Pharmaceutical composition for treating or preventing t cell-related disorders

Abstract

The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related disorders, the pharmaceutical composition containing induced regulatory T cells having high functionality and a stable immunosuppression action.　The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related disorders, the composition containing, as an active ingredient, induced regulatory T cells having at least one property selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity.

Description

Pharmaceutical composition for treating or preventing T cell-related diseases

[0001] The present disclosure relates to pharmaceutical compositions for treating or preventing T cell-related diseases.

[0002] An important feature of CD25+CD4+ regulatory T cells inherent in the immune system is their specific expression of the transcription factor FoxP3, and FoxP3 deficiency or mutation can impair the development, differentiation, and suppressive function of regulatory T cells. Regulatory T cells suppress the immune system by expressing a variety of genes, including FoxP3, CTLA4, and IL-10. Epigenetic conditions such as DNA demethylation are thought to contribute to the comprehensive gene expression control of regulatory T cells, including stable FoxP3 expression, and these conditions correlate with the functional phenotype of regulatory T cells.

[0003] The present inventors have found for the first time that, when inducing regulatory T cells from human peripheral T cells, stable induced T cells are induced from human peripheral T cells by stimulation with an anti-CD3 antibody, followed by resting culture, and then stimulation with an anti-CD3 antibody, followed by culture, and then further resting culture, thereby enabling the induction of regulatory T cells with high expression of inhibitory molecules and high inhibitory function. Furthermore, the present inventors have found that the induced regulatory T cells thus obtained have sufficient immunosuppressive activity to function as a pharmaceutical.

[0004] Accordingly, the present disclosure provides the following: (Item X1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient inducible regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. (Item X1A) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient inducible regulatory human T cells that are NT5E-positive. (Item X1B) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient inducible regulatory human T cells that are ITGAE (CD103)-positive. (Item X1C) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient inducible regulatory human T cells that are AREG-positive. (Item X1D) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CD172g-positive inducible human regulatory T cells as an active ingredient. (Item X1E) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CD26-positive inducible human regulatory T cells as an active ingredient. (Item X1F) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CTLA4-positive inducible human regulatory T cells as an active ingredient. (Item X1G1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive inducible human regulatory T cells as an active ingredient. (Item X1G2) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising, as an active ingredient, inducible human regulatory T cells having at least three characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.(Item X1G3) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as active ingredients inducible regulatory human T cells having at least four characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. (Item X1G4) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as active ingredients inducible regulatory human T cells having at least five characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. (Item X1G5) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising, as an active ingredient, induced regulatory human T cells having all of the following characteristics: CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. (Item X2) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory human T cells are at least CTLA4-positive and FoxP3-positive. (Item X3) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory human T cells are at least CD172g-positive and / or CD26-positive. (Item X4) The pharmaceutical composition according to any one of the above items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory human T cells is demethylated. (Item X5) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory human T cells are CD4-positive or CD8-positive. (Item X6) The pharmaceutical composition according to any one of the above items, wherein the inducible human regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.(Item X7) The pharmaceutical composition according to any one of the preceding items, wherein the inducible human regulatory T cells are obtained by a method comprising: (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. (Item X8) The pharmaceutical composition according to any one of the preceding items, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8A) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of NT5E positivity of about 50% or more. (Item X8B) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of ITGAE (CD103) positivity of about 50% or more. (Item X8C) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has an AREG-positive rate of about 50% or more. (Item X8D) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has an CD172g-positive rate of about 50% or more. (Item X8E) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has an CD26-positive rate of about 50% or more.(Item X8F) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population comprising inducible human regulatory T cells as an active ingredient, wherein the cell population has a CTLA4 positivity rate of about 50% or more. (Item X8G1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population comprising inducible human regulatory T cells as an active ingredient, wherein the cell population has a CTLA4 positivity rate of about 50% or more for at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (Item X8G2) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a proportion of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G3) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a proportion of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G4) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a proportion of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G5) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a proportion of all of the characteristics CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.(Item X9) The pharmaceutical composition according to any one of the above items, wherein the proportion of at least CTLA4-positive and FoxP3-positive cells in the cell population is each about 50% or more. (Item X10) The pharmaceutical composition according to any one of the above items, wherein the proportion of at least CD172g-positive and / or CD26-positive cells in the cell population is each about 50% or more. (Item X11) The pharmaceutical composition according to any one of the above items, wherein the proportion of at least one characteristic in the cell population is about 60% or more. (Item X12) The pharmaceutical composition according to any one of the above items, wherein the proportion of at least one characteristic in the cell population is about 80% or more. (Item X12a) The pharmaceutical composition according to any one of the above items, wherein the proportion of strongly FoxP3-positive cells in the cell population is about 50% or more. (Item X13) The pharmaceutical composition according to any one of the above items, wherein about 90% or more of the cell population are T cells. (Item X14) The induced regulatory human T cells are administered at a dose of about 10 per administration. ８ ~about 10 ９ pieces, or about 10 ７The pharmaceutical composition according to any one of the preceding items, wherein the T cell-related disease comprises an autoimmune disease, an infectious disease, a cancer, an allergy, an inflammatory disease, and ALS that can be treated by immunosuppression. The autoimmune disease is selected from the group consisting of Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis, Raynaud's syndrome, The pharmaceutical composition according to any one of the preceding items, wherein the disease is selected from the group consisting of preeclampsia, myocarditis, glaucoma, hypertension, pulmonary hypertension, malignant hypertension, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune disease, Graves' disease, autoimmune hepatitis, primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis, polymyalgia rheumatica, adult Still's disease, Behcet's disease, and ulcerative colitis. (Item X17) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is administered by injection. (Item X18) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is additionally administered to patients for whom the initial administration of the pharmaceutical composition is ineffective or insufficient. (Item X19) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is additionally administered at least about two weeks after the initial administration.

[0005] The present disclosure also provides the following: (Item 1) A pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising, as an active ingredient, induced regulatory T cells having at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Item 2) The pharmaceutical composition according to the above items, wherein the induced regulatory T cells have at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Item 3) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are at least CTLA4-positive. (Item 4) The pharmaceutical composition according to any one of the above items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory T cells is demethylated. (Item 5) The pharmaceutical composition of any one of the preceding items, wherein the induced regulatory T cells are CD4-positive or CD8-positive. (Item 6) The pharmaceutical composition of any one of the preceding items, wherein the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells. (Item 7) The pharmaceutical composition of any one of the preceding items, wherein the induced regulatory T cells are obtained by a method comprising: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days, (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. (Item 8) The pharmaceutical composition according to any one of the preceding items, comprising a T cell population in which about 50% or more of the cells in the T cell population are the inducible regulatory T cells. (Item 9) The pharmaceutical composition according to any one of the preceding items, comprising a T cell population in which about 80% or more of the cells in the T cell population are the inducible regulatory T cells. (Item 10) The pharmaceutical composition according to any one of the preceding items, wherein the T cell population is a regulatory T cell population.(Item 11) The pharmaceutical composition according to any one of the above items, wherein the cell population is about 90% or more T cells. (Item 12) The induced regulatory T cells are administered at a concentration of about 10 per administration. ８ ~about 10 ９ pieces, or about 10 ７ The pharmaceutical composition according to any one of the preceding items, wherein the T cell-related disease comprises an autoimmune disease treatable by immunosuppression, an infectious disease, a cancer, an allergy, an inflammatory disease, and ALS. (Item 14) The pharmaceutical composition according to any one of the preceding items, wherein the autoimmune disease comprises systemic lupus erythematosus, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and cardiomyopathy. (Item 15) The pharmaceutical composition according to any one of the preceding items, wherein the pharmaceutical composition is administered by injection. (Item 16) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is additionally administered to a patient for whom the initial administration of the pharmaceutical composition is ineffective or insufficient. (Item 17) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is additionally administered at least about two weeks after the initial administration.

[0006] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated, and further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] Note that features and significant actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings.

