR gene (iHCD19CAR / IL-15γ9δ2T) on mice implanted with luciferase-expressing human tumors.

[0010] (Detailed Description of the Invention) As used herein, "gene expression" includes both the synthesis of mRNA from a specific nucleotide sequence of the gene (also referred to as transcription or mRNA expression) and the synthesis of a protein based on the information in the mRNA (also referred to as translation or protein expression). However, unless otherwise specified, "gene expression" or simply "expression" refers to protein expression.

[0011] As used herein, "positive" means that the protein or mRNA is expressed in a detectable amount by techniques known in the art. Protein detection can be performed using immunological assays using antibodies, such as ELISA, immunostaining, and flow cytometry. In addition, in the case of proteins that are expressed intracellularly and not present on the cell surface (e.g., transcription factors or their subunits), the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. mRNA detection can be performed using nucleic acid amplification and / or nucleic acid detection methods, such as RT-PCR, microarrays, biochips, and RNAseq.

[0012] As used herein, "negative" means that the expression level of the protein or mRNA is below the lower limit of detection by all or any of the above-mentioned known techniques. The lower limit of detection of the expression of the protein or mRNA may vary depending on the technique.

[0013] In this specification, a positive result is also referred to as "there is expression of the protein or mRNA," and a negative result is also referred to as "there is no expression of the protein or mRNA." Therefore, adjusting the "presence or absence of expression" means adjusting the cell to either a state where the expression amount of the target protein or mRNA is equal to or greater than the lower limit of detection (positive) or a state where the expression amount is less than the lower limit of detection (negative).

[0014] As used herein, "culturing" refers to maintaining, expanding (growing), and / or differentiating cells in an in vitro environment. "Culturing" refers to maintaining, expanding (growing), and / or differentiating cells outside a tissue or body, for example, in a cell culture plate, dish, or flask.

[0015] As used herein, "enriching" refers to increasing the proportion of a particular component in a composition, such as a composition of cells, and "enriched," when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a particular component is increased compared to the proportion of such component in the cell population prior to enrichment. For example, a composition, such as a cell population, can be enriched for a target cell type, thus increasing the proportion of the target cell type compared to the proportion of target cells present in the cell population prior to enrichment. Cell populations can also be enriched for a target cell type by cell selection and sorting methods known in the art. Cell populations can also be enriched by certain culture methods, sorting, or selection processes described herein. In certain embodiments of the present invention, a method of enriching a target cell population results in a cell population that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% enrichment for the target cell population.

[0016] As used herein, "expansion culture" refers to culturing a desired cell population for the purpose of expanding the cell population and increasing the cell number. The increase in cell number may be achieved by the increase in cell number due to cell proliferation exceeding the decrease in cell number due to cell death, and does not require proliferation of all cells in the cell population. The increase in cell number may be 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 3000-fold, 5000-fold, 10000-fold, 100000-fold, or 1000000-fold or more compared to before the start of expansion culture.

[0017] As used herein, the term "stimulation" means that a substance binds to various receptors and activates downstream signal pathways.

[0018] As used herein, the term "cell population" refers to two or more cells of the same or different types. The term "cell population" also refers to a mass of cells of the same or different types.

[0019] 1. Method for producing γδ T cells from induced pluripotent stem cells The present invention provides methods for producing γδ T cells from induced pluripotent stem cells and cell populations containing the γδ T cells (hereinafter abbreviated as "production methods of the present invention"). The production methods of the present invention include a step of differentiating induced pluripotent stem cells into T cells. The induced pluripotent stem cells used in the production methods of the present invention may be cells that have already been established and stored, or may be induced pluripotent stem cells established from cells other than αβ T cells. Thus, in one embodiment of the present invention, the production methods of the present invention include (1) a step of establishing induced pluripotent stem cells from cells other than αβ T cells, and (2) a step of differentiating the induced pluripotent stem cells established in step (1) into T cells.

[0020] In the present invention, a "T cell receptor (TCR)" is composed of a dimer of TCR chains (α chain, β chain, γ chain, and δ chain). "γδ T cells" refer to cells that express CD3 and a TCR (hereinafter sometimes referred to as "γδ TCR") composed of a TCRγ chain (γ TCR) and a TCRδ chain (δ TCR). "αβ T cells" refer to cells that express CD3 and a TCR (hereinafter sometimes referred to as "αβ TCR") composed of a TCRα chain (α TCR) and a TCRβ chain (β TCR). Most αβ T cells recognize antigen peptide-MHC (major histocompatibility complex, in humans, HLA: human leukocyte antigen) complexes using their αβ TCR (this is called MHC restriction). In contrast, γδ T cells recognize a variety of molecules expressed by cells using their γδ TCR, regardless of MHC molecules. Each TCR chain is composed of a variable region and a constant region, and the variable region contains three complementarity-determining regions (CDR1, CDR2, and CDR3). The TCR gene is composed of numerous V (variable), D (diversity), J (joining), and C (constant) gene segments on the genome. Gene rearrangement occurs during the differentiation and maturation process of T cells, and in the β-chain gene, one D and one J are randomly selected and joined, followed by gene rearrangement between the V-DJ segments. During this process, random insertions and deletions of bases occur between the VD and DJ segments, increasing gene diversity. RNA splicing occurs in the TCR precursor mRNA at the VDJ region and the common C region, resulting in expression of a functional TCR gene.

[0021] Examples of γTCRs include Vγ1TCR, Vγ2TCR, Vγ3TCR, Vγ4TCR, Vγ5TCR, Vγ6TCR, Vγ7TCR, Vγ8TCR, and Vγ9TCR, and examples of δTCRs include Vδ1TCR, Vδ2TCR, Vδ3TCR, Vδ4TCR, Vδ5TCR, Vδ6TCR, Vδ7TCR, Vδ8TCR, and Vδ9TCR. Specific combinations of γTCR and δTCR include, but are not limited to, Vγ3Vδ1TCR, Vγ4Vδ1TCR, Vγ9Vδ1TCR, and Vγ9Vδ2TCR.

[0022] (1) Establishing induced pluripotent stem cells In the present invention, "induced pluripotent stem cells" (hereinafter sometimes referred to as "iPS cells") refer to stem cells established by introducing reprogramming factors into somatic cells, which have pluripotency and the ability to differentiate into many cells present in the body, as well as the ability to proliferate, and include at least any cells that can be induced to become the hematopoietic progenitor cells used in the present invention. Induced pluripotent stem cells are preferably derived from mammals (e.g., mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans), and more preferably from humans.

[0023] Methods for establishing induced pluripotent stem cells are known in the art, and can be established by introducing reprogramming factors into any somatic cells. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors can be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al.(2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat. Cell Biol. 11:197-203、RL Judson et al., (2009), Nat. Biotechnol., 27:459-461、Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0024] Somatic cells include, but are not limited to, fetal (baby) somatic cells, neonatal (baby) somatic cells, and adult somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Furthermore, the aforementioned cells may be healthy cells or diseased cells. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic progenitor cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as blood cells (e.g., peripheral blood cells, umbilical cord blood cells, etc.), mononuclear cells (e.g., lymphocytes (NK cells, B cells, T cells other than αβ T cells (e.g., γδ T cells, etc.), monocytes, dendritic cells, etc.)), granulocytes (e.g., eosinophils, neutrophils, basophils), megakaryocytes), epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (e.g., exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes. Among these, mononuclear cells other than αβ T cells are preferred, and more specifically, monocytes or γδ T cells are preferred.

[0025] When the reprogramming factor is in the form of DNA, the reprogramming factor can be introduced into somatic cells by, for example, calcium phosphate coprecipitation, PEG, electroporation, microinjection, or lipofection. For example, methods described in Cell Engineering, Special Issue 8, New Cell Engineering Experimental Protocols, 263-267 (1995) (Shujunsha Publishing), Virology, Vol. 52, 456 (1973), and Folia Pharmacol. Jpn., Vol. 119 (No. 6), 345-351 (2002) can be used. When a viral vector is used, the nucleic acid can be introduced into a suitable packaging cell (e.g., Plat-E cells) or a complementing cell line (e.g., 293 cells), the viral vector produced in the culture supernatant can be collected, and the viral vector can be introduced into cells by infecting the cells with the vector using a method appropriate for each viral vector. For example, specific means using retroviral vectors as vectors are disclosed in International Publication No. 2007 / 69666, Cell, 126, 663-676 (2006), and Cell, 131, 861-872 (2007), etc. In particular, when using retroviral vectors, highly efficient gene transfer into various cells is possible by using a recombinant fibronectin fragment, CH-296 (manufactured by Takara Bio Inc.).

[0026] The reprogramming factor may be directly introduced into cells in the form of RNA, and the reprogramming factor may be expressed in the cells. A known method for introducing RNA can be used, and for example, lipofection or electroporation can be suitably used. Furthermore, when the reprogramming factor is in the form of a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (e.g., HIV-derived TAT and polyarginine), or microinjection.

