Method for producing γ δ t cells

Abstract

There is provided a method of producing γ δ T cells from induced pluripotent stem cells, wherein the induced pluripotent stem cells are derived from cells other than α β T cells.SOLUTION: Disclosed is a method for efficiently obtaining γ δ T cells by inducing induced pluripotent stem cells from cells other than α β T cells and further inducing the cells 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, ... and a method for producing γδ T cells from induced pluripotent stem cells. The present invention relates to γδ T 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 γδT cell therapy, it is necessary to efficiently produce the cells and stably The development of a manufacturing method for supplying these cells is desirable, and it is expected that only γδ T cells in the patient's blood will be selected. Although a method for producing γδ T cells from stem cells has been reported (a method for culturing blood cells in a medium containing zoledronic acid and IL-2 (Patent Document 1)), as far as the present inventors know, no method for producing γδ T cells from stem cells has been reported. It has not been announced. [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. The present invention aims to provide γδ T cells differentiated from stem cells and cell populations containing said cells. The title is: [Means for solving the problem]

[0006] As a result of extensive research to solve the above problems, the present inventors have discovered that cells other than αβT cells can be used to treat inflammatory bowel diseases. The present inventors have found that γδ T cells can be efficiently obtained by inducing induced pluripotent stem cells from the γδ T cells and then inducing the cells to T cells. By introducing an antigen receptor (CAR) gene, we generated γδT cells expressing the CAR. Furthermore, it was shown that the γδ T cells exhibited high cytotoxicity against cancer cells that were difficult for the γδ T cells to recognize and damage before the introduction. Based on these findings, the present inventors conducted further research, and as a result, they completed 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 cells other than αβ T cells are mononuclear cells other than αβ T cells. [1] or [2] The method described below. [4] The method according to any one of [1] to [3], wherein the cells other than αβT cells are monocytes. [5] The cells obtained in either of the steps (1) and (2) are treated with a tumor-specific antigen or Recognizes and binds to tumor-associated antigens (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], 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). [8] γδT cells derived from induced pluripotent stem cells, wherein the induced pluripotent stem cells are αβT cells. A cell that is derived from a cell other than the host. [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 cell of any one of [8] to

[12] , wherein the γδ T cell expresses a CAR. Cell.

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

[14] or the cell population according to

[15] Medicine.

[17] The pharmaceutical agent according to

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

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

[14] or the cell population according to

[15] , a cell killing agent.

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

[14] for use in the prevention or treatment of tumors. A cell or a cell population described in

[15] .

[20] The method according to any one of [8] to

[14] for producing a preventive or therapeutic agent for tumors. Use of cells or cell populations as described in

[15] .

[21] Administering the cells according to any one of [8] to

[14] or the cell population according to

[15] . A method for preventing or treating a tumor, comprising: [Effects of the Invention]

[0008] According to the present invention, a method for producing γδ T cells from induced pluripotent stem cells, induced pluripotent stem cells, It is possible to provide γδ T cells differentiated from the above-mentioned cells and cell populations containing the 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 a method known in the art. Protein detection can be performed using an immunological assay using an antibody, such as ELISA, immunostaining, or flow cytometry. Furthermore, in the case of proteins that are expressed intracellularly but not 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 can be detected using nucleic acid amplification and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0012] As used herein, the term "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 for mRNA expression may vary depending on the method.

[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 a method for producing γδ T cells from induced pluripotent stem cells, and a cell population containing the γδ T cells (hereinafter abbreviated as "the production method of the present invention"). The production method of the present invention comprises a step of differentiating induced pluripotent stem cells into T cells. The induced pluripotent stem cells used in the production method of the present invention are derived from previously isolated γδ T cells. The cells may be established and stocked, or may be established from cells other than αβ T cells. Therefore, in one embodiment of the present invention, the method of the present invention comprises the steps of: (1) establishing induced pluripotent stem cells from cells other than αβ T cells; (1) The method includes a step of differentiating the induced pluripotent stem cells established in (1) into T cells.

[0020] In the present invention, the term "T cell receptor (TCR)" refers to a group of TCR chains (α chain, β chain, γ chain, δ chain). "γδ T cells" are composed of T cells that express CD3 and the TCR γ chain (γTCR) and "αβ T cells" refer to cells that express a TCR (hereinafter sometimes referred to as "γδ TCR") composed of a TCR δ chain (δ TCR). "αβ T cells" refer to cells that express CD3 and have a TCR α chain (α TCR) and a TCR They express a TCR (hereinafter sometimes referred to as "αβTCR") composed of a β chain (βTCR). Most αβ T cells bind antigen peptides to MHC (major histocompatibility complex) via αβ TCR. In contrast, γδ T cells recognize the MHC complex, which in humans is called the HLA (human leukocyte antigen) complex (this is called MHC restriction). Each TCR chain is composed of a variable region and a constant region. There are three complementarity determining regions (CDR1, CDR2, and CDR3) in the TCR region. Above are a number of V (variable), D (diversity), J (joining), and C (constant) genes. During the differentiation and maturation of T cells, gene rearrangement occurs, resulting in the formation of the β chain gene. In this case, one D and one J are randomly selected and combined, and then gene recombination occurs between V and DJ. During this process, random insertions and deletions of bases occur between the VD and DJ regions, increasing genetic diversity. In the TCR pre-mRNA, RNA splicing occurs between the VDJ region and the common region, the C region, and the functional TCR gene is expressed.

[0021] Examples of γTCRs include Vγ1TCR, Vγ2TCR, Vγ3TCR, Vγ4TCR, Vγ5TCR, Vγ6TCR, Vγ7TCR, Vγ8TCR, and Vγ9TCR, and examples of δTCRs include Vδ1TCR, Vδ2TCR, Vδ3TCR, and V δ4TCR, Vδ5TCR, Vδ6TCR, Vδ7TCR, Vδ8TCR, and Vδ9TCR. Examples of 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") are The term "induced pluripotent stem cells" refers 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, and also have the ability to proliferate. This term encompasses at least any cell 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, and Glis1. These reprogramming factors can be used alone or as a single reprogramming factor. Combinations of reprogramming factors include those described in 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, and Maekawa M, et al. (2011), Nature. 474:225-9.

[0024] Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature 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) neural stem cells, hematopoietic progenitor cells, mesenchymal stem cells, and the like. Examples include (1) tissue stem cells (somatic stem cells) such as αβ T cells and dental pulp stem cells, (2) tissue progenitor cells, (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, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes. Mononuclear cells other than cells are preferred, more particularly monocytes or γδ T cells.

