WO2022168959A1 - 人工多能性幹細胞由来γδＴ細胞及びその作製方法 - Google Patents

人工多能性幹細胞由来γδＴ細胞及びその作製方法 Download PDF

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WO2022168959A1
WO2022168959A1 PCT/JP2022/004542 JP2022004542W WO2022168959A1 WO 2022168959 A1 WO2022168959 A1 WO 2022168959A1 JP 2022004542 W JP2022004542 W JP 2022004542W WO 2022168959 A1 WO2022168959 A1 WO 2022168959A1
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cells
cell
γδt
derived
ips
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French (fr)
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貴之 青井
信幸 村井
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Kobe University NUC
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Kobe University NUC
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Priority to CA3206400A priority Critical patent/CA3206400A1/en
Priority to US18/274,725 priority patent/US20240100093A1/en
Priority to CN202280013673.3A priority patent/CN116964194A/zh
Priority to JP2022579629A priority patent/JP7793209B2/ja
Priority to EP22749830.0A priority patent/EP4289940A4/en
Priority to KR1020237025924A priority patent/KR20230128324A/ko
Publication of WO2022168959A1 publication Critical patent/WO2022168959A1/ja
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Priority to US19/019,930 priority patent/US12576149B2/en
Priority to JP2025124240A priority patent/JP2025163076A/ja
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Definitions

  • the present invention relates to ⁇ T cells derived from induced pluripotent stem cells (iPS cells) and methods for producing them. Specifically, the present invention relates to iPS cell-derived ⁇ T cells that act in an MHC-unrestricted manner and a method for producing the same. Furthermore, it relates to a cell population containing the produced iPS cell-derived ⁇ T cells.
  • iPS cells induced pluripotent stem cells
  • ⁇ T cells Human mature T cells are roughly divided into two groups: ⁇ T cells whose T cell receptors are composed of ⁇ and ⁇ chains, and ⁇ T cells whose T cell receptors are composed of ⁇ and ⁇ chains.
  • ⁇ T cells are extremely diverse, and while the types of cells that can be attacked by one type of ⁇ T cells are MHC-restricted and few, one type of ⁇ T cells is MHC-unrestricted and many ⁇ T cells can attack. It is known to attack different types of cancer cells.
  • ⁇ T cells are a type of T cell receptor (TCR) that recognizes and directly kills many types of cancer cells.
  • ⁇ T cells are usually present in only 1-5% of peripheral blood, even if a small amount of blood is collected to activate and/or proliferate ⁇ T cells, sufficient purity and cell numbers for treatment should be ensured. There is a problem that it is not possible to In addition, there is also the problem that if the amount of blood collected from a patient is increased in order to ensure sufficient purity and cell count for treatment, the patient will suffer a great burden. ⁇ T cells isolated from the patient's peripheral blood have already been treated by in vitro expansion and infusion into the patient. No activation was obtained.
  • Patent Document 1 A method for producing iPS cells ( ⁇ TCR-type iPS cells) having a ⁇ TCR rearrangement gene is disclosed (Patent Document 1, Non-Patent Document 1).
  • Patent Document 1 and Non-Patent Document 1 further disclose that ⁇ TCR-type iPS cells were induced to differentiate into blood cell progenitor cells. However, it has not been disclosed that the hematopoietic progenitor cells were further induced to differentiate into T cells.
  • Patent Document 2 A method for inducing the differentiation of T cell-derived iPS cells into T cells is disclosed (Patent Document 2).
  • Patent Document 3 A method for inducing the differentiation of T cell-derived iPS cells into T cells.
  • T cells with the same rearrangement as the original cells can be obtained.
  • all of them are reports on ⁇ T cells, and ⁇ T cells are not disclosed. Since all of the ⁇ T cells have a specific ⁇ TCR, there are few types of cancers that express the antigen, and they are MHC-restricted, which limits the number of patients who can be treated. .
  • Non-Patent Document 4 T cells induced to differentiate from stem cells such as ES cells or iPS cells exhibit a ⁇ T cell-like phenotype.
  • T cells shown in the above literature have a gene expression pattern that resembles the phenotype characteristic of ⁇ T, they actually express ⁇ T cell receptors, thereby recognizing antigens and damaging target cells. That is, it cannot be said that they are ⁇ T cells.
  • a method for effectively preparing T cells capable of attacking various types of cancer cells in an MHC-unrestricted manner is desired.
  • ⁇ T cells normally exist in only 1-5% of peripheral blood, there was the problem of not being able to secure sufficient purity and cell numbers for treatment. In addition, there is also a problem that if a large amount of blood is collected in order to ensure sufficient purity and cell count for treatment, a great burden is placed on the person receiving the blood. In vitro expansion of ⁇ T cells isolated from peripheral blood was difficult to obtain, and sufficient expansion and activation were not achieved due to cell exhaustion.
  • An object of the present invention is to effectively produce and provide ⁇ T cells. More specifically, the object is to provide excellent ⁇ T cells that are homogenous ⁇ T cells and are not affected by cell exhaustion.
  • the present inventors focused on iPS cells and extensively studied differentiation-inducing treatment methods. We have completed the present invention.
  • the present invention consists of the following.
  • An iPS cell-derived ⁇ T cell which is an induced pluripotent stem cell (iPS cell)-derived T cell, wherein the T cell has antigen-specific cytotoxic activity in an MHC-unrestricted manner.
  • the iPS cell-derived ⁇ T cell according to the preceding item 1 wherein the iPS cell is an iPS cell not derived from ⁇ T cell.
  • 3. The iPS cell-derived ⁇ T cell according to the preceding item 1 or 2, wherein the iPS cell is an iPS cell having a ⁇ TCR rearrangement gene.
  • Blood cell progenitor cells obtained by differentiation induction treatment of iPS cells with ⁇ TCR reconstruction gene were added to the basal medium with FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), IL-2, IL-7, TPO (thrombopoietin ), a method for producing iPS cell-derived ⁇ T cells, comprising the step of culturing using a medium to which one or more selected from L-ascorbic acid is added. 6.
  • a ⁇ T cell stimulating agent 6 After the step of culturing using a medium in which one or more selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid is added to a basal medium, a ⁇ T cell stimulating agent 6.
  • the step of culturing using a medium in which one or more selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid is added to a basal medium is coculturing with feeder cells 7.
  • the step of culturing without co-culturing with feeder cells is VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or DLL1 (Delta-Like Protein 1) using a culture substrate coated with 9.
  • the method for producing iPS cell-derived ⁇ T cells according to the preceding item 8 comprising the step of culturing. 10.
  • the method for producing iPS cell-derived ⁇ T cells according to the preceding item 8 or 9, wherein the step of culturing without coculturing with feeder cells further comprises a step of culturing using a medium containing DKK1 and/or AZA (Azelaic acid). . 11. 11.
  • the iPS cell-derived ⁇ T according to any one of 6 to 10 above, wherein the medium containing the ⁇ T cell stimulating agent is a medium containing one or more selected from a ⁇ T cell stimulating agent, IL-2 and IL-15.
