WO2024204553A1 - ヒト誘導性制御性ｔ細胞およびその作製方法、およびｔ細胞関連疾患を治療または予防するための医薬組成物 - Google Patents

ヒト誘導性制御性ｔ細胞およびその作製方法、およびｔ細胞関連疾患を治療または予防するための医薬組成物 Download PDF

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Publication number
WO2024204553A1
WO2024204553A1 PCT/JP2024/012680 JP2024012680W WO2024204553A1 WO 2024204553 A1 WO2024204553 A1 WO 2024204553A1 JP 2024012680 W JP2024012680 W JP 2024012680W WO 2024204553 A1 WO2024204553 A1 WO 2024204553A1
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Prior art keywords
cells
positive
cell population
cell
regulatory
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Ceased
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PCT/JP2024/012680
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English (en)
French (fr)
Japanese (ja)
Inventor
統久 三上
志文 坂口
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Regcell Co Ltd
University of Osaka NUC
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Regcell Co Ltd
Osaka University NUC
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Priority to EP24780653.2A priority Critical patent/EP4692327A1/en
Priority to CN202480023635.5A priority patent/CN121057812A/zh
Priority to JP2025511160A priority patent/JPWO2024204553A1/ja
Priority to KR1020257035941A priority patent/KR20250166296A/ko
Publication of WO2024204553A1 publication Critical patent/WO2024204553A1/ja
Priority to MX2025011478A priority patent/MX2025011478A/es
Anticipated expiration legal-status Critical
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Definitions

  • the present disclosure relates to highly functional human inducible regulatory T cells and a method for producing the same, as well as a pharmaceutical composition containing the cells for treating or preventing T cell-related diseases.
  • CD25+CD4+ regulatory T cells present in the immune system are that they specifically express the transcription factor FoxP3, and FoxP3 deficiency or mutation can impair the development and differentiation of regulatory T cells, as well as their suppressive function.
  • Regulatory T cells suppress the immune system by expressing a variety of genes, including FoxP3, CTLA4, and IL-10. It is believed that epigenetic conditions such as DNA demethylation contribute to the comprehensive gene expression control of regulatory T cells, including the stable expression of FoxP3, and these are correlated with the functional phenotype of regulatory T cells.
  • the present inventors have found for the first time that when inducing regulatory T cells from human peripheral T cells, they can induce stable induced T cells from human peripheral T cells by stimulating them with anti-CD3 antibodies, subjecting the cells to resting culture, stimulating them again with anti-CD3 antibodies, culturing them, and then subjecting them to resting culture again, thereby inducing regulatory T cells with high expression of inhibitory molecules and high inhibitory function.
  • the present inventors have also found that the induced regulatory T cells obtained in this way have sufficient immunosuppressive ability to function as a medicine.
  • the present disclosure provides: (Item X1) An inducible regulatory human T cell having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item X1G5) Inducible human regulatory T cells with all the following characteristics: CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item X2) The inducible human regulatory T cell according to any one of the preceding items, which is at least CTLA4 positive and FoxP3 positive.
  • (Item X2a) The inducible human regulatory T cell according to any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item X2b) The inducible regulatory human T cell according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item X2c) The inducible human regulatory T cell according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item X2d) The inducible regulatory human T cell according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item X3) The inducible regulatory human T cell according to any one of the preceding items, which is at least CD172g positive and/or CD26 positive.
  • (Item X4) The inducible human regulatory T cell according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene is demethylated.
  • the inducible human regulatory T cell according to any one of the preceding items which is CD4 positive or CD8 positive.
  • (Item X7) (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) subjecting the cells obtained in step (c) to resting culture in a medium containing IL-2 for at least about 1 to about 3 days.
  • (Item X8) A cell population comprising the induced regulatory human T cells according to any one of the preceding items, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item X8A) A cell population comprising an inducible human regulatory T cell, the cell population having an NT5E positive rate of about 50% or more.
  • (Item X8B) A cell population comprising induced human regulatory T cells, the cell population having an ITGAE (CD103) positive rate of about 50% or more.