[0025] As a method for introducing reprogramming factors into somatic cells, when the reprogramming factors are in the form of DNA, for example, , calcium phosphate co-precipitation method, PEG method, electroporation method, microinjection For example, the method described in Cell Engineering Special Issue 8 Methods that can be used include those described in 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). When using a viral vector, the nucleic acid is introduced into an appropriate packaging cell (e.g., Plat-E cells) or a complementation cell line (e.g., 293 cells). The virus vectors are then introduced into cells, and the virus vectors produced in the culture supernatant are collected and then infected into cells using an appropriate method depending on the virus vector. For example, specific methods using retroviral vectors as vectors are disclosed in International Publication No. 2007 / 69666, Cell, 126, 663-676 (2006), and Cell, 131, 861-872 (2007), among others. In particular, when using retroviral vectors, the use of recombinant fibronectin fragment CH-296 (Takara Bio Inc.) enables highly efficient gene transfer into various cells.

[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. As a method for introducing RNA, known methods can be used, for example, lipofection. Suitable methods for use include the transfection method, electroporation, etc. 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), and 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, medium for primate ES cells (culture medium for primate ES / iPS cells, ReproCell (Inc.), mouse ES cell medium (TX-WES medium, ThromboX), serum-free medium (mTeSR, Stemcell Technology), ReproFF, StemSpan (registered trademark) SFEM, StemSpan (registered trademark) H3000 , Stemline II, ESF-B medium, ESF-C medium, CSTI-7 medium, Neurobasal medium (Life Techno Examples of suitable media include, but are not limited to, StemPro-34 medium, StemFit (registered trademark) (e.g., StemFit AK03N, StemFit AK02N), and the like. Depending on the circumstances, they may be mixed and used, for example, DMEM / F12 medium.

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

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

[0030] In the present invention, the mammal from which the somatic cells are collected is not particularly limited, but is preferably a human. From the viewpoint of preventing rejection reactions, autologous cells, cells with the same HLA type or or substantially identical allogeneic cells, or other cells with adjusted HLA expression and / or expression level. Preferably, the cells are homeostatic cells, etc. Preferably, the presence or absence of expression and / or the expression level of at least some of the subunits contained in HLA class I and / or class II is 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 can differentiate induced pluripotent stem cells into γδ T cells. In one embodiment of the present invention, however, induced pluripotent stem cells are differentiated into T The step of differentiating the induced pluripotent stem cells into hematopoietic progenitor cells includes (2-1) a step of differentiating the induced pluripotent stem cells into hematopoietic progenitor cells; and (2-2) 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)" means CD34 positive cells, preferably CD34 / CD43 dual positive (DP) cells. In this context, 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 can differentiate into hematopoietic progenitor cells. For example, as described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, and Liu S. et al., Cytotherapy, 17 (2015); 344-358, One example is a method of culturing pluripotent stem cells in a medium for inducing hematopoietic progenitor cells.

[0034] In the present invention, the medium for inducing hematopoietic progenitor cells is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C ( 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-alpha Ranyl-L-glutamine (e.g., Glutamax®), non-essential amino acids, vitamins, growth Factors, low molecular weight compounds, antibiotics (eg, penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like may also be included.

[0035] In the present invention, vitamin C means L-ascorbic acid and its derivatives. A scorbic acid derivative is a substance that becomes vitamin C through an enzymatic reaction in the body. The derivatives of ascorbic acid used in the present invention include vitamin C phosphate (e.g., ascorbic acid 2-phosphate), ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, tetrahydrofuran, and the like. Ascorbyl tetrahexyldecanoate, ascorbyl stearate and diphosphate ascorbate Preferred is vitamin C phosphate (e.g., ascorbic acid 2-phosphate), including, for example, L-ascorbic acid phosphate salts such as sodium L-ascorbate phosphate or magnesium L-ascorbate phosphate.

[0036] When vitamin C is used, it is preferable to separately add (supplement) vitamin C every four days, every three days, every two days, or every day, and it is more preferable to add it every day. In this state, the vitamin C compounds are present in the culture medium in an amount equivalent to 5 ng / ml to 500 ng / ml (e.g., 5 In another embodiment, the vitamin C analogue is added in an amount equivalent to 5 μg / ml to 500 μg / ml (e.g., 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) in the culture medium. (amount required) is added.

[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. These are small molecule inhibitors that interfere with the signal transduction of myristates, such as SB431542 and SB202190 ( , RK Lindemann et al., Mol. Cancer 2:20(2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), etc. For example, when the TGFβ inhibitor is SB431542, the medium The concentration of the medium is preferably 0.5 μM to 100 μM.

[0040] The culture of induced pluripotent stem cells may be either adherent culture or suspension culture. In the case of adherent culture, the culture may be performed using a culture vessel coated with an extracellular matrix component, or co-culture with feeder cells. The feeder cells are not particularly limited, but examples thereof include fibroblasts (mouse embryonic fibroblasts (MEF), mouse fibroblasts (STO), etc.). It is preferable that the feeder cells are inactivated by a method known per se, for example, irradiation (such as gamma rays) or treatment with an anticancer drug (such as mitomycin C). Examples of extracellular matrix components include Matrigel (Niwa A, et al. PLoS One.6(7):e22261, 2011), gelatin, collagen, Examples include fibrous proteins such as 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. This includes, but is not limited to, culture vessels that have not been artificially treated to improve cell adhesion (for example, coating with an extracellular matrix, etc.), or culture vessels that have not been artificially treated to suppress adhesion (for example, coating with polyhydroxyethyl methacrylate (poly-HEMA) or non-ionic boundary layer). This can be done using a culture vessel coated with a surface-active polyol (such as Pluronic F-127). When performing suspension culture, it is preferable to form embryoid bodies (EBs) and then culture them.

[0042] In the present invention, hematopoietic progenitor cells can also be prepared from a net-like structure (also referred to as ES-sac or iPS-sac) obtained by culturing pluripotent stem cells. The "artificial structure" is a three-dimensional sac-like structure (with a space inside) derived from pluripotent stem cells, formed from endothelial cell groups, etc., and containing hematopoietic progenitor cells inside.

[0043] The culture temperature is not particularly limited, but is preferably about 37°C to 42°C, and more preferably about 37°C to 39°C. The culture period can be appropriately determined by those skilled in the art while monitoring the number of hematopoietic progenitor cells. 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, and is preferably 14 days. Long culture periods are not usually problematic in the production of hematopoietic progenitor cells, but are preferably 35 days or less, and more preferably 21 days or less. Culture may also be performed under hypoxic conditions. In the present invention, hypoxic conditions refer to oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or less. Concentrations are exemplified.