  • a method for making cells 12. 6 to 11 above, wherein the ⁇ T cell stimulating agent is a phosphoric acid compound or derivative thereof that is an isoprenoid biosynthetic pathway metabolite, or a specific inhibitor of FPP (farnesyl pyrophosphate) synthase that is the rate-limiting enzyme in the isoprenoid biosynthetic pathway. 3.
  • the method for producing iPS cell-derived ⁇ T cells according to any one of 1. 13. 13.
  • the cell population according to 16 above wherein the cell population containing iPS cell-derived ⁇ T cells has high antigen-specific cytotoxic activity compared to the cell population of ⁇ T cells isolated from peripheral blood. 18.
  • a cell population containing ⁇ T cells, characterized in that ⁇ T cells having the same nucleotide sequence in the CDR3 region of the TCR gene comprise 90% or more of the ⁇ T cells constituting the cell population. group. 19.
  • 20. A cell population containing ⁇ T cells, wherein 90% or more of the ⁇ T cells constituting the cell population are ⁇ T cells that exhibit higher expression levels of CD7 and CD8a than ⁇ T cells isolated from peripheral blood.
  • 21. The cell population according to any one of the preceding items 18 to 20, which is a cell population containing ⁇ T cells, wherein undifferentiated cells account for 10% or less of the ⁇ T cells constituting the cell population.
  • 22. An antigen-specific cellular immunotherapeutic agent comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
  • 23. 16 The method for culturing iPS cell-derived ⁇ T cells according to any one of the above items 1 to 4 and 15, wherein the culture is performed using a medium containing a bead-like carrier in a liquid medium. 24.
  • a therapeutic agent for diseases such as cancer, infectious diseases, and autoimmune disorders comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
  • a pharmaceutical composition comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
  • An antigen-specific cell-mediated immune cell therapy method comprising administering the iPS cell-derived ⁇ T cells selected from 1 to 4 and 15 above.
  • ⁇ T cells can be effectively produced without burdening the blood recipient and without being affected by cell exhaustion. Furthermore, according to the method for producing iPS cell-derived ⁇ T cells of the present invention, excellent ⁇ 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 ⁇ T cells of the present invention have excellent functions of non-MHC-restricted antigen-specific cytotoxic activity, and are homogeneous and more effective ⁇ T cell populations than ⁇ T cells isolated from peripheral blood. was made.
  • FIG. 1A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
  • FIG. 1B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 31 of induction of differentiation.
  • FIG. 2A shows the results of flow cytometry evaluation of the expression of CD7 (T cell differentiation marker) in cells on day 17 of induction of differentiation.
  • FIG. 2B shows the results of evaluating the expression of CD3/ ⁇ TCR/CD45RA in cells on day 54 of differentiation induction by flow cytometry.
  • FIG. 3A shows the results of evaluation of CD7 expression by flow cytometry on cells on day 17 of induction of differentiation.
  • FIG. 1A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
  • FIG. 1B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 31 of induction of differentiation.
  • FIG. 2A shows the results of flow cytometry evaluation of
  • FIG. 3B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 55 of induction of differentiation.
  • FIG. 3C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 55 of induction of differentiation.
  • Fig. 2 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
  • Example 4) 3 shows the results of evaluation by flow cytometry for the expression of CD3/ ⁇ TCR in cells on days 33, 35, and 37 of induction of differentiation under conditions in which feeder cells were not used.
  • Fig. 2 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
  • FIG. 5 shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 55 of induction of differentiation.
  • FIG. 7A shows the results of observation of cells on day 37 of differentiation induction using a phase-contrast microscope.
  • FIG. 7B shows the results of evaluating CD3/ ⁇ TCR expression by flow cytometry.
  • Example 5 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
  • Example 6 Shown are the results of phase-contrast microscopy observation of cells on day 32 of induction of differentiation when cultured in each medium without feeder cells.
  • Example 3 shows that cells on day 35 of induction of differentiation have cytotoxicity against Jurkat cells when cultured in each medium without feeder cells.
  • Example 6 The results of confirming the cytotoxic activity of the cells on day 35 of induction of differentiation with Jurkat cells 1 day and 4 days after the start of mixed culture when cultured in each medium without feeder cells are shown.
  • Example 6) 24 shows the results of flow cytometry evaluation of the expression of CD7, a T cell differentiation marker, in cells on the 24th day of induction of differentiation under conditions in which feeder cells were not used.
  • Example 7 In the condition that feeder cells are not used, instead of coating the culture dish, magnetic beads coated with VCAM1 and DLL4 are co-cultured to induce differentiation into T cells.
  • FIG. 10 shows a protocol for inducing differentiation of ⁇ T cells produced in Example 9 from iPS cells.
  • FIG. 16A shows the results of phase-contrast microscopy observation of the shape of cells in the process of differentiation.
  • FIG. 16B shows the results of confirmation of cell surface markers by flow cytometry for cells in the process of differentiation.
  • Fig. 3 shows the results of confirming the antitumor activity against various tumor cells of ⁇ T cells on day 38 of induction of differentiation.
  • FIG. 17A shows the results of confirming the cytotoxic activity against Jurkat cells.
  • FIG. 17B shows the results of confirming the cytotoxic activity against Huh-7 cells.
  • FIG. 17C shows the results of confirming the cytotoxic activity against SW480 cells.
  • FIG. 17D shows the viability of iPS cell-derived ⁇ T cells (E) and Jurkat cells (T) in mixed culture when the E:T ratio was changed stepwise.
  • Fig. 3 shows the results of confirming the retention of TCR reconstitution and the cytotoxic mechanism of ⁇ T cells on day 36 of differentiation induction.
  • FIG. 18A shows the results of evaluating the expression of ⁇ TCR on the cell surface of unpurified ⁇ T cells (igdT) and peripheral blood mononuclear cells (PB).
  • FIG. 18B shows the results of confirming the rearrangement of TCR genes (V ⁇ 9, V ⁇ 2) by genomic PCR.
  • FIG. 18C shows the results confirming that ⁇ T cells express GranzymeB and Perforin.
  • FIG. 18D shows the results of confirming the cytotoxic activity of purified ⁇ T cells (igdT). There was no significant difference in the dead cell rate between ⁇ T cells with and without purification.
  • Fig. 2 shows results of confirming gene expression patterns in iPS cell-derived ⁇ T cells and ⁇ T cells isolated from peripheral blood by single-cell RNA-seq analysis.
  • FIG. 1 shows the results of analysis by flow cytometry for CD25 among the cell surface expression markers for ⁇ T cells derived from iPS cells and ⁇ T cells isolated from peripheral blood.
  • FIG. 1 shows a protocol for inducing differentiation of ⁇ T cells from iPS cells to confirm the activation method of iPS cell-derived ⁇ T cells.
  • Fig. 2 shows the results of investigations on IL-2 and/or IL-15 regarding the method of activating iPS cell-derived ⁇ T cells.
  • FIG. 22A shows the results of confirming the number of viable cells
  • FIG. 22B shows the results of evaluating CD3 + / ⁇ TCR + cells by flow cytometry.
  • ⁇ T cells obtained by inducing differentiation from the ⁇ T cell-derived iPS cell line 121-3 are shown.
