Induced pluripotent stem cell-derived γδt cells and method for producing the same

Abstract

To provide superior γδ T cells that are homogeneous and unaffected by cell exhaustion to ensure sufficient purity and cell numbers for treatment, and to provide methods for producing the γδ T cells.SOLUTION: The present invention relates to γδ T cells obtained by inducing the differentiation of induced pluripotent stem cells (iPS cells). Specifically, the γδ T cells are produced by inducing the differentiation of iPS cells harboring a γδ TCR rearrangement gene (γδ TCR-type iPS cells). The method for producing γδ T cells of the present invention has the excellent function of possessing antigen-specific cytotoxic activity in an MHC-independent manner, and has made it possible to provide γδ T cells and γδ T cell populations that are more homogeneous and effective than γδ T cells isolated from peripheral blood.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to induced pluripotent stem cell (iPS cell)-derived γδ T cells and a method for producing the same, specifically to iPS cell-derived γδ T cells that act in an MHC-unrestricted manner and a method for producing the same, as well as to a cell population containing the produced iPS cell-derived γδ T cells.

[0002] This application claims priority from Japanese Patent Application No. 2021-017831, which is incorporated herein by reference. [Background technology]

[0003] Human mature T cells are broadly divided into two groups: αβ T cells, whose T cell receptors are composed of α and β chains, and γδ T cells, whose T cell receptors are composed of γ and δ chains. αβ T cells are highly diverse, and a single type of αβ T cell can attack only a limited number of cells due to MHC restriction. In contrast, a single type of γδ T cell is known to attack multiple types of cancer cells in an MHC-unrestricted manner. γδ T cells recognize and directly damage multiple types of cancer cells using a single T cell receptor (TCR). However, because γδ T cells typically account for only 1–5% of peripheral blood, activating and / or expanding γδ T cells using a small amount of blood cannot ensure sufficient purity and cell numbers for treatment. Furthermore, drawing large amounts of blood from patients to ensure sufficient purity and cell numbers for treatment places a significant burden on the patient. A treatment in which γδ T cells isolated from a patient's peripheral blood are expanded ex vivo and then infused back into the patient has already been performed, but this method has not been able to achieve sufficient expansion and activation due to the difficulty in securing the required number of cells and cell exhaustion.

[0004] Methods for producing iPS cells carrying a γδ TCR rearrangement gene (γδ TCR-type iPS cells) have been disclosed (Patent Document 1, Non-Patent Document 1). Patent Document 1 and Non-Patent Document 1 further disclose that γδ TCR-type iPS cells have been induced to differentiate into blood cell progenitor cells. However, they do not disclose that these blood cell progenitor cells have been further induced to differentiate into T cells.

[0005] A method for inducing differentiation of T cell-derived iPS cells into T cells has been disclosed (Patent Document 2). It has also been reported that T cells with the same rearrangement as the original cells can be obtained by producing iPS cells from T cells with cancer antigen-specific TCR gene rearrangements and inducing their differentiation. + It has been reported that human tumor antigen-specific αβ T cells were reconstituted by inducing differentiation of human iPS cells derived from αβ T cells (Non-Patent Document 2), and that the human tumor antigen-specific αβ T cells obtained by differentiation induction exhibited antigen-specific cytotoxicity (Non-Patent Document 3, Patent Documents 3 and 4). It has also been reported that T cells with the same rearrangement as the original cells can be obtained by producing iPS cells from T cells with tumor antigen-specific TCR gene rearrangements and inducing their differentiation. However, all reports relate to αβ T cells, and no disclosure is made regarding γδ T cells. Because all of the above-mentioned αβ T cells have a specific αβ TCR, the number of patients who can be treated is limited due to the limited number of cancer types that express the antigen and the MHC restriction.

[0006] There have been reports that T cells induced to differentiate from stem cells such as ES cells or iPS cells exhibit a γδ T cell-like phenotype (Non-Patent Document 4, Patent Document 5). However, although the T cells shown in these documents show some similarities to the γδ T cell characteristic phenotype in terms of gene expression patterns, etc., they cannot be said to actually express γδ T cell receptors and thereby recognize antigens and damage target cells, i.e., they are γδ T cells.

[0007] There is a need for an effective method for preparing T cells that can attack various types of cancer cells in an MHC-unrestricted manner. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2018 / 143243 (PCT / JP2018 / 003120) [Patent Document 2] International Publication No. WO2011 / 096482 [Patent Document 3] International Publication No. WO2016 / 010153 [Patent Document 4] International Publication No. WO2016 / 010155 [Patent Document 5] International Publication No. WO2014 / 165707 [Non-patent literature]

[0009] [Non-Patent Document 1] Stem cells translational medicine, 7(1), 34-44 (2018) [Non-patent document 2] Cell Stem Cell, 12, 31-36 (2013), [Non-patent document 3] Cancer Research, 76(23), 6839 (2016) [Non-patent document 4] Nat Biotechnol, 31, 928-3 (2013) Summary of the Invention [Problem to be solved by the invention]

[0010] Because gamma delta T cells typically account for only 1-5% of peripheral blood, there have been problems with ensuring sufficient purity and cell numbers for treatment. Furthermore, drawing large amounts of blood to ensure sufficient purity and cell numbers for treatment places a significant burden on the recipient. Ex vivo expansion of gamma delta T cells isolated from peripheral blood not only poses difficulties in ensuring sufficient cell numbers, but also leads to insufficient expansion and activation due to cell exhaustion.

[0011] An objective of the present invention is to effectively produce and provide γδ T cells, more specifically, to provide superior γδ T cells that are homogeneous and not affected by cell exhaustion. [Means for solving the problem]

[0012] In order to solve the above problems, the inventors focused on iPS cells and conducted extensive research into differentiation induction treatment methods. As a result, they succeeded in producing superior γδ T cells that retain the functions of γδ T cells, thereby completing the present invention.

[0013] That is, the present invention comprises the following. 1. iPS cell-derived γδ T cells, which are T cells derived from induced pluripotent stem cells (iPS cells), characterized in that the T cells have antigen-specific cytotoxic activity in an MHC-nonrestricted manner. 2. iPS cell-derived γδ T cells according to the preceding item 1, wherein the iPS cells are not derived from αβ T cells. 3. iPS cell-derived γδ T cells according to the preceding item 1 or 2, wherein the iPS cells are iPS cells having a γδ TCR rearrangement gene. 4. iPS cell-derived γδ T cells generated by differentiation induction treatment of iPS cells carrying γδ TCR rearrangement genes. 5. A method for producing iPS cell-derived γδ T cells, comprising the step of culturing blood progenitor cells obtained by differentiation induction treatment of iPS cells carrying a γδ TCR rearrangement gene in a medium containing a basal medium supplemented with one or more selected from FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), IL-2, IL-7, TPO (thrombopoietin), and L-ascorbic acid. 6. A method for producing iPS cell-derived γδ T cells according to the preceding paragraph 5, comprising the step of culturing the cells in a medium to which one or more members selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid have been added to a basal medium, followed by the step of culturing the cells in a medium containing a γδ T cell stimulant. 7. The method for generating iPS cell-derived γδ T cells according to the preceding paragraph 5 or 6, wherein the step of culturing using a medium to which one or more members selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid have been added to a basal medium is a step of culturing the cells in co-culture with feeder cells. 8. The method for generating iPS cell-derived γδ T cells according to the preceding paragraph 5 or 6, wherein the step of culturing the cells in a medium containing a basal medium to which one or more members selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid have been added is a step of culturing the cells without co-culturing them with feeder cells. 9. The method for generating iPS cell-derived γδ T cells according to the preceding paragraph 8, wherein the step of culturing without co-culturing with feeder cells comprises a step of culturing using a culture substrate coated with VCAM1 (vascular cell adhesion molecule-1), and DLL4 (Delta-Like Protein 4) or DLL1 (Delta-Like Protein 1). 10. The method for generating iPS cell-derived γδ T cells according to the preceding item 8 or 9, wherein the step of culturing without co-culturing with feeder cells further comprises a step of culturing using a medium containing DKK1 and / or AZA (Azelaic acid). 11. The method for producing iPS cell-derived γδ T cells according to any one of the preceding paragraphs 6 to 10, wherein the medium containing a γδ T cell stimulating agent is a medium containing one or more species selected from the group consisting of a γδ T cell stimulating agent, IL-2, and IL-15. 12. A method for producing iPS cell-derived γδ T cells according to any one of the preceding paragraphs 6 to 11, wherein the γδ T cell stimulator is a phosphate compound or a derivative thereof, which is a metabolite in the isoprenoid biosynthetic pathway, or a specific inhibitor of FPP (farnesyl pyrophosphate) synthase, a rate-limiting enzyme in the isoprenoid biosynthetic pathway. 13. A method for producing iPS cell-derived γδ T cells according to any one of the preceding items 6 to 12, wherein the cells are cultured under serum-free conditions. 14. A method for producing iPS cell-derived γδ T cells according to any one of the preceding items 6 to 13, which comprises culturing the cells under hypoxic conditions. 15. iPS cell-derived γδ T cells prepared by the method for preparing iPS cell-derived γδ T cells according to any one of the preceding items 5 to 14. 16. A cell population comprising iPS cell-derived γδ T cells according to any one of items 1 to 4 and 15 above. 17. A cell population according to the preceding item 16, characterized in that the cell population containing iPS cell-derived γδ T cells has high antigen-specific cytotoxic activity compared to a cell population of γδ T cells isolated from peripheral blood. 18. A cell population of γδ T cells, characterized in that γδ T cells having the same base sequence in the CDR3 region of the TCR gene account for 90% or more of the γδ T cells constituting the cell population. 19. γδ T cells 1 × 10 5 19. The cell population according to item 18, comprising at least one of the cells. 20. A cell population of γδ T cells, characterized in that γδ T cells that express higher levels of CD7 and CD8a than γδ T cells isolated from peripheral blood account for 90% or more of the γδ T cells constituting the cell population. 21. The cell population according to any one of the preceding items 18 to 20, which is a cell population containing γδ T cells, wherein undifferentiated cells account for 10% or less of the γδ T cells constituting the cell population. 22. An antigen-specific cellular immunotherapeutic agent, comprising the iPS cell-derived γδ T cells according to any one of the preceding items 1 to 4 and 15 as an active ingredient. 23. A method for culturing iPS cell-derived γδ T cells according to any one of items 1 to 4 and 15 above, wherein the cells are cultured in a liquid medium containing a beaded carrier. 24. A therapeutic agent for diseases such as cancer, infectious diseases, and autoimmune disorders, which comprises, as an active ingredient, iPS cell-derived γδ T cells selected from any one of the above items 1 to 4 and 15. 25. A pharmaceutical composition comprising, as an active ingredient, the iPS cell-derived γδ T cells according to any one of items 1 to 4 and 15 above. 26. A method for antigen-specific cellular immune cell therapy, which comprises administering iPS cell-derived γδ T cells according to any one of the preceding paragraphs 1 to 4 and 15. 27. A method for treating diseases such as cancer, infectious diseases, and autoimmune disorders, which comprises administering iPS cell-derived γδ T cells according to any one of the preceding paragraphs 1 to 4 and 15. [Effects of the Invention]

