Induced regulatory t cells containing chimeric antigen receptor (CAR)

Abstract

The present invention provides a pharmaceutical composition for treating or preventing T cell-related diseases, which contains an induced regulatory T cell having high functionality and a stable immunosuppressive function, and further provides an induced regulatory T cell containing a chimeric antigen receptor (CAR). The present invention provides a pharmaceutical composition for treating or preventing T cell-related diseases, which contains, as an active ingredient, an induced regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, and AREG positive. Provided in the present invention is an induced regulatory T cell comprising a chimeric antigen receptor (CAR).

Description

Technical Field

[0001] The present invention relates to inducible regulatory T cells containing chimeric antigen receptors (CARs) and pharmaceutical compositions for treating or preventing T cell-related diseases. Background Art

[0002] As an important feature of CD25-positive CD4-positive regulatory T cells in the immune system, there is a situation where the production, differentiation and inhibitory function of regulatory T cells are impaired due to the knockout or mutation of FoxP3. In addition to FoxP3, regulatory T cells suppress the immune system through the expression of multiple genes such as CTLA4 and IL-10. In the global gene expression regulation of regulatory T cells, led by the stable expression of FoxP3, epigenetic states such as DNA demethylation are believed to contribute, and they are related to the phenotype of functional regulatory T cells. Summary of the invention Problem that the invention aims to solve

[0003] The inventors studied whether a chimeric antigen receptor (CAR) could be introduced into the induced regulatory T cells developed by themselves, and completed the present invention by containing it, and found that an induced regulatory T cell containing a chimeric antigen receptor (CAR) can be provided as an epoch-making drug. The induced regulatory T cells used in the present invention are induced by human peripheral blood T cells when regulatory T cells are induced by using anti-CD3 antibody stimulation to induce stable induced T cells from human peripheral blood T cells, and then dormant culture is performed, and then cultured after anti-CD3 antibody stimulation again, and further dormant culture is performed to generate, and it has the expression of high inhibitory molecules and high inhibitory function.

[0004] Furthermore, the present inventors have found that chimeric antigen receptors (CARs) can be further expressed and / or maintained in such induced regulatory T cells, thereby obtaining induced regulatory T cells containing chimeric antigen receptors (CARs) that can be used as drugs.

[0005] Therefore, the present invention provides the following. (Item 1) An inducible regulatory T cell containing a chimeric antigen receptor (CAR). (Item 2) According to the induced regulatory T cells described in the above item, the CAR is expressed in the induced regulatory T cells. (Item 3) The inducible regulatory T cell according to any one of the above items, which has at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive and AREG-positive. (Item 4) The inducible regulatory T cell according to any one of the above items, which has at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive and AREG-positive. (Item 5) The inducible regulatory T cell according to any one of the above items, which is at least CTLA4-positive. (Item 6) The inducible regulatory T cell according to any one of the above items, wherein the CNS2 site of the FOXP3 gene is demethylated. (Item 7) The induced regulatory T cell according to any one of the above items, which is CD4-positive or CD8-positive. (Item 8) The induced regulatory T cells according to any one of the above items are obtained or induced from human peripheral blood T cells or human tissue-derived T cells. (Item 8A) The inducible regulatory T cell according to any one of the above items, wherein the CAR is a second-generation or third-generation CAR. (Item 8B) The inducible regulatory T cell according to any one of the above items, wherein the CAR is a second-generation CAR. (Item 8C) The inducible regulatory T cell according to any one of the above items, wherein the CAR is a third-generation CAR. (Item 9) The inducible regulatory T cells according to any one of the above items are obtained by a method comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). (Item 10) A cell population comprising T cells, wherein about 50% or more of the T cells in the cell population are the inducible regulatory T cells according to any one of the above items. (Item 11) The inducible regulatory T cells according to item 10, wherein about 80% or more of the T cells in the cell population are the inducible regulatory T cells according to any one of the above items. (Item 12) The cell population according to any one of the above items, wherein the T cells in the cell population are regulatory T cells. (Item 13) The cell population according to any one of the above items, wherein about 90% or more of the cell population are T cells. (Project A1) A pharmaceutical composition comprising inducible regulatory T cells containing a chimeric antigen receptor (CAR). (Project A2) According to any one of the above items, the CAR is expressed in the induced regulatory T cells. (Item A3) The pharmaceutical composition according to any one of the above items, wherein the inducible regulatory T cells have at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Item A4) The pharmaceutical composition according to any one of the above items, wherein the inducible 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 A5) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are at least CTLA4 positive. (Item A6) The pharmaceutical composition according to any one of the above items, wherein the CNS2 site of the FOXP3 gene of the inducible regulatory T cells is demethylated. (Item A7) In the pharmaceutical composition according to any one of the above items, the induced regulatory T cells are CD4-positive or CD8-positive. (Item A8) According to any one of the above items, the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells. (Project A8A) According to any one of the above items, the CAR is a second-generation or third-generation CAR. (Project A8B) According to the pharmaceutical composition described in any one of the above items, the CAR is a second-generation CAR. (Project A8C) According to the pharmaceutical composition described in any one of the above items, the CAR is a third-generation CAR. (Item A9) The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). (Item A10) The pharmaceutical composition according to any one of the above 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 A11) The pharmaceutical composition according to any one of the above 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 A12) The pharmaceutical composition according to any one of the above items, wherein the T cell population is a regulatory T cell population. (Item A13) The pharmaceutical composition according to any one of the above items, wherein about 90% or more of the T cell population are T cells. (Item A14) The pharmaceutical composition according to any one of the above items is administered at about 10 8 ~ About 10 9 or about 10 7 The induced regulatory T cells are contained at a concentration of 100 cells / kg. (Item A15) The pharmaceutical composition according to any one of the above items, which is used for treating or preventing T cell-related diseases. (Item A16) According to any one of the above items, the T cell-related diseases include autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, inflammatory diseases, infections, cancer and amyotrophic lateral sclerosis. (Item A17) The pharmaceutical composition according to any one of the above items, wherein the autoimmune disease includes systemic lupus erythematosus, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis and cardiomyopathy. (Item A18) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is administered by injection. (Item A19) The pharmaceutical composition according to any one of the above items, when the pharmaceutical composition is administered to a patient, is additionally administered to a patient for whom the effect is ineffective or insufficient. (Item A20) The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is administered additionally at least about 2 weeks after the initial administration. (Item A21) A therapeutic drug, which is a therapeutic drug for a T cell-related disease comprising the induced regulatory T cells described in any one of the above items or the cell population described in any one of the above items, diagnosing the T cell-related disease of the disease subject, and selecting a suitable CAR contained in the induced regulatory T cells or the cell population based on the diagnosis. (Project AA1) A method for treating or preventing a disease, injury or symptom that can be treated by the induced regulatory T cells in the subject, comprising the step of administering to the subject an effective amount of induced regulatory T cells containing a chimeric antigen receptor (CAR). (Project AA2) According to the method of any one of the above items, the CAR is expressed in the induced regulatory T cells. (Project AA3) The method according to any one of the above items, wherein the inducible regulatory T cells have at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Project AA4) The method according to any one of the above items, wherein the inducible 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 AA5) According to the method according to any one of the above items, the induced regulatory T cells are at least CTLA4 positive. (Item AA6) The method according to any one of the above items, wherein the CNS2 site of the FOXP3 gene of the inducible regulatory T cells is demethylated. (Item AA7) The method according to any one of the above items, wherein the induced regulatory T cells are CD4-positive or CD8-positive. (Item AA8) According to the method of any one of the above items, the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells. (Project AA8A) According to the method of any one of the above items, the CAR is a second-generation or third-generation CAR. (Project AA8B) According to the method of any one of the above items, the CAR is a second-generation CAR. (Project AA8C) According to the method of any one of the above items, the CAR is a third-generation CAR. (Item AA9) The method according to any one of the above items, further comprising the step of obtaining the induced regulatory T cells by a method comprising the following steps, (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). (Item AA10) The method according to any one of the above items, wherein the T cell population comprises at least about 50% of the cells in the T cell population being the inducible regulatory T cells. (Project AA11) The method according to any one of the above items, wherein the T cell population comprises at least about 80% of the cells in the T cell population being the inducible regulatory T cells. (Item AA12) According to the method according to any one of the above items, the T cell population is a regulatory T cell population. (Item AA13) The method according to any one of the above items, wherein about 90% or more of the cell population are T cells. (Project AA14) The method according to any one of the above items, wherein about 10 8 ~ About 10 9 or about 10 7 The induced regulatory T cells are contained at a concentration of 100 cells / kg. (Project AA15) According to the method of any one of the above items, the treatment or prevention of the disease, injury or symptom includes the treatment or prevention of T cell-related diseases. (Project AA16) According to the method of any one of the above items, the disease, injury or condition includes autoimmune disease, allergy, transplant rejection, graft-versus-host disease, inflammatory disease, infection, cancer and amyotrophic lateral sclerosis. (Project AA17) According to the method described in any one of the above items, the autoimmune diseases include systemic lupus erythematosus, Crohn's disease, diabetes (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, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis and cardiomyopathy. (Project AA18) According to the method of any one of the above items, the administration comprises administration by injection. (Project AA19) The method according to any one of the above items, comprising the step of additionally administering the inducible regulatory T cells to a subject in which the inducible regulatory T cells have no effect or have an insufficient effect when administered to the subject. (Project AA20) The method according to any one of the above items, wherein in the case of additional administration, the pharmaceutical composition is additionally administered at least about 2 weeks after the initial administration. (Project AA21) A method, the method further comprising: diagnosing the disease, injury or symptom in the subject, and selecting a suitable CAR contained in the induced regulatory T cell or the cell population based on the diagnosis. (Project AAA1) Use of induced regulatory T cells containing chimeric antigen receptors (CARs) in the manufacture of drugs for treating or preventing diseases, injuries or symptoms that can be treated by the induced regulatory T cells. (Project AAA2) According to the use of any one of the above items, the CAR is expressed in the induced regulatory T cells. (Project AAA3) The use according to any one of the above items, wherein the inducible regulatory T cells have at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Project AAA4) The use according to any one of 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. (Project AAA5) The use according to any one of the above items, wherein the induced regulatory T cells are at least CTLA4 positive. (Project AAA6) The use according to any one of the above items, wherein the CNS2 site of the FOXP3 gene of the inducible regulatory T cells is demethylated. (Project AAA7) The use according to any one of the above items, wherein the induced regulatory T cells are CD4-positive or CD8-positive. (Project AAA8) The use according to any one of the above items, wherein the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells. (Project AAA8A) The use according to any one of the above items, wherein the CAR is a second-generation or third-generation CAR. (Project AAA8B) The use according to any one of the above items, wherein the CAR is a second generation CAR. (Project AAA8C) The use according to any one of the above items, wherein the CAR is a third generation CAR. (Project AAA9) The use according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). (Project AAA10) The use according to any one of the above items, wherein the T cell population comprises at least about 50% of the cells in the T cell population are the inducible regulatory T cells. (Project AAA11) The use according to any one of the above items, wherein the T cell population comprises at least about 80% of the cells in the T cell population are the inducible regulatory T cells. (Project AAA12) The use according to any one of the above items, wherein the T cell population is a regulatory T cell population. (Project AAA13) The use according to any one of the above items, wherein about 90% or more of the T cell population are T cells. (Project AAA14) The use according to any one of the above items, wherein about 10 8 ~ About 10 9 or about 10 7 The induced regulatory T cells are contained in the amount of 100 cells / kg. (Project AAA15) The use according to any one of the above items, wherein the disease, injury or symptom comprises a T cell-related disease. (Project AAA16) The use according to any one of the above items, wherein the disease, injury or symptom comprises autoimmune disease, allergy, transplant rejection, graft-versus-host disease, inflammatory disease, infection, cancer and amyotrophic lateral sclerosis. (Project AAA17) According to the use described in any one of the above items, the autoimmune diseases include systemic lupus erythematosus, Crohn's disease, diabetes (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, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis and cardiomyopathy. (Project AAA18) The use according to any one of the above items, wherein the administration is by injection. (Project AAA19) The use according to any one of the above items, wherein the use is administered to a patient as an additional administration to a patient for whom the effect is ineffective or insufficient. (Project AAA20) The use according to any one of the above items, wherein the booster administration is performed at least about 2 weeks after the initial administration. (Project AAA21) A use, characterized in that the drug diagnoses the disease, injury or symptom, and based on the diagnosis, selects the induced regulatory T cells or the appropriate CAR contained in the cell population. (Item B1) A method for producing an inducible regulatory T cell containing a chimeric antigen receptor (CAR), comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). (Item B2) According to any one of the above items, the introduction of the CAR gene is performed at least about twice in total in any one of steps (a) to (d). (Item B3) According to any one of the above items, the introduction of the CAR gene is performed at least once in step (a). (Item B4) The method according to any one of the above items, wherein the first basal medium contains at least one factor selected from the group consisting of anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors and ascorbic acid. (Item B5) According to the method of any one of the above items, the first basal culture medium contains anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor and ascorbic acid. (Item B6) The method according to any one of the above items, wherein the second basal medium contains at least one factor selected from the group consisting of anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, and CDK8 / 19 inhibitors. (Item B7) According to the method of any one of the above items, the second basal culture medium contains anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor and a CDK8 / 19 inhibitor. (Item B8) The method according to any one of the above items, wherein in step (a), the CD4-positive T cells or CD8-positive T cells are stimulated with the first basal medium for about 3 days. (Item B9) The method according to any one of the above items, wherein in step (b), the cells obtained in step (a) are dormantly cultured in a medium containing the IL-2 for at least about 2 days. (Item B10) The method according to any one of the above items, wherein in step (c), the cells obtained in step (b) are stimulated with a second basal medium for about 3 days. (Item B11) The method according to any one of the above items, wherein in step (d), the cells obtained in step (c) are dormantly cultured in a medium containing the IL-2 for at least about 2 days. (Item B12) An induced regulatory T cell or a cell population comprising the induced regulatory T cell, produced by the method according to any one of the above items.