[0008] The present disclosure provides a method for inducing highly functional regulatory T cells from human peripheral T cells in vitro. The regulatory T cells induced by the method of the present disclosure highly express genes associated with regulatory T cells and have strong suppressive ability. Therefore, the regulatory T cells can be used for the treatment and prevention of various immune diseases and inflammatory diseases such as autoimmune diseases.

[0009] The method of the present disclosure enables in vitro induction of functional regulatory T cells from human peripheral T cells. The regulatory T cells obtained by the method of the present disclosure have high suppressive function and are stable as regulatory T cells. That is, the method of the present disclosure enables induction of highly functional regulatory T cells that stably express FoxP3, the master gene of regulatory T cells, from human peripheral T cells.

[0010] FIG. 1 shows an outline of a method for producing one embodiment of mouse induced regulatory T cells (also referred to as mouse highly functional stable iTreg (HSF iTreg) cells) and the results of flow cytometry of the induced regulatory T cells. FIG. 1A shows an outline of a protocol for inducing iTreg cells from mouse CD4-positive naive T cells by various stimulation methods. FIG. 1B shows the results of flow cytometry analysis of FoxP3 and CD25 expression in iTreg cells, activated T cells, and activated nTreg cells produced in FIG. 1A. FIG. 2 shows the results of analysis of FoxP3 expression by flow cytometry and the demethylation state of the Treg-specific demethylated region by the bisulfite method when SF-iTregs were produced from mouse CD4-positive naive T cells or effector T cells based on the method for producing iTregs (SF-iTregs in the figure) according to one embodiment of the present disclosure shown in FIG. 1. Figure 3 shows the results of one embodiment in which the global gene expression patterns of each cell in Figure 1 were analyzed by RNA sequencing. Figure 3A shows a PCA analysis plot, and Figure 3B shows a heat map of Treg-related genes. Figure 4 shows the results of one embodiment in which the in vitro suppressive ability of each cell in Figure 1 was analyzed. Mouse CD4-positive naive T cells were stained with Cell Trace Violet reagent and co-cultured with each cell in Figure 1 at a fixed ratio in the presence of anti-mouse CD3 antibody + MHC-II-positive cells or Dynabeads T-activator (Veritas) (5 μL / well) for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry. Figure 5 shows the results of one embodiment in which the single-stimulation CD28 antibody-naive group in Figure 1 and the iTregs of the present disclosure were administered to wild-type mice, and Foxp3 expression was analyzed in the transferred regulatory T cells two weeks later. Figure 6 shows the results of one embodiment in which a colitis model was created by transferring mouse CD4-positive naive T cells into RAG2-deficient mice, and iTreg cells of the present disclosure were administered to analyze the therapeutic effect. Figure 6A shows the change in body weight (g), Figure 6B shows an HE-stained image of colon tissue, and Figure 6C shows the results of flow cytometry analysis of CD69 expression in lymph node T cells. Figure 7 shows the results of analysis of one embodiment of iTregs of the present disclosure derived from a human Crohn's disease patient.Figure 7A shows the results of flow cytometry analysis of the expression of FOXP3, CTLA4, and Helios in induced regulatory T cells (HSF-iTregs) prepared from CD4+ T cells derived from a human Crohn's disease patient. Cells stimulated with conventional nTregs (CD4+CD25+ T cells) were compared with conventional iTregs (CD4+ T cells stimulated with CD3 / CD28 for 3 days in the presence of IL-2 and TGF-β1). Figure 7B shows the results of an analysis of the in vitro suppressive ability of each cell type shown in Figure 7A. Human CD4+ T cells were stained with Cell Trace Violet reagent and co-cultured with each cell type shown in Figure 1 at a fixed ratio in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 μL / well) for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry. FIG. 8 is a diagram showing phenotypic analysis (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure prepared from human CD8-positive T cells. Highly functional inducible regulatory T cells were prepared from human CD8-positive T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry. FIG. 9 is a diagram showing CD25 expression and FOXP3 expression (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure. The expression intensities of CD25 and FOXP3 were analyzed by flow cytometry using a BD FACSLyric flow cytometer. FIG. 10 is a diagram showing phenotypic analysis of one embodiment of iTreg (HSF iTreg) cells of the present disclosure. Inducible regulatory T cells (HSF-iTreg) of the present disclosure were prepared, and the expression of CD172g, CD26, and the like was analyzed. 11 is a diagram showing the phenotype analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure prepared from CD4-positive T cells derived from an SLE patient. Highly functional inducible regulatory T cells were prepared from CD4-positive T cells derived from an SLE patient, and the expression of FoxP3, CD4, and CTLA4 was analyzed by flow cytometry.12 is a diagram showing the phenotype analysis (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure prepared from CD4-positive T cells derived from a rheumatoid arthritis patient. Highly functional inducible regulatory T cells were prepared from CD4-positive T cells derived from a rheumatoid arthritis patient, and the expression of FoxP3, CD4, and CTLA4 was analyzed by flow cytometry.

[0011] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0012] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0013] As used herein, "about" means ±10% of the numerical value that follows. For example, "about 20" includes "18 to 22." Numerical ranges include all values ​​between and at the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0014] In this specification, when a gene name and its product are written in all capital letters, contrary to the usual usage, it may refer to both the gene and the protein. For example, the FOXP3 gene and the FOXP3 protein may be used interchangeably, and the term FoxP3 refers to both the concept and entity (whole) of the gene or protein.

[0015] As used herein, "regulatory T cells" refer to T cells that are positive for FoxP3 expression. Herein, they may also be referred to as "Tregs." Tregs include naturally occurring regulatory T cells (nTregs) and inducible regulatory T cells (iTregs). Regulatory T cells can generally have various functions (e.g., immunosuppressive function).

[0016] As used herein, the term "inducible regulatory T cells (iTregs)" refers to regulatory T cells that are negative for IKZF2 (Helios) expression. iTregs are typically obtained by inducing differentiation from naive CD4-positive T cells or the like.

[0017] As used herein, "naturally occurring regulatory T cells (nTreg)" refer to regulatory T cells that are positive for the expression of IKZF2 (Helios) and CTLA4. These cells are normally present in vivo.

[0018] As used herein, "peripheral T cells" refers to T cells present outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues. The term "peripheral T cells" as used herein simply refers to a cell population containing peripheral T cells, and does not require that the T cells be isolated. Cell fractions containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMCs), may also be used.

[0019] As used herein, "flow cytometry" refers to a technique for measuring the number of cells, solids, and other biological particles suspended in a liquid, as well as their individual physical, chemical, and biological properties. A device using this technique is called a "flow cytometer." In this disclosure, the "positive" and "negative" status of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) is determined by flow cytometry, as commonly used in the art. More specifically, in flow cytometry, cells are lined up and flowed, and the number of cells is counted using spectroscopic techniques. For example, target cells are counted by irradiating cells labeled with fluorescent or luminescent enzymes with laser light, and the resulting fluorescent or luminescent signals are detected by a detector such as a photodiode. Furthermore, the detection results from the detector can be input into a computer and displayed as a two-dimensional plot. This allows for easy identification of the presence and number of target cells.

[0020] As used herein, "demethylation" refers to the removal of methylation modifications from typically methylated adenines (e.g., m6A at position 6, m1A at position 1) and cytosines (e.g., m5C at position 5, m3C at position 3). Demethylation can be determined using techniques known in the art, and can be measured, for example, using the bisulfite method.

[0021] As used herein, a "cell population" refers to a population containing two or more cells, and may be, for example, a collection of cells in a planar state, or a cell mass formed by cells adhering to each other in a three-dimensional manner. Furthermore, a "cell population" may be formed by a single type of cell, or may contain multiple types of cells.

[0022] As used herein, "stimulation" refers to stimulation via TCR. For example, stimulation of T cells includes stimulation with an anti-CD3 antibody or a complex containing the same, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation using an anti-CD3 antibody and antigen-presenting cells simultaneously, and stimulation using a peptide or protein and antigen-presenting cells simultaneously.