[0014] According to the method of the present invention for producing iPS cell-derived γδ T cells by iPS cell differentiation induction treatment, γδ T cells can be produced effectively without burdening the recipient and without being affected by cell exhaustion. Furthermore, according to the method of the present invention for producing iPS cell-derived γδ T cells, excellent γδ T cells can be produced even under conditions that do not contain feeder cells and / or serum, or that do not contain animal-derived components. The γδ T cells of the present invention have excellent functionality, possessing antigen-specific cytotoxic activity in an MHC-nonrestricted manner, and have been able to form a γδ T cell population that is more homogeneous and more effective than γδ T cells isolated from peripheral blood. [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1A shows the results of flow cytometry evaluation of CD34 / CD43 expression in cells on day 10 of differentiation induction. Figure 1B shows the results of flow cytometry evaluation of CD3 / γδTCR expression in cells on day 31 of differentiation induction. (Example 1) [Figure 2] Figure 2A shows the results of flow cytometry evaluation of CD7 (T cell differentiation marker) expression in cells on day 17 of differentiation induction. Figure 2B shows the results of flow cytometry evaluation of CD3 / γδTCR / CD45RA expression in cells on day 54 of differentiation induction. (Example 2) [Figure 3] Figure 3A shows the results of flow cytometry evaluation of CD7 expression in cells on day 17 of differentiation induction. Figure 3B shows the results of flow cytometry evaluation of CD3 / γδTCR expression in cells on day 55 of differentiation induction. Figure 3C shows the results of confirming the cytotoxic activity of cells on day 55 of differentiation induction against Jurkat cells. (Example 3) [Figure 4] Example 4 shows a protocol for inducing differentiation from iPS cells without using feeder cells. [Figure 5] 1 shows the results of flow cytometry evaluation of CD3 / γδTCR expression in cells on days 33, 35, and 37 after differentiation induction without the use of feeder cells (Example 4). [Figure 6] Example 5 shows a protocol for inducing differentiation from iPS cells without using feeder cells. [Figure 7] Figure 7A shows the results of observing cells on day 37 of differentiation induction using a phase-contrast microscope. Figure 7B shows the results of further evaluation of CD3 / γδTCR expression by flow cytometry. (Example 5) [Figure 8] Example 6 shows a protocol for inducing differentiation from iPS cells without using feeder cells. [Figure 9] The cells were cultured in each medium without using feeder cells on day 32 after differentiation induction, and the results of observing the cells under a phase contrast microscope are shown (Example 6). [Figure 10]6 shows the results of flow cytometry evaluation of CD3 / γδTCR expression when cells were cultured in each medium without the use of feeder cells on day 32 of differentiation induction (Example 6). [Figure 11] This shows that cells on day 35 after differentiation induction have cytotoxicity against Jurkat cells when cultured in each medium without the use of feeder cells (Example 6). [Figure 12] The cells on day 35 of differentiation induction were cultured in each medium without using feeder cells, and the cytotoxic activity was confirmed 1 day and 4 days after the start of mixed culture with Jurkat cells (Example 6). [Figure 13] 7 shows the results of flow cytometry evaluation of the expression of CD7, a T cell differentiation marker, for cells on day 24 after differentiation induction without the use of feeder cells (Example 7). [Figure 14] Example 8 shows the results of inducing differentiation into T cells by co-culturing magnetic beads coated with VCAM1 and DLL4 instead of coating the culture dish without using feeder cells, and evaluating the expression of CD7, a T cell differentiation marker, in the cells 24 days after differentiation induction using flow cytometry (Example 8). [Figure 15] This shows a protocol for inducing differentiation of γδ T cells from iPS cells prepared in Example 9. (Example 9) [Figure 16] Figure 16A shows the results of observing the shape of cells during differentiation using a phase-contrast microscope. Figure 16B shows the results of confirming cell surface markers for cells during differentiation using flow cytometry. (Example 9) [Figure 17] The figures show the results of confirming the antitumor activity of γδ T cells on day 38 of differentiation induction against various tumor cells. Figure 17A shows the results of confirming cytotoxic activity against Jurkat cells. Figure 17B shows the results of confirming cytotoxic activity against Huh-7 cells. Figure 17C shows the results of confirming cytotoxic activity against SW480 cells. Figure 17D shows the viability of iPS cell-derived γδ T cells (E) and Jurkat cells (T) when the E:T ratio was changed stepwise during co-culture (Example 9). [Figure 18] Figure 18 shows the results of confirming the maintenance of TCR rearrangement and the cytotoxic mechanism of γδ T cells on day 36 of differentiation induction. Figure 18A shows the results of evaluating the expression of αβ TCR on the cell surface of unpurified γδ T cells (igdT) and peripheral blood mononuclear cells (PB). Figure 18B shows the results of confirming the rearrangement of TCR genes (Vγ9, Vδ2) by genomic PCR. Figure 18C shows the results of confirming the expression of Granzyme B and Perforin in γδ T cells. Figure 18D shows the results of confirming the cytotoxic activity of purified γδ T cells (igdT). There was no significant difference in the cell death rate between purified and unpurified γδ T cells. (Example 9) [Figure 19] Example 10 shows the results of single-cell RNA-seq analysis of gene expression patterns in iPS cell-derived γδ T cells and γδ T cells isolated from peripheral blood. [Figure 20]

[0049] Figure 10 shows the results of flow cytometry analysis of CD25, one of the cell surface expression markers in iPS cell-derived γδ T cells and γδ T cells isolated from peripheral blood (Example 10). [Figure 21] Example 11 shows a protocol for inducing differentiation of γδ T cells from iPS cells to confirm the activation of iPS cell-derived γδ T cells. [Figure 22] Figure 22 shows the results of examining the effect of IL-2 and / or IL-15 on the activation of iPS cell-derived γδ T cells. Figure 22A shows the results of counting viable cells, and Figure 22B shows the results of flow cytometry evaluation of CD3+ / γδ TCR+ cells (Example 11). [Figure 23] This figure shows γδ T cells obtained by inducing differentiation from the 121-3 strain, which is a γδ T cell-derived iPS cell. Figure 23A shows the results of genomic PCR confirming the rearrangement of TCR genes (Vγ9, Vγ2) in undifferentiated iPS cells (121-3 strain) and γδ T cells obtained by inducing differentiation from them. Figure 23B shows the results of next-generation sequencing confirming the sequences of TCRγ and TCRδ in γδ T cells obtained by amplifying and culturing γδ T cells and peripheral blood mononuclear cells (Example 12). [Figure 24]Example 13 shows the results of flow cytometry evaluation of IFN-γ expression after 4 hours of co-culture of iPS cell-derived γδ T cells on day 39 of differentiation induction or γδ T cells obtained by expanding and culturing peripheral blood mononuclear cells with Jurkat cells. [Figure 25] Example 14 shows the results of flow cytometry evaluation of the expression of various surface markers in a cell population containing iPS cell-derived γδ T cells obtained 40 days after differentiation induction using a method using feeder cells, and in a cell population containing γδ T cells (CD3-positive or TCRγ9-positive) obtained by expanding and culturing peripheral blood mononuclear cells. [Figure 26] Figure 26A shows the protocol for stimulating γδ T cells starting from day 17. Figure 26B shows the results of flow cytometry evaluation of CD3 / γδ TCR expression in cells on day 17 of differentiation induction. Figure 26C shows the results of flow cytometry evaluation of CD3 / CD7 expression in cells on day 24 of differentiation induction. (Example 15) [Figure 27] Figure 27 shows the results of examining IL-2 or IL-15, or IL15 or IL-15 + HMBPP, as a method for activating iPS cell-derived γδ T cells. Figure 27A shows the results of flow cytometry evaluation of CD3 / γδ TCR expression in cells on day 37 or day 33 of differentiation induction. Figure 27B shows the results of flow cytometry evaluation of CD3 / CD7 expression in cells on day 23 of differentiation induction (Example 16). [Figure 28]

[0062] Figure 17 shows the results of freeze-thawing cells on day 24 of differentiation induction without the use of feeder cells, and then confirming their cytotoxic activity against Jurkat cells (Example 17). [Figure 29] Figure 29A shows the results of flow cytometry evaluation of CD34 / CD43 expression in cells on day 10 of differentiation induction. Figure 29B shows the results of freeze-thawing the cells on day 10 of differentiation induction, followed by flow cytometry evaluation of CD3 / γδTCR expression in cells on day 37 of differentiation induction. Figure 29C shows the results of confirming the cytotoxic activity of the cells on day 37 of differentiation induction against Jurkat cells (Example 18). [Figure 30]Figure 30A shows a protocol for inducing differentiation of iPS cell-derived blood progenitor cells after freezing and thawing them without feeder cells or serum. Figure 30B shows the results of flow cytometry analysis of CD3 / γδ TCR expression in cells on day 17 of differentiation induction. Figure 30C shows the results of confirming the cytotoxic activity of cells on day 24 of differentiation induction against Jurkat cells. (Example 19) [Figure 31] Figure 31A shows a protocol for inducing differentiation of blood progenitor cells into γδ T cells under hypoxic conditions. Figure 31B shows the results of flow cytometry analysis of CD3 / CD7 expression in cells on day 17 of differentiation induction. Figure 31C shows the results of confirming the cytotoxic activity of cells on day 29 of differentiation induction against Jurkat cells. (Example 20) [Figure 32] Figure 32 shows that iPS cell-derived γδ T cells were induced to differentiate under conditions that did not contain animal-derived components. Figure 32A shows the results of flow cytometry analysis of CD3 / CD7 expression in cells on day 17 of differentiation induction. Figure 32B shows the results of confirming the cytotoxic activity of cells on day 31 of differentiation induction against Jurkat cells. (Example 21) [Figure 33] This shows the absence of undifferentiated cells in the cell population. Figure 33A shows the results of flow cytometry evaluation of the expression of the undifferentiated marker TRA-1-85 in a cell population on day 35 of differentiation induction under serum-free conditions without the use of feeder cells. Figure 33B shows the protocol used to confirm whether colonies of undifferentiated cells appear in the cell population. Figure 33C shows the absence of colonies of undifferentiated cells in the cell population. (Example 22) [Figure 34] Figure 34A shows the purification of CD3 / γδ T-positive cells from a cell population under serum-free conditions without the use of feeder cells. Figure 34B further shows the results of confirming the cytotoxic activity of the purified cells against Jurkat cells (Example 23). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to iPS cell-derived γδ T cells, which are T cells derived from iPS cells and characterized in that the T cells have antigen-specific cytotoxic activity in an MHC-non-restricted manner.

[0017] Human mature T cells are divided into two groups: αβ T cells, whose T cell receptors (TCRs) are composed of α and β chains, and γδ T cells, whose T cell receptors are composed of γ and δ chains. In this specification, "γδ T cells" refers to γδ T cells. While αβ T cells account for the majority of blood, γδ T cells account for only 1-5% of all T cells. Because they undergo TCR gene rearrangement to bind to diverse antigens and retain memory cells, γδ T cells can be considered a component of the adaptive immune system. Furthermore, γδ T cells do not require antigen recognition by TCRs and can attack, for example, tumor cells through antigen recognition similar to that of innate immune cells (NK cells). γδ T cells are therefore thought to possess both innate and adaptive immune functions. Meanwhile, αβ T cell-derived cytotoxic T cells (CTLs) can be considered adaptive immune systems that require antigen information from dendritic cells to target tumor antigens. Thus, it is known that γδ T cells and αβ T cells not only differ in the proportions present in the blood, but also in their functions, and that the differentiation processes of these cells are also different (Non-Patent Document 3).

[0018] As used herein, "iPS cells" refers to undifferentiated cells established by reprogramming somatic cells using various methods. The iPS cells that serve as the starting material for the present invention are preferably iPS cells that are not iPS cells that have an αβTCR rearrangement. Most preferably, they are iPS cells that have a γδTCR rearrangement. iPS cells that have the γδTCR rearrangement are also referred to simply as "γδTCR iPS cells" below. As used herein, "γδTCR rearrangement" refers to a gene encoding a TCR in which both rearrangements of the TCRG region and the TCRD region have occurred. The TCRG region is composed of Vγ-Jγ, and the TCRD region is composed of Vδ-Dδ-Jδ.

[0019] The iPS cells referred to herein can be produced by any known method or any method to be developed in the future, for example, based on the description in Patent Document 1 and Non-Patent Document 1.

[0020] (Method for producing iPS cells) The iPS cells used to generate the γδ T cells of the present invention can be generated by any method known per se or any method to be developed in the future. Specifically, they can be generated by the methods described in Patent Document 1 or Non-Patent Document 1. For example, they can be generated by a method for generating iPS cells that includes the following steps 1) to 3): 1) stimulating the collected blood cells with IL-2 and a bisphosphonate (e.g., one or more selected from zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, minodronic acid, salts thereof, and hydrates thereof, preferably zoledronic acid); 2) introducing at least four types of genes capable of expressing cell reprogramming factors (e.g., OCT3 / 4, SOX2, KLF4, and c-MYC) into the blood cells using a Sendai virus (SeV) vector; 3) A step of culturing the cells into which the gene has been introduced.

[0021] (iPS cell culture) StemFit is a basal medium that can be used for the maintenance culture of iPS cells. (R) AK02N (product name), StemFit (R) Various stem cell maintenance media such as AK03N (trade name), ReproStem (trade name), iPSellon (trade name), Essential 8 (trade name), and TeSR-E8 (trade name) can be used. StemFit is particularly preferred. (R)AK02N (trade name). The amount of substances added to each medium can be increased or decreased as needed depending on the purpose. An example of a substance that can be added is the Rho-Associated Coil Kinase (ROCK) inhibitor Y27632. To promote cell adhesion and proliferation, laminin 511-E8 fragments can be used in culture substrates such as culture dishes. Specifically, iMatrix-511 silk (trade name) or iMatrix-511 (trade name) can be used. The manufacturer and distributor of the reagents used are not particularly limited, as long as they perform equivalent functions. When passaging iPS cells, proteases such as trypsin can be used to detach cells from the culture vessel; for example, TrypLE Select (trade name) can be used.

