Immune cells

Abstract

Disclosed are: CD3low / -, CD25+ and FOXP3+ immune cells; a method for producing a cell population in which CD3low / -, CD25+ and FOXP3+ immune cells are proliferated, the method including a step (1) in which a cell population including CD4+ T cells derived from pluripotent stem cells is cultured in the presence of interleukin (IL)-2 and IL-33; a method for proliferating CD3low / -, CD25+ and FOXP3+ immune cells, the method including a step (1A) in which a cell population including CD3low / -, CD25+ and FOXP3+ immune cells derived from pluripotent stem cells is cultured in the presence of IL-2 and IL-33; a method for producing a cell population including CD3low / -, CD25+ and FOXP3+ immune cells, the method including a step (1B) in which a cell population including CD4+ / CD25- / FOXP3- T cells derived from pluripotent stem cells is cultured in the presence of IL-2 and IL-33; a cell population which is produced by the method and includes CD3low / -, CD25+ and FOXP3+ immune cells; and a medicine which contains a cell population including such regulatory T cells.

Description

immune cells

[0001] The present invention relates to immune cells, a method for producing a cell population in which the immune cells have proliferated, a method for proliferating the immune cells, a cell population containing the immune cells obtained by the method, a medicine containing the cell population, etc. (Background of the invention)

[0002] In recent years, because regulatory T cells (Treg) have the function of suppressing immune responses, research and development of cell therapy (Treg therapy) using regulatory T cells has been progressing worldwide for the purpose of treating various autoimmune diseases. Thus, regulatory T cells are expected to be used in the treatment of graft-versus-host disease (GvHD), autoimmune diseases, inflammatory diseases, and allergic diseases, as well as for the treatment and prevention of these diseases.

[0003] Regulatory T cells are broadly classified into two types: endogenous regulatory T cells (naturally occurring regulatory T cells (nTreg): or thymic Treg: tTreg) and inducible regulatory T cells (inducible T cells (induced Treg): iTreg). It is known that nTregs are naturally generated in the thymus, while iTregs are differentiated and induced from naive T cells in peripheral blood by antigen stimulation and cytokines such as IL-2 and TGF-β. These regulatory T cells are further subdivided according to the types of markers expressed on the cells.

[0004] When regulatory T cells are removed from the body, various organ-specific autoimmune diseases spontaneously develop. Transplantation of regulatory T cells in such cases prevents the onset of autoimmune diseases. Therefore, regulatory T cells were thought to play an important role in maintaining immune self-tolerance in the periphery. It has since become clear that regulatory T cells can suppress not only autoimmunity, but also most immune responses, including inflammation caused by foreign antigens, transplant rejection, infection immunity, allergy, and tumor immunity. Furthermore, the transcription factor FOXP3 has now been identified as the master regulator of regulatory T cells.

[0005] FOXP3 is a master regulator of Tregs, and it is known that it is constitutively expressed in Tregs, but not in conventional T cells (also known as Tconv (conventional T cells), effector T cells, or inflammatory T cells) that do not have suppressive function. Therefore, studies have been conducted on the maintenance of FOXP3 expression in primary Tregs and the induction of FOXP3 expression in primary Tconv or bulk CD4 single positive (SP) T cells, and compounds, cytokines, and combinations thereof that have the activity of maintaining and inducing FOXP3 expression have been reported. Examples of such compounds and cytokines include IL-2 (Non-Patent Documents 1-7).

[0006] Further, such compounds, cytokines, and combinations thereof include IL-4 (Non-Patent Documents 2, 8, 9), TNFR2 agonist antibodies (Non-Patent Documents 4, 5, 7, 10), IL-3 (Non-Patent Document 1), TGF-β (Non-Patent Documents 1, 3, 6, 8, 11), and rapamycin (Non-Patent Documents 3, 5, 6, 12).

[0007] Although the effect of IL-2 on primary Tregs has been reported as described above, there have been no reports that a combination of IL-2 and IL-33 (together with IL-4, IL-3, a TNFR2 agonist, TGF-β, or rapamycin) is particularly preferable.

[0008] With regard to regulatory T cells derived from pluripotent stem cells, compounds and cytokines known to maintain FOXP3 expression or induce FOXP3 expression in T cells that do not express FOXP3 include TNFR2 agonists, TGF-β, rapamycin, and combinations thereof (Patent Document 1). However, there have been no reports that a combination of IL-2 and IL-33 (to which IL-4, IL-3, TNFR2 agonists, TGF-β, and rapamycin have been added) is particularly preferable. Furthermore, while an industrially viable production method with high production efficiency (yield) is desirable, it is known that it is difficult to proliferate regulatory T cells, particularly while maintaining their suppressive function, and an efficient production method for them is desired.

[0009] Non-Patent Document 13 describes IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, which is caused by mutations in FOXP3. To restore FOXP3 expression, a lentiviral vector was used to introduce a gene into autologous hematopoietic stem cells, and the cells were then administered to a mouse model of IPEX syndrome. As a result, it was reported that the hematopoietic stem cells differentiated into functional regulatory T cells, resulting in recovery from the autoimmune phenotype. Patent Document 2 also describes a recombinant lentiviral vector containing a nucleotide sequence encoding a similar human FOXP3 protein.

[0010] Patent Document 3 discloses that expression of Mcl-1 together with Foxp3 is important for improving the survival of Treg. ＋ Genetically engineered mammalian cells containing a transgene encoding a lineage-committing factor that promotes differentiation into Tregs have been disclosed. Patent Document 5 discloses a method for preparing a composition of T cells from stem or progenitor cells by culturing three-dimensional cell aggregates containing a selected population of stromal cells that express a Notch ligand and a selected population of stem or progenitor cells.

[0011] International Publication No. WO 2023 / 182328 International Publication No. WO 2019 / 040655 International Publication No. WO 2014 / 180943 International Publication No. WO 2021 / 092581 International Publication No. WO 2017 / 075389

[0012] J Immunol (2011) 186:2262-2272 Immunology. 2009 Jul;127(3):338-344PLoS One. , 11(2):e0148474. (2016) PLoS One. , 11(5), e0156311 (2016) Front. Immunol. 9:573 (2018) Clin. Exp. Immunol. , 197(1):52-63. (2019) Scientific Reports 3:3153 (2013) Immunology (2009) 128:e670-e678Front. Immunol. 8:1508. doi:10.3389 / fimmu. 2017.01508Sci. Signal. 13, eaba9600 (2020) J Exp Med (2003) 198(12):1875-1886 Nat. Immunol. 20(9), 1208-1219 (2019) Cell Stem Cell 24, 309-317, February 7, 2019

[0013] An object of the present invention is to provide a method for producing novel suppressive immune cells and a cell population in which the immune cells are expanded.

[0014] As a result of extensive research to achieve the above object, the present inventors have discovered that CD4 T cells derived from pluripotent stem cells (particularly iPS cells) can be expressed in the presence of IL-2 and IL-33. ＋ By culturing a cell population containing T cells, CD3 ｌｏｗ／－ The present inventors have found that it is possible to produce novel suppressive immune cells that are

[0015] The present invention was completed based on these findings and through further investigation, and provides the following immune cells, a method for producing a cell population in which immune cells have proliferated, a method for proliferating immune cells, a cell population containing immune cells, a pharmaceutical, etc.

[0016] [1] CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3＋ [2] CD4 ＋ [3] The immune cell according to [1], which is a CD3 cell. [3] The immune cell according to [1] or [2], which is derived from a pluripotent stem cell. [4] The immune cell according to [3], wherein the pluripotent stem cell is an iPS cell. [4a] The immune cell according to any one of [1] to [4], which is a cell into which a foreign gene (particularly a gene for expressing a chimeric antigen receptor) has been introduced. [5] CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A method for producing a cell population in which immune cells that are expanded are: (1) in the presence of interleukin (IL)-2 and IL-33, CD4 ＋ A method comprising a step of culturing a cell population containing T cells. [6] The method according to [5], wherein the step (1) is further performed in the presence of an IL-4 and / or a TNFR2 agonist. [7] The method according to [5] or [6], wherein the step (1) is further performed in the presence of an IL-3 and / or a TGF-βR agonist. [8] The method according to any of [5] to [7], wherein the step (1) is further performed in the presence of an mTOR inhibitor. [9] The method according to any of [5] to [8], wherein the step (1) is further performed in the presence of IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, and an mTOR inhibitor.

[10] The method according to any of [5] to [9], wherein the culture in the step (1) is further performed in the presence of at least one selected from the group consisting of a CD30 agonist and a CD3 agonist. [10a] The method according to any one of [5] to [9], wherein the culture in the step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist.

[11] The method according to any one of [5] to [10a], wherein the culture in the step (1) is carried out repeatedly.

[12] The method according to any one of [6] to

[11] , wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

[13] The method according to any one of [7] to

[12] , wherein the TGF-βR agonist is TGF-β.

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

[13] , wherein the mTOR inhibitor is rapamycin.

[15] The method according to any one of

[16] to

[17] , wherein the CD4＋ T cells express CD25 ＋ / FOXP3 ＋ Cells and CD25 － / FOXP3 －

[16] The method according to any one of [5] to

[14] , wherein the CD4 ＋

[17] The method according to any one of [5] to

[15] , wherein the T cells are cells into which an expression construct comprising: (a) Conserved Non-coding Sequence (CNS) 1, CNS 2, and CNS 3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3 has been introduced.

[17] Prior to the step (1), (2) the pluripotent stem cells are transfected with CD4 ＋

[18] The method according to any one of [5] to

[16] , further comprising a step of inducing differentiation into a cell population containing T cells.

[18] The method according to

[17] , further comprising, before the step (2), a step of introducing into pluripotent stem cells an expression construct comprising: (a) CNS1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3. [18a] (4) Pluripotent stem cells (particularly iPS cells), CD4 ＋ T cells or CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method according to any one of [5] to

[18] , comprising a step of introducing a foreign gene (particularly a gene for expressing a chimeric antigen receptor) into a cell population containing immune cells that are CD3.