  • FIG. 23A shows the results of genomic PCR confirming the rearrangement of TCR genes (V ⁇ 9, V ⁇ 2) in undifferentiated iPS cells (121-3 strain) and ⁇ T cells obtained by differentiation induction therefrom.
  • FIG. 23B shows the results of confirming the sequences of TCR ⁇ and TCR ⁇ of ⁇ T cells obtained by expanding and culturing ⁇ T cells and peripheral blood mononuclear cells using a next-generation sequencer.
  • ⁇ T cells derived from iPS cells on day 39 of differentiation induction or ⁇ T cells obtained by expanding and culturing peripheral blood mononuclear cells were co-cultured with Jurkat cells for 4 hours, and then the expression of IFN ⁇ was evaluated by flow cytometry. show.
  • Example 13 A cell population containing ⁇ T cells derived from iPS cells on day 40 of differentiation induction obtained by differentiation induction using a feeder cell and a cell population containing ⁇ T cells obtained by expanding and culturing peripheral blood mononuclear cells (CD3 It shows the results of flow cytometry evaluation of the expression of various surface markers in TCR ⁇ 9-positive or TCR ⁇ 9-positive).
  • FIG. 26A shows a protocol in which the step of stimulating ⁇ T cells is performed from day 17.
  • FIG. 26B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 17 of induction of differentiation.
  • FIG. 26C shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 24 of induction of differentiation.
  • FIG. 1 shows the results of investigations of IL-2 or IL-15, or IL15 or IL-15+HMBPP regarding the method of activating iPS cell-derived ⁇ T cells.
  • FIG. 27A shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 37 or 33 of induction of differentiation.
  • FIG. 27B shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 23 of induction of differentiation.
  • 24 shows the results of confirming the cytotoxic activity against Jurkat cells after freezing and thawing the cells on the 24th day of induction of differentiation under the condition that no feeder cells were used.
  • FIG. 17 shows the results of confirming the cytotoxic activity against Jurkat cells after freezing and thawing the cells on the 24th day of induction of differentiation under the condition that no feeder cells were used.
  • FIG. 29A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
  • FIG. 29B shows the results of freezing and thawing the cells on day 10 of differentiation induction, and evaluating the expression of CD3/ ⁇ TCR on the cells on day 37 of differentiation induction by flow cytometry.
  • FIG. 29C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 37 of induction of differentiation. (Example 18)
  • FIG. 30A shows a protocol for inducing differentiation under serum-free conditions without using feeder cells after freezing and thawing iPS cell-derived progenitor cells.
  • FIG. 30B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 17 of induction of differentiation.
  • FIG. 31A shows a protocol for inducing the differentiation of blood cell progenitor cells into ⁇ T cells under hypoxic conditions.
  • FIG. 31B shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 17 of induction of differentiation.
  • FIG. 31C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 29 of induction of differentiation.
  • Fig. 10 shows that iPS cell-derived ⁇ T cells were induced to differentiate under animal-derived component-free conditions.
  • FIG. 10 shows that iPS cell-derived ⁇ T cells were induced to differentiate under animal-derived component-free conditions.
  • FIG. 32A shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 17 of induction of differentiation.
  • FIG. 32B shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 31 of induction of differentiation. (Example 21) It shows the absence of undifferentiated cells in the cell population.
  • FIG. 33A shows the results of flow cytometry evaluation of the expression of the undifferentiated marker TRA-1-85 in the cell population on day 35 of induction of differentiation under serum-free conditions without using feeder cells.
  • FIG. 33B shows a protocol for confirming the appearance of colonies of undifferentiated cells for a cell population.
  • FIG. 33C shows that no colonies of undifferentiated cells appear for the cell population.
  • FIG. 34A shows purification of CD3/ ⁇ T-positive cells from a cell population under serum-free conditions without using feeder cells.
  • FIG. 34B further shows the results of confirming the cytotoxic activity against Jurkat cells of the purified cells.
  • the present invention relates to iPS cell-derived ⁇ T cells, which are iPS cell-derived T cells characterized by having antigen-specific cytotoxic activity in an MHC-unrestricted manner.
  • ⁇ -type T cells whose T cell receptor (TCR) is composed of ⁇ and ⁇ chains
  • TCR T cell receptor
  • ⁇ -type T cells composed of ⁇ and ⁇ chains.
  • TCR T cell receptor
  • ⁇ T cells refers to ⁇ T cells.
  • ⁇ T cells In the blood, ⁇ T cells account for the majority, whereas ⁇ T cells are a minority, accounting for 1-5% of all T cells.
  • ⁇ T cells can be regarded as an element of the adaptive immune system because of the rearrangement of the TCR gene to bind to various antigens and the presence of memory cells. It also has the function of attacking, for example, tumor cells by antigen recognition similar to NK cells of innate immune cells.
  • ⁇ T cells are considered to have both innate and adaptive immune system functions.
  • ⁇ T cell-derived cytotoxic T cells (CTL) against tumor antigens can be said to be an adaptive immune system that requires antigen information from dendritic cells.
  • CTL cytotoxic T cells
  • iPS cells refer to undifferentiated cells established by reprogramming somatic cells by various methods.
  • the iPS cells that are the starting material for the present invention are preferably iPS cells that are not iPS cells having the ⁇ TCR rearrangement gene. Most preferred are iPS cells having a ⁇ TCR rearrangement gene.
  • the iPS cells having the ⁇ TCR-rearranged gene are hereinafter simply referred to as “ ⁇ TCR-type iPS cells”.
  • ⁇ TCR-rearranged gene refers to a TCR-encoding gene in which both the TCRG region and the TCRD region are rearranged.
  • the TCRG region consists of V ⁇ -J ⁇ and the TCRD region consists of V ⁇ -D ⁇ -J ⁇
  • the iPS cells in the present specification can be produced by a method known per se or any method that will be developed in the future. For example, it can be produced based on the descriptions of Patent Document 1 and Non-Patent Document 1.
  • the iPS cells used for producing the ⁇ T cells of the present invention can be produced by a method known per se or any method that will be developed in the future. Specifically, it can be produced by the method described in Patent Document 1 or Non-Patent Document 1, for example. For example, they can be produced by a method for producing iPS cells, including the following steps 1) to 3).
  • IL-2 and bisphosphonates e.g., zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, minodronic acid, their salts and their hydrates stimulating with one or more, preferably zoledronic acid
  • IL-2 and bisphosphonates e.g., zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, minodronic acid, their salts and their hydrates stimulating with one or more, preferably zoledronic acid
  • introducing at least four genes capable of expressing cell reprogramming factors e.g., OCT3/4, SOX2, KLF4 and c-MYC
  • SeV Sendai virus
  • StemFit (R) AK02N (trade name), StemFit (R) AK03N (trade name), ReproStem (trade name), iPSellon (trade name), and Essential 8 (trade name) are basal media that can be used to maintain and culture iPS cells. ), TeSR-E8 (trade name), and various other stem cell maintenance media can be used. Especially preferred is StemFit (R) AK02N (trade name). Substances added to each medium can be appropriately increased or decreased depending on the purpose. Y27632, which is a Rho-Associated Coil Kinase (ROCK) inhibitor, can be used as an example of the substance to be added.
