Methods for producing regulatory t cells

Abstract

Disclosed are: a method for producing a cell population containing regulatory T cells, the method comprising the step (1) of culturing a cell population containing pluripotent stem cell-derived CD4 + T cells in the presence of at least one substance selected from the group consisting of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor, and a TGF-[beta] R agonist; a cell population containing regulatory T cells obtained by said method; and a drug comprising said population of cells comprising regulatory T cells.

Description

Technical Field

[0001] The present invention relates to a method for producing a cell population containing regulatory T cells, a cell population containing regulatory T cells obtained by the method, a drug containing the cell population containing regulatory T cells, and the like. Background of the Invention

[0003] In recent years, due to the immune response suppression function of regulatory T cells (Treg), the research and development of cell therapy (Treg therapy) using regulatory T cells has been promoted worldwide for the purpose of treating various autoimmune diseases. Therefore, regulatory T cells are expected to be used in the treatment of graft-versus-host disease (GvHD) and autoimmune diseases, as well as in the treatment and prevention of inflammatory diseases and allergic diseases.

[0004] Regulatory T cells are roughly divided into two types: naturally occurring regulatory T cells (natural Treg: nTreg or thymic Treg: tTreg) and induced regulatory T cells (induced Treg: iTreg), and it is known that nTreg naturally exists in the thymus, and iTreg is induced to differentiate from the initial T cells in the peripheral blood by antigen stimulation and cytokines (such as IL-2, TGF-β). These regulatory T cells are further subdivided according to the type of markers to be expressed in the cells.

[0005] When regulatory T cells are removed from an organism, various organ-specific autoimmune diseases develop spontaneously, and when regulatory T cells are transplanted at this time, the development of autoimmune diseases is prevented, so that regulatory T cells are considered to play an important role in maintaining peripheral immune self-tolerance. Since then, it has become clear that regulatory T cells can not only suppress autoimmunity, but also most immune responses, such as inflammation caused by foreign antigens, rejection caused by transplantation, infection immunity, allergy and tumor immunity. In addition, it has now been shown that the transcription factor FOXP3 is a master regulator of regulatory T cells.

[0006] FOXP3 is the main regulator of Treg, and it is known that FOXP3 is constitutively expressed in Treg, and constitutively expressed is not observed in normal T cells (also referred to as Tconv (conventional T cells), effector T cells or inflammatory T cells) without inhibitory function. Therefore, research on maintaining FOXP3 expression in primary Treg and inducing FOXP3 expression in primary Tconv or a large number of CD4 single positive (SP) T cells has been carried out, and such compounds and cytokines and their combinations with the activity of maintaining and inducing FOXP3 expression have been reported. Such compounds, cytokines and their combinations include AS2863619 ((NPL 1 and PTL 1)) as CDK8 / 19 inhibitors, rapamycin (rapamycin) (NPL 2, NPL 3, NPL 4 and NPL 5) as mTOR inhibitors, TNFR2 agonist antibodies (NPL 4, NPL 6 and NPL 7), cytokine TGF-β (NPL 1 and NPL 5), etc.

[0007] However, for pluripotent stem cell-derived regulatory T cells, compounds and cytokines that maintain FOXP3 expression, and combinations thereof; compounds and cytokines that induce T cells that do not express FOXP3 to express FOXP3, and combinations thereof; etc. are still unknown. Regulatory T cells are known to be difficult to proliferate, but an industrial production method with high production efficiency (yield) is desired, and an efficient induction method thereof is desired.

[0008] In NPL 8, regarding IPEX (X-linked polyendocrine enteropathy with immune dysregulation) syndrome caused by mutations in FOXP3, the following has been described: Gene transduction into autologous hematopoietic stem cells using a lentiviral vector to restore FOXP3 expression, and the cells were administered to model mice of IPEX syndrome. As a result, it was reported that the hematopoietic stem cells differentiated into functional regulatory T cells and recovered from the autoimmune phenotype. In addition, PTL 2 also describes a similar recombinant lentiviral vector containing a nucleotide sequence encoding a human FOXP3 protein.

[0009] PTL 3 discloses that simultaneous expression of Mcl-1 and Foxp3 is important for improving Treg survival. In addition, PTL 4 discloses that genetically engineered mammalian cells containing transgenes encoding lineage commitment factors can promote the differentiation of lineage commitment factors into CD4 + Differentiation of Tregs. PTL 5 discloses a method for preparing a T cell composition derived from stem cells or progenitor cells by culturing three-dimensional cell aggregates, the three-dimensional cell aggregates comprising a selected Notch ligand-expressing stromal cell population and a selected stem cell or progenitor cell population.

[0010] Citation List

[0011] Patent Literature

[0012] PTL 1: WO2018 / 139660

[0013] PTL 2: WO2019 / 040655

[0014] PTL 3: WO2014 / 180943

[0015] PTL 4: WO2021 / 092581

[0016] PTL 5: WO2017 / 075389

[0017] Non-patent literature

[0018] NPL 1: Akamatsu et al., Sci.Immunol.4, eaaw2707 (2019)

[0019] NPL 2: Charbonnier et al., Nat. Immunol. 20(9), 1208-1219 (2019)

[0020] NPL 3: MacDonald et al., Clin. Exp. Immunol., 197(1): 52-63. (2019)

[0021] NPL 4: Urbano et al., Front. Immunol. 9:573 (2018)

[0022] NPL 5: Schmid et al., PLoS One., 11(2): e0148474. (2016)

[0023] NPL 6: He et al., PLoS One. 11(5), e0156311 (2016)

[0024] NPL 7: Torrey et al., Sci. Signal. 13, eaba9600 (2020)

[0025] NPL 8: Cell Stem Cell 24, 309-317, February 7, 2019 Summary of the invention

[0026] Technical issues

[0027] An object of the present invention is to provide a method for producing a cell population containing regulatory T cells, wherein the method can efficiently produce regulatory T cells.

[0028] Solution to the problem

[0029] The present inventors have conducted intensive studies to achieve the above-mentioned object and have found that by culturing CD4+ derived cells containing pluripotent stem cells (particularly, iPS cells) in the presence of at least one substance selected from the group consisting of the following: + T cell populations that can efficiently generate cell populations containing regulatory T cells: CDK8 and / or CDK19 inhibitors, TNFR2 agonists, mTOR inhibitors, and TGF-βR agonists.

[0030] The present invention was completed by further conducting studies based on these findings, and provides the following methods for producing a cell population containing regulatory T cells, a cell population containing regulatory T cells, a drug, and the like.

[0031] [1] A method for producing a cell population containing regulatory T cells, the method comprising:

[0032] Step (1) culturing CD4 T cells derived from pluripotent stem cells in the presence of at least one substance selected from the group consisting of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor, and a TGF-βR agonist. + T cell population.

[0033] [1a] A method for producing a cell population containing regulatory T cells, the method comprising:

[0034] Step (1) culturing CD8 containing pluripotent stem cells in the presence of at least one substance selected from the group consisting of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor, and a TGF-βR agonist. + T cell population.

[0035] [2] The method according to [1] or [1a], wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor and an mTOR inhibitor.

[0036] [3] The method according to [1] or [1a], wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor, an mTOR inhibitor and a TGF-βR agonist.

[0037] [4] The method according to [1] or [1a], wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor and a TGF-βR agonist.

[0038] [5] The method according to any one of [1] to [4], wherein the CDK8 and / or CDK19 inhibitor is at least one selected from the group consisting of: 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine and siRNA of CDK8 and / or CDK19.

[0039] [6] The method according to any one of [1] to [5], wherein the mTOR inhibitor is rapamycin.

[0040] [7] The method according to any one of [1], [1a] and [4] to [6], wherein the TNFR2 agonist is a TNFR2 agonist antibody.

[0041] [8] The method according to any one of [1], [1a] and [3] to [7], wherein the TGF-βR agonist is TGF-β.

[0042] [9] The method according to any one of [1] to [8], wherein the regulatory T cells are CD25 + / FOXP3 + cell.

[0043] [9a] The method according to any one of [1] to [8], wherein the regulatory T cells are CD25 + / CD127 - cell.

[0044]

[10] The method according to any one of [1] to [9], wherein the CD4 + T cells are CD4 + / CD8 - T cells.

[0045] [10a] The method according to any one of [1a] to [9a], wherein the CD8 + T cells are CD4 - / CD8 + T cells.

[0046]

[11] The method according to any one of [1] to

[10] , further comprising, before step (1), step (2) inducing pluripotent stem cells to differentiate into cells containing CD4 + T cell population.

[0047] [11a] The method according to any one of [1a] to [9a] and [10a], which further comprises, before step (1), step (2) inducing pluripotent stem cells to differentiate into cells containing CD8 + T cell population.

[0048] [11b] The method according to any one of [1] to [11a], comprising (3) introducing a foreign gene (especially a gene for expressing a chimeric antigen receptor) into a cell containing pluripotent stem cells (especially iPS cells), CD4 + T cells, CD8 + T cells or regulatory T cells.

[0049]

[12] The method according to any one of [1] to [11b], wherein the pluripotent stem cells are iPS cells.

[0050]

[13] The method according to any one of

[11] , [11b] or

[12] , wherein in step (2), a three-dimensional cell aggregate is cultured, wherein the three-dimensional cell aggregate contains CD4 T cells that can differentiate into the pluripotent stem cells. + T cells and stromal cells expressing Notch ligands.

[0051] [13a] The method according to any one of [11a], [11b] or

[12] , wherein in step (2), a three-dimensional cell aggregate is cultured, wherein the three-dimensional cell aggregate contains CD8 T cells that can differentiate into the pluripotent stem cells. + T cells and stromal cells expressing Notch ligands.

[0052]

[14] A cell population containing regulatory T cells, obtained by the method according to any one of [1] to [13a].

[0053]

[15] A medicament comprising the cell population containing regulatory T cells according to

[14] .

[0054]

[16] The drug according to

[15] , which is used for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection.

[0055]

[17] A method for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection, comprising administering the cell population containing regulatory T cells according to

[14] to a subject in need thereof.

[0056]

[18] The cell population containing regulatory T cells according to

[14] , which is used for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection.

[0057]

[19] Use of the cell population containing regulatory T cells according to

[14] in the preparation of a medicament for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection.

[0058]

[20] The method according to any one of [1] to [13a], wherein the CD4 + A T cell is a cell into which has been introduced an expression construct comprising:

[0059] (1)(a) Conserved noncoding sequences (CNS) 1, CNS2, and CNS3 of the Foxp3 gene;

[0060] (b) a promoter; and

[0061] (c) Nucleic acid encoding FOXP3.

[0062] [20a] A method for proliferating a cell population containing regulatory T cells, the method comprising

[0063] (A) A cell population containing regulatory T cells, which are primary cultured cells, was cultured in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, and a TGF-βR agonist.

[0064] [20b] The method according to [20a], wherein step (A) is performed in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, and a TGF-βR agonist, and an mTOR inhibitor.

[0065] [20c] The method according to [20a] or [20b], wherein the CDK8 and / or CDK19 inhibitor is at least one selected from the group consisting of: 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine and siRNA of CDK8 and / or CDK19.

[0066] [20d] The method according to [20b] or [20c], wherein the mTOR inhibitor is rapamycin.

[0067] [20e] The method of any one of [20a] to [20d], wherein the TNFR2 agonist is a TNFR2 agonist antibody.

[0068] [20f] The method according to any one of [20a] to [20e], wherein the TGF-βR agonist is TGF-β.

[0069] [20g] The method according to any one of [20a] to [20f], wherein the regulatory T cells are CD25 + / FOXP3 + cell.

[0070] [20h] The method according to any one of [20a] to [20f], wherein the regulatory T cells are CD25 + / CD127 - cell.

[0071] Advantageous Effects of the Invention

[0072] According to the present invention, regulatory T cells can be produced efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Shown are flow cytometry (FACS) graphs showing cells containing ATO derived from 4GAD1-4 at 9 weeks (top) and cells containing ATO derived from FFI01s04 at 6 weeks (bottom).

[0074] Figure 2 Shown are FACS plots showing the profiles of PBMC-derived Tconv and 4GAD1-4-derived CD4SP T cells after initial expansion culture using basal medium.

[0075] Figure 3 Shown are FACS plots showing bulk (CD4(+) sorted fraction), Tconv (CD4(+)CD25(-) sorted fraction), and Treg (CD4(+)CD25(+)CD127(-) sorted fraction) in PBMCs from healthy volunteer donors.

[0076] Figure 4 The results of expansion culture of PBMC-derived Tregs in the presence of a basal medium or a basal medium supplemented with AMT or AMRT are shown (FACS graphs showing FOXP3 expression).

[0077] Figure 5 The results of expansion culture of PBMC-derived Tregs in the case where the IL-2 concentration in the basal medium was changed to 100 ng / mL or in the case where the basal medium was supplemented with AMT or AMRT are shown (FACS plots showing FOXP3 expression (left) and graphs showing expansion ratios (right)).

[0078] Figure 6 The results of expansion culture of 4GAD1-4-derived CD4SP T cells in the presence of a basal medium or a basal medium supplemented with AR, ART, AMT, or AMRT are shown (FACS graphs showing the FOXP3 expression rate).

[0079] Figure 7 FACS plots of PBMC-derived Tregs expanded by culturing with basal medium supplemented with AMT and IL-2 concentration changed to 100 ng / mL (upper row), PBMC-derived Tconv expanded by culturing in basal medium (middle row), and PBMC-derived Tconv expanded by culturing with basal medium supplemented with ART (lower row) are shown.

[0080] Figure 8 Shown are FACS graphs of 4GAD1-4-derived CD4SP T cells expanded by culturing in a basal medium (upper row) and 4GAD1-4-derived CD4SP T cells expanded by culturing in a basal medium supplemented with AMRT (lower row).

[0081] Fig. 9 This is a graph showing the ratio of the number of copies of the demethylated FOXP3 TSDR region to the number of copies of the demethylated GAPDH gene in each cell type. Since the GAPDH gene is demethylated at a substantially constant rate in any cell, it can be interpreted as a value indicating what percentage (%) of cells in a cell population have demethylated TSDR regions.

[0082] Fig.10 The structure of the CAR molecule used in Example 4 is shown.

[0083] Fig.11 Shown are FACS graphs showing PBMC-derived Tregs expanded by culturing with a basal medium supplemented with AMT with (upper row) and without (lower row) CAR gene transduction, and iPS cell-derived CD4SP T cells expanded by culturing with a basal medium supplemented with AMRT.