[0006] In the present invention, it is intended that in addition to the explicit combinations, one or more of the above features may be further combined and provided. In addition, more embodiments and advantages of the present invention may be recognized by those skilled in the art if the following detailed description is read and understood as needed.

[0007] In addition, features and significant actions and effects of the present invention other than those described above will become apparent to those skilled in the art by referring to the following sections of the embodiments of the present invention and the accompanying drawings. Effects of the Invention

[0008] The regulatory T cells of the present invention can enable chimeric antigen receptors (CARs) to function efficiently, and as highly functional regulatory T cells, can be used for the treatment and prevention of various immune diseases, inflammatory diseases such as autoimmune diseases, and the like.

[0009] In addition, according to the present invention, since the gene can be introduced into the inducible regulatory T cells of the present invention in a manner that can efficiently function using lentivirus, the gene can be introduced into the inducible regulatory T cells using the same method without being limited to the chimeric antigen receptor (CAR) gene, and any gene can be introduced into the inducible regulatory T cells using the same method. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] [ Figure 1 ] Figure 1 The present invention provides an overview of a method for producing one embodiment of mouse induced regulatory T cells (also referred to as mouse highly functional stable iTreg (HSFiTreg) cells) and flow cytometry results on the induced regulatory T cells. Figure 1 A shows the outline of the protocol for inducing iTreg cells from mouse CD4-positive naive T cells by various stimulation methods. Figure 1 B is analyzed by flow cytometry Figure 1 Results of the expression of FoxP3 and CD25 in the iTreg cells, activated T cells, and activated nTreg cells prepared in A. [ Figure 2 ] Figure 2 To display based on Figure 1A method for producing one embodiment of iTreg (SF-iTreg in the figure) of the present invention, wherein the expression of FoxP3 when SF-iTreg is produced from mouse CD4-positive primary T cells or effector T cells is analyzed by flow cytometry, and the demethylation state of Treg-specific demethylation regions is analyzed by bisulfite method. [ Figure 3 ] Figure 3 For Figure 1 Results of one embodiment of analyzing comprehensive gene expression patterns of individual cells using RNA sequencing. Figure 3 A shows the PCA analysis plot. Figure 3 B shows a heatmap of Treg-related genes. [ Figure 4 ] Figure 4 For analysis Figure 1 The results of one embodiment of the in vitro suppressive ability of each cell. Mouse CD4 positive naive T cells were stained with CellTrace Violet reagent to compare with Figure 1 Each cell was mixed and cultured at a certain 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 ] Figure 5 For Figure 1 The results of one embodiment of analyzing Foxp3 expression in regulatory T cells introduced 2 weeks after administration of the CD28 antibody group without primary stimulation and the iTreg of the present invention to wild-type mice. [ Figure 6 ] Figure 6 Results of one embodiment in which a colitis model was prepared by introducing mouse CD4-positive naive T cells into RAG2 knockout mice, administering the iTreg cells of the present invention, and analyzing the therapeutic effect. Figure 6 A shows the change in body weight (g), Figure 6 B shows the HE staining image of colon tissue. Figure 6 C shows the results of analyzing CD69 expression in lymph node T cells by flow cytometry. [ Figure 7 ] Figure 7 This is a diagram showing the analysis results of one embodiment of the iTreg of the present invention derived from a human Crohn's disease patient. Figure 7A is the result of analyzing the expression of FOXP3, CTLA4, and Helios by flow cytometry using CD4-positive T cells derived from human Crohn's disease patients to produce the inducible regulatory T cells of the present invention (HSF-iTreg in the figure). The cells that stimulated conventional nTreg (CD4-positive CD25-positive T cells) and conventional iTreg (Conventional iTreg: cells that stimulated CD4-positive T cells with CD3 / CD28 for 3 days in the presence of IL-2 and TGF-β1) were compared. Figure 7 B is Figure 7 A. Human CD4-positive T cells were stained with Cell Trace Violet reagent and incubated with Treg Suppression Inspector (Miltenyi Biotec) (5 μL / well) at a certain ratio. Figure 1 The cells were mixed and cultured for 3 days, and the intensity of Cell Trace Violet was analyzed by flow cytometry. [ Figure 8 ] Figure 8 This is a diagram showing the phenotypic analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present invention prepared from human CD8-positive T cells. High-function 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 ] Fig. 9 The schematic diagram shown in the upper part is a diagram showing the process of making highly functional inducible regulatory T cells (HSF-iTreg) from human CD4 positive T cells and the timing of infection with a lentivirus carrying a CAR expression virus vector in the process. As shown by the arrows, 1 / 20 of the total culture volume of lentivirus (Lenti-CD19 CAR (scFv-CD28, FMC63) Viral Particle, Cat. No.: VP-CAR-LC61) was added at the timing of Day1, Day2, Day4, and Day8, and FCM analysis of FOXP3 and CAR expression was performed on the final day. Fig. 9 The graph shown in the lower part of shows the expression of FOXP3 and CAR genes on Day 13. It is shown that CAR can be efficiently expressed by introducing genes during a single stimulation period. [ Fig.10 ] Fig.10In one embodiment of the present invention, the results of FCM analysis of CTLA4 expression in the Day 1 transfection sample of Example 7 are shown. It was confirmed that CTLA4 was also highly expressed in the HSF-iTreg cell population expressing CAR. [ Fig.11 ] Fig.11 In the process of making highly functional inducible regulatory T cells (HSF-iTreg) from human CD4-positive T cells, lentivirus (Lenti-HLA-A2 CAR (scFv-28ζ, BB7.2)-VP (VP-CAR-LC809) or Lenti-EpCAM CAR (scFv-28ζ, M13-57)-VP (VP-CAR-LC847)) was added at 1 / 50 or 1 / 25 of the total culture volume at the above timing, and the expression of CAR (GFP), FOXP3 and CTLA4 was analyzed by FCM on the final day. It shows that by introducing genes during a single stimulation period, any type of CAR can be efficiently expressed without compromising the properties and induction efficiency of Treg. DETAILED DESCRIPTION

[0011] The present invention is described below while showing the best embodiment. Throughout this specification, the expression of the singular form should be understood as also including the concept of its plural form unless otherwise mentioned. Therefore, the singular article (such as "a", "an", "the", etc. in English) should be understood as also including the concept of its plural form unless otherwise mentioned. In addition, the terms used in this specification should be understood as being used with the meaning commonly used in the art unless otherwise mentioned. Therefore, all professional terms and scientific and technological terms used in this specification have the same meaning as those commonly understood by those skilled in the art in the field to which the present invention belongs. In the event of a conflict, this specification (including definitions) takes precedence.