[0027] Examples of basal media include Dulbecco's medium (e.g., IMDM), Eagle's medium (e.g., DMEM, EMEM, BME, MEM, αMEM), Ham's medium (e.g., F10 medium, F12 medium), RPMI medium (e.g., RPMI-1640 medium, RPMI-1630 medium), MCDB medium (e.g., MCDB104, 107, 131, 151, 153 medium), Fischer's medium, 199 medium, primate ES cell medium (primate ES / iPS cell culture medium, ReproCell), mouse ES cell medium (TX-WES culture medium, ThromboX), serum-free medium (mTeSR, Stemcell Examples of suitable media include, but are not limited to, StemSpan (registered trademark) SFEM, StemSpan (registered trademark) H3000, Stemline II, ESF-B medium, ESF-C medium, CSTI-7 medium, Neurobasal medium (Life Technologies), StemPro-34 medium, and StemFit (registered trademark) (e.g., StemFit AK03N, StemFit AK02N). Furthermore, these media can be mixed and used as needed, for example, to produce DMEM / F12 medium.

[0028] The basal medium may be supplemented with 10% to 20% serum (fetal bovine serum (FBS), human serum, horse serum) or serum substitutes (e.g., KSR), insulin, various vitamins, L-glutamine, various amino acids such as non-essential amino acids, 2-mercaptoethanol, various cytokines (e.g., interleukins (IL-2, IL-7, IL-15), stem cell factor (SCF), activin), various hormones, various growth factors (e.g., leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β), various extracellular matrices, various cell adhesion molecules, antibiotics such as penicillin / streptomycin and puromycin, pH indicators such as phenol red, and the like, as appropriate.

[0029] The culture is preferably carried out, for example, in an atmosphere of 1% to 10%, preferably 2% to 5% CO2, at about 37°C to 42°C, preferably about 37°C to 39°C, for about 25 to 50 days.

[0030] In the present invention, the mammal from which somatic cells are collected is not particularly limited, but is preferably a human. From the viewpoint of preventing rejection reactions, autologous cells, allogeneic cells with identical or substantially identical HLA types, and allogeneic cells in which the presence or absence of HLA expression and / or the expression level has been adjusted are preferred. As for HLA, it is preferred that the presence or absence of expression and / or the expression level of at least some subunits contained in class I and / or class II are adjusted.

[0031] (2) Differentiating induced pluripotent stem cells into T cells The method for differentiating induced pluripotent stem cells into T cells is not particularly limited as long as it allows the induced pluripotent stem cells to be differentiated into γδ T cells. In one embodiment of the present invention, the step of differentiating induced pluripotent stem cells into T cells may comprise: (2-1) a step of differentiating the induced pluripotent stem cells into hematopoietic progenitor cells; and (2-2) a step of differentiating the hematopoietic progenitor cells into CD3-positive T cells.

[0032] (2-1) Differentiating induced pluripotent stem cells into hematopoietic progenitor cells In the present invention, "hematopoietic progenitor cells (HPCs)" refer to CD34-positive cells, preferably CD34 / CD43 dual-positive (DP) cells. In the present invention, hematopoietic progenitor cells and hematopoietic stem cells are not distinguished from each other and refer to the same cells unless otherwise specified.

[0033] The method for differentiating induced pluripotent stem cells into hematopoietic progenitor cells is not particularly limited as long as it allows differentiation into hematopoietic progenitor cells. For example, methods include culturing pluripotent stem cells in a medium for inducing hematopoietic progenitor cells, as described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, and Liu S. et al., Cytotherapy, 17 (2015); 344-358.

[0034] In the present invention, the medium for inducing hematopoietic progenitor cells is not particularly limited, and a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include those used in step (1) above. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax®), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like.

[0035] In the present invention, vitamin C refers to L-ascorbic acid and its derivatives, and L-ascorbic acid derivatives refer to those that become vitamin C through an enzymatic reaction in vivo. Examples of ascorbic acid derivatives used in the present invention include vitamin C phosphate (e.g., ascorbic acid 2-phosphate), ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6 palmitate. Vitamin C phosphate (e.g., ascorbic acid 2-phosphate) is preferred, including L-ascorbate phosphates such as sodium L-ascorbate phosphate and magnesium L-ascorbate phosphate.

[0036] When vitamin C is used, it is preferably added (supplemented) every 4 days, every 3 days, every 2 days, or every day, and more preferably added every day. In one embodiment, the vitamin C is added in an amount equivalent to 5 ng / ml to 500 ng / ml in the culture medium (e.g., an amount equivalent to 5 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml). In another embodiment, the vitamin C compound is added in an amount equivalent to 5 μg / ml to 500 μg / ml in the culture medium (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0037] The medium used in step (2-1) may further contain at least one cytokine selected from the group consisting of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Flt-3 Ligand (FLT-3L), and basic fibroblast growth factor (bFGF). Cultures supplemented with BMP4, VEGF, and bFGF are more preferred, and cultures supplemented with BMP4, VEGF, SCF, and bFGF are even more preferred.

[0038] When cytokines are used, the concentrations in the medium may be, for example, 5 ng / ml to 500 ng / ml for BMP4, 5 ng / ml to 500 ng / ml for VEGF, 5 ng / ml to 100 ng / ml for SCF, 1 ng / ml to 100 ng / ml for TPO, 1 ng / ml to 100 ng / ml for FLT-3L, and 5 ng / ml to 500 ng / ml for bFGF.

[0039] The medium may also contain a TGFβ inhibitor. TGFβ inhibitors are small molecule inhibitors that interfere with signal transduction of the TGFβ family, and include, for example, SB431542, SB202190 (RK Lindemann et al., Mol. Cancer 2:20(2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories). For example, when the TGFβ inhibitor is SB431542, its concentration in the medium is preferably 0.5 μM to 100 μM.

[0040] Induced pluripotent stem cells may be cultured in either adherent or suspension culture. Adherent culture may be performed using a culture vessel coated with an extracellular matrix component, or co-culture with feeder cells. Examples of feeder cells include, but are not limited to, fibroblasts (mouse embryonic fibroblasts (MEF) and mouse fibroblasts (STO)). Feeder cells are preferably inactivated by known methods, such as irradiation (e.g., gamma rays) or treatment with an anticancer drug (e.g., mitomycin C). Examples of extracellular matrix components include Matrigel (Niwa A, et al., PLoS One. 6(7):e22261, 2011), fibrous proteins such as gelatin, collagen, and elastin, glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesive proteins such as fibronectin, vitronectin, and laminin.

[0041] Suspension culture is the cultivation of cells in a non-adherent state to a culture vessel, and can be carried out using, but is not limited to, a culture vessel that has not been artificially treated to improve adhesion to the cells (e.g., coated with an extracellular matrix, etc.), or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or a nonionic surface-active polyol (e.g., Pluronic F-127)). When performing suspension culture, it is preferable to form and cultivate embryoid bodies (EBs).

[0042] In the present invention, hematopoietic progenitor cells can also be prepared from a net-like structure (also called ES-sac or iPS-sac) obtained by culturing pluripotent stem cells. Here, the term "net-like structure" refers to a three-dimensional sac-like structure (with an internal space) derived from pluripotent stem cells, formed from an endothelial cell population or the like, and containing hematopoietic progenitor cells.

[0043] The culture temperature is not particularly limited, but is, for example, approximately 37°C to 42°C, preferably approximately 37°C to 39°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of hematopoietic progenitor cells, etc. The number of days is not particularly limited as long as hematopoietic progenitor cells are obtained, but is, for example, at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, preferably 14 days. Long culture periods are not usually problematic in the production of hematopoietic progenitor cells, but a period of, for example, 35 days or less is preferred, and 21 days or less is more preferred. Culture may also be performed under hypoxic conditions. In the present invention, hypoxic conditions include oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0044] (2-2) Differentiating hematopoietic progenitor cells into CD3-positive T cells The method for differentiating hematopoietic progenitor cells into CD3-positive T cells is not particularly limited as long as it allows hematopoietic progenitor cells to be differentiated into CD3-positive T cells. Examples include methods of culturing hematopoietic progenitor cells under culture conditions similar to those used for methods of inducing T cells from hematopoietic progenitor cells, as described in WO 2016 / 076415 or WO 2017 / 221975.

[0045] In the present invention, the medium for inducing differentiation into CD3-positive T cells is not particularly limited, and a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include those used in step (1) above. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax®), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like.

[0046] When vitamin C is used in step (2-2), the vitamin C may be the same as those described in step (2-1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0047] In step (2-2), it is preferable to use a p38 inhibitor and / or SDF-1 (stromal cell-derived factor 1). In the present invention, the term "p38 inhibitor" refers to a substance that inhibits the function of p38 protein (p38 MAP kinase), and examples thereof include, but are not limited to, chemical inhibitors of p38, dominant-negative mutants of p38, and nucleic acids encoding the same.

[0048] Examples of chemical inhibitors of p38 used in the present invention include, but are not limited to, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol) and derivatives thereof, SB220025 and derivatives thereof, PD169316, RPR200765A, AMG-548, BIRB-796, SCIO-469, SCIO-323, VX-702, and FR167653. These compounds are commercially available, for example, SB203580, SB202190, SC239063, SB220025, and PD169316 are available from Calbiochem, and SCIO-469 and SCIO-323 are available from Scios. Preferred chemical inhibitors of P38 include SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives.

[0049] Dominant-negative mutants of p38 used in the present invention include p38T180A, in which threonine at position 180 in the DNA-binding domain of p38 is mutated to alanine, and p38Y182F, in which tyrosine at position 182 of human and mouse p38 is mutated to phenylalanine. The p38 inhibitor is contained in the medium at a concentration ranging from about 1 μM to about 50 μM. When SB203580 is used as the p38 inhibitor, it can be contained in the medium at a concentration ranging from 1 μM to 50 μM, 5 μM to 30 μM, or 10 μM to 20 μM.