[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 can differentiate hematopoietic progenitor cells into CD3-positive T cells. For example, the method described in International Publication No. 2016 / 076415 or International Publication No. Examples of such methods include culturing hematopoietic progenitor cells under culture conditions similar to those used in methods for inducing T cells from hematopoietic progenitor cells, such as those described in US Pat. No. 2017 / 221975.

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

[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 one embodiment, the vitamin C compound is present in a concentration of 5 μg / ml to 500 μg / ml in the culture medium. amount (e.g. 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 400 μg / ml, 500 μg / ml) are added.

[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] Chemical inhibitors of p38 used in the present invention include SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivative, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole). azole) and its derivatives, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxyphenyl)-2-methyl-2-propanol) Examples include, but are not limited to, [(4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SC1O-469, SC1O-323, VX-702, and FR167653. These compounds are commercially available. Chemical inhibitors of P38 include SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonyl) ... Phenyl)-5-(4-pyridyl)-1H-imidazole), and derivatives thereof are preferred.

[0049] Dominant negative mutants of p38 used in the present invention include p38T180A, in which threonine at position 180, located in the DNA binding domain of p38, is point mutated to alanine, and p38Y182F, in which tyrosine at position 182 of human and mouse p38 is point mutated to phenylalanine. is contained in the medium in a range of about 1 μM to about 50 μM. When SB203580 is used as the P38 inhibitor, it can be contained in the medium in a range of 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. It may also be a mixture of these in any ratio. Preferably, SDF-1α is used. SDF-1 is also called CXCL-12 or PBSF.

[0051] In the present invention, SDF-1 is a chemokine having an amino acid sequence similar to that of SDF-1, as long as it has chemokine activity. In the sequence, one or several amino acids are substituted, deleted, added and / or inserted. (SDF-1 having such amino acid substitutions, deletions, additions and / or insertions may be used.) Similarly, in SDF-1 or SDF-1 mutants, the sugar chain The SDF-1 mutant may be substituted, deleted and / or added. Examples of SDF-1α 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 amino acid sequence of the native SDF-1α, but are not limited to these amino acid mutations. The SDF-1α may be derived from a mammal, such as a human, or a non-human mammal, such as a monkey, sheep, cow, horse, pig, dog, cat, rabbit, rat, or mouse. The protein registered under GenBank accession number: NP_954637 can be used as SDF-1β. The protein registered under GenBank accession number: NP_000600 can be used.

[0052] SDF-1 may be commercially available or purified from natural sources. Alternatively, SDF-1 produced by peptide synthesis or genetic engineering techniques may be used. SDF-1 is contained in the medium, for example, in a range 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) contains SCF, TPO (thrombopoietin), FLT-3L, and IL-7. At least one, preferably all, of the cytokines selected from the group consisting of SCF, TPO, IL-7, and FLT-3L may be added to the culture medium at concentrations of, for example, 10 ng / ml to 100 ng / ml, 10 ng / ml to 200 ng / ml, 1 ng / ml to 100 ng / ml, and 1 ng / ml to 200 ng / ml. The average concentration is 100 ng / ml.

[0054] In step (2-2), the hematopoietic progenitor cells may be cultured in an adherent culture or a suspension culture. In this case, the culture vessel may be coated before use, or the cells may be co-cultured with feeder cells or the like. An example of the feeder cells 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 that constitutively express DLL4 or DLL1 (see, for example, Holmes R1 and Zuniga-Pfluc In the present invention, when OP9 cells are used as feeder cells, separately prepared DLL4 or DLL1, or a combination of DLL4 or DLL1 and Fc This may be achieved by adding a fusion protein with, for example, a soluble factor (e.g., soluble factor) to the medium as appropriate. When feeder cells are used, it is preferable to appropriately replace the feeder cells during culture. Feeder cell replacement can be achieved by transferring the target cells being cultured onto previously seeded feeder cells. This 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, it is preferable to dissociate them into single cells and then perform adhesion culture. Co-culture with feeder cells is possible, but preferably, culture is performed without using feeder cells. In the case of adherent culture, examples of coating agents for coating a culture vessel 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 a fusion protein of DLL4 or DLL1 with an Fc region of an antibody (hereinafter sometimes referred to as Fc) (e.g., DLL4 / Fc chimera), entactin, and / or the like. Among these combinations, a combination of Retronectin and a fusion protein of DLL4 with Fc or the like is preferred.

[0055] In step (2-2), the culture temperature conditions are not particularly limited, but may be, for example, about 37°C to about 42°C. Preferably, the culture temperature is 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. However, for example, the method may be carried out under the same culture conditions as in the process of inducing CD8-positive T cells from CD4CD8-positive T cells, as described in International Publication Nos. 2016 / 076415 and 2017 / 221975. and methods for culturing CD3-positive T cells.

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

[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 one embodiment, the vitamin C compound is present in a concentration of 5 μg / ml to 500 μg / ml in the culture medium. amount (e.g. 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 400 μg / ml, 500 μg / ml) are added.

[0060] When a hormone is used in step (2-3), the hormone may be an adrenocortical hormone. The adrenal cortical 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 adrenal cortical 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. By specifically binding to the CD3 / TCR complex, the CD3 / TCR complex is released into the CD3-positive cells. There are no particular limitations on the molecule as long as it can transmit a signal to the CD3 / TCR complex. Examples of agonists include CD3 agonists and / or TCR agonists. CD3 agonists include anti-CD3 agonist antibodies (also simply referred to as "anti-CD3 antibodies") or Binding fragments, anti-TCR agonist antibodies (also simply called "anti-TCR antibodies") as TCR agonists or a binding fragment thereof, an MHC / antigen peptide complex or a multimer thereof, and an MHC / superantigen complex or a multimer thereof. When antibodies are used, anti-CD3 antibodies are available in both polyclonal and monoclonal forms. The antibody may be any of IgG, IgA, IgM, and the like, but is preferably a monoclonal antibody. The anti-CD3 antibody may belong to either the IgD or IgE immunoglobulin class, but is preferably IgG. Examples of the anti-CD3 antibody include an antibody produced from the OKT3 clone (OKT3) and an antibody produced from the UCHT1 clone (UCHT1), with UCHT1 being preferred. The concentration of the anti-CD3 antibody in the medium is, for example, 10 ng / ml to 1000 ng / ml, and is preferably The concentration is 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, etc.