[0023] As used herein, "dormant culture" refers to culturing cells in the absence of the above-mentioned stimuli.

[0024] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0025] The present disclosure relates to medical uses of highly functional stable induced regulatory T cells (highly functional stable iTregs, also referred to as HSF iTregs).

[0026] In one aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising, as an active ingredient, induced regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. In one embodiment of the present disclosure, the induced regulatory T cells in the pharmaceutical composition of the present disclosure may have at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. In another embodiment of the present disclosure, the induced regulatory T cells in the pharmaceutical composition of the present disclosure may be at least CTLA4-positive. Without intending to be limited thereto, the induced regulatory T cells in the pharmaceutical composition of the present disclosure may be CD4-positive or CD8-positive.

[0027] In one embodiment of the present disclosure, the induced regulatory T cells in the pharmaceutical composition of the present disclosure may be at least CTLA4-positive and FoxP3-positive. In one embodiment of the present disclosure, the induced regulatory T cells in the pharmaceutical composition of the present disclosure may be at least CD172g-positive and / or CD26-positive.

[0028] The present disclosure provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising CD172g-positive inducible regulatory human T cells as an active ingredient. CD172g is a tyrosine kinase-related protein involved in cell adhesion of neurons and other cells (adhesion of cerebellar neurons, neurite outgrowth, and glial cell attachment). Therefore, CD172g-positive inducible regulatory human T cells are expected to activate functions related to adhesiveness and other properties, and to be highly effective against various diseases related to these diseases.

[0029] The present disclosure provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising CD26-positive inducible human regulatory T cells as an active ingredient. CD26 is a gene also known as DPP4, and is associated with glucose metabolism, insulin metabolism, and immune function (notably in infectious diseases). Therefore, CD26-positive inducible human regulatory T cells are expected to be highly effective against various diseases associated with these.

[0030] The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related diseases, comprising NT5E-positive inducible human regulatory T cells as an active ingredient. NT5E (CD73) is a cell membrane protein that catalyzes the conversion of extracellular nucleotides into membrane-permeable nucleosides. The encoded protein is used as a determinant for lymphocyte differentiation. Defects in this gene can cause calcification of joints and arteries, and therefore NT5E-positive inducible human regulatory T cells are expected to be highly effective against various diseases related to these conditions.

[0031] The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related diseases, comprising ITGAE (CD103)-positive induced regulatory human T cells as an active ingredient. ITGAE (CD103) encodes an I-domain α integrin that undergoes post-translational cleavage in the extracellular domain to generate disulfide-linked heavy and light chains. This protein binds to β7 integrin to form an E-cadherin-binding integrin known as human mucosal lymphocyte-1 antigen. This protein is preferentially expressed on human intestinal intraepithelial lymphocytes (IELs) and may function as an accessory molecule for IEL activation in addition to its adhesive role. Therefore, ITGAE (CD103)-positive induced regulatory human T cells are expected to be highly effective against various diseases related to IELs.

[0032] The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related diseases, comprising AREG-positive induced regulatory human T cells as an active ingredient. AREG is a member of the epidermal growth factor family and is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). This protein interacts with the EGF / TGF-α receptor to promote the growth of normal epithelial cells and inhibit the growth of certain aggressive cancer cell lines. It also functions in the development of mammary glands, oocytes, and bone tissue. This gene is associated with a psoriasis-like skin phenotype and is also associated with other pathological diseases, including various types of cancer and inflammatory conditions. Therefore, AREG-positive induced regulatory human T cells are expected to be highly effective against various related diseases.

[0033] The present disclosure provides a pharmaceutical composition for treating or preventing T cell-related diseases, comprising CTLA4-positive inducible regulatory human T cells as an active ingredient. CTLA4 belongs to the immunoglobulin superfamily and is a protein that transmits inhibitory signals to T cells. Membrane-bound CTLA4 functions as a disulfide-bonded homodimer, while soluble CTLA4 functions as a monomer. Mutations in this gene are said to be associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases. Therefore, CTLA4-positive inducible regulatory human T cells are expected to be highly effective against various related diseases. CTLA4-positive inducible regulatory human T cells are also expected to be highly effective against autoimmune diseases such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis.

[0034] Expression of FoxP3 in regulatory T cells can be induced in vitro by culturing CD4-positive T cells in a medium containing IL2 and TGFβ in the presence of anti-CD3 and anti-CD28 antibodies, followed by culturing in a medium containing IL2 and TGFβ. Although several methods for inducing regulatory T cells from peripheral T cells are known, FoxP3 expression in induced regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has not been confirmed.

[0035] Recently, culture methods have been developed to induce DNA demethylation in induced regulatory T cells, such as by adding ascorbic acid to the culture medium (Kasahara et al. Int. Immunol. (2017) 29(10):457-469) and by using no CD28 antibody stimulation (Mikami et al. Proc Natl Acad Sci U S A. (2020) 117(22):12258-12268). However, the expression of functional molecules has not been confirmed in induced regulatory T cells generated using these methods, and sufficient immunosuppressive activity has not been achieved. One clinical trial using induced regulatory T cells has also been conducted for the treatment of GVHD, but the percentage of regulatory T cells used was low, and no efficacy has been demonstrated for preventing GVHD (MacMillan et al., Blood Adv. (2021) 5(5):1425-1436).

[0036] The present disclosure can provide a pharmaceutical composition containing, as an active ingredient, induced regulatory T cells that stably express FoxP3 and advantageously stably retain immunosuppressive activity. Such induced regulatory T cells can be produced, for example, by a method for producing induced regulatory T cells described elsewhere in this specification. For example, the present disclosure can provide a pharmaceutical composition in which the induced regulatory T cells are obtained by a method comprising the steps of: (a) stimulating CD4+ T cells or CD8+ T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0037] The induced regulatory T cells in the pharmaceutical composition of the present disclosure have an induced regulatory T cell-specific demethylation state. That the obtained regulatory T cells are in a regulatory T cell-specific demethylation state can be confirmed, for example, by demethylation of the CNS2 site of the FoxP3 gene (FOXP3 (all italics)). Such a demethylation state can be an indicator of a stable type, and therefore, by confirming the demethylation state, the induced regulatory T cells in the pharmaceutical composition of the present disclosure can be shown to be stable induced regulatory T cells.

[0038] Herein, the immunosuppressive activity or immunosuppressive effect of the induced regulatory T cells in the pharmaceutical composition of the present disclosure can be confirmed, for example, by measuring the Cell Trace Violet intensity in responder T cells. The induced regulatory T cells in the pharmaceutical composition of the present disclosure can stably provide immunosuppressive activity or immunosuppressive effect; for example, the induced regulatory T cells of the present disclosure can provide immunosuppressive activity or immunosuppressive effect for at least about two weeks. As shown in the Examples below, the induced regulatory T cells in the pharmaceutical composition of the present disclosure can have a stronger immunosuppressive effect than conventional regulatory T cells (including induced and endogenous). Therefore, the induced regulatory T cells in the pharmaceutical composition of the present disclosure can also be referred to as functional or highly functional induced regulatory T cells.

[0039] In one embodiment, the induced regulatory T cells in the pharmaceutical composition of the present disclosure can stably express FoxP3. Therefore, the induced regulatory T cells in the pharmaceutical composition of the present disclosure can also be referred to as stable induced regulatory T cells. In one aspect, the induced regulatory T cells in the pharmaceutical composition of the present disclosure are highly functional and stable, and can be referred to as highly functional stable induced regulatory T cells (HSF iTreg).

[0040] In one embodiment of the present disclosure, whether or not a marker in an induced regulatory T cell of the present disclosure is positive can be determined by measuring the positive rate using flow cytometry. For example, analysis can be performed using a flow cytometer, and positive or negative can be determined based on the percentage of cells that express antigens at or above a certain level. Depending on the expression intensity of the cell surface marker, the cells can be classified as negative, weakly positive, moderately positive, or strongly positive (weakly positive, moderately positive, and strongly positive are collectively referred to as "positive"). For example, depending on the instrument settings, the ratio of the median fluorescence intensity of each marker to the median fluorescence intensity of the negative control (stained with an isotype control antibody) can be determined as negative, weakly positive, moderately positive, or strongly positive, respectively, when the ratio is less than 5, 5 to less than 10, 10 to less than 30, or 30 or more. Such determinations can be made as exemplified in (measuring the positive rate using flow cytometry), but the present disclosure is not limited thereto.