[0050] The SDF-1 used in the present invention may be not only SDF-1α or its mature form, but also isoforms such as SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, or SDF-1φ, or their mature forms, or a mixture of these in any proportion. Preferably, SDF-1α is used. SDF-1 is also referred to as CXCL-12 or PBSF.

[0051] In the present invention, SDF-1 may have one or more amino acid substitutions, deletions, additions, and / or insertions in its amino acid sequence, so long as it retains its chemokine activity (SDF-1 with such amino acid substitutions, deletions, additions, and / or insertions is also referred to as an "SDF-1 mutant"). Similarly, SDF-1 or an SDF-1 mutant may have a sugar chain substitution, deletion, and / or addition. Examples of the SDF-1 mutant include those that retain at least four cysteine ​​residues (Cys30, Cys32, Cys55, and Cys71 in the case of human SDF-1α) and have 90% or more identity to the native amino acid sequence, but are not limited to these amino acid mutations. SDF-1 may be from mammals, such as humans, or non-human mammals, such as monkeys, sheep, cows, horses, pigs, dogs, cats, rabbits, rats, and mice. For example, the protein registered under GenBank accession number NP_954637 can be used as human SDF-1α, and the protein registered under GenBank accession number NP_000600 can be used as SDF-1β.

[0052] SDF-1 may be commercially available, purified from nature, or produced by peptide synthesis or genetic engineering techniques. SDF-1 is contained in the medium, for example, at a concentration of about 10 ng / ml to about 100 ng / ml. Alternatively, SDF-1 substitutes having SDF-1-like activity may be used instead of SDF-1. Examples of such SDF-1 substitutes include CXCR4 agonists, and low-molecular-weight compounds having CXCR4 agonistic activity may be added to the medium instead of SDF-1.

[0053] The medium used in step (2-2) may further contain at least one, preferably all, of cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT-3L, and IL-7, with concentrations of, for example, 10 ng / ml to 100 ng / ml for SCF, 10 ng / ml to 200 ng / ml for TPO, 1 ng / ml to 100 ng / ml for IL-7, and 1 ng / ml to 100 ng / ml for FLT-3L.

[0054] In step (2-2), hematopoietic progenitor cells may be cultured in an adherent or suspension culture. In the case of adherent culture, the culture vessel may be coated, or the cells may be co-cultured with feeder cells or the like. An example of a feeder cell to be co-cultured is the bone marrow stromal cell line OP9 cells (available from the RIKEN BioResource Center). The OP9 cells are preferably OP9-DL4 cells or OP9-DL1 cells, which constitutively express DLL4 or DLL1 (e.g., Holmes R1 and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009(2)). When OP9 cells are used as feeder cells in the present invention, they may be appropriately added to the medium with separately prepared DLL4 or DLL1, or a fusion protein of DLL4 or DLL1 with Fc or the like. When feeder cells are used, it is preferable to appropriately replace the feeder cells during culture. Feeder cell replacement can be performed by transferring the target cells during culture onto previously seeded feeder cells. The replacement can be performed every 5 days, 4 days, 3 days, or 2 days. Furthermore, when hematopoietic progenitor cells are obtained by suspension culture of embryoid bodies, they are preferably dissociated into single cells and then cultured in an adherent manner. Co-culture with feeder cells is also possible, but preferably, the culture is performed without feeder cells. In the case of adherent culture, examples of coating agents for coating culture vessels include Matrigel (Niwa A, et al. PLos One, 6(7):e22261, 2011), collagen, gelatin, laminin, heparan sulfate proteoglycan, RetroNectin (registered trademark), DLL4 or DLL1, or fusion proteins of DLL4 or DLL1 with the Fc region of an antibody (hereinafter sometimes referred to as Fc) (e.g., DLL4 / Fc chimera), entactin, and / or combinations thereof, with a combination of RetroNectin and a fusion protein of DLL4 with Fc or the like being preferred.

[0055] In step (2-2), the culture temperature conditions are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of γδ T cells, etc. The number of days is not particularly limited as long as γδ T cells are obtained, but is typically at least 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, or 20 days or more, and preferably 21 days. Furthermore, 90 days or less is preferred, and 42 days or less is more preferred.

[0056] The CD3-positive T cell population obtained by the above steps contains γδ T cells, and step (2) may further include the following step (2-3).

[0057] (2-3) Enriching CD3-positive T cells The method for enriching CD3-positive T cells is not particularly limited as long as it enriches γδ T cells. For example, methods include culturing CD3-positive T cells under culture conditions similar to those used in the process of inducing CD8-positive T cells from CD4CD8-positive T cells, as described in WO 2016 / 076415 and WO 2017 / 221975.

[0058] In the present invention, the medium used for enriching CD3-positive T cells is not particularly limited, and a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include those used in step (1) above. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax®), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, hormones, and the like. In one embodiment of the present invention, vitamin C such as ascorbic acid, insulin, transferrin, selenium compounds (eg, sodium selenite), and cytokines such as IL-7 may be contained.

[0059] When vitamin C is used in step (2-3), the vitamin C may be the same as those described in step (2-1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0060] When a hormone is used in step (2-3), the hormone may be an adrenocortical hormone. The adrenocortical hormone is a glucocorticoid or a derivative thereof, and examples thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. Dexamethasone is preferred. When the adrenocortical hormone is dexamethasone, its concentration in the medium is 1 nM to 100 nM.

[0061] In step (2-3), a CD3 / TCR complex agonist is contained in the medium. The CD3 / TCR complex agonist is not particularly limited, as long as it is a molecule capable of specifically binding to the CD3 / TCR complex and transmitting a signal from the CD3 / TCR complex into CD3-positive cells. Examples of CD3 / TCR complex agonists include CD3 agonists and / or TCR agonists. Examples of CD3 agonists include anti-CD3 agonist antibodies (also simply referred to as "anti-CD3 antibodies") or binding fragments thereof, and examples of TCR agonists include at least one selected from the group consisting of anti-TCR agonist antibodies (also simply referred to as "anti-TCR antibodies") or binding fragments thereof, MHC / antigen peptide complexes or multimers thereof, and MHC / superantigen complexes or multimers thereof. When an anti-CD3 antibody is used, the anti-CD3 antibody encompasses both polyclonal and monoclonal antibodies, but is preferably a monoclonal antibody. The antibody may belong to any immunoglobulin class, including IgG, IgA, IgM, IgD, or IgE, with IgG being preferred. Examples of anti-CD3 antibodies include the antibody produced from the OKT3 clone (OKT3) and the antibody produced from the UCHT1 clone (UCHT1), with UCHT1 being preferred. The concentration of the anti-CD3 antibody in the culture medium is, for example, 10 ng / ml to 1000 ng / ml, preferably 50 ng / ml to 800 ng / ml, and more preferably 250 ng / ml to 600 ng / ml. The CD3 / TCR complex agonist may be commercially available, purified from nature, or produced by peptide synthesis, genetic engineering, or chemical synthesis. For example, OKT3 and UCHT1 can be purchased from ThermoFisher, GeneTex, or other companies.

[0062] When a cytokine is used in step (2-3), examples of the cytokine include IL-2 and IL-7. When the cytokine is IL-2, its concentration in the medium is 10 U / ml to 1000 U / mL, and when the cytokine is IL-7, its concentration in the medium is 1 ng / ml to 1000 ng / mL.

[0063] In step (2-3), the culture temperature conditions are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of γδ T cells, etc. The number of days is not particularly limited as long as γδ T cells are obtained, but is, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, or more, and preferably 6 days. Furthermore, 28 days or less is preferred, and 14 days or less is more preferred.

[0064] The CD3-positive T cell population obtained by the above steps contains γδ T cells, which can be further enriched. Step (2) may further include the following step (2-4).

[0065] (2-4) Expanding CD3-positive T cells including γδ T cells The method for expanding CD3-positive T cells, including γδ T cells, is not particularly limited as long as it allows the γδ T cells to proliferate. For example, methods include culturing CD3-positive T cells, including γδ T cells, under culture conditions similar to those used in the process of expanding CD8α+β+ cytotoxic T cells, as described in WO 2016 / 076415 and WO 2018 / 135646.

[0066] In the present invention, the medium used for expanding CD3+ T cells, including γδ T cells, is not particularly limited, and a medium used for culturing animal cells can be prepared as the basal medium. Examples of the basal medium include those used in step (2-3) above. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax®), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, hormones, and the like. In one embodiment of the present invention, vitamin C such as ascorbic acid, insulin, transferrin, selenium compounds (eg, sodium selenite), and cytokines such as IL-7 may be contained.