[0012] The definitions of terms used in this specification and / or basic technical contents are described as follows:

[0013] In this specification, "about" means ±10% of the value that follows. For example, "about 20" includes the range of "18 to 22". The range of values ​​includes all values ​​between the two endpoints and the values ​​of the two endpoints. "About" in relation to a range applies to both endpoints of the range. Therefore, for example, "about 20 to 30" includes the range of "18 to 33".

[0014] In this specification, when describing gene names and their products, unlike the usual usage, when both are described in capital letters, there are cases where both genes and proteins are referred to. For example, there are cases where FOXP3 gene and FOXP3 protein are used separately, and when described as FoxP3, both the concept and entity (whole) of the gene or protein are referred to.

[0015] In this specification, "regulatory T cells" are T cells that are positive for FoxP3 expression. In this specification, they are sometimes referred to as "Tregs". Tregs include endogenous regulatory T cells (Naturally Occurring Regulatory T cells: nTregs), inducible regulatory T cells (Inducible Regulatory T cells: iTregs), etc. Regulatory T cells generally have various functions (e.g., immunosuppressive function).

[0016] In the present specification, "inducible regulatory T cells (iTreg)" refers to cells in which the expression of IKZF2 (Helios) is negative among regulatory T cells. iTregs are generally obtained by induction of differentiation from naive CD4-positive T cells and the like.

[0017] In the present specification, "Naturally Occurring Regulatory T cells (nTreg)" refers to cells that are positive for IKZF2 (Helios) and CTLA4 expression among regulatory T cells. These are cells that are usually present in a living body.

[0018] In this specification, "peripheral blood T cells" refer to T cells present outside the thymus, which can be obtained from peripheral blood, lymph nodes, and other tissues. In this specification, when referred to as "peripheral blood T cells", as long as the cell population contains peripheral blood T cells, it is not necessary to separate the T cells. Cell components containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMC), can also be used.

[0019] In this specification, "flow cytometry" refers to the technology of measuring the physical, chemical and biological properties of the number of particles of cells, individuals and other biological particles suspended in a liquid. The device using this technology is called a "flow cytometer". In the present invention, the "positive" and "negative" of cell markers (such as FoxP3, CTLA4, Helios, CD103, etc.) can be determined by flow cytometry in a manner commonly used in the art. In more detail, in flow cytometry, cells are arranged in a row for flow, and the number of cells is calculated by spectral technology. For example, laser is irradiated to cells marked by fluorescence or luminescent enzymes, and the fluorescence or luminescent signals emitted from the cells are detected by detectors such as photodiodes, thereby calculating the number of target cells. In addition, the detection results in the detector can also be input to a computer to generate a two-dimensional graph for display. It is thus easy to grasp the presence or absence of target cells and their quantity, etc.

[0020] In this specification, "demethylation" means that the methylation modification of adenine (e.g., position 6: m6A; position 1: m1A) and cytosine (e.g., position 5: m5C; position 3: m3C) that are typically methylated is removed. Demethylation can be identified using techniques known in the art, such as the bisulfite method.

[0021] In this specification, "cell group" refers to a group containing two or more cells, for example, it can be a state where cells are aggregated on a plane, or it can be a cell mass composed of cells bonded three-dimensionally. In addition, a "cell group" can be formed by a single type of cell, or it can contain multiple types of cells.

[0022] In this specification, "chimeric antigen receptor (CAR)" refers to a modified receptor that can confer antigen specificity to cells (eg, Treg). It is known as CAR and artificial T cell receptors, chimeric T cell receptors or chimeric immune receptors. Preferably, the CAR of the present invention comprises at least one extracellular domain that can bind to an antigen, at least one transmembrane domain, and at least one intracellular domain. In the case of use in this specification, when used in combination with T cells, there are cases called CAR-T cells, etc. As a usable CAR, there may be first generation, second generation, and third generation, etc. Among them, "first generation CAR" refers to a CAR that combines a fragment (scFv) that connects the VH chain and VL chain of a monoclonal antibody variable region specific for a tumor-associated antigen in series with a ζ chain of a T cell receptor. "Second generation CAR" refers to a costimulatory factor such as CD28 (related to PI3K) and 4-1BB (related to TRAFs) that is considered to be important for T cell activation, and is assembled into the first generation CAR. The third generation CAR refers to the incorporation of multiple co-stimulatory factors such as CD28 (related to PI3K) and 4-1BB (related to TRAFs) that are considered important for T cell activation into the first generation CAR.

[0023] In this specification, "T cell-related disease" refers to any disease, injury or symptom directly or indirectly related to the state of T cells. Therefore, it is expected that the inducible regulatory T cells of the present invention can be used to treat or prevent T cell-related diseases. Examples of T cell-related diseases include autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, inflammatory diseases, infections, cancers, and amyotrophic lateral sclerosis (ALS). In addition, in one embodiment, the autoimmune diseases are not limited to these, but examples thereof include: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (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 (strong skin disease), Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas cardiomyopathy, Chagas megacolon, Chagas megaesophagus , Chagas nerve damage, benign prostatic hyperplasia, scleroderma (scleroderma), psoriasis, Raynaud's syndrome, preeclampsia, myocarditis, glaucoma, hypertension, pulmonary hypertension, malignant hypertension, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid antibody syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune diseases, Basedow's disease, autoimmune hepatitis, primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's hemorrhage-nephritis syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu'saortitis, giant cell arteritis, rheumatic polymyopathy, adult-onset Still's disease, Behcet's disease, sarcoidosis, ulcerative colitis, etc.

[0024] In the present specification, "diseases, injuries or symptoms that can be treated by the induced regulatory T cells" include any disease, injury or symptom that can be treated or prevented thereby, and may also include any disease, injury or symptom that is generally effective even if there are few regulatory T cells, but is not limited to this, and it can be understood that: even when normal regulatory T cells do not show an effect, these are also included because the use of induced regulatory T cells may have an effect.

[0025] (Preferred embodiment) The preferred embodiments of the present invention are described below. The embodiments provided below are provided in order to better understand the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate changes within the scope of the present invention with reference to the records in this specification. In addition, the following embodiments of the present invention can be used alone, or these can be used in combination.

[0026] <Inducible regulatory T cells containing chimeric antigen receptors (CARs) and their use> In one aspect, the present invention provides inducible regulatory T cells containing chimeric antigen receptors (CARs), methods for making and using the same, and other related technologies.

[0027] The present invention utilizes highly functional stable inducible regulatory T cells (also referred to as highly functional stable iTreg, HSFiTreg).

[0028] In one aspect of the present invention, an induced regulatory T cell containing a chimeric antigen receptor (CAR) is provided as a pharmaceutical composition for treatment or prevention. The induced regulatory T cells used in the present invention have at least one feature selected from a group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive and AREG positive. In one embodiment of the present invention, the induced regulatory T cells of the present invention may have at least 2 features selected from a group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive and AREG positive. In addition, in one embodiment of the present invention, the induced regulatory T cells in the present invention may also be at least CTLA4 positive. Although not intended to be limited, the induced regulatory T cells in the present invention may also be CD4 positive or CD8 positive.

[0029] In one embodiment, CAR is expressed in inducible regulatory T cells. In one embodiment, the gene of CAR is contained in inducible regulatory T cells. The CAR used can be in any form, and any one of the first generation, the second generation, and the third generation can be preferably used. Although it is not desired to be bound by theory, when used in the present invention, it is advantageous to have a suitable stimulation intensity by the relative relationship with the iTreg as the object, but it is not limited thereto. From this viewpoint, it is preferred to have the second generation or the third generation, but it is not limited thereto. In a certain embodiment, the second generation may be advantageous, and in other embodiments, the third generation may be advantageous. By studying the compatibility with iTreg separately, it can be selected according to a suitable stimulation intensity (for example, as an indicator of stimulation intensity, the nutritional metabolic state, the intensity of phosphorylation, etc. can be measured in vitro, and the stability of FOXP3 expression as a result can also be measured in vivo or in vitro), but the present invention is not limited thereto.

[0030] The expression of FoxP3 in regulatory T cells can be induced in a test tube by culturing CD4-positive T cells in a medium containing IL2 and TGFβ in the presence of anti-CD3 antibodies and anti-CD28 antibodies, and then culturing them in a medium containing IL2 and TGFβ. In addition, although various methods for inducing regulatory T cells from peripheral blood T cells are known, the expression of FoxP3 in induced regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has not been confirmed.

[0031] In recent years, a method for adding ascorbic acid to the culture medium in induced regulatory T cells has been developed (Kasahara et al. Int. Immunol. (2017) 29 (10): 457-469), and a method for not using CD28 antibody stimulation (Mikami et al. Proc Natl Acad Sci USA. (2020) 117 (22): 12258-12268.), which causes a culture method for DNA demethylation induction. However, in the induced regulatory T cells produced by this method, the expression of functional molecules was not confirmed, and sufficient immunosuppressive activity was not obtained. Although there is a clinical trial using induced regulatory T cells in the clinic, GVHD is implemented on the subject, but the proportion of regulatory T cells used in the cells is low, and the effectiveness of GVHD prevention, etc. has not been confirmed at present (MacMillan et al., Blood Adv. (2021) 5 (5): 1425-1436).