[0062] When a cytokine is used in step (2-3), the cytokine may be IL-2, IL-7, or the like. 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. be.

[0063] In step (2-3), the culture temperature conditions are not particularly limited, but may be, for example, about 37°C to about 42°C. The culture temperature is 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 or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more, and preferably 6 days. 28 days or less is preferable, and 14 days or less is more preferable.

[0064] The CD3-positive T cell population obtained through the above steps contains γδ T cells, which can be further enriched. However, 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 the γδ T cells proliferate. For example, the method may be a culture method similar to the process for expanding CD8α+β+ cytotoxic T cells, as described in WO 2016 / 076415 and WO 2018 / 135646. For example, a method for culturing CD3-positive T cells, including γδ T cells, under nutrient-enriched conditions is provided.

[0066] In the present invention, the medium used for the expansion of CD3-positive T cells including γδ T cells is particularly The basal medium can be prepared from a medium used for culturing animal cells, but is not limited to the above. Examples of the basal medium include the same medium as that used in step (2-3) above. The basal 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, etc. 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 (registered trademark) The supplement may contain non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, hormones, etc. In one embodiment of the present invention, the supplement may contain vitamin C such as ascorbic acid, insulin, transferrin, selenium compounds (e.g., sodium selenite), etc. and cytokines such as IL-7.

[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 one embodiment, the vitamin C compound is present in a concentration of 5 μg / ml to 500 μg / ml in the culture medium. amount (e.g. 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 400 μg / ml, 500 μg / ml) are added.

[0068] In step (2-4), a CD3 / TCR complex agonist is contained in the medium. By specifically binding to the CD3 / TCR complex, the CD3 / TCR complex is released into the CD3-positive cells. There are no particular limitations on the molecule as long as it can transmit a signal to the CD3 / TCR complex. Examples of agonists include CD3 agonists and / or TCR agonists. CD3 agonists include anti-CD3 agonist antibodies (also simply referred to as "anti-CD3 antibodies") or Binding fragments, anti-TCR agonist antibodies (also simply called "anti-TCR antibodies") as TCR agonists or a binding fragment thereof, an MHC / antigen peptide complex or a multimer thereof, and an MHC / superantigen complex or a multimer thereof. When antibodies are used, anti-CD3 antibodies are available in both polyclonal and monoclonal forms. The antibody may be any of IgG, IgA, IgM, and the like, but is preferably a monoclonal antibody. The anti-CD3 antibody may belong to either the IgD or IgE immunoglobulin class, but is preferably IgG. Examples of the anti-CD3 antibody include an antibody produced from the OKT3 clone (OKT3) and an antibody produced from the UCHT1 clone (UCHT1), with UCHT1 being preferred. The concentration of the anti-CD3 antibody in the medium is, for example, 0.3 ng / ml to 10,000 ng / ml, and is preferably The concentration is preferably 50 ng / ml to 5000 ng / ml, and more preferably 200 ng / ml to 4000 ng / ml. The CD3 / TCR complex agonist may be a commercially available product or may be purified from a natural source. Alternatively, those produced by peptide synthesis, genetic engineering techniques, or chemical synthesis methods may be used. For example, OKT3 and UCHT1 can be purchased from ThermoFisher, GeneTex, etc.

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

[0070] When fibronectin or a variant thereof is contained in the medium, the medium contains the CD3 / TCR complex. The medium may be the same as that containing the agonist. The presence or absence of serum, additives, etc. may also be the same as that of the medium containing the CD3 / TCR complex agonist. When the modified form is contained in the medium, the concentration of fibronectin or its modified form is 10 ng / ml or more as a lower limit, preferably 100 ng / ml or more, and 10,000 μg / ml or more as an upper limit. The concentration may be below 1000 μg / ml, preferably below 1000 μg / ml.

[0071] In step (2-4), it is also preferable that a CD30 agonist is present in the medium. The 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 contain a CD3 / TCR complex agonist. The medium may be the same as that used for the CD3 / TCR complex agonist. The concentration of the CD30 agonist in the medium may be the same as that of the medium containing the CD30 agonist. When the medium contains a CD30 agonist, 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] In addition, when a CD30 agonist is immobilized on a culture vessel, the culture vessel is also used for the CD3 / TCR complex agonist. The method for immobilizing the CD30 agonist on the culture vessel may be the same as the method for immobilizing the CD3 / TCR complex agonist on the culture vessel. The concentration of the CD30 agonist solution when immobilizing the agonist on the culture vessel is 0.1 ng / ml or more as a lower limit, preferably 1 ng / ml or more, and 10000 ng / ml or less as an upper limit, preferably 10000 ng / ml or less. Preferably, it may be 1000 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. Only one of these cytokines may be used, or multiple cytokines may be used. A variety of cytokines (preferably all types) may be used. When the cytokine is IL-2, its concentration in the medium may be 10 U / ml to 1000 U / ml, and 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, and the concentration of IL-15 in the medium may be 1 ng / ml to 100 ng / ml. Preferably, 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, and phospholipase A (PTA). Examples include photoxin α, Fas ligand, TRAIL, TWEAK, TL1A, RANK ligand, OX40 ligand, APRIL, AITRL, BAFF, 4-1BBL, and CD40 ligand, with TL1A being preferred. When used, the 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] In step (2-4), the medium may further contain an apoptosis inhibitor. Examples of apoptosis inhibitors include protease inhibitors, such as caspase inhibitors. Examples of caspase inhibitors include Pan Caspase FMK inhibitor Z-VAD (N-benzoxazole). Z-VAD-FMK is preferred, and its concentration in the medium is 1 μM to 1000 μM. The concentration is preferably 1 μM to 500 μM, more preferably 1 μM to 200 μM, and particularly preferably 1 μM to 50 μM. Preferred.