[19] ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ (1A) growing CD3 cells derived from pluripotent stem cells in the presence of IL-2 and IL-33; ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋[19a] The method according to

[19] , wherein the step (1A) is further carried out in the presence of IL-4 and / or a TNFR2 agonist. [19b] The method according to

[19] or [19a], wherein the step (1A) is further carried out in the presence of IL-3 and / or a TGF-βR agonist. [19c] The method according to any one of

[19] to [19b], wherein the step (1A) is further carried out in the presence of an mTOR inhibitor. [19d] The method according to any one of

[19] to [19c], wherein the step (1A) is further carried out in the presence of IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, and an mTOR inhibitor. [19e] The method of any of

[19] to [19d], wherein the culture in step (1A) is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist and a CD3 agonist. [19f] The method of any of

[19] to [19d], wherein the culture in step (1A) is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. [19g] The method of any of

[19] to [19f], wherein the step (1A) is repeatedly performed. [19h] The method of any of [19a] to [19g], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody. [19i] The method of any of [19b] to [19h], wherein the TGF-βR agonist is TGF-β. [19j] The method according to any one of [19c] to [19i], wherein the mTOR inhibitor is rapamycin. [19k] The method according to any one of

[19] to [19j], wherein the immune cells are cells into which an expression construct comprising: (a) Conserved Non-coding Sequence (CNS) 1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3 has been introduced. [19l] The method according to any one of

[19] to [19j], wherein, prior to the step (1A), the step (2A) comprises: (i) transfecting the pluripotent stem cells with CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋[19m] The method of any of

[19] to [19k], further comprising a step of inducing differentiation into a cell population containing immune cells which are: [19m] The method of [19l], further comprising, before step (2A), a step of introducing into pluripotent stem cells an expression construct comprising: (3A) (a) CNS1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3. [19n] (4A) Pluripotent stem cells (particularly iPS cells) or CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method according to any one of

[19] to [19m], comprising a step of introducing a foreign gene (particularly a gene for expressing a chimeric antigen receptor) into a cell population containing immune cells that are CD3.

[20] ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ (1B) in the presence of IL-2 and IL-33, CD4 ＋ / CD25 － / FOXP3 －A method comprising a step of culturing a cell population containing T cells. [20a] The method according to

[20] , wherein the step (1B) is further carried out in the presence of IL-4 and / or a TNFR2 agonist. [20b] The method according to

[20] or [20a], wherein the step (1B) is further carried out in the presence of IL-3 and / or a TGF-βR agonist. [20c] The method according to any of

[20] to [20b], wherein the step (1B) is further carried out in the presence of an mTOR inhibitor. [20d] The method according to any of

[20] to [20c], wherein the step (1B) is further carried out in the presence of IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, and an mTOR inhibitor. [20e] The method of any of

[20] to [20d], wherein the culture in step (1B) is further performed in the presence of at least one selected from the group consisting of a CD30 agonist and a CD3 agonist. [20f] The method of any of

[20] to [20d], wherein the culture in step (1B) is further performed in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. [20g] The method of any of

[20] to [20f], wherein the step (1B) is repeatedly performed. [20h] The method of any of [20a] to [20g], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody. [20i] The method of any of [20b] to [20h], wherein the TGF-βR agonist is TGF-β. [20j] The method according to any one of [20c] to [20i], wherein the mTOR inhibitor is rapamycin. [20k] Prior to the step (1B), (2B) the pluripotent stem cells are transfected with CD4 ＋ / CD25 － / FOXP3 － The method according to any one of

[20] to [20j], further comprising a step of inducing differentiation into a cell population containing T cells. [20l] (4B) Pluripotent stem cells (particularly iPS cells), CD4 ＋ / CD25 － / FOXP3 － T cells or CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋

[21] The method according to any one of [5] to

[201] , wherein the pluripotent stem cells are iPS cells.

[22] The method according to any one of [5] to

[21] , wherein the pluripotent stem cells are iPS cells.

[23] The method according to any one of [5] to

[23] , wherein the pluripotent stem cells are iPS cells.

[24] The method according to any one of [5] to

[24] , wherein the pluripotent stem cells are iPS cells.

[25] The method according to any one of [5] to

[25] , wherein the pluripotent stem cells are iPS cells.

[26] The method according to any one of [5] to

[26] , wherein the pluripotent stem cells are iPS cells. ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋

[23] A cell population comprising immune cells which are any of [1] to [4a] and / or the cell population of

[22] .

[24] The medicament of

[23] , for use in preventing and / or treating an abnormally enhanced immune response.

[25] A method for preventing and / or treating an abnormally enhanced immune response, comprising administering the immune cells of any of [1] to [4a] and / or the cell population of

[22] to a subject in need thereof.

[26] The immune cells of any of [1] to [4a] and / or the cell population of

[22] , for use in preventing and / or treating an abnormally enhanced immune response.

[27] Use of the immune cells of any of [1] to [4a] and / or the cell population of

[22] in the manufacture of a medicament for preventing and / or treating an abnormally enhanced immune response.

[0017] According to the present invention, CD3 ｌｏｗ／－ It is therefore possible to produce novel suppressive immune cells.

[0018] CD4 from iPS cells ＋ FIG. 1 shows the results of flow cytometry analysis of the CD3 positivity rate in cells after induction of differentiation into T cells. ＋ This figure shows the results of examining the expression levels of each protein in cells that were expanded in a medium containing IL-2 and IL-33 after differentiation induction into T cells. ＋ This figure shows the results of examining the expression levels of each protein in cells that were expanded in a medium containing IL-2 and IL-33 after differentiation induction into T cells. ＋This figure shows the change in cell number over time when cells after differentiation induction into T cells were expanded in a medium containing IL-2 and IL-33. ＋ This figure shows the results of flow cytometry analysis of cells induced to differentiate into T cells, which were expanded in a medium containing IL-2 and IL-33, and then co-cultured with allogeneic T cells derived from human PBMC. The values ​​shown as % inhibition in the figure indicate the rate of inhibition of target cell division. E:T is CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ The ratio of immune cells to human T cells is shown. Left: CD4 cells cultured in a 7-day expansion culture cycle, right: CD4 cells cultured in a 14-day expansion culture cycle. ＋ This figure shows the change in cell number over time when cells after differentiation induction into T cells were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, and TGF-β1. The cells were also cultured under a medium condition in which one or two of the cytokines IL-2, IL-3, IL-4, IL-33, and TGF-β1 were removed. ＋ This figure shows the results of examining the expression levels of each protein in cells that were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, and TGF-β1 after differentiation induction into T cells. Cells were also cultured under medium conditions in which one or two of the cytokines IL-2, IL-3, IL-4, IL-33, and TGF-β1 were removed. ＋ This figure shows the results of examining the CD3 expression levels of cells that were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, and TGF-β1 after differentiation induction into T cells. The cells were also cultured in a medium from which one or two of the cytokines IL-2, IL-3, IL-4, IL-33, and TGF-β1 were removed.

[0019] Hereinafter, embodiments of the present invention will be described in detail.

[0020] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" or "consisting essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0021] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0022] As used herein, "culturing a cell population in the presence of a substance" refers to, for example, culturing a cell population in a medium containing the substance. Examples of such culturing include culturing in a medium containing the substance alone, or in a medium containing the substance together with other differentiation-inducing factors. When the substance is added to the medium, it can be added directly to the medium, or the substance can be dissolved in an appropriate solvent just before use and then added to the medium. The substance can also be immobilized on the surface of a substrate or carrier during culturing and then cultured.

[0023] As used herein, "positive (+)" means that the protein or gene is expressed in an amount that can be detected by a method known in the art. In addition, in the case of a protein that is expressed intracellularly and not present on the cell surface (e.g., a transcription factor or a subunit thereof), the protein of interest can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Gene detection can be carried out using, for example, nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0024] As used herein, "negative (-)" means that the expression level of a protein or gene is below the lower limit of detection by all or any of the above-mentioned known techniques. The lower limit of detection of protein or gene expression may vary depending on the technique.

[0025] As used herein, the term "marker" refers to a protein or its gene that is specifically expressed on the cell surface, in the cytoplasm, in the nucleus, etc., in a specific cell type. The marker is preferably a "cell surface marker." A "cell surface marker" refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance, and that facilitates the detection, concentration, isolation, etc., of cells expressing the cell surface marker. The cell surface marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a specific cell type, specifically a substance that is produced (positive marker) or not produced (negative marker) as mRNA by transcription of the gene in the genome, or as a protein by translation of that mRNA.

[0026] Such cell surface markers can be detected by immunological assays using antibodies specific to the cell surface markers, such as ELISA, immunostaining, and flow cytometry.

[0027] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0028] As used herein, the term "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification).

[0029] As used herein, "hematopoietic stem cells (HSCs)" refer to multipotent stem cells that can differentiate into blood cells. In the human body, hematopoietic stem cells are primarily present in the bone marrow and differentiate into white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages), red blood cells, platelets, mast cells, dendritic cells, etc. As used herein, hematopoietic stem cells (HSCs) may be CD34 positive and CD7 negative (CD34 ＋ / CD7 － In the present specification, "CD34 ＋ / CD7 － The " / " used in expressions such as " means "and."

[0030] As used herein, "hematopoietic progenitor cells" (HPCs) are cells that have the ability to differentiate into blood cells but do not have the self-renewal ability of stem cells. In the human body, hematopoietic progenitor cells are primarily present in the bone marrow. As used herein, hematopoietic progenitor cells (HPCs) are CD34-positive and may be CD43-positive (CD34 ＋ / CD43 ＋ Additionally, HPCs may 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 WO 2018 / 199186.