[0041] In one embodiment of the present disclosure, the expression intensity of a cell surface marker in the induced regulatory T cells of the present disclosure is determined based on the results of analysis using a BD FACSLyric flow cytometer (BD Biosciences) using a sample prepared as described in the Examples of the present disclosure, and the expression intensity is determined to be 10 ２ Above is weak positive, 10 ３ Above 10 is medium positive, ４ or more may be considered strongly positive, or 10 ３ A value of 10 or more may be considered strongly positive, and a value of 10 or less may be considered weakly positive. ２ A value above this level may be considered strongly positive, and a value below this level may be considered weakly positive. Such a determination can be appropriately set depending on the experimental conditions, experimental purpose, cell type, instrument setting conditions, etc., and the present disclosure is not limited thereto. Furthermore, even when analysis is performed using a flow cytometer instrument other than the BD FACSLyric flow cytometer, the expression intensity on the other instrument corresponding to the expression intensity when analyzed using the BD FACSLyric flow cytometer can be calculated, and a value indicating the expression intensity can be appropriately found depending on the instrument used.

[0042] In one embodiment of the present disclosure, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can be induced from any cells, but are preferably obtained by induction from human peripheral blood T cells or human tissue-derived T cells.

[0043] In one embodiment of the present disclosure, the induced regulatory T cells or cell populations thereof in the pharmaceutical composition of the present disclosure can be human cells, and can include induced human regulatory T cells or cell populations thereof induced by a specific method using T cells obtained from human peripheral blood. Human cells and mouse cells have distinctly different properties, and even if the same cell surface markers are present, the properties of the cells cannot be considered identical. For example, in the field of induced regulatory T cells, the applicable techniques differ between mice and humans. In mice, the majority of lymphocytes are antigen-naïve, while in humans, T cells in peripheral blood vary in the degree of antigen sensitization and activation. Therefore, while highly functional and stable induced regulatory T cells can be directly generated from T cells collected from mouse lymphoid tissues in mice, this is difficult to achieve in humans, and the cells cannot be considered similar simply based on the presence of cell surface markers.

[0044] In one embodiment of the present disclosure, the pharmaceutical composition of the present disclosure can comprise a T cell population in which about 50% or more of the cells in the T cell population are inducible regulatory T cells as described elsewhere herein. In one embodiment, the cell population of the present disclosure can have about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the T cells in the cell population be inducible regulatory T cells as described elsewhere herein.

[0045] In one embodiment of the present disclosure, the T cells in the cell population of the present disclosure can be regulatory T cells, in which case about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the regulatory T cells in the cell population of the present disclosure can be inducible regulatory T cells as described elsewhere herein.

[0046] In one embodiment of the present disclosure, the cell population of the present disclosure may contain cells other than T cells, but preferably, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the cell population of the present disclosure may be T cells, and preferably, about 90% or more may be T cells.

[0047] In one aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a CTLA4-positive and FoxP3-positive percentage of at least about 50%. In one embodiment, the CTLA4-positive and FoxP3-positive percentages of the cell population in the pharmaceutical composition of the present disclosure can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%.

[0048] In one embodiment of the present disclosure, the cell population in the pharmaceutical composition of the present disclosure can have at least the CD172g-positive and / or CD26-positive percentages of about 50% or more. In one embodiment, the cell population in the pharmaceutical composition of the present disclosure can have a CD172g-positive and / or CD26-positive percentage of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0049] In one embodiment of the present disclosure, the percentage of strongly FoxP3 positive cells in the cell population in the pharmaceutical composition of the present disclosure can be about 50% or more. In one embodiment, the percentage of strongly FoxP3 positive cells in the cell population in the pharmaceutical composition of the present disclosure can be about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more. Without intending to be limited thereto, measurement of the expression intensity of cell surface markers in the cell population in such a pharmaceutical composition can be performed by measuring the positivity rate by flow cytometry as described above.

[0050] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a FoxP3-positive percentage of about 50% or more. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of FoxP3-positive inducible human regulatory T cells, based on the cell number.

[0051] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a CTLA4-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain CTLA4-positive inducible human regulatory T cells in an amount, by cell number, of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0052] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has an NT5E-positive rate of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain NT5E-positive inducible human regulatory T cells in an amount, by cell number, of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0053] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has an ITGAE (CD103)-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain ITGAE (CD103)-positive inducible human regulatory T cells in an amount of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more by cell number.

[0054] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population containing inducible human regulatory T cells, wherein the cell population has an AREG-positive rate of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain AREG-positive inducible human regulatory T cells in an amount, by cell number, of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0055] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a CD172g-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD172g-positive inducible human regulatory T cells, based on the cell number.

[0056] In another aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a CD26-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD26-positive inducible human regulatory T cells, based on the cell number.

[0057] As described above, the properties of human cells and mouse cells are clearly different, and therefore, even if the induction conditions are the same, the induction rate of specific cells will differ between mouse cells and human cells. In the present disclosure, when induced using a predetermined induction method, a cell population in which the rate of positive cells for a predetermined cell surface marker is about 50% or more can be obtained, and preferably a cell population in which "the rate of CTLA4 positive and FoxP3 positive cells is each about 50% or more" can be obtained.

[0058] CTLA4 is a molecule localized intracellularly, at least not at a level that can be used as an indicator for enrichment or purification. Furthermore, FoxP3 is a transcription factor and is a molecule that is entirely present intracellularly; therefore, it cannot be detected on the surface at all and cannot be used for purification. That is, because CTLA4 and FoxP3 are markers expressed "inside" T cells, it is not possible to obtain a cell population in which CTLA4 and / or FoxP3 are about 50% or more positive for CTLA4 and / or FoxP3 by sorting and enriching using CTLA4 and / or FoxP3 as indicators. Therefore, it is also not possible to obtain a cell population in which the proportions of CTLA4 and FoxP3 are less than about 50%, respectively, and then use such a cell population as a starting material for enrichment using CTLA4 and / or FoxP3 as indicators to obtain a cell population in which the respective cells are about 50% or more positive for CTLA4 and / or FoxP3.

[0059] In one embodiment of the present disclosure, the inducible regulatory human T cells in the pharmaceutical composition of the present invention are primarily responsible for the immunosuppressive function mediated by Tregs, and when these cells account for more than half of a certain cell population, a medically effective immunosuppressive effect can be stably achieved, which is important in terms of technical and medical effects. In other words, when the composition contains 50% or more T cells that exert a medically effective immunosuppressive effect, a stable cell preparation can be provided. This effect is an extremely important point from a medical perspective, and is not merely a matter of differences in quantity or numerical value, but is an important point in terms of quality, i.e., whether the preparation can be established.

[0060] In one embodiment of the present disclosure, the induced regulatory human T cells in the cell population in the pharmaceutical composition of the present disclosure can further be characterized as ITGAE (CD103)-positive, NT5E-positive, and / or AREG-positive. These cell markers are genes that play important roles in immunosuppressive function, and high expression of these markers allows the cells to maintain greater functional stability than conventional induced regulatory T cells after administration. For example, CD103 is important for regulatory T cell migration to inflammatory sites, NT5E (CD73) is important for the production of adenosine, which has immunosuppressive properties, and AREG is important for tissue regeneration by regulatory T cells. The cell population of the present disclosure can achieve the effect of maintaining functional stability due to the high expression of these markers.