  • (Item X8C) A cell population comprising induced regulatory human T cells, the cell population having an AREG positive rate of about 50% or more.
  • (Item X8D) A cell population comprising induced regulatory human T cells, the cell population having a CD172g positive rate of about 50% or more.
  • (Item X8E) A cell population comprising an inducible human regulatory T cell, the cell population having a CD26 positive rate of about 50% or more.
  • (Item X8F) A cell population comprising an induced human regulatory T cell, the cell population having a CTLA4 positive rate of about 50% or more.
  • (Item X8G) A cell population comprising an inducible human regulatory T cell, the cell population having a stable FoxP3 positive rate of about 50% or more.
  • (Item X8G1) A cell population comprising induced regulatory human T cells, the cell population having a percentage of at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item X8G2) A cell population comprising induced regulatory human T cells, the cell population having a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item X8G3) A cell population comprising induced regulatory human T cells, the cell population having a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item X8G4) A cell population comprising induced regulatory human T cells, the cell population having a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item X8G5) A cell population comprising induced regulatory human T cells, the cell population having a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • ITGAE CD103
  • AREG positivity AREG positivity
  • CD172g positivity CD26 positivity of about 50% or more.
  • (Item X9a) The cell population according to any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item X9b) The cell population according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item X9c) The cell population according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item X9d) The cell population according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item X10) The cell population according to any one of the preceding items, wherein at least the CD172g positive and/or CD26 positive proportions are each about 50% or more.
  • (Item X11) The cell population according to any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 60% or more.
  • (Item X12) The cell population according to any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 80% or more.
  • (Item X12a) The cell population according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells is about 50% or more.
  • (Item X12b) The cell population according to any one of the preceding items, wherein the proportion of cells stably strongly positive for FoxP3 is about 50% or more.
  • (Item X13) The cell population of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • (Item X14) A pharmaceutical composition comprising an inducible regulatory human T cell according to any one of the preceding items or a cell population according to any one of the preceding items.
  • (Item X15) A regenerative medical material or product comprising the inducible regulatory human T cell described in any one of the above items or the cell population described in any one of the above items. (Item X16) 1.
  • a method for producing inducible human regulatory T cells comprising: (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days as a dormant culture.
  • the first basal medium comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the second basal medium comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, ascorbic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the second basal medium comprises an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, ascorbic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • TGF- ⁇ contained in the second basal medium includes TGF- ⁇ 1 and/or TGF- ⁇ 3.
  • TGF- ⁇ contained in the first basal medium includes TGF- ⁇ 1.
  • TGF- ⁇ contained in the second basal medium includes TGF- ⁇ 1.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, and Selexin A.
  • the second basal medium contains an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, Selexin A, and ascorbic acid.
  • the first basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8/19 inhibitor, and ascorbic acid.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8/19 inhibitor, and ascorbic acid.
  • the second basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor.
  • step (a) The method according to any one of the preceding items, wherein the second basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor.
  • step (a) The method according to any one of the preceding items, wherein step (a) stimulates the CD4-positive T cells or CD8-positive T cells with the first basal medium for about 3 days.
  • step (b) comprises dormantly culturing the cells obtained in the step (a) in a medium containing the IL-2 for at least about 2 days.
  • step (c) stimulates the cells obtained in step (b) with the second basal medium for about 3 days.
  • step (d) comprises dormantly culturing the cells obtained in the step (c) in a medium containing the IL-2 for at least about 2 days.
  • step (d) An induced regulatory human T cell or a cell population containing the induced regulatory human T cell, produced by the method according to any one of the preceding items.
  • the present disclosure also provides: (Item 1) An induced regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. (Item 2) The induced regulatory T cells according to the above item, having at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. (Item 3) The induced regulatory T cell according to any one of the preceding items, which is at least CTLA4 positive. (Item 4) The induced regulatory T cell of any one of the preceding items, wherein the CNS2 site of the FOXP3 gene is demethylated.