[0084] Fig.12Shown is a graph of the extent of proliferation of cytotoxic T cells (CTLs) when CTLs (sorted from the same PBMCs used and cultured as in the in vivo experiments described below) are co-cultured with iPS cell-derived CD4SP T cells expanded with IL-2 alone, iPS cell-derived CD4SP T cells expanded with AMRT, or PBMC-derived Tregs expanded with AMT (with (upper row) and without (lower row) CAR gene transduction). The more right the peak detected, the more severe the inhibition of cell proliferation.

[0085] Fig.13 : is a graph showing the proliferation inhibition rate (% inhibition) of CTL when one of the following six types of cells is co-cultured with CTL at different ratios: iPS cell-derived CD4SP T cells expanded by culturing with IL-2 alone without CAR gene transduction (iPS-CD4T, no CAR, cultured with IL-2), iPS cell-derived CD4SP T cells expanded by culturing with IL-2 alone with CAR gene transduction (iPS-CD4T, CAR, cultured with IL-2), iPS cell-derived CD4SP T cells expanded by culturing with AMRT without CAR gene transduction (iPS-CD4T, no CAR, cultured with AMRT), iPS cell-derived CD4SP T cells expanded by culturing with AMRT with CAR gene transduction. T cells (iPS-CD4T, CAR, cultured with AMRT), and PBMC-derived Treg cells expanded by culture with AMT without CAR gene transduction (nTreg, no CAR, cultured with AMT), or PBMC-derived Treg cells expanded by culture with AMT with CAR gene transduction (nTreg CAR, CAR, cultured with AMT). Data values ​​are mean ± SEM. n = 3

[0086] Fig.14It is a graph showing the weight (upper row) and overall survival (lower row) after transplanting one of the following four types of cells into each group of mice: iPS cell-derived CD4SP T cells expanded with AMRT without CAR gene transduction, iPS cell-derived CD4SP T cells expanded with AMRT with CAR gene transduction, PBMC-derived Treg cells expanded with AMT without CAR gene transduction, or PBMC-derived Treg cells expanded with AMT with CAR gene transduction, and HLA-A2-positive PBMC. Data values ​​are mean ± SEM. n = 5 (n = 4 only in iPS cell-derived CD4SP T cells without CAR gene transduction)

[0087] Fig.15 Figure 2 is a graph showing body weight (upper row) and overall survival rate (lower row) after transplantation of one of the following four types of cells into each group of mice: iPS cell-derived CD4SP T cells expanded by culture with AMRT without CAR gene transduction, iPS cell-derived CD4SP T cells expanded by culture with AMRT with CAR gene transduction, PBMC-derived Treg cells expanded by culture with AMT without CAR gene transduction, PBMC-derived Treg cells expanded by culture with AMT with CAR gene transduction, and HLA-A2-positive PBMCs (derived from mice with Fig.14 The HLA-A2 positive PBMCs in the samples were from different donors). Data values ​​are mean ± SEM. n = 5

[0088] Fig.16 A graph showing the expansion ratio of CNS-Foxp3-transduced iPS cell-derived Tregs cultured in a medium containing or not containing AMRT.

[0089] Fig.17 Shown are FACS plots of CNS-Foxp3-transduced iPS cell-derived Tregs cultured with medium containing or not containing AMRT.

[0090] Fig.18 The graph shows the extent of proliferation of donor-derived PBMCs when they were co-cultured with CNS-Foxp3-transduced iPS cell-derived Tregs expanded by culturing with medium supplemented with or without AMRT. The more to the right the detected peak is, the more severe the inhibition of cell proliferation is.

[0091] Fig.19AResults of a pseudo-temporal analysis of an integrated single-cell RNA-seq dataset of cells in organoid cultures and thymocytes are shown (Trajectory Analysis of Integration of Single-Cell RNA-seq Data from 3D Organoid Cultures with Single-Cell RNA-seq Data from Human Thymocytes).

[0092] Fig.19B Shown are the results of a pseudo-temporal analysis of an integrated single-cell RNA-seq dataset of cells in organoid culture and thymocytes (extracted T-cell lineage differentiated cells).

[0093] Fig.19C Results of pseudo-temporal analysis of integrated single-cell RNA-seq datasets of cells in organoid culture and thymocytes are shown (heat map of DEG modules based on pseudo-temporal analysis).

[0094] Fig.19D Results of pseudo-temporal analysis of integrated single-cell RNA-seq datasets of cells in organoid culture and thymocytes are shown (top 10 significant genes per module cluster).

[0095] Fig. 20A Shown are transcriptome profiles of differentiated cells in 2D and 3D cultures obtained by single-cell RNA sequencing analysis (integrated UMAP of 3D organoids, 2D cultures and human thymocytes).

[0096] Fig. 20B Shown are transcriptome profiles of differentiated cells in 2D and 3D culture obtained by single-cell RNA sequencing analysis (signature plots showing CD4, CD8A, and CD8B).

[0097] Fig. 20C Shown are the transcriptome profiles of differentiated cells in 2D and 3D cultures obtained by single-cell RNA sequencing analysis (CD4 signatures of 3D organoid culture samples, thymocytes, and 2D mature cells).

[0098] Fig.21A is a view showing the characteristic plots of the indicated gene sets for 3D culture, thymocytes, and 2D culture.

[0099] Fig. 21B is a view showing the characteristic plots of the indicated gene sets for 3D culture, thymocytes, and 2D culture.

[0100] Fig. 21C is a view showing the characteristic plots of the indicated gene sets for 3D culture, thymocytes, and 2D culture. DETAILED DESCRIPTION

[0101] Hereinafter, embodiments of the present invention will be described in detail.

[0102] The term "comprise (comprise(s) / comprising)" means including but not limited to the elements following the word. Therefore, this indicates that one or more elements following the word are included, but does not indicate the exclusion of any other elements. The phrase "consist(s) of / consisting of" means including and limited to all one or more elements following the phrase. Therefore, the phrase "consist(s) of / consisting of" means that the one or more elements listed are required or necessary, and there are basically no other elements. The phrase "consist(s) essentially of / consistingessentially of" means including any element following the phrase, and limiting other elements to those elements that do not affect the activity or effect of the one or more elements listed in detail in the present disclosure. Therefore, the phrase "essentially consisting of..." indicates that one or more elements listed are required or necessary, while other elements are optional and may be present or absent, depending on whether they affect the activity or effect of the one or more elements listed.

[0103] In this specification, the term "culture" refers to the maintenance and / or proliferation of cells in an in vitro environment. The term "culture" refers to the maintenance and / or proliferation of cells outside a tissue or in vitro (eg, in a cell culture dish or flask).

[0104] In this specification, the phrase "culturing a cell population in the presence of a substance" means, for example, culturing a cell population in a culture medium containing the substance. Examples of such culturing include culturing in a culture medium containing only the substance or in a culture medium containing the substance, other differentiation inducing factors, and the like. When a substance is added to a culture medium, it may be added directly to the culture medium, or it may be dissolved in an appropriate solvent before use and then added to the culture medium. In addition, during the culture period, the substance may also be cultured while being fixed on a substrate or a carrier surface.

[0105] In this specification, the term "positive (+)" means that the protein or gene is expressed in an amount detectable by methods known in the art. In the case where the protein is expressed intracellularly and does not appear on the cell surface (e.g., transcription factors or their subunits), a reporter protein is expressed together with the protein, and the reporter protein is detected, whereby the target protein can be detected. Gene detection can be performed using nucleic acid amplification and / or nucleic acid detection methods, such as RT-PCR, microarrays, biochips, and RNAseq.

[0106] In this specification, the term "negative (-)" means that the expression level of protein or gene is less than the detection limit of all or any one of the known methods described above, or their expression level is low. The detection limit of protein or gene expression may vary depending on the method. By comparing with the result of the control cells measured under the same conditions, the degree (low expression or high expression) of protein or gene expression can be determined. The example of control cells includes the CD4SP T cells of the Treg and iPS cell sources of the PBMCs described in the embodiment section. For example, the expression level of CD25 in a certain cell group can be compared with the expression level of CD25 in the Tconv (control: known CD25 expression is low) derived from PBMC using flow cytometry to determine the expression level of CD25 in the cell group, and when an expression comparable to the control is observed, the expression level can be determined as low expression, and when the expression is higher than the control cells, the expression level can be determined as high expression.

[0107] In this specification, the term "marker" refers to a protein or its gene specifically expressed on the cell surface, in the cytoplasm or in the nucleus of a given cell type. Marker is preferably a "cell surface marker". "Cell surface marker" refers to a protein expressed on the cell surface, which can be labeled (stained) with a fluorescent substance and promotes the detection, concentration, separation, etc. of cells expressing cell surface markers. Cell surface markers refer to genes specifically expressed (positive markers) or not specifically expressed (negative markers) in a given cell type, and specifically refer to a substance that is produced (positive markers) or not produced (negative markers) by the transcription of the gene in the genome as mRNA or produced (positive markers) or not produced (negative markers) as protein by the translation of mRNA.

[0108] Such cell surface markers can be detected by immunological assays such as ELISA, immunostaining, and flow cytometry using antibodies specific for the cell surface markers.

[0109] In this specification, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter in a cell.

[0110] In this specification, "pluripotent stem cells" refer to embryonic stem cells (ES cells) and cells with similar pluripotency, that is, cells with the potential to differentiate into various tissues (endoderm, mesoderm and ectoderm) of a living body. Examples of cells having the same pluripotency as ES cells include "induced pluripotent stem cells" (also referred to as "iPS cells" in this specification).

[0111] In this specification, "hematopoietic stem cells (HSC)" are multipotent stem cells that can differentiate into blood cells. Hematopoietic stem cells are mainly present in the bone marrow of living humans, and differentiate into white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes and macrophages), red blood cells, platelets, mast cells, dendritic cells, etc. In this specification, hematopoietic stem cells (HSC) can be CD34 positive and CD7 negative (CD34 + / CD7 - ). In this specification, the phrase "CD34 + / CD7 - " / " used in " means "and".

[0112] In this specification, "hematopoietic progenitor cells" (HPC) are cells that have the ability to differentiate into blood cells, but do not have the self-renewal ability like stem cells. Hematopoietic progenitor cells are mainly present in the bone marrow of living humans. In this specification, hematopoietic progenitor cells (HPC) can be CD34 positive and CD43 negative (CD34 + / CD43 + ). In addition, HPCs can be positive for CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, and CD325, and negative for CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c, as described in WO2018 / 199186.

[0113] In this specification, "hemogenic endothelial cells (HEC)" are cells that express CD34 but do not express CD43, CD184, and CD73 (CD34 + / CD43 - / CD184 - / CD73 - )(CD34 + / CD43 - / CD184 - / CD73 - Cells do not express CD7, so CD34 + / CD43 - / CD184 - / CD73 - Cells are also called "CD34 + / CD7 - / CD43 - / CD184- / CD73 - cell").

[0114] In this specification, "pro-T cells (proT)" refer to hematopoietic cells generated in the process of differentiation from hematopoietic stem cells into CD3-positive T cells in human living bodies, and are CD34 and CD7 (CD34 + / CD7 + In addition, in the present specification, proT may be negative for CD43, CD1a and / or CD116.

[0115] In this specification, "mesoderm progenitor cells" refer to cells expressing at least one marker gene selected from the group consisting of T (synonymous with Brachyury), KDR, FOXF1, FLK1, BMP4, MOX1 and SDF1. Mesodermal progenitor cells are not distinguished from mesodermal cells, and cells expressing the above marker genes weakly can be referred to as "mesoderm progenitor cells".

[0116] In this specification, "regulatory T cells" refer to T cells that have the ability to inhibit the activation of effector T cells when stimulated via T cell receptors and are responsible for suppressing immune responses (immune tolerance). Regulatory T cells are usually CD25 + / FOXP3 + CD25 + / CD127 - cells, and the presence of CD4 + or CD8 + In the present invention, regulatory T cells can be CD4 + cells (ie, CD4 + / CD25 + / FOXP3 + or CD4 + / CD25 + / CD127 - ) or CD8 + cells (i.e., CD8 + / CD25 + / FOXP3 + or CD8 + / CD25 + / CD127 - ), and more preferably CD4 + In addition, the transcription factor FOXP3 is known to be a CD4 + / CD25 + Master regulator of regulatory T cells.

[0117] In this manual, "CD4 +"T cells" refers to cells that are positive for the surface antigen CD4 in T cells. Preferably, CD4 + T cells are CD8 negative (CD4 + / CD8 - , CD4 single positive (SP)), and in this case, since T cells can be identified by surface antigens CD3 and CD45 positivity, CD4 + T cells can be identified as CD8 negative and CD4, CD3, and CD45 positive (CD4 + / CD8 - / CD3 + / CD45 + cells). CD4 + T cells are CD25 and FOXP3 negative and CD127 positive. + Examples of T cells include helper T cells.

[0118] In this manual, "CD8 + "T cells" refers to cells that are positive for the surface antigen CD8 in T cells. Preferably, CD8 + T cells are CD4 negative (CD4 - / CD8 + , CD8 single positive (SP)), and in this case, since T cells can be identified by surface antigens CD3 and CD45 positivity, CD8 + T cells can be identified as CD4 negative and CD8, CD3, and CD45 positive cells (CD4 - / CD8 + / CD3 + / CD45 + cells). CD8 + T cells are CD25 and FOXP3 negative and CD127 positive. + Examples of T cells include cytotoxic T cells.

[0119] In this specification, "stromal cells" are cells constituting connective tissue supporting parenchymal cells in living tissues, and particularly refer to stromal cells capable of producing ATO (producing T cells), usually, stromal cells are contained in hematopoietic tissues (such as bone marrow or thymus), and their examples include fibroblasts, blood cells, mesenchymal stem cells, endothelial cells, smooth muscle cells, epithelial cells and tissue stem cells. Stromal cells can be established cells, such as MS-5, OP9 and S17 (they are mouse bone marrow cell strains), or HS-5 and HS-27a (they are human stromal cell strains), or can be primary cells collected from people, cells differentiated from pluripotent stem cells (iPS cells, etc.), and in the case of cells induced to differentiate from pluripotent stem cells such as people, stromal cells induced to differentiate from human iPS cells (especially fibroblasts induced to differentiate from human iPS cells) are preferred.

[0120] In this specification, "CD4CD8 double positive T cells" refers to cells that are positive for both surface antigens CD4 and CD8 (CD8 + / CD4 + ), and since T cells can be identified by surface antigens CD3 and CD45 positivity, CD4CD8 double-positive T cells can be identified as cells positive for CD4, CD8, CD3, and CD45 (CD8 + / CD4 + / CD3 + / CD45 + cell).

[0121] In this specification, "CD4CD8 double negative T cells" refers to cells whose surface antigens CD4 and CD8 are both negative (CD8 - / CD4 - ), and since T cells can be identified by surface antigens CD3 and CD45 positivity, CD4CD8 double-negative T cells can be identified as cells that are CD4 and CD8 negative and CD3 and CD45 positive (CD8 - / CD4 - / CD3 + / CD45 + cell).