[0032] In the present invention, the expression of FoxP3 is stable, and the advantage is that a pharmaceutical composition containing induced regulatory T cells that stably maintain immunosuppressive effects as an active ingredient can be provided. Such induced regulatory T cells can be prepared, for example, by the method for producing induced regulatory T cells described elsewhere in the specification of this application. For example, the present invention can provide a pharmaceutical composition obtained by a method comprising the following steps: (a) a step of stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal culture medium for about 1 to about 5 days, (b) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days with a culture medium containing IL-2, (c) a step of stimulating the cells obtained in step (b) with a second basal culture medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days with a culture medium containing IL-2. The induced regulatory T cells containing a chimeric antigen receptor (CAR) of the present invention can be generated in such a method by a step including introducing CAR.

[0033] The inducible regulatory T cells of the present invention have an inducible regulatory T cell-specific demethylation state. The obtained regulatory T cells are in a regulatory T cell-specific demethylation state, for example, by demethylating the CNS2 site of the FoxP3 gene (FOXP3 (all in italics)) to confirm. Since such a demethylation state can be used as an indicator of a stable type, it is possible to show that the inducible regulatory T cells of the present invention are stable inducible regulatory T cells by confirming the demethylation state.

[0034] In this specification, the immunosuppressive activity or immunosuppressive effect of the inducible regulatory T cells in the present invention can be confirmed, for example, by measuring the Cell Trace Violet intensity in the responding T cells. The inducible regulatory T cells in the present invention can stably provide immunosuppressive activity or immunosuppressive effect, for example, the inducible regulatory T cells of the present invention can provide immunosuppressive activity or immunosuppressive effect for at least 2 weeks. As shown in the embodiments described later, the inducible regulatory T cells in the present invention have a higher immunosuppressive effect than existing regulatory T cells (including inducibility and intrinsic). Therefore, the inducible regulatory T cells in the present invention can also be referred to as functional or highly functional inducible regulatory T cells.

[0035] In one embodiment, the induced regulatory T cells of the present invention can stably express FoxP3. Therefore, the induced regulatory T cells of the present invention can also be referred to as stable induced regulatory T cells. The induced regulatory T cells of the present invention are highly functional and stable on one side, and can be referred to as highly functional stable induced regulatory T cells (HSFiTreg).

[0036] In one embodiment of the present invention, whether the marker in the inducible regulatory T cells of the present invention is positive can be determined by using the positive rate determination of flow cytometry. For example, flow cytometry can be used for analysis, and whether it is positive can be determined by the proportion of cells showing an antigen expression amount above the benchmark. According to the expression intensity of the cell surface marker, it can also be divided into negative, weak positive, medium positive, strong positive (weak positive, medium positive and strong positive are summarized and set as "positive"), etc. For example, according to the setting of the machine, when the value of the central value of the fluorescence intensity of each marker / the central value of the fluorescence intensity of the negative control (using the staining of the isotype control antibody) is lower than 5, 5 or more and lower than 10, 10 or more and lower than 30, 30 or more, it can also be set as negative, weak positive, medium positive, and strong positive respectively. Such a judgment can be determined as exemplified in (positivity determination using flow cytometry), but the present invention is not limited to this.

[0037] In one embodiment of the present invention, the inducible regulatory T cells of the present invention can be induced from any cells, but can preferably be induced from human peripheral blood T cells or human tissue-derived T cells.

[0038] In one embodiment of the present invention, the present invention may include a T cell group containing a chimeric antigen receptor (CAR) in which about 50% or more of the cells in the T cell group are T cell groups of inducible regulatory T cells described elsewhere in the specification. In one embodiment, for the cell group of the present invention, 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 group can be used as inducible regulatory T cells recorded in other locations of the specification of the application.

[0039] In one embodiment of the present invention, the T cells in the cell population of the present invention can be used as regulatory T cells. At this time, among the regulatory T cells in the cell population of the present invention, 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 can be used as inducible regulatory T cells recorded in other locations of the specification of this application.

[0040] In one embodiment of the present invention, the cell population of the present invention may also contain cells other than T cells, but it may be preferred that 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 invention are T cells, and it may also be preferred that about 90% or more are T cells.

[0041] In one aspect of the present invention, a pharmaceutical composition comprising an inducible regulatory T cell or cell group containing a chimeric antigen receptor (CAR) of the present invention is provided. In addition, in other aspects, a regenerative medical material or product containing an inducible regulatory T cell or cell group of the present invention is provided. The pharmaceutical composition, regenerative medical material or product can be used in autoimmune diseases, inflammatory diseases, and allergies. These drugs, regenerative medical materials or products can be used together with culture media and any other additives used in this field. As such a culture medium, a culture medium to which essential factors have been added to a basal culture medium for animal cell culture for cell culture can be used. Examples of such culture media are described in detail elsewhere in this specification. Examples of components added to the culture medium are also described in detail elsewhere in this specification. When such a product is provided, DMSO and the like may also be contained.

[0042] In one embodiment, the pharmaceutical composition of the present invention can be used to treat or prevent T cell-related diseases. Examples of T cell-related diseases include autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, inflammatory diseases, infections, cancer, and amyotrophic lateral sclerosis. In addition, in one embodiment, the autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (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, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardiomyopathy, Chagas' cardiomyopathy (chagasic myocardiopathy), Chagas megacolon, Chagas megaesophagus, Chagas nerve injury, 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 antibody syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Crescentic glomerulonephritis, organ-specific autoimmune diseases, Basedow's disease, autoimmune hepatitis, primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's hemorrhage-nephritis syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis, rheumatic polymyopathy, adult-onset Still's disease, Behcet's disease, sarcoidosis, ulcerative colitis, etc.

[0043] In one embodiment, the pharmaceutical composition of the present invention can be administered in various dosages and methods, preferably by injection. In one embodiment, the pharmaceutical composition of the present invention can be administered in a dosage of about 10 8 ~ About 10 9 or about 10 7 The induced regulatory T cells described elsewhere in the specification are contained at a concentration of 100 cells / kg.

[0044] In one embodiment, when administering to a patient, the pharmaceutical composition of the present invention may be administered additionally to patients who have no effect or insufficient effect. Whether there is no effect or insufficient effect may be determined, for example, by the degree of inhibition of inflammation in the target disease as an indicator. In one embodiment, when additional administration is performed, administration may be performed at least about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after the initial administration.

[0045] <Method for producing inducible regulatory T cells> The method for producing the induced regulatory T cells of the present invention is a novel method for producing highly functional and stable induced regulatory T cells, and is described in detail below in this specification.

[0046] In one aspect of the present invention, a method is provided, which is a method for producing induced regulatory T cells, comprising (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood for about 1 to about 5 days with a first basal medium, (b) culturing the cells obtained in step (a) in a dormant manner for at least about 1 to about 3 days with a medium containing IL-2, (c) stimulating the cells obtained in step (b) for about 1 to about 5 days with a second basal medium, and (d) culturing the cells obtained in step (c) in a dormant manner for at least about 1 to about 3 days with a medium containing IL-2. In the present invention, the induced regulatory T cells of the present invention can also be produced using any one of CD4-positive T cells and CD8-positive T cells as a raw material, and (e) CAR can be introduced by a process including introducing a chimeric antigen receptor (CAR) into the T cells. In one embodiment, the induced regulatory T cells of the present invention can also be produced using mixed cells of CD4-positive T cells and CD8-positive T cells as a raw material.

[0047] In one embodiment, the method of the present invention can also be a method for producing regulatory T cells from human peripheral blood T cells (including CD4-positive T cells or CD8-positive T cells), which method includes the step of culturing human peripheral blood T cells using a culture medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation, the step of culturing human peripheral blood T cells using a culture medium containing IL-2 in the absence of anti-CD3 antibodies, and the step of culturing human peripheral blood T cells again using a culture medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation.

[0048] In this specification, "anti-CD3 antibody stimulation" refers to specific stimulation of CD3 receptors on cells. As CD3 stimulation, anti-CD3 agonist antibodies are exemplified. Anti-CD3 agonist antibodies can use products commercially available as research reagents, or can be prepared using conventional methods. Anti-CD3 antibodies can use antibodies derived from animals such as mice, rabbits, goats, and cattle, and antibodies derived from humans, for example.

[0049] In one embodiment, the culture medium used in each step may further contain retinoic acid and / or ascorbic acid. Preferably, the culture medium may contain ascorbic acid. In one embodiment, the culture medium may further contain a CDK8 inhibitor, a CDK19 inhibitor and / or a CDK8 / 19 inhibitor. Preferably, the culture medium contains a CDK8 inhibitor, a CDK19 inhibitor and / or a CDK8 / 19 inhibitor.

[0050] In one embodiment of the present invention, 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 factors selected from the group consisting of anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors and ascorbic acid. In addition, in other embodiments, the first basal medium and the second basal medium may each independently contain anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors and ascorbic acid. The concentrations contained in these components may be the usual concentrations used in this field. In one embodiment, the concentration of the CDK8 inhibitor, CDK19 inhibitor and / or CDK8 / 19 inhibitor that can be used can be any suitable concentration that can be used in this field. For example, when using SenexinA, it can be set to 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 are not limited to these concentrations, and those skilled in the art can make appropriate changes according to other culture medium compositions.

[0051] In one embodiment, in the present invention, regulatory T cells are produced from human peripheral blood T cells. Peripheral blood T cells include initial regulatory T cells, CD4-positive T cells, CD8-positive T cells, etc. Regulatory T cells can be induced from a culture containing a variety of T cells, or after specific cells such as CD4-positive T cells and CD8-positive T cells are separated from these cells, regulatory T cells can be induced. In addition, regulatory T cells can also be induced after specific antigen-specific T cells are separated. Therefore, the induced regulatory T cells of the present invention include CD4-positive regulatory T cells and CD8-positive regulatory T cells. In addition, in this specification, "CD4-positive" or "CD4-positive" is referred to as "CD4-positive" or "CD8-positive" + ", unless otherwise specified, refers to a single positive cell that is CD4-positive and CD8-negative. In addition, in the production method of the present invention, any T cell that is CD4-positive or CD8-positive can be used as a raw material, and after the induced regulatory T cells are generated, the CD4-positive or CD8-positive characteristics can be maintained without special operations. In the present invention, the same properties are also maintained in the induced regulatory T cells containing the chimeric antigen receptor (CAR), so it is preferred.