[0078] In the present invention, the obtained γδ T cells may be isolated and used, or may be used as they are (i.e., other In the case of isolation, the cells may be isolated using at least one molecule selected from the group consisting of γTCR, δTCR, and CD3 as an index. The isolation method can be a method well known to those skilled in the art. For example, antibodies to γTCR, δTCR, and CD3 (bound to magnetic beads, etc., as needed) can be used to isolate the cells. Examples of methods include, but are not limited to, isolation by cytometry or magnetic cell separation, and purification using an affinity column on which a desired antigen is immobilized. When used as is, it is possible to determine the proportion of γδ T cells in a cell population using a method well known to those skilled in the art. Methods for increasing the proportion of γδT cells in a cell population include those described in Front. Immunol., 5:636 (2014), JP2017-537625, and JP2003-529363. This includes, but is not limited to:

[0079] Furthermore, the cells used in the production method of the present invention may have a nucleic acid encoding an exogenous TCR and / or chimeric antigen receptor (CAR) that recognizes and binds to an antigen or the antigen-HLA complex. Thus, in one embodiment of the present invention, a method for producing induced pluripotent stem cells (ISTS) comprises (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. The method may include a step of introducing a nucleic acid encoding the TCR (i.e., (i) αTCR and βTCR, (ii) γTCR and δTCR) and / or (iii) a nucleic acid encoding the CAR into cells (e.g., pluripotent stem cells, hematopoietic progenitor cells, etc.) obtained at any time during the step of inducing the TCR. ) The nucleic acids encoding αTCR and βTCR are used in the process of differentiating induced pluripotent stem cells into T cells. The nucleic acid encoding a TCR is introduced into γδ T cells obtained during any of the steps. As used herein, the nucleic acid encoding a TCR refers to a nucleic acid comprising a base sequence encoding one of the chains that form a TCR and a base sequence encoding the other chain. The nucleic acid encoding a TCR also refers to a combination of a nucleic acid containing a base sequence encoding one of the chains that form a TCR and a nucleic acid containing a base sequence encoding the other chain. That is, a nucleic acid encoding a TCR ((i) αTCR and βTCR) is introduced into a cell. When inserting a TCR fragment, a single fragment containing both the αTCR and βTCR coding sequences is required. Alternatively, a nucleic acid containing a nucleotide sequence encoding an αTCR and a nucleotide sequence encoding a βTCR may be introduced separately. When these nucleic acids are introduced separately, they may be introduced simultaneously or sequentially. (ii) The same applies to the case of a γTCR and a δTCR.

[0080] The TCR used in the present invention includes not only heterodimers in which the α and β chains of the TCR form a heterodimer (i.e., αβTCR) or in which the γ and δ chains of the TCR form a heterodimer (i.e., γδTCR), but also homodimers. Alternatively, those in which the entire TCR is deleted or those in which the amino acid sequence is modified may be used. Among these, γδTCR is preferred, and Vγ9Vδ2TCR is particularly preferred.

[0081] Furthermore, the constant region of the TCR chain may be modified in a specific manner in the constant region of the TCR chain of the cytotoxic T cell (CTL) clone from which it is derived. For example, this modification may involve substituting a specific amino acid residue in the TCR constant region of the CTL clone with a cysteine ​​residue to enhance the efficiency of dimer formation due to disulfide bonds between the TCR chains. Not limited to these.

[0082] Antigens targeted by the TCR include, but are not limited to, tumor antigens. The tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). Specific examples of such tumor antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, as well as WT1, Glypican-3, MAGE-1, and MAGE-3. tumor-specific multilineage antigens such as 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 Epstein-Barr virus antigens (EBV) and human papillomavirus (HPV). Other tumor antigens include one or more antigens selected from the group consisting of viral antigens such as 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, p15, p16, 43-9F, 5T4, 791Tgp72, and α-fetoprotein. Protein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 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 These include proteins such as cyclophilin C-associated proteins, TAAL6, TAG72, TLP, and TPS. Not limited to these.

[0083] As shown in the examples below, in one embodiment, γδ T cells obtained by the method of the present invention Among these cells, cells expressing a chimeric antigen receptor (CAR) have been shown to have 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 of measuring cytotoxic activity against cells expressing the target antigen of the CAR by a chromium release assay or the like. Examples include the law.

[0084] In the present invention, a "chimeric antigen receptor (CAR)" refers to a chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain. The term "CAR" refers to a fusion protein comprising an antigen-binding domain and an intracellular signaling domain. The fusion domain is a linker (e.g., a linker consisting of G and S (GS linker) (e.g., GGGS, GGGGS, or a linker combining these)) that connects the light chain (VL) and heavy chain (VH) of the variable region of an antibody. The CAR comprises single-chain antibodies (scFv) linked in series via a spacer such as a CAR-like antibody (e.g., SEQ ID NO: 4 or 5). γδ T cells expressing CAR recognize antigens via the scFv region and then transmit the recognition signal into the T cell via the intracellular signaling domain. By introducing CAR, it is possible to confer specificity to the target antigen. In addition, since CAR can directly recognize antigen molecules without relying on HLA class I or class II, it can induce a strong immune response even against cells with reduced expression of HLA class I or class II genes. Antigens targeted by the CAR include the same antigens as those targeted by the TCR.

[0085] Examples of the transmembrane domain of CAR include the α chain, β chain, or ζ chain of TCR, CD28, and CD3ε. Examples of suitable transmembrane domains include, but are not limited to, transmembrane domains derived from one or more proteins selected from the group consisting of CD28, 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 to be linked to the antigen-binding domain is derived may also be used. For example, if the molecule from which the first intracellular signaling domain to be 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 the 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, the intracellular signaling domain derived from the CD3 ζ chain is preferred. Preferably, the intracellular signaling domain may further include the intracellular domain of a costimulatory molecule, and examples of such costimulatory molecules include 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 activity of CAR can be controlled by selecting the type and number of costimulatory molecules to be bound. The intensity and duration of the stimulation can be controlled (e.g., Mol Ther. 2009;17:1453-1464.).

[0087] A spacer may be incorporated 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 usually consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. Specific examples include, but are not limited to, a hinge region derived from IgG1, or a peptide containing the CH2CH3 region of immunoglobulin and a portion of CD3. do not have.

[0088] Specific examples of CARs include first-generation CARs in which scFV and 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 the activation ability of 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 intracellular domain of CD28 and the CD3ζ chain of the second-generation CAR. Not limited.

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

[0090] Furthermore, the intracellular domain derived from CD30 may be, for example, one or more (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 50) amino acids of SEQ ID NO: 3. More preferably, the amino acid sequence includes an amino acid sequence in which about 1 to 10, and particularly preferably 1 to several (2, 3, 4, or 5) amino acids have been substituted, deleted, added, and / or inserted. 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 CD30 intracellular domain is maintained.

[0091] The above TCR and / or CAR (hereinafter sometimes abbreviated as "TCR, etc.") specifically bind to the antigen. The ability of the antibody to recognize and bind to the target protein can be confirmed by known methods, and suitable methods include, for example, a dextramer assay or an ELISPOT assay. By performing this assay, T cells expressing TCR etc. on the cell surface can bind to target antigens via the TCR etc. It is possible to recognize the origin and confirm that the signal has been transmitted into the cell.