[0022] (Induction of differentiation of iPS cells into blood progenitor cells) In the process of inducing differentiation of iPS cells into γδ T cells, iPS cells are first induced to differentiate into blood progenitor cells. In the method of producing iPS cell-derived γδ T cells of the present invention, cells induced to differentiate into blood progenitor cells from iPS cells are used as the starting material, and the process of inducing differentiation of blood progenitor cells into γδ T cells can be used as the method of producing iPS cell-derived γδ T cells. Furthermore, the method of producing iPS cell-derived γδ T cells can include a process of converting iPS cells into blood progenitor cells. Cells obtained by freezing and thawing iPS cell-derived blood progenitor cells can also be used in the method of the present invention. The freezing period is not particularly limited, but can be, for example, 2 weeks to 1 year. In either case, the iPS cells of the present invention are preferably iPS cells other than those having an αβ TCR rearrangement. Most preferably, they are γδ TCR iPS cells.

[0023] The process for inducing differentiation of iPS cells into blood progenitor cells is not particularly limited, and any known method or any process developed in the future can be used. In the process for inducing differentiation into blood progenitor cells, the medium can be supplemented with one or more cytokines selected from the group consisting of tyrosine kinase 3 ligand (FLT3L), stem cell factor (SCF), bone morphogenetic protein-4 (BMP4), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), IL-6, insulin-like growth factors (IGF-1), IL-7, IL-11, erythropoietin (EPO), thrombopoietin (TPO), IL-15, and IL-3. The medium may also contain fetal bovine serum (FBS) or fetal calf serum (FCS).

[0024] The iPS cells of the present invention can be differentiated into blood progenitor cells by culturing them in the media shown in the following 1-1) to 1-4) and inducing differentiation under conditions without using feeder cells. To differentiate into blood progenitor cells, the ROCK inhibitor Y27632 can be used at a final concentration of 0 to 50 μM, preferably 1 to 30 μM, and more preferably 10 μM, and 0 to 50 μl, preferably 1 to 30 μl, and more preferably about 5 μl of laminin-511 E8 fragment, such as iMatrix-511 (trade name), can be used. The next day, StemFit containing no ROCK inhibitor or laminin-511 E8 can be used. (R)The medium can be changed to AK02N and cultured by changing it every few days, for example, every two days. The frequency and amount of medium change are not particularly limited and can be determined as appropriate. The number of cells seeded can be increased or decreased as appropriate. The reagents used are not particularly limited to those manufactured or sold by any manufacturer, as long as they perform the same function. All cultures can be performed at 37±0.5°C and 5% CO2. For subculture, a protease such as trypsin, such as TrypLE Select (trade name), can be used to detach the cells from the culture vessel.

[0025] 1-1) Day 0 of differentiation induction StemFit (R) AK02N (trade name) can be used as the basal medium. The cells can be cultured in a culture system further containing a GSK-3α / β inhibitor (CHIR99021, CAS number: 252917-06-9) at 0 to 20 μM, preferably 0.5 to 10 μM, and more preferably 4 μM, BMP4 at 0 to 400 ng / ml, preferably 10 to 200 ng / ml, and more preferably 80 ng / ml, and VEGF at 0 to 400 ng / ml, preferably 10 to 200 ng / ml, and more preferably 80 ng / ml.

[0026] 1-2) 2nd day of differentiation induction The basal medium can be Advanced DMEM / F12 (trade name) or Essential 6 (trade name). Furthermore, the culture system can contain a selective ALK5, 4, 7 inhibitor (SB431542) at 0-20 μM, preferably 0.5-10 μM, more preferably 2-4 μM; bFGF at 0-200 ng / ml, preferably 1-100 ng / ml, more preferably 50 ng / ml; SCF at 0-200 ng / ml, preferably 1-100 ng / ml, more preferably 50 ng / ml; and VEGF at 0-400 ng / ml, preferably 10-200 ng / ml, more preferably 80 ng / ml. In addition to the above, L-glutamine, penicillin / streptomycin, differentiation-inducing supplements for iPS / ES cells (e.g., StemFit (trade name) For Differentiation: hereinafter referred to as "AS401"), etc., can be appropriately selected and added. The optimal amount can be determined as needed.

[0027] 1-3) Day 4 of differentiation induction The basal medium can be Advanced DMEM / F12 (trade name) or StemPro-34 SFM (trade name). Furthermore, the cells can be cultured in a culture system containing L-Glutamine 0 to 20 mM, preferably 0.5 to 10 mM, and more preferably 2 mM, IL-3 0 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 50 ng / ml, IL-6 0 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 50 ng / ml, FLT3L 0 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 50 ng / ml, SCF 0 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 50 ng / ml, VEGF 0 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 20 ng / ml, and EPO 0 to 100 IU / ml, preferably 1 to 50 IU / ml, and more preferably 10 IU / ml. In addition to the above, penicillin / streptomycin, differentiation-inducing supplements for iPS / ES cells (e.g., AS401), etc. may be appropriately selected and added. The optimal amount to be added can be determined appropriately.

[0028] 1-4) Day 6-8 of differentiation induction The basal medium can be Advanced DMEM / F12 (trade name) or StemPro-34 SFM (trade name). The culture can be performed in a culture system containing 0-50 mM, preferably 1-20 mM, and more preferably 2 mM, L-glutamine, 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 50 ng / ml, IL-3, 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 50 ng / ml, IL-6, 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 50 ng / ml, SCF, 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 50 ng / ml, and EPO, 0-100 IU / ml, preferably 1-50 IU / ml, and more preferably 10 IU / ml. In addition to the above, penicillin / streptomycin, differentiation-inducing supplements for iPS / ES cells (e.g., AS401), and the like can be appropriately selected and added. The optimal amount can be determined as needed.

[0029] (feeder cells) Feeder cells can be co-cultured when culturing iPS cells or inducing their differentiation. Examples of feeder cells that can be used include one or more cell lines selected from the group consisting of MEF (mouse embryonic fibroblasts) and OP9, OP9 / DLL1, OP9-DL4, and 10T1 / 2 / DL4 cells. Meanwhile, when cells obtained by inducing differentiation of iPS cells are to be administered to humans for cell therapy or other purposes, a stable production method that does not contain animal-derived substances is desired. In the present invention, differentiation into the γδ T cells of the present invention can also be induced without the use of feeder cells by modifying the laminin-511 E8 fragment and medium components described above.

[0030] (Induction of differentiation of iPS cell-derived blood progenitor cells into gamma delta T cells) The differentiation of iPS cell-derived hematopoietic progenitor cells into γδ T cells can be induced by co-culture with feeder cells or by culturing without feeder cells. Furthermore, the cells can be cultured under serum-free conditions or animal-derived component-free conditions. The differentiation of iPS cell-derived hematopoietic progenitor cells into γδ T cells can also be induced under hypoxic conditions. Hypoxic conditions refer to culture conditions in which the O2 concentration during differentiation of iPS cell-derived hematopoietic progenitor cells into γδ T cells is lower than the O2 concentration used in normal culture. The O2 concentration used for culturing under hypoxic conditions is not particularly limited, but is, for example, less than 20% (v / v), preferably less than 10% (v / v).

[0031] Furthermore, to obtain the desired γδ T cells, a γδ T cell stimulator may be added, or may not be added depending on the culture conditions. Examples of γδ T cell stimulators include phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway, as well as derivatives thereof. Examples of phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway include (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl diphosphate (IPP). Examples of such derivatives include bromohydrin diphosphate (BrHBP). Other γδ T cell stimulators include specific inhibitors of FPP (farnesyl pyrophosphate) synthase, the rate-limiting enzyme of the biosynthetic pathway. Specific inhibitors of FPP synthase promote the intracellular accumulation of such phosphate compounds. Examples of FPP synthase-specific inhibitors include nitrogen-containing bisphosphonates (N-BPs), specifically zoledronic acid and pamidronate. Furthermore, IL-15 and IL-1L-2 also function as γδT cell stimulators.

[0032] A. Co-culture with feeder cells A-1) Day 10 of differentiation induction For example, αMEM (trade name) can be used as the basal medium for culturing iPS cells on or after day 10 (blood cell progenitor cells) after differentiation induction from iPS cells by the above-mentioned steps 1-1) to 1-4). The culture can be performed in a culture system further containing 0-30% FBS, preferably 0-20%, more preferably 10-20%, 0-100 ng / ml SCF, preferably 1-50 ng / ml, more preferably 10 ng / ml IL-7, 0.1-20 ng / ml, preferably 0.5-10 ng / ml, more preferably 5 ng / ml FLT3L, 0.1-50 ng / ml, preferably 1-20 ng / ml, more preferably 5 ng / ml L-ascorbic acid, and 1-1000 μg / ml, preferably 10-500 μg / ml, more preferably 100 μg / ml. The medium may further contain IL-2 at 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 10 ng / ml, or TPO at 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 10 ng / ml. In addition to the above, penicillin / streptomycin, etc., can be added as appropriate. Furthermore, 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement, etc., can be used instead of FBS. The manufacturer and distributor of the reagents used are not particularly limited, as long as they perform equivalent functions. The optimal amount can be determined appropriately. The cells (blood cell precursor cells) on day 10 after differentiation induction can be seeded on a culture substrate such as a culture dish seeded with feeder cells and cultured. The medium is changed, for example, every two days. On days 12, 18, and 24 after differentiation induction, the supernatant is collected by pipetting and transferred to new feeder cells for continued culture. The frequency of medium exchange, the amount of medium exchange, etc. are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.

[0033] A-2) From day 30 or 31 of differentiation induction Cells cultured using the above medium from day 10 to day 30 or 31 after differentiation induction can be cultured without feeder cells. For such culture conditions, RPMI 1640 medium can be used as the basal medium. Furthermore, the cells can be cultured in a medium containing 0-30%, preferably 0-20%, and more preferably 10-20% FBS. Instead of FBS, 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement or the like can be used. Furthermore, the cells can be cultured in a culture system containing 0-200 ng / ml, preferably 1-100 ng / ml, and more preferably 10 ng / ml, of IL-2 and / or IL-15, or in a culture system containing 0-1000 IU / ml, 10-500 IU / ml, or preferably 100 IU / ml, of Immunace (trade name) and 0-100 μM, 1-50 μM, or preferably 10 μM, of 2-Me (2-Mercaptoethanol). In addition to the above, penicillin / streptomycin etc. may be added as appropriate.

[0034] Furthermore, as a γδ T cell stimulant, for example, HMBPP may be added. The concentration of the added agent is not particularly limited as long as it stimulates γδ T cells without causing cytotoxicity, and may be, for example, 0 to 100 nM, preferably 0.01 to 20 nM, and more preferably 1 nM.

[0035] B. A culture system without feeder cells B-1) Day 10 of differentiation induction ~ For example, after the 10th day (blood cell precursor cells) of differentiation induction from iPS cells by the above-mentioned treatments 1-1) to 1-4), the cells can be cultured on a culture substrate coated with VCAM1 (vascular cell adhesion molecule-1), DLL4 (Delta-Like Protein 4), or DLL1 (Delta-Like Protein 1). From the 10th to 24th day of differentiation induction, the cells can be cultured using, for example, StemSpan TMThey can be cultured in Lymphoid Progenitor Expansion Medium (trade name) included in the T cell generation kit (trade name). Medium changes are performed using StemSpan TM The protocol of the kit was followed. Specifically, additional medium was added on day 13 of differentiation induction, and the medium was replaced on days 17 and 20 of differentiation induction. Between days 17 and 24 of differentiation induction, the medium was replaced with T cell progenitor Maturation Medium (product name) included in the kit. Additional medium was added on day 27 of differentiation induction, and thereafter the medium was replaced approximately twice a week, for example, on days 31 and 34 of differentiation induction. The frequency and amount of medium replacement are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.

[0036] B-2) From day 17 to day 24 of differentiation induction Although the cells can be continuously cultured using the method described in B-1), they can also be cultured in a medium supplemented with a γδ T cell stimulator from around 17 to 24 days after differentiation induction. A tendency for a decrease in cell number may be observed from around 17 to 24 days after differentiation induction, and this can be improved by adding a γδ T cell stimulator. Specifically, the cells can be cultured in the medium shown in A-2, supplemented with IL-2 and / or IL-15 and a γδ T cell stimulator such as HMBPP and an FPP synthase-specific inhibitor. The cells can also be cultured in a medium similarly supplemented with HMBPP, which is an FBS-free medium shown in A-2. Instead of the medium shown in A-2, the cells can also be cultured in RPMI1640 medium containing AS401, supplemented with IL-2 and / or IL-15 and HMBPP.