[0052] In one embodiment, in the method of the present invention, antibodies that are immobilized on the inner wall of the culture container or the surface of an insoluble carrier even when added to the culture medium can also be used. An insoluble carrier is a component that can physically or chemically bind to an anti-CD3 antibody, and a carrier that is insoluble in an aqueous solution can be used. Materials that can physically adsorb anti-CD3 antibodies include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, glass, and the like. The shape of the insoluble carrier is not particularly limited, and for example, a plate-like, bead-like, or container-like shape can be used. The amount of anti-CD3 antibodies can be varied by the titer and source of the antibody used, but can be appropriately set to provide sufficient stimulation for inducing regulatory T cells.

[0053] In one embodiment, in the method of the present invention, a medium in which essential factors are added to a basal medium for animal cell culture can be used in the culture of cells. Examples of basal medium for animal cell culture that can be used in the method of the present invention include Iscove's modified Eagle's Medium, Ham's F12, MEM Zinc Option, IMEM Zinc Option, IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), RPMI 1640, Fischer's, mixed mediums thereof, or mediums with modified compositions.

[0054] The basal medium may also contain serum (e.g., fetal bovine serum (FBS)), or serum-free. The serum-free medium may contain, as required, one or more serum substitutes such as albumin, bovine serum albumin (BSA), transferrin, apo-transferrin, KnockOut Serum Replacement (KSR) (a serum substitute when culturing ES cells) (Thermo Fisher Scientific), N2 additive (Thermo Fisher Scientific), B27 additive (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-mercaptoglycerol, and monothioglycerol. The basal medium may also contain one or more substances including lipids (e.g., chemically defined lipid concentrates), amino acids, L-glutamine, GlutaMAX (ThermoFisher Scientific), non-essential amino acids (NEAA), vitamins (e.g., niacinamide, ascorbic acid), growth factors, antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and equivalents of these substances.

[0055] In one embodiment, the basal medium may be RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0056] In the method of the present invention, the cells can be cultured under normal animal cell culture conditions. The culture temperature is defined as follows, about 30 to 40° C., preferably about 37° C. The culture is preferably carried out in an atmosphere of air containing CO 2 , and the CO 2 concentration is preferably about 2 to 5%.

[0057] In the method of the present invention, the anti-CD3 antibody can be directly added to the culture medium, or the antibody immobilized on the inner wall of the culture container or the surface of an insoluble carrier can be used. The amount of the anti-CD3 antibody can be changed by the titer and source of the antibody used, but it can be appropriately set so as to provide sufficient stimulation for inducing regulatory T cells.

[0058] Examples of TGFβ used include TGFβ1, TGFβ2, and TGFβ3, for example, TGFβ1. The concentration of TGFβ is not particularly limited as long as a person skilled in the art can appropriately set it. When TGFβ1 or TGFβ3 is used as TGFβ, the concentration in the culture medium is not particularly limited, and may be set to 0.25 to 25 ng / mL, for example, about 10 ng / mL.

[0059] The concentration of IL-2 in the medium to be used is not limited, but can be set to about 5 U / mL to about 500 U / mL, for example, about 100 U / mL.

[0060] Retinoic acid and / or ascorbic acid may be further added to the culture medium used in the present invention. Preferably, the culture 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.

[0061] CDK8 inhibitors, CDK19 inhibitors and / or CDK8 / 19 inhibitors may be further added to the culture medium used in the present invention. As CDK8 inhibitors, CDK19 inhibitors and / or CDK8 / 19 inhibitors, any inhibitors may be used, for example: 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, etc. of these substances, or U.S. Patent No. 8598344, WO2013 / 001310, WO2013 / 040153, WO2013 / 040154, WO2013 / 001315 13 / 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, etc. As an example, SenexinA or AS2863619 is exemplified, but the present invention is not limited thereto. The concentration 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 this field, or any suitable concentration described in the above-mentioned literature, and those skilled in the art may make appropriate changes according to other culture medium compositions.

[0062] In the method of the present invention, human peripheral blood T cells can be stimulated with anti-CD3 antibodies using a culture medium containing TGFβ and IL-2 to induce regulatory T cells, and further, after dormant culture using a culture medium containing IL-2 without anti-CD3 antibodies, they are stimulated again with anti-CD3 antibodies to induce regulatory T cells with high immunosuppressive function. The obtained induced regulatory T cells have high immunosuppressive function, which can be confirmed by, for example, comprehensive gene expression analysis by RNA sequencing and in vitro cell proliferation inhibition test.

[0063] In one embodiment of the present invention, the number of days for 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 set by those skilled in the art without particular limitation, but can be set to about 3 days, for example.

[0064] Furthermore, in one embodiment, the number of days for culturing in the dormant culturing step of step (b) can be appropriately set by those skilled in the art and is not particularly limited, but may be set to, for example, about 2 days.

[0065] Furthermore, in one embodiment, the number of days for culturing in the culturing step of the second basal medium in step (c) can be appropriately set by those skilled in the art and is not particularly limited, but may be set to, for example, about 3 days.

[0066] In addition, in one embodiment, for the number of days of culture in the dormant culture step of step (d), those skilled in the art can appropriately set, without particular limitation, but for example, can also be set to about 2 days. In one embodiment, in the presence of IL-2, by culturing in a non-stimulated state, regulatory T cells with induced regulatory T cell-specific demethylation state can be proliferated. The culture medium can also further contain ascorbic acid, and in addition, by further culturing with a culture medium containing IL-2, a stable regulatory T cell culture of induced regulatory T cells can be obtained.

[0067] To isolate regulatory T cells from a cell culture containing the obtained regulatory T cells, it is sufficient to separate the cells based on cell surface markers specific to the regulatory T cells according to conventional methods, for example, by selecting FoxP3-positive fractions using a cell sorter. In addition, regulatory T cells having specific antigenic properties can be isolated as desired.

[0068] The induced regulatory T cells obtained by the method of the present invention are expected to be used for the treatment of human inflammatory diseases such as autoimmune diseases and allergies.

[0069] (Positive rate determination by flow cytometry) In one embodiment, the positivity rate determination by flow cytometry can be performed as follows.

[0070] Prepare Fixation / Permeabilization Concentrate (hereinafter referred to as buffer) (eBio Science, 00-5123-43) Fixation / Permeabilization Diluent (eBio Science, 00-5223-56) Permeabilization Buffer (10×) (eBio Science, 00-833-56) ·FOXP3 Monoclonal Antibody (236A / E7), PE (hereinafter referred to as anti-FOXP3 antibody) (eBioScience, 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 antibody (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) ·0.5mol / l-EDTA solution (pH 8.0) (NACALAI TESQUE, 06894-14) ·MilliQ water Centrifuge Safety cabinet Micropipette (P200, P1000) 5mL polystyrene round tube (hereinafter referred to as a special test tube) (Falcon, 352008) Nylon mesh (65μm) (Kyojin Riko, PP-65N)

[0071] Reagent preparation ※Fixation Buffer (100 μL per sample) Mix the buffer and diluent in a 1:3 ratio. ※Perm Buffer Dilute the Permeabilization Buffer 10-fold (10×) with MilliQ water. ※FACS Bufer (when preparing 500mL) D-PBS 489mL FBS 10mL (final concentration 2%) 0.5 mol / l EDTA solution 1 mL (final concentration 1 mM) The above reagents were stored at 4°C or on ice after preparation.

[0072] method (1) Add 500 μL of FACS buffer to a 1.5 mL test tube. (2) Add 1x10 6 The final product is gently resuspended using a micropipette. (3) Centrifuge at 500 × g at 4°C for 5 minutes. (4) After removing the supernatant using a pipette, add 100 μL of Fixation buffer and pipette gently. Be careful not to create bubbles. (5) Fix by leaving the sample on ice in the dark for 30 minutes or more. → Alternatively, the sample may be fixed for 45 minutes. (6) After fixation, add 1 mL of Perm buffer to the test tube and pipette gently. (7) Prepare several new 1.5 mL test tubes for the samples. (8) Inject 500 μL of each of the control sample and the antibody staining sample. (9) Centrifuge at 500 × g at 4°C for 5 minutes. (10) During the centrifugation process, 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) were prepared by 100-fold dilution using Perm buffer (hereinafter referred to as antibody preparation solution). When used as a control sample, 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) were adjusted to the same concentration as the antibody of the pigment (hereinafter referred to as control solution). *CTLA4 staining was not performed during the in-process management test. (11) After removing the supernatant using a pipette, add 100 μL of the antibody preparation solution to the antibody-stained sample and 100 μL of the control solution to the control sample, and pipette gently. → Be careful not to create bubbles. (12) Fix the sample by leaving it on ice in the dark for at least 60 minutes. (13) Start the measuring instrument during the staining process. (14) After staining, add 1 mL of FACS buffer and pipette gently. (15) Centrifuge at 500 × g for 5 minutes at 4°C. → Remove the supernatant with a pipette and repeat (14) and (15) for washing. (16) After removing the supernatant using a pipette, add 500 μL of FACS buffer and gently pipette. (17) Use tweezers to place the nylon filter on a special test tube and filter the suspension. (18) Analyze using any flow cytometer. The number of cells for data collection is set to 10,000 or more. For target antigen positivity calculation, a baseline is drawn when the positivity of the control sample is less than 5%, and the percentage of cells expressing antigen above the baseline is taken as the positivity.

[0073] Remark The reagents used for fixation and staining can be purchased as a set of 3 as "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.: 00-5523). From a general scientific point of view, any setting of the equipment is acceptable as long as it does not deviate to the extent that it is judged that normal measurement cannot be performed. Obvious deviation refers to the situation where the values ​​of various signals of the control sample or the sample to be measured fall below or exceed the limit value that can be normally measured by the equipment when the fluorescence threshold value set for various signals of the target cell group drops below the set value.

[0074] (Immunosuppressive activity assay) The cells of the present invention may have immunosuppressive activity. Immunosuppressive activity can be measured by various methods.