[0122] In the present specification, "cell population" means two or more cells of the same type or different types. "Cell population" also means a large number of cells of the same type or different types.

[0123] From the perspective of therapeutic application, the various cells used in the present invention are preferably cells that have passed Good Manufacturing Practice (GMP) certification.

[0124] Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), embryonic germ stem cells (EG cells) and Muse cells; and preferably iPS cells (more preferably human iPS cells). When the pluripotent stem cell is an ES cell or any cell derived from a human embryo, cells can be produced by destroying the embryo or not, and preferably the cells produced without destroying the embryo.

[0125] Regarding “ES cells”, various mouse ES cell strains established by Ingenious Targeting Laboratory, RIKEN, etc. can be used as mouse ES cells, and various human ES cell strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used as human ES cells. Available examples of human ES cell strains include CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc. provided by ESI Bio; H1 strains, H9 strains, etc. provided by WiCell Research; and KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc. provided by RIKEN.

[0126] "Induced pluripotent stem cells" are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are many different types of induced pluripotent stem cells. Available examples include iPS cells established by Yamanaka et al. by introducing four factors Oct3 / 4, Sox2, Klf4 and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), and human cell-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al. Cell, (2007) 131: 861-872.), Nanog-iPS cells established by introducing the above four factors and then screening using Nanog expression as an index (Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells generated by a method that does not include c-Myc (Nakagawa M, Yamanaka S. et al. Nature Biotechnology, (2008) 26, 101-106) and iPS cells established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al. Stem Cells. 31 (3): 458-66.). Other available examples include induced pluripotent stem cells (Yu J., Thomson JA et al., Science (2007) 318: 1917-1920.) produced by Thomson et al. and established by introducing four factors OCT3 / 4, SOX2, NANOG and LIN28, induced pluripotent stem cells (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells (JP 2008-307007A) produced by Sakurada et al., etc.

[0127] Other available examples are known in the art and are described in all published articles (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, No. 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7, 795-797) or any induced pluripotent stem cell described in patents (e.g., JP2008-307007A, JP2008-283972A, US2008 / 2336610, US2009 / 047263, WO2007 / 069666, WO2008 / 118220, WO2008 / 124133, WO2008 / 151058, WO2009 / 006930, WO2009 / 006997 and WO2009 / 007852).

[0128] As induced pluripotent stem cell strains, various iPS cell strains established by NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPS cell strains include HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain (RIKEN); Ff-I01s04 strain, QHJI strain, RWMH strain, DRXT strain, RJWI strain, YZWJ strain, ILCL strain, GLKV strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain (Kyoto University), etc.

[0129] "Nucleic acid" may be any molecule obtained by polymerizing nucleotides and molecules having the same function as nucleotides, examples of which include RNA as a polymer of ribonucleotides, DNA as a polymer of deoxyribonucleotides, a polymer as a mixture of ribonucleotides and deoxyribonucleotides, and a nucleotide polymer containing nucleotide analogs, and the nucleic acid may also be a nucleotide polymer containing a nucleic acid derivative. The nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. Double-stranded nucleic acids include double-stranded nucleic acids in which one chain hybridizes with the other chain under stringent conditions.

[0130] Nucleotide analogs may be any molecules as long as they are obtained by modifying ribonucleotides, deoxyribonucleotides, RNA or DNA to improve nuclease resistance, stabilization, increase affinity with complementary strand nucleic acid, enhance cell permeability or visualization compared to RNA or DNA. Nucleotide analogs may be naturally occurring molecules or non-natural molecules. Examples include sugar-modified nucleotide analogs (e.g., 2'-O-methylribose substituted nucleotide analogs, 2'-O-propylribose substituted nucleotide analogs, 2'-methoxyethoxyribose substituted nucleotide analogs, 2'-O-methoxyethylribose substituted nucleotide analogs, 2'-O-[2-(guanidinium)ethyl]ribose substituted nucleotide analogs, 2'-fluororibose substituted nucleotide analogs, bridged nucleic acids (BNA), locked nucleic acids (LNA), ethylene bridged nucleic acids (ENA), peptide nucleic acids (PNA), oxygen peptide nucleic acids (OPNA) and peptide ribonucleic acids (PRNA)), phosphodiester bond-modified nucleotide analogs (e.g., phosphorothioate bond-substituted nucleotide analogs and N3'-P5' phosphoramidate bond-substituted nucleotide analogs), and the like.

[0131] The nucleic acid derivative may be any molecule as long as it is a molecule obtained by adding another chemical substance to nucleic acid, and the nucleic acid derivative has improved nuclease resistance, is more stable, has increased affinity with complementary chain nucleic acid, has enhanced cell permeability or increased visualization compared to the nucleic acid, and specific examples include 5'-polyamine adduct derivatives, cholesterol adduct derivatives, steroid adduct derivatives, bile acid adduct derivatives, vitamin adduct derivatives, Cy5 adduct derivatives, Cy3 adduct derivatives, 6-FAM adduct derivatives, biotin adduct derivatives and the like.

[0132] The method for producing a cell population containing regulatory T cells of the present invention (also referred to as "the production method of the present invention" in this specification) characteristically comprises the following steps:

[0133] 1) culturing a CD4+ TGF-βR-containing cell derived from a pluripotent stem cell in the presence of at least one substance selected from the group consisting of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor, and a TGF-βR agonist; + T cell population.

[0134] A CDK8 (cyclin-dependent kinase 8) and / or CDK19 inhibitor is defined as a substance that inhibits the function of CDK8 and / or CDK19, in particular, a substance that inhibits the kinase activity of CDK8 and / or CDK19, and may be an inhibitor of both CDK8 and CDK19, or may be an inhibitor of either one of them. Examples of CDK8 and / or CDK19 inhibitors include 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine (AS2863619) (hereinafter also referred to as "Compound 1"); 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine (AS3334366) (hereinafter also referred to as "Compound 2"); siRNA, shRNA, dsRNA, miRNA and antisense nucleic acids encoding CDK8 and / or CDK19 genes and / or their transcripts, and expression vectors expressing them; US8598344, WO2013 / 116786, Proc.Natl.Acad.Sci.USA109 13799-13804(2012), WO2013 / 001310, WO2013 / 040153, WO2014 / 029726, WO2014063778, WO2014 / 072435, WO2014 / 09 0692、WO2014 / 106606、WO2014 / 123900、WO2014 / 154723、WO2014 / 194245、WO2015 / 049325、WO2015 / 100420、WO2015 / Compounds described in WO2015 / 159937 and WO2015 / 159938 (especially (2E)-N-(2-fluoro-4-(morpholin-4-ylmethyl)phenyl)-3-(4-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)acrylamide (hereinafter also referred to as “Compound 3”) and (2E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(1-methyl-1H-indol-5-yl)acrylamide (hereinafter also referred to as “Compound 4”));Compounds described in WO2016 / 009076 and WO2018 / 159805 (especially 8-(2,4-difluorophenoxy)-1-methyl-4,5-dihydro-1H-thieno[3,4-g]indazole-6-carboxamide (hereinafter also referred to as "Compound 5"), 4-((4-fluorophenyl)sulfonyl)-3-(2-(imidazo[1,2-b]pyridazin-6-ylsulfanyl)ethyl)-3,4-dihydroquinoxaline -2(1H)-one (hereinafter also referred to as "Compound 6"), 2-(benzylamino)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzamide (hereinafter also referred to as "Compound 7"), 3-(3-(benzyloxy)phenyl)-1H-pyrrolo[2,3-b]pyridine (hereinafter also referred to as "Compound 8"), 4-(4-(2,3-dihydro-1,4-benzodioxin-6-yl)-1H-pyrazol-3-yl)benzene- 1,3-diol (especially its trifluoroacetate) (hereinafter also referred to as "Compound 9"), N-butyl-8-(4-methoxyphenyl)-1,6-naphthyridine-2-carboxamide and 8-(4-methylphenyl)-N,N-dipropyl-1,6-naphthyridine-2-carboxamide (especially its trifluoroacetate) (hereinafter also referred to as "Compound 10")); compounds described in WO2022 / 107877 (especially (E)-(4-(3-( diethyl 5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamide)benzyl)phosphonate (hereinafter also referred to as "Compound 11"), 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide (BI-1347) and 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile (Senexin B)); MSC2530818 (CAS No.: 1883423-59-3) and CCT251921 (CAS No.: 1607837-31-9), etc. Preferred among these substances are compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, BI-1347, Senexin B, MSC2530818, CCT251921, and siRNAs against genes encoding CDK8 and / or CDK19 and / or their transcripts; more preferred are compound 1, compound 2, and siRNAs against genes encoding CDK8 and / or CDK19 and / or their transcripts;And compound 1 is also preferred. CDK8 and / or CDK19 inhibitors can be used alone or in combination of two or more thereof. In addition, as a method for inhibiting CDK8 and / or CDK19, the method can also be performed by knocking out genes encoding CDK8 and / or CDK19. As a method for knocking out such a gene, for example, methods known in the art, such as genome editing using ZFN, TALEN, CRISPR / Cas system, etc., can be used. ;

[0135] TNFR2 (tumor necrosis factor receptor-2) agonists are defined as substances that can bind to TNFR2 to activate TNFR2 signaling, and their examples include antibodies (e.g., anti-TNFR2 antibodies), peptides, low molecular weight compounds, proteins, etc. Examples of TNFR2 agonist antibodies include monoclonal antibodies that bind to TNFR2, such as clone MR2-1 (HycultBiotech) and clone MAB2261 (R&D Systems). TNFR2 agonists can be TNF-α mutant proteins that only bind to TNFR2 as agonists. TNFR2 agonists can be used alone or in combination of two or more thereof.

[0136] The TNFR2 agonist is preferably a TNFR2 agonist antibody. The antibody may be a functional fragment thereof, and examples of functional fragments include Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies and minibodies. Examples of antibodies include those derived from animals such as mice, rats, cattle, rabbits, goats, sheep and guinea pigs. The isotype of the antibody is not particularly limited, and examples thereof include IgG (IgG1, IgG2, IgG3 and IgG4), IgA, IgD, IgE and IgM. The antibody may be a monoclonal antibody or a polyclonal antibody, and preferably a monoclonal antibody, and the antibody may be a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody) and the like. The antibody may be produced by a known method, for example, by constructing an expression vector containing a nucleic acid encoding the antibody, and culturing a transformant into which the nucleic acid is introduced, or culturing a hybridoma producing the antibody.

[0137] mTOR (mechanistic target of rapamycin) inhibitors are defined as substances that inhibit the function of mTOR, and these substances include substances that inhibit the function of mTOR itself and substances that inhibit the function of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (which are mTOR complexes). Examples of mTOR inhibitors include rapamycin or its derivatives, everolimus, temsirolimus, ridaforolimus, sirolimus, KU0063794, AZD805, AZD8055, WYE-354, WAY-600, WYE-687, Pp121, Pp242, Dactolisib, Sapanisertib, Omipalisib, Vistusertib, Torin 1, Torin 2, etc. Other examples of mTOR inhibitors include siRNA, shRNA, dsRNA, miRNA and antisense nucleic acids encoding mTOR genes and / or their transcripts, and expression vectors expressing them; and also include siRNA, shRNA, dsRNA, miRNA and antisense nucleic acids encoding genes and / or their transcripts of enzymes that phosphorylate and activate mTOR, and expression vectors expressing them. Among them, the mTOR inhibitor is preferably rapamycin or a derivative thereof, and more preferably rapamycin. The mTOR inhibitor can be used alone or in combination of two or more thereof.

[0138] TGF-βR (transforming growth factor-β receptor) agonist is defined as a substance that can bind to TGF-βR to activate TGF-βR signaling. Examples of TGF-βR agonists include factors belonging to the TGF-β superfamily, and the TGF-β superfamily includes the TGF-β family, the activin family, and the BMP (bone morphogenetic protein) family. Examples of factors belonging to the TGF-β superfamily include TGF-β (TGF-β1, TGF-β2, and TGF-β3), activin A, activin B, GDF-8, GDF-11, and the like. TGF-βR agonists are preferably TGF-β. TGF-βR agonists can be used alone or in combination of two or more thereof.

[0139] In addition, CDK8 and / or CDK19 inhibitors, TNFR2 agonists, mTOR inhibitors and TGF-βR agonists can be used in free form or in salt form. Examples of salts include salts formed with inorganic bases, such as sodium salts, magnesium salts, potassium salts, calcium salts and aluminum salts; salts formed with organic bases, such as methylamine salts, ethylamine salts and ethanolamine salts; salts formed with basic amino acids (such as lysine, ornithine and arginine); and ammonium salts. These salts may be acid addition salts, and specific examples of such salts include addition salts formed with the following acids: inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid; organic acids, such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid and ethanesulfonic acid; and acidic amino acids, such as aspartic acid and glutamic acid. CDK8 and / or CDK19 inhibitors, TNFR2 agonists, mTOR inhibitors and TGF-βR agonists also include hydrates, solvates, polymorphs and the like.

[0140] When the CDK8 and / or CDK19 inhibitors, TNFR2 agonists, mTOR inhibitors and TGF-βR agonists contain an asymmetric carbon, these include R-isomers, S-isomers and mixtures (eg, racemates) containing the two isomers in any ratio.

[0141] siRNA (small interfering RNA) is a double-stranded oligo RNA containing RNA having a sequence complementary to the nucleotide sequence of the mRNA of the target gene or a partial sequence thereof (target nucleotide sequence) and its complementary chain. siRNA also includes: RNA as a single-stranded RNA (small hairpin RNA (shRNA)), in which the sequence complementary to the target nucleotide sequence (first sequence) and its complementary sequence (second sequence) are partially connected by a hairpin loop, in which the first sequence forms a double-stranded structure with the second sequence by adopting a hairpin loop structure; and dumbbell-shaped nucleic acid, in which the two ends of the double-stranded structure of the first sequence and the second sequence are closed with a ring structure.

[0142] siRNA may have an overhang at the 5'-end or 3'-end of one or both of the sense strand and the antisense strand. The overhang is formed by adding one to several (e.g., 1, 2, or 3) bases at the end of the sense strand and / or the antisense strand. The base length of siRNA is not particularly limited as long as RNA interference can be induced, for example, one side chain has 10 to 50 bases, 15 to 30 bases, or 21 to 27 bases.

[0143] siRNA is a siRNA targeting mRNA encoding CDK8 and / or CDK19, mTOR, etc., and can be designed according to the information on the base sequence of mRNA, and the sequence is not particularly limited as long as RNA interference can be induced. siRNA can be obtained by chemical synthesis using known methods, or produced using genetic recombination technology.