[0037] B-3) Day 10 of differentiation induction ~ The culture can also be continued using a method in which the medium conditions described in B-1) are further supplemented with DKK1 (Dickkopf-1) and / or AZA (Azelaic acid). Furthermore, from about 17 to 24 days after differentiation induction, the cells can be cultured in a medium supplemented with a γδ T cell stimulant. Specifically, from about 17 to 24 days after differentiation induction, the cells can be cultured in the medium shown in A-2 to which HMBPP has been added. The cells can also be cultured in a medium similarly supplemented with HMBPP, which is a medium not containing FBS, as shown in A-2. Instead of the medium shown in A-2, the cells can also be cultured in RPMI1640 medium containing AS401, to which IL-2 and / or IL-15 and a γδ T cell stimulant such as HMBPP have been added.

[0038] C. Culture with beads The cells cultured using the differentiation induction method of the present invention can be cultured using beads. The size of the beads is not particularly limited and may be smaller than the size of the cells or larger than the size of the cells. For example, when culturing cells on day 10 after differentiation induction under the various conditions described above, beads can be mixed into the medium and cultured. The beads may be made of any material that can be used for cell culture, and are not particularly limited. Specifically, Dynabeads Protein G (trade name) can be used. By coating the beads with, for example, VCAM1 and DLL4, culture can also be performed under conditions without the use of feeder cells.

[0039] D. Culture using animal-derived component-free media D-1) Day 10 of differentiation induction ~ Cells cultured using the differentiation-inducing method of the present invention can also be cultured under conditions using a medium free of animal-derived components. For example, after 10 days (blood cell progenitor cells) of differentiation-inducing iPS cells by the above-mentioned treatments 1-1) to 1-4), the cells can be cultured using a culture substrate coated with VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or DLL1 (Delta-Like Protein 1). From about 10 to 24 days after differentiation induction, an animal-derived component-free medium, such as RPMI1640 containing AS401, can be used as the basal medium. Furthermore, the medium may contain SCF, IL-7, FLT3L, L-ascorbic acid, IL2, TPO, etc., as shown in A-1.

[0040] D-2) Differentiation induction from day 17 to day 24 From about day 17 to day 24 after differentiation induction, the cells can be cultured in a medium supplemented with IL-2, IL-15, and a γδ T cell stimulant as shown in A-2. RPMI1640 containing AS401 can be used as the basal medium for such a medium. Specifically, from about day 17 to day 24 after differentiation induction, the cells can be cultured in a medium supplemented with one or more of IL-2, IL-15, and HMBPP.

[0041] (γδT cells obtained by differentiation induction) The γδ T cells generated by the differentiation induction method of the present invention are T cells that possess a unique T cell receptor (TCR) consisting of a γ chain and a δ chain on their surface. The expression of cell markers such as CD3, CD7, CD8a, CD45RA, and γδ TCR can be confirmed on the cell surface. The γδ T cells of the present invention preferably express one or more markers selected from CD7, CD8a, and CD45RA, while preferably do not express one or more markers selected from CD25, IFNγ, CD5, and CD27. The obtained iPS cell-derived γδ T cells are characterized by their antigen-specific cytotoxic activity in an MHC-nonrestricted manner. Furthermore, differences are observed in the cell surface marker patterns between γδ T cells generated by inducing differentiation of iPS cells of the present invention and γδ T cells isolated from peripheral blood. For example, iPS cell-derived γδ T cells tend to express CD7 and CD8a at higher levels, while IL2RA (CD25), CD5, and IFNγ at higher levels are observed in γδ T cells isolated from peripheral blood. Furthermore, for example, iPS cell-derived γδ T cells tend to express CD45RA at a higher level, and γδ T cells isolated from peripheral blood tend to express CD27 at a higher level.

[0042] The γδ T cells induced to differentiate in this manner can be isolated by an appropriate known method. Examples of such known methods include flow cytometry using antibodies against cell surface markers and a cell sorter, as described in the Examples below. When isolating "T cells with a desired antigen specificity" from a human, a purification method using an affinity column onto which the desired antigen is immobilized can also be employed.

[0043] A purified γδ T cell population is composed of homogeneous cells and is distinguishable from a cell population composed of γδ T cells isolated from peripheral blood. The γδ T cell population of the present invention has higher antigen-specific cytotoxic activity than a γδ T cell population isolated from peripheral blood.

[0044] The cell population containing γδ T cells contains many cells having identical base sequences in, for example, the complementarity determining region (CDR) of the TCR gene. γδ T cells having identical base sequences, particularly in the CDR3 region, among the CDRs, are contained in a large proportion of the γδ T cells constituting the cell population, for example, at a proportion of 90% or more. The cell population containing γδ T cells of the present invention contains 1×10 5 It is possible to include more than one.

[0045] Furthermore, the cell population containing γδ T cells of the present invention contains γδ T cells that express CD7 and / or CD8a at a higher level than γδ T cells isolated from peripheral blood, accounting for 90% or more of the γδ T cells constituting the cell population. Furthermore, the cell population contains γδ T cells that express one or more of CD25, INFγ, and CD5 at a lower level than γδ T cells isolated from peripheral blood, accounting for 90% or more of the γδ T cells constituting the cell population. Furthermore, the cell population contains γδ T cells that express CD45RA at a higher level than γδ T cells isolated from peripheral blood and expanded in vitro, and that express CD27 at a lower level than γδ T cells isolated from peripheral blood and expanded in vitro, accounting for 70% or more of the γδ T cells constituting the cell population.

[0046] Furthermore, the cell population containing γδ T cells of the present invention is characterized in that undifferentiated cells account for 10% or less of the γδ T cells constituting the cell population, and it is further preferred that undifferentiated cells are not present among the γδ T cells constituting the cell population. Whether a cell is an undifferentiated cell can be determined using a marker that indicates undifferentiation, such as TRA-1-85.

[0047] γδ T cells produced by the differentiation-inducing treatment method of the present invention have excellent immune functions and can be used for the treatment or prevention of diseases such as tumors, infectious diseases (e.g., viral infections), and autoimmune disorders. Furthermore, the γδ T cell population produced by the method of the present invention can be used as an antigen-specific cellular immunotherapy agent or pharmaceutical composition containing the cell population as an active ingredient. γδ T cells produced by the differentiation-inducing treatment method of the present invention can be used for these formulations even after freeze-thawing. It is expected that the γδ T cell population can also be applied to immune cell therapy methods using the γδ T cell population. The γδ T cell population of the present invention is expected to further enhance the effects of γδ T cells when used in combination with an immune checkpoint inhibitor. Immune checkpoint inhibitors include, but are not limited to, known or future developments, and include, for example, drugs targeting immune checkpoints such as PD-1, PD-L1, and CTLA-4. Furthermore, like NK cells, they are expected to have antibody-dependent cellular cytotoxicity (ADCC) effects that enhance the effects of molecular targeted therapeutic drugs and antibody preparations (e.g., Herceptin, Rituxan, etc.) used to treat various cancers, and when used in combination with these antibody preparations, high therapeutic effects can be expected. The pharmaceutical composition containing the γδ T cell population of the present invention can be prepared by formulation using known pharmaceutical methods.

[0048] In preparing these formulations, the pharmaceutical composition of the present invention may be appropriately combined with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, solubilizing agent, or other additive. It may also be used in combination with known pharmaceutical compositions or immunostimulants used in the treatment or prevention of the aforementioned diseases. When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected depending on the age, weight, symptoms, health condition, type of composition, and the like of the subject.

[0049] The present invention also includes a method for antigen-specific cellular immunotherapy by administering the iPS cell-derived γδ T cells of the present invention. Furthermore, the present invention also includes a method for treating diseases such as cancer, infectious diseases, and autoimmune disorders by administering the iPS cell-derived γδ T cells of the present invention. In the methods of the present invention, the dosage of the active ingredient administered to a subject varies depending on the subject's weight, age, symptoms, and administration method, but can be appropriately selected by one skilled in the art. [Example]

[0050] To better understand the present invention, the present invention will be specifically explained below by showing examples, but it goes without saying that the present invention is not limited to these examples.

[0051] (Example 1) Differentiation induction from iPS cells In this example, a differentiation induction treatment method for γδ T cells prepared from γδ TCR-type iPS cells prepared by the method described in Non-Patent Document 1 is described.

[0052] (1-1) Cultivation of γδTCR iPS cells (62B3 strain) 2 x 10 γδTCR iPS cells (62B3 strain) cultured without feeder cells 3 The cells were subcultured in a 6-well plate at 1.6 μg / well in StemFit containing iMatrix-511 (Nippon). (R) AK02N (Ajinomoto Co., Inc.) was used. Cell detachment and dispersion during subculture were performed using 0.5x TrypLE. TM select (ThermoFisher) was used, and StemFit was used for subculture. (R) The medium used was AK02N supplemented with Y27632 (Wako Pure Chemical Industries, Ltd.) at a final concentration of 10 μM and 3.2 μl of iMatrix-511. (R) The medium was then replaced with AK02N, and thereafter replaced every two days. 1.5 ml of medium was added per well. All of the following steps and the Examples described later were cultured at 37±0.5°C and 5% CO2.

[0053] (1-2) Differentiation induction day 0 (Day 0) Seven days after the passage in (1-1) above, the medium (Step 1) shown in Table 1 was replaced at 2 ml / well. [Table 1]

[0054] (1-3) Day 2 of differentiation induction (Day 2) Two days after (1-2) above, the medium (Step 2) shown in Table 2 was replaced at 2 ml / well. [Table 2]

[0055] (1-4) Day 4 of differentiation induction (Day 4) Two days after the above (1-3), the medium shown in Table 3 (Step 3) was replaced at 2 ml / well. [Table 3]

[0056] (1-5) Day 6 of differentiation induction (Day6) Two days after the above (1-4), the medium shown in Table 4 (Step 4) was replaced at 2 ml / well. [Table 4]

[0057] (1-6) Day 8 of differentiation induction (Day8) Two days after the above (1-5), the medium was replaced with the same medium as shown in Table 4 (Step 4) at 2 ml / well.

[0058] (1-7) Cell evaluation on day 10 of differentiation induction (Day 10) CD34 / CD43 expression was assessed by flow cytometry. + / CD43 + Cells and CD34 - / CD43+ A large number of cells were detected, which indicated that they had differentiated into blood cell precursor cells (Fig. 1A).

[0059] (1-8) Day 10 of differentiation induction (Day 10) Cells other than those subjected to flow cytometry in (1-7) above were seeded onto 12-well culture dishes seeded with OP9 / N-DLL1 feeder cells. The medium had the composition shown in Table 5, and the volume was 1 ml / well. Half of the medium was replaced every two days. [Table 5]

[0060] (1-9) Evaluation of cells on day 31 of differentiation induction (Day 31) CD3 / γδTCR expression was assessed by flow cytometry. + / TCR + A large number of cells were detected, confirming their differentiation into TCR cells (FIG. 1B). The obtained cells are hereinafter referred to as "iPS cell-derived γδ T cells" in this example.

[0061] (1-10) Evaluation of cells on day 31 of differentiation induction (Day 31) A cytotoxicity assay was performed against Jurkat cells (derived from human leukemia T cells). The E:T (effector:target) ratio was 2:1, and 5 × 10 Jurkat cells (T) stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish. 5 iPS cell-derived γδ T cells (E) were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed for many Jurkat cells (CFSE-positive cells). In other words, it was confirmed that iPS cell-derived γδ T cells have cytotoxic function. In this example, cytotoxic activity was confirmed even though γδ T cells were not subjected to activation stimulation culture.

[0062] (Example 2) Differentiation induction from iPS cells In this example, γδ T cells were produced by differentiation induction treatment from γδ TCR-type iPS cells produced by the method of Non-Patent Document 1. The medium components from day 10 onwards and from day 31 onwards of differentiation induction differ from those in Example 1. In particular, the medium components from day 31 onwards of differentiation induction contain HMBPP, a γδ T cell stimulant.