[0061] In one aspect of the present disclosure, a pharmaceutical composition comprising the induced regulatory T cells or cell population of the present disclosure is provided. In another aspect, a regenerative medicine material or product comprising the induced regulatory T cells or cell population of the present disclosure is provided. This pharmaceutical composition or regenerative medicine material or product can be used for autoimmune diseases, inflammatory diseases, and allergies that can be treated by immunosuppressive activity. These pharmaceuticals, regenerative medicine materials, or products can be used together with culture media and any other additives used in the relevant field. Such media can be a medium prepared by adding necessary factors to a basal animal cell culture medium used for cell culture. Examples of such media are described in detail elsewhere herein. Examples of components added to the medium are also described in detail elsewhere herein. When provided as such a product, it may contain DMSO, etc.

[0062] In one embodiment, the pharmaceutical composition of the present disclosure can be used to treat or prevent a T cell-related disease. The T cell-related disease is not particularly limited as long as it is a disease that can be treated by immunosuppressive action, and examples of T cell-related diseases include autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS. Also in one embodiment, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (Type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, and Chagas' neuropathy. , benign prostatic hyperplasia, scleroderma, psoriasis, Raynaud's syndrome, preeclampsia, myocarditis, glaucoma, hypertension, pulmonary hypertension, malignant hypertension, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune diseases, Graves' disease, autoimmune hepatitis, primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis, polymyalgia rheumatica, adult Still's disease, Behcet's disease, and ulcerative colitis can be mentioned. The autoimmune disease is preferably autoimmune hepatitis, primary biliary cholangitis, or primary sclerosing cholangitis, more preferably primary biliary cholangitis or primary sclerosing cholangitis, and even more preferably primary sclerosing cholangitis.

[0063] In one embodiment, the pharmaceutical composition of the present disclosure can be administered in a variety of dosage forms, but is preferably administered by injection. In one embodiment, the pharmaceutical composition of the present disclosure contains induced regulatory T cells, as described elsewhere herein, at a concentration of about 10 per administration. ８ ~about 10 ９ pieces, or about 10 ７ The compound may be administered in an amount of 100 mg / kg.

[0064] In one embodiment, the pharmaceutical composition of the present disclosure can be further administered to a patient for whom the administration is ineffective or insufficient. Whether the administration is ineffective or insufficient can be determined, for example, by the degree of inflammation suppression in the target disease. In one embodiment, the additional administration can be administered at least about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after the first administration.

[0065] (Production Method) The method for producing the induced regulatory T cells in the pharmaceutical composition of the present disclosure is a novel method that can produce highly functional and stable induced regulatory T cells, and is described in detail herein below.

[0066] In one aspect of the present disclosure, there is provided a method for producing inducible regulatory T cells, the method comprising the steps of: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. In the present disclosure, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can be produced using either CD4-positive T cells or CD8-positive T cells as a starting material, and in one embodiment, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can also be produced using a mixture of CD4-positive T cells and CD8-positive T cells as a starting material.

[0067] In one embodiment, the method of the present disclosure can also be a method for producing regulatory T cells from human peripheral T cells (including CD4-positive T cells or CD8-positive T cells), comprising the steps of culturing human peripheral T cells in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation, culturing them in a medium containing IL-2 in the absence of anti-CD3 antibody, and again culturing them in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation.

[0068] In one embodiment, the stimulation of T cells refers to stimulation when obtaining Tregs, and is not particularly limited as long as it is stimulation via TCR. For example, stimulation of T cells can include stimulation with an anti-CD3 antibody or a complex containing the antibody, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation using an anti-CD3 antibody and antigen-presenting cells simultaneously, stimulation using a peptide or protein and antigen-presenting cells simultaneously, etc. However, the medium and conditions are not particularly limited as long as Tregs can be obtained.

[0069] In one embodiment, the medium and conditions for dormant culture are not particularly limited, as long as the cells are cultured in the absence of the above-mentioned stimuli.

[0070] As used herein, "anti-CD3 antibody stimulation" refers to specific stimulation of the CD3 receptor on a cell. An example of a CD3 stimulation is an anti-CD3 agonist antibody. The anti-CD3 agonist antibody may be a commercially available product as a research reagent, or may be prepared by conventional methods. Anti-CD3 antibodies may be derived from animals such as mice, rabbits, goats, and cows, or from humans.

[0071] In one embodiment, the method of the present disclosure involves first inducing iTregs (stimulating T cells to induce demethylation) (step a), then changing the medium and resting culture for 1 to 3 days to recover the cells (step b), and then stimulating the T cells once more to induce demethylation (step c), thereby obtaining functional and stable inducible regulatory human T cells. While it is common technical knowledge that T cells typically undergo apoptosis after two stimulations, the method of the present disclosure has been found to obtain functional and stable inducible regulatory human T cells without undergoing apoptosis by performing "resting culture" and "second stimulation."

[0072] In other words, in the method of the present disclosure, functional and stable inducible regulatory human T cells can be obtained by performing T cell stimulation, resting culture, and subsequent re-stimulation, and therefore the desired effects of the present disclosure can be achieved with inducible regulatory human T cells obtained in this manner or a cell population containing such inducible regulatory human T cells. Therefore, in one embodiment of the present disclosure, the medium for culture and the type of stimulus are not particularly limited, and functional and stable inducible regulatory human T cells can be obtained by using a medium of any composition and any type of stimulus.

[0073] In one embodiment, the medium used in each step may further contain retinoic acid and / or ascorbic acid. Preferably, the medium can contain ascorbic acid. In one embodiment, the medium may further contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor. Preferably, the medium can contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor.

[0074] In one embodiment of the present disclosure, the first basal medium and the second basal medium may each independently contain at least one, at least two, at least three, at least four, at least five, or all of the factors selected from the group consisting of an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. In another embodiment, the first basal medium and the second basal medium may each independently contain an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. The concentrations of each of these components may be conventional concentrations used in the art. In one embodiment, the concentration of the CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor that can be used may be any appropriate concentration that can be used in the art. For example, when Senexin A is used, the concentration can be about 0.1 μM or more, about 0.5 μM or more, about 1 μM or more, about 2 μM or more, about 3 μM or more, about 4 μM or more, about 5 μM or more, about 6 μM or more, about 7 μM or more, about 8 μM or more, about 9 μM or more, about 10 μM or more, about 12 μM or more, about 14 μM or more, about 16 μM or more, about 18 μM or more, about 20 μM or more, etc., but is not limited to these concentrations, and can be changed as appropriate by a person skilled in the art depending on other medium compositions.

[0075] In one embodiment, the method of the present disclosure produces regulatory T cells from human peripheral T cells. Peripheral T cells include naive regulatory T cells, CD4+ T cells, CD8+ T cells, etc. Regulatory T cells may be induced from a culture containing multiple types of T cells, or specific cells such as CD4+ T cells or CD8+ T cells may be isolated from these cells and then induced. Alternatively, regulatory T cells may be induced after isolating T cells specific to a specific antigen. Therefore, the induced regulatory T cells of the present disclosure include both CD4+ and CD8+ cells. Note that, in this specification, "CD4+" or "CD4+" refers to single-positive CD4+CD8-negative cells unless otherwise specified. Furthermore, in the production method of the present disclosure, either CD4+ or CD8+ T cells can be used as the starting material, and even after the generation of induced regulatory T cells, the CD4+ or CD8+ characteristics can be maintained unless special manipulation is performed.

[0076] In one embodiment, in the method of the present disclosure, the antibody may be added to the culture medium or may be immobilized on the inner wall of a culture vessel or the surface of an insoluble carrier. The insoluble carrier may be a material capable of physically or chemically binding an anti-CD3 antibody and insoluble in aqueous solution. Examples of materials capable of physically adsorbing an anti-CD3 antibody include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, and glass. The shape of the insoluble carrier is not particularly limited, and may be, for example, a plate, bead, or container. The amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately determined to provide sufficient stimulation for the induction of regulatory T cells.

[0077] In one embodiment, the method of the present disclosure can be used to culture cells in a medium prepared by adding necessary factors to a basal animal cell culture medium. Examples of basal animal cell culture media that can be used in the method of the present disclosure include Iscove's modified Eagle's Medium medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, RPMI 1640 medium, Fischer's medium, and mixtures or media with modified compositions thereof.