[0075] In one embodiment, as described in the examples, it can be determined whether the cell proliferation caused by the immune response caused by the responding T cells is suppressed. It is preferably ideal to use an in vivo model to confirm in the present invention. For example, as described in the examples, when used as a colitis model mouse, after a certain period of administration, tissues are recovered from the colitis model mice administered with the inducible regulatory T cells of the present invention, and its activity can be determined by whether immunosuppression is confirmed in the tissue. Confirmation of immunosuppression in the tissue can be achieved by various methods, for example, the presence or absence of immunosuppression can also be confirmed by staining analysis of the tissue.

[0076] <Chimeric Antigen Receptor (CAR)> In one aspect of the present invention, an inducible regulatory T cell containing a chimeric antigen receptor (CAR) is provided. The CAR contained in the inducible regulatory T cells of the present invention can be contained as a protein as long as it can function as a CAR, or it can also contain a nucleic acid molecule expressing CAR. In one embodiment, the CAR of the present invention can be expressed in an inducible regulatory T cell.

[0077] The CAR disclosed in the present specification comprises at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0078] Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody linked to a T cell signaling domain via a transmembrane domain. As a feature of CAR, it can be cited that it is independent of MHC and utilizes the antigen binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to the selected target. Antigen recognition independent of MHC can give CAR-expressing T cells the ability to recognize antigens independently of antigen processing.

[0079] The intracellular T cell signaling domain of CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a part of CAR containing the intracellular domain of the T cell receptor, such as the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain refers to a part of CAR containing the intracellular domain of a co-stimulatory molecule that is a cell surface molecule other than an antigen receptor or its ligand, which is required for the efficient response of lymphocytes to antigens.

[0080] (Extracellular domain) In one embodiment, the CAR used in the inducible regulatory T cells disclosed in this specification contains an antigen binding domain or a portion thereof. The antigen binding domain or a portion thereof can be appropriately selected according to the type and number of ligands on the surface of the target cell. For example, the antigen binding domain can be selected in a manner that can identify a ligand that acts as a cell surface marker on a target cell associated with a specific disease symptom. Therefore, as examples of cell surface markers that can act as ligands relative to the antigen binding domain in the CAR of the present invention, there can be cited: tissue-specific markers, cell tumor-specific markers, and markers associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0081] In one embodiment, the antigen-binding domain portion of the CAR of the present invention can target (1) alloantigens including MHC class I and MHC class II; (2) extracellular self-antigens including TSHR (thyroid stimulating hormone receptor stimulating antibody), DSG3 (desmoglein 3) and cytokeratin 8; (3) foreign antigens including gliadin and Ara h2; (4) antigens of targeting molecules including CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin) and MadCam1 (mucosal vascular addressin cell adhesion molecule 1), but the antigen-binding domain portion of the CAR of the present invention can target antigens not limited to these.

[0082] In one embodiment, the CAR of the present invention can be modified to contain an antigen binding domain specific to the desired antigen target, depending on the desired antigen being targeted. For example, when CD19 is the target antigen, an antibody to CD19 can be used as the antigen binding domain in the CAR.

[0083] (Transmembrane domain) The CAR used in the inducible regulatory T cells disclosed in the present specification may contain one or more transmembrane domains fused to the extracellular domain.

[0084] In one embodiment, a linker domain derived from an extracellular domain may also be linked to a transmembrane domain. The transmembrane domain may be natural or synthetic, and as a natural transmembrane domain, may be from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present invention may be from the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0085] In one embodiment, the CAR used in the inducible regulatory T cells disclosed in this specification may also be configured with a spacer domain between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain preferably promotes the binding of the CAR to the antigen and may have a sequence that enhances signal transduction in the cell.

[0086] (Intracellular domain) The cytoplasmic signaling domain (or intracellular signaling domain) of CAR is involved in the activation of at least one of the normal effector functions of the immune cells expressed by CAR. "Effect function" refers to the specialized function of the cell. For example, in Treg, the effector function can be the secretion of immunosuppressive cytokines such as IL-10, IL-35, TGF-β, or the inhibitory activity or control activity of the expression of inhibitory molecules such as TIGIT, CTLA4, and competitive cytokine receptors such as IL-2 receptors. The intracellular signaling domain refers to the protein portion that transmits the effector function signal and then instructs the cell expressing CAR to implement the specialized function. The intracellular signaling domain may contain any completely mutant or cut-off portion of the intracellular signaling domain of the protein that is sufficient to transmit the signal that excites or blocks the effector function of the immune cell.

[0087] In one embodiment, examples of intracellular signaling domains for CAR include cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that stimulate signaling after antigen receptor binding.

[0088] (Alloantigens, allergens and haptens associated with rejection) CARs used in the inducible regulatory T cells disclosed in the present specification include CARs related to alloantigens, allergens, and haptens associated with rejection reactions.

[0089] <Method for producing inducible regulatory T cells containing chimeric antigen receptor (CAR)> In one aspect of the present invention, a method is provided, which is a method for producing an inducible regulatory T cell containing a chimeric antigen receptor (CAR), 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) culturing the cells obtained in step (a) in a dormant culture 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, (d) culturing the cells obtained in step (c) in a dormant culture medium containing IL-2 for at least about 1 to about 3 days, (e) introducing a CAR gene at least once in any one of steps (a) to (d). In one embodiment, in the method for producing an inducible regulatory T cell containing a chimeric antigen receptor (CAR) of the present invention, when producing the inducible regulatory T cell of the present invention, it can be carried out by introducing a CAR gene.

[0090] In one embodiment, the introduction of the CAR gene can be performed at least twice in total in any of the above steps (a) to (d), and by doing so, CAR can be stably expressed. In one embodiment, the introduction of the CAR gene can be performed at least once in the above step (a).

[0091] In one embodiment of the present invention, a method for producing an induced regulatory T cell containing a chimeric antigen receptor (CAR) may have one or more features of a method for producing an induced regulatory T cell.

[0092] <Medical use of inducible regulatory T cells containing chimeric antigen receptors (CAR)> In one aspect of the present invention, a pharmaceutical composition comprising an induced regulatory T cell containing a chimeric antigen receptor (CAR) is provided. In one embodiment of the present invention, the induced regulatory T cell of the present invention may have one or more characteristics of the above-mentioned induced regulatory T cell.

[0093] In addition, in one aspect, a therapeutic drug is provided, which is a therapeutic drug for a T cell-related disease containing the induced regulatory T cells or cell population of the present invention, and the T cell-related disease of the subject of the disease is diagnosed, and based on the diagnosis, a suitable CAR contained in the induced regulatory T cells or the cell population is selected.

[0094] In other aspects of the present invention, a method is provided, comprising the step of administering to a subject an effective amount of an induced regulatory T cell containing a chimeric antigen receptor (CAR) or a cell population containing the same, wherein the subject is treated or prevented from a disease, injury or symptom that can be treated by the induced regulatory T cell. In one embodiment of the present invention, the induced regulatory T cell of the present invention may have one or more characteristics of the above-mentioned induced regulatory T cell.

[0095] The method of the present invention can treat any disease, injury or symptom as a target for treatment or prevention if it is a disease, injury or symptom that can be treated by inducible regulatory T cells. Any disease, injury or symptom that is generally effective even if there are few regulatory T cells can be used as a target, but it is not limited to this. It can be understood that even when the usual regulatory T cells do not show an effect, since the inducible regulatory T cells can have an effect, these are also included.

[0096] A pharmaceutical composition comprising inducible regulatory T cells containing the chimeric antigen receptor (CAR) of the present invention can be used as a pharmaceutical composition for treating or preventing chronic inflammatory diseases such as autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, inflammatory bowel disease, or chronic infections of a subject caused by viruses, bacteria, or parasites.

[0097] In one embodiment, the pharmaceutical composition of the present invention comprising an inducible regulatory T cell containing a chimeric antigen receptor (CAR) can be used for cell therapy. In cell therapy, the inducible regulatory T cells containing the chimeric antigen receptor (CAR) of the present invention can be used as a pharmaceutical composition, or as a preparation of a therapeutically effective cell group expressing the CAR of the present invention, and injected into an object in need thereof. The Treg cells injected into the object can suppress the inflammatory immune response of the object, or at least reduce the impact and / or symptoms of the damage in the treatment. The object can be the same object (autologous cell therapy) from which the cell is obtained, or it can be other objects of the same species (homologous cell therapy) from which the cell is derived.

[0098] In one embodiment, Treg cells or groups thereof containing the CAR of the present invention can be formulated for administration to an object using techniques known to those skilled in the art. In one embodiment, a preparation comprising a therapeutically effective Treg cell or group thereof containing a CAR of the present invention may contain a pharmaceutically acceptable excipient (carrier or diluent). The excipients contained in the preparation, for example, depend on the properties of the antigen binding domain of the CAR of the present invention, and have different purposes. For examples of commonly used excipients, the following physiological saline, buffered physiological saline, dextrose, water for injection, glycerol, ethanol, and combinations of these substances, stabilizers, solubilizers, surfactants, buffers, and preservatives, permeabilizers, bulking agents, and lubricants can be cited without limitation.

[0099] The preparation containing the therapeutically effective Treg cells or their groups containing CAR of the present invention can be administered to the subject using methods and techniques known to those skilled in the art. Illustrative examples include intravenous injection, but are not limited to this. For other methods, it is not limited to: intratumor, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular (joint), synovial cyst (synovial fluid area), intracranial, intraspinal and intramedullary (medullary fluid) and the like.

[0100] (General Technology) The molecular biology techniques, biochemical techniques, and microbiological techniques that can be used in the present specification are well known and commonly used in the relevant fields, 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, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Part 1: Experimental Medicine "Gene Introduction & Expression Analysis Experimental Methods", Yangtu Publishing House, 1997, etc., the relevant parts (may be all) of these in this specification are cited as references.

[0101] DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes include, for example, gene synthesis and fragment synthesis services provided by GeneArt, GenScript, Integrated DNA Technologies (IDT), etc. Others are described in, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. Let 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). Nucleic Acids in Chemistry and Biology, Oxford University Press. Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., the relevant parts of which are incorporated herein by reference.