[0063] (2-1) Differentiation induction treatment Up to the 10th day, the same treatments as in (1-1) to (1-6) of Example 1 were carried out. (2-2) Day 10 of differentiation induction (Day 10) The cells prepared in (1-6) of Example 1 above were seeded onto a 12-well culture dish seeded with OP9 / N-DLL1 feeder cells. 1 ml / well of medium (Step 5) with the composition shown in Table 6 was replaced every 7 days. [Table 6]

[0064] (2-3) Evaluation of cells on day 17 of differentiation induction (Day 17) Expression of CD7 (a T cell differentiation marker) was evaluated by flow cytometry. CD7-positive cells were observed, demonstrating the progression of differentiation into T cells (Figure 2A).

[0065] (2-4) Day 31 of differentiation induction (Day 31) Half of the medium was replaced every two days with the γδ T cell stimulation medium shown in Table 7. The γδ T cell stimulation medium contained HMBPP. [Table 7]

[0066] (2-5) Evaluation of cells on day 54 of differentiation induction (Day 54) (2-5) Evaluation of cells on day 54 of differentiation induction (Day 54) CD3 / γδTCR expression was assessed by flow cytometry. + / TCR +A large number of cells were detected, and differentiation into TCR cells was confirmed. In other words, the obtained cells were confirmed to be iPS cell-derived γδ T cells. Furthermore, when the expression of CD45RA, which is generally used as an indicator of T cell maturation, was evaluated, the expression of CD3 + The cells are CDRA + Cells and CDRA - It was revealed that both cells were involved (Fig. 2B).

[0067] (Example 3) Induction of differentiation from iPS cells using feeder cells Similar to Example 1, this Example describes γδ T cells generated by differentiation induction treatment from γδ TCR-type iPS cells generated by the method described in Non-Patent Document 1. Differentiation induction treatment was performed as in Example 1, and from day 31 onwards, half of the γδ T cell stimulation medium (containing HMBPP and FBS) was replaced every two days as in (2-4) of Example 2. Then, marker expression was evaluated and a cytotoxicity assay was performed.

[0068] (3-1) Differentiation Induction Treatment Up to day 10, the same treatments as in (1-1) to (1-6) and (1-8) shown in Example 1 were carried out. (3-2) Evaluation of cells on day 17 of differentiation induction (Day 17) Expression of CD7 (a T cell differentiation marker) was evaluated by flow cytometry. CD7-positive cells were detected, demonstrating the progression of differentiation into T cells (Figure 3A). (3-3) Evaluation of cells on day 55 of differentiation induction (Day 55) CD3 / γδTCR expression was assessed by flow cytometry. + / TCR + A large number of cells were detected, confirming their differentiation into γδ T cells (FIG. 3B). The obtained cells are hereinafter referred to as "iPS cell-derived γδ T cells" in this example. (3-4) Evaluation of cells on day 55 of differentiation induction (Day 55) A cytotoxicity assay against Jurkat cells was performed. 5 × 10 CFSE-stained Jurkat cells were plated per well of a 96-well culture dish. 4 1 × 10 cells were added, and 1 × 10 cells were added on the 55th day of differentiation induction.5 iPS cell-derived γδ T cells were added and cultured at an E:T ratio of 2:1 for 16 hours, after which 7-AAD staining (dead cell staining) was performed. Many Jurkat cells (CFSE-positive cells) were 7-AAD-positive, and many dead cells were confirmed. This confirms that iPS cell-derived γδ T cells have cytotoxic activity against tumor cells (Figure 3C).

[0069] (Example 4) Induction of differentiation from iPS cells without using feeder cells Similar to Example 1, this Example demonstrates a method for inducing differentiation without the use of feeder cells for γδ T cells produced by differentiation induction treatment from γδ TCR-type iPS cells produced by the method described in Non-Patent Document 1. In this Example, differentiation induction treatment was carried out in the following manner, according to the protocol shown in Figure 4.

[0070] (4-1) Differentiation Induction Treatment Up to the 8th day, the same treatments as in (1-1) to (1-6) shown in Example 1 were carried out. (4-2) Day 10 of differentiation induction (Day 10) A 48-well culture dish coated with VCAM1 and DLL4 was used, and StemSpan TM 1.2 × 10 cells were added to 250 μl of Lymphoid Progenitor Expansion Medium included in the T cell generation kit (Stem Cell Technologies) on the 10th day of differentiation induction. 4 A suspension of 5 μg / ml VCAM1 and 10 μg / ml DLL4 in PBS(-) was added at 100 μl per well to a commercially available 48-well culture dish (non-treated cell culture), which had not been hydrophilized for cell adhesion. The dish was left standing overnight at 4°C, the solution was removed, and the dish was washed once with PBS(-) to prepare a VCAM1- and DLL4-coated culture dish. In the process using Lymphoid Progenitor Expansion Medium, the dish was cultured without feeder cells or serum.

[0071] (4-3) After that, the medium was replaced with StemSpan. TM The incubation was carried out according to the kit's protocol. Specifically, on day 13 of differentiation induction (Day 13), an additional 250 μl of medium was added, and half of the medium was replaced on days 17 and 20 of differentiation induction (Day 20). On day 24 of differentiation induction (Day 24), the medium was replaced with the T cell progenitor maturation medium included in the kit. On day 27 of differentiation induction (Day 27), additional medium was added, and thereafter, half of the medium was replaced twice a week, for example, on days 31 and 34 of differentiation induction.

[0072] (4-4) Evaluation of cells on days 33, 35, and 37 of differentiation induction CD3 / γδTCR expression was assessed by flow cytometry. + / TCR + A large number of cells were detected, and differentiation into TCR cells was confirmed, confirming that they were iPS cell-derived γδ T cells (Figure 5). The results show the results of three independent differentiation induction experiments. The day of evaluation (the start of differentiation induction is day 0) is indicated in the figure.

[0073] (Example 5) Differentiation induction from iPS cells without using feeder cells This example demonstrates a method for inducing differentiation without the use of feeder cells for γδ T cells produced by differentiation induction treatment from γδ TCR-type iPS cells, as in Example 4. In this example, differentiation induction treatment was carried out in the following manner, according to the protocol shown in Figure 6.

[0074] (5-1) The same treatments as in (4-1) to (4-3) of Example 4 were carried out, and from day 10 to day 24 of differentiation induction, the cells were cultured without the use of feeder cells or serum. (5-2) Day 24 of differentiation induction (day 24) The medium was replaced with the γδT cell stimulation medium (containing HMBPP and FBS) shown in Table 7 of Example 2 (2-4), and thereafter half of the medium was replaced every three days. (5-3) Evaluation of cells on day 37 of differentiation induction The number of cells was observed using a phase-contrast microscope. The cells prepared in Example 4 by culturing in a medium that did not contain a γδ T cell stimulating agent (HMBPP) were also observed in the same way. As a result, the number of cells observed was clearly higher when cultured in the γδ T cell stimulating medium (Figure 7A). Furthermore, CD3 / γδ TCR expression was evaluated by flow cytometry, and CD3 + / TCR + A large number of cells were detected, confirming their differentiation into TCR cells (Figure 7B). The obtained cells were confirmed to be iPS cell-derived γδ T cells.

[0075] (Example 6) Induction of differentiation from iPS cells without using feeder cells This example demonstrates a method for inducing differentiation without the use of feeder cells for γδ T cells produced by differentiation induction treatment from γδ TCR-type iPS cells, as in Example 4. In this example, differentiation induction treatment was carried out in the following manner, according to the protocol shown in Figure 8.

[0076] (6-1) The same treatments as in (4-1) to (4-3) of Example 4 were carried out, and from day 10 to day 24 of differentiation induction, the cells were cultured without the use of feeder cells or serum. (6-2) Day 24 of differentiation induction (day 24) On day 24 of differentiation induction, a. γδ T cell stimulation medium (containing HMBPP and FBS) shown in Table 7 of Example 2 (2-4), b. RPMI1640 (containing HMBPP) medium containing AS401 instead of the basal medium (10% FBS / RPMI1640) of the γδ T cell stimulation medium shown in Table 7, and c. StemSpan TM The medium was replaced with the Lymphoid Progenitor Expansion Medium included in the kit, and the medium was replaced in the same manner as in Example 5 (5-2). (6-3) Evaluation of cells on day 32 of differentiation induction 2 The number of cells was observed using a phase contrast microscope on day 32 of differentiation induction. TMWhile the cell count was clearly low in the medium included in the kit, the serum-free medium (b) had a cell density equivalent to that of the serum-containing medium (a) (Figure 9). Furthermore, the expression of CD3 / γδTCR was evaluated by flow cytometry for the above cells. + / TCR + A large number of cells were detected, and they were confirmed to be iPS cell-derived γδ T cells (FIG. 10). The obtained cells are hereinafter referred to as "iPS cell-derived γδ T cells" in this example. (6-4) Evaluation of cells on day 35 of differentiation induction (day 35) 1 For the cells obtained under the medium conditions a. and b. on day 35 of differentiation induction, a cytotoxicity assay against Jurkat cells was performed using the same method as in (1-10) of Example 1. 5 × 10 CFSE-stained Jurkat cells were placed in each well of a 96-well culture dish. 4 1 × 10 cells were added, and 1 × 10 cells were added on the 35th day of differentiation induction. 5 iPS cell-derived γδ T cells were added at an E:T ratio of 2:1, and evaluation was performed 1 day (d1) and 4 days (d4) after the start of mixed culture. Cell clusters indicating T cell activation were observed. Compared to the control (ctrl) without effector cells (iPS cell-derived γδ T cells), the number of cells obtained under medium conditions a and b was significantly lower (Figure 11). Although the number was lower than that in medium condition a, cytotoxicity was evident in serum-free medium condition b after 1 day of mixed culture, and even more pronounced cytotoxic activity was observed after 4 days (Figure 12).

[0077] (Example 7) Induction of differentiation from iPS cells without using feeder cells In this example, γδ TCR iPS cells were subjected to differentiation induction treatment in the same manner as in Example 4 to produce γδ T cells.

[0078] (7-1) The same treatments as in (4-1) and (4-2) of Example 4 were carried out and the cells were cultured. (7-2) However, on the 10th day of differentiation induction, in addition to the same conditions as (4-2) (i), the following conditions were also performed: (ii) DKK1 (Dickkopf-1) was added to a final concentration of 30 ng / ml; (iii) AZA (Azelaic acid) was added to a final concentration of 5 mM; and (iv) both DKK1 (Dickkopf-1) and AZA were added at the same concentrations as (ii) and (iii), respectively. (7-3) After that, the medium was replaced with StemSpan. TM The procedure was carried out according to the kit's protocol. That is, on day 13 of differentiation induction, 250 μl of each medium shown in (7-2) was added, and half of each medium was replaced on days 17 and 20 of differentiation induction. (7-4) Evaluation of cells on day 24 of differentiation induction (Day 24) The expression of CD7, a T cell differentiation marker, was evaluated by flow cytometry on day 24 of differentiation induction in the cells. The results showed that DKK1 and AZA each had a positive effect on differentiation induction efficiency, and that combined treatment was even more effective (Figure 13).

[0079] (Example 8) Differentiation induction method using magnetic beads In this example, differentiation into T cells was induced by co-culturing magnetic beads coated with VCAM1 and DLL4 instead of coating the culture dish under conditions without using feeder cells.

[0080] (8-1) Differentiation Induction Treatment Up to the 8th day, the same treatments as in (1-1) to (1-6) of Example 1 were carried out. (8-2) Preparation of VCAM1 and DLL4-coated magnetic beads Magnetic beads (Dynabeads TMProtein G (Invitrogen) was stirred using a vortex mixer, and 5 μl of this and 1 ml of PBS were placed in a tube and allowed to stand for 1 minute on a magnetic stand for capturing magnetic beads. The PBS was removed and the tube was removed from the stand, and 200 μl of PBS, 4.26 μl of VCAM1 (100 μg / ml solution), and 4.26 μl of DLL4 (100 μg / ml solution) were added, followed by allowing the tube to stand at room temperature for 15 minutes. The tube was then left to stand on the magnetic stand for 1 minute, after which the above solutions were removed and the tube was removed from the stand. StemSpan TM 500 μl of Lymphoid Progenitor Expansion Medium included in the kit was added and suspended by pipetting. (8-3) Day 10 of differentiation induction (Day 10) 4.75 × 10 cells prepared in (8-1) above 5 The cells were suspended in 500 μl of the magnetic bead solution prepared in (8-2) and placed in a 24-well low-adhesion culture dish (PrimeSurface TM ) and cultured. (8-4) On day 13 of differentiation induction, 500 μl of the medium was added, and half of the medium was replaced on days 17 and 20 of differentiation induction. (8-5) Evaluation of cells on day 24 of differentiation induction (Day 24) The expression of CD7, a T cell differentiation marker, was evaluated by flow cytometry on day 24 of differentiation induction. The results confirmed the presence of CD7-positive cells, although the percentage was low at 0.3%, compared to the control (isotype control). This demonstrates that differentiation into T cells is possible using this method of co-culturing with magnetic beads (Figure 14).