[0078] The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. The serum-free medium may optionally contain one or more serum substitutes, such as albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and monothioglycerol. The basal medium may also contain one or more substances such as lipids (e.g., chemically defined lipid concentrate), amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), growth factors, antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.

[0079] In one embodiment, the basal medium is exemplified by RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0080] In the method of the present disclosure, cells may be cultured under general animal cell culture conditions. The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. The culture is preferably carried out under CO ２ The culture was carried out under an atmosphere of CO ２ The concentration is preferably about 2-5%.

[0081] In the method of the present disclosure, the anti-CD3 antibody may be added directly to the culture medium, or may be immobilized on the inner wall of a culture vessel or on the surface of an insoluble carrier. The amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately determined so as to provide sufficient stimulation for the induction of regulatory T cells.

[0082] Examples of TGFβ that can be used include TGFβ1, TGFβ2, and TGFβ3, for example, TGFβ1. The concentration of TGFβ can be determined appropriately by those skilled in the art and is not particularly limited. When TGFβ1 or TGFβ3 is used as TGFβ, the concentration in the medium is not particularly limited, but may be 0.25 to 25 ng / mL, for example, about 10 ng / mL.

[0083] The concentration of IL-2 in the medium used is not limited, but can be about 5 U / mL to about 500 U / mL, for example, about 100 U / mL.

[0084] The medium used in the present disclosure may further contain retinoic acid and / or ascorbic acid. Preferably, the medium contains ascorbic acid. The concentration of ascorbic acid is not limited, but is about 1 to about 100 μg / mL, for example, about 10 μg / mL.

[0085] The medium used in the present disclosure may further contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor. Any CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor can be used, and examples thereof include 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine, or salts, hydrates, solvates thereof, or compounds described in U.S. Patent No. 8,598,344, WO2013 / 001310, WO 2013 / 040153, WO2013 / 116786, WO2014 / 029726, WO2014 / 063778, WO2014 / 072435, WO2014 / 090692, WO2014 / 106606, WO2014 / 123900, WO2014 / 154723, WO2014 / 194245, WO2015 / 049325, WO2015 / 100420, WO2015 / 144290, WO2015 / 159937, WO2015 / 159938, WO2016 / 009076, or WO2018 / 139660. Examples include Senexin A and AS2863619, but the present disclosure is not limited thereto. The concentrations of the CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor that can be used may be any suitable concentration that can be used in the art or any suitable concentration described in the above-mentioned documents, and can be appropriately changed by a person skilled in the art depending on other medium compositions.

[0086] In the method of the present disclosure, regulatory T cells are induced by stimulating human peripheral T cells with an anti-CD3 antibody in a medium containing TGFβ and IL-2, and regulatory T cells with high immunosuppressive function can be induced by performing resting culture in an IL-2-containing medium without the anti-CD3 antibody and then stimulating the cells again with the anti-CD3 antibody. The high immunosuppressive function of the resulting induced regulatory T cells can be confirmed, for example, by comprehensive gene expression analysis using RNA sequencing or by an in vitro cell proliferation inhibition test.

[0087] In one embodiment of the present disclosure, the number of days of culture in the step (a) of stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days can be appropriately determined by those skilled in the art and is not particularly limited, and can be, for example, about 3 days.

[0088] In one embodiment, the number of days for culture in the dormant culture step (b) can be appropriately determined by those skilled in the art and is not particularly limited, but can be, for example, about 2 days.

[0089] In one embodiment, the number of days for the culture step in the second basal medium in step (c) can be appropriately determined by a person skilled in the art and is not particularly limited, but can be, for example, about 3 days.

[0090] In one embodiment, the number of days for culture in the resting culture step (d) can be appropriately determined by those skilled in the art and is not particularly limited, but can be, for example, about 2 days. In one embodiment, regulatory T cells having the induced regulatory T cell-specific demethylation state can be expanded by culturing the cells under unstimulated conditions in the presence of IL-2. The medium may further contain ascorbic acid, and a stable regulatory T cell culture of the induced regulatory T cells can be obtained by culturing the cells in a medium further containing IL-2.

[0091] To isolate regulatory T cells from the resulting cell culture containing regulatory T cells, the cells may be isolated by a conventional method based on cell surface markers specific to regulatory T cells, for example, by extracting FoxP3-positive fractions using a cell sorter. Furthermore, if desired, regulatory T cells having specific antigenic properties may be isolated.

[0092] The inducible regulatory T cells obtained by the method of the present invention are expected to be used in the treatment of human inflammatory diseases, such as autoimmune diseases and allergies.

[0093] (Measurement of Positive Rate by Flow Cytometry) In one embodiment, measurement of positive rate by flow cytometry can be performed as follows.

[0094] Preparation: Fixation / Permeabilization Concentrate (hereinafter referred to as Buffer) (eBio Science, 00-5123-43) Fixation / Permeabilization Diluent (hereinafter referred to as Diluent) (eBio Science, 00-5223-56) Permeabilization Buffer (10x) (eBio Science, 00-833-56) FOXP3 Monoclonal Antibody (236A / E7), PE (hereinafter referred to as anti-FOXP3 antibody) (eBio Science, 12-4777-42) Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE (hereinafter referred to as PE control) (eBio Science) BV421, Mouse, Anti-Human, CD152 (hereinafter referred to as anti-CTLA4 antibody) (BD) BV421 Mouse IgG2a, k Isotype Control (hereinafter referred to as BV421 control) (BD) CD4 Monoclonal Antibody (RPA-T4), APC (hereinafter referred to as anti-CD4 antibody) (eBio Science) Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), APC (hereinafter referred to as APC control) (eBio Science) D-PBS (Nacalai Tesque, 14249-95) FBS (HyClone, SH30084.03) 0.5 mol / l-EDTA solution (pH 8.0) (Nacalai Tesque, 06894-14) MilliQ water Centrifuge Safety cabinet Micropipettes (P200, P1000) 5 mL polystyrene round tube (hereinafter referred to as dedicated tube) (Falcon, 352008) Nylon mesh (65 μm) (Kyoshin Riko, PP-65N)

[0095] Reagent preparation *Fixation Buffer (use 100 μL per sample) Mix Buffer and Diluent in a 1:3 ratio. *Perm Buffer Dilute Permeabilization Buffer (10x) 10 times with MilliQ water. *FACS Buffer (for preparing 500 mL) D-PBS 489 mL FBS 10 mL (final concentration 2%) 0.5 mol / l EDTA solution 1 mL (final concentration 1 mM) After preparation, store the above reagents at 4°C or on ice.

[0096] Method: (1) Add 500 μL of FACS buffer to a 1.5 mL tube. (2) Add 1x10 6Add 100 μL of the final product and gently suspend with a micropipette. (3) Centrifuge at 500×g, 4°C, for 5 minutes. (4) Remove the supernatant with an aspirator, then add 100 μL of Fixation Buffer and gently pipette. → Be careful not to create bubbles. (5) Fix by leaving the tube on ice, protected from light, for at least 30 minutes. → 45 minutes is also acceptable. (6) After fixation, add 1 mL of Perm Buffer to the tube and gently pipette. (7) Prepare new 1.5 mL tubes for the number of samples. (8) Dispense 500 μL of the sample into each tube for control and antibody staining. (9) Centrifuge at 500×g, 4°C, for 5 minutes. (10) During centrifugation, prepare a 100-fold dilution of anti-FOXP3 antibody (stock concentration = 0.05 mg / ml), anti-CTLA4 antibody (Lot: 0030269 stock concentration = 0.2 mg / ml), and anti-CD4 antibody (stock concentration = 0.1 mg / ml) in Perm Buffer (hereafter referred to as the antibody preparation). For control samples, prepare PE control (stock concentration = 0.1 mg / ml), BV421 control (stock concentration = 0.2 mg / ml), and APC control (stock concentration = 0.1 mg / ml) at the same concentrations as the corresponding dye antibodies (hereafter referred to as the control solutions). *CTLA4 staining is not performed during in-process control testing. (11) After removing the supernatant with an aspirator, add 100 μL of antibody preparation solution to the antibody-stained sample and control solution to the control sample, and gently pipette. → Be careful not to create bubbles. (12) Allow to set on ice, protected from light, for 60 minutes. (13) Start up the measuring equipment during staining. (14) After staining, add 1 mL of FACS Buffer and gently pipette. (15) Centrifuge at 500 x g, 4°C, for 5 minutes. → After removing the supernatant with an aspirator, repeat steps (14) and (15) once more to wash. (16) After removing the supernatant with an aspirator, add 500 μL of FACS Buffer and gently pipette. (17) Place a nylon mesh in a dedicated tube with tweezers and filter the suspension. (18) Analyze using any flow cytometer. The number of cells to collect data should be 10,000 or more.In calculating the target antigen positive rate, a reference line is drawn so that the positive rate of the control sample is 5% or less, and the percentage of cells showing an antigen expression level equal to or higher than the reference line is taken as the positive rate.