  • the inducible regulatory T cell according to any one of the preceding items which is CD4 positive or CD8 positive.
  • the inducible regulatory T cells of any one of the preceding items which are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.
  • (Item 7) (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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) rest-culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.
  • 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 preceding items.
  • (Item 9) The cell population according to any one of the preceding items, wherein about 80% or more of T cells in the cell population are the induced regulatory T cells according to any one of the preceding items.
  • (Item 10) The cell population according to any one of the preceding items, wherein the T cells in the cell population are regulatory T cells.
  • (Item 11) The cell population of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • a pharmaceutical composition 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.
  • (Item 13) A regenerative medical material or product comprising the induced regulatory T cell according to any one of the above items or the cell population according to any one of the above items.
  • (Item A1) A method for producing an inducible regulatory T cell, 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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days as a dormant culture.
  • the first basal medium comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the second basal medium comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, ascorbic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • the second basal medium comprises an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, ascorbic acid, and a CDK8 inhibitor, a CDK19 inhibitor, or a CDK8/19 inhibitor.
  • TGF- ⁇ contained in the second basal medium includes TGF- ⁇ 1 and/or TGF- ⁇ 3.
  • TGF- ⁇ contained in the first basal medium includes TGF- ⁇ 1.
  • TGF- ⁇ contained in the second basal medium includes TGF- ⁇ 1.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, and Selexin A.
  • the second basal medium contains an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, Selexin A, and ascorbic acid.
  • the first basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8/19 inhibitor, and ascorbic acid.
  • the first basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8/19 inhibitor, and ascorbic acid.
  • the second basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor.
  • step (Item A5) The method according to any one of the preceding items, wherein the second basal medium comprises an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor.
  • step (a) The method according to any one of the preceding items, wherein step (a) stimulates the CD4-positive T cells or CD8-positive T cells with the first basal medium for about 3 days.
  • step (b) comprises dormantly culturing the cells obtained in the step (a) in a medium containing the IL-2 for at least about 2 days.
  • step (c) stimulates the cells obtained in step (b) with the second basal medium for about 3 days.
  • step (d) comprises dormantly culturing the cells obtained in the step (c) in a medium containing the IL-2 for at least about 2 days.
  • step (d) An induced regulatory T cell or a cell population containing the induced regulatory T cell, produced by the method according to any one of the preceding items.
  • the present disclosure further provides: (Item CX1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising, as an active ingredient, inducible regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • (Item CX1B) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising ITGAE (CD103)-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1C) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising AREG-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1D) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CD172g-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1E) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CD26-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1F) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising CTLA4-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1G) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising stably FoxP3-positive inducible human regulatory T cells as an active ingredient.
  • (Item CX1G1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising, as an active ingredient, inducible regulatory human T cells having at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising, as an active ingredient, inducible regulatory human T cells having at least three characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising, as an active ingredient, inducible regulatory human T cells having at least four characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising, as an active ingredient, inducible regulatory human T cells having at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity.
  • inducible human regulatory T cells having all of the following characteristics: CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • (Item CX2) The pharmaceutical composition according to any one of the preceding items, wherein the induced regulatory human T cells are at least CTLA4 positive and FoxP3 positive.
  • (Item CX2a) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item CX2b) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item CX2c) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item CX2d) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item CX3) The pharmaceutical composition according to any one of the preceding items, wherein the induced regulatory human T cells are at least CD172g positive and/or CD26 positive.
  • (Item CX4) The pharmaceutical composition according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the inducible regulatory human T cells is demethylated.
  • (Item CX5) The pharmaceutical composition according to any one of the preceding items, wherein the inducible regulatory human T cells are CD4 positive or CD8 positive.
  • the induced regulatory human T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) subjecting the cells obtained in step (c) to dormant culture in a medium containing IL-2 for at least about 1 to about 3 days.
  • the pharmaceutical composition according to any one of the preceding items comprising a cell population containing the inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having an NT5E positive rate of about 50% or more.