[0081] (Example 9) γδ T cells generated from γδ TCR iPS cells In this example, we confirmed the characteristics of γδ T cells generated from γδ TCR-type iPS cells (iPS cell-derived γδ T cells). First, we will show the method for generating iPS cell-derived γδ T cells, and then we will show various characteristics of the cells.

[0082] (9-1) Method for generating iPS cell-derived γδ T cells γδ T cells were generated by the method shown in FIG. Establishment of iPS cells We used γδTCR iPS cells prepared by the method described in Non-Patent Document 1. Stemfit iPS cells were used for maintenance culture. (R) AK02N (Ajinomoto) was used. 0.5x TrypLE was used for subculture. TM For the differentiation induction treatment into hematopoietic progenitor cells, 6-well culture plates were used, and 2 × 10 3 Every other day, the medium was aspirated and replaced with 2.0 ml / well of the entire medium.

[0083] Day 0 of differentiation induction (Day 0): γδTCR iPS cell status (HPC1) Stemfit AK02N (Ajinomoto, Tokyo, Japan, AK02N) CHIR99021 (Tocris, Bristol, UK, 4423) 4μM BMP4 (R&D, Minneapolis, MN, 314-BP) 80 ng / ml VEGF (R&D, Minneapolis, MN, 293-VE) 80 ng / ml

[0084] Day 2 of differentiation induction (HPC2) Essential6 (Thermofisher, Waltham, MA, A1516501) SB431542 (WAKO, Osaka, Japan, 033-24631)2μM bFGF(WAKO, Osaka, Japan,060-04543) 50 ng / ml SCF (R&D, Minneapolis, MN, 255-SC) 50 ng / ml VEGF (R&D, Minneapolis, MN, 293-VE) 80 ng / ml

[0085] ·Day4:(HPC3) StemPRO34SFM (Thermofisher, Waltham, MA, 10639-011) L-Glutamine (Life Technologies, 25036-081) 2 mM IL-3(Peprotech, Cranbury, NJ, AF-200-03) 50 ng / ml IL-6(R&D, Minneapolis, MN, 206-IL) 50 ng / ml FLT3L(R&D, Minneapolis, MN, 308-FK) 50 ng / ml SCF (R&D, Minneapolis, MN, 255-SC) 50 ng / ml VEGF (R&D, Minneapolis, MN, 293-VE) 20 ng / ml EPO(Kyowa Kirin, Tokyo, Japan) 10IU / ml

[0086] ·Day 6:8:(HPC4) StemPRO34SFM (Thermofisher, Waltham, MA, 10639-011) L-Glutamine (Life Technologies, 25036-081) 2 mM IL-6(R&D, Minneapolis, MN, 206-IL) 50 ng / ml SCF (R&D, Minneapolis, MN, 255-SC) 50 ng / ml EPO(Kyowa Kirin, Tokyo, Japan) 10IU / ml

[0087] ·Observation at 10 Days~(Day10~): T cell depletion of the mammalian antigen (OP9 / N-DLL1) in the presence of T cell genes The cells were passaged using Accutase (Nacalai Tesque, Kyoto, Japan, 12679-54). Half of the medium was replaced every two days. On days 12, 18, and 24, the supernatant was collected by pipetting and seeded onto new feeder cells (OP9 / N-DLL1). (T cell differentiation medium) αMEM (Gibco, 11900-016) FBS(Sigma-Aldrich, St. Louis, MO, F7524) 20% SCF (R&D, Minneapolis, MN, 255-SC) 10 ng / ml TPO(R&D, Minneapolis, MN, ) 10 ng / ml IL-7 (R&D, Minneapolis, MN, 207-IL) 5 ng / ml FLT3L (R&D, Minneapolis, MN, 308-FK) 5 ng / ml L-ascorbic acid (Nacalai Tesque, Kyoto, Japan, 30264-56) 100μg / ml

[0088] Day 30 of differentiation induction (Day 30 onwards): Culture in γδT activation medium Accutase-treated cells were suspended in the γδT activation medium described below and cultured in a medium without feeder cells. Half of the medium was replaced every two days. Cells cultured for 7 to 14 days were subjected to a cytotoxicity assay.

[0089] (γδT activation medium) RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56) FBS(Sigma-Aldrich, St. Louis, MO, F7524) 10% HMBPP (Cayman chemical, Ann Arbor, MI, 13580) 1 nM Immunace(Shionogi pharmaceuticals, Osaka, Japan) 100IU / ml 2-Me (Nacalai Tesque, Kyoto, Japan) 10μM

[0090] (9-2) Differentiation process from γδTCR iPS cells to γδT cells The cell morphology during differentiation was observed using a phase-contrast microscope (Fig. 16A), and cell surface markers were confirmed by flow cytometry (Fig. 16B). d0: Differentiation induction Day 0: γδTCR type iPS cells d10: Day 10 of differentiation induction: cells differentiated into blood cell progenitor cells d30: Day 30 of differentiation induction: γδ T cells before activation stimulation d51: Day 51 of differentiation induction: γδ T cells after γδ T cell activation stimulation

[0091] (9-3) Antitumor effect The antitumor activity of iPS cell-derived γδ T cells on day 38 of differentiation induction against various tumor cells was confirmed (Figure 17). These experiments used unpurified γδ T cells. As a control, tumor cells alone were cultured without γδ T cells. A. Cytotoxicity assay was performed against Jurkat cells (derived from human leukemia T cells). E:T (effector:target) ratio = 2:1, 5 x 10 Jurkat cells stained with the fluorescent dye CFSE. 4 Add 1 x 10 cells to one well of a 96-well culture dish. 5 After adding 10 iPS cell-derived γδ T cells and culturing for 16 hours, dead cells were stained with 7-AAD. Compared to the control, the γδ T cells of the present invention clearly had higher cytotoxic activity against Jurkat cells (Figure 17A).

[0092] B. Cytotoxicity assay was performed on Huh-7 cells (derived from human hepatoma cells). E:T (effector:target) ratio = 2:1. 5 × 10 Huh-7 cells stained with the fluorescent dye CFSE were used. 4Add 1 x 10 cells to one well of a 96-well culture dish. 5 After adding iPS cell-derived γδ T cells and culturing for 16 hours, the tumor areas were observed under a phase-contrast microscope and measured. Compared to the control, the γδ T cells of the present invention clearly had higher cytotoxic activity against Huh-7 cells (Figure 17B).

[0093] C. A cytotoxicity assay was performed on SW480 cells (derived from human colon carcinoma). E:T (effector:target) ratio = 2:1. 5 × 10 SW480 cells were stained with the fluorescent dye CFSE. 4 Add 1 x 10 cells to one well of a 96-well culture dish. 5 After adding 10 iPS cell-derived γδ T cells and culturing for 16 hours, the tumor areas were observed under a phase-contrast microscope and measured. Compared to the control, the γδ T cells of the present invention clearly had higher cytotoxic activity against SW480 cells (Figure 17C).

[0094] D. iPS cell-derived γδ T cells (E) were cocultured with Jurkat cells (T) at a stepwise E:T ratio. The viability of cells was compared, with the viability of a 0:1 ratio set at 100% (Figure 17D).

[0095] (9-4) Maintenance of TCR rearrangement and cytotoxicity Using iPS cell-derived γδ T cells on day 36 of differentiation induction, the maintenance of TCR rearrangement and the mechanism of cytotoxicity were confirmed (FIG. 18). A. Cell surface expression of αβ TCR was evaluated for unpurified iPS cell-derived γδ T cells (igdT) and peripheral blood mononuclear cells (PB). αβ TCR expression was detected in PB, but not in the γδ T cells (igdT) of the present invention (Figure 18A). B. Genomic PCR of TCR gene rearrangement TCR gene (Vg9, Vd2) rearrangements were confirmed by genomic PCR. Flow cytometry-sorted γδ T cells (igdT) were confirmed to retain TCR gene rearrangements, similar to undifferentiated cells (Figure 18B). Peripheral blood mononuclear cells (PBMCs) were used as a positive control. C. iPS cell-derived γδ T cells (igdT) that had been previously labeled with CD3 were co-cultured with Jurkat cells in the presence of 3 μg / ml Brefeldin A. The iPS cell-derived γδ T cells were confirmed to express Granzyme B and Perforin (Figure 18C). Granzyme B and Perforin are the molecular entities responsible for the cytotoxic function of T cells, confirming that the iPS cell-derived γδ T cells of the present invention possess cytotoxicity. D. A cytotoxicity assay was performed on γδ T cells (igdT) purified by flow cytometry (FACS). The cytotoxicity assay was performed under the conditions described in (9-3)A. In Figure 18D, a control (ctrl) was used in which Jurkat cells were cultured alone without iPS cell-derived γδ T cells. Unpurified iPS cell-derived γδ T cells are designated "bulk," and purified iPS cell-derived γδ T cells are designated "sort." There was no significant difference in the cell death rate between purified and unpurified iPS cell-derived γδ T cells (Figure 18D).

[0096] E. HLA typing of iPS cell lines and tumor cells Table 8 shows the results of confirming the HLA types of the iPS cells used to generate the iPS cell-derived γδ T cells of the present invention, and of the tumor cells used in Examples 3 and 6 and this Example. Although the HLA types of the iPS cells did not match those of the tumor cells, antitumor activity was observed against each tumor cell (Examples A to C). This confirmed that the iPS cell-derived γδ T cells of the present invention have antigen-specific cytotoxic activity in a non-MHC-restricted manner. [Table 8]

[0097] Example 10: Comparison of iPS cell-derived γδ T cells and γδ T cells isolated from peripheral blood In this example, we compared the cell surface expression marker genes of iPS cell-derived γδ T cells (igdT) generated by inducing differentiation of iPS cells with those of γδ T cells present in peripheral blood (PB-gdT). The iPS cell-derived γδ T cells in this example were cultured by the methods described in Example 1 and Example 9 (9-1), and cells on days 36 to 42 after differentiation induction were used. Mononuclear cells isolated from peripheral blood were cultured in the γδ T activation medium described in Example 9 (9-1), and the resulting cells were used as the γδ T cells isolated from peripheral blood in this example.

[0098] (10-1) Single-cell RNA-seq analysis We analyzed the differences in marker gene expression between iPS cell-derived γδ T cells, γδ T cells isolated from peripheral blood, and non-γδ T cells in peripheral blood using single-cell RNA-seq analysis.The results showed distinct expression patterns for CD7, CD8a, IL18R1, IL2RA (CD25), IL2RB, and IFNγ (Figure 19, Table 9). [Table 9]

[0099] (10-2) Analysis of CD25 by flow cytometry We compared CD25 expression in iPS cell-derived γδ T cells and γδ T cells isolated from peripheral blood by flow cytometry. Most iPS cell-derived TCR-Vγ9-positive cells were CD25-negative, whereas most TCR-Vγ9-positive cells isolated from peripheral blood were CD25-positive (Figure 20). From the above, it was confirmed that the cell surface marker patterns differ between iPS cell-derived γδ T cells and γδ T cells isolated from peripheral blood.

[0100] (Example 11) Method for activating iPS cell-derived γδ T cells In this example, we investigated methods for activating iPS cell-derived γδ T cells. Specifically, we investigated whether the addition of IL-2 and / or IL-15 to the following γδ T activation medium could more effectively generate iPS cell-derived γδ T cells using cells on day 30 of differentiation induction using the production method described in Example 9 (9-1) (see Figure 21). (activation medium) RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56) FBS(Sigma-Aldrich, St. Louis, MO, F7524) 10% HMBPP (Cayman chemical, Ann Arbor, MI, 13580) 1 nM 2-Me (Nacalai Tesque, Kyoto, Japan) 10μM

[0101] Viable cell count and CD3 + As a result of evaluating γδT cells, the addition of IL-15 was preferable to IL-2, and IL-15 alone was more effective than the combination of IL-2 (FIG. 22).

[0102] (Example 12) Characteristics of γδ T cells generated from γδ TCR iPS cells (121-3 line) In this example, the characteristics of γδ T cells generated from γδ TCR iPS cells (121-3 strain) were confirmed.

[0103] (12-1) In this Example, γδ TCR iPS cells were cultured using the 121-3 strain instead of the 62B3 strain by the method described in Example 9 (9-1), and the cells were used on day 36 of differentiation induction.