[0144] The concentration of the CDK8 and / or CDK19 inhibitor in the culture medium is not particularly limited as long as regulatory T cells can be produced, and is appropriately adjusted according to the type of CDK8 and / or CDK19 inhibitor used, etc. The concentration of the CDK8 and / or CDK19 inhibitor is, for example, 0.1 to 300 nM, and preferably 0.5 to 150 nM.

[0145] The concentration of the TNFR2 agonist in the culture medium is not particularly limited as long as regulatory T cells can be produced, and is appropriately adjusted depending on the type of TNFR2 agonist used, etc. The concentration of the TNFR2 agonist is, for example, 0.0001 to 100 μg / mL, and preferably 0.01 to 10 μg / mL.

[0146] The concentration of the mTOR inhibitor in the culture medium is not particularly limited as long as regulatory T cells can be produced, and is appropriately adjusted depending on the type of mTOR inhibitor used, etc. The concentration of the mTOR inhibitor is, for example, 1 to 100 nM, and preferably 10 to 100 nM.

[0147] The concentration of the TGF-βR agonist in the culture medium is not particularly limited as long as regulatory T cells can be produced, and is appropriately adjusted depending on the type of TGF-βR agonist used, etc. The concentration of the TGF-βR agonist is, for example, 0.1 to 100 ng / mL, and preferably 1 to 50 ng / mL.

[0148] In step (1), the culture is carried out in the presence of at least one substance selected from the group consisting of: CDK8 and / or CDK19 inhibitors (hereinafter also abbreviated as "A"), TNFR2 agonists (hereinafter also abbreviated as "M"), mTOR inhibitors (hereinafter also abbreviated as "R") and TGF-βR agonists (hereinafter also abbreviated as "T"). These four substances can be used alone or in combination with AM, AR, AT, MR, MT, RT, AMR, ART, AMT, MRT or AMRT. These four substances are preferably used in combination with AR, ART or AMRT, and more preferably in combination with AMRT.

[0149] The culture medium used in the culture of step (1) uses a culture medium for culturing animal cells as a basal culture medium, and contains at least one substance selected from the group consisting of the following: CDK8 and / or CDK19 inhibitors, TNFR2 agonists, mTOR inhibitors, and TGF-βR agonists described above. The basal culture medium is not particularly limited as long as it can be used to culture animal cells, and their examples include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow's MEM, Improved MEM Zinc Option, IMDM, 199 culture medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, Fischer culture medium, etc. These culture media can be used alone or in a mixture of two or more thereof.

[0150] The culture medium may contain serum or may not contain serum. The culture medium may also contain serum substitutes (e.g., albumin, transferrin, Knockout serum substitute (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, ITS-supplements, B27 (trademark) supplements, etc.). Serum substitutes may be used alone or in combination of two or more thereof.

[0151] The culture medium may further contain one or more substances selected from lipids, amino acids (non-essential amino acids, etc.), L-glutamine, vitamins, growth factors, cytokines (IL-2, etc.), beads bound with CD3 antibodies and / or CD28 antibodies, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc., and particularly preferably contains IL-2. As the culture medium, it is desirable to use a chemically defined culture medium (such as serum) that does not contain substances with unclear components, because this can reduce the difference in the culture medium between batches and can prepare cells of stable quality.

[0152] When the culture medium contains IL-2, the concentration of IL-2 is, for example, 1 to 500 ng / mL, preferably 1 to 300 ng / mL. +When the cell population of T cells is cultured in step (1), its concentration is, for example, preferably 1 to 100 ng / mL, more preferably 1 to 50 ng / mL, further preferably 3 to 30 ng / mL, and particularly preferably 5 to 15 ng / mL. In addition, when the cell population containing regulatory T cells (as primary culture cells) is cultured and expanded using a combination of AMT in step (A) described below, in order to maintain a high FOXP3 expression rate, the IL-2 concentration contained in the culture medium is set to, for example, preferably 30 to 300 ng / mL, more preferably 50 to 200 ng / mL.

[0153] The pH of the medium is usually 7.0 to 7.8, preferably 7.2 to 7.6. In order to prevent contamination before use, the medium is preferably sterilized by a method such as filtration, UV irradiation, heat sterilization or radiation.

[0154] The culture is performed in the presence or absence of feeder cells. Since the production method of the present invention can stably produce regulatory T cells having uniform properties without mixing substances of unknown composition, it is desirable to perform the production method in the absence of feeder cells.

[0155] The culture conditions of the production method of the present invention are not particularly limited, and the culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C, and the CO2 concentration is, for example, 2% to 10%, preferably 2% to 5%, and the oxygen concentration is, for example, 1% to 20%, preferably 5% to 20%.

[0156] The culture time is not particularly limited either, and can be appropriately determined by those skilled in the art when monitoring the number of regulatory T cells, and is, for example, 10 days or longer, preferably 1 week or longer, more preferably 2 weeks or longer. The upper limit of the culture time is not particularly limited, and is, for example, 35 days or shorter, preferably 28 days or shorter, and more preferably 21 days or shorter. In the culture of the present invention, multiple passages can be performed as needed to obtain the regulatory T cells of the desired amount, or the addition and replacement of the culture medium can be performed. The culture in the present invention can be carried out using known CO2 incubators. The culture container is not particularly limited, and can be appropriately selected from plates, dishes, culture dishes, flasks, bags, bottles, grooves (culture tanks), bioreactors, etc.

[0157] The pluripotent stem cells used in the present invention and the cells for producing pluripotent stem cells can be derived from humans, or can be derived from mammals other than humans (non-human mammals), and are preferably derived from humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cattle, horses, sheep and monkeys. In addition, universal iPS cells (with specific HLA that does not cause immune rejection in a large number of patients, or HLA is knocked out) can also be used.

[0158] The CD4 + The cell population of T cells is a cell population containing CD4 T cells differentiated from pluripotent stem cells (especially iPS cells (especially human iPS cells)). + T cells. A cell population containing CD4 + CD4 + The proportion of T cells (the number of cells) is, for example, 5% or more, preferably 50% or more, more preferably 90% or more, and the upper limit is, for example, 100% or less.

[0159] The production method of the present invention may further include isolating regulatory T cells to concentrate the obtained regulatory T cells. Isolation of regulatory T cells may be performed by known methods such as a method using flow cytometry or a magnetic cell separation method.

[0160] According to the production method of the present invention, a cell population containing regulatory T cells with a high content ratio of regulatory T cells can be produced. The ratio of regulatory T cells contained in the cell population containing regulatory T cells (the number of cells) is, for example, 10% or more, preferably 40% or more, more preferably 80% or more, and the upper limit is, for example, 90% or less, more preferably 100% or less.

[0161] According to the production method of the present invention, a cell population containing regulatory T cells having a high content ratio of regulatory cells showing high inhibitory function (for example, Helios-positive cells as a functional marker of suppressibility, cells that actually show high suppressibility in vitro and / or in vivo) can be produced. The proportion of regulatory T cells contained in the cell population containing regulatory T cells (the number of cells) is, for example, 10% or more, preferably 50% or more, more preferably 80% or more, and the upper limit is, for example, 90% or less, more preferably 100% or less.

[0162] The production method of the present invention can be applied to + In addition to T cell populations, it can also be applied to CD8 + T cell population.

[0163] In another embodiment of the present invention, a method for proliferating a cell population containing regulatory T cells (also referred to as "the proliferating method of the present invention" in this specification) characteristically comprises the following steps:

[0164] A) Cell populations containing regulatory T cells (as primary culture cells) are cultured in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, and a TGF-βR agonist, or in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, a TGF-βR agonist, and an mTOR inhibitor.

[0165] Unless otherwise specified, the proliferation method of the present invention can be performed in the same manner as the production method of the present invention described above, but using a cell population containing regulatory T cells as primary culture cells, etc.

[0166] The cell group containing regulatory T cells used in the proliferation method of the present invention is a primary culture cell separated from a biological tissue (such as bone marrow, umbilical cord blood and blood). The proportion of regulatory T cells contained in the cell group containing regulatory T cells (the number of cells) is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and the upper limit is, for example, 100% or less.

[0167] The cell population containing regulatory T cells used in the proliferation method of the present invention can be derived from humans, or can be derived from mammals other than humans (non-human mammals), and preferably derived from humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cattle, horses, sheep and monkeys.

[0168] In step (A), the culture is carried out in the presence of (i) a CDK8 and / or CDK19 inhibitor (A), a TNFR2 agonist (M), an mTOR inhibitor (R) and a TGF-βR agonist (T), or in the presence of (ii) a CDK8 and / or CDK19 inhibitor (A), a TNFR2 agonist (M) and a TGF-βR agonist (T). More preferably, the culture is carried out in the presence of AMT.

[0169] The production method of the present invention may further include step (2) before step (1) of inducing pluripotent stem cells to differentiate into cells containing CD4 + T cell population.

[0170] Differentiation of pluripotent stem cells into cells containing CD4 + T cell populations can be induced according to known methods. Examples of such methods include inducing pluripotent stem cells to differentiate into CD4 + T cells can then be induced to differentiate into CD4 + T cells differentiate into cells containing CD4 + A population of T cells that can differentiate into CD4 + Examples of T cells include CD34 +cells, mesodermal progenitor cells, CD4CD8 double negative T cells, CD4CD8 double positive T cells, etc., CD34 + CD34 cells or mesodermal progenitor cells are preferred, hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic progenitor cells, pro-T cells or mesodermal progenitor cells are more preferred, and hematopoietic endothelial cells are particularly preferred. + The cells express CD34 (CD34 + ) cells, especially those expressing CD34 but not CD7 (CD34 + / CD7 - ) cells. + Examples of cells include hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells.

[0171] Differentiation of pluripotent stem cells into CD4 + T cells can be induced according to known methods (see, for example, WO2021 / 085576, etc.). For example, CD4 + T cells can be produced by a feeder-free method, in particular the "embryoid body method (EB method)" (see AE Grigoriadis et al. (2010). Blood, 115(14): 2769-2776.), wherein it is preferred to form embryoid bodies without using feeder cells that produce hematopoietic progenitor cells.

[0172] Pluripotent stem cells can also be cultured on feeder cells to induce differentiation into hematopoietic stem cells and / or hematopoietic progenitor cells (see WO2011 / 096482 and WO2013 / 176197). From the viewpoint of being easy to induce differentiation into mesodermal lineages, feeder cells are preferably stromal cells. From the viewpoint of being helpful for differentiation into hematopoietic lineages, stromal cells are preferably OP9 cells, 10T1 / 2 cells (C3H10T1 / 2 cells), and the like.

[0173] Differentiation of pluripotent stem cells into CD34 + Cells can be differentiated according to known methods, and when the pluripotent stem cells are iPS cells, for example, differentiation of hematopoietic progenitor cells can be induced by the methods described in WO2017 / 221975, WO2018 / 135646, and Cell Reports 2 (2012) 1722-1735 to produce CD34 + cell.

[0174] Used to induce differentiation into CD4 + T cells differentiate into cells containing CD4 +Examples of methods for producing a cell population of T cells include the artificial thymic organoid (ATO) method (see WO2017 / 075389, etc.). The ATO method includes culturing cells containing cells that can differentiate into CD4 + Three-dimensional cell aggregates of T cells and stromal cells expressing Notch ligands, thereby + The differentiation of T cells can be induced efficiently. The ATO method is a known method and can be performed with reference to the descriptions of WO2017 / 075389, WO2021 / 085576, etc. Other methods include the differentiation method described in WO2021 / 092581 (e.g., with MS5-DLL1 / 4 stromal cells, OP9 or OP9-DLL1 stromal cells, or EpCAM-CD56 + stromal cell co-culture).

[0175] When the ATO method was performed, the isolated cells could be differentiated into CD4 + The ATO method may not be performed on cells that can differentiate into CD4 T cells before the ATO method is performed, and the ATO method may also be performed on cells that can differentiate into CD4 T cells separated by known methods (e.g., flow cytometry or magnetic cell separation methods). + T cells are carried out.

[0176] Preferably, the nucleic acid for expressing Notch ligand is introduced into the matrix cell. The matrix cell expressing Notch ligand can be produced by introducing the nucleic acid into the matrix cell. In addition, the matrix cell expressing Notch ligand can be produced by introducing the nucleic acid into pluripotent stem cells (especially iPS cells (especially human iPS cells)), and then differentiating the pluripotent stem cells into matrix cells.

[0177] Examples of the matrix cells used in the production method of the present invention include matrix cells induced to differentiate from pluripotent stem cells (especially iPS cells (especially human iPS cells)), wherein it is desirable to use cells obtained by inducing differentiation of pluripotent stem cells (especially iPS cells (especially human iPS cells)) into fibroblasts. The differentiation of pluripotent stem cells to matrix cells can be carried out according to known methods, and when the pluripotent stem cells are human iPS cells, for example, fibroblasts induced to differentiate from iPS cells can be produced by the method described in PLoS ONE 8 (10): e77673, 2013.

[0178] Notch ligands are not particularly limited, and include canonical Notch ligands and non-canonical Notch ligands described in WO2017 / 075389. Examples of canonical Notch ligands include DLL4 (delta-like ligand 4), DLL1 (delta-like ligand 1), JAG1 (Jagged 1), JAG2 (Jagged 2), etc. These can be used alone or in combination of two or more thereof. Examples of non-canonical Notch ligands include contactin-1, NOV / CCN3, contactin-6, periostin / OSF-2, DLK2 / EGFL9, Pref-1 / DLK1 / FA1, DNER, thrombospondin-2, MAGP-1 / MFAP2, thrombospondin-3, MAGP-2 / MFAP5, thrombospondin-4, and spindle protein-1 (Netrin-1). Human DLL4 is preferably used as a Notch ligand.

[0179] The method for introducing a nucleic acid for expressing a Notch ligand into a stromal cell or a pluripotent stem cell is not particularly limited, and various known or general methods can be used. Typically, an expression vector is used to introduce a nucleic acid for expressing a Notch ligand into a stromal cell, and the nucleic acid is expressed. The expression vector may be linear or circular, and may be a non-viral vector (such as a plasmid), a viral vector, or a transposon vector.

[0180] The method by which expression vector is introduced into stroma cells can be carried out according to the embodiment. For example, expression vector can be introduced into stroma cells by known methods such as virus infection method, calcium phosphate method, lipid transfection method, microinjection method or electroporation method. Expression vector can be prepared into the form suitable for use in each method by known means or suitably using commercially available test kit (according to its specification sheets).