[0102] In this specification, "or" is used when "at least one or more" of the items listed in the article can be used. The same applies to "or". When "within the range" of "two values" is clearly stated in this specification, the range also includes the two values ​​themselves. The entirety of the references such as scientific literature, patents, and patent applications cited in the present specification are incorporated herein by reference to the same extent as if each was specifically described.

[0103] Above, the preferred embodiments are shown to illustrate the present invention for easy understanding. Below, the present invention is described based on examples, but the above description and the following examples are provided only for the purpose of illustration, and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in this description, but is limited only by the claims. Example

[0104] In the following examples, glutamine-free RPMI1640 (containing 10% FCS (v / v), 60 μg / mL penicillin G, 100 μg / mL streptomycin, and 10 mM HEPES) was used as a basal medium. If necessary, 30 to 300 mg / L of L-glutamine was added to the medium.

[0105] Bisulfite sequencing Genomic DNA was obtained from the induced cells and treated with MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures) to investigate the demethylation of regulatory T cell-specific genes. Demethylation of each gene was analyzed using known methods 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 the analysis of the human Foxp3 CNS2 region, 5'-TTGGGTTAAGTTTGTTGTAGGATAG-3' (SEQ ID NO: 1) was used on the forward side, and 5'-ATCTAAACCCTATTATCACAACCCC-3' (SEQ ID NO: 2) was used on the reverse side.

[0107] Obtaining Mouse T Cell Fractions Foxp3-eGFP (eFox) reporter mice (Ito et al. (2014) Science 346, 363-368) were used to obtain lymph node cells of Foxp3-eGFP (eFox) reporter mice, and CD4 + GFP + Cell sorting was used to obtain the intrinsic regulatory T cell (nTreg) fraction. + GFP - CD44低 CD62L 高 Cell sorting to obtain the initial T cell component. Further, obtain CD4 + GFP - CD44 高 CD62L 低 Cell fractions for use as effector / memory T cells.

[0108] Obtaining human T cell fractions Human CD4 isolated from PBMC of patients with Crohn's disease + T cells were purified from concentrated 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 anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), and SenexinA (5 μM). After that, the cells were cultured for 2 days in a dormant state using a basal medium containing hIL-2 (100 U / ml). After 2 days, the culture medium was replaced in the same manner, and the cells were further cultured in a dormant state for 2 days. After 2 days, the cells were stimulated again for 2 days using a basal medium containing anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), SenexinA (5 μM), and ascorbic acid (10 μg / ml). Then, the cells were cultured in a basal medium containing hIL-2 (100 U / ml) for 2 days. After 2 days, the medium was replaced in the same manner and the cells were cultured in a basal medium for 2 days. After 2 days, the cells were analyzed by flow cytometry ( Figure 1 ) The expression of FoxP3 and CD25 in the obtained regulatory T cells was analyzed by the bisulfite method ( Figure 2 ) to analyze the demethylation status of Treg-specific demethylation regions by RNA-seq ( Figure 3 ) to analyze comprehensive gene expression patterns.

[0110] In conventional iTregs, functional molecules (at this time according to Figure 3For example, the expression of CTLA4, Entpd1, Nt5e, Itgae, etc.) is insufficient, and the FoxP3 induction efficiency is also low, but it can be confirmed that the highly functional inducible regulatory T cells obtained by the preparation method of the present application highly express FoxP3 and functional genes (at this time according to Figure 3 For example, CTLA4, Entpd1, Nt5e, Itgae, etc.), with Treg-specific demethylation state ( Figures 1 to 3 ).

[0111] (Example 2: In vitro suppressive activity of regulatory T cells) Regulatory T cells were obtained by the same experimental system as in Example 1. In the presence of anti-CD3 antibody (1 μg / ml) + antigen presenting cells (MHC-II positive cells: 1×10 4 ) or Dynabeads T-activator (VERITAS) (5 μL / well), Cell Trace Violet-labeled responder T cells (5×10 4 ), regulatory T cells (5×10 4 ) were mixed and cultured for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry. When the responding T cells caused an immune response, the Cell Trace Violet intensity showed multiple weaker peaks due to cell proliferation, but when the immune response was suppressed, the Cell Trace Violet intensity remained in a single strong peak state. It was confirmed that the regulatory T cells obtained by the preparation method of the present application strongly inhibited the immune response of the responding T cells. Therefore, it was confirmed that the obtained regulatory T cells had a high inhibitory function compared to the known regulatory T cell population ( Figure 4 ).

[0112] (Example 3: In vivo stability of regulatory T cells) Regulatory T cells were prepared from Thy1.2 / eFox reporter mice according to the method of Example 1, and Foxp3-positive cells (2×10 5 ) were administered to Thy1.1 / wild-type mice via the tail vein. Two weeks later, the transplanted Thy1.2 cells were recovered from the lymph nodes and the expression of Foxp3 was analyzed ( Figure 5 ). It was confirmed that regulatory T cells induced and stably cultured without CD28 stimulation stably expressed Foxp3 even after 2 weeks in vivo.

[0113] (Example 4: Inhibitory effect on colitis model) RAG knockout mice were given 2x10 5CD45RB-high-expressing CD4-positive naive T cells from wild-type mice were used to establish a colitis model. 2x10 5 The highly functional stable iTreg cells prepared by the method of Example 1 were used to measure the body weight changes over time ( Figure 6 A). Then, colon tissue and lymph nodes were collected 1 month after administration, and HE staining analysis of colon tissue was performed ( Figure 6 B) Analysis of CD69 expression in lymph node T cells ( Figure 6 C: Flow cytometry). The results confirmed that the administration of highly functional stable iTregs suppressed colitis. 5 The therapeutic effect of the dose can be confirmed at 1x10 7 The effectiveness of / kg.

[0114] Example 5: Production of regulatory T cells from human peripheral blood T cells CD4-positive T cells from Crohn's disease patients were stimulated for 3 days with a basal medium containing anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and SenexinA (5 μM). After that, the cells were cultured in a dormant state for 2 days with a basal medium containing hIL-2 (100 U / ml). After 2 days, the culture medium was replaced in the same manner, and the cells were further cultured in a dormant state for 2 days. After 2 days, the cells were stimulated again with a basal medium containing anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), SenexinA (5 μM), and ascorbic acid (10 μg / ml) for 2 days. After that, the cells were cultured in a basal medium containing hIL-2 (100 U / ml) for 2 days. After 2 days, the medium was replaced in the same manner and the cells were cultured in a quiescent state for another 2 days. After 2 days, the expression of FoxP3 and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry ( Figure 7 A).

[0115] In conventional iTregs, expression of functional molecules (here CTLA4) is insufficient and FoxP3 induction efficiency is also low, but it can be confirmed that the highly functional iTregs obtained by the production method of the present application are a group containing many cells expressing FoxP3 and CTLA4.

[0116] (Example 6: In vitro suppressive activity of regulatory T cells) Regulatory T cells were obtained by the same experimental system as in Example 1. Cell Trace Violet-labeled responder T cells (5×10 4 ), regulatory T cells (5×10 4 ) were mixed and cultured for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry. When the responding T cells caused an immune response, due to cell proliferation, the Cell Trace Violet intensity showed multiple weaker peaks, but when the immune response was suppressed, the Cell Trace Violet intensity maintained a single strong peak state. It was confirmed that the regulatory T cells obtained by the preparation method of the present application strongly inhibited the immune response of the responding T cells. Therefore, it was confirmed that the obtained regulatory T cells had a high inhibitory function compared to the known regulatory T cell population.

[0117] (Example 7: Production of regulatory T cells from CD8-positive human peripheral blood T cells) CD8-positive T cells were stimulated for 3 days with a basal medium containing anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and SenexinA (5 μM). After that, the cells were cultured in a dormant state for 2 days with a basal medium containing hIL-2 (100 U / ml). After 2 days, the culture medium was replaced in the same manner, and the cells were further cultured in a dormant state for 2 days. After 2 days, the cells were stimulated again with a basal medium containing anti-CD3 antibodies (100 μL of 10 μg / ml antibody was added to each well, and the cells were allowed to stand at room temperature for 60 minutes to become stationary in the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), SenexinA (5 μM), and ascorbic acid (10 μg / ml) for 2 days. After that, the cells were cultured in a basal medium containing hIL-2 (100 U / ml) for 2 days. After 2 days, the medium was replaced in the same way and the cells were cultured in a quiescent state for another 2 days. After 2 days, the expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained was analyzed by flow cytometry ( Figure 8 ).

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

[0119] (Example 7: Introduction of chimeric antigen receptor into inducible regulatory T cells) In this example, the process of producing highly functional inducible regulatory T cells (HSF-iTreg) from human CD4-positive T cells and the timing of infecting them with a lentivirus carrying a CAR-expressing viral vector during the process were studied. (Materials and methods) Detailed plan such as Fig. 9 (Top) shows the process of producing highly functional induced regulatory T cells (HSF-iTreg) from human CD4 positive T cells and the timing of infecting them with lentivirus carrying a CAR viral vector during the process. In this example, highly functional inducible regulatory T cells (HSF-iTreg) were prepared from human CD4 positive T cells. Fig. 9 As shown in the schematic diagram above, during the manufacturing process of HSF-iTreg, it is infected with a lentivirus carrying a CAR viral vector. Fig. 9 At the timing indicated by the arrow in the figure, 1 / 20 of the total volume of the culture medium was added with lentivirus (Lenti-CD19CAR (scFv-CD28, FMC63) Viral Particle, Cat. No.: VP-CAR-LC61), and the expression of FOXP3 and CAR was analyzed by FCM on the final day.

[0120] (result) The results are as follows Fig. 9 As shown below. Fig. 9 At the timing indicated by the arrow in (above), 1 / 20 of the total volume of the culture medium was added with lentivirus Lenti-CD19 CAR (scFv-CD28, FMC63) Viral Particle, Cat. No.: VP-CAR-LC61), and on the final day, FCM analysis of the expression of FOXP3 and CAR was performed. Fig.11 (Bottom) shows the expression of FOXP3 and CAR genes on Day 13. It was confirmed that CAR can be efficiently expressed by introducing genes during a single stimulation period. Fig. 9 The lower part shows the results of FCM analysis of CTLA4 expression for the Day 1 introduced sample. As shown, the expression of FOXP3 and CAR genes on Day 13 was shown, and it was confirmed that CTLA4 was also highly expressed in the HSF-iTreg cell group expressing CAR. By introducing the gene during a single stimulation period, CAR can be expressed efficiently.