  • ROCK Rho-Associated Coil Kinase
  • a laminin 511-E8 fragment can be used in culture substrates such as culture dishes to promote cell attachment and proliferation.
  • culture substrates such as culture dishes to promote cell attachment and proliferation.
  • iMatrix-511 silk (trade name) and iMatrix-511 (trade name) can be used. Manufacturers and distributors of reagents and the like to be used are not particularly limited as long as they can exhibit equivalent functions.
  • a protease such as trypsin can be used to detach the cells from the culture vessel.
  • TrypLE Select (trade name) can be used.
  • iPS cells are first induced to differentiate into blood cell progenitor cells.
  • iPS cell-derived ⁇ T cells are produced by using cells that have been induced to differentiate from iPS cells into ⁇ T cells as starting materials, and performing a differentiation-inducing treatment step from hemocyte progenitor cells to ⁇ T cells. can be a method. Furthermore, it can be a method for producing iPS cell-derived ⁇ T cells that includes a step of converting iPS cells into blood cell progenitor cells.
  • iPS cells obtained by freezing and thawing iPS cell-derived progenitor cells can also be used in the method of the present invention.
  • the freezing period is not particularly limited, it may be, for example, 2 weeks to 1 year.
  • the iPS cells of the present invention are preferably iPS cells other than iPS cells having the ⁇ TCR rearrangement gene. ⁇ TCR-type iPS cells are most preferred.
  • the process of inducing differentiation from iPS cells to blood cell progenitor cells is not particularly limited, and any process known per se or any process that will be developed in the future can be adopted.
  • the medium contains, for example, FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), BMP4 (bone morphogenetic protein-4), bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), IL-6, IGF-1 (insulin-like growth factors), IL-7, IL-11, EPO (erythropoietin), TPO (thrombopoietin), IL-15, IL-3, etc. It is possible to appropriately select and add one or a plurality of types.
  • FBS fetal bovine serum
  • FCS fetal calf serum
  • the iPS cells of the present invention can be cultured, for example, in the media shown in the following 1-1) to 1-4) and subjected to differentiation induction treatment under conditions that do not use feeder cells.
  • a ROCK inhibitor at a final concentration of 0-50 ⁇ M, preferably 1-30 ⁇ M, more preferably 10 ⁇ M
  • Laminin-511 E8 fragment such as iMatrix-511 (trade name)
  • 0-50 ⁇ l preferably 1 ⁇ 30 ⁇ l , more preferably around 5 ⁇ l
  • the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
  • the number of cells to be seeded can be increased or decreased as appropriate.
  • the reagents and the like to be used are not particularly limited in terms of manufacturers and distributors, as long as they can exhibit equivalent functions. All cultures can be performed at 37 ⁇ 0.5°C and 5% CO2 conditions.
  • a protease such as trypsin, such as TrypLE Select (trade name)
  • TrypLE Select trade name
  • Day 0 of Differentiation Induction StemFit (R) AK02N (trade name) can be used as a basal medium.
  • Further GSK-3 ⁇ / ⁇ inhibitor (CHIR99021, CAS number: 252917-06-9) 0-20 ⁇ M, preferably 0.5-10 ⁇ M, more preferably 4 ⁇ M, BMP4 0-400 ng/ml, preferably 10-200 ng/ ml, more preferably 80 ng/ml, VEGF 0-400 ng/ml, preferably 10-200 ng/ml, more preferably 80 ng/ml.
  • Day 2 of Differentiation Induction Advanced DMEM/F12 (trade name) or Essential 6 (trade name) can be used as a basal medium.
  • Further selective ALK5, 4, 7 inhibitor (SB431542) 0-20 ⁇ M, preferably 0.5-10 ⁇ M, more preferably 2-4 ⁇ M, bFGF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, SCF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml and VEGF 0-400 ng/ml, preferably 10-200 ng/ml, more preferably It can be cultured in a culture system containing 80 ng/ml.
  • L-Glutamine, penicillin/streptomycin, differentiation-inducing supplements for iPS/ES cells for example, StemFit (trade name) For Differentiation: hereinafter "AS401"
  • AS401 StemFit
  • the optimum addition amount can be determined as appropriate.
  • Day 4 of Differentiation Induction Advanced DMEM/F12 (trade name) or StemPro-34 SFM (trade name) can be used as a basal medium.
  • Days 6 to 8 of Differentiation Induction Advanced DMEM/F12 (trade name) or StemPro-34 SFM (trade name) can be used as a basal medium.
  • L-Glutamine 0-50 mM, preferably 1-20 mM, more preferably 2 mM, IL-3 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, IL -6 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, SCF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml and EPO 0-100 IU/ml, preferably 1-50 IU/ml, more preferably 10 IU/ml.
  • penicillin/streptomycin, differentiation-inducing supplements for iPS/ES cells for example, AS401
  • the optimum addition amount can be determined
  • Feeder cells can be co-cultured when iPS cells are cultured or iPS cells are subjected to differentiation induction treatment.
  • feeder cells for example, MEF (mouse embryonic fibroblast), OP9, OP9/DLL1, OP9-DL4, and 10T1/2/DL4 cell line selected from one or more cell lines to use can be done.
  • MEF mouse embryonic fibroblast
  • OP9, OP9/DLL1, OP9-DL4, and 10T1/2/DL4 cell line selected from one or more cell lines to use can be done.
  • OP9 embryonic fibroblast
  • OP9/DLL1 OP9-DL4 cell line selected from one or more cell lines to use
  • 10T1/2/DL4 cell line selected from one or more cell lines to use
  • cells obtained by differentiation induction of iPS cells are administered to humans by cell therapy or the like, a stable production method that does not contain animal-derived substances is desired.
  • differentiation into the ⁇ T cells of the present invention can be induced without
  • induction of differentiation from iPS cell-derived progenitor cells to ⁇ T cells In the process of inducing the differentiation of iPS cell-derived blood cell progenitor cells into ⁇ T cells, co-culturing with feeder cells may be performed, or culture may be performed under conditions in which feeder cells are not used. Furthermore, the cells may be cultured under serum-free conditions or animal-derived components-free conditions. In addition, the process of inducing differentiation from iPS cell-derived blood cell progenitor cells to ⁇ T cells can also be performed by culturing under hypoxic conditions.
  • hypoxic conditions mean that the O 2 concentration in the culture conditions during the differentiation induction process from iPS cell-derived blood progenitor cells to ⁇ T cells is lower than the O 2 concentration in normal culture.
  • the O 2 concentration for culturing under hypoxic conditions is not particularly limited, but is, for example, less than 20% (v/v), preferably less than 10% (v/v).
  • a ⁇ T cell stimulating agent may be added, or may not be added depending on the culture conditions.
  • ⁇ T cell stimulators include phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway, or derivatives thereof.
  • phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway include, for example, HMBPP ((E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate) and IPP ( isopentenyl diphosphate).
  • the derivative include BrHBP (bromohydrin diphosphate).
  • ⁇ T cell stimulating agents also include specific inhibitors of FPP (farnesyl pyrophosphate) synthase, which is the rate-limiting enzyme in the biosynthetic pathway.