[0104] (12-2) Maintenance of TCR rearrangement A. Rearrangement of TCR genes (Vγ9, Vγ2) in γδ T cells (iγδ T) obtained by differentiation induction from γδ TCR iPS cells (line 121-3) was confirmed by genomic PCR. iγδ T cells sorted by flow cytometry were confirmed to retain TCR gene rearrangement, similar to undifferentiated cells (Figure 23A). B. We analyzed the TCRγ and TCRδ sequences of γδ T cells (iγδ T) obtained by differentiation induction from γδ TCR-type iPS cells (line 121-3) and γδ T cells (PBγδ T) obtained by expansion and culture of peripheral blood mononuclear cells using next-generation sequencing. The nucleotide and amino acid sequences of the CDR3 regions of each TCRγ and TCRδ were identified, and the frequency of each sequence is shown in a pie chart (Figure 23B). We confirmed that the PBγδ T cell population is composed of cells with diverse sequences, while the iγδ T cell population is composed entirely of cells with a single type of TCRγ and TCRδ gene rearrangement.

[0105] (Example 13) Characteristics of iPS-derived γδ T cells generated from γδ TCR iPS cells (62B3 strain) In this example, iPS-derived γδ T cells were prepared by the method described in Example 9. The cells on day 39 after differentiation induction were co-cultured with Jurkat cells for 4 hours, and the expression of IFNγ was evaluated by flow cytometry.

[0106] The expression of IFNγ (interferon gamma) in iPS cell-derived γδT cells (iγδT) and γδT cells obtained by expanding and culturing peripheral blood mononuclear cells (PBγδT) was evaluated using a flow cytometer, along with the expression of Granzyme B. As a result, it was confirmed that Granzyme B was expressed in both cell populations, whereas IFNγ was expressed only in PBγδT, but not in iγδT (Figure 24).

[0107] Example 14 Comparison of iPS cell-derived γδ T cells and γδ T cells obtained by expanding peripheral blood In this example, we compared the cell surface expression markers of iPS cell-derived γδ T cells (igdT) produced by inducing differentiation of γδ TCR-type iPS cells (62B3 strain or 121-3 strain) and γδ T cells (PB-gdT) obtained by expanding peripheral blood.

[0108] (14-1) The same treatment as in (9-1) of Example 9 was carried out, and the cells were cultured using a method using feeder cells. (14-2) The expression of various cell surface markers (CD25, CD7, CD5, CD45RA, and CD27) was evaluated using a flow cytometer in cell populations containing γδT cells (iγδT) on day 40 of differentiation induction and cell populations containing γδT cells (PBγδT) obtained by expanding and culturing peripheral blood mononuclear cells (CD3-positive or TCRγ9-positive). Compared to PBγδT, iPS cell-derived γδT cells (iγδT CD3-positive or TCRγ9-positive cells) had a higher percentage of cells expressing CD7, a lower percentage of cells expressing CD5 and CD25, and a higher percentage of cells expressing CD45RA. + CD27 - It was confirmed that the characteristic of this product was that the proportion of

[0109] Example 15: Study of the step of stimulating γδ T cells This Example demonstrates a differentiation induction method for γδ T cells generated by differentiation induction treatment from γδ TCR-type iPS cells, similar to Example 5, under conditions in which neither feeder cells nor serum are used, and the step of stimulating γδ T cells is performed on day 17, rather than from day 24. In this Example, differentiation induction was performed according to the protocol shown in Figure 26A (New protocol) as follows.

[0110] (15-1) The cells were cultured by the same treatment as in (5-1) of Example 5. However, the step of stimulating γδ T cells was started from day 17 after differentiation induction.

[0111] (15-2) Evaluation of cells on day 17 of differentiation induction (Day 17) On day 17 of differentiation induction, the expression of CD3 / γδTCR (gdTCR) was evaluated by flow cytometry.+ / TCR + The cells were detected, and differentiation into TCR cells was confirmed, confirming that they were iPS cell-derived γδ T cells ( Figure 26B ). The obtained cells are hereinafter referred to as "iPS cell-derived γδ T cells" in this example.

[0112] (15-3) Day 17 of differentiation induction RPMI1640 containing 20% ​​AS401 was used as the basal medium, which was then replaced with a medium supplemented with 1 nM HMBPP (Cayman Chemical, Ann Arbor, MI 13580) and 100 ng / ml IL2 (Reprotech, 200-02). Half of the medium was then replaced every three days.

[0113] (15-4) Day 24 of differentiation induction (Day 24) Furthermore, the expression of CD3 / CD7 was evaluated by flow cytometry for cells on day 24 of differentiation induction (Figure 26C). iPS cell-derived γδ T cells were obtained even when the γδ T cell stimulation step was shortened.

[0114] (Example 16) Method for activating iPS cell-derived γδ T cells without using feeder cells In this example, a method for activating iPS cell-derived γδ T cells without using feeder cells or serum was investigated.

[0115] A. The same treatments as in (15-1) and (15-3) of Example 15 were performed, and the cells were cultured under conditions without the use of feeder cells or serum. However, for cells on day 17 of differentiation induction, the same conditions as in (15-3) of Example 15 were used, except that IL-2 in (15-3) was replaced with IL-15. On day 33 or 37 of differentiation induction, CD3 / γδTCR expression was evaluated by flow cytometry to determine whether iPS cell-derived γδT cells could be generated more effectively. CD3 + / TCR +The cells were detected and differentiation into TCR cells was confirmed, confirming that they were iPS cell-derived γδ T cells (Figure 27A). It was confirmed that iPS cell-derived γδ T cells could be generated using either IL-2 or IL-15 in the γδ T cell stimulation process. Furthermore, the addition of IL-15 yielded more iPS cell-derived γδ T cells than IL-2. B. In the γδ T cell stimulation step of the above A, differentiation induction treatment was performed using IL-15, and the possibility of generating iPS cell-derived γδ T cells was examined with or without the addition of HMBPP. The expression of CD3 / CD7 was evaluated by flow cytometry for cells on day 23 of differentiation induction. CD3 + / TCR + The cells were detected, and differentiation into TCR cells was confirmed, confirming that they were iPS cell-derived γδ T cells. iPS cell-derived γδ T cells were obtained even without the addition of HMBPP, a γδ TCR stimulant (Figure 27B).

[0116] (Example 17) Cytotoxic activity of iPS cell-derived γδ T cells after freezing and thawing In this example, iPS cell-derived γδ T cells were frozen and thawed under conditions without feeder cells or serum, and a cytotoxicity assay was performed.

[0117] (17-1) The same treatments as in (15-1) and (15-3) of Example 15 were carried out, and the cells were cultured under conditions without the use of feeder cells or serum. However, IL-2 in (15-3) of Example 15 was replaced with IL-15. On day 24 of differentiation induction, the cells were frozen using CS10 (Cosmo Bio).

[0118] (17-2) Evaluation of cells on day 24 of differentiation induction (Day 24) The frozen cells were thawed after 2 weeks and subjected to a cytotoxicity assay against Jurkat cells. The E:T (effector:target) ratio was 2:1, and 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 24 of differentiation induction. 5iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed for many Jurkat cells (CFSE positive cells) (Figure 28). This indicates that iPS cell-derived γδ T cells retain cytotoxic function even after freeze-thawing.

[0119] Example 18: Differentiation induction of iPS cell-derived blood progenitor cells after freezing and thawing In this example, iPS cell-derived blood progenitor cells were frozen and thawed, and then induced to differentiate to produce γδ T cells.

[0120] (18-1) Evaluation of cells on day 10 of differentiation induction (Day 10) The same treatments as in (1-1) to (1-6) shown in Example 1 were carried out, and the cells on day 10 after differentiation induction were evaluated by flow cytometry, confirming that they were at the stage of blood cell precursor cells (FIG. 29A).

[0121] (18-2) Day 10 of differentiation induction (Day 10) The cells were frozen using CS10 (Cosmo Bio) and thawed about one year later. After thawing, differentiation was induced using the same treatment as in Example 9 (9-1) using feeder cells.

[0122] (18-3) Evaluation of cells on day 37 of differentiation induction (Day 37) 1 On day 37 of differentiation induction (the differentiation induction culture period including before and after freezing was 37 days), CD3 / γδTCR expression was evaluated by flow cytometry. + / TCR + Since γδ T cells were detected, they were confirmed to be iPS cell-derived γδ T cells (Figure 29B). Furthermore, a cytotoxicity assay against Jurkat cells was performed on the cells on day 37 of differentiation induction. At an E:T (effector:target) ratio of 2:1, 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were cultured. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 24 of differentiation induction. 5iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed in many Jurkat cells (CFSE positive cells) (Figure 29C). This indicates that iPS cell-derived γδ T cells retain cytotoxic function even after freeze-thawing.

[0123] Example 19: Differentiation induction of iPS cell-derived blood progenitor cells after freezing and thawing without the use of feeder cells or serum In this example, iPS cell-derived blood progenitor cells were frozen and thawed, followed by differentiation induction without the use of feeder cells or serum to produce γδ T cells. In this example, differentiation induction was carried out according to the protocol shown in Figure 30A, as follows. In this example, freezing was carried out for 18 days.

[0124] (19-1) Up to day 8 of differentiation induction, the same treatments as in (1-1) to (1-6) shown in Example 1 were carried out. On day 10 of differentiation induction, the cells were frozen using CS10 (Cosmo Bio) and thawed 18 days later.

[0125] (19-2) Day 10 of differentiation induction (Day 10) The thawed cells were cultured in a 48-well culture dish coated with VCAM1 and DLL4, and each well contained StemSpan TM 1.2 × 10 cells were cultured in the Lymphoid Progenitor Expansion Medium included in the T cell generation kit (Stem Cell Technologies) supplemented with DKK1 (final concentration: 30 ng / ml) and azelaic acid (AZA) (final concentration: 5 mM) on the 10th day after differentiation induction. 4 A suspension of 5 μg / ml VCAM1 and 10 μg / ml DLL4 in PBS(-) was added at 100 μl per well to a commercially available 48-well culture dish (non-treated cell culture), and the dish was left to stand overnight at 4°C. The solution was then removed and the dish was washed once with PBS(-) to prepare a VCAM1- and DLL4-coated culture dish.

[0126] (19-3) After the medium change, use StemSpan. TM The procedure was carried out according to the kit's protocol. Specifically, on day 13 of differentiation induction, 250 μl of medium was added.

[0127] (19-4) Day 17 of differentiation induction On the 17th day of differentiation induction, IL-2 in Example 15 (15-3) was replaced with IL-15, and the same procedure as in Example 15 (15-3) was performed, without the use of feeder cells or serum, to produce γδ T cells.

[0128] (19-5) Evaluation of cells on day 17 of differentiation induction (Day 17) On day 17 of differentiation induction (the differentiation induction culture period including before and after freezing was 17 days), CD3 / γδTCR expression was evaluated by flow cytometry. + / TCR + Since the cells were detected, they were confirmed to be iPS cell-derived γδ T cells (FIG. 30B).

[0129] (19-6) Evaluation of cells on day 24 of differentiation induction (Day 24) A cytotoxicity assay against Jurkat cells was performed on the cells on day 24 of differentiation induction (the differentiation induction culture period including before and after freezing was 24 days). At an E:T (effector:target) ratio of 2:1, 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 24 of differentiation induction. 5 iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed in many Jurkat cells (CFSE positive cells) (Figure 30C).

[0130] (Example 20) Induction of differentiation from blood cell progenitor cells under hypoxic conditions In this example, neither feeder cells nor serum were used. However, differentiation induction from blood progenitor cells was performed under hypoxic conditions to produce γδ T cells. In this example, differentiation induction was performed according to the protocol shown in Figure 31A, as follows.

[0131] (20-1) The same treatments as in (4-1) and (4-2) of Example 4 were carried out. (20-2) However, in cells on the 10th day after differentiation induction, the StemSpan shown in (4-2) of Example 4 was used. TM The cells were cultured in the Lymphoid Progenitor Expansion Medium included in the T cell generation kit (Stem Cell Technologies) supplemented with DKK1 (final concentration: 30 ng / ml) and azelaic acid (AZA) (final concentration: 5 mM) without feeder cells or serum, and the O2 concentration was changed from 20% to 5%. (20-3) After the medium change, use StemSpan TM The procedure was carried out according to the kit's protocol. Specifically, on day 13 of differentiation induction, 250 μl of the medium shown in (20-2) was added.