[0031] As used herein, "hemogenic endothelial cells (HECs)" refer to cells that express CD34 but do not express CD43, CD184, or CD73 (CD34 ＋ / CD43 － / CD184 － / CD73 － ) cells (Note: CD34 ＋ / CD43 － / CD184 － / CD73 － The cells do not express CD7, so CD34 ＋ / CD43 － / CD184 － / CD73 － The cells are CD34 ＋ / CD7 － / CD43 － / CD184 － / CD73 － (also referred to as cells).

[0032] As used herein, the term "proT cells" refers to hematopoietic cells that are generated in the human body during the process of differentiation from hematopoietic stem cells into CD3-positive T cells, and are CD34 and CD7 positive (CD34 ＋ / CD7 ＋ Also, as used herein, proT may be negative for CD43, CD1a, and / or CD116.

[0033] As used herein, the term "mesodermal progenitor cells" refers to cells expressing at least one marker gene selected from the group consisting of, for example, T (synonymous with Brachyury), KDR, FOXF1, FLK1, BMP4, MOX1, and SDF1. Mesodermal progenitor cells are not to be distinguished from mesodermal cells, and cells that weakly express the above marker genes may also be referred to as mesodermal progenitor cells.

[0034] As used herein, the term "regulatory T cells" refers to T cells that, when stimulated via a T cell receptor, have the ability to inhibit the activation of effector T cells and are responsible for suppressing immune responses (immune tolerance). Regulatory T cells are generally CD25 ＋ / FOXP3 ＋cells or CD25 ＋ / CD127 － cells, among which CD4 ＋ or CD8 ＋ In the present invention, regulatory T cells are CD4 ＋ cells (i.e., CD4 ＋ / CD25 ＋ / FOXP3 ＋ or CD4 ＋ / CD25 ＋ / CD127 － ) or CD8 ＋ cells (i.e., CD8 ＋ / CD25 ＋ / FOXP3 ＋ or CD8 ＋ / CD25 ＋ / CD127 － ), and CD4 ＋ More preferred are CD4 ＋ / CD25 ＋ The transcription factor FOXP3 is known as a master regulator of regulatory T cells. Regulatory T cells may also be positive for Helios, CTLA4 (Cytotoxic T Lymphocyte (associated) Antigen 4) (CD152), CD39, and CD73, which are known to be indicators of the suppressive function of regulatory T cells. Helios is a member of the Ikaros transcription factor family and has been shown to bind to the FOXP3 promoter and increase FOXP3 expression. CTLA4 is an immune checkpoint protein, and when CTLA4 expressed on the surface of T cells binds to CD80 or CD86 on the surface of antigen-presenting cells, T cell activation is suppressed. Furthermore, it is known that CD39 and CD73 hydrolyze ATP and AMP, respectively, to produce adenosine as the final product, and that when adenosine acts on T cells, activation of the T cells is suppressed.

[0035] As used herein, "CD4 ＋ "T cells" refers to T cells that are positive for the surface antigen CD4. Preferably, CD4 ＋ T cells were CD8 negative (CD4＋ / CD8 － , CD4 single positive (SP)). In this case, T cells can be recognized by the positivity of the surface antigens CD3 and CD45. ＋ T cells can be identified as cells that are CD8 negative and CD4, CD3, and CD45 positive (CD4 ＋ / CD8 － / CD3 ＋ / CD45 ＋ cell). CD4 ＋ Examples of T cells include helper T cells and regulatory T cells. ＋ As for "T cells," CD4 ＋ / CD25 － / FOXP3 － cells or CD4 ＋ / CD25 ＋ / FOXP3 ＋ Cells are preferred.

[0036] As used herein, "CD8 ＋ "T cells" refers to T cells that are positive for the surface antigen CD8. Preferably, CD8 ＋ T cells are CD4 negative (CD4 － / CD8 ＋ , CD8 single positive (SP)). In this case, T cells can be recognized by the positivity of the surface antigens CD3 and CD45. ＋ T cells can be identified as cells that are CD4 negative and CD8, CD3, and CD45 positive (CD4 － / CD8 ＋ / CD3 ＋ / CD45 ＋ cell). CD8 ＋ Examples of T cells include cytotoxic T cells and regulatory T cells. ＋ As for "T cells," CD8 ＋ / CD25 － / FOXP3 － cells or CD8 ＋ / CD25 ＋ / FOXP3 ＋Cells are preferred.

[0037] As used herein, the term "stromal cells" refers to cells that constitute connective tissue that supports parenchymal cells in biological tissues, and in particular to stromal cells capable of producing ATO (artificial thymic organoid) that produces T cells, representative stromal cells contained in hematopoietic tissues such as bone marrow and thymus. Examples include fibroblasts, blood cells, mesenchymal stem cells, endothelial cells, smooth muscle cells, epithelial cells, and tissue stem cells. Stromal cells may be established cell lines such as mouse bone marrow cell lines MS-5, OP9, and S17, and human stromal cell lines HS-5 and HS-27a, primary cells collected from humans, and cells induced to differentiate from pluripotent stem cells (e.g., iPS cells) of humans, etc. In the case of cells induced to differentiate from pluripotent stem cells of humans, etc., stromal cells induced to differentiate from human iPS cells (particularly fibroblasts induced to differentiate from human iPS cells) are preferred.

[0038] As used herein, the term "CD4CD8 dual positive T cells" refers to T cells that are positive for both the surface antigens CD4 and CD8 (CD8 ＋ / CD4 ＋ ) and T cells can be recognized by their surface antigens CD3 and CD45 positivity. Therefore, CD4CD8 co-positive T cells can be identified as cells that are positive for CD4, CD8, CD3, and CD45 (CD8 ＋ / CD4 ＋ / CD3 ＋ / CD45 ＋ cell).

[0039] As used herein, the term "CD4CD8 double negative T cells" refers to T cells that are negative for both the surface antigens CD4 and CD8 (CD8 － / CD4 － ) and T cells can be recognized by their surface antigens CD3 and CD45 positivity. Therefore, CD4CD8-negative T cells can be identified as cells that are CD4 and CD8 negative and CD3 and CD45 positive (CD8 － / CD4 － / CD3 ＋ / CD45 ＋ cell).

[0040] As used herein, the term "cell population" refers to two or more cells of the same or different types. The term "cell population" also refers to a mass of cells of the same or different types.

[0041] As used herein, "immune cells proliferated" and "immune cell proliferation" refer to the proliferation of immune cells (CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The term "immune cells" refers to an increase in the number (absolute number) of immune cells (which is at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 40-fold, 50-fold, or 100-fold per cycle (repeated culture unit) compared to before the culture or the control, and also refers to an increase in the number (absolute number) of immune cells (which is at least 1 x 10) over the entire culture period compared to before the initial culture or the control by repeated culture. １ times 1 x 10 ２ times 1 x 10 ３ times 1 x 10 4 times 1 x 10 5 times 1 x 10 ６ times 1 x 10 7 times 1 x 10 8 times 1 x 10 ９ times 1 x 10 １０ times 1 x 10 １１ times 1 x 10 １２ times 1 x 10 １３ times 1 x 10 1４ times 1 x 10 15 times 1 x 10 20 In a specific embodiment of the present invention, the CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋The number (absolute number) of immune cells is increased by at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 40-fold, 50-fold, or 100-fold per cycle (repeated culture unit) compared to the number of cells before culture or the control, and by repeated culture, the number (absolute number) of immune cells is increased by at least 1 x 10 over the entire culture period compared to the number of cells before the initial culture or the control. １ times 1 x 10 ２ times 1 x 10 ３ times 1 x 10 4 times 1 x 10 5 times 1 x 10 ６ times 1 x 10 7 times 1 x 10 8 times 1 x 10 ９ times 1 x 10 １０ times 1 x 10 １１ times 1 x 10 １２ times 1 x 10 １３ times 1 x 10 1４ times 1 x 10 15 times 1 x 10 20 It is manufactured to double the amount.

[0042] From the viewpoint of therapeutic application, the various cells used in the present invention are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0043] Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor 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 cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, and preferably cells produced without destroying an embryo.

[0044] As for "ES cells," in the case of mouse ES cells, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (Institute of Physical and Chemical Research), etc. can be used, and in the case of human ES cells, various human ES cell lines established by University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used. For example, human ES cell lines that can be used include CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. distributed by ESI Bio, H1 strain, H9 strain, etc. distributed by WiCell Research, and KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. distributed by RIKEN.

[0045] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including 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), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872), and Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using 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.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open Publication No. 2008-307007), and the like can also be used.

[0046] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholeer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, U.S. Patent Application Publication No. 2008 / 2336610, U.S. Patent Application Publication No. 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) Any of the induced pluripotent stem cells known in the art can be used.

[0047] As induced pluripotent stem cell lines, various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain, and Kyoto University's 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.

[0048] The term "nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules having functions equivalent to those nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analogue. Nucleic acids may also be single-stranded or double-stranded nucleic acids. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0049] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acid (BNA), locked artificial nucleic acid (LNA), ethylene bridged artificial nucleic acid (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0050] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0051] The immune cells of the present invention are CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ cells (referred to herein as "CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ The immune cells of the present invention are characterized in that they are immune cells of the present invention.