[0067] When vitamin C is used in step (2-4), the vitamin C may be the same as those described in step (2-1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0068] In step (2-4), a CD3 / TCR complex agonist is contained in the medium. The CD3 / TCR complex agonist is not particularly limited, as long as it is a molecule capable of specifically binding to the CD3 / TCR complex and transmitting a signal from the CD3 / TCR complex into CD3-positive cells. Examples of CD3 / TCR complex agonists include CD3 agonists and / or TCR agonists. Examples of CD3 agonists include anti-CD3 agonist antibodies (also simply referred to as "anti-CD3 antibodies") or binding fragments thereof, and examples of TCR agonists include at least one selected from the group consisting of anti-TCR agonist antibodies (also simply referred to as "anti-TCR antibodies") or binding fragments thereof, MHC / antigen peptide complexes or multimers thereof, and MHC / superantigen complexes or multimers thereof. When an anti-CD3 antibody is used, the anti-CD3 antibody encompasses both polyclonal and monoclonal antibodies, with monoclonal antibodies being preferred. The antibody may belong to any immunoglobulin class, including IgG, IgA, IgM, IgD, or IgE, with IgG being preferred. Examples of anti-CD3 antibodies include the antibody produced from the OKT3 clone (OKT3) and the antibody produced from the UCHT1 clone (UCHT1), with UCHT1 being preferred. The concentration of the anti-CD3 antibody in the culture medium is, for example, 0.3 ng / ml to 10,000 ng / ml, preferably 50 ng / ml to 5,000 ng / ml, and more preferably 200 ng / ml to 4,000 ng / ml. The CD3 / TCR complex agonist may be commercially available, purified from nature, or produced by peptide synthesis, genetic engineering, or chemical synthesis. For example, OKT3 and UCHT1 can be purchased from ThermoFisher, GeneTex, or other companies.

[0069] In step (2-4), it is preferable that fibronectin or a variant thereof is present in the medium. Such fibronectin is not particularly limited, as long as it is a molecule capable of binding to CD3-positive cells. The fibronectin variant is not particularly limited, as long as it is a molecule capable of binding to VLA-5 and VLA-4 on the surface of CD3-positive cells, and examples thereof include retronectin. Fibronectin or a variant thereof may be present in any form in the medium. For example, it may be contained in the medium during culture or may be immobilized on a culture vessel, but is preferably immobilized on a culture vessel.

[0070] When fibronectin or a variant thereof is contained in a medium, the medium may be the same as the medium containing a CD3 / TCR complex agonist. Furthermore, the presence or absence of serum, additives, etc. may also be the same as the medium containing a CD3 / TCR complex agonist. When fibronectin or a variant thereof is contained in a medium, the concentration of the fibronectin or variant thereof may be, at the lower limit, 10 ng / ml or more, preferably 100 ng / ml or more, and at the upper limit, 10,000 μg / ml or less, preferably 1,000 μg / ml or less.

[0071] In step (2-4), it is also preferable that a CD30 agonist is present in the medium. Such a CD30 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD30 into the cell by specifically binding to CD30. Examples of the CD30 agonist include at least one selected from the group consisting of an anti-CD30 agonist antibody (also simply referred to as an "anti-CD30 antibody") or a binding fragment thereof, and a CD30 ligand or a binding fragment thereof.

[0072] As with the CD3 / TCR complex agonist, the CD30 agonist used in step (2-4) may be present in any form so long as it is capable of contacting CD30 during culture. For example, it may be contained in the culture medium during culture or may be immobilized on a culture vessel, but is preferably contained in the culture medium.

[0073] When a CD30 agonist is contained in the medium, the medium may be the same as the medium containing a CD3 / TCR complex agonist. Furthermore, the presence or absence of serum, additives, and the like may be the same as the medium containing a CD3 / TCR complex agonist. When a CD30 agonist is contained in the medium, the concentration of the CD30 agonist in the medium may be appropriately determined by those skilled in the art depending on the CD30 agonist. For example, when the CD30 agonist is an anti-CD30 agonist antibody or a binding fragment thereof, the concentration of the anti-CD30 agonist antibody or a binding fragment thereof in the medium is usually 1 ng / ml to 10,000 ng / ml, and preferably 30 ng / ml to 300 ng / ml.

[0074] Furthermore, when a CD30 agonist is immobilized on a culture vessel, the culture vessel may be the same as the culture vessel on which the CD3 / TCR complex agonist is immobilized. Furthermore, the method for immobilizing the CD30 agonist on a culture vessel may be the same as the method for immobilizing the CD30 agonist on a culture vessel. The concentration of the CD30 agonist solution when immobilizing the CD30 agonist on a culture vessel may be, at the lower limit, 0.1 ng / ml or more, preferably 1 ng / ml or more, and, at the upper limit, 10,000 ng / ml or less, preferably 1,000 ng / ml or less.

[0075] When a cytokine is used in step (2-4), examples of the cytokine include IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. These cytokines may be used singly or in combination (preferably all of them). When the cytokine is IL-2, its concentration in the medium may be 10 U / ml to 1000 U / ml. When the cytokine is IL-7, its concentration in the medium may be 1 ng / ml to 1000 ng / ml. Furthermore, the concentration of IL-12 in the medium may be 5 ng / ml to 500 ng / ml, the concentration of IL-15 in the medium may be 1 ng / ml to 100 ng / ml, the concentration of IL-18 in the medium may be 5 ng / ml to 500 ng / ml, and the concentration of IL-21 in the medium may be 2 ng / ml to 200 ng / ml.

[0076] In step (2-4), the medium may further contain a TNF family cytokine as a cytokine. Examples of TNF family cytokines include TNF-α, TNF-β, lymphotoxin α, Fas ligand, TRAIL, TWEAK, TL1A, RANK ligand, OX40 ligand, APRIL, AITRL, BAFF, 4-1BBL, and CD40 ligand, with TL1A being preferred. When TL1A is used, its concentration in the medium may be 5 ng / ml to 500 ng / ml, preferably 10 ng / ml to 300 ng / ml, and more preferably 20 ng / ml to 200 ng / ml.

[0077] Furthermore, in step (2-4), the medium may further contain an apoptosis inhibitor. Examples of the apoptosis inhibitor include protease inhibitors, such as caspase inhibitors. A preferred caspase inhibitor is the Pan Caspase FMK inhibitor Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone) (hereinafter, sometimes referred to as "Z-VAD-FMK"). The concentration of the inhibitor in the medium may be 1 μM to 1000 μM, preferably 1 μM to 500 μM, more preferably 1 μM to 200 μM, and particularly preferably 1 μM to 50 μM.

[0078] In the present invention, the obtained γδ T cells may be isolated and used, or they may be used as is (i.e., as a cell population that may contain other cell types). When isolating, they can be isolated using at least one molecule selected from the group consisting of γTCR, δTCR, and CD3 as an indicator, and methods well known to those skilled in the art can be used for the isolation method. Examples include, but are not limited to, isolation methods using flow cytometry or magnetic cell separation using antibodies to γTCR, δTCR, and CD3 (optionally bound to magnetic beads, etc.), and purification methods using an affinity column on which a desired antigen is immobilized. When using the cells as they are, the proportion of γδ T cells in the cell population may be increased using methods well known to those skilled in the art, including, but not limited to, the methods described in Front. Immunol., 5:636 (2014), JP 2017-537625, and JP 2003-529363.

[0079] Furthermore, the cells used in the production methods of the present invention may have nucleic acids encoding exogenous TCRs and / or chimeric antigen receptors (CARs) that recognize and bind to antigens or the antigen-HLA complexes. Thus, one embodiment of the present invention may include the steps of (1) establishing induced pluripotent stem cells from cells other than αβ T cells, and (2) introducing nucleic acids encoding the TCRs (i.e., (i) αTCR and βTCR, (ii) γTCR and δTCR) and / or (iii) nucleic acids encoding the CARs into cells (e.g., pluripotent stem cells, hematopoietic progenitor cells, etc.) obtained at any time during the step of differentiating the induced pluripotent stem cells established in step (1) into T cells. Among these, (i) nucleic acids encoding αTCR and βTCR are introduced into γδ T cells obtained during any step of the step of differentiating the induced pluripotent stem cells into T cells. As used herein, a nucleic acid encoding a TCR refers to a nucleic acid containing a nucleotide sequence encoding one chain forming a TCR and a nucleotide sequence encoding the other chain. Furthermore, a nucleic acid encoding a TCR also refers to a combination of a nucleic acid containing a nucleotide sequence encoding one of the chains forming a TCR and a nucleic acid containing a nucleotide sequence encoding the other chain. That is, when nucleic acids encoding TCRs ((i) αTCR and βTCR) are introduced into cells, a single nucleic acid containing both a nucleotide sequence encoding an αTCR and a nucleotide sequence encoding a βTCR may be introduced, or a nucleic acid containing a nucleotide sequence encoding an αTCR and a nucleotide sequence encoding a βTCR may be introduced separately. When introduced separately, these nucleic acids may be introduced simultaneously or sequentially. The same applies to (ii) γTCR and δTCR.

[0080] The TCRs used in the present invention include not only heterodimers formed by the α and β chains of TCR (i.e., αβTCRs) or heterodimers formed by the γ and δ chains of TCR (i.e., γδTCRs), but also homodimers. Furthermore, TCRs lacking part or all of the constant region or those with recombinant amino acid sequences may also be used. Among these, γδTCRs are preferred, with Vγ9Vδ2TCRs being particularly preferred.

[0081] Furthermore, the constant regions of the above-mentioned TCR chains may be modified in a specific manner in the constant regions of the TCR chains of the cytotoxic T cell (CTL) clones from which they are derived, such that, for example, specific amino acid residues in the TCR constant regions of the CTL clones are substituted with cysteine ​​residues to enhance the efficiency of dimer formation through disulfide bonds between the TCR chains, but this modification is not limited to this.