[0181] The expression vector can be introduced into the matrix cells by a viral infection method. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors. When these viral vectors are used, the corresponding commercially available kits can be used to transfect the vector containing the nucleic acid for expressing Notch ligands and the packaging vector (plasmid) of each virus into the host cell to produce recombinant viruses, and then the recombinant viruses obtained can be used to infect matrix cells.

[0182] In addition to the nucleic acid for expressing the Notch ligand, the expression vector may contain sequences such as a nuclear localization signal (NLS) and a multiple cloning site (MCS), if necessary. The expression vector may also contain nucleic acids (base sequences) encoding "functional genes", such as reporter genes (e.g., genes encoding fluorescent proteins of various colors), drug selection genes (e.g., kanamycin resistance gene, ampicillin resistance gene, and puromycin resistance gene), and suicide genes (e.g., genes encoding: diphtheria A toxin, herpes simplex virus thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9, etc.).

[0183] Three-dimensional cell aggregates can be differentiated into CD4 + The method is formed by combining cells that can differentiate into CD4 T cells and stromal cells that express Notch ligands. Preferably, the nucleic acid for expressing Notch ligands is introduced into the stromal cells. + The ratio of cells that can differentiate into CD4 T cells can be determined by a person skilled in the art according to the stromal cells used and the cells that can differentiate into CD4 + The type of cells of T cells and the like are appropriately adjusted, and are, for example, 100:1 to 1:100, 90:1 to 1:90, 80:1 to 1:80, 70:1 to 1:70, 60:1 to 1:60, 50:1 to 1:50, 40:1 to 1:40, 30:1 to 1:30, 20:1 to 1:20, 10:1 to 1:10, etc. For example, when the stromal cells are mouse bone marrow cells (especially MS-5), the ratio is preferably 20:1 to 1:4, and when the stromal cells are pluripotent stem cell-derived stromal cells (especially fibroblasts induced to differentiate from iPS cells (especially human iPS cells)), the ratio is preferably 4:1 to 1:4, and further preferably 1:1.

[0184] The medium used for culturing the three-dimensional cell aggregates is not particularly limited, and examples thereof include serum-containing medium, serum-free medium or xeno-free medium, and preferably includes serum-free medium, particularly insulin-containing serum-free medium (also containing biotin, transferrin and albumin).

[0185] Examples of basal culture media for culturing three-dimensional cell aggregates include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, ImprovedMEM Zinc Option, IMDM, 199 medium, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, Fischer medium, etc. These culture media may be used alone or as a mixture of two or more thereof.

[0186] The culture medium may contain serum or may not contain serum. The culture medium may also contain serum substitutes (e.g., albumin, transferrin, Knockout serum substitute (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, ITS-supplements, B27 (trademark) supplements, etc.). Serum substitutes may be used alone or in combination of two or more thereof.

[0187] The culture medium may additionally contain one or more substances in lipids, amino acids (non-essential amino acids, etc.), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. As a culture medium, it is desirable to use a chemically defined culture medium (such as serum) that does not contain substances with unclear components, because this can reduce the differences in culture medium between batches and can prepare cells with stable quality. In addition, the culture medium components described in WO2017 / 075389, etc. can also be appropriately blended.

[0188] The culture temperature of the three-dimensional cell aggregate is, for example, 20 to 40° C., preferably about 37° C., and the CO2 concentration is, for example, 2% to 10%, preferably 2% to 5%, and the oxygen concentration is, for example, 1% to 20%, preferably 5% to 20%. The cell density at the start of the culture can be, for example, about 1.0×10 4 to 1.0×10 10 cells / mL.

[0189] The culture time of the three-dimensional cell aggregates can be determined by a person skilled in the art according to, for example, the cell types used (such as stromal cells and cells that can differentiate into CD4 + The culture time is appropriately adjusted for cells (cells of T cells), and is, for example, 4 weeks or longer, preferably 5 weeks or longer, more preferably 6 weeks or longer, and further preferably 7 weeks or longer. The upper limit of the culture time is not particularly limited, and is preferably 16 weeks or shorter, more preferably 14 weeks or shorter, further preferably 12 weeks or shorter, particularly preferably 9 weeks or shorter.

[0190] Due to the removal of CD4+ Cells other than T cells can be included in the CD4 + T cell population, so CD4 + T cells can be sorted, for example, by flow cytometry (usually fluorescence activated cell sorting: FACS) using fluorescently labeled anti-CD4 antibodies and a cell sorter targeting cells containing CD4 + T cell populations can be separated and recovered. In addition, CD4SP T cells can be separated and recovered. In addition, effector T cells (Tconv) separated as CD25 negative can be separated and recovered.

[0191] When containing CD8 + When the cell population of T cells is used in step (1), step (2) is performed to induce CD8 + Differentiation of T cell populations.

[0192] In the production method of the present invention, as CD4 + T cells can be used in which an expression construct (also referred to as "CNS-Foxp3" in this specification) is introduced, wherein the expression construct comprises:

[0193] (a) CNS1, CNS2, and CNS3 of the Foxp3 gene;

[0194] (b) a promoter; and

[0195] (c) Nucleic acid encoding FOXP3.

[0196] The cells into which the expression construct is introduced are not particularly limited and may be at any stage of differentiation, and examples thereof include CD4 + T cells, pluripotent stem cells or can differentiate into CD4 + T cells. For the introduction of expression constructs into CD4 + T cells, pluripotent stem cells or can differentiate into CD4 + Examples of methods for expressing T cells include the methods described above.

[0197] The expression construct of the present invention is used to express FOXP3, and comprises: (a) CNS1 (conserved non-coding sequence 1), CNS2 (conserved non-coding sequence 2) and CNS3 (conserved non-coding sequence 3) of the Foxp3 gene, (b) a promoter and (c) a nucleic acid encoding FOXP3. By introducing the expression construct into CD4 + T cells, pluripotent stem cells or can differentiate into CD4 +Regulatory T cells can be efficiently produced in T cell cells. The expression construct is not particularly limited as long as it can express FOXP3. In addition to the above (a), (b) and (c), the expression construct preferably also contains a terminator, a polyadenylation signal, Foxp3 3'UTR, etc., and more preferably, its components (such as these components) function in the cell into which the expression construct is introduced.

[0198] The base sequence of the human Foxp3 gene is registered as RefSeq accession number NM_001114377 (SEQ ID No. 1) and NM_014009 (SEQ ID No. 2), and its amino acid sequence is also registered as RefSeq accession number NP_001107849 (SEQ ID No. 3) and NP_054728 (SEQ ID No. 4). These RefSeq IDs are registered on the NCBI website. In the present invention, the above-mentioned genes also include degenerate products and variants of genes other than genes having base sequences registered in the above-mentioned databases, and the desired variants are variants encoding proteins having biological activities equivalent to proteins composed of the above-mentioned amino acid sequences. Examples of variants include FOXP3 variants having amino acid sequences having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the natural amino acid sequence. In the present specification, FOXP3 refers to a protein, and Foxp3 refers to a gene; however, when such an interpretation is appropriate, FOXP3 and Foxp3 refer to a gene and a protein, respectively.

[0199] The nucleic acid encoding FOXP3 is not particularly limited as long as it is a nucleic acid encoding FOXP3 protein, and is preferably cDNA of FOXP3.

[0200] CNS1, CNS2 and CNS3 used in the present invention are all derived from Foxp3 gene. CNS1, CNS2 and CNS3 are Foxp3 enhancer elements. The promoter used in the present invention is not particularly limited, and examples include CAG promoter, ubiquitin gene promoter, Foxp3 gene promoter, EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, Moloney murine leukemia virus (MoMuLV) LTR, herpes simplex virus thymidine kinase (HSV-TK) promoter, etc.; and preferably Foxp3 gene promoter. Preferred examples of the base sequences of CNS1, CNS2, CNS3 and promoters of human Foxp3 gene include base sequences represented by SEQ ID No.5 to 8, respectively. Promoters, CNS1, CNS2, CNS3 include variants even if they do not have the base sequences described above, and desired variants are variants having biological activities comparable to those of promoters, CNS1, CNS2, CNS3 consisting of the above base sequences. Examples of variants include those having base sequences that have 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the natural base sequence.

[0201] The arrangement (order) of the promoter, CNS1, CNS2, CNS3 and the nucleic acid encoding FOXP3 in the expression construct is not particularly limited, and preferably, CNS1, CNS2 and CNS3 are positioned upstream of the promoter; and the order of CNS1, CNS2, CNS3, the promoter and the nucleic acid encoding FOXP3 from the 5' end side is more preferred.

[0202] The cells into which the expression construct is introduced are not particularly limited as long as they are CD4 + T cells, pluripotent stem cells or can differentiate into CD4 + Any cell may be a T cell, and examples thereof include pluripotent stem cells, hematopoietic stem cells, hematopoietic endothelial cells, hematopoietic progenitor cells, progenitor T cells, mesodermal progenitor cells, helper T cells, and the like, pluripotent stem cells are preferred, and iPS cells are more preferred.

[0203] The regulatory T cells obtained by the production method and proliferation method of the present invention may be regulatory T cells into which foreign genes are introduced.

[0204] The "foreign gene" is a gene introduced from the outside to express a desired protein in regulatory T cells, and can be appropriately selected depending on the purpose of the regulatory T cells.

[0205] Exogenous gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR), and may also contain a gene for expressing a cytokine and / or a chemokine. As with general or known CAR, the CAR expressed by regulatory T cells is substantially configured so that the peptide at the following site is connected via a spacer as needed: (i) antigen recognition sites (e.g., single-chain antibodies) for recognizing cell surface antigens of cancer cells, (ii) transmembrane regions, and (iii) signal transduction regions for inducing T cell activation. Exogenous gene can also be, for example, a gene for expressing an exogenous T cell receptor (TCR). Exogenous TCR means that it is exogenous for the T cell into which the nucleic acid encoding exogenous TCR is to be introduced. The amino acid sequence of exogenous TCR may be identical or different from the amino acid sequence of the endogenous TCR of T cells. One or two or more exogenous genes (e.g., CAR and exogenous TCR) may be introduced.

[0206] Examples of methods for introducing foreign genes into cells include the above methods. The cells into which foreign genes are introduced are not particularly limited and may be at any stage of differentiation, and examples thereof include pluripotent stem cells (especially iPS cells), CD4 + T cells, regulatory T cells, etc.

[0207] The cell group containing regulatory T cells produced by the production method and proliferation method of the present invention can be used to treat animals (especially humans) with abnormally enhanced immune responses, and can be used, for example, to treat and prevent X-linked polyendocrine enteropathy with immune dysregulation (IPEX) syndrome, graft-versus-host disease (GVHD), organ transplant rejection, autoimmune diseases, inflammatory diseases, allergic diseases (hay fever, asthma, atopic dermatitis, eczema, food allergies, food hypersensitivity, urticaria, allergic rhinitis, allergic conjunctivitis and drug allergies), etc., but are not limited to this. The regulatory T cells produced by the production method of the present invention can be used for autologous transplantation or allogeneic transplantation. In addition, regulatory T cells can be used in combination with other drugs.

[0208] When such cell therapy is performed, the subject from which cells are isolated to produce regulatory T cells preferably has the same HLA type as the subject to which the regulatory T cells are to be administered, and is more preferably the same as the subject to which the regulatory T cells are to be administered, from the viewpoint that no rejection occurs.

[0209] According to the present invention, a drug (hereinafter also referred to as "the drug of the present invention") comprising a cell population containing regulatory T cells can be prepared. The drug of the present invention is preferably produced as a parenteral preparation by mixing an effective amount of regulatory T cells with a pharmaceutically acceptable carrier according to a known method (e.g., a method described in the Japanese Pharmacopoeia). The drug of the present invention is preferably prepared as a parenteral preparation, such as an injection, a suspension or an infusion. Examples of parenteral administration methods include intravenous, intra-arterial, intramuscular, intraperitoneal or subcutaneous administration. Examples of pharmaceutically acceptable carriers include solvents, substrates, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonic agents, surfactants, solubilizing agents, etc.

[0210] The dosage of the drug of the present invention can be appropriately determined according to various conditions such as the patient's weight, age, sex and symptoms. Usually, the drug of the present invention is administered so that the number of cells per administration is usually 1×10 6 Up to 1×10 10 cells, preferably 1×10 7 Up to 1×10 9 cells, more preferably 5×10 7 Up to 5×10 8 Cells. The medicine of the present invention can be administered once or several times. The medicine of the present invention may have a known form suitable for parenteral administration, such as injection or infusion. In addition, the medicine of the present invention may contain physiological saline, phosphate buffered saline (PBS), culture medium, etc. to stably maintain cells. Examples of culture medium include RPMI, AIM-V, X-VIVO10 and other culture mediums, but are not limited thereto. In addition, for the purpose of stability, a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. may be added to the medicine. The medicine of the present invention is applied to mammals, including humans.

[0211] As used in this specification and claims, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Therefore, unless otherwise expressly indicated, singular articles (e.g., in the case of English, "a / an", "the", etc.) should also be understood to include the concept of their plural forms.

[0212] Example

[0213] Examples will be given below to describe the present invention in more detail. However, the present invention is not limited to these examples at all.

[0214] Example 1 Differentiation of iPS cells into CD4SP T cells and isolation of CD4SP T cells

[0215] Step i: Differentiate the T cell-derived iPS cell line (4GAD1-4) or the non-T cell-derived iPS cell line (FFI01s04) into a cell population containing hematopoietic endothelial cells (HEC) and hematopoietic progenitor cells (HPC) by the embryoid body method (EB method) (hereinafter also referred to as the "EB method"). Steps i to v of the present invention are performed according to the method described in WO2021 / 085576, and the differences are extracted and described.

[0216] Step ii: In the T cell-derived iPS cell line 4GAD1-4, the date when the undifferentiated cells started to differentiate was defined as day 0, and the EB product on day 5 was subjected to the ATO differentiation culture method. In the non-T cell-derived iPS cell line FFI01s04, the EB product on day 7 was subjected to the ATO differentiation culture method.

[0217] Step iii: In this example, the EB product recovered without sorting was mixed with MS5hDLL4 cells so that the ratio of the number of cells was the total number of cells contained in the EB product: the total number of MS5hDLL4 cells = 4:1 (in 4GAD1-4) and 1:20 (in FFI01s04), and the droplet of the suspension was placed on an insert culture dish (Merck Millipore, MILLICELL INSERT 30MM ORGANOTYPIC PTFE 0.4UM 50 / PK) and floated on the culture medium together with the insert culture dish to start the ATO differentiation culture method. The composition of the culture medium is the same as that described in WO2021 / 085576.

[0218] Step iv: When the mixture of iPS cell-derived EB method product and MS5hDLL4 is cultured on an insert culture dish floating on the medium for 6 to 9 weeks, the cells in the mixture differentiate into CD4SP T cells and CD8SP T cells, such as Figure 1 shown.