  • FPP farnesyl pyrophosphate
  • a specific inhibitor of FPP synthase promotes intracellular accumulation of the phosphate compound.
  • FPP synthase-specific inhibitors include nitrogen-containing bisphosphonates (N-BPs), specifically zoledronic acid and pamidronate.
  • N-BPs nitrogen-containing bisphosphonates
  • IL-15 and 1L-2 also function as ⁇ T cell stimulators.
  • ⁇ MEM (trade name) can be used as a basal medium for culturing 10 days after induction of differentiation from iPS cells (blood cell progenitor cells) by the above treatments 1-1) to 1-4).
  • penicillin/streptomycin and the like can be appropriately selected and added.
  • 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement may be used instead of FBS. Manufacturers and distributors of reagents and the like to be used are not particularly limited as long as they can exhibit equivalent functions. The optimum addition amount can be determined as appropriate.
  • Culture can be performed by seeding cells (blood cell progenitor cells) 10 days after induction of differentiation on a culture substrate such as a culture plate seeded with feeder cells.
  • the medium can be replaced, for example, every two days, and the supernatant can be recovered by pipetting 12 days, 18 days and 24 days after induction of differentiation, transferred to new feeder cells, and culture continued.
  • the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
  • A-2) 30 days or 31 days after differentiation induction Cells cultured in the above medium from day 10 to day 30 or 31 after induction of differentiation can be cultured under conditions in which feeder cells are not used.
  • RPMI1640 medium can be used as a basal medium for culture under such conditions.
  • it can be cultured in a medium containing 0-30%, preferably 0-20%, more preferably 10-20% FBS. 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement may be used instead of FBS.
  • IL-2 and/or IL-15 0 to 200 ng/ml, preferably 1 to 100 ng/ml, more preferably 10 ng/ml, or Immunace (trade name ) 0-1000 IU/ml, 10-500 IU/ml, preferably 100 IU/ml and 2-Me (2-Mercaptoethanol) 0-100 ⁇ M, 1-50 ⁇ M, preferably 10 ⁇ M. good.
  • penicillin/streptomycin and the like can be added as appropriate.
  • HMBPP may be added as a ⁇ T cell stimulant.
  • concentration to be added is not particularly limited as long as it stimulates ⁇ T cells and does not cause cytotoxicity.
  • Day 10 of differentiation induction For example, culture after 10 days (blood cell progenitor cells) after induction of differentiation from iPS cells by the above treatments 1-1) to 1-4), VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or can be cultured using a culture substrate coated with DLL1 (Delta-Like Protein 1). From 10 to 24 days after induction of differentiation, the cells can be cultured, for example, in Lymphoid progenitor Expansion Medium (trade name) included in the StemSpan TM T cell generation kit (trade name). Medium exchange was performed according to the StemSpan TM kit protocol.
  • Lymphoid progenitor Expansion Medium (trade name) included in the StemSpan TM T cell generation kit (trade name). Medium exchange was performed according to the StemSpan TM kit protocol.
  • an additional medium can be added on day 13 of differentiation induction, and the medium can be replaced on days 17 and 20 of differentiation induction, respectively. It can be replaced with T cell progenitor maturation medium (trade name) included in the above-mentioned kit at around 17 to 24 days of differentiation induction. On the 27th day of differentiation induction, the above medium is additionally added, and thereafter, the medium can be replaced about twice a week, such as on the 31st and 34th days of differentiation induction.
  • the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
  • the cells can be continuously cultured by the method described in B-1), they can be cultured in a medium supplemented with a ⁇ T cell stimulating agent from around 17 to 24 days after differentiation induction. From around 17th to 24th day of induction of differentiation, the number of cells tends to decrease, which can be improved by adding a ⁇ T cell stimulant.
  • a medium shown in A-2 in which IL-2 and/or IL-15 and ⁇ T cell stimulating agents such as HMBPP and FPP synthase-specific inhibitors are added.
  • HMBPP and FPP synthase-specific inhibitors can.
  • RPMI1640 medium containing AS401 in which IL-2 and/or IL-15 and HMBPP are added.
  • the cells can be cultured in a medium supplemented with a ⁇ T cell stimulating agent.
  • the cells can be cultured in the medium shown in A-2, in which HMBPP is added. It is also possible to culture in a medium containing no FBS among the medium shown in A-2 and similarly containing HMBPP.
  • RPMI1640 medium containing AS401 which is a medium supplemented with IL-2 and/or IL-15 and a ⁇ T cell stimulant such as HMBPP, can also be used.
  • Cells cultured by 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 cell size or larger than the cell size.
  • beads can be mixed in the culture medium and cultured.
  • the beads are not particularly limited as long as they are made of a material that can be used for cell culture. Specifically, Dynabeads Protein G (trade name) can be used.
  • VCAM1 and DLL4 the cells can be cultured under feeder-free conditions.
  • Cells cultured by the differentiation induction method of the present invention can also be cultured under conditions using a medium that does not contain animal-derived components. For example, culture after 10 days (blood cell progenitor cells) after induction of differentiation from iPS cells by the above treatments 1-1) to 1-4), VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or can be cultured using a culture substrate coated with DLL1 (Delta-Like Protein 1).
  • RPMI1640 containing AS401 can be used as a basal medium as a medium containing no animal-derived components.
  • SCF, IL-7, FLT3L, L-ascorbic adid, IL2, TPO, etc. shown in A-1 may be included.
  • the cells can be cultured in a medium supplemented with IL-2, IL-15, and ⁇ T cell stimulating agent shown in A-2.
  • a medium can be RPMI1640 containing AS401 as a basal medium.
  • cells can be cultured in a medium supplemented with one or more of IL-2, IL-15 and HMBPP.
  • ⁇ T cells produced by the method of inducing differentiation of the present invention are T cells that have a unique T cell receptor (TCR) consisting of ⁇ and ⁇ chains on their surface. Such cell surfaces can be checked for expression of cell markers such as CD3, CD7, CD8a, CD45RA and ⁇ TCR.
  • the ⁇ T cells of the present invention preferably express one or more selected from CD7, CD8a and CD45RA, while not expressing one or more selected from CD25, IFN ⁇ , CD5 and CD27. is preferred.
  • the obtained iPS cell-derived ⁇ T cells are characterized by having non-MHC-restricted antigen-specific cytotoxic activity.
  • iPS cell-derived ⁇ T cells tend to express CD7 and CD8a higher
  • ⁇ T cells isolated from peripheral blood tend to express IL2RA (CD25), CD5, and IFN ⁇ higher.
  • CD45RA tends to be expressed more in iPS cell-derived ⁇ T cells
  • CD27 tends to be expressed more in ⁇ T cells isolated from peripheral blood.
  • T cells induced to differentiate in this way can be isolated by appropriately selecting known techniques.
  • known techniques include, for example, flow cytometry using antibodies against cell surface markers and a cell sorter, as shown in Examples below.
  • T cells having desired antigen specificity are isolated from humans, a method of purification using an affinity column or the like on which the desired antigen is immobilized can also be employed.
  • a cell population of purified ⁇ T cells is composed of homogeneous cells and is distinguished from a cell population composed of ⁇ T cells isolated from peripheral blood, and the ⁇ T cell population of the present invention is composed of ⁇ T cells isolated from peripheral blood. It has a high antigen-specific cytotoxic activity compared to the husk.