[0082] Antigens targeted by the TCR include, but are not limited to, tumor antigens, which may be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). Specific examples of such tumor antigens include one or more antigens selected from the group consisting of differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2; tumor-specific multilineage antigens such as WT1, Glypican-3, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; fetal antigens such as CEA; overexpressed oncogenes or mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA These include, but are not limited to, 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0083] As shown in the Examples below, in one embodiment, among the γδ T cells obtained by the production method of the present invention, cells expressing a chimeric antigen receptor (CAR) exhibited specific cytotoxic activity and antitumor activity (also referred to simply as "cytotoxic activity" herein) against cells expressing the target antigen of the CAR. Therefore, from the viewpoint of antigen-specific cytotoxic activity, it is preferable that the γδ T cells obtained by the production method of the present invention express a CAR. Whether the cells have cytotoxic activity can be confirmed by known methods, and a suitable method is, for example, a method in which cytotoxic activity against cells expressing the target antigen of the CAR is measured by a chromium release assay or the like.

[0084] In the present invention, the term "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain of the CAR comprises a single-chain fragment (scFv) consisting of the light (VL) and heavy (VH) variable chains of an antibody linked in tandem via a spacer such as a linker (e.g., a linker consisting of G and S (GS linker) (e.g., GGGS, GGGGS, or a combination thereof (e.g., SEQ ID NO: 4 or 5)). γδ T cells expressing the CAR recognize antigens via the scFv domain and then transmit the recognition signal into the T cell via the intracellular signaling domain. Introducing a CAR into a γδ T cell can confer specificity for a target antigen. Furthermore, because CARs can directly recognize antigen molecules independently of HLA class I or class II, they can elicit a strong immune response even against cells with reduced expression of HLA class I or class II genes. The antigens targeted by the CAR include the same antigens targeted by the TCR.

[0085] Examples of the transmembrane domain of the CAR include, but are not limited to, transmembrane domains derived from one or more proteins selected from the group consisting of the α chain, β chain, or ζ chain of TCR, CD28, CD3ε chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB (CD137), and CD154. The transmembrane domain of the molecule from which the first intracellular signaling domain linked to the antigen-binding domain is derived may be used. For example, if the molecule from which the first intracellular signaling domain linked to the antigen-binding domain is derived is CD28, the transmembrane domain may also be derived from CD28. Alternatively, an artificially designed transmembrane domain may be used.

[0086] Examples of the intracellular signaling domain of a CAR include, but are not limited to, intracellular domains derived from one or more proteins selected from the group consisting of CD3 ζ chain (TCR ζ chain), FcR γ chain, FcR β chain, CD3 γ chain, CD3 δ chain, CD3 ε chain, CD5, CD22, CD79a, CD79b, and CD66d. Among these, an intracellular signaling domain derived from the CD3 ζ chain is preferred. The intracellular signaling domain may further include the intracellular domain of a costimulatory molecule, such as the intracellular domain of one or more proteins selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. The strength and duration of CAR activity can be controlled by selecting the type and number of costimulatory molecules to be bound (e.g., Mol Ther. 2009;17:1453-1464.).

[0087] A spacer may be inserted between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular signaling domain and the transmembrane domain of the CAR. The spacer may be a peptide typically consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. Specific examples of the spacer include, but are not limited to, a hinge region derived from IgG1, or a peptide containing the CH2CH3 region of an immunoglobulin and a portion of CD3.

[0088] Specific examples of CARs include, but are not limited to, first-generation CARs in which an scFV and a CD3ζ chain are linked via a spacer; second-generation CARs in which a transmembrane domain and an intracellular domain derived from CD28 are incorporated between the scFV and CD3ζ chain of the first-generation CAR to enhance its ability to activate T cells; and third-generation CARs in which an intracellular domain of a costimulatory molecule other than CD28 (4-1BB or OX40) is incorporated between the CD28 intracellular domain and the CD3ζ chain of the second-generation CAR.

[0089] More specifically, the CAR used in the present invention includes a chimeric antigen receptor comprising an scFv that recognizes CD19 as the antigen-binding domain, a CD8 transmembrane domain as the transmembrane domain, and an intracellular signaling domain derived from CD28, CD30, 4-1BB, or CD3ζ chain. The order of the intracellular domains contained in the intracellular signaling domain is not particularly limited, but may be, for example, the order of the intracellular domain derived from CD28, the intracellular domain derived from CD30, or the intracellular domain derived from 4-1BB, and the intracellular domain derived from CD3ζ chain. More specifically, the chimeric antigen receptor of the present invention comprises, for example, the amino acid sequence represented by SEQ ID NO: 1 or 2, or an amino acid sequence in which one or more (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to several (2, 3, 4, or 5)) amino acids have been substituted, deleted, added, and / or inserted within the amino acid sequence represented by SEQ ID NO: 1 or 2.

[0090] Furthermore, the intracellular domain derived from CD30 may be, for example, an amino acid sequence in which one or more (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to several (2, 3, 4, or 5)) amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 3. When amino acids have been substituted, deleted, added, and / or inserted as described above, the position of the substitution, deletion, addition, and / or insertion is not particularly limited, as long as the function of the intracellular domain of CD30 is maintained.

[0091] Whether the above-mentioned TCR and / or CAR (hereinafter sometimes abbreviated as "TCR, etc.") can specifically recognize and bind to an antigen can be confirmed by known methods, and suitable methods include, for example, a dextramer assay or an ELISPOT assay. By performing an ELISPOT assay, it can be confirmed that a T cell expressing a TCR, etc. on its cell surface recognizes a target antigen via the TCR, etc., and that the signal is transmitted into the cell.

[0092] Furthermore, the present inventors have found that cells expressing a fusion protein containing IL-15 and IL-15Rα together with the CAR (hereinafter sometimes abbreviated as "IL-15 / IL-15Rα") have increased cytotoxic activity compared to cells expressing only the CAR. Therefore, from the viewpoint of cytotoxic activity, γδ T cells obtained by the production method of the present invention preferably express IL-15 / IL-15Rα, and more preferably also express the CAR. Therefore, to obtain γδ T cells expressing IL-15 / IL-15Rα, the production method of the present invention may include a step of introducing a nucleic acid encoding IL-15 / IL-15Rα into cells obtained during either step (1) or (2) of 1 above (e.g., CD3-positive T cells obtained in step (2-2), CD3-positive T cells enriched in step (2-3), etc.).

[0093] In the IL-15 signal transduction system, IL-15Rα expressed on antigen-presenting cells typically binds to IL-15 and presents IL-15 to the IL-15 receptor, consisting of IL-15Rβ and the common gamma chain (γc), on CD8+CD4-negative cells (trans-presentation), thereby maintaining the cytotoxic activity of CD8+CD4-negative cells. Therefore, when CD3+ cells expressing IL-15 / IL-15Rα are CD8+CD4-negative, they can transmit the IL-15 signal into their own cells via the IL-15 receptor. Alternatively, CD3+ cells expressing IL-15 / IL-15Rα can transmit the IL-15 signal into other CD8+CD4-negative cells via the IL-15 receptor. As described above, IL-15 / IL-15Rα can maintain the cytotoxic activity of CD8+CD4-negative cells, and thus a continuous cytotoxic effect against cells targeted by CAR can be expected.

[0094] IL-15 / IL-15Rα may be a transmembrane protein or a secreted protein. It is known that the IL-15-binding domain, consisting of amino acids 1-65 from the N-terminus of the mature IL-15Rα protein, is responsible for binding to IL-15 (Wei X. et al., J. Immunol., 167:277-282, 2001). Therefore, a transmembrane protein may be any protein that retains the IL-15-binding domain and the transmembrane domain of IL-15Rα. On the other hand, a secreted protein may be any protein that retains the IL-15-binding domain but lacks the transmembrane domain of IL-15Rα (e.g., a protein consisting of amino acid residues 1-65, 1-85, or 1-182 of IL-15Rα, or a peptide containing an amino acid sequence 85% or more identical to said amino acid sequence).

[0095] IL-15 / IL-15Rα may incorporate a spacer between IL-15 and IL-15Rα, and the spacer may be a peptide generally consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 20 to 50 amino acids. Specific examples of the spacer include, but are not limited to, the GS linker described above.

[0096] IL-15 / IL-15Rα is not particularly limited as long as it is a fusion protein of IL-15 and IL-15Rα, and a specific example is the peptide consisting of SEQ ID NO: 6. Alternatively, IL-15 / IL-15Rα is not particularly limited as long as it binds to the IL-15 receptor and transduces the IL-15 signal into cells, and examples include peptides comprising an amino acid sequence that has about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 6. Here, "homology" or "identity" refers to the percentage (%) of identical and similar amino acid residues (in the case of identity, identical amino acid residues) relative to the total overlapping amino acid residues in the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). "Similar amino acids" refer to amino acids similar in physicochemical properties, and include, for example, amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitution with such similar amino acids is expected to not change the phenotype of the protein (i.e., conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).The homology or identity of amino acid sequences herein can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0097] As used herein, the term "capable of binding" means "having an ability to bind" and refers to the ability to form a non-covalent complex with one or more other molecules. Various methods and assays for determining binding ability are known in the art. Binding is typically high affinity, with an affinity measured by a KD value preferably less than 1 μM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, and even more preferably less than 1 pM. The term "KD" or "KD value" refers to the equilibrium dissociation constant, as known in the art.