[0052] As used herein, "immune cells" refer to cells involved in an immune response. The term "low" used in connection with CD3 is well known in the art and refers to a cell marker expression level that is lower (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower) than the expression level of that cell marker in the cell population analyzed as a whole. Generally, the level of protein or gene expression (whether low or high) can be determined by comparing it with the results of control cells measured under the same conditions. The level of CD3 expression in a certain cell population can be determined, for example, using flow cytometry, by comparing the CD3 expression level in the cell population with the CD3 expression level in control cells known to be low CD3 expressers. If expression is equivalent to that of the control, it is determined to be low expression, and if expression is higher than that of the control cells, it is determined to be high expression. Alternatively, for example, when comparing the CD3 expression level in a positive control (e.g., T cells) known to have high CD3 expression, if expression is lower than that of the control cells (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower), it can be determined that the expression is low, and if expression is equivalent to that of the control, it can be determined that the expression is high. ｌｏｗ means that CD3 is expressed but at a low level. ｌｏｗ refers to a type of cell that is only weakly or dimly stained when contacted with a labeled anti-CD3 antibody. The immune cells of the present invention are CD3 ｌｏｗ／－ That is, the immune cells of the present invention are cells in which CD3 expression is reduced or below the lower limit of detection, and are novel suppressive immune cells that have not been known so far and have the same function as Tregs to suppress immune responses (immune tolerance). － , CD25 ＋ and FOXP3 ＋Conventionally, Tregs are CD3 positive (=TCRαβ positive), and thus there has been concern about side effects due to non-specific antigen recognition in cell therapy. However, the immune cells of the present invention are TCRαβ negative, and therefore such side effects are expected to be reduced. Furthermore, the immune cells of the present invention further contain CD4 ＋ In addition, the immune cells of the present invention may be CD8 negative, and may be GZMB negative or positive (preferably GZMB negative). Furthermore, the immune cells of the present invention may be positive for Helios, CTLA4 (Cytotoxic T Lymphocyte (associated) Antigen 4) (CD152), CD39, and CD73, which are known as indicators of the suppressive function of regulatory T cells.

[0053] The immune cells of the present invention are preferably derived from pluripotent stem cells (particularly iPS cells (especially human iPS cells)). The pluripotent stem cells may be derived from humans or from mammals other than humans (non-human mammals), preferably humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys. Universalized iPS cells (i.e., iPS cells with a specific HLA type that does not cause immune rejection in many patients, or iPS cells in which HLA has been knocked out) can also be used. The method for obtaining the immune cells of the present invention is not particularly limited, and can be obtained, for example, by the production and proliferation methods described below.

[0054] CD3 of the present invention ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method for producing a cell population in which immune cells that are the above-mentioned IL-2 and IL-33 are expanded (hereinafter, simply referred to as the "production method of the present invention") is characterized by comprising the following steps: (1) Producing pluripotent stem cell-derived CD4 ＋ Culturing the cell population comprising T cells.

[0055] Step (1) can also be carried out in the presence of IL-4 and / or a TNFR2 agonist in addition to IL-2 and IL-33. That is, step (1) can be carried out in the presence of IL-2, IL-33, and IL-4, in the presence of IL-2, IL-33, and a TNFR2 agonist, or in the presence of IL-2, IL-33, IL-4, and a TNFR2 agonist, etc.

[0056] Furthermore, step (1) can also be carried out in the presence of IL-3 and / or a TGF-βR agonist, particularly in the presence of IL-3 and a TGF-βR agonist, in addition to the above components. That is, step (1) can be carried out in the presence of IL-2, IL-33, and IL-3, in the presence of IL-2, IL-33, and a TGF-βR agonist, in the presence of IL-2, IL-33, IL-3, and a TGF-βR agonist, in the presence of IL-2, IL-33, IL-4, and IL-3, in the presence of IL-2, IL-33, IL-4, and a TGF-βR agonist, in the presence of IL-2, IL-33, IL-4, IL-3, and a TGF-βR agonist, in the presence of IL-2, IL-33, a TNFR2 agonist, and IL-3, or in the presence of IL-2, IL-33, a TNFR2 agonist, or IL-3. The incubation can also be carried out in the presence of IL-2, IL-33, a TNFR2 agonist, and a TGF-βR agonist, in the presence of IL-2, IL-33, a TNFR2 agonist, IL-3, and a TGF-βR agonist, in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, and IL-3, in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, and a TGF-βR agonist, or in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, IL-3, and a TGF-βR agonist.

[0057] Step (1) can also be carried out in the presence of an mTOR inhibitor in addition to the above components. That is, step (1) can be carried out in the presence of IL-2, IL-33 and an mTOR inhibitor, in the presence of IL-2, IL-33, IL-4 and an mTOR inhibitor, in the presence of IL-2, IL-33, a TNFR2 agonist and an mTOR inhibitor, in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist and an mTOR inhibitor, or in the presence of IL-2, IL-33, IL-3 and an mTOR inhibitor. , in the presence of IL-2, IL-33, TGF-βR agonist and mTOR inhibitor, IL-2, IL-33, IL-3, TGF-βR agonist and mTOR inhibitor, IL-2, IL-33, IL-4, in the presence of IL-3 and mTOR inhibitor, IL-2, IL-33, IL-4, TGF-βR agonist and mTOR inhibitor, IL-2, IL-33, IL-4 In the presence of IL-3, a TGF-βR agonist and an mTOR inhibitor; In the presence of IL-2, IL-33, a TNFR2 agonist, IL-3 and an mTOR inhibitor; In the presence of IL-2, IL-33, a TNFR2 agonist, a TGF-βR agonist and an mTOR inhibitor; In the presence of IL-2, IL-33, a TNFR2 agonist, a TGF-βR agonist and an mTOR inhibitor; The treatment can also be carried out in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, IL-3 and an mTOR inhibitor, in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, a TGF-βR agonist and an mTOR inhibitor, or in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, IL-3, a TGF-βR agonist and an mTOR inhibitor. Of these, the treatment is most preferably carried out in the presence of IL-2, IL-33, IL-4, a TNFR2 agonist, IL-3, a TGF-βR agonist and an mTOR inhibitor.

[0058] IL-4 (interleukin)-3, IL-2, and IL-33 are preferably derived from mammals, particularly humans. IL-4, IL-3, IL-2, and IL-33 may be natural products isolated and purified from mammals (particularly humans), or may be artificially produced by genetic engineering techniques (which may include amino acid substitution, deletion, insertion, and / or addition).

[0059] A TGF-βR (Transforming Growth Factor-β Receptor) agonist is defined as a substance that can bind to TGF-βR and activate TGF-βR signal transduction. Examples of TGF-βR agonists include factors belonging to the TGF-β superfamily, which 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, TGF-β3), activin A, activin B, GDF-8, and GDF-11. The TGF-βR agonist is preferably TGF-β, and more preferably TGF-β1. One type of TGF-βR agonist can be used alone, or two or more types can be used in combination.

[0060] A TNFR2 (Tumor Necrosis Factor Receptor-2) agonist is defined as a substance that can bind to TNFR2 and activate TNFR2 signal transduction, and examples of such an agonist include antibodies (e.g., anti-TNFR2 antibodies), peptides, small molecules, and proteins. Examples of anti-TNFR2 agonist antibodies include monoclonal antibodies that bind to TNFR2, such as clone MR2-1 (Hycult Biotech) and clone MAB2261 (R&D Systems). The TNFR2 agonist may also be a TNF-α mutein that binds only to TNFR2 as an agonist. TNFR2 agonists can be used singly or in combination of two or more.

[0061] The TNFR2 agonist is preferably an anti-TNFR2 agonist antibody. The antibody may be a functional fragment thereof, and examples of the functional fragment include Fd, Fv, Fab, F(ab'), and F(ab). ２ , F(ab') ２ Examples of antibodies include single-chain Fvs (scFvs), diabodies, triabodies, tetrabodies, and minibodies. Antibodies may be derived from animals such as mice, rats, cows, rabbits, goats, sheep, and guinea pigs. The antibody isotype is not particularly limited, and examples of isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM. The antibody may be either a monoclonal or polyclonal antibody, preferably a monoclonal antibody. The antibody may also be a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), or the like. Antibodies can be produced by known methods, for example, by constructing an expression vector containing a nucleic acid encoding the antibody, culturing a transformant into which the nucleic acid has been introduced, or culturing a hybridoma that produces the antibody.

[0062] An mTOR (mechanistic target of rapamycin) inhibitor is defined as a substance that inhibits the function of mTOR, and includes substances that inhibit the function of mTOR itself, as well as substances that inhibit the function of mTOR complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Examples of mTOR inhibitors include rapamycin or a derivative thereof, everolimus, temsirolimus, ridaforolimus, sirolimus, KU0063794, AZD805, AZD8055, WYE-354, WAY-600, WYE-687, Pp121, Pp242, Dactolisib, Sapanisertib, Omipalisib, Vistusertib, Torin 1, and Torin 2. Other examples of mTOR inhibitors include siRNA, shRNA, dsRNA, miRNA, antisense nucleic acids against a gene encoding mTOR and / or its transcription product, and expression vectors that express these, as well as siRNA, shRNA, dsRNA, miRNA, antisense nucleic acids against a gene encoding an enzyme that phosphorylates and activates mTOR and / or its transcription product, and expression vectors that express these. Among these, rapamycin or a derivative thereof is preferred as an mTOR inhibitor, and rapamycin is more preferred. One mTOR inhibitor can be used alone, or two or more can be used in combination.

[0063] Furthermore, IL-2, IL-33, IL-4, IL-3, TGF-βR agonists, TNFR2 agonists, and mTOR inhibitors can be used in their free form or in the form of a salt. Examples of salts include salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts with organic bases such as methylamine salts, ethylamine salts, and ethanolamine salts; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. The salts may be acid addition salts, and specific examples of such salts include acid addition salts with mineral 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. IL-2, IL-33, IL-4, IL-3, TGF-βR agonists, TNFR2 agonists, and mTOR inhibitors also include hydrates, solvates, crystalline polymorphs, and the like.

[0064] The concentration of IL-2 in the medium is not particularly limited and is, for example, 0.1 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0065] The concentration of IL-33 in the medium is not particularly limited and is, for example, 0.1 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0066] The concentration of IL-4 in the medium is not particularly limited and is, for example, 0.01 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0067] The concentration of IL-3 in the medium is not particularly limited and is, for example, 0.01 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0068] The concentration of the TGF-βR agonist in the medium is not particularly limited 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, preferably 1 to 50 ng / mL.