[0092] Furthermore, the present inventors have demonstrated that cells expressing a fusion protein containing IL-15 and IL-15Rα together with the CAR (hereinafter sometimes abbreviated as "IL-15 / IL-15Rα") express only the CAR. Therefore, from the viewpoint of cytotoxic activity, it is preferable that the γδ T cells obtained by the method of the present invention express IL-15 / IL-15Rα. It is preferable that the IL-15 / IL-15Rα is expressed, and it is even more preferable that the CAR is expressed. In order to obtain γδ T cells expressing γδ T cells, the production method of the present invention comprises either step (1) or (2) of 1. above. The method may also include a step of introducing a nucleic acid encoding IL-15 / IL-15Rα into cells obtained during the step (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 pathway, IL-15Rα, which is normally expressed on antigen-presenting cells, binds to IL-15 and presents IL-15 to the IL-15 receptor, which consists of IL-15Rβ and the common γ chain (γc), on CD8+CD4-cells (trans-presentation), thereby promoting the cytotoxic activity of CD8+CD4-cells. Therefore, when CD3-positive cells expressing IL-15 / IL-15Rα are CD8-positive and CD4-negative, they are unable to transmit IL-15 signals into their own cells via the IL-15 receptor. Alternatively, CD3+ cells expressing IL-15 / IL-15Rα can transmit IL-15 signals to other CD8+CD4-cells via the IL-15 receptor. α can maintain the cytotoxic activity of CD8+CD4-cells, making it a target cell for CAR. It is expected to have a continuous cytotoxic effect against

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

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

[0096] As for IL-15 / IL-15Rα, there is no particular restriction on a fusion protein of IL-15 and IL-15Rα. IL-15 / IL-15Rα is not limited as long as it binds to the IL-15 receptor and transmits the IL-15 signal into cells, but a specific example is a peptide consisting of SEQ ID NO: 6. Alternatively, IL-15 / IL-15Rα is not limited as long as it binds to the IL-15 receptor and transmits the IL-15 signal into cells, but for example, it may have a similar identity to the amino acid sequence shown in SEQ ID NO: 6 by about 90% or more, preferably by about 95% or more, more preferably by about 97% or more, particularly preferably by about 98% or more, and most preferably by about 99% or more. Examples include peptides containing amino acid sequences having homology or identity. Here, "homology" or "identity" refers to the percentage (%) of identical and similar amino acid residues (in the case of identity, identical amino acid residues) to the total overlapping amino acid residues in the optimal alignment (preferably, the algorithm can consider the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, for example, Aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids Acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), water Amino acids with acid groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met ) and other amino acids that belong to the same group. Substitution with such similar amino acids is expected to not cause any change in the phenotype of the protein (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)). The homology or identity of amino acid sequences in the specification is 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; fill This can be calculated with tallying = 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 usually with high affinity, with an affinity measured by a KD value preferably less than 1 μM, more preferably less than 100 nM, and even more preferably less than 1 μM. 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 value" relates to the equilibrium dissociation constant as known in the art.

[0098] The above TCRs and the like are introduced into cells in the form of nucleic acids encoding the TCRs and the like. Fusion proteins containing IL-15Rα and IL-15Rα are also introduced into cells in the form of nucleic acids encoding the fusion proteins. The nucleic acids may be DNA or RNA, or may be DNA / RNA chimeras. The nucleic acid may be double-stranded or single-stranded, but is preferably DNA. In the case of double strands, 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 should 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 a cell.

[0099] The above nucleic acids can be constructed by known methods. Based on the amino acid or nucleic acid sequence of CAR, DNA strands are chemically synthesized, or synthesized overlapping oligo DNA strands are assembled using PCR or Gibson assembly. By connecting these DNA fragments, it is possible to construct DNA fragments encoding the full length or part of a TCR or CAR. A nucleic acid encoding a fusion protein containing IL-15 and IL-15Rα can also be synthesized in the same manner. It can be constructed as follows.

[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 EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Morovirus) promoter, and Murine leukemia virus (HSV) long terminal repeat (LTR), herpes simplex virus (HSV) thymidine kinase (HSV-TK) promoter promoter, TCR V α gene promoter, TCR V β gene promoter, etc. Among these, the EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, and S The Rα promoter and the like are preferred.

[0102] In addition to the promoter, the vector may optionally contain transcriptional and translational regulatory sequences, ribosome binding sites, enhancers, replication origins, poly(A) addition signals, and selectable marker genes. Examples of the selectable marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.

[0103] In one embodiment of the present invention, a nucleic acid encoding the α chain of a TCR and a nucleic acid encoding the β chain and an expression vector containing the above is introduced into target cells, and the α and β chains of TCR are expressed within the target cells or on the cell surface. In this case, the α chain of the TCR can be encoded by the The nucleic acid encoding the β 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 these two types of nucleic acids are incorporated into one expression vector, they are preferably incorporated via a sequence that allows polycistronic expression. By using a sequence that allows polycistronic expression, it is possible to incorporate these two types of nucleic acids into one type of expression vector. This allows for more efficient expression of multiple genes embedded in the vector. Examples of sequences that enable polycistronic expression include the 2A sequence (e.g., the 2A sequence of the foot-and-mouth disease virus (FMDV)). Examples of such sequences include the 2A sequence derived from the Equine Rhinitis A Virus (ERAV) (F2A), the 2A sequence derived from the Porcine Teschovirus (PTV-1) (P2A), the 2A sequence derived from the Thosea asigna virus (TaV) (T2A) (PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), and the internal ribosome entry site (IRES) (US Patent No. 4,937,190). From the viewpoint of quantity, the P2A sequence and the T2A sequence are preferred. The same applies when an expression vector containing a nucleic acid encoding the

[0104] The expression vector is not particularly limited as long as it can express TCR and the like for a period of time sufficient for the prevention or treatment of a disease when introduced into a cell. Examples of the expression vector 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] There is no particular limitation on the method for introducing the nucleic acid or vector into cells, and any known method can be used. When introducing a nucleic acid or a plasmid vector, in step 1.(1) above, The same method as described above can be used. Alternatively, the nucleic acid can be introduced into the genome of a 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 TCRs and the like in the cells. As a method for introducing RNA, known methods can be used, for example, lipof The injection method, electroporation method, etc. can be suitably used.