[0098] The above-mentioned TCRs and the like are introduced into cells in the form of nucleic acids encoding the TCRs and the like. A fusion protein containing IL-15 and IL-15Rα is also introduced into cells in the form of a nucleic acid encoding the fusion protein. The nucleic acid may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. When the nucleic acid is RNA, T in the RNA sequence is to be read as U. The nucleic acid may contain natural nucleotides, modified nucleotides, nucleotide analogs, or a mixture thereof, as long as it is capable of expressing a polypeptide in vitro or in cells.

[0099] The above nucleic acids can be constructed by known methods. For example, DNA encoding the full length or a portion of a TCR or CAR can be constructed by chemically synthesizing a DNA strand based on the amino acid sequence or nucleic acid sequence of a known TCR or CAR, or by connecting synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson assembly. Nucleic acids encoding fusion proteins containing IL-15 and IL-15Rα can also be constructed in a similar manner.

[0100] The nucleic acid can be incorporated into an expression vector. The vector may or may not be integrated into the genome of the target cell. In one embodiment, the vector that is not integrated into the genome can replicate outside the genome of the target cell. The vector may exist in multiple copies outside the genome of the target cell. In another embodiment of the present invention, the vector is integrated into the genome of the target cell. In a preferred embodiment, the vector is integrated into a predetermined location in the genome of the target cell.

[0101] Examples of promoters used in the above vectors include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR Vα gene promoter, and TCR Vβ gene promoter. Of these, the EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, and SRα promoter are preferred.

[0102] In addition to the promoter, the vector may optionally contain transcriptional and translational regulatory sequences, a ribosome binding site, an enhancer, a replication origin, a poly(A) addition signal, a selection marker gene, etc. Examples of selection marker genes include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.

[0103] In one embodiment of the present invention, an expression vector containing a nucleic acid encoding a TCR α chain and a nucleic acid encoding a β chain can be introduced into a target cell to form a heterodimer of the TCR α chain and β chain within the target cell or on the cell surface. In this case, the nucleic acid encoding the TCR α chain and the nucleic acid encoding the β chain may be incorporated into separate expression vectors or may be incorporated into a single expression vector. When incorporated into a single expression vector, these two types of nucleic acids are preferably incorporated via a sequence that enables polycistronic expression. The use of a sequence that enables polycistronic expression enables more efficient expression of multiple genes incorporated into a single expression vector. Examples of sequences that enable polycistronic expression include 2A sequences (e.g., 2A sequences (F2A) derived from foot-and-mouth disease virus (FMDV), 2A sequences (E2A) derived from equine rhinitis A virus (ERAV), 2A sequences (P2A) derived from porcine teschovirus (PTV-1), and 2A sequences (T2A sequences) derived from Thosea asigna virus (TaV) (PLoS ONE 3, e2532, 2008; Stem Cells 25, 1707, 2007), and internal ribosome entry sites (IRES) (US Patent No. 4,937,190). From the viewpoint of uniform expression levels, however, P2A and T2A sequences are preferred. The same applies when using an expression vector containing a nucleic acid encoding a TCR γ chain and a nucleic acid encoding a TCR δ chain.

[0104] The expression vector is not particularly limited as long as it can express TCR or the like for a period of time sufficient for disease prevention or treatment when introduced into cells, and examples include viral vectors and plasmid vectors. Examples of viral vectors include retroviral vectors (including lentiviral vectors and pseudotype vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai virus, and episomal vectors. Transposon expression systems (PiggyBac systems) may also be used. Examples of plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo).

[0105] The method for introducing the nucleic acid or vector into cells is not particularly limited, and known methods can be used. When introducing a nucleic acid or a plasmid vector, the same method as described in step 1.(1) above can be used. Alternatively, the nucleic acid can be introduced into the genome of the cell by genome editing (e.g., CRISPR system, TALEN, ZFN, etc.).

[0106] The above nucleic acids may also be directly introduced into cells in the form of RNA and used to express TCR etc. in the cells. As a method for introducing RNA, known methods can be used, and for example, lipofection and electroporation can be preferably used.

[0107] In the above steps (1) and (2), the timing of introducing the nucleic acid is not particularly limited as long as the introduced TCR and the like can be expressed in γδ T cells. For example, the timing of introducing the nucleic acid is not particularly limited as long as the introduced TCR and the like can be expressed in γδ T cells. + / CD43 + ), ProT cells (CD4 - / CD8 - ), CD3 + / CD4 + / CD8 + T cells, CD3 + / CD4 - / CD8 + T cells or other cells (e.g., CD3- / CD4 + / CD8 + It can be introduced at the stage of the gene expression vector (cells, etc.).

[0108] When the above-mentioned nucleic acid is introduced into a cell, it is preferable to suppress the expression of the endogenous TCR chain that the cell naturally expresses using siRNA, from the viewpoint of increasing the expression of the introduced TCR, suppressing the appearance of mispaired TCRs, or suppressing non-autoreactivity. When the above-mentioned nucleic acid is applied to the method, it is preferable to use a nucleotide sequence of the nucleic acid encoding the TCR that is different from the nucleotide sequence corresponding to the RNA on which the siRNA that suppresses the expression of the endogenous TCR chain acts (a codon-altered sequence) in order to avoid the effect of the siRNA on the TCR. These methods are described, for example, in WO 2008 / 153029. The nucleotide sequence can be prepared by introducing silent mutations into a nucleic acid encoding a naturally occurring TCR or by chemically synthesizing an artificially designed nucleic acid. Alternatively, to avoid mispairing with the endogenous TCR chain, part or all of the constant region of the introduced nucleic acid encoding the TCR may be replaced with a constant region derived from a non-human animal, such as a mouse.

[0109] 2. γδ T cells or cell populations containing said γδ T cells The present invention also provides γδ T cells or cell populations comprising the γδ T cells, wherein the γδ T cells are differentiated from induced pluripotent stem cells derived from cells other than αβ T cells. The percentage of γδ T cells in the cell population (number of γδ T cells in the cell population / total number of cells in the cell population) is preferably 90% or higher (e.g., 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). Such cell populations can be obtained, for example, by the methods of the present invention. The percentages can be calculated by measuring the percentages of cells expressing γTCR, δTCR, and CD3 by flow cytometry. Accordingly, in one embodiment, the present invention provides γδ T cells and / or cell populations comprising the γδ T cells produced by the methods of the present invention. The γδ T cells may comprise a nucleic acid encoding an exogenous TCR, a nucleic acid encoding a CAR, and / or a nucleic acid encoding a fusion protein comprising IL-15 and IL-15Rα, as described in 1 above. The γδ T cells mentioned herein, or a cell population containing the γδ T cells, may hereinafter be abbreviated as "the cells of the present invention, etc."

[0110] 3. Medicines containing the cells of the present invention The present invention provides a pharmaceutical comprising the cells, etc. of the present invention as an active ingredient (hereinafter, sometimes referred to as the "pharmaceutical of the present invention"). The cells, etc. of the present invention can exhibit cytotoxic activity against, for example, cancer cells, cancer stem cells, tumor cells, etc., and therefore, pharmaceuticals comprising the cells, etc. of the present invention can be used for the prevention or treatment of tumors such as cancer, and can be administered to, for example, mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans), preferably humans. Thus, in one aspect of the present invention, the cells, etc. of the present invention are provided for use in the prevention or treatment of tumors. Also provided is a method for preventing or treating tumors, which comprises administering the cells, etc. of the present invention, preferably in the form of a pharmaceutical comprising the cells, etc.

[0111] Tumors such as cancer that can be prevented or treated by the medicament of the present invention or the cells of the present invention are described, for example, in "Daniel Baumhoer et al., Am J. Clin Pathol, 2008, 129, 899-906," and tumors include benign tumors, malignant tumors (also referred to as "cancer"), and tumors that can be diagnosed or determined to be benign or malignant. Specific examples of tumors include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., ovarian clear cell adenocarcinoma), childhood cancer, lung cancer (e.g., squamous cell carcinoma, small cell lung carcinoma), testicular cancer (e.g., non-seminomatous germ cell tumor), soft tissue tumor (e.g., liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g., cervical intraepithelial neoplasia, cervical squamous cell carcinoma), melanoma, adrenal tumor (e.g., adrenal adenoma), neural tumor (e.g., schwannoma), gastric cancer (e.g., gastric adenocarcinoma), kidney cancer (e.g., Grawitz tumor), breast cancer (e.g., invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g., medullary carcinoma), laryngeal cancer (e.g., squamous cell carcinoma), and bladder cancer (e.g., invasive transitional cell carcinoma).

[0112] The cells contained in the pharmaceutical composition of the present invention may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before administration to a subject. Examples of stimulatory molecules include, but are not limited to, cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, and IL-2 is preferred. The concentration of IL-2 in the medium is not particularly limited, but is preferably 0.01 U / ml to 1 x 10 5 U / ml, more preferably 1 U / ml to 1×10 4U / ml. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, lymphocyte stimulating factors such as lectin can also be added. Furthermore, serum or plasma can be added to the medium. The amount of these to be added to the medium is not particularly limited, but examples include 0% to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources, but from the viewpoint of safety, autologous sources are preferred.