[0219] Step v: ATO at this stage was suspended in 2% FBS / 1mM EDTA / PBS, CD4-positive CD8b-negative cells were sorted out from CD3-positive TCRab-positive T cells using a cell sorter, and the separated CD4SP T cells were expanded by culture. For expansion culture, Dynabeads human T-activator CD3 / CD28 (Thermo Fisher Scientific, hereinafter referred to as "Dynabeads") was used, which is a bead combined with CD3 antibody and CD28 antibody. The ratio of beads to cells in the first expansion culture was only 4:1 (the second and subsequent expansion cultures were 1:1). The culture medium (hereinafter referred to as "basal medium") was prepared as follows: IL-2 (final concentration: 10 ng / mL) and PAA (phosphorylated ascorbic acid) (100 μg / mL) were added to α-MEM (Thermo Fisher Scientific, MEM-a, nucleosides, prepared as powder) supplemented with 15% fetal bovine serum (FBS), PSG (penicillin, streptomycin and L-glutamine) supplemented in a 100-fold dilution, and ITS (Thermo Fisher Scientific, insulin-transferrin-selenium (ITS-G) (100×)) supplemented in a 100-fold dilution.

[0220] CD4SP T cells derived from iPS cells showed a proliferation of hundreds to thousands of times in the initial expansion culture. The cytokine production capacity of these CD4SP T cells was evaluated by intracellular staining and flow cytometry. Figure 2 As shown, in the presence of monensin (2 μM), the cells were stimulated with PMA (40 ng / mL) and ionomycin (4 μg / mL) for 4 hours. The results showed that after stimulation, 13.24% of the CD4SP T cells derived from 4GAD1-4 produced interferon-γ (IFN-γ) and 23.77% produced interleukin-4 (IL-4) (the two were partially overlapping).

[0221] As a control, inflammatory T cells (Tconv) further separated in a CD25-negative state from CD4SP T cells were sorted from the peripheral blood mononuclear cell fraction (PBMC) of a living human body, and the cells expanded by culture were placed in a similar manner, and the results showed that 43.6% of Tconv also produced interferon-γ (IFN-γ) and 38.8% of Tconv produced interleukin-4 (IL-4) (again, there was a partial overlap between the two). This result shows that the cells obtained by expanding CD4SP T cells (obtained from ATO by culture using Dynabeads and IL-2) are FOXP3-negative cells (close to Tconv that secretes inflammatory cytokines).

[0222] Example 2 Induction of Treg from CD4SP T cells

[0223] The T cells contained in the peripheral blood mononuclear cell fraction (PBMC) of living human beings are TCRab-positive and CD4-positive CD4SP T cells, and can be further separated into the above-mentioned CD25-negative inflammatory T cells (Tconv) and CD25-positive CD127-negative regulatory T cells (Treg) ( Figure 3 ).

[0224] Experiments to maintain or induce FOXP3 were performed using a large number of CD4SP T cells, Tconv or Tregs derived from PBMC (derived from healthy volunteer donors). 32.52nM AS2863619 (MedChemExpress), 100nM rapamycin (LKT Labs), 2.5μg / mL TNFR2 antibody (Hycult Biotech, clone MR2-1) and 5ng / mL TGF-β (PeproTech, HEK293-derived) were added to the basal medium in the following combination to expand each cell by culture, and the FOXP3 positivity rate after 14 days was analyzed by intracellular staining and flow cytometry.

[0225] AR: AS2863619 + rapamycin

[0226] ART: AS2863619+rapamycin+TGF-β

[0227] AMT: AS2863619+TNFR2 antibody+TGF-β

[0228] AMRT: AS2863619 + rapamycin + TNFR2 antibody + TGF-β

[0229] First, Tregs were sorted from PBMCs, mixed with Dynabeads to a bead: cell ratio of 4:1, suspended in basal medium, and expanded by culture for 14 days. At the same time, for each group of basal medium only, AR, ART, AMT, and AMRT, the expansion ratio and FOXP3 positivity were analyzed by cell number counting and by intracellular staining and flow cytometry. Figure 4 The results are shown in .

[0230] When cultured with IL-2 alone, FOXP3 could not be maintained at sufficiently high levels, and when cultured with AR or ART, sufficient cell proliferation could not be achieved. On the other hand, when cultured with AMT or AMRT, FOXP3 expression was highly maintained.

[0231] The results showed that when cultured with AMT and 10 ng / mL IL-2, the FOXP3 positive rate was lower than when cultured with AMRT, but when cultured with AMT and 100 ng / mL IL-2, a comparable FOXP3 expression rate was maintained. Therefore, when PBMC-derived Tregs were expanded by culture, the IL-2 concentration was set to 100 ng / mL. The results of this case are shown in Figure 5 As shown. When cultured with AMT, the ratio of FOXP3-positive cells was 85.6%, and when cultured with AMRT, it was 85.3%. In addition, compared with cultured with IL-2 alone, when cultured with AMRT, the expansion ratio increased several times (2.4 times this time), and when cultured with higher levels of AMT, the expansion ratio increased by tens to hundreds of times (31.5 times this time).

[0232] Then, CD4SP T cells derived from iPS cell line 4GAD1-4 were used to induce FOXP3 by each combination described above. Similarly, for each culture condition group of basal medium only, containing AR, containing ART, containing AMT, containing AMRT, the FOXP3 positive rate was analyzed by intracellular staining and flow cytometry. At this time, CD4 positive CD8b negative cells (CD4SP T cells) sorted from ATO and cultured and expanded once using basal medium (IL-2 10ng / mL) were used.

[0233] The results are as follows Figure 6 As shown. Cultivation with each combination of AR, ART, and AMRT induced high FOXP3 expression, with a FOXP3 positivity rate greater than 50%. Among these groups, the group containing AMRT had an extremely high FOXP3 positivity rate of 84.8%.

[0234] Example 3 Evaluation of Treg

[0235] Study on marker expression pattern and inflammatory cytokine secretion capacity

[0236] It was verified whether the cells induced to express FOXP3 under the conditions described in Example 2 showed the CD25-positive CD127-negative CTLA-4-positive expression pattern characteristic of Treg, and whether the cells had the property of hardly secreting inflammatory cytokines even when stimulated. The analysis was performed by intracellular staining and flow cytometry under the conditions of PMA and ionomycin stimulation.

[0237] Figure 7Shown are FACS plots comparing PBMC-derived Tconv expanded by culturing under conditions of IL-2 alone (basal medium) or under conditions of basal medium supplemented with ART, using PBMC-derived Tregs as a control.

[0238] Treg highly expressed FOXP3, and was CD25 positive (positive rate: 93.15%), CD127 negative (positive rate: 2.09%), and CTLA-4 positive (positive rate: 90.9%). The positive rate of IFN-γ was 2.84%, the positive rate of IL-4 was 3.01%, and Treg almost did not secrete inflammatory cytokines.

[0239] Compared with culturing in a basal medium supplemented with ART, changes in the expression rate of markers (such as CD25) were observed in the PBMC-derived Tconv expanded by culturing in a basal medium. Specifically, the CD25 positivity rate changed from 1.23% to 88.09%, the CD127 positivity rate changed from 39.52% to 6.80%, and the CTLA-4 positivity rate changed from 16.49% to 60.10%. In addition, the IFN-γ positivity rate decreased from 50.12% to 5.06%, and the IL-4 positivity rate decreased from 39.5% to 5.04%. Based on this result, it can be confirmed that for PBMC-derived Tconv, the addition of ART has the effect of inducing traits close to Treg in addition to FOXP3 expression.

[0240] It was also verified by the same method whether the expression pattern of marker molecules characteristic of Treg other than FOXP3 was observed even when FOXP3 expression was induced in iPS cell-derived CD4SP T cells by AMRT addition. Figure 8 Shown are FACS graphs comparing 4GAD1-4-derived CD4SP T cells expanded by culturing under conditions of IL-2 alone (basal medium) and under conditions of basal medium supplemented with AMRT.

[0241] In addition, for 4GAD1-4 derived CD4SP T cells, by culturing with a basal medium supplemented with AMRT, it was observed that the CD25 positive rate (increased from 28.89% to 92.85%), the CD127 expression level decreased (decreased from 13.03% to 4.87%), and the CTLA-4 expression level increased (increased from 2.65% to 5.15%). In addition, the positive rate of IFN-γ decreased from 19.02% to 0.100%, and the positive rate of IL-4 decreased from 27.6% to 0.802%.

[0242] This confirmed that, in iPS cell-derived CD4SP T cells, the addition of AMRT to the basal medium has the effect of inducing Treg-like characteristics in addition to FOXP3 expression.

[0243] Demethylation study in CNS region

[0244] The transcription factor binding site (called CNS (conserved non-coding DNA sequence)) with the function of stabilizing the expression of the FOXP3 gene is present in the untranslated region of the FOXP3 gene locus (Zheng et al. Nature, 463 (7282), 808-812 (2010)), and is demethylated in Treg. In addition, the gene regions that are specifically demethylated in Treg have also been identified, and these regions are called TSDR (Treg-specific demethylation regions).

[0245] Therefore, PureQuant Treg assay (Thermo Fisher Scientific, original method described in Wieczorek et al. Cancer Res., 69(2), 599-608 (2009)) was used to analyze whether a large number of PBMC-derived CD4SP T cells expanded by culturing with basal medium supplemented with AMRT and iPS cell-derived CD4SP T cells expanded by culturing with basal medium supplemented with AMRT were associated with demethylation of TSDR (herein referred to as FOXP3 CNS2). Fig. 9 The results are shown in .

[0246] In a large number of PBMC-derived CD4SP T cells and iPS cell-derived CD4SP T cells expanded by culture with IL-2 alone, TSDR was hardly demethylated. In other words, the constitutive expression of FOXP3 (a transcription factor that controls immunosuppressive function) was strongly inhibited by methylation. This is also consistent with the Figure 2 This is consistent with the fact that both types of cells showed a strong tendency to secrete inflammatory cytokines.

[0247] In addition, a large number of PBMC-derived CD4SP T cells expanded by culturing with a basal medium supplemented with AMRT showed a demethylation rate of 43.3%, which was higher than that in the case of expansion culture with IL-2 alone, and iPS cell-derived CD4SP T cells expanded by culturing with a basal medium supplemented with AMRT showed a very high demethylation rate of 86.6%.

[0248] These facts indicate that expansion culture using a basal medium supplemented with a combination of these compounds and cytokines is not a method for transiently expressing FOXP3 by activation, but a method for inducing stable expression of Treg traits by demethylation of TSDR.

[0249] Example 4 Evaluation of immunosuppressive function

[0250] We verified in vitro and in vivo whether PBMC-derived Tregs expanded by culture with AMT and iPS cell-derived CD4SP T cells expanded by culture with AMRT had immunosuppressive function.

[0251] In these experiments, as iPS cells, the non-T cell-derived iPS cell line FFI01s04 that does not have a restored TCR gene was used. In this case, the CD4SP T cells obtained from ATO do not have a specific TCR library. Then, antigen specificity is imparted by gene transduction of a chimeric antigen receptor (CAR). CAR has a chimeric structure in which a single-chain antibody (scFv) in which the light chain (VL) and heavy chain (HL) of the variable region of a monoclonal antibody are tandemly bound to an antigen molecule is used as an extracellular domain, and a costimulatory molecule and a CD3z chain are used as an intracellular domain. This time, HLA-A2 was used as an antigen, and CD28 was used as a costimulatory molecule (see Fig.10 ). The molecule is expressed by gene transduction, and T cells expressing the molecule (CAR-T cells) can directly recognize cell surface antigens (here HLA-A2) in an HLA-unrestricted manner.

[0252] The gene (SEQ ID NO: 9) of the above-mentioned CAR molecule was transduced into PBMC-derived Treg and iPS cell-derived CD4SP T cells using a retroviral vector. Then, CAR-expressing cells were marked using a PE-conjugated antibody (CellSignaling Technology, clone: ​​9B11) for the myc tag, and anti-PE microbeads and MS columns (both from Miltenyi Biotec) were used to concentrate the cells by the MACS method.

[0253] The CAR expression rate and FOXP3 expression rate of the obtained cells were analyzed by flow cytometry. Fig.11 The results are shown in . In cells that have been transduced with the CAR gene, as a result of concentration by MACS, PBMC-derived Tregs amplified by culture with AMT and iPS cell-derived CD4SP T cells amplified by culture with AMRT both showed a CAR expression rate close to 100%. In addition, it was confirmed that FOXP3 expression did not disappear or significantly decrease due to CAR gene transduction.

[0254] As a representative method for determining the function of Treg, a method for evaluating the function of Treg has been established, which is performed by transplanting HLA-A2-positive human PBMCs into super immunodeficient mice, and determining the extent to which the graft-versus-host disease (GvHD) reaction produced against a foreign antigen can be suppressed by Tregs (in which CAR targeting HLA-A2 has been transduced), and as supporting evidence, determining the extent to which the proliferation of the same PBMCs transplanted into mice or cytotoxic T cells (CTLs) derived therefrom can be suppressed by Tregs in vitro (MacDonald et al., J. Clin. Invest., 126(4), 1413-1424 (2016), Dawson et al., Sci. Transl. Med. 12, eaaz3866 (2020), Lamarthe et al., Nat. Commun., 12(1), 6446 (2021)).

[0255] The same PBMC used in the in vivo experiment described below (derived from a different donor than the donor of the iPS cells and a different donor than the donor of the PBMC from which the Tregs were sorted) was co-cultured with the sorted and amplified CD8b-positive CTLs and PBMC-derived Tregs or iPS cell-derived CD4SP T cells (without or with CAR gene transduction, respectively). The degree of inhibition of CTL proliferation was then analyzed by flow cytometry using the Cell Trace Violet cell proliferation kit (Thermo Fisher Scientific). Before co-culture began, CTLs were stained with Cell Trace Violet (CTV) dye, and the degree of proliferation was assessed by the fact that each daughter cell produced by cell division had half the fluorescence intensity of its parent cell. The results obtained directly by flow cytometry are shown in Figure 2. Fig.12 shown.

[0256] First, the iPS cell-derived CD4SP T cells amplified by culturing with IL-2 alone showed no inhibitory effect on the proliferation of CTLs, regardless of the presence or absence of CAR expression. Under conditions where the number of CD4SP T cells is superior to the number of CTLs, such as CD4SP T cells: CTL = 4:1, the number of CTLs is reduced when CAR-transduced cells are used, indicating that CD4SP T cells have a cell-killing effect on CTLs. On the other hand, regardless of whether CAR gene transduction is performed, iPS cell-derived CD4SP T cells amplified by culturing with basal medium supplemented with AMRT show an inhibitory effect on CTL proliferation.