[0107] In the above steps (1) and (2), the timing of introducing the nucleic acid is γδT There are no particular limitations as long as the cells can express the introduced TCR, etc., but for example, iPS cells, HPCs (CD34 + / CD43 + ), ProT cells (CD4 - / CD8 - ), CD3 + / CD4 + / CD8 + T cells, CD3 + / CD4 - / CD8 + T cells, or are 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 nucleic acid is introduced into a cell, the expression of the endogenous TCR chain that the cell naturally expresses is suppressed from the viewpoint of increasing the expression of the introduced TCR, suppressing the appearance of mispaired TCRs, or suppressing non-autoreactivity. It is preferable to suppress the expression by siRNA. When the above nucleic acid is applied to the method, In order to avoid the effect of siRNA on TCR, the base sequence of the nucleic acid encoding the TCR is The siRNA that suppresses the expression of the TCR chain has a different sequence (codon) from the base sequence corresponding to the RNA that acts on it. These methods are described, for example, in WO 2008 / 153029. The base sequence is a sequence encoding a TCR obtained from nature. These can be produced by introducing an innate mutation or by chemically synthesizing an artificially designed nucleic acid. Alternatively, to avoid mispairing with endogenous TCR chains, part or all of the constant region of the introduced TCR-encoding nucleic acid 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 relates to a γδ T cell or a cell population comprising said γδ T cell, are cells differentiated from induced pluripotent stem cells derived from cells other than αβ T cells, or A cell population is provided. The percentage of γδ T cells contained in the cell population (the percentage of γδ T cells contained in the cell population) is The ratio (number of γδ T cells in the cell population / total number of cells in the cell population) is preferably 90% or more (e.g., 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%). Such a cell population can be obtained, for example, by the production method of the present invention. The ratio can be calculated by measuring the ratio of cells expressing γTCR, δTCR, and CD3 by flow cytometry. Therefore, in one embodiment, the present invention provides γδ T cells produced by the production method of the present invention and / or a cell population containing the γδ T cells. The nucleic acid may include the nucleic acid encoding the exogenous TCR described in 1., the nucleic acid encoding the CAR, and / or the nucleic acid encoding a fusion protein comprising IL-15 and IL-15Rα. The γδ T cells mentioned above or a cell population containing the γδ T cells may hereinafter be abbreviated as "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 cancers 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 the like. Tumors include benign tumors, malignant tumors (also called "cancers"), and tumors that can be diagnosed or determined as 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 of the present invention may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before administration to a subject. 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, IFN-γ, etc., and preferred examples include IL-2, IL-7, IL-12, IL-15, IFN-γ, etc. Preferably, IL-2 can be used. There is no particular limitation on the concentration of IL-2 in the medium. However, for example, preferably 0.01 U / ml to 1×10 5 U / ml, more preferably 1 U / ml to 1×10 4 U / ml Suitable proteins include, for example, 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 may 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 depends 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 x10 7 ~1×10 9 5×10 7 ~5×10 8 Administered to achieve The pharmaceutical composition of the present invention 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 medium, etc., in order to stably maintain the cells. The medium is not particularly limited, but may be RPMI, AIM-V, X-VIVO10, etc. The media include, but are not limited to, the following: Furthermore, the pharmaceutical may contain pharmaceutically acceptable carriers (e.g., human serum albumin), preservatives, etc. added for stabilization purposes.

[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 As a cell population containing hematopoietic progenitor cells, 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 were differentiated according to known methods (e.g., the methods described in Cell Reports 2 (2012) 1722-1735 and WO 2017 / 221975) to obtain a suspension cell population. Specifically, the Ff-I01s04 strain was cultured at 3 x 10 in a 6-well plate treated with ultra-low attachment. 5 On Day 0, cells were seeded at 100 cells / well and incubated 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) at 10 ng / ml BMP4, 50 ng / ml bFGF, 15 ng / ml VEGF, and 2 μM SB431542 were added, and the cells were cultured 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 to 9 days (~Day 14), to obtain a floating cell population. During the culture period, the medium was changed every 2 or 3 days. The staining was performed using a set of antibodies.

[0118] [Table 1]

[0119] The stained cell population was subjected to sorting using FACSAria. The obtained cell fraction was differentiated into lymphoid cells according to known methods (for example, the methods described in Journal of Leukocyte Biology 96 (2016) 1165-1175 and WO 2017 / 221975). For the study, hematopoietic progenitor cell populations were seeded at 2000 cells / well onto 48-well plates coated with recombinant h-DLL4 / Fc chimera (Sino Biological) and Retronectin (Takara Bio), and cultured under 5% CO2 at 37°C. The medium was changed every 2 or 3 days during the culture period. The medium contained 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 55 μM 2-mercaptoethanol, and 50 μg / ml ascorbic acid. 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml The αMEM medium was supplemented with 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 subcultured 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 TM The cells were confirmed to be resistant to HIV-1 by ELISA using a 24-well plate (BD Biosciences Fusion). The obtained cells were seeded in 24-well plates and cultured under 5% CO2 and 37°C conditions. The medium contained 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 (enriched erythrocyte selenite). αMEM medium containing 100 U / ml IL-2 and 10 ng / mL IL-7 (Shisei Pharmaceutical Co., Ltd.: 10171-H02H) On the 27th day (Day 41) after the start of culture, all cells were collected and the cell count was determined using a hemocytometer. After counting, cells were stained with the following antibody set:

[0120] [Table 2]

[0121] As a result of staining, cells expressing γδTCR were isolated from hematopoietic progenitor cells derived from iPS cells (Ff-I01s04 line). It was shown that it was possible to prepare γδTCR-positive cells (Figure 1).

[0122] Furthermore, the γδ TCR-positive cells include Vδ1-positive γδ T cells and Vδ2-positive γδ T cells. These results indicate that Vδ1 and Vδ2 γδ T cells can be prepared (Figure 2 ).

[0123] [Example 2] Examination of cytotoxic activity of γδT cells Evaluation of the cytotoxic activity of γδT cells derived from iPS cells (Ff-I01s04 cell line) obtained in [Example 1] The mesothelioma cell line NCI-H226 was used as target cells and reacted with DELFIA BATDA Reagent (Perkin Elmer) at 37°C for 30 minutes. After washing the reaction solution, a population of γδ T cells derived from iPS cells (Ff-I01s04 line) 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 line) was assessed based on target cell death after 2 hours.

[0124] As a result of the evaluation, it was found that γδT cells derived from iPS cells (Ff-I01s04 line) were able to inhibit the growth of tumor cells against the tumor cell line NCI-H226. It was shown to have cytotoxic activity (Figure 3).