[0113] The pharmaceutical agent of the present invention is preferably administered parenterally to a subject. Examples of parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but typically, the number of cells is 1×10 per administration for a subject weighing 60 kg. 6 ~1×10 10 Preferably 1 x 10 7 ~1×10 9 5×10 7 ~5×10 8 The pharmaceutical composition of the present invention is administered so that the total number of cells reaches 100. The pharmaceutical composition may be administered once or multiple times. The pharmaceutical composition of the present invention may be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition of the present invention may contain a pharmacologically acceptable excipient as appropriate. The pharmaceutical composition of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc., in order to stably maintain the cells. Examples of the culture medium include, but are not limited to, RPMI, AIM-V, X-VIVO10, etc. Furthermore, the pharmaceutical composition may contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. for the purpose of stabilization.

[0114] Furthermore, the cells etc. of the present invention can kill cells expressing target antigens such as the above-mentioned tumor antigens, and therefore can be used as a killing agent for cells expressing the antigens (e.g., cancer cells, cancer stem cells, tumor cells, etc.). Such killing agents can be prepared and used in the same manner as the above-mentioned pharmaceuticals.

[0115] The present invention also encompasses an embodiment of using the cells of the present invention in the manufacture of a tumor preventive or therapeutic agent, similar to a pharmaceutical comprising the cells of the present invention. The tumor preventive or therapeutic agent can be manufactured by a method known per se. For example, similar to the above-described method for preparing the pharmaceutical of the present invention, it can be manufactured in a known form suitable for parenteral administration, such as an injection or infusion.

[0116] The present invention will be explained in more detail in the following examples, but the scope of the present invention is not limited to these examples. [Example]

[0117] [Example 1] Examination of methods for producing cells expressing γδTCR The cell population containing hematopoietic progenitor cells was a suspension cell population obtained by differentiating iPS cells (Ff-I01s04 line: derived from peripheral blood mononuclear cells of a healthy individual) provided by the Center for iPS Cell Research and Application, Kyoto University, according to known methods (e.g., the methods described in Cell Reports 2 (2012) 1722-1735 and WO 2017 / 221975). Specifically, 3 x 10 Ff-I01s04 lines were cultured in an ultra-low-attachment 6-well plate. 5Cells were seeded at 100 cells / well (Day 0) and cultured in EB medium (StemPro34 supplemented with 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 2 mM L-glutamine, 45 mM α-monothioglycerol, and 50 μg / ml ascorbic acid 2-phosphate) containing 10 ng / ml BMP4, 50 ng / ml bFGF, 15 ng / ml VEGF, and 2 μM SB431542 under hypoxic conditions (5% O2) for 5 days (Day 5). Subsequently, 50 ng / ml SCF, 30 ng / ml TPO, and 10 ng / ml FLT-3L were added, and the cells were cultured for an additional 5–9 days (up to Day 14) to obtain a suspension cell population. The medium was changed every 2–3 days during the culture period. The above-mentioned floating cell population containing HPCs was stained with the following antibody set.

[0118] [Table 1]

[0119] The stained cell population was subjected to sorting using a FACSAria. The resulting cell fraction was differentiated into lymphoid cells according to known methods (e.g., the methods described in Journal of Leukocyte Biology 96 (2016) 1165-1175 and WO 2017 / 221975). Specifically, the hematopoietic progenitor cell population was seeded at 2000 cells / well onto a 48-well plate coated with recombinant h-DLL4 / Fc chimera (Sino Biological) and Retronectin (Takara Bio) and cultured at 5% CO2 and 37°C. The medium was changed every 2 or 3 days during the culture period. The culture medium was αMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 55 μM 2-mercaptoethanol, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 50 ng / ml SCF, 50 ng / ml IL-7, 50 ng / ml FLT-3L, 100 ng / ml TPO, 15 μM SB203580, and 30 ng / ml SDF-1α. On days 7 and 14, the cells were passaged onto similarly coated 48-well plates. On day 21 (day 35), all cells were harvested and the presence of CD45(+) and CD3(+) fractions was confirmed by flow cytometry (BD FACSAria). TMThe cells were confirmed to be resistant to HIV-1 by ELISA using a 24-well plate (BD Biosciences Fusion). The resulting cells were seeded into 24-well plates and cultured at 37°C under 5% CO2 conditions. The culture medium used was αMEM medium containing 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 500 ng / ml anti-CD3 antibody (OKT3), 10 nM dexamethasone (Fuji Pharma Co., Ltd.: 10171-H02H), 100 U / ml IL-2, and 10 ng / ml IL-7. On day 27 (Day 41) after the start of culture, all cells were collected, the number of cells was counted using a hemocytometer, and then stained with the following antibody set.

[0120] [Table 2]

[0121] The staining results showed that cells expressing γδTCR (γδTCR-positive cells) could be prepared from hematopoietic progenitor cells derived from iPS cells (Ff-I01s04 line) (Figure 1).

[0122] Furthermore, the γδTCR-positive cells included Vδ1-positive γδT cells and Vδ2-positive γδT cells, demonstrating that Vδ1-type and Vδ2-type γδT cells can be prepared (Figure 2).

[0123] [Example 2] Examination of cytotoxic activity of γδT cells The cytotoxic activity of γδ T cells derived from iPS cells (Ff-I01s04 strain) obtained in Example 1 was evaluated. Using the mesothelioma cell line NCI-H226 as target cells, DELFIA BATDA Reagent (Perkin Elmer) was incubated at 37°C for 30 minutes. After washing the reaction solution, a population of γδ T cells derived from iPS cells (Ff-I01s04 strain) containing Vδ1-positive and Vδ2-positive γδ T cells was mixed with the target cells at a ratio of 0.5, 1, 2, 4, 8, or 16 times. The cytotoxic activity of γδ T cells derived from iPS cells (Ff-I01s04 strain) was evaluated based on target cell death after 2 hours.

[0124] The evaluation results showed that iPS cell (Ff-I01s04 line)-derived γδT cells had cytotoxic activity against the tumor cell line NCI-H226 (Figure 3).

[0125] (Expansion and functional evaluation of γδT cells) [Example 3] Production of iγδT cells iPS cell (Ff-I01s04 strain)-derived γδ T cells (iγδ T cells) were produced in the same manner as in Example 1, except that UCHT1 (GeneTex) was used as the anti-CD3 antibody.

[0126] [Example 4] Expansion of iγδT cells The γδT cells obtained in Example 3 were suspended at 2,000,000 cells / mL in α-MEM medium containing 15% FBS and supplemented with the cytokines listed in Table 3. The cells were then seeded onto plates coated with anti-CD3 antibody (UCHT1) and retronectin and cultured for 3 days at 5% CO₂ and 37°C. On day 3 of culture, the cells were harvested from the plates and counted using a NucleoCounter® NC-200 (ChemoMetec). The cells were then resuspended in an appropriate volume of α-MEM medium containing 15% FBS and supplemented with the cytokines listed in Table 4, added to non-coated G-Rex® 6-well plates (WILSONWOLF), and cultured at 5% CO₂ and 37°C. Subsequently, aliquots of cells were harvested from the plates 4-6 times on days 5, 6, 7, 8, 9, 10, 11, 14, and 17 of culture, and cell counts were measured using a hemocytometer. Anti-CD3 antibody and Retronectin were immobilized on culture plates as follows: Anti-CD3 antibody (UCHT1, final concentration 3000 ng / mL) and Retronectin (final concentration 150 μg / mL) dissolved in PBS at the required concentrations were added to the plate and then left to stand overnight at 4°C. After washing with PBS, the plate was subjected to the test.

[0127] [Table 3]

[0128] [Table 4]

[0129] Stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody resulted in proliferation of iγδT cells (FIG. 4).

[0130] [Example 5] Production of iPS cell-derived Vγ9Vδ2 T cells 1. iPS Cell Preparation The iPS cells used were the Ff-I01s04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, as in Example 1. iPS cell culture was performed according to the protocol "Feeder-Free Culture of Human iPS Cells" distributed by CiRA.

[0131] 2. Differentiation of iPS cells into HPCs Differentiation of iPS cells into hematopoietic progenitor cells (HPCs) was carried out in accordance with a known method (WO2017 / 221975), as in [Example 1].

[0132] 3.Vγ9Vδ2 gene The Vγ9Vδ2 T cell receptor (Vγ9Vδ2TCR G115) derived from the G115γδT cell clone was used. As a nucleic acid containing a gene encoding Vγ9Vδ2TCR G115, an oligo DNA encoding a polypeptide (SEQ ID NO: 7) designed to be arranged in the order of Table 5 from the N-terminus was artificially synthesized.

[0133] [Table 5]

[0134] 4. Construction of retroviral vector carrying the Vγ9Vδ2 gene The lentiviral vector used was pLVSIN-Ub, which was prepared by removing the sequence encoding the neomycin resistance gene from pLVSIN-CMV Neo (Clontech) and replacing the CMV promoter with a human ubiquitin promoter. The artificial oligo DNA synthesized in Example 5, section 3, was integrated into the multicloning site of the pLVSIN-Ub retroviral vector. This plasmid and Clontech's Lenti-X TM 293T cell line and Lenti-X TM Lentiviral vectors were produced using Packaging Single Shots (VSV-G).