[0257] In order to quantitatively evaluate the cell growth inhibitory effect of various cells on CTL, six types of cells, i.e., iPS cell-derived CD4SP T cells (without or with CAR gene transduction) cultured with IL-2 alone, iPS cell-derived CD4SP T cells (without or with CAR gene transduction) cultured with AMRT, and PBMC-derived Tregs (without or with CAR gene transduction) cultured with AMT were mixed at various ratios of iPS cell-derived CD4SP T cells (or Treg): CTL = 1:16, 1:8, 1:4, 1:2, 1:1, 2:1 and 4:1, and co-cultured. After 96 hours of co-culture, the fluorescence intensity of CTV was measured by flow cytometry, and the ratio of CTL division was calculated. Then, % inhibition = {(CTL division ratio (%) when CD4SP T cells were not added) - (CTL division ratio (%) when CD4SP T cells were added at each ratio) / CTL division ratio (%) when CD4SP T cells were not added} × 100 was further calculated and graphed. Fig.13 The results are shown in . Higher values ​​indicate stronger inhibition of CTL proliferation.

[0258] All four types of cells amplified by culture with AMRT or AMT showed an inhibitory effect on CTL proliferation in a cell number ratio-dependent manner. The inhibitory effect of iPS cell-derived CD4SP T cells did not depend on whether CAR gene transduction was performed, and there was a significant difference. In addition, for PBMC-derived Tregs, the inhibitory effect was equal regardless of whether CAR gene transduction was performed. In addition, iPS cell-derived CD4SP T cells showed a stronger inhibitory effect on CTL growth than PBMC-derived Tregs.

[0259] From the above, it was confirmed that CD4SP T cells derived from iPS cells expanded by culture with AMRT and Tregs derived from PBMCs expanded by culture with AMT have the function of suppressing the proliferation of CTLs in vitro.

[0260] Subsequently, the same HLA-A2-positive PBMCs were used for in vivo experiments. HLA-A2-positive PBMCs were mixed with one of four types of iPS cell-derived CD4SP T cells (without or with CAR gene transduction) expanded by culture with AMRT and PBMC-derived Tregs (without or with CAR gene transduction) expanded by culture with AMT to form 5.0×10 6The cells were irradiated with 2.2 Gy and then transplanted into NSG mice by intravenous injection. Subsequently, the transplantation day was set as day 0, body weight was measured every day until day 29, and overall survival was evaluated. Fig.14 The results are shown in .

[0261] In in vivo functional assays, cell transplantation without CAR gene transduction did not reduce weight loss and showed prolonged overall survival compared to transplantation of PBMC alone, regardless of whether the cells were iPS cell-derived CD4SP T cells or PBMC-derived Tregs. On the other hand, in both iPS cell-derived CD4SP T cells and PBMC-derived Tregs, CAR gene-transduced cells significantly reduced the weight loss of mice and significantly prolonged overall survival, indicating that GvHD was suppressed. In addition, HLA-A2-positive human PBMCs were confirmed in the peripheral blood of surviving NSG mice at this time, indicating that the inhibitory mechanism of GvHD is not only due to the cytotoxicity of CAR-positive T cells to HLA-A2-positive human PBMCs.

[0262] As described above, it was confirmed that PBMC-derived Tregs expanded by culture with AMT have the expected immunosuppressive ability, and that iPS cell-derived CD4SP T cells expanded by culture with AMRT have the function of suppressing immunity in vivo in the same manner as PBMC-derived Tregs.

[0263] The same experiment was also repeated using HLA-A2 positive PBMCs from another donor. Fig.15 The results are shown in . Similarly, the results showed that iPS cell-derived CD4SP T cells that had been transduced with the CAR gene reduced weight loss and prolonged overall survival of mice, and the same was true for PBMC-derived Tregs that had been transduced with the CAR gene.

[0264] Example 5 Evaluation by iPS cells containing CNS-Foxp3

[0265] (1) Introducing the construct into iPS cells and differentiating the iPS cells into HECs

[0266] The cell population containing hemogenic endothelial cells used is a floating cell population differentiated from iPS cells (Ff-I01s04 strain: derived from healthy peripheral blood mononuclear cells) provided by the iPS Cell Research and Application Center of Kyoto University by a known method (e.g., the method described in Cell Reports 2 (2012) 1722-1735 and WO2017 / 221975).

[0267] Specifically, the method is as follows. The plasmid sequence was designed and synthesized in the following manner: a DNA sequence (obtained by changing the mStrawberry protein sequence in the sequence registered in GenBank (MK012431) to the dTomato sequence) was introduced into a transfer plasmid to produce a third-generation lentiviral vector. The plasmid was transfected into HEK293 lineage cells together with a packaging plasmid and an envelope plasmid to produce a lentiviral vector, and the supernatant containing the produced lentiviral vector was collected and then concentrated by high-speed centrifugation to obtain a lentiviral vector carrying CNS-Foxp3 to be introduced into iPS cells. The Ff-I01s04 strain was cultured at 1×10 4 Cells / well were seeded in a 24-well plate, protamine was added at a final concentration of 10 μg / mL, and then directly added to the lentiviral suspension carrying the CNS-Foxp3 gene to generate virus-infected iPS cells. The virus-infected Ff-I01s04 strain was plated at 1×10 6 Cells / well were seeded in EB medium (StemPro34 (Gibco), supplemented with 50 ng / mL BMP4 (R&D Systems), 50 ng / mL bFGF (FUJIFILM WakoPure Chemical Corporation), 50 ng / mL VEGF (R&D Systems) and 2 μM SB431542 (FUJIFILM Wako Pure Chemical Corporation), 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / mL sodium selenite (ITS, Gibco), 2 mM L-glutamine (Sigma-Aldrich), 45 mM α-monothioglycerol (NacalaiTesque, Inc.) and 50 μg / ml ascorbic acid 2-phosphate (Sigma-Aldrich)) in ultra-low attachment 6-well plates (Corning) (Day 0), and then the cells were cultured under hypoxic conditions (5% O2) for 4 days (Day 4). Subsequently, the cells were further cultured in a medium supplemented with 50 ng / mL bFGF, 50 ng / mL VEGF, and 50 ng / mL SCF (R&D Systems) for 4 days (Day 8), thereby obtaining a cell population containing HECs.

[0268] (2) Differentiation of HEC to Treg

[0269] See the method described in WO2017 / 075389, etc., allowing the HEC-containing cell population obtained in (1) to differentiate into Treg (CD25 + / FOXP3 + ).

[0270] Specifically, the method is as follows. iPS cells (Ff-I01s04 strain: derived from healthy peripheral blood mononuclear cells) provided by the iPS Cell Research Application Center of Kyoto University were induced to differentiate into fibroblasts to use them as feeder cells when inducing differentiation from iPS cells into T cells. Subsequently, lentivirus was used to generate cells that forcibly expressed DLL4 (as a Notch ligand under the EF1α promoter). More specifically, the Ff-I01s04 strain was inoculated at 1×10 6 10 cells / well were seeded in ultra-low attachment 6-well plates (Corning) in a medium (day 0) prepared by adding Y-27632 (FUJIFILM WakoPure Chemical Corporation) and CHIR990211 (Tocris Bioscience) to AK03N (Ajinomoto Co., Inc.) and on the next day, the cells were seeded in 10 cm culture dishes (Corning) coated with 0.1% gelatin (Nacalai Tesque) in fibroblast medium (α-MEM (Invitrogen), supplemented with 20% FBS (Corning), 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / mL sodium selenite (ITS, Gibco), 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin (PSG, Sigma-Aldrich) and 50 μg / ml ascorbic acid 2-phosphate (Sigma-Aldrich)) (Day 1). The medium was changed twice a week and cultured for 7 days to obtain a cell population containing fibroblasts (Day 8). The iPS cell-derived fibroblasts were cultured at 1×10 5 Cells / well were seeded in a gelatin-coated 24-well plate, and on the next day, a lentiviral suspension carrying the human DLL4 gene downstream of the EF1α promoter and protamine at a final concentration of 10 μg / mL were added to generate iPS cell-derived fibroblast / DLL4 cells (iFibro / DLL4).

[0271] The generated iFibro / DLL4 was used as a support and co-cultured with the CNS-Foxp3 transduced human iPS cell-derived HEC generated in (1) at a cell ratio of 1:1. The culture medium was RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation), which contained 2×B27 supplement (Invitrogen), 1×PSG (Sigma-Aldrich), 1×Glutamax (Invitrogen), 5 ng / mL IL-7 (PeproTech), 5 ng / mL F1T3L (PeproTech), and 50 μg / mL ascorbic acid (Sigma-Aldrich) in final concentrations, and the cells were co-cultured on 30 mm Millicell (hydrophilic PTFE, pore size: 0.4 μm, height: 5 mm, Merck Millipore) in a 6-well plate (TPP) and cultured for 7 weeks, while the culture medium was replaced every three or four days.

[0272] (3) Expansion and culture of iPS-Treg

[0273] The Tregs differentiated from iPS cells obtained in (2) were cultured and expanded in a medium containing 10 nM rapamycin (Merck) and 3 μg / mL anti-TNFR2 antibody (Hycult Biotech, clone MR2-1).

[0274] Specifically, the CNS-Foxp3-transduced human iPS cell-derived Treg obtained in (2) was cultured in a 48-well cell culture plate bound to an anti-CD3 antibody (eBioscience) for 3 days, and then re-seeded and cultured in a 24-well G-Rex cell culture plate. The culture medium was α-MEM (Invitrogen), which contained FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich), and IL-2 (10 ng / mL, PeproTech) in final concentrations. In the first three days, anti-CD28 antibody (1.5 μg / mL, BioLegend), anti-CD30 antibody (300 ng / mL, R&D Systems) and caspase inhibitor (10 μM, R&D Systems) were further added to the above-mentioned preparation. The culture was carried out for 9 or 10 days, and the culture medium was replaced on the 3rd day after the start of the culture, and the culture medium was replaced every three or four days thereafter. When the cells obtained after the first amplification culture were recovered, TregCD4 microbeads (MyltenyiBiotec) were used to separate the CD4 positive fraction. Further amplification culture was carried out twice in succession, and amplification culture was carried out three times in total.

[0275] (4) iPS-Treg expansion and culture using AMRT

[0276] The iPS-Treg obtained in (3) was further cultured and expanded in a medium supplemented with AS2863619 (as a CDK8 / 19 inhibitor) and TGF-β (AMRT).

[0277] Specifically, the iPS-Treg obtained in (3) was further cultured and expanded under the same conditions as (3) (control) or in a culture medium supplemented with AS2863619 (MedChemExpress, 30 nM) and TGF-β (Peprotech, 5 ng / mL) (AMRT). The culture time was 14 days, and the expansion culture was repeated three times. The cell proliferation results at this time are as follows: Fig.16 On the last day of expansion, each cell population was stained with the following antibodies (Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, FITC FOXP3, and PE Helios). Fig.17 The results are shown in . Fig.17 Zombie NIR is shown- CD3 + CD4 + CD8β - cell.

[0278] In the group expanded by culture under AMRT conditions, no significant difference in cell proliferation was observed compared with the control group, but CD25 + FOXP3 + Fractions and FOXP3 + Helios + The obtained amounts of the fractions were 76.5% and 47.4%, respectively, both higher than those of the control group (28.6% and 24.7%, respectively).

[0279] Example 6 Evaluation of the suppressive function of Tregs derived from iPS cells transduced with CNS-Foxp3

[0280] The Tregs cultured and expanded in Example 5 were co-cultured with allogeneic T cells derived from human PBMCs and then analyzed by flow cytometry.

[0281] Specifically, in the presence or absence of anti-CD3 antibodies, PBMCs from donors different from Treg donors were treated and stained with Cell Trace Violet (Thermo Fisher Scientific) to be used as target cells, and PBMCs were co-cultured with Tregs (control group in Example 5 or group expanded by culture under AMRT conditions): PBMCs at a cell number ratio of 0:1, 1:1 and 2:1. The culture medium was α-MEM (Invitrogen), which contained FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen) and ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich) at final concentrations, and the cells were cultured for 5 days. Each cell population was stained with the following antibodies (Zombie NIR, PE / Cy7 CD3 and PE HLA-A24). exist Fig.18 The results are shown in .

[0282] In the group co-cultured with iPS-Treg, the cell division of target cells was inhibited in a Treg cell number-dependent manner. In addition, in terms of inhibitory function, a stronger effect was observed in Treg cultured under AMRT conditions than in the control group. When Treg: target = 2: 1, the inhibition rate of the control group was 58.9%, while in the cell group cultured under AMRT conditions, the inhibition rate observed was 75.7%.

[0283] Example 7 Candidate genes for inducing differentiation of CD4SP T cells

[0284] Human induced pluripotent stem cell lines

[0285] The human iPS cell line TKT3V1-7 was obtained from antigen-nonspecific CD3 T cells of healthy volunteers using retroviral vectors carrying OCT3 / 4, KLF-4, SOX-2 and C-MYC (Molecular Therapy 29 10, 2013kanekoS).

[0286] Cell lines

[0287] To generate MS5-hDLL4, MS5 cells (K Itoh et al., 1989. Exp Hematol 17, 145-153) were transformed with a retroviral vector encoding full-length human DLL4. Up to 5% of cells expressing DLL4 were sorted by FACS using an anti-DLL4 antibody and the cells were passaged in DMEM / 10% fetal calf serum (FCS). After several weeks in culture, stable expression of DLL4 was confirmed by flow cytometry.