[0125] (Expansion and functional evaluation of γδT cells) [Example 3] Production of iγδT cells The same method as in Example 1 was used, except that UCHT1 (GeneTex) was used as the anti-CD3 antibody. We produced γδT cells (iγδT cells) derived from iPS cells (Ff-I01s04 strain) by the above procedure.

[0126] [Example 4] Expansion of iγδT cells The γδT cells obtained in Example 3 were cultured in α-MEM medium containing 15% FBS and the cytokines in Table 3. The cells were suspended at 2,000,000 cells / mL in medium containing the following additives, and then retroviral immunolabeling with anti-CD3 antibody (UCHT1) was performed. The cells were seeded onto a plate on which nectin had been solidified and cultured for 3 days under 5% CO2 / 37°C. On the third day of culture, the cells were collected from the plate, counted using a NucleoCounter (registered trademark) NC-200 (ChemoMetec), and analyzed by adding α-MEM medium containing 15% FBS and the cytokines listed in Table 4. The cells were suspended in an appropriate amount of medium containing the agent, added to a non-solid-phase G-Rex (registered trademark) 6-well plate (WILSONWOLF), and cultured under 5% CO2 / 37°C. After that, 4-6 times on the 5th, 6th, 7th, 8th, 9th, 10th, 11th, 14th, and 17th days of culture, some of the cells were collected from the plate and counted using a hemocytometer. It was measured using. Anti-CD3 antibody and retronectin were immobilized on the culture plate by the following method. Anti-CD3 antibody (UCHT1, final concentration 3000 ng / mL) and Retronec® were dissolved in PBS at the required concentration. After adding 150 μg / mL of chloramphenicol (final concentration) to the plate, the plate was left standing 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 G115 γδ T cell clone was used. The nucleic acid containing the gene encoding Vγ9Vδ2TCR G115 was arranged in the order of Table 5 from the N-terminus. An oligo DNA encoding the designed polypeptide (SEQ ID NO: 7) was artificially synthesized.

[0133] [Table 5]

[0134] 4. Construction of retroviral vector carrying the Vγ9Vδ2 gene The lentiviral vector was pLVSIN-CMV Neo (Clontech) with the neomycin resistance gene sequence removed, and the CMV promoter replaced with the human ubiquitin promoter. The artificial oligo DNA synthesized in [Example 5] 3 was used as the pLVSIN-Ub vector. The plasmid was then inserted into the multiple cloning site of a Streptococcus aureus virus vector. 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 [Example 5] The retroviral vector carrying the Vγ9Vδ2 gene prepared in 4. was used in [ The iPS cells prepared in Example 5, 1. and the iPS cell-derived hematopoietic progenitor cells (HPCs) prepared in Example 5, 2. were infected with the vectors. These cells were infected with the vectors using a known method, similar to that used in Example 1. iPS cell-derived Vγ9Vδ2 T cells were generated by differentiation into T cells according to the method described in (WO2017 / 221975). 500 ng / mL UCHT1 (GeneTex) was used as the anti-CD3 antibody in the differentiation process. (Hereinafter, iPS cell-derived Vγ9Vδ2 T cells generated from iPS cells are referred to as "iγ9δ2 T cells.") iPS cell-derived Vγ9Vδ2 T cells generated from iPS cell-derived HPCs are referred to as "iHγ9δ2 T cells." The expression of CD3, γδTCR, 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 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. were transfected into the iγδT cells obtained in [Example 4] and the iHγ9δ2T cells prepared in [Example 5] 5. The iPS cell-derived anti-CD19-CAR / IL-15γδ T cells were generated by infection. (Hereinafter, the iPS cell-derived anti-CD19-CAR / IL-15γδ T cells generated from iγδ T cells are referred to as "iCD19CAR / IL-15γδ T cells.") iPS cell-derived anti-CD19-CAR / IL-15γδT cells generated from iHγ9δ2T cells are sometimes referred to as "iHCD19CAR / IL-15γ9δ2T cells."

[0143] [Example 7] Expansion of iPS cell-derived anti-CD19-CAR / IL-15γδ T cells 1. Expansion of iCD19CAR / IL-15γδ T cells 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 increased the proliferation of iCD19CAR / IL-15γδT cells. was observed (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. iCD19CAR / IL-15γδ T cells or iHCD19CAR / IL-15γ9δ2 T cells were mixed with the target cells at a ratio of 0.5, 1, 2, 4, 8, or 16 times. 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 revealed that iCD19CAR / IL-15γδT cells and iHCD19CAR / IL-15γ9δ2T cells were CD19-positive It was shown that the antibody had cytotoxic activity against CD19-negative Raji cells but not against CD19-negative CCRF-CEN cells (Figs. 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 cells (ATCC) were transplanted into the tail vein to generate Nalm6 xenograft mice. On day 4 after transplantation, iCD19CAR / IL-15γδT cells (5x10 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. Mice in which CD19-positive Nalm6 cancer cells were transplanted via the tail vein showed a significant improvement in the control group after 3 weeks. All patients died within 6 weeks of receiving iCD19CAR / IL-15γδT cells, whereas all patients died within 6 weeks of receiving iCD19CAR / IL-15γδT cells. All patients survived until 3 days after surgery (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 obtained The cells 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) The process of establishing induced pluripotent stem cells from cells other than αβ T cells, (2) A step of differentiating the induced pluripotent stem cells established in step (1) into T cells to obtain γδ T cells. Herein, the method further includes the step of introducing nucleic acids encoding γTCR and nucleic acids encoding δTCR into cells obtained during either step (1) or (2).

2. The method according to claim 1, wherein the cells other than αβT cells are mononuclear cells other than αβT cells.

3. The method according to claim 1 or 2, wherein the cells other than αβT cells are monocytes.

4. The method according to any one of claims 1 to 3, further comprising the step of introducing nucleic acids encoding CAR into cells obtained during either step (1) or (2).

5. The method according to any one of claims 1 to 4, wherein the γTCR is Vγ9TCR and the δTCR is Vδ2TCR.

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

7. γδ T cells produced by the method described in any one of Claims 1 to 6.

8. A pharmaceutical product comprising the cells described in Claim 7.

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

10. A cell killer comprising the cells described in Claim 7.

11. The cells according to claim 7, for use in the prevention or treatment of tumors.

12. Use of the cells according to claim 7 in the manufacture of a tumor preventive or therapeutic agent.

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