[0069] The concentration of the TNFR2 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of TNFR2 agonist used, etc. The concentration of the TNFR2 agonist is, for example, 0.0001 to 100 μg / mL, preferably 0.01 to 10 μg / mL.

[0070] The concentration of the mTOR inhibitor in the medium is not particularly limited and is adjusted appropriately depending on the type of mTOR inhibitor used, etc. The concentration of the mTOR inhibitor is, for example, 1 to 100 nM, preferably 10 to 100 nM.

[0071] The medium used for the culture in step (1) is a basal medium that is used for culturing animal cells and contains the above-mentioned IL-2 and IL-33 (as well as IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, an mTOR inhibitor, etc.). The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium. Any one of these media may be used alone, or two or more may be used in combination.

[0072] The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). One or more types of serum substitutes may be used.

[0073] Furthermore, the medium may also contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD28 antibodies, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. It is desirable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows the preparation of cells of stable quality.

[0074] The pH of the medium is usually 7.0 to 7.8, preferably 7.2 to 7.6. Before use, the medium is preferably sterilized by filtration, ultraviolet irradiation, heat sterilization, radiation irradiation, or other methods to prevent contamination.

[0075] The culture may be carried out in the presence or absence of feeder cells. The production method of the present invention is preferably carried out in the absence of feeder cells, since it allows stable production of immune cells of the present invention having uniform properties without contamination with unknown components.

[0076] The culture conditions for the production method of the present invention are not particularly limited. The culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C, and CO ２ The 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%.

[0077] The culture period is also not particularly limited, and can be appropriately determined by a person skilled in the art while monitoring the number of immune cells of the present invention, and may be, for example, 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, 49 days or more, 56 days or more, 63 days or more, 70 days or more, 77 days or more, 84 days or more, 91 days or more, 98 days or more, 105 days or more, 112 days or more, etc. The upper limit of the culture period is not particularly limited, and may be, for example, 400 days or less, 300 days or less, 200 days or less, 150 days or less, etc. In the culture of the present invention, passage may be performed as many times as necessary to obtain a desired amount of immune cells, and the medium may be added and replaced. The culture of the present invention can be performed using known CO ２The culture can be carried out using an incubator. The culture vessel is not particularly limited and can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture vessels), bioreactors, etc. A vessel to which an anti-CD3 antibody is bound can be used as the culture vessel.

[0078] The CD30 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD30 into a cell by specifically binding to CD30. Examples of the CD30 agonist include an anti-CD30 agonist antibody or a functional fragment thereof, a CD30 ligand or a functional fragment thereof, etc. The concentration of the CD30 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD30 agonist used, etc. The concentration of the CD30 agonist is, for example, 10 to 1000 ng / mL, preferably 100 to 500 ng / mL.

[0079] The CD3 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD3 into a cell by specifically binding to CD3. Examples of CD3 agonists include an anti-CD3 agonist antibody or a functional fragment thereof, a CD3 ligand or a functional fragment thereof, etc. The concentration of the CD3 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD3 agonist used, etc. The concentration of the CD3 agonist is, for example, 10 to 10,000 ng / mL, preferably 100 to 10,000 ng / mL, and more preferably 1,000 to 5,000 ng / mL.

[0080] The CD28 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD28 into a cell by specifically binding to CD28. Examples of the CD28 agonist include an anti-CD28 agonist antibody or a functional fragment thereof, a CD30 ligand or a functional fragment thereof, etc. The concentration of the CD28 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD28 agonist used, etc. The concentration of the CD28 agonist is, for example, 10 to 10,000 ng / mL, preferably 100 to 10,000 ng / mL, and more preferably 500 to 5,000 ng / mL.

[0081] In step (1), culture (cell stimulation) may be initiated initially in the presence of at least one selected from the group consisting of a CD30 agonist and a CD3 agonist (preferably a CD30 agonist) in the presence of IL-2 and IL-33 (and IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, or an mTOR inhibitor may also be added), followed by culture in their absence. Step (1) may be repeatedly performed (by subculturing), or culture (cell stimulation) may be initiated initially in the presence of at least one selected from the group consisting of a CD30 agonist and a CD3 agonist (preferably a CD30 agonist), followed by culture in their absence. The culture period for this repetition is, for example, 5 to 16 days, preferably 6 to 14 days, more preferably 6 to 10 days, and even more preferably 6 to 8 days. Specifically, the cells are cultured (stimulated) for the first three days or so in the presence of at least one selected from the group consisting of a CD30 agonist and a CD3 agonist (preferably a CD30 agonist), and then cultured for about four to eleven days in a medium that does not contain these agonists, and this unit of culture is repeated. The number of times the culture is repeated is not particularly limited, and can be determined appropriately by a person skilled in the art while monitoring the number of immune cells of the present invention, etc., and may be, for example, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more. The upper limit of the number of times the culture can be repeated is, for example, 20 times. Repeating the culture of step (1) in this manner enables long-term culture and allows the proliferation of the immune cells of the present invention (the immune cells of the present invention (CD3) contained in the cell population). ｌｏｗ／－ The proportion of cells in the sera can be increased.

[0082] The pluripotent stem cells and cells for producing pluripotent stem cells used in the present invention may be derived from humans or mammals other than humans (non-human mammals), preferably humans. Non-human mammals include, for example, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys. Universalized iPS cells (i.e., cells with a specific HLA type or knockout HLA that do not cause immune rejection in many patients) can also be used.

[0083] CD4 used in the production method of the present invention ＋ The cell population containing T cells is a CD4 T cell-derived cell that has been differentiated from pluripotent stem cells (particularly iPS cells (especially human iPS cells)). ＋ A cell population that includes T cells. CD4 ＋ CD4 in a cell population containing T cells ＋ The proportion (cell number) of T cells is, for example, 5% or more, preferably 10% or more, more preferably 30% or more, and the upper limit is, for example, 100% or less.

[0084] The production method of the present invention may further comprise a step of separating the immune cells of the present invention in order to enrich the obtained immune cells of the present invention. The immune cells of the present invention can be separated by known methods such as a method using flow cytometry or a magnetic cell separation method.

[0085] By the production method of the present invention, a cell population containing the immune cells of the present invention can be produced by expanding the immune cells. ＋ For T cells, CD25 ＋ / FOXP3 ＋ Cells and CD25 － / FOXP3 － Any of the cells can be used, and in either case, these cells can be induced to differentiate into the immune cells of the present invention, thereby producing a cell population containing the immune cells of the present invention at a high ratio.

[0086] CD4 ＋ CD25 on T cells － / FOXP3 －When cells are used, the proportion (cell number) of the immune cells contained in a cell population containing the immune cells of the present invention obtained by the production method of the present invention is, for example, 10% or more, preferably 50% or more, more preferably 80% or more, and the upper limit is, for example, 100% or less.

[0087] In addition, in an embodiment of the present invention, CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method for expanding immune cells is characterized by comprising the following steps: (1A) growing CD3 cells derived from pluripotent stem cells in the presence of IL-2 and IL-33; ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A step of culturing a cell population comprising immune cells which are

[0088] CD4 derived from pluripotent stem cells ＋ For T cells, CD25 － / FOXP3 － In embodiments of the invention where T cells are used, CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method for producing a cell population containing immune cells comprising: (1B) generating CD4 cells derived from pluripotent stem cells in the presence of IL-2 and IL-33; ＋ / CD25 － / FOXP3 － Culturing the cell population comprising T cells.

[0089] Steps (1A) and (1B) can be carried out in the same manner as step (1). ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A method for expanding immune cells that are CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The method for producing a cell population containing immune cells is encompassed by the production method of the present invention, and the explanation regarding the production method of the present invention is applied.

[0090] The production method and proliferation method of the present invention comprise the steps of: (2) transfecting pluripotent stem cells with CD4 ＋The method may further comprise the step of inducing differentiation into a cell population containing T cells.

[0091] CD4 in pluripotent stem cells ＋ The differentiation of pluripotent stem cells into a cell population containing T cells can be carried out according to known methods. ＋ After inducing differentiation into cells capable of differentiating into T cells, the CD4 ＋ CD4 cells that can differentiate into T cells ＋ Induction of differentiation into a cell population containing CD4 T cells. ＋ Cells that can differentiate into T cells include CD34 ＋ cells, mesodermal progenitor cells, CD4CD8 double negative T cells, CD4CD8 double positive T cells, etc., and preferably CD34 ＋ The CD34 is preferably a hematopoietic endothelial cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell precursor, or a mesodermal progenitor cell, and more preferably a hematopoietic endothelial cell. ＋ The cells express CD34 (CD34 ＋ ) cells, which specifically express CD34 but do not express CD7 (CD34 ＋ / CD7 － ) cells. ＋ Examples of cells include hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells.

[0092] Pluripotent stem cells were transduced with CD4 ＋ The differentiation of cells capable of differentiating into T cells can be carried out according to known methods (see, for example, International Publication No. WO 2021 / 085576). ＋ Cells that can be differentiated into T cells can be produced by a feeder-free method, and among these, the "embryonic body method (EB method)" (see A.E. Grigoriadis et al. (2010). Blood, 115(14):2769-2776) is preferred, which involves forming embryonic bodies to produce hematopoietic progenitor cells without using feeder cells.

[0093] Pluripotent stem cells can also be cultured on feeder cells to induce differentiation into hematopoietic stem cells and / or hematopoietic progenitor cells (see International Publication Nos. WO 2011 / 096482 and WO 2013 / 176197). From the viewpoint of facilitating induction of differentiation into mesodermal lineages, the feeder cells are preferably stromal cells. From the viewpoint of facilitating induction of differentiation into hematopoietic lineages, the stromal cells are preferably OP9 cells, 10T1 / 2 cells (C3H10T1 / 2 cells), or the like.