[0097] Notes: The reagents used for fixation and staining can also be purchased as a set of three, known as the "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.: 00-5523). Instrument settings, etc., can be set arbitrarily as long as they do not deviate to the extent that they are clearly inaccurate from a general or scientific standpoint. Clear deviations include when the various signals of the target cell population fall below the set fluorescence threshold value, or when the various signal values ​​of the control sample or the sample being measured fall below or above the limit of what can be measured normally by the instrument.

[0098] (Measurement of immunosuppressive activity) The cells of the present disclosure may have immunosuppressive activity. The immunosuppressive activity can be measured by various techniques.

[0099] In one embodiment, as described in the Examples, whether or not immunosuppression is observed can be determined by measuring cell proliferation due to an immune response induced by responder T cells. Preferably, the present disclosure is demonstrated using an in vivo model. For example, as described in the Examples, in the case of a colitis model mouse, tissues can be collected from a colitis model mouse to which the induced regulatory T cells of the present disclosure have been administered after a certain period of administration, and the activity can be measured by determining whether or not immunosuppression is confirmed in the tissue. Confirmation of immunosuppression in tissue can be achieved by various techniques, and for example, the presence or absence of immunosuppression can be confirmed by tissue staining analysis.

[0100] (General Techniques) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press;Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). These are described in PCR Applications: Protocols for Functional Genomics, Academic Press, a special edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and other publications, the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0101] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) and the like can be used. Other examples include Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.; M. et al. (1996). These are described in "Nucleic Acids in Chemistry and Biology," Oxford University Press; and "Bioconjugate Techniques," Academic Press, Hermanson, G. T. (1996), the relevant portions of which are incorporated herein by reference.

[0102] As used herein, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." When "within the range of" two values ​​is specified herein, the range includes the two values ​​themselves. References cited herein, such as scientific literature, patents, patent applications, etc., are incorporated herein by reference in their entirety to the same extent as if each were specifically set forth.

[0103] The present disclosure has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0104] In the following examples, glutamine-free RPMI 1640 (containing 10% FCS (v / v), 60 μg / mL penicillin G, 100 μg / mL streptomycin, and 10 mM HEPES) was used as the basal medium. 30 to 300 mg / L of L-glutamine was added to the medium as needed.

[0105] Genomic DNA was isolated from the induced cells and treated with the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (Human Genetic Signatures). The demethylation of genes characteristic of regulatory T cells was analyzed using a previously published method and primers (Floess et al. (2007) PloS Biology Volume 5, Issue 2, e38, and Ohkura et al. (2012) Immunity 37(5) 785-799).

[0106] In addition, in the analysis of the human Foxp3 CNS2 region, 5'-TTGGGTTAAGTTTGTTGTAGGATAG-3' (SEQ ID NO: 1) was used in the forward side, and 5'-ATCTAAACCCTATTATCACAACCCC-3' (SEQ ID NO: 2) was used in the reverse side.

[0107] Obtaining mouse T cell fractions: Foxp3-eGFP (eFox) reporter mice (Ito et al. (2014) Science 346, 363-368) were used. Lymph node cells from Foxp3-eGFP (eFox) reporter mice were obtained and analyzed by FACSAria II (BD). ＋ GFP＋ The endogenous regulatory T cell (nTreg) fraction was obtained by sorting the cells. ＋ GFP － CD44 ｌｏｗ CD62L ｈｉｇｈ The cells were sorted to obtain the naive T cell fraction. ＋ GFP － CD44 ｈｉｇｈ CD62L ｌｏｗ A cell fraction was obtained and used as effector / memory T cells.

[0108] Obtaining human T cell fractions Human CD4 ＋ T cells were isolated from PBMCs of patients with Crohn's disease and purified from enriched PBMCs using CliniMACS CD4 GMP MicroBeads according to the manufacturer's protocol.

[0109] (Example 1: Method for producing mouse highly functional stable regulatory T cells) Mouse CD4-positive T cells were stimulated for 3 days with a basal medium containing an anti-CD3 antibody (100 μL of the antibody at a concentration of 10 μg / ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), and senexin A (5 μM). The cells were then cultured for two days in a basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same manner, and the cells were cultured for two more days in a basal medium containing hIL-2 (100 U / ml). Two days later, the cells were stimulated again for two days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody per well was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml). Then, the cells were resting cultured for two days in basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same way, and the cells were resting cultured for another two days. The regulatory T cells obtained after two days were analyzed for FoxP3 and CD25 expression using flow cytometry (Figure 1), the demethylation status of Treg-specific demethylated regions using the bisulfite method (Figure 2), and the global gene expression pattern using RNA-seq (Figure 3).

[0110] Conventional induced regulatory T cells (conventional iTregs) show insufficient expression of functional molecules (in this case, for example, CTLA4, Entpd1, Nt5e, Itgae, etc., as shown in Figure 3 ) and low FoxP3 induction efficiency. However, the highly functional induced regulatory T cells obtained by the production method of the present application highly express FoxP3 and functional genes (in this case, for example, CTLA4, Entpd1, Nt5e, Itgae, etc., as shown in Figure 3 ), and were confirmed to have a demethylation state characteristic of Tregs ( Figures 1 to 3 ).

[0111] (Example 2: In vitro suppressive activity of regulatory T cells) Regulatory T cells were obtained using the same experimental system as in Example 1. Cell Trace Violet-labeled responder T cells (5 × 10 ４ ), and the prepared regulatory T cells (5 × 10 ４ ), anti-CD3 antibody (1 μg / ml) + antigen presenting cells (MHC-II positive cells: 1 x 10 ４ The cells were co-cultured for 3 days in the presence of IgG4-dependent T cells (IgG4-dependent T cells) or Dynabeads T-activator (Veritas) (5 μL / well), and the Cell Trace Violet intensity was analyzed by flow cytometry. When responder T cells initiate an immune response, the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation; however, when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak. The regulatory T cells obtained by the method of the present application were confirmed to strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a stronger suppressive function than existing regulatory T cell populations (Figure 4).

[0112] (Example 3: In vivo stability of regulatory T cells) Regulatory T cells were prepared from Thy1.2 / eFox reporter mice by the method of Example 1, and Foxp3-positive cells (2 × 10 ５The Thy1.2 cells were isolated by FACS and administered via the tail vein to Thy1.1 / wild-type mice. Two weeks later, the transferred Thy1.2 cells were recovered from lymph nodes and analyzed for Foxp3 expression (Figure 5). Regulatory T cells induced without CD28 stimulation and subjected to stabilization culture were confirmed to stably express Foxp3 even after two weeks in vivo.