  • (Item CX8B) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having an ITGAE (CD103) positive rate of about 50% or more.
  • (Item CX8C) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having an AREG positive rate of about 50% or more.
  • (Item CX8D) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having a CD172g positive rate of about 50% or more.
  • (Item CX8E) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having a CD26 positive rate of about 50% or more.
  • (Item CX8F) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having a CTLA4 positive rate of about 50% or more.
  • (Item CX8G) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising a cell population containing inducible human regulatory T cells as an active ingredient, the cell population having a stable FoxP3 positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising a cell population containing inducible human regulatory T cells as an active ingredient, wherein the cell population has a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • AREG positivity CD172g positivity
  • CD26 positivity CD26 positivity of about 50% or more.
  • Item CX9a The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item CX9b) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item CX9c) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item CX9d) The pharmaceutical composition according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item CX10) The pharmaceutical composition according to any one of the preceding items, wherein the proportion of at least the CD172g positive and/or CD26 positive cells in the cell population is about 50% or more, respectively.
  • (Item CX11) The pharmaceutical composition of any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 60% or more.
  • (Item CX12) The pharmaceutical composition of any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 80% or more.
  • (Item CX12a) The pharmaceutical composition according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells in the cell population is about 50% or more.
  • (Item CX12b) The pharmaceutical composition according to any one of the preceding items, wherein the proportion of stably strongly FoxP3 positive cells in the cell population is about 50% or more.
  • T cell-related disease comprises an autoimmune disease, an infectious disease, a cancer, an allergy, an inflammatory disease, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic
  • the present disclosure also provides: (Item C1) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising, as an active ingredient, an inducible regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity.
  • the induced regulatory T cells are (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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;
  • the pharmaceutical composition according to any one of the preceding items comprising a T cell population in which about 50% or more of the cells are the inducible regulatory T cells.
  • the pharmaceutical composition according to any one of the preceding items comprising a T cell population in which about 80% or more of the cells are the inducible regulatory T cells.
  • the pharmaceutical composition according to any one of the preceding claims wherein the T cell population is a regulatory T cell population.
  • the pharmaceutical composition of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • T cell-related disease comprises an autoimmune disease, an infectious disease, a cancer, an allergy, an inflammatory disease, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardio
  • the present disclosure further provides: (Item AX14) The inducible regulatory human T cell according to any one of the preceding items or the cell population according to any one of the preceding items for use as a medicament. (Item AX15) The inducible regulatory human T cell according to any one of the preceding items or the cell population according to any one of the preceding items for use as a regenerative medical material or product. (Item ACX1) Inducible regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive for use in treating or preventing a T cell-related disease.
  • Item ACX1D Inducible regulatory human T cells that are CD172g positive for use in treating or preventing T cell-related diseases.
  • (Item ACX1F) Inducible human regulatory T cells that are CTLA4 positive for use in the treatment or prevention of T cell-related diseases.
  • (Item ACX1G) A stable FoxP3 positive inducible regulatory human T cell for use in the treatment or prevention of T cell related diseases.
  • (Item ACX1G1) An inducible regulatory human T cell having at least two characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive, for use in treating or preventing a T cell-related disease.
  • the present invention relates to an inducible regulatory human T cell having at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity, for use in treating or preventing a T cell-related disease.
  • An inducible regulatory human T cell having at least four characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive for use in treating or preventing a T cell-related disease.
  • (Item ACX1G4) An inducible regulatory human T cell having at least five characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive for use in treating or preventing a T cell-related disease.
  • (Item ACX1G5) Inducible human regulatory T cells having the characteristics of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive for use in the treatment or prevention of T cell-related diseases.
  • (Item ACX2) The inducible regulatory human T cells for use according to any one of the preceding items, wherein the inducible regulatory human T cells are at least CTLA4 positive and FoxP3 positive.