[0094] CD34 in pluripotent stem cells ＋ The differentiation of hematopoietic progenitor cells can be carried out according to known methods. When the pluripotent stem cells are iPS cells, for example, hematopoietic progenitor cells can be induced to differentiate into CD34 cells by the methods described in International Publication Nos. 2017 / 221975, 2018 / 135646, and Cell Reports 2 (2012) 1722-1735. ＋ The cells can be manufactured.

[0095] CD4 ＋ CD4 cells that can differentiate into T cells ＋ Examples of methods for inducing differentiation into a cell population containing T cells include the ATO (artificial thymic organoid) method (see International Publication No. 2017 / 075389, etc.). ＋ The goal is to culture three-dimensional cell aggregates containing cells that can differentiate into T cells and stromal cells that express Notch ligands, which allows for highly efficient CD4 ＋ The ATO method is a known method and can be carried out with reference to the descriptions in WO 2017 / 075389, WO 2021 / 085576, etc. Other methods include the differentiation induction method described in WO 2021 / 092581 (MS5-DLL1 / 4 stromal cells, OP9 or OP9-DLL1 stromal cells, or EpCAM-CD56 ＋ and co-culturing with stromal cells.

[0096] When performing the ATO method, CD4＋ The step of separating cells that can be differentiated into T cells may not be performed, or CD4 cells separated according to known methods (e.g., flow cytometry, magnetic cell separation) may be used. ＋ The ATO method can also be performed on cells that can be differentiated into T cells.

[0097] It is preferable that a nucleic acid for expressing a Notch ligand has been introduced into the stromal cells. Stromal cells expressing a Notch ligand can be produced by introducing the nucleic acid into stromal cells. Alternatively, the stromal cells can be produced by introducing the nucleic acid into pluripotent stem cells (particularly iPS cells (especially human iPS cells)) and then differentiating the pluripotent stem cells into stromal cells.

[0098] Examples of stromal cells used in the production method of the present invention include stromal cells induced to differentiate from pluripotent stem cells (particularly iPS cells (especially human iPS cells)). Among these, it is desirable to use cells obtained by inducing differentiation of pluripotent stem cells (particularly iPS cells (especially human iPS cells)) into fibroblasts, as described in International Publication No. WO 2023 / 149555, etc. Pluripotent stem cells can be induced to differentiate into stromal cells according to known methods. When the pluripotent stem cells are human iPS cells, fibroblasts induced to differentiate from iPS cells can be produced, for example, by the method described in PLoS ONE 8(10):e77673, 2013.

[0099] The Notch ligand is not particularly limited and includes canonical Notch ligands and non-canonical Notch ligands described in WO 2017 / 075389. Examples of canonical Notch ligands include DLL4 (delta-like ligand 4), DLL1 (delta-like ligand 1), JAG1 (Jagged 1), and JAG2 (Jagged 2). These can be used alone or in combination of two or more. 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 netrin-1. As the Notch ligand, human DLL4 is preferably used.

[0100] The means for introducing the nucleic acid for expressing the Notch ligand into stromal cells or pluripotent stem cells is not particularly limited, and various known or common means can be used. Typically, the nucleic acid for expressing the Notch ligand is introduced into stromal cells using an expression vector, and then 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-based vector.

[0101] The technique for introducing the expression vector into stromal cells can be selected appropriately depending on the embodiment. For example, the expression vector can be introduced into stromal cells by known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or, as appropriate, using a commercially available kit (following the instructions).

[0102] The expression vector can be introduced into stromal cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a vector containing a nucleic acid for expressing the Notch ligand and a packaging vector (plasmid) for each virus can be transfected into host cells using a corresponding commercially available kit to produce a recombinant virus, and then the resulting recombinant virus can be used to infect stromal cells.

[0103] 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 multicloning site (MCS) as necessary. The expression vector may further comprise a reporter gene (e.g., a gene encoding a fluorescent protein of each color), a drug selection gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene), a suicide gene (e.g., a diphtheria A toxin, herpes simplex 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 (inducible caspase 9)), or a gene encoding a cytochrome P450 (cyt-450). The nucleic acid sequence may include a nucleic acid (base sequence) encoding a "functional gene" such as a gene encoding a nucleotide sequence of ...

[0104] The three-dimensional cell aggregates may be, for example, CD4 ＋ The cells can be formed by centrifuging cells that can differentiate into T cells and stromal cells that express a Notch ligand. The stromal cells are preferably transfected with a nucleic acid that expresses a Notch ligand. Stromal cells: CD4 ＋ The ratio of cells that can differentiate into T cells is determined by the ratio of the stromal cells and CD4＋ The ratio can be appropriately adjusted by those skilled in the art depending on the type of cells that can be differentiated into T cells, and examples include 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, and 10:1 to 1:10. For example, when the stromal cells are mouse bone marrow cells (particularly MS-5), the ratio is preferably 20:1 to 1:4, and when the stromal cells are pluripotent stem cell-derived stromal cells (particularly fibroblasts induced to differentiate from iPS cells (especially human iPS cells)), the ratio is preferably 4:1 to 1:4, and more preferably 1:1.

[0105] The medium for culturing the three-dimensional cell aggregates is not particularly limited, and may be, for example, a serum-containing medium, a serum-free medium, or a xeno-free medium, preferably a serum-free medium, particularly a serum-free medium containing insulin (and further biotin, transferrin, and albumin).

[0106] Examples of basal media for culturing three-dimensional cell aggregates include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium. Any one of these media may be used alone, or two or more may be used in combination.

[0107] The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). One or more types of serum substitutes may be used.

[0108] Furthermore, the medium may contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. It is desirable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows the preparation of cells of stable quality. Other medium components, such as those described in International Publication No. 2017 / 075389, may also be appropriately blended.

[0109] The culture temperature of the three-dimensional cell aggregate is, for example, 20 to 40°C, preferably about 37°C, and CO ２ The 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 culture is, for example, 1.0 × 10 ４ ~1.0~10 １０ The concentration can be about 100 cells / mL.

[0110] The culture period of the 3D cell aggregates depends on the stromal cells and CD4 ＋ The culture period can be appropriately adjusted by those skilled in the art depending on the type of cells that can be differentiated into T cells, and is, for example, 4 weeks or more, preferably 5 weeks or more, and more preferably 6 weeks or more. The upper limit of the culture period is not particularly limited, and is preferably 16 weeks or less, more preferably 14 weeks or less, even more preferably 12 weeks or less, and particularly preferably 9 weeks or less.

[0111] CD4 obtained by culturing three-dimensional cell aggregates ＋ The cell population containing T cells includes CD4 ＋ Because cells other than T cells may be included, CD4 ＋ For example, a cell population containing T cells is subjected to flow cytometry (typically, fluorescence-activated cell sorting: FACS) using a fluorescently labeled anti-CD4 antibody and a cell sorter to detect CD4 ＋ T cells may be isolated and collected. CD4SP T cells may be isolated and collected. Furthermore, effector T cells (Tconv), which are CD25-negative, may be isolated and collected.

[0112] CD4 obtained by culturing three-dimensional cell aggregates ＋ Prior to step (1), the cell population containing T cells may be isolated by a known method (e.g., by isolating CD4 T cells in a medium containing an anti-CD3 antibody and IL-2). ＋ The cell population containing the T cells may be subjected to an expansion step by culturing the resulting cell population.

[0113] In the production method of the present invention, CD4 ＋ T cells may be used that have been transfected with an expression construct comprising: (a) CNS1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3 (sometimes referred to herein as CNS-FOXP3) (see WO 2023 / 048275).

[0114] The cells into which the expression construct is introduced are not particularly limited and may be at any stage of differentiation. For example, CD4 ＋ T cells, pluripotent stem cells or CD4 ＋ The expression construct may be used to express CD4 ＋ T cells, pluripotent stem cells or CD4 ＋ Methods for introducing the antibody into cells that can differentiate into T cells include the methods described above.

[0115] The expression construct of the present invention is for expressing FOXP3 and includes (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. ＋ T cells, pluripotent stem cells or CD4 ＋By introducing the expression construct into cells capable of differentiating into T cells, it becomes possible to produce the immune cells of the present invention with high efficiency. The expression construct is not particularly limited as long as it is capable of expressing FOXP3, and preferably includes, in addition to (a), (b), and (c) above, a terminator, a polyadenylation signal, a FOXP3 3'UTR, etc., and more preferably, these are functional in the cells into which they are introduced.

[0116] The nucleotide sequence of the human FOXP3 gene has been registered as RefSeq Accession No. NM_001114377 (SEQ ID NO: 1) and NM_014009 (SEQ ID NO: 2), and the amino acid sequence has been registered as RefSeq Accession No. NP_001107849 (SEQ ID NO: 3) and NP_054728 (SEQ ID NO: 4). The RefSeq IDs here are registered on the NCBI website. In the present invention, the above-mentioned genes include degenerates and mutants thereof, even if they have a nucleotide sequence registered in the aforementioned database. Desirably, the mutants encode proteins having biological activity equivalent to that of the proteins consisting of the above-mentioned amino acid sequences. Variants include mutant forms of FOXP3 having an amino acid sequence that is 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 identical to the naturally occurring amino acid sequence. As used herein, when it is appropriate to interpret FOXP3 as a gene or protein, this refers to the gene or protein.

[0117] The nucleic acid encoding FOXP3 is not particularly limited as long as it encodes the FOXP3 protein, and is preferably a FOXP3 cDNA.