[0132] (20-4) Evaluation of cells on day 17 of differentiation induction (Day 17) On day 17 of differentiation induction, CD3 / D7 expression was assessed by flow cytometry (Figure 31B). + / CD7 + The cells were detected, confirming that they were iPS cell-derived γδ T cells. Compared to 20% O2, the proportion and absolute number of iPS cell-derived γδ T cells were higher under hypoxic (5% O2) conditions.

[0133] (20-5) Day 17 of differentiation induction Culture was carried out in the same manner as in Example 19 (19-4), except that the O2 concentration was changed from 20% to 5%.

[0134] (20-6) Day 29 of differentiation induction On day 29 of differentiation induction, cells were subjected to a cytotoxicity assay against Jurkat cells. At an E:T (effector:target) ratio of 2:1, 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 29 of differentiation induction. 5 iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed in many Jurkat cells (CFSE positive cells) (Figure 31C). Thus, cytotoxic activity was more effective under hypoxic conditions than under normoxic conditions.

[0135] Example 21: Differentiation induction from iPS cells under culture conditions that do not contain animal-derived components In this example, iPS cell-derived γδ T cells were produced under medium conditions that did not contain animal-derived components.

[0136] (21-1) The same treatments as in (4-1) and (4-2) of Example 4 were carried out. (21-2) However, on day 10 of differentiation induction, γδ T cells were produced by the same method as in (4-2) of Example 4, except that, instead of using feeder cells, the basal medium shown in Table 6 of Example 2 was replaced with 20% FBS / αMEM for 20% AS401 / RPMI1640 (a medium condition that does not contain animal-derived components) and this was used instead of the Lymphoid Progenitor Expansion Medium shown in (4-2) of Example 4. On day 13 of differentiation induction, 250 μl of medium was added.

[0137] (21-3) Evaluation of cells on day 17 of differentiation induction (Day 17) On day 17 of differentiation induction, the expression of CD3 / CD7 was assessed by flow cytometry. + / CD7 + Since the cells were detected, they were confirmed to be iPS cell-derived γδ T cells (FIG. 32A).

[0138] (21-4) Day 17 of differentiation induction On the 17th day after differentiation induction, the basal medium 20% FBS / αMEM in Table 7 of Example 2 (2-4) was replaced with 20% AS401 / RPMI1640, and IL-2 was replaced with IL-15, and differentiation was induced and cultured using the same method as in Example 2 (2-4).

[0139] (21-5) Day 31 of differentiation induction (Day 31) On day 31 of differentiation induction, cells were subjected to a cytotoxicity assay against Jurkat cells. At an E:T (effector:target) ratio of 2:1, 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 31 of differentiation induction. 5 iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D). Cell death (7-AAD positive) was confirmed in many Jurkat cells (CFSE positive cells) (Figure 32B). Significant cytotoxic activity was confirmed.

[0140] (Example 22) Identification of undifferentiated cells relative to iPS cell-derived γδ T cells In this example, the experiment was carried out under conditions in which neither feeder cells nor serum were used. In this example, undifferentiated cells were confirmed for iPS cell-derived γδ T cells.

[0141] (22-1) The same treatments as in (4-1) and (4-2) of Example 4 were carried out. (22-2) However, in cells on the 10th day after differentiation induction, the StemSpan shown in (4-2) of Example 4 was used. TM The culture was carried out in the Lymphoid Progenitor Expansion Medium included in the T cell generation kit (Stem Cell Technologies), supplemented with DKK1 at a final concentration of 30 ng / ml and azelaic acid (AZA) at a final concentration of 5 mM, without the use of feeder cells or serum.

[0142] (22-3) After that, the medium was replaced with StemSpan.TM The procedure was carried out according to the kit's protocol. Specifically, on day 13 of differentiation induction, an additional 250 μl of medium was added, and from day 17 onward, half of the medium was replaced twice a week with RPMI 1640 containing 20% ​​AS401 as the basal medium, supplemented with 1 nM HMBPP (Cayman Chemical, Ann Arbor, MI 13580) and 100 ng / ml IL-15.

[0143] (22-4) Evaluation of cell populations on day 35 of differentiation induction (Day 35) 1 The expression of the undifferentiated marker TRA-1-85 in the cell population differentiated without feeder cells or serum was evaluated by flow cytometry. It was confirmed that the cell population on day 35 contained no TRA-1-85-positive cells (Figure 33A).

[0144] (22-5) Cell population 2 on day 35 of differentiation induction (Day 35) The protocol for confirming the appearance of colonies of undifferentiated cells using a cell population on day 35 differentiated under conditions without feeder cells or serum is shown (Figure 33B). 4 The iPS-derived γδ T cell population was seeded under the maintenance culture conditions for undifferentiated iPS cells (Example 1 (1-1)), and the appearance of colonies of undifferentiated cells was examined. 2 Undifferentiated iPS cells were mixed. After 11 days, alkaline phosphatase staining (AP staining) was performed. Colonies of undifferentiated cells stain red with AP staining. In the positive control condition where iPS cells were added, many AP-positive colonies were observed, whereas in the cell population after differentiation induction where no iPS cells were added, not a single AP-positive colony was observed (Figure 33C).

[0145] (Example 23) Cytotoxicity assay of CD3 / γδ T-positive cells In this example, CD3 / γδT positive cells were purified from the cell population obtained by the same treatment as in Example 22, and a cytotoxicity assay was carried out.

[0146] On day 35 of differentiation induction, cells were evaluated by flow cytometry before and after FACS (Figure 34A). CD3 / γδTCR (gdTCR)-positive cells were detected, confirming successful purification.

[0147] The purified cells were subjected to a cytotoxicity assay against Jurkat cells. At an E:T (effector:target) ratio of 0.2:1, 5 × 10 Jurkat cells stained with the fluorescent dye CFSE were used. 4 Add 1 x 10 cells to one well of a 96-well culture dish on day 35 of differentiation induction. 5 iPS cell-derived γδ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D) and graphed (Figure 34B). Despite the E:T ratio of 0.2:1, which represents a very low number of effector cells against tumor cells, the cells exhibited strong cytotoxic activity. Previously, cytotoxicity assays performed using unpurified cell populations revealed that the target cells possessed cytotoxic activity: CD3 / γδ T-positive cells (i.e., γδ T cells). [Industrial Applicability]

[0148] As described above in detail, the method for producing iPS cell-derived γδ T cells of the present invention allows for effective production of γδ T cells without burdening the recipient and without being affected by cell exhaustion. Furthermore, the production method of the present invention allows for the production of excellent iPS cell-derived γδ T cells even without the use of feeder cells. Furthermore, the production method of the present invention allows for the production of excellent iPS cell-derived γδ T cells even without the use of feeder cells or serum, or even with a medium containing no animal-derived components. Furthermore, the production method of the present invention allows for the production of excellent iPS cell-derived γδ T cells even if the cells are frozen and thawed during production.

[0149] The iPS cell-derived γδ T cells of the present invention overcome the problems of peripheral blood γδ T cells not being able to ensure sufficient purity and cell numbers for therapy, and the burden on the recipient of blood collection when large amounts of blood are drawn to ensure sufficient purity and cell numbers for therapy. Furthermore, the method of ex vivo expansion of γδ T cells isolated from peripheral blood overcomes the problems of difficulty in obtaining sufficient cell numbers and insufficient expansion and activation due to cell exhaustion, making it extremely useful. The γδ T cell populations generated by the present methods are more homogeneous and effective than γδ T cell populations isolated from peripheral blood, and exhibit superior functionality, including antigen-specific cytotoxic activity in a more effective, non-MHC-restricted manner. Furthermore, the γδ T cell populations generated by the present methods are free of residual undifferentiated cells, making them suitable for clinical applications.

Claims

1. iPS cell-derived gamma delta T cells are T cells derived from induced pluripotent stem cells (iPS cells), characterized in that the T cells have antigen-specific cytotoxic activity in an MHC-nonrestricted manner.

2. The iPS cell-derived γδ T cells according to claim 1, wherein the iPS cells are not derived from αβ T cells.

3. The iPS cell-derived γδ T cell according to claim 1 or 2, wherein the iPS cell is an iPS cell having a γδ TCR rearrangement gene.

4. iPS cell-derived γδ T cells were generated by inducing differentiation of iPS cells carrying γδ TCR rearrangements.

5. A method for producing iPS cell-derived γδ T cells, comprising the step of culturing blood progenitor cells obtained by differentiation induction treatment of iPS cells having a γδ TCR rearrangement gene in a medium containing a basal medium supplemented with one or more selected from FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), IL-2, IL-7, TPO (thrombopoietin), and L-ascorbic acid.

6. 6. The method for producing iPS cell-derived γδ T cells according to claim 5, comprising the steps of culturing the cells in a medium containing a γδ T cell stimulator after culturing the cells in a medium containing a basal medium to which one or more members selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid have been added.

7. 7. The method for generating iPS cell-derived γδ T cells according to claim 5 or 6, wherein the step of culturing the iPS cell-derived γδ T cells in a medium containing one or more selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid in a basal medium is a step of culturing the iPS cell-derived γδ T cells in co-culture with feeder cells.

8. 7. The method for generating iPS cell-derived γδ T cells according to claim 5 or 6, wherein the step of culturing the cells in a medium containing a basal medium to which one or more members selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid are selected is a step of culturing the cells without co-culturing them with feeder cells.

9. 9. The method for generating iPS cell-derived γδ T cells according to claim 8, wherein the step of culturing the cells without co-culturing with feeder cells comprises a step of culturing the cells using a culture substrate coated with VCAM1 (vascular cell adhesion molecule-1), and DLL4 (Delta-Like Protein 4) or DLL1 (Delta-Like Protein 1).

10. The method for producing iPS cell-derived γδ T cells according to claim 8 or 9, wherein the step of culturing without co-culturing with feeder cells further comprises a step of culturing using a medium containing DKK1 and / or AZA (Azelaic acid).

11. The method for producing iPS cell-derived γδ T cells according to any one of claims 6 to 10, wherein the medium containing a γδ T cell stimulator is a medium containing one or more species selected from the group consisting of a γδ T cell stimulator, IL-2, and IL-15.

12. The method for producing iPS cell-derived γδ T cells according to any one of claims 6 to 11, wherein the γδ T cell stimulator is a phosphate compound or a derivative thereof, which is a metabolite in the isoprenoid biosynthetic pathway, or a specific inhibitor of FPP (farnesyl pyrophosphate) synthase, which is a rate-limiting enzyme in the isoprenoid biosynthetic pathway.

13. The method for producing iPS cell-derived γδ T cells according to any one of claims 6 to 12, characterized in that the cells are cultured under serum-free conditions.

14. A method for producing iPS cell-derived γδ T cells according to any one of claims 6 to 13, comprising culturing the cells under hypoxic conditions.

15. iPS cell-derived γδ T cells prepared by the method for preparing iPS cell-derived γδ T cells according to any one of claims 5 to 14.

16. A cell population comprising iPS cell-derived γδ T cells according to any one of claims 1 to 4 and claim 15.

17. The cell population described in claim 16, characterized in that the cell population containing iPS cell-derived γδ T cells has high antigen-specific cytotoxic activity compared to a cell population of γδ T cells isolated from peripheral blood.

18. A cell population of γδ T cells, characterized in that γδ T cells having the same base sequence in the CDR3 region of the TCR gene account for 90% or more of the γδ T cells constituting the cell population.

19. 1 × 10 γδ T cells 5 The cell population according to claim 18, characterized in that it comprises more than one cell.

20. A cell population of γδ T cells, characterized in that γδ T cells that express higher levels of CD7 and / or CD8a than γδ T cells isolated from peripheral blood account for 90% or more of the γδ T cells constituting the cell population.

21. The cell population according to any one of claims 18 to 20, which comprises γδ T cells, wherein undifferentiated cells account for 10% or less of the γδ T cells constituting the cell population.

22. An antigen-specific cellular immunotherapy agent comprising the iPS cell-derived γδ T cells according to any one of claims 1 to 4 and claim 15 as an active ingredient.

23. 16. A method for culturing iPS cell-derived γδ T cells according to any one of claims 1 to 4 and 15, characterized in that the cells are cultured in a liquid medium containing a beaded carrier.

24. A therapeutic agent for diseases such as cancer, infectious diseases, and autoimmune disorders, comprising the iPS cell-derived γδ T cells according to any one of claims 1 to 4 and claim 15 as an active ingredient.

25. A pharmaceutical composition comprising the iPS cell-derived γδ T cells according to any one of claims 1 to 4 and claim 15 as an active ingredient.

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