  • (Item ACX2a) The inducible regulatory human T cell for use according to any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item ACX2b) The inducible regulatory human T cell for use according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item ACX2c) The inducible regulatory human T cell for use according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item ACX2d) The inducible regulatory human T cell for use according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item ACX3) The inducible regulatory human T cells for use according to any one of the preceding items, wherein the inducible regulatory human T cells are at least CD172g positive and/or CD26 positive.
  • (Item ACX4) The inducible regulatory human T cell for use according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the inducible regulatory human T cell is demethylated.
  • the induced regulatory human T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) a step of rest-culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.
  • Item ACX8A A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, said cell population having an NT5E positive rate of about 50% or more.
  • (Item ACX8B) A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, the cell population having an ITGAE (CD103) positive rate of about 50% or more.
  • (Item ACX8C) A cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having an AREG positive rate of about 50% or more.
  • (Item ACX8D) A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, the cell population having a CD172g positive rate of about 50% or more.
  • (Item ACX8E) A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, the cell population having a CD26 positive rate of about 50% or more.
  • (Item ACX8F) A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, the cell population having a CTLA4 positive rate of about 50% or more.
  • (Item ACX8G) A cell population comprising inducible human regulatory T cells for use in treating or preventing a T cell-related disease, said cell population having a stable FoxP3 positive rate of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having a percentage of at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item ACX8G5) A cell population comprising inducible regulatory human T cells for use in treating or preventing a T cell-related disease, said cell population having a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity CTLA4 positivity
  • NT5E positivity NT5E positivity
  • AREG positivity AREG positivity
  • CD172g positivity CD26 positivity of about 50% or more.
  • (Item ACX9) The cell population for use according to any one of the preceding items, wherein the proportion of CTLA4 positive and FoxP3 positive in the cell population is about 50% or more, respectively.
  • (Item ACX9a) The cell population for use according to any one of the preceding claims, in which FoxP3 is stably expressed.
  • (Item ACX9b) The cell population for use according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item ACX9c) The cell population for use according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item ACX9d) The cell population for use according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item ACX10) A cell population for use according to any one of the preceding items, wherein at least the proportion of CD172g positive and/or CD26 positive cells in the cell population is about 50% or more, respectively.
  • (Item ACX11) A cell population for use according to any one of the preceding claims, wherein the proportion of said at least one characteristic in said cell population is about 60% or more.
  • (Item ACX12) A cell population for use according to any one of the preceding claims, wherein the proportion of said at least one characteristic in said cell population is about 80% or more.
  • (Item ACX12a) The cell population for use according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells in the cell population is about 50% or more.
  • (Item ACX12b) The cell population for use according to any one of the preceding items, wherein the proportion of stably strongly FoxP3 positive cells in the cell population is about 50% or more.
  • T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic
  • (Item ACX17) The cell population for use according to any one of the preceding items, wherein the cell population is administered by injection.
  • (Item ACX18) The cell population for use according to any one of the preceding items, wherein the pharmaceutical composition is additionally administered to a patient for whom administration of the cell population to the patient is ineffective or insufficient.
  • (Item ACX19) The cell population for use according to any one of the preceding items, wherein the cell population is administered additionally at least about two weeks after the initial administration.
  • the present disclosure also provides: (Item AC1) An inducible regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, and AREG positive, for use in treating or preventing a T cell-related disease.
  • (Item AC2) The induced regulatory T cells for use according to the above item, wherein the induced regulatory T cells have at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity.
  • (Item AC3) The induced regulatory T cells for use according to any one of the preceding items, wherein the induced regulatory T cells are at least CTLA4 positive.
  • the induced regulatory T cells are (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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;
  • (Item AC8) The cell population for use according to any one of the preceding items, comprising a T cell population in which about 50% or more of the cells are said induced regulatory T cells.
  • (Item AC9) The cell population for use according to any one of the preceding items, comprising a T cell population in which about 80% or more of the cells are said induced regulatory T cells.
  • (Item AC10) The cell population for use according to any one of the preceding items, wherein the T cell population is a regulatory T cell population.
  • (Item AC11) The cell population for use according to any one of the preceding items, wherein about 90% or more of the cell population are T cells.