  • the cell population containing the ⁇ T cells contains, for example, many cells having the same nucleotide sequence in the complementarity determining regions (CDRs) of the TCR gene.
  • CDRs complementarity determining regions
  • ⁇ T cells having the same nucleotide sequence in the CDR3 region in particular are included in a large proportion of the ⁇ T cells constituting the cell population, for example, 90% or more.
  • a cell population containing ⁇ T cells of the present invention can contain 1 ⁇ 10 5 or more ⁇ T cells.
  • ⁇ T cells exhibiting a higher expression level of CD7 and/or CD8a than ⁇ T cells isolated from peripheral blood are ⁇ T cells that constitute the cell population. contained at a rate of 90% or more of Furthermore, 90% of the ⁇ T cells constituting the cell population are ⁇ T cells that exhibit a lower expression level of one or more expression levels selected from CD25, INF ⁇ , and CD5 than the ⁇ T cells isolated from peripheral blood. It is included in the ratio above.
  • ⁇ T cells exhibiting a higher expression level of CD45RA than ⁇ T cells isolated from peripheral blood and expanded in vitro and a lower expression level of CD27 than ⁇ T cells isolated from peripheral blood and expanded in vitro are Contained in 70% or more of the ⁇ T cells that make up the cell population.
  • the cell population containing ⁇ T cells of the present invention is characterized in that undifferentiated cells account for 10% or less of the ⁇ T cells that make up the cell population, and the undifferentiated cells are present in the ⁇ T cells that make up the cell population. preferably not. Whether a certain cell is an undifferentiated cell can be determined by a marker indicating undifferentiation such as TRA-1-85.
  • ⁇ T cells produced by treatment with the differentiation-inducing treatment method of the present invention have excellent immune functions, and therefore are used for the treatment or prevention of diseases such as tumors, infectious diseases (e.g., viral infections), and autoimmune disorders. can be used. Furthermore, it can be used as an antigen-specific cellular immunotherapeutic agent or pharmaceutical composition containing the ⁇ T cell population produced by the method of the present invention as an active ingredient.
  • the ⁇ T cells produced by the differentiation-inducing treatment method of the present invention can be used for these formulations even after freezing and thawing. It is expected that the ⁇ T cell population can also be applied to immune cell therapy methods.
  • the ⁇ T cell population of the present invention is expected to further enhance the effects of ⁇ T cells when used in combination with an immune checkpoint inhibitor.
  • Immune checkpoint inhibitors are not limited to those known per se or those to be developed in the future, but include, for example, drugs targeting immune checkpoints such as PD-1, PD-L1 and CTLA-4.
  • Antibody-dependent cellular cytotoxicity (ADCC) is expected to enhance the effects of molecular targeted drugs and antibody preparations (e.g. Herceptin, Rituxan, etc.) that are used to treat various cancers, similar to NK cells.
  • a high therapeutic effect can be expected when used in combination with a preparation.
  • a pharmaceutical composition containing the ⁇ T cell population of the present invention can be prepared by formulating with a known pharmaceutical method.
  • pharmacologically acceptable carriers or media specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, Appropriate combination with preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, coloring agents, solubilizers, or other additives. can be done. In addition, it may be used in combination with known pharmaceutical compositions, immunostimulants, and the like used for treatment or prevention of the aforementioned diseases. When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected according to the subject's age, body weight, symptoms, health condition, type of composition, and the like.
  • the present invention also includes an antigen-specific cellular immunotherapy method by administering the iPS cell-derived ⁇ T cells of the present invention. Furthermore, the present invention also includes therapeutic methods for diseases such as cancer, infectious diseases, and autoimmune disorders by administering the iPS cell-derived ⁇ T cells of the present invention.
  • the dosage of the active ingredient to the subject varies depending on the body weight, age, symptoms, administration method, etc. of the subject, and can be appropriately selected by those skilled in the art.
  • Example 1 Induction of Differentiation from iPS Cells
  • Example 2 Induction of Differentiation from iPS Cells
  • a method for inducing differentiation of ⁇ T cells produced from ⁇ TCR-type iPS cells produced by the method of Non-Patent Document 1 will be described.
  • 0.5 ⁇ TrypLE TM select (manufactured by ThermoFisher) was used for detachment and dispersion of cells during passage, and for subculture, StemFit (R) AK02N with Y27632 (manufactured by Wako Pure Chemical Industries) at a final concentration of 10 ⁇ M and iMatrix-511 A culture medium added to 3.2 ⁇ l was used. The next day, the medium was replaced with StemFit (R) AK02N containing no Y27632 and iMatrix-511, and thereafter the medium was replaced every two days. A medium was added at 1.5 ml/well. All cultures, including the following steps and examples described later, were performed under conditions of 37 ⁇ 0.5° C. and 5% CO 2 .
  • Example 2 Differentiation induction from iPS cells
  • medium components and differentiation induction after 10 days of differentiation induction The medium components on and after the 31st day are different from those in Example 1.
  • medium components after 31 days of induction of differentiation contain HMBPP, which is a ⁇ T cell stimulant.
  • Example 3 Differentiation induction from iPS cells under conditions using feeder cells
  • ⁇ TCR-type iPS cells prepared by the method of Non-Patent Document 1 were prepared by differentiation induction treatment. ⁇ T cells are shown. Differentiation induction treatment was performed in the same manner as in Example 1, and after the 31st day, half of the ⁇ T cell stimulation medium (containing HMBPP and FBS) was replaced every 2 days in the same manner as in (2-4) of Example 2. A cytotoxicity assay was then performed along with evaluation of marker expression.
  • cytotoxicity assay was performed on Jurkat cells. 5 ⁇ 10 4 CFSE-stained Jurkat cells were added per well of a 96-well culture dish, and 1 ⁇ 10 5 iPS cell-derived ⁇ T cells on day 55 of differentiation induction were added. After culturing at 2:1 for 16 hours, 7-AAD staining (dead cell staining) was performed. Many Jurkat cells (CFSE-positive cells) were 7-AAD-positive, and many dead cells were confirmed. That is, it was confirmed that the iPS cell-derived ⁇ T cells have a cytotoxic function against tumor cells (Fig. 3C).
  • Example 4 Differentiation induction from iPS cells under conditions without feeder cells
  • ⁇ TCR-type iPS cells prepared by the method of Non-Patent Document 1 were prepared by differentiation induction treatment.
  • ⁇ T cells a method of inducing differentiation without using feeder cells is shown.
  • the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
  • Example 5 Induction of differentiation from iPS cells under conditions without feeder cells
  • ⁇ T cells prepared from ⁇ TCR-type iPS cells by differentiation induction treatment in the same manner as in Example 4 were treated under conditions without feeder cells.
  • the differentiation induction method in is shown.
  • the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
  • Example 6 Induction of differentiation from iPS cells under conditions without feeder cells
  • ⁇ T cells prepared from ⁇ TCR-type iPS cells by differentiation induction treatment in the same manner as in Example 4 were treated under conditions without feeder cells.