[0118] CNS1, CNS2, and CNS3 used in the present invention are all derived from the FOXP3 gene. CNS1, CNS2, and CNS3 are FOXP3 enhancer elements. The promoter used in the present invention is not particularly limited, and examples include the CAG promoter, the ubiquitin gene promoter, the FOXP3 gene promoter, the EF1α promoter, the SRα promoter, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney murine leukemia virus) LTR, and the HSV-TK (herpes simplex virus thymidine kinase) promoter, with the FOXP3 gene promoter being preferred. Preferred nucleotide sequences of CNS1, CNS2, CNS3, and the promoter of the human FOXP3 gene include the nucleotide sequences set forth in SEQ ID NOs: 5 to 8, respectively. The promoters CNS1, CNS2, and CNS3 include mutants other than those having the above-mentioned base sequences, and mutants preferably have biological activity equivalent to that of the promoters CNS1, CNS2, and CNS3 consisting of the above-mentioned base sequences. Examples of mutants include those having a base sequence that is 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 identical to the natural base sequence.

[0119] The arrangement (order) of the promoter and the nucleic acid encoding CNS1, CNS2, CNS3, and FOXP3 in the expression construct is not particularly limited, but it is preferable that CNS1, CNS2, and CNS3 are located upstream of the promoter, and it is more preferable that the nucleic acid encoding FOXP3 is arranged in the following order from the 5' end: CNS1-CNS2-CNS3-promoter-FOXP3.

[0120] The cells into which the expression construct is introduced include CD4 ＋ T cells, pluripotent stem cells or CD4 ＋The cells are not particularly limited as long as they can be differentiated into T cells, and examples thereof include pluripotent stem cells, hematopoietic stem cells, hematopoietic endothelial cells, hematopoietic progenitor cells, T progenitor cells, mesodermal progenitor cells, and helper T cells. Pluripotent stem cells are preferred, and iPS cells are more preferred. Among these, pluripotent stem cells (particularly iPS cells) are used as cells into which the expression construct is introduced, and these cells are transfected with CD34 ＋ cells (particularly hemogenic endothelial cells), and ＋ Cells (especially hemogenic endothelial cells) were subjected to the ATO method, and CD4 ＋ It is desirable to differentiate into T cells.

[0121] The immune cells of the present invention, and the CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The immune cells may be immune cells into which a foreign gene has been introduced.

[0122] The "foreign gene" is a gene introduced from the outside to cause the immune cells of the present invention to express a desired protein, and can be selected appropriately depending on the intended use of the immune cells of the present invention.

[0123] The foreign gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR), which may further include a gene for expressing a cytokine and / or a chemokine. Similar to common or known CARs, the CAR expressed by the immune cells of the present invention is basically composed of peptides from each of the following sites linked via a spacer as needed: (i) an antigen recognition site (e.g., a single-chain antibody) that recognizes a cell surface antigen of a cancer cell; (ii) a cell membrane-spanning domain; and (iii) a signal transduction domain that induces T cell activation. The foreign gene can also be, for example, a gene for expressing an exogenous T cell receptor (TCR). The term "exogenous TCR" refers to a nucleic acid encoding the exogenous TCR being exogenous to the T cell into which the exogenous TCR is introduced, and the amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR of the T cell. One or more types of foreign genes can be introduced (e.g., a CAR and an exogenous TCR).

[0124] The methods for introducing a foreign gene into cells include the methods described above. The cells into which the foreign gene is introduced are not particularly limited and may be at any stage of differentiation, for example, pluripotent stem cells (particularly iPS cells), CD4 ＋ T cells, CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ Examples include immune cells.

[0125] Immune cells of the present invention, and CD3 produced by the production method and proliferation method of the present invention. ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A cell population containing immune cells of the formula (I) is useful for treating animals (particularly humans) with abnormally enhanced immune responses, and is useful for treating and preventing, for example, X-linked immunodysregulation-polyendocrinopathy-enteropathy (IPEX) syndrome, graft-versus-host disease (GVHD), rejection in organ transplants, autoimmune diseases, inflammatory diseases, allergic diseases (hay fever, asthma, atopic dermatitis, eczema, food allergies, food hypersensitivity, urticaria, allergic rhinitis, allergic conjunctivitis, drug allergies), and the like, but is not limited to these. ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ The immune cells may be used for autologous transplantation or allogeneic transplantation. They may also be used in combination with other drugs. Conventionally, Tregs are CD3 positive (=TCRαβ positive), and therefore there have been concerns about side effects due to non-specific antigen recognition. However, the immune cells of the present invention are CD3 negative (=TCRαβ negative), and therefore such side effects are expected to be reduced.

[0126] When carrying out such cell therapy, from the viewpoint of preventing rejection reactions, it is preferable that the subject from which the cells to be used in producing the immune cells of the present invention are isolated has an HLA type that matches that of the subject to which the immune cells of the present invention are administered, and it is more preferable that the subject is the same as the subject to which the immune cells of the present invention are administered.

[0127] According to the present invention, CD3 ｌｏｗ／－ , CD25＋ and FOXP3 ＋ It is possible to produce a pharmaceutical comprising a cell population containing immune cells of the present invention (hereinafter, sometimes referred to as the pharmaceutical of the present invention). The pharmaceutical of the present invention is preferably produced as a parenteral formulation by mixing an effective amount of the immune cells of the present invention with a pharmaceutically acceptable carrier according to known means (e.g., the method described in the Japanese Pharmacopoeia, etc.). The pharmaceutical of the present invention is preferably produced as a parenteral formulation such as an injection, suspension, or infusion. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. Examples of pharmaceutically acceptable carriers include solvents, bases, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonic agents, surfactants, and solubilizing agents.

[0128] The dosage of the pharmaceutical of the present invention can be appropriately determined depending on various conditions such as the patient's weight, age, sex, and symptoms. Generally, the number of cells administered per administration is 1×10 for a subject weighing 60 kg. ６ ~1 x 10 １０ Preferably, 1×10 ７ ~1 x 10 ９ 5×10 ７ ~5 x 10 ８ The pharmaceutical composition of the present invention is administered so that the number of cells reaches 100. The pharmaceutical composition may be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition of the present invention may also contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc., in order to stably maintain the cells. The culture medium is not particularly limited, but includes media such as RPMI, AIM-V, and X-VIVO10, but is not limited to these. Furthermore, a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. may be added to the pharmaceutical composition for stabilization purposes. The pharmaceutical composition of the present invention is intended to be used in mammals, including humans.

[0129] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

[0130] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0131] (1) iPSC-CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Creation of immune cells: CD4 from iPS cells ＋ The cell population containing cells after induction of differentiation into T cells was expanded in a medium containing IL-2 and IL-33.

[0132] (1-1) CD4 ＋ Generation of T cells from iPS cells ＋ Specifically, the differentiation induction into T cells is as follows. A DNA sequence in which the mStrawberry protein sequence in the sequence registered in GenBank (MK012431) was changed to a dTomato sequence was incorporated into a transfer plasmid for producing a third-generation lentiviral vector, and a plasmid sequence was designed and synthesized. The received plasmid was transfected into HEK293 cells together with a packaging plasmid and an envelope plasmid for producing a lentiviral vector. The supernatant containing the produced lentiviral vector was collected and then concentrated by high-speed centrifugation to obtain the lentiviral vector to be introduced into iPS cells. Next, the Ff-I01s04 strain was placed in a 24-well plate at 1 x 10 ４ After adding protamine to a final concentration of 10 μg / mL, a lentivirus solution incorporating the CNS-FOXP3 gene was directly added to generate virus-infected iPS cells.

[0133] 1 × 10 Ff-I01s04 strain infected with the virus was placed in an ultra-low attachment treated 6-well plate (Corning). ６The cells were seeded at 1000 cells / well (Day 0) and cultured in EB medium (StemPro34 (Gibco) supplemented with 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 (Nacalai Tesque, Inc.), and 50 μg / ml ascorbic acid 2-phosphate (Sigma-Aldrich)) containing 50 ng / ml BMP4 (R&D systems), 50 ng / ml bFGF (Fujifilm Wako Pure Chemical Industries, Ltd.), 50 ng / ml VEGF (R&D systems), 2 μM SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the cells were incubated under hypoxic conditions (5% O ２ ) for 4 days (Day 4). Subsequently, they were cultured for an additional 4 days in medium supplemented with 50 ng / ml bFGF, 50 ng / ml VEGF, and 50 ng / ml SCF (R&D Systems) (Day 8), yielding a cell population containing HECs. Furthermore, iPS cell-derived fibroblast cell line iFibroblast, which was forced to express human DLL4 protein, was used as a support and co-cultured with CNS-FOXP3-introduced human iPS cell-derived HECs at a cell ratio of 10:1. The medium used was RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) containing final concentrations of 2x B27 supplement (Invitrogen), 1x PSG (Sigma-Aldrich), 1x Glutamax (Invitrogen), 5 ng / mL IL-7 (PeproTech), 5 ng / mL FIT3L (PeproTech), and 50 μg / mL ascorbic acid (Sigma-Aldrich). The cells were co-cultured in G-Rex 24-well plates and cultured for 7 weeks with medium changes every 3-4 days.

[0134] (1-2) CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Preparation of immune cells CD4 obtained above ＋ The cell population containing T cells was plated at 2 × 10 cells in a 48-well cell culture plate conjugated with anti-CD3 antibody (3 μg / mL, eBioscience). ５ cells / well and２ Incubator at 37°C and 5.0% CO ２ After culturing for 3 days under these conditions, the cells were reseeded onto a 24-well G-Rex cell culture plate and culture was continued. The medium contained, at final concentrations, 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), IL-2 (10 ng / mL, Peprotech), rapamycin (10 nM, Merck Millipore), and anti-TNFR2 antibody (3 μg / mL, Hycult). The medium used was α-MEM (Invitrogen) containing IL-3 (60 ng / mL, BioLegend), IL-4 (30 ng / mL, BioLegend), IL-33 (30 ng / mL, BioLegend), and TGF-β1 (5 ng / mL, BioLegend). For the first three days, anti-CD28 antibody (1.5 μg / mL, BioLegend) and anti-CD30 antibody (300 ng / mL, R&D systems) were further added to the above composition. The cells were cultured for 7 or 14 days, with medium changes on day 3 and every 2–3 days thereafter. Furthermore, a portion of the cells on day 7 or 14 of culture was plated at 2 × 10 in a 48-well cell culture plate conjugated with the above anti-CD3 antibody. ５ The cells were seeded at 1000 x g / well and cultured for 7 or 14 days in the same manner, and the culture was continued for a total of 70 days (in the group where the 14-day culture process was repeated, anti-CD28 antibody was added only in the first culture process, but not in the second or subsequent culture processes).