[0135] 5. Production of iPS cell-derived Vγ9Vδ2 T cells The retroviral vector carrying the Vγ9Vδ2 gene prepared in [Example 5] 4. was infected into the iPS cells prepared in [Example 5] 1. and the iPS cell-derived hematopoietic progenitor cells (HPCs) prepared in Example [Example 5] 2. As in [Example 1], these cells were differentiated into T cells according to a known method (WO 2017 / 221975) to produce iPS cell-derived Vγ9Vδ2 T cells. The anti-CD3 antibody used in the differentiation process was 500 ng / mL UCHT1 (GeneTex). (Hereinafter, iPS cell-derived Vγ9Vδ2 T cells generated from iPS cells will be referred to as "iγ9δ2 T cells," and iPS cell-derived Vγ9Vδ2 T cells generated from iPS cell-derived HPCs will be referred to as "iHγ9δ2 T cells.") The expression of CD3, γδ TCR, Vγ9, and Vδ2 on the cell membrane surface of the obtained iγ9δ2 T cells and iHγ9δ2 T cells was analyzed using a flow cytometer (BD FACSAria TM Fusion, BD Biosciences) (Figs. 5 and 6).

[0136] [Example 6] Production of iPS cell-derived anti-CD19-CAR / IL-15γδ T cells 1. Anti-CD19-CAR gene As a nucleic acid containing the anti-CD19-CAR gene, an oligo DNA encoding a polypeptide (SEQ ID NO: 2) designed to be arranged in the order shown in Table 6 from the N-terminus was artificially synthesized.

[0137] [Table 6]

[0138] 2. Preparation of retroviral vector carrying anti-CD19-CAR gene [Example 6] The artificial oligo DNA synthesized in 1. was inserted into the multicloning site of the pMY retroviral vector. A viral vector was produced using FRY-RD18 cells for producing retroviral vectors.

[0139] 3.IL-15Rα / IL-15 gene As a nucleic acid containing the IL-15Rα / IL-15 gene, an oligoDNA encoding a polypeptide (SEQ ID NO: 6) designed to be arranged in the order shown in Table 7 from the N-terminus was artificially synthesized.

[0140] [Table 7]

[0141] 4. Construction of retroviral vector carrying IL-15Rα / IL-15 gene The artificial oligo DNA synthesized in [Example 6] 3. was inserted into the multicloning site of the pMY retroviral vector. A viral vector was produced using FRY-RD18 cells for producing retroviral vectors.

[0142] 5. Production of iPS cell-derived anti-CD19-CAR / IL-15γδ T cells The iγδ T cells obtained in [Example 4] and the iHγ9δ2 T cells prepared in [Example 5], 5. were infected with the retroviral vector carrying the anti-CD19-CAR gene prepared in [Example 6], 2. and the retroviral vector carrying the IL-15Rα / IL-15 gene prepared in [Example 6], 4., to produce iPS cell-derived anti-CD19-CAR / IL-15γδ T cells. (Hereinafter, iPS cell-derived anti-CD19-CAR / IL-15γδ T cells prepared from iγδ T cells may be referred to as "iCD19CAR / IL-15γδ T cells," and iPS cell-derived anti-CD19-CAR / IL-15γδ T cells prepared from iHγ9δ2 T cells may be referred to as "iHCD19CAR / IL-15γ9δ2 T cells.")

[0143] [Example 7] Expansion of iPS cell-derived anti-CD19-CAR / IL-15γδ T cells 1. Expansion of iCD19CAR / IL-15γδ T cells The iCD19CAR / IL-15γδT cells obtained in [Example 6] were expanded in the same manner as in [Example 4]. However, a medium containing an additive containing a cytokine in Table 8 was used instead of the additive containing a cytokine in Table 3, and a medium containing an additive containing a cytokine in Table 9 was used instead of the additive containing a cytokine in Table 4.

[0144] [Table 8]

[0145] [Table 9]

[0146] Stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody resulted in proliferation of iCD19CAR / IL-15γδT cells (Figure 7).

[0147] 2. Expansion of iHCD19CAR / IL-15γ9δ2 T Cells The iHCD19CAR / IL-15γ9δ2 T cells obtained in [Example 6] were expanded in the same manner as in [Example 7] 1. However, no anti-human CD30 antibody was added. Stimulation with anti-CD3 antibody (UCHT1) resulted in proliferation of iHCD19CAR / IL-15γ9δ2 T cells (Figure 8).

[0148] [Example 8] Examination of the cytotoxic activity of iPS cell-derived anti-CD19-CAR / IL-15γδ T cells The cytotoxic activity of iCD19CAR / IL-15γδ T cells and iHCD19CAR / IL-15γ9δ2 T cells obtained in Example 7 was evaluated. CD19-positive Raji cells and CD19-negative CCRF-CEN cells were used as target cells, and iCD19CAR / IL-15γδ T cells or iHCD19CAR / IL-15γ9δ2 T cells were mixed at a ratio of 0.5, 1, 2, 4, 8, or 16 times the target cells. The cytotoxic activity of iCD19CAR / IL-15γδ T cells and iHCD19CAR / IL-15γ9δ2 T cells was evaluated based on the rate of target cell death after 2 hours.

[0149] The evaluation results showed that iCD19CAR / IL-15γδT cells and iHCD19CAR / IL-15γ9δ2T cells had cytotoxic activity against CD19-positive Raji cells, but not against CD19-negative CCRF-CEN cells (Figures 9 and 10).

[0150] [Example 9] Effect of iCD19CAR / IL-15γδT cells on extending survival time 5x10 NOD / Shi-scid, IL-2RγKO (NOG) mice (Central Institute for Experimental Animals, female, 7-8 weeks old) 5 Nalm6 xenograft mice were generated by transplanting 100 cells of Nalm6 cells (ATCC) into the tail vein. Four days after transplantation, iCD19CAR / IL-15γδT cells (5x10) prepared in [Example 6] were added. 6 A suspension of 100 cells (1000 cells) in 0.1 mL of HBSS-buffer solution or an equal volume of HBSS-buffer solution (control) was administered via the tail vein, and the survival time was then determined. In mice that received CD19-positive Nalm6 cancer cells via the tail vein, all mice in the control group died within 3 weeks, whereas all mice in the iCD19CAR / IL-15γδT cell group survived for at least 6 weeks (Figure 11).

[0151] [Example 10] In vivo antitumor effect of iHCD19CAR / IL-15γ9δ2 T cells 5x10 NOD / Shi-scid, IL-2RγKO (NOG) mice (Central Institute for Experimental Animals, female, 7-8 weeks old) 5 Luciferase-expressing Nalm6 cells (ATCC) were transplanted into the tail vein to generate luciferase-expressing Nalm6 xenograft mice. Four days after transplantation, iHCD19CAR / IL-15γ9δ2 T cells (5×10) prepared in [Example 6] were added. 6 A suspension of Nalm6 cells in 0.1 mL of HBSS buffer or an equal volume of HBSS buffer (control) was administered via the tail vein. Two weeks after administration, luciferin was administered via the tail vein, and the luciferase activity expressed by Nalm6 cells was measured using an IVIS Imaging System (IVIS LUMINA II, CaliperLS). In the control administration group, luminescence derived from Nalm6 cells was confirmed throughout the body, whereas in the iHCD19CAR / IL-15γ9δ2 T cell administration group, almost no luminescence was detected (Figure 12). [Industrial Applicability]

[0152] According to the present invention, γδ T cells can be efficiently obtained, and the cells thus obtained are useful for preventing or treating diseases such as tumors.

[0153] This application is based on Japanese Patent Application No. 2018-133727 (filing date: July 13, 2018) and Japanese Patent Application No. 2019-117891 (filing date: June 25, 2019), the contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for producing γδ T cells from induced pluripotent stem cells derived from cells other than αβ T cells, comprising the following steps (1) and (2): (1) Establishment of induced pluripotent stem cells from cells other than αβ T cells (2) differentiating the induced pluripotent stem cells established in step (1) into T cells to obtain γδ T cells or a cell population containing the γδ T cells; Here, the γδ T cells or the cell population containing γδ T cells contain CD8α+β+ cells.

2. The method of claim 1, wherein the cells other than αβ T cells are mononuclear cells other than αβ T cells.

3. The method of claim 1 or 2, wherein the cells other than αβ T cells are monocytes.

4. The cells obtained in either step (1) or (2) above are treated with a compound that recognizes and binds to a tumor-specific antigen or a tumor-associated antigen. (i) a nucleic acid encoding an αTCR and a nucleic acid encoding a βTCR; (ii) a nucleic acid encoding a γTCR and a nucleic acid encoding a δTCR, and / or (iii) a nucleic acid encoding a CAR The method according to any one of claims 1 to 3, comprising the step of introducing:

5. The method of claim 4, wherein the γTCR is a Vγ9TCR and the δTCR is a Vδ2TCR.

6. The method according to any one of claims 1 to 5, comprising a step of introducing a nucleic acid encoding a fusion protein comprising IL-15 and IL-15Rα into cells obtained in either step (1) or (2).

7. A cell population in which at least 90% or more of the total cells are γδ T cells, wherein the γδ T cells are differentiated from induced pluripotent stem cells derived from cells other than αβ T cells, and the cell population contains γδ TCR+ and CD8α+β+ cells.

8. A pharmaceutical comprising the cell population described in claim 7.

9. The pharmaceutical composition according to claim 8, for use in the prevention or treatment of tumors.

10. A cell killing agent comprising the cell population described in claim 7.

11. The cell population of claim 7 for use in the prevention or treatment of tumors.

12. Use of the cell population according to claim 7 in the manufacture of a preventive or therapeutic agent for tumors.

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