[0121] (Example 8: Expression analysis of functional molecules in CAR Treg) In this example, FCM analysis was performed on the expression of CTLA4 in the Day 1 transfection sample of Example 7. The results confirmed that CTLA4 was also highly expressed in the HSF-iTreg cell population expressing CAR.

[0122] As an example, when the expression of CD103 molecules encoded by ITGAE is analyzed by flow cytometry, it can be confirmed that the inducible regulatory T cells obtained by the preparation method of the present application have high CD103 expression compared to other regulatory T cell populations. As a comprehensive investigation, it can be expected that the inducible regulatory T cells obtained by the preparation method of the present application will exert high inhibitory ability compared to known regulatory T cells.

[0123] In addition, the expression of Helios can be analyzed by flow cytometry. In the classical inducible regulatory T cells (conventional iTreg), the expression of functional molecules (such as CTLA4 here) is insufficient, and the FoxP3 induction efficiency is also low. On the other hand, CTLA4 and FoxP3 are expressed in intrinsic regulatory T cells (nTreg), and Helios, a representative nTreg marker molecule, is expressed. On the other hand, the highly functional inducible regulatory T cells (CAR Treg) expressing FoxP3 obtained by the preparation method of the present application are Helios negative and contain a group of cells expressing many FoxP3 and CTLA4.

[0124] (Example 9: Evaluation of Antigen Specificity of CAR Treg) CAR-expressing regulatory T cells produced using CD19-positive cells or hCD19 recombinant protein were stimulated, and the expressions of FOXP3 and CTLA4 in the stimulated CAR-expressing regulatory T cells were analyzed by flow cytometry.

[0125] (Example 10: Evaluation of the immunosuppressive function of CAR Treg) The immunosuppressive capacity against CD19-positive cells was evaluated in vitro. CD19-positive cells were co-cultured with CAR-expressing regulatory T cells, and the immunological properties of CD19-positive cells were evaluated by flow cytometry.

[0126] (Example 11: FCM analysis of highly functional inducible regulatory T cells (HSF-iTreg) from human CD4-positive T cells) In this example, FCM analysis of highly functional inducible regulatory T cells (HSF-iTreg) from human CD4-positive T cells was performed.

[0127] Specifically, Fig.11As shown, highly functional induced regulatory T cells (HSF-iTreg) were prepared from human CD4-positive T cells, wherein lentivirus (Lenti-HLA-A2 CAR (scFv-28ζ, BB7.2) -VP (VP-CAR-LC809) or Lenti-EpCAM CAR (scFv-28ζ, M13-57) -VP (VP-CAR-LC847)) was added at 1 / 50 or 1 / 25 of the total culture medium at the timing described in the above examples, and the expression of CAR (GFP), FOXP3 and CTLA4 was analyzed by FCM on the final day.

[0128] (result) like Fig.11 As shown, it was shown that by introducing the gene during a single stimulation period, any type of CAR can be efficiently expressed without impairing the properties and induction efficiency of Treg.

[0129] (Notes) As described above, it is understood that although the preferred embodiments of the present invention are used to illustrate the present invention, the scope of the present invention should be interpreted only according to the claims. For the patents, patent applications and other documents cited in this specification, it is understood that their contents themselves should be cited as references to the present specification in the same way as the contents specifically recorded in this specification. This application claims priority to Special Application No. 2022-152882 filed in Japan on September 26, 2022, which is cited as a reference in this application as a whole. Industrial Applicability

[0130] The cell population of the present invention can be used for the treatment and prevention of various immune diseases, autoimmune diseases and other inflammatory diseases. In addition, by the method for preparing the cell population of the present invention, highly functional regulatory T cells can be stably induced from peripheral blood T cells, and it can be expected to be applied in the medical field. Sequence Listing Free Text

[0131] Sequence number 1: Forward primer used in the examples Sequence number 2: Reverse primer used in the examples.

Claims

1. An inducible regulatory T cell comprising a chimeric antigen receptor CAR.

2. The inducible regulatory T cells according to claim 1, wherein the CAR is expressed in the inducible regulatory T cells. 3 . The inducible regulatory T cell according to claim 1 , which has at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity and AREG positivity. The inducible regulatory T cell according to any one of claims 1 to 3, which has at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive and AREG-positive. The inducible regulatory T cell according to any one of claims 1 to 4, which is at least CTLA4 positive. The inducible regulatory T cell according to any one of claims 1 to 5, wherein the CNS2 site of the FOXP3 gene is demethylated. 7 . The inducible regulatory T cell according to claim 1 , which is CD4-positive or CD8-positive. 8 . The inducible regulatory T cells according to claim 1 , which are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

9. The inducible regulatory T cell according to any one of claims 1 to 8, which is obtained by a method comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). 10 . A cell population comprising T cells, wherein about 50% or more of the T cells in the cell population are the inducible regulatory T cells according to any one of claims 1 to 9. The cell population according to claim 10, wherein about 80% or more of the T cells in the cell population are the inducible regulatory T cells according to any one of claims 1 to 9. The cell population according to claim 10 or 11, wherein the T cells in the cell population are regulatory T cells. 13 . The cell population according to claim 10 , wherein about 90% or more of the cell population are T cells.

14. A pharmaceutical composition comprising inducible regulatory T cells containing a chimeric antigen receptor CAR.

15. The pharmaceutical composition of claim 14, wherein the CAR is expressed in the inducible regulatory T cells. 16 . The pharmaceutical composition according to claim 14 or 15 , wherein the inducible regulatory T cells have at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. 17 . The pharmaceutical composition according to any one of claims 14 to 16 , wherein the inducible regulatory T cells have at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. 18 . The pharmaceutical composition according to claim 14 , wherein the inducible regulatory T cells are at least CTLA4 positive. 19 . The pharmaceutical composition according to claim 14 , wherein the CNS2 site of the FOXP3 gene of the inducible regulatory T cells is demethylated. 20 . The pharmaceutical composition according to claim 14 , wherein the induced regulatory T cells are CD4-positive or CD8-positive. 21 . The pharmaceutical composition according to any one of claims 14 to 20, wherein the inducible regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

22. The pharmaceutical composition according to any one of claims 14 to 21, wherein the inducible regulatory T cells are obtained by a method comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d). 23 . The pharmaceutical composition according to any one of claims 14 to 22, 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. 24 . The pharmaceutical composition according to any one of claims 14 to 23 , 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.

25. The pharmaceutical composition according to claim 23 or 24, wherein the T cell population is a regulatory T cell population. 26 . The pharmaceutical composition according to claim 23 , wherein about 90% or more of the cell population are T cells.

27. The pharmaceutical composition according to any one of claims 14 to 26, wherein each administration is about 10 8 ~ About 10 9 or about 10 7 The induced regulatory T cells are contained at a concentration of 100 cells / kg.

28. The pharmaceutical composition according to any one of claims 14 to 27, which is used for treating or preventing T cell-related diseases.

29. The pharmaceutical composition according to claim 28, wherein the T cell-related diseases include autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, inflammatory diseases, infections, cancer and amyotrophic lateral sclerosis.

30. The pharmaceutical composition of claim 29, wherein the autoimmune disease comprises systemic lupus erythematosus, Crohn's disease, type I diabetes, 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, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and cardiomyopathy.

31. The pharmaceutical composition according to any one of claims 14 to 30, which is administered by injection.

32. The pharmaceutical composition according to any one of claims 14 to 31, wherein when the pharmaceutical composition is administered to a patient, the pharmaceutical composition is additionally administered to a patient for whom the effect is ineffective or insufficient.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is administered additionally at least about 2 weeks after the initial administration.

34. A therapeutic drug, which is a therapeutic drug for a T cell-related disease comprising the induced regulatory T cells described in any one of claims 1 to 9 or the cell population described in any one of claims 10 to 13, wherein the T cell-related disease of the disease subject is diagnosed, and based on the diagnosis, a suitable CAR contained in the induced regulatory T cells or the cell population is selected.

35. A method for producing an inducible regulatory T cell containing a chimeric antigen receptor CAR, comprising the following steps: (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) a step of culturing the cells obtained in step (a) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, (d) a step of culturing the cells obtained in step (c) in a dormant state for at least about 1 to about 3 days using a medium containing IL-2, (e) A step of introducing the CAR gene at least once in any one of steps (a) to (d).

36. The method according to claim 35, wherein the introduction of the CAR gene is performed at least about twice in total in any one of steps (a) to (d).

37. The method according to claim 35 or 36, wherein the introduction of the CAR gene is performed at least once in step (a).

38. The method according to any one of claims 35 to 37, wherein the first basal medium contains at least one factor selected from the group consisting of anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors and ascorbic acid.

39. The method according to any one of claims 35 to 38, wherein the first basal culture medium contains anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor and ascorbic acid.

40. The method according to any one of claims 35 to 39, wherein the second basal medium contains at least one factor selected from the group consisting of anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor. The method according to any one of claims 35 to 40, wherein the second basal medium contains anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor.

42. The method according to any one of claims 35 to 41, wherein step (a) comprises stimulating the CD4-positive T cells or CD8-positive T cells for about 3 days using the first basal medium.

43. The method according to any one of claims 35 to 42, wherein the step (b) comprises culturing the cells obtained in the step (a) in a dormant state for at least about 2 days using a medium containing the IL-2.

44. The method according to any one of claims 35 to 43, wherein the step (c) comprises stimulating the cells obtained in the step (b) with a second basal medium for about 3 days.

45. The method according to any one of claims 35 to 44, wherein the step (d) comprises culturing the cells obtained in the step (c) in a dormant state for at least about 2 days using a medium containing the IL-2. An induced regulatory T cell or a cell population comprising the induced regulatory T cell, produced by the method according to any one of claims 35 to 45.

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