[0135] Flow cytometric analysis of the cells obtained in (1-1) is shown in Figure 1. Specifically, a cell population immediately after differentiation induction was stained with the following antibody set and analyzed by flow cytometry: APC / Cy7 CD3, FITC TCRαβ, BV421 CD4, PE / Cy7 CD8β, APC CD7, and BV510 CD5.

[0136] The CD3 and TCRαβ positivity rates of the cells obtained above were 73.3%.

[0137] (2) iPSC-CD3 expansion ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Examination of protein expression levels in immune cells The cell population obtained in (1) was expanded and cultured, and the expression levels of proteins expressed in Treg were examined.

[0138] Cell populations expanded as described in (1) were sampled over time, and the CD3 positivity rate and the expression rate of proteins known to be expressed in Tregs were monitored. The results are shown in Figures 2-1 and 2-2. Specifically, each cell population expanded as described above was stained with the following two antibody sets and analyzed by flow cytometry. The antibodies used were a set of APC / Cy7 CD3, FITC TCRαβ, BV421 CD4, and PE / Cy7 CD8β, and a set of Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, and FITC FOXP3.

[0139] The CD3 positivity rate of cells expanded every 7 days decreased over time, reaching 1.59% by day 55. On the other hand, the FOXP3 and CD25 positivity rates remained at 67.5% even on day 55. A similar trend was observed in cells expanded every 14 days, although the decrease in CD3 positivity was slower than in cells expanded every 7 days.

[0140] (3) iPSC-CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Evaluation of the proliferation ability of immune cells Expansion culture was performed under the conditions shown in (1) and (2). For the group in which the expansion culture cycle was repeated every 7 days, the cell count was measured on days 3, 5, and 7 of each repeated culture step. For the group in which the expansion culture cycle was repeated every 14 days, the cell count was measured on days 3, 5, 7, 10, 12, and 14 of each repeated culture step, and a line graph was created. The results are shown in Figure 3.

[0141] The cell population obtained through the expansion culture cycle every 7 days could be cultured without any change in proliferation potential, and showed a proliferation potential of approximately 10 to the power of 14 on the 70th day of culture. On the other hand, the cell population obtained through the expansion culture cycle every 14 days could also be cultured while maintaining its proliferation potential, and showed a proliferation potential of approximately 10 to the power of 10 on the 70th day.

[0142] (4) iPSC-CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Evaluation of the suppressive function of immune cells The cell population expanded in (1) was used to perform analysis by flow cytometry after co-culture with allogeneic T cells derived from human PBMC.

[0143] Specifically, PBMCs derived from a donor other than the iPS cells, untreated or treated with anti-CD3 antibody, were stained with CellTrace Violet (Thermo Fisher) and used as target cells. These were co-cultured with cell populations on day 70 of expansion culture in (1) and (2). The medium used was α-MEM (Invitrogen) containing final concentrations of 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), and the cells were cultured for 5 days. Each cell population was stained with the following antibody set: Zombie NIR, PE / Cy7 CD3, FITC HLA-A24. The results are shown in Figure 4.

[0144] iPSC-CD3 ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋ Cell division of target cells was strongly suppressed in a cell number-dependent manner by immune cells. Furthermore, no significant difference was observed between the 7-day and 14-day expansion culture cycles.

[0145] (5) iPSC-CD25 ＋ / FOXP3 ＋ Evaluation of immune cell proliferation ability CD4 obtained in (1-1) ＋The cell population containing T cells was plated at 2 × 10 cells in a 48-well cell culture plate conjugated with anti-CD3 antibody (3 μg / mL, eBioscience). ５ cells / well and ２ Incubator at 37°C and 5.0% CO ２ After culturing for 3 days under these conditions, the cells were reseeded onto a 24-well G-Rex cell culture plate and culture was continued. The medium contained, at final concentrations, 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), IL-2 (10 ng / mL, Peprotech), rapamycin (10 nM, Merck Millipore), and anti-TNFR2 antibody (3 μg / mL, Hycult). The cells were cultured in α-MEM (Invitrogen) containing IL-2 (60 ng / mL, BioLegend), IL-3 (60 ng / mL, BioLegend), IL-4 (30 ng / mL, BioLegend), IL-33 (30 ng / mL, BioLegend), and TGF-β1 (5 ng / mL, BioLegend). For the first three days, anti-CD30 antibody (300 ng / mL, R&D systems) was added to the above composition. Furthermore, a medium condition was used in which one or two of the cytokines IL-2, IL-3, IL-4, IL-33, and TGF-β1 were removed from the above medium conditions. The medium was changed on day 3 of culture and every 2–3 days thereafter for 7 days. Furthermore, on day 7 of culture, a portion of the cells was cultured at 2 × 10 cells per well in a 48-well cell culture plate pre-coated with the above anti-CD3 antibody. ５ The cells were seeded at 1000 cells / well and cultured again for 7 days in the same manner. This process was repeated three times. The cell counts were counted on the third, fifth, and seventh days of culture for each repeated step, and a line graph was created. The results are shown in Figure 5.

[0146] (6) iPSC-CD3 expansion ｌｏｗ／－ / CD25 ＋ / FOXP3 ＋Examination of protein expression levels in immune cells The cell population obtained in (5) was used to examine the expression levels of proteins expressed in Tregs.

[0147] Cell populations were sampled on day 21 of the expansion culture described in (5), and the CD3 positivity rate and the expression rate of proteins known to be expressed in Tregs were monitored. The results are shown in Figures 6-1 and 6-2. Specifically, each cell population expanded as described above was stained with the following two antibody sets and analyzed by flow cytometry. The antibodies used were a set of APC / Cy7 CD3, FITC TCRαβ, BV421 CD4, and PE / Cy7 CD8β, and a set of Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, and FITC FOXP3.

[0148] Of the cell populations obtained through the above expansion culture cycle, cells cultured under conditions in which IL-2 was omitted showed no cell proliferation at all, and therefore data could not be obtained on day 21 of culture.

[0149] This application is based on Japanese Patent Application No. 2024-048803 filed on March 25, 2024, the contents of which are incorporated in their entirety herein.

Claims

1. CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ immune cells.

2. CD4 ＋ The immune cell of claim 1 .

3. The immune cells of claim 1, which are derived from pluripotent stem cells.

4. The immune cell of claim 3, wherein the pluripotent stem cell is an iPS cell.

5. CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A method for producing a cell population in which immune cells that are expanded are: (1) in the presence of interleukin (IL)-2 and IL-33, CD4 ＋ A method comprising the step of culturing a cell population comprising T cells.

6. The method according to claim 5, wherein step (1) is carried out further in the presence of IL-4 and / or a TNFR2 agonist.

7. The method according to claim 5, wherein step (1) is carried out further in the presence of IL-3 and / or a TGF-βR agonist.

8. The method according to claim 5, wherein step (1) is further carried out in the presence of an mTOR inhibitor.

9. The method according to claim 5, wherein step (1) is carried out further in the presence of IL-4, IL-3, a TGF-βR agonist, a TNFR2 agonist, and an mTOR inhibitor.

10. The method according to claim 5, wherein the culturing in step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist and a CD3 agonist.

11. The method according to claim 5, wherein step (1) is repeated.

12. The method of claim 6, wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

13. The method of claim 7, wherein the TGF-βR agonist is TGF-β.

14. The method of claim 8, wherein the mTOR inhibitor is rapamycin.

15. CD4 ＋ T cells express CD25 ＋ / FOXP3 ＋ Cells and CD25 － / FOXP3 － The method of claim 5 , wherein the cell is at least one type of cell selected from the group consisting of cells.

16. CD4 ＋ 6. The method of claim 5, wherein the T cell is a cell into which an expression construct comprising: (a) Conserved Non-coding Sequence (CNS) 1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3.

17. Before the step (1), (2) pluripotent stem cells are transfected with CD4 ＋ The method according to claim 5 , further comprising the step of inducing differentiation into a cell population containing T cells.

18. The method of claim 17, further comprising, prior to step (2), the step of: (3) introducing into the pluripotent stem cells an expression construct comprising: (a) CNS1, CNS2, and CNS3 of the FOXP3 gene; (b) a promoter; and (c) a nucleic acid encoding FOXP3.

19. CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ (1A) growing CD3 cells derived from pluripotent stem cells in the presence of IL-2 and IL-33; ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A method comprising culturing a cell population comprising immune cells which are 20. CD3 ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ (1B) in the presence of IL-2 and IL-33, CD4 ＋ / CD25 － / FOXP3 － A method comprising the step of culturing a cell population comprising T cells.

21. The method of claim 5, 19 or 20, wherein the pluripotent stem cells are iPS cells.

22. CD3 obtained by the method of claim 5, 19 or 20. ｌｏｗ／－ , CD25 ＋ and FOXP3 ＋ A cell population containing immune cells.

23. A pharmaceutical comprising the immune cells of claim 1 and / or the cell population of claim 22.

24. The pharmaceutical composition according to claim 23 for use in the prevention and / or treatment of abnormally enhanced immune responses.

25. A method for preventing and / or treating abnormally enhanced immune responses, comprising administering the immune cells of claim 1 and / or the cell population of claim 22 to a subject in need thereof.

26. Immune cells according to claim 1 and / or cell populations according to claim 22 for use in the prevention and / or treatment of abnormally enhanced immune responses.

27. Use of the immune cells of claim 1 and / or the cell population of claim 22 in the manufacture of a medicament for the prevention and / or treatment of abnormally enhanced immune responses.

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