[0113] Example 4: Suppressive effect on colitis model RAG-deficient mice were inoculated with 2 x 10 wild-type mouse-derived CD45RB highly expressing CD4-positive naive T cells. ５ After one week, 2 × 10 highly functional stable iTreg cells prepared by the method of Example 1 were administered to the mice. ５ The mice were administered 2 x 10 iTregs and their body weight changes were measured over time (Fig. 6A). One month after administration, colon tissues and lymph nodes were collected and analyzed by HE staining of the colon tissues (Fig. 6B) and by CD69 expression analysis in lymph node T cells (Fig. 6C: flow cytometry). The results confirmed that administration of highly functional stable iTregs suppressed colitis. The dose was 2 x 10 iTregs per mouse (approximately 20 g). ５ The therapeutic effect was confirmed at 1 x 10 ７ The efficacy was confirmed at a dose of 1 / kg.

[0114] (Example 5: Production of regulatory T cells from human peripheral T cells) CD4-positive T cells derived from a patient with Crohn's disease were stimulated for 3 days with a basal medium containing an anti-CD3 antibody (100 μL of the antibody at a concentration of 10 μg / ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and senexin A (5 μM). The cells were then cultured for two days in a basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same manner, and the cells were cultured for two more days in a basal medium containing hIL-2 (100 U / ml). Two days later, the cells were stimulated again for two days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody per well was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml). Then, the cells were resting cultured for two days in basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same manner, and the cells were resting cultured for another two days. The expression of FoxP3 and CTLA4 in the regulatory T cells obtained after two days was analyzed by flow cytometry (Figure 7A).

[0115] Conventional induced regulatory T cells (conventional iTregs) show insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained by the production method of the present application have been confirmed to be a population containing many cells expressing FoxP3 and CTLA4.

[0116] (Example 6: In vitro suppressive activity of regulatory T cells) Regulatory T cells were obtained using the same experimental system as in Example 1. Cell Trace Violet-labeled responder T cells (5 × 10 ４ ), and the prepared regulatory T cells (5 × 10 ４) were co-cultured for 3 days in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 μL / well), and the Cell Trace Violet intensity was analyzed by flow cytometry. When responder T cells initiate an immune response, the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak. The regulatory T cells obtained by the method of the present application were confirmed to strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a stronger suppressive function than existing regulatory T cell populations.

[0117] (Example 7: Production of regulatory T cells from CD8-positive human peripheral T cells) CD8-positive T cells were stimulated for 3 days with a basal medium containing an anti-CD3 antibody (100 μL of the antibody at a concentration of 10 μg / ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and senexin A (5 μM). The cells were then cultured for two days in a basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same manner, and the cells were cultured for two more days in a basal medium containing hIL-2 (100 U / ml). Two days later, the cells were stimulated again for two days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody per well was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml). Then, the cells were resting cultured for two days in basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same manner, and the cells were resting cultured for another two days. The regulatory T cells obtained after two days were analyzed for expression of FoxP3, CTLA4, and Helios by flow cytometry (Figure 8).

[0118] When induced regulatory T cells were prepared from CD8-positive T cells, it was also confirmed that the population was Helios-negative and contained many cells expressing FoxP3 and CTLA4.

[0119] (Example 8: Expression intensity analysis) The expression intensities of CD25 and FOXP3 for the cell population of induced regulatory T cells produced in Example 1 were analyzed by flow cytometry using a BD FACSLyric flow cytometer. The results are shown in Figure 9. In all cells tested, FOXP3 Ｈｉ It was confirmed that a high proportion of the induced regulatory T cell population was present.

[0120] (Example 9: Antibody panel analysis of induced regulatory T cells) Antibody panel analysis was performed to comprehensively analyze the cell surface markers in the induced regulatory T cells produced in Example 1. Using BioLegend's LEGENDScreen Human PE Kit, the induced regulatory T cells produced in Example 1 were stained, and antibodies reactive with the induced regulatory T cells of the present disclosure were analyzed by flow cytometry. Some of the results (those in which clear and significant expression was confirmed) are shown in Tables 1 to 4. When stained with an antibody panel containing just under 400 types, the positive / negative characteristics of the cell surface markers in the induced regulatory T cells of the present disclosure could be confirmed by at least 185 types of surface marker molecules.

[0121] (Example 10: Analysis of cell surface markers in induced regulatory T cells) Characteristic results from the antibody screening performed in Example 9 are shown in Figure 10. It was found that the induced regulatory T cells of the present disclosure were positive for specific cell surface markers such as CD172g and CD26.

[0122] (Example 11: Production of regulatory T cells from T cells derived from a systemic lupus erythematosus (SLE) patient) Using CD4-positive T cells derived from an SLE patient, regulatory T cells were obtained using the same experimental system as in Example 1. The expression of FoxP3, CD4, and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry ( FIG. 11 ).

[0123] Conventional induced regulatory T cells (conventional iTregs) show insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained by the production method of the present invention have been confirmed to be a population containing many cells expressing FoxP3 and CTLA4.

[0124] (Example 12: Production of regulatory T cells from T cells derived from rheumatoid arthritis (RA) patients) Using CD4-positive T cells derived from RA patients, regulatory T cells were obtained using the same experimental system as in Example 1. The expression of FoxP3, CD4, and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry ( FIG. 12 ).

[0125] Conventional induced regulatory T cells (conventional iTregs) show insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained by the production method of the present invention have been confirmed to be a population containing many cells expressing FoxP3 and CTLA4.

[0126] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and other documents cited herein are to be incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2021-190125, filed on November 24, 2021, with the Japan Patent Office, the contents of which are incorporated by reference in their entirety as if they constitute the contents of this application.

[0127] The cell population disclosed herein can be used for the treatment and prevention of various immune diseases, autoimmune diseases, and other inflammatory diseases. Furthermore, the method for producing the cell population disclosed herein makes it possible to stably induce highly functional regulatory T cells from peripheral T cells, which is expected to be applied in the medical field.

[0128] SEQ ID NO: 1: Forward primer used in the examples SEQ ID NO: 2: Reverse primer used in the examples

Claims

1. A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population including inducible regulatory human T cells, wherein the cell population is FoxP3-positive and CTLA4-positive, and Helios-negative inducible regulatory human T cells accounting for 50% or more of the cell population.

2. The pharmaceutical composition described in claim 1, wherein the CNS2 site of the FOXP3 gene is demethylated.

3. The pharmaceutical composition described in claim 1, wherein the inducible regulatory human T cells maintain their immunosuppressive activity or immunosuppressive effect for at least approximately two weeks.

4. The pharmaceutical composition described in claim 1, wherein the inducible regulatory human T cells are CD4 positive.

5. A method comprising the steps of: (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) in a second basal medium for about 1 to about 5 days; and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days, the first basal medium comprises an anti-CD3 antibody, TGF-β, IL-2, retinoic acid, and a CDK8 / 19 inhibitor; A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a population of inducible human regulatory T cells produced by a method wherein the second basal medium comprises an anti-CD3 antibody, TGF-β, IL-2, retinoic acid, ascorbic acid, and a CDK8 / 19 inhibitor.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the proportion of said at least one characteristic in said cell population is about 60% or more.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the proportion of said at least one characteristic in said cell population is about 80% or more.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the proportion of strongly FoxP3 positive cells in the cell population is about 50% or more.

9. The pharmaceutical composition of any one of claims 1 to 5, wherein about 90% or more of the cell population are T cells.

10. The induced regulatory human T cells are administered in an amount of about 10 ８ ~about 10 ９ pieces, or about 10 ７ The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is administered in an amount of 1 / kg.

11. 6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the T cell-related disease comprises an autoimmune disease, an infectious disease, a cancer, an allergy, an inflammatory disease, and ALS that can be treated by immunosuppressive effects.

12. The autoimmune disease may be Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas' cardiomyopathy, Chagas' megacolon, Chagas' megaesophagus, Chagas' neuropathy, benign prostatic hyperplasia, scleroderma, psoriasis, Raynaud's syndrome 12. The pharmaceutical composition of claim 11, wherein the inflammatory bowel disease is selected from the group consisting of inflammatory bowel disease (IGD), inflammatory bowel disease (IGF ...

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is administered by injection.

14. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is additionally administered to a patient for whom administration of the pharmaceutical composition to the patient is ineffective or insufficient.

15. 15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is administered additionally at least about two weeks after the initial administration.