[0288] Generation of iHPCs from iPSCs

[0289] The method of differentiating iPSCs into T cells has been previously reported (Nishimura et al., 2013, Cell Stem Cell 12, 114-126). iPSCs were cultured and expanded on iMatrix-511 of StemFit AK02N for 6 to 7 days and separated into single cells using 0.5×TryPLE select (Thermo Fisher Scientific). 3 to 6×10 5All cells were resuspended in StemFit AK02N supplemented with 10 μM Y-27632 (FUJIFILM Wako Pure Chemical Corporation) and 10 μM CHIR99021 (Tocirs Bioscience) and cultured in 6-well ultra-low attachment plates (Corning) for 24 h. Subsequently, EBs were recovered and precipitated at the bottom of the tube and resuspended in 2 mL / well of "EB basal medium" (StemPro-34 (Thermo Fisher Scientific), supplemented with 10 ng / mL penicillin / streptomycin (Sigma-Aldrich), 2 mM Glutamax (Thermo Fisher Scientific), 50 μg / mL ascorbic acid-2-phosphate (Sigma-Aldrich), 4 × 10 -4 50 ng / mL recombinant human (rh) BMP-4 (R&D Systems), 50 ng / mL rhVEGF (R&D Systems) and 50 ng / mL bFGF (FUJIFILM Wako Pure Chemical Corporation) were further added to 1× monothioglycerol (MTG, Nacalai Tesque) and 1× insulin-transferrin-selenium solution (ITS-G, ThermoFisher Scientific). After 24 hours, 6 μM SB431542 (FUJIFILM Wako Pure Chemical Corporation) was added. After 4 days, the differentiated EBs were collected, washed, and then resuspended in 2 mL / well of EB basal medium supplemented with 50 ng / mL rhVEGF, 50 ng / mL rhbFGF, 50 ng / mL rhSCF (R&D Systems) and cultured for 2 days. After 7 days, the differentiated EBs were collected again, washed, and resuspended in 2 mL / well of EB basal medium supplemented with 50 ng / mL rhVEGF, 50 ng / mL rhbFGF, 50 ng / mL rhSCF, 30 ng / mL rhTPO (PeproTech), and 10 ng / mL FLT3L (PeproTech). Starting from the 7th day, cells were harvested every 2 to 3 days and replaced with fresh medium. The culture was maintained in a 5% CO2 / 5% O2 / 90% N2 environment for the first 7 days and then in a 5% CO2 environment. The differentiated iHPCs were filtered through a 40 μm cell strainer and cryopreserved at -80°C using a TC-protectant (Bio-Rad antibody).

[0290] 2D feeder-free differentiation of iHPCs into progenitor T cells

[0291] iHPCs derived from TKT3v1-7 as previously reported were used (Iriguchi et al., 2021, Nat Commun 12, 430.10.1038 / s41467-020-20658-3). T cell differentiation was induced on rhDL4 coated plates prepared one day before iHPC inoculation. The rhDL4 / Fc chimeric protein solution (5 μg / mL, Sino Biological) was diluted with an equal amount of retronectin (5 μg / mL, Takara Bio Inc.) and added to a 48-well plate at 150 μL / well, and the plate was incubated overnight at 4 °C. The coating solution was removed immediately before adding T cell differentiation medium.

[0292] For seeding iHPCs, 11 to 14 EBs were collected and isolated into single cells by TryPLE Select (Thermo Fisher Scientific). 2000 CD34 + / CD43 + Cells were sorted by FACS directly into wells of DL4-coated plates containing T cell differentiation medium consisting of αMEM (Thermo Fisher Scientific) supplemented with 15% FBS (Corning), 100× ITS-G (1×), 55 μM 2-mercaptoethanol (Thermo Fisher Scientific), 50 μg / mL ascorbic acid-2-phosphate, 2 mM Glutamax, 50 ng / mL rhSCF, 50 ng / mL rhTPO, 50 ng / mL rhIL-7, 50 ng / mL FLT3L, 30 nM rhSDF-1α (PeproTech), and 15 μM SB203580 (Tocris Bioscience). Most of the medium (80%) was replaced every other day. Differentiated cells were transferred to new DL4-coated plates on day 7, and 1 to 2×10 5 The cells / well were transferred to a new DL4 coated plate and the culture was maintained in a 5% CO2 environment.

[0293] Progenitor T cells mature into CD8 single positive T cells

[0294] On day 21, DL4 cells were stimulated with 500 ng / mL anti-CD3 monoclonal antibody (clone: ​​OKT3, eBioscience) in a maturation medium consisting of αMEM, 15% FBS, 100×ITS-G (1×), 50 μg / mL ascorbic acid-2-phosphate, 100×PSG (1×, Sigma-Aldrich), 10 ng / mL rhIL-7, 10 ng / mL rhIL-2 (PeproTech), and 10 nM dexamethasone (Fuji Pharma Co., Ltd.). After 3 days, the cells were recovered, washed, resuspended in maturation medium without OKT3, and cultured at 37° C. in an environment containing 5% CO 2 for 4 days.

[0295] iHPCs mature into CD4 and CD8 single positive T cells

[0296] The previously reported method was used with some modifications (Seet et al., 2017, Nat Methods 14, 521-530.10.1038 / nmeth.4237). Briefly, 1×10 6 MS5-hDLL4 cells and 1×10 5 Each purified iHPC was added to a 1.5 mL microcentrifuge tube to prepare a dense MS5-hDLL4-iHPC mixture. The mixture was adjusted to 15 μL per well. For each culture, a 0.4 μm Millicell Transwell insert (EMD Millipore, Billerica, MA; Catalog No. PICMORG50) was placed in a 6-well plate, with each well containing 1.5 mL of organoid culture. The culture medium was completely replaced every 3 to 4 days.

[0297] Single-cell RNA-seq

[0298] The isolated single cell suspension was subjected to droplet-based massively parallel scRNA-seq using the Chromium Single Cell 50 kit (10xGenomics) according to the manufacturer's instructions. Briefly, the cell suspension was added at 700 to 1,200 cells / μL so that 3,000 cells were captured per well. GEM droplets were generated on a 10x Chromium controller, each cell was labeled with a specific barcode, and each transcript was labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated using a Dynabeads MyOne SILANE bead cleanup mixture. The cDNA was amplified by PCR and purified by SPRI bead cleanup.

[0299] For gene expression libraries, 50 ng of amplified cDNA was used and library preparation was performed according to the manufacturer's instructions, which consisted of fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR. The library was sequenced using a NovaSeq sequencer (Illumina, Inc.).

[0300] Single-cell RNA-seq data processing

[0301] Sequence reads of organoid culture samples were aligned to the human GRCh38 genome reference, and the label counts of genes and antibodies were quantified as UMIs using CellRanger v3.1.0 software (10x Genomics). The UMI count matrix was imported into the Seurat v3.1.1 package of R v3.6.1 software, and the Seurat package was used for further data processing. Cells with a mitochondrial gene number ratio of 20% or more were excluded from further analysis as dead cells or damaged cells. The number of genes and label counts derived from antibodies were normalized by the global scale normalization method and the central logarithm ratio normalization method, respectively. The genes differentially expressed in the cells were selected by the "vst" method in the Seurat package, and principal component analysis was performed. The calculated principal components were used for dimensionality reduction and graph-based clustering.

[0302] BAM files of human thymocyte samples were downloaded from the SRA repository (GSE148978). BAM files were converted to FASTQ sequence files using bamtofastq software v1.3.2 (10x Genomics). Sequence reads were aligned to the human GRCh38 genome reference, and gene tag counts were quantified as UMIs using Cell Ranger v5.0.1 software. Cells with less than 200 or 10,000 or more UMIs, or cells with a mitochondrial gene number ratio of 5% or more were excluded from further analysis. The human thymocyte dataset was integrated and data processed by the Seurat standard integration method. Clusters consisting of cells with the same number of UMIs or cells derived from one donor were deleted.

[0303] Sequence reads of 2D culture samples were aligned to the human GRCh38 genome reference and gene tag counts were quantified as UMIs using CellRanger software v5.0.1. Data processing was performed using Seurat v3.2.3. package.

[0304] Data sets for organoid cultures, 2D cultures, and human thymocyte samples were integrated and processed using Seurat standard integration methods.

[0305] Genes with different expression levels in a cluster relative to other clusters were defined as those with a fold change of 2 or greater and a p value less than 0.05 as determined by the Wilcoxon rank sum test.

[0306] Pseudo-time analysis

[0307] The integrated dataset was converted to the monocle3 v0.2.3.0 package (Cao et al., 2019, Nature 566, 496-502.10.1038 / s41586-019-0969-x; Qiu et al., 2017, Nat Methods 14, 309-315.10.1038 / nmeth.4150; Qiu et al., 2017, Nat Methods 14, 979-982.10.1038 / nmeth.4402; Trapnell et al., 2014, Nat Biotechnol 32, 381-386.10.1038 / nbt.2859) format and data processing was performed. The Seurat embedding space of UMAP was used for further analysis. The embedding space of the main UMAP was fitted to obtain a trajectory map. The pseudo time was determined using the node of the DN cell as the starting point. According to the Moran I test, genes with different expression levels were extracted in the paths of different trajectories, and the identified genes with different expression levels were classified into the gene set module of co-regulated genes together with the trajectory and pseudo-time.

[0308] Pseudotemporal analysis was used to identify gene candidates involved in the transition to CD4SP T cell differentiation in organoid cultures.

[0309] In order to identify the + CD8 + Pseudo-temporal analysis of integrated single-cell RNA-seq datasets of all cells in organoid cultures and thymocytes ( Fig.19A ). Clusters specific to organoid cultures were excluded from the integrated dataset. The results of trajectory analysis showed that the cell dataset was divided into three branches ( Fig.19B These branches divide cells into immature cells (red), CD4SP lineage (green), and CD8SP lineage (blue) ( Fig.19B ). Hierarchical clustering analysis was further performed on the significantly controlled genes, and 17 modules were obtained ( Fig.19C ; The top 10 genes in each module cluster are as follows Fig.19D). To investigate genes that are differentially expressed during T cell differentiation from DP to CD4SP, we focused on module clusters #7, #8, and #9, which contain genes whose expression increases during differentiation from the immature stage to CD4SP, and module clusters #2, #5, and #6, which contain genes whose expression decreases during relatively early stages of differentiation to CD8SP. The genes contained in these module clusters are considered candidate genes for the transition from DP to CD4SP T cells.

[0310] Sensitized helper population included in 2D differentiation samples

[0311] To identify potential genes / pathways for CD4SP T cell differentiation via feeder-free culture, single-cell transcriptomes of cells obtained from 2D culture (CD8SP only) and organoid culture (CD4SP and CD8SP cells) were compared.

[0312] Data sets from 6-week organoid cultures, 2D cultures, and thymocytes were integrated and processed as UMAP plots ( Fig. 20A , left). The non-integrated datasets also show common clustering ( Fig. 20A , right). Thymocytes show various clusters. Clusters #1 and #3 show very strong increases in CD8 expression, while clusters #0 and #2 show CD4 expression. These cluster gene expression is higher in the thymocyte dataset than in the culture data. The 2D and organoid culture clusters show T cell-related gene expression. Unexpectedly, some cells from the 2D culture samples also showed increased expression in clusters ( Fig. 20C ) shows CD4 expression in thymocytes and organoid cultures data ( Fig. 20B and 20C ). In addition, SELL associated with Tscm (Cheng-Chi Chao et al., 1997, J Immunol 159, 1686-1694) was expressed in cluster #0, and BACH2 associated with T cells (Rahul Roychoudhuri et al., 2013, Nature 498, 506-512.10.1038 / nature12199) was expressed in cluster #2. These results suggest that some cells in 2D culture have auxiliary functions.

[0313] To elucidate the mechanism of low CD4 expression in 2D cultures, we compared the kinetics of gene expression during the transition to mature CD4SP T cells. Using the regulated gene clusters identified in pseudo-time analysis ( Fig.19D ), and assuming that the expression difference was related to CD4 expression in organoid cultures, the expression of the top 10 genes of module clusters #2, #5, #6, #7, #8, and #9 of the 2D culture dataset was evaluated. For the potential auxiliary function clusters ( Fig. 20C ), SELENOW (ENSG00000178980) and SYTL2 (ENSG00000137501) of module cluster #2, S100A11 (ENSG00000163191) and STAT1 (ENSG00000115415) of module cluster #5, GIMAP4 (ENSG00000133574), GIMAP7 (ENSG00000179144), IFITM1 (ENSG00000163191) of module cluster #6. 00185885) and IL7R (ENSG00000168685) and LZTFL1 (ENSG00000163818), SATB1 (ENSG00000182568), SLAMF1 (ENSG00000117090) and SOX4 (ENSG00000124766) of module cluster #8 showed higher expression levels in organoid cultures than in 2D cultures (collective gene ID is shown in brackets) (Figure 21). It is envisioned that by controlling these genes, differentiation from pluripotent stem cells (especially iPS cells) and the like to desired T cells (especially CD4SP T cells) can be induced.

[0314] This application is based on Japanese patent application No. 2022-47437 filed in Japan on March 23, 2022 and Japanese patent application No. 2022-152604 filed in Japan on September 26, 2022, and the contents of these two patent applications are incorporated herein in their entirety.

Claims

1. A method for producing a cell population containing regulatory T cells, the method comprising: Step (1) culturing CD4 T cells derived from pluripotent stem cells in the presence of at least one substance selected from the group consisting of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor, and a TGF-βR agonist. + T cell population.

2. The method according to claim 1, wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor and an mTOR inhibitor.

3. The method according to claim 1, wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor, an mTOR inhibitor and a TGF-βR agonist.

4. The method according to claim 1, wherein step (1) is performed in the presence of a CDK8 and / or CDK19 inhibitor, a TNFR2 agonist, an mTOR inhibitor and a TGF-βR agonist.

5. The method according to claim 1, wherein the CDK8 and / or CDK19 inhibitor is at least one selected from the group consisting of: 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine, and siRNA of CDK8 and / or CDK19.

6. The method of claim 1, wherein the mTOR inhibitor is rapamycin.

7. The method of claim 1, wherein the TNFR2 agonist is a TNFR2 agonist antibody.

8. The method of claim 1, wherein the TGF-βR agonist is TGF-β.

9. The method of claim 1, wherein the regulatory T cells are CD25 + / FOXP3 + cell.

10. The method according to claim 1, wherein the CD4 + T cells are CD4 + / CD8 - T cells.

11. The method according to claim 1, further comprising before step (1): Step (2) inducing pluripotent stem cells to differentiate into CD4 + T cell population.

12. The method according to claim 1, wherein: The pluripotent stem cells are iPS cells.

13. The method according to claim 11, wherein: In step (2), a three-dimensional cell aggregate is cultured, wherein the three-dimensional cell aggregate contains CD4 T cells that can differentiate into the pluripotent stem cell-derived cells. + T cells and stromal cells expressing Notch ligands.

14. A cell population containing regulatory T cells, obtained by the method according to claim 1. 15 . A medicament comprising the cell population containing regulatory T cells according to claim 14 .

16. The medicament according to claim 15, which is used for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection. 17 . A method for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection, the method comprising administering the cell population containing regulatory T cells according to claim 14 to a subject in need thereof. The cell population containing regulatory T cells according to claim 14, which is used for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection.

19. Use of the cell population containing regulatory T cells according to claim 14 in the preparation of a medicament for preventing and / or treating autoimmune diseases, graft-versus-host disease or transplant rejection.

20. The method of claim 1, wherein the CD4 + A T cell is a cell into which has been introduced an expression construct comprising: (1)(a) Conserved noncoding sequences (CNS) 1, CNS2, and CNS3 of the Foxp3 gene; (b) a promoter; and (c) Nucleic acid encoding FOXP3.

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