  • the differentiation induction method in is shown.
  • the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
  • the cells were treated in the same manner as (4-1)-(4-3) in Example 4, and cultured under the condition that neither feeder cells nor serum was used on days 10 to 24 of induction of differentiation.
  • (6-2) Day 24 of differentiation induction (day24) On day 24 of differentiation induction, a. ⁇ T cell stimulation medium (containing HMBPP and FBS) shown in Table 7 of Example 2 (2-4), b. ⁇ T cell stimulation medium shown in Table 7 (10% FBS/RPMI1640) instead of RPMI1640 (including HMBPP) medium containing AS401 and c. Lymphoid progenitor expansion medium included in the StemSpan TM kit. was replaced.
  • Example 7 Induction of Differentiation from iPS Cells without Feeder Cells
  • ⁇ TCR-type iPS cells were differentiated in the same manner as in Example 4 to produce ⁇ T cells.
  • Example 8 Differentiation induction method using magnetic beads
  • VCAM1 and DLL4-coated magnetic beads were mixed and cultured under conditions that did not use feeder cells, thereby producing T cells. Differentiation was induced.
  • Example 9 ⁇ T cells produced from ⁇ TCR-type iPS cells
  • the characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells were confirmed.
  • a method for producing iPS cell-derived ⁇ T cells is shown, and then various characteristics of the cells are shown.
  • ⁇ Day 0 of differentiation induction (Day0): State of ⁇ TCR-type iPS cells (HPC1) Stemfit AK02N (Ajinomoto, Tokyo, Japan, AK02N) CHIR99021 (Tocris, Bristol, UK, 4423) 4 ⁇ M BMP4 (R&D, Minneapolis, MN, 314-BP) 80 ng/ml VEGF (R&D, Minneapolis, MN, 293-VE) 80ng/ml
  • ⁇ From day 30 of differentiation induction (Day 30-): Cultivation in ⁇ T active medium
  • ⁇ T active medium Accutase-treated cells were suspended in the following ⁇ T active medium and cultured in a feeder cell-free medium. Thereafter, half of the medium was replaced every 2 days. Cells at 7-14 days of active culture were subjected to a cytotoxicity assay.
  • ⁇ T activity 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) 10 ⁇ M
  • ⁇ T cells 9-3) Antitumor effect Antitumor activity against various tumor cells was confirmed using iPS cell-derived ⁇ T cells on day 38 of differentiation induction (Fig. 17). Unpurified ⁇ T cells were used in these experiments. As a control, conditions were used in which only tumor cells were cultured without addition of ⁇ T cells.
  • a cytotoxicity assay was performed on Huh-7 cells (derived from human hepatoma cells).
  • E:T (effector:target) ratio 2:1, 5 x 10 4 Huh-7 cells stained with fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 x 10 5 iPS cells were added to each well.
  • the tumor area was measured by observing with a phase-contrast microscope.
  • the ⁇ T cells of the present invention clearly had higher cytotoxic activity against Huh-7 cells than the control (Fig. 17B).
  • a cytotoxicity assay was performed on SW480 cells (derived from human colon cancer).
  • E:T (effector:target) ratio 2:1, 5 ⁇ 10 4 SW480 cells stained with the fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 ⁇ 10 5 iPS cell-derived ⁇ T cells were added to each well. After adding the cells and culturing for 16 hours, the tumor area was measured by observing with a phase-contrast microscope.
  • the ⁇ T cells of the present invention clearly had higher cytotoxic activity against SW480 cells than the control (Fig. 17C).
  • iPS cell-derived ⁇ T cells on day 36 of differentiation induction, maintenance of TCR rearrangement and cytotoxic mechanism were confirmed (Fig. 18).
  • A. Non-purified iPS cell-derived ⁇ T cells (igdT) and peripheral blood mononuclear cells (PB) were evaluated for cell surface ⁇ TCR expression. Expression of ⁇ TCR was detected in PB, but not in ⁇ T cells (igdT) of the present invention (Fig. 18A).
  • igdT Non-purified iPS cell-derived ⁇ T cells
  • PB peripheral blood mononuclear cells
  • ⁇ TCR was detected in PB, but not in ⁇ T cells (igdT) of the present invention (Fig. 18A).
  • B. Genomic PCR for TCR gene rearrangements The rearrangement of TCR genes (Vg9, Vd2) was confirmed by genomic PCR.
  • ⁇ T cells sorted by flow cytometry were confirmed to retain the TCR gene rearrangement as in the undifferentiated state (Fig. 18B).
  • Peripheral blood mononuclear cells PBMC
  • Cytotoxicity assays were performed on ⁇ T cells (igdT) purified by flow cytometry (FACS). The conditions for the cytotoxicity assay are the same as A. of (9-3). was performed by the method shown in .
  • As a control (ctrl) in FIG. 18D only Jurkat cells were cultured without adding iPS cell-derived ⁇ T cells.
  • iPS cell-derived ⁇ T cells that have not been purified are indicated as bulk, and purified iPS cell-derived ⁇ T cells are indicated as sort.
  • iPS cell-derived ⁇ T cells there was no significant difference in cell death rate between the presence and absence of purification (Fig. 18D).
  • HLA types of iPS cell lines and tumor cells Table 8 shows the results of confirming the HLA types of the iPS cells used in the iPS cell-derived ⁇ T cells of the present invention and the tumor cells used in Examples 3 and 6 and this example. show. Although the HLA type of iPS cells did not match the HLA type of each tumor cell, an antitumor effect was observed for each tumor cell (this example AC). As a result, it was confirmed that the iPS cell-derived ⁇ T cells of the present invention have antigen-specific cytotoxic activity in an MHC-unrestricted manner.
  • Example 10 Comparison of iPS cell-derived ⁇ T cells and ⁇ T cells isolated from peripheral blood
  • iPS cell-derived ⁇ T cells prepared by inducing the differentiation of iPS cells and ⁇ T present in peripheral blood Cell surface expression marker genes in cells (PB-gdT) were compared.
  • the iPS cell-derived ⁇ T cells of this example were cultured by the method shown in Example 1 and the method shown in Example 9 (9-1), and cells after 36 to 42 days of induction of differentiation were used.
  • Cells obtained by culturing mononuclear cells isolated from peripheral blood in the ⁇ T active medium shown in Example 9 (9-1) were used as ⁇ T cells isolated from peripheral blood in this example.
  • Example 11 Method for activating iPS cell-derived ⁇ T cells
  • a method for activating iPS cell-derived ⁇ T cells was examined. Specifically, in the production method shown in (9-1) of Example 9, for cells on day 30 of differentiation induction, either IL-2 and/or IL-15 is added to the following ⁇ T activity medium. It was examined whether it is possible to produce iPS cell-derived ⁇ T cells more effectively (see FIG. 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
  • Example 12 Characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells (121-3 strain) In this example, characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells (121-3 strain) were confirmed.
  • TCR reconstructionA Retention of TCR reconstructionA.
  • the nucleotide sequences and amino acid sequences of the CDR3 regions of each of TCR ⁇ and TCR ⁇ were identified, and the frequencies for each sequence were shown in a pie chart (Fig. 23B). It was confirmed that the PB ⁇ T cell population was composed of cells with diverse sequences, whereas the i ⁇ T cell population was composed of cells with a single type of TCR ⁇ and TCR ⁇ gene rearrangement.
  • IFN ⁇ interferon gamma
  • i ⁇ T iPS cell-derived ⁇ T cells
  • PB ⁇ T ⁇ T cells
  • Example 14 Comparison of iPS cell-derived ⁇ T cells and ⁇ T cells obtained by amplifying peripheral blood
  • iPS prepared by inducing differentiation of ⁇ TCR-type iPS cells (62B3 strain or 121-3 strain) Cell surface expression markers were compared between cell-derived ⁇ T cells (igdT) and ⁇ T cells obtained by expanding peripheral blood (PB-gdT).
  • iPS cell-derived ⁇ T cells (i ⁇ T CD3-positive or TCR ⁇ 9-positive cells) had a higher percentage of cells expressing CD7, a lower percentage of cells expressing CD5 and CD25, and a higher percentage of CD45RA + CD27 ⁇ cells. It was confirmed to have the characteristic of having a high ratio (Fig. 25).
  • Example 15 Examination of the process of stimulating ⁇ T cells
  • ⁇ T cells prepared by differentiation induction treatment from ⁇ TCR-type iPS cells in the same manner as in Example 5 were treated under conditions in which neither feeder cells nor serum were used, and ⁇ T cells were A method of inducing differentiation under the condition that the step of stimulating is performed on the 17th day instead of the 24th day is shown.
  • differentiation was induced by the following procedure according to the protocol shown in FIG. 26A (New protocol).
  • Example 5 The same treatment as in Example 5 (5-1) was performed and cultured. However, the step of stimulating ⁇ T cells was performed from day 17 of induction of differentiation.
  • Example 16 Method for activating iPS cell-derived ⁇ T cells without feeder cells
  • a method for activating iPS cell-derived ⁇ T cells without using feeder cells or serum was examined. .
  • Example 15 The same treatments as in Example 15 (15-1) and (15-3) were performed, and the cells were cultured under conditions in which neither feeder cells nor serum were used. However, cells on the 17th day of induction of differentiation were tested under the same conditions as in Example 15 (15-3) and under the conditions in (15-3) in which IL-2 was replaced with IL-15. On day 33 or 37 of differentiation induction, the expression of CD3/ ⁇ TCR was evaluated by flow cytometry to determine whether iPS cell-derived ⁇ T cells could be produced more effectively. CD3 + /TCR + cells were detected, differentiation into TCR cells was confirmed, and they were confirmed to be iPS cell-derived ⁇ T cells (Fig. 27A).
  • iPS cell-derived ⁇ T cells can be produced with either IL-2 or IL-15 in the step of ⁇ T cell stimulation. In addition, more iPS cell-derived ⁇ T cells were obtained with the addition of IL-15 than with IL-2.
  • the expression of CD3/CD7 was evaluated by flow cytometry in cells on day 23 of induction of differentiation. CD3 + /TCR + cells were detected, differentiation into TCR cells was confirmed, and they were confirmed to be iPS cell-derived ⁇ T cells.
  • iPS cell-derived ⁇ T cells were obtained even under the condition that HMBPP, which is a ⁇ TCR stimulant, was not added (Fig. 27B).
  • Example 17 Cytotoxic Activity of iPS Cell-Derived ⁇ T Cells After Freezing and Thawing
  • iPS cell-derived ⁇ T cells were freeze-thawed under the condition that neither feeder cells nor serum were used, and a cytotoxicity assay was performed.
  • Example 15-1) The same treatment as (15-1) and (15-3) of Example 15 was performed, and culture was performed under the condition that neither feeder cells nor serum was used. However, IL-2 in (15-3) of Example 15 was replaced with IL-15. On the 24th day of differentiation induction, the cells were frozen using CS10 (manufactured by Cosmo Bio).
  • Example 18 Induction of differentiation after freezing and thawing of iPS cell-derived progenitor blood cells
  • differentiation was induced after freezing and thawing of iPS cell-derived progenitor blood cells to prepare ⁇ T cells.
  • iPS cell-derived ⁇ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D) staining. Cell death (7-AAD positive) was confirmed for many Jurkat cells (CFSE positive cells) (Fig. 29C). That is, it was confirmed that the iPS cell-derived ⁇ T cells have a cytotoxic function even after freezing and thawing.
  • Example 19 Differentiation induction under conditions in which neither feeder cells nor serum was used after iPS cell-derived progenitor cells were frozen and thawed
  • iPS cell-derived progenitor cells were freeze-thawed under conditions in which neither feeder cells nor serum were used. was induced to differentiate into ⁇ T cells.
  • differentiation was induced by the following procedure according to the protocol shown in FIG. 30A. The freezing in this example was performed for 18 days.
  • Example 20 Differentiation induction from blood cell progenitor cells under hypoxic conditions
  • the conditions were carried out without using feeder cells or serum.
  • we generated ⁇ T cells by inducing differentiation from blood cell progenitor cells under hypoxic conditions.
  • differentiation was induced by the following procedure according to the protocol shown in FIG. 31A.
  • Example 21 Induction of differentiation from iPS cells under animal-derived component-free medium
  • iPS cell-derived ⁇ T cells were produced under animal-derived component-free medium.
  • Example 22 Confirmation of Undifferentiated Cells for iPS Cell-Derived ⁇ T Cells
  • the conditions were carried out without using feeder cells or serum.
  • undifferentiated cells were confirmed for iPS cell-derived ⁇ T cells.
  • Example 23 Cytotoxicity Assay for CD3/ ⁇ T-Positive Cells
  • CD3/ ⁇ T-positive cells were purified from the cell population obtained by the same treatment as in Example 22, and a cytotoxicity assay was performed.
  • E:T (effector:target) ratio 0.2:1, 5 ⁇ 10 4 Jurkat cells stained with the fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 ⁇ 10 5 cells on day 35 of induction of differentiation were added. iPS cell-derived ⁇ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D) staining and graphed (Fig. 34B). It exhibited strong cytotoxic activity in spite of the condition of an E:T ratio of 0.2:1, in which the number of attacking (effector) cells to tumor cells was extremely small. In the cytotoxicity assay, which was previously performed using unpurified cell populations, it was clarified that the target cells, CD3/ ⁇ T-positive cells (i.e., ⁇ T cells), possessed cytotoxic activity. Became.
  • ⁇ T cells can be effectively produced without burdening the blood recipient and without being affected by cell exhaustion. Furthermore, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced even by a method that does not use feeder cells. Furthermore, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced by a method that does not use feeder cells or serum, or even a method that uses a medium that does not contain animal-derived components. Further, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced even after freezing and thawing during production.
  • the ⁇ T cell population produced by the method of the present invention can be a ⁇ T cell population that is more homogeneous and highly effective than the ⁇ T cell population isolated from peripheral blood, and is more effective in producing MHC-free cells. It has an excellent function of restricted antigen-specific cytotoxic activity. Furthermore, the ⁇ T cell population produced by the method of the present invention can be a ⁇ T cell population with no remaining undifferentiated cells, which is excellent for clinical application.

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