  • T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic cardio
  • (Item AC15) The induced regulatory T cell or cell population for use according to any one of the preceding items, wherein the induced regulatory T cell or cell population is administered by injection.
  • (Item AC16) The induced regulatory T cells or cell population for use according to any one of the preceding items, wherein the pharmaceutical composition is additionally administered to a patient for whom administration of the induced regulatory T cells or cell population to the patient is ineffective or insufficient.
  • (Item AC17) The induced regulatory T cell or cell population for use according to any one of the preceding items, wherein the induced regulatory T cell or cell population is administered additionally at least about two weeks after the initial administration.
  • the present disclosure also provides the following.
  • (Item BX14) A method for treatment or prevention, comprising administering to a subject an effective amount of the inducible regulatory human T cell described in any one of the preceding items or the cell population described in any one of the preceding items as a pharmaceutical.
  • (Item BX15) A method for treatment or prevention comprising administering to a subject an effective amount of the inducible regulatory human T cell described in any one of the preceding items or the cell population described in any one of the preceding items as a regenerative medical material or product.
  • (Item BCX1) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX1A) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible human regulatory T cells that are NT5E positive.
  • (Item BCX1B) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells that are ITGAE (CD103) positive.
  • (Item BCX1C) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells that are AREG positive.
  • (Item BCX1D) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells that are CD172g positive.
  • (Item BCX1E) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible human regulatory T cells that are CD26 positive.
  • (Item BCX1F) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible human regulatory T cells that are CTLA4 positive.
  • (Item BCX1G) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of stably FoxP3-positive inducible regulatory human T cells.
  • (Item BCX1G1) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having at least two characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX1G2) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having at least three characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX1G3) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having at least four characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX1G4) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having at least five characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX1G5) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of inducible regulatory human T cells having all of the following characteristics: CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • (Item BCX2) The method according to any one of the preceding items, wherein the induced regulatory human T cells are at least CTLA4 positive and FoxP3 positive.
  • (Item BCX2a) Item 11. The method of any one of the preceding items, wherein the FoxP3 is stably expressed.
  • (Item BCX2b) The method according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item BCX2c) The method according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item BCX2d) The method according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing the cells.
  • (Item BCX3) The method according to any one of the preceding items, wherein the induced regulatory human T cells are at least CD172g positive and/or CD26 positive.
  • (Item BCX4) The method according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory human T cells is demethylated.
  • (Item BCX5) The method according to any one of the preceding items, wherein the induced regulatory human T cells are CD4 positive or CD8 positive.
  • (Item BCX6) The method of any one of the preceding claims, wherein the inducible human regulatory T cells are obtained or derived from human peripheral blood T cells or human tissue-derived T cells.
  • the induced regulatory human T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) subjecting the cells obtained in step (c) to dormant culture in a medium containing IL-2 for at least about 1 to about 3 days.
  • (Item BCX8) The method according to any one of the preceding items, comprising a step of administering to the subject an effective amount of a cell population containing the induced regulatory human T cells, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item BCX8A) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has an NT5E positive rate of about 50% or more.
  • (Item BCX8B) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has an ITGAE (CD103) positive rate of about 50% or more.
  • (Item BCX8C) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has an AREG positive rate of about 50% or more.
  • (Item BCX8D) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a CD172g positive rate of about 50% or more.
  • (Item BCX8E) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible human regulatory T cells, wherein the cell population has a CD26 positive rate of about 50% or more.
  • (Item BCX8F) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a CTLA4 positive rate of about 50% or more.
  • (Item BCX8G) 1. A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible human regulatory T cells, wherein the cell population has a stable FoxP3 positive rate of about 50% or more.
  • (Item BCX8G1) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of at least one of the following characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a method for treating or preventing a T cell-related disease comprising administering to a subject an effective amount of a cell population comprising inducible human regulatory T cells, wherein the cell population has a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a method for treating or preventing a T cell-related disease comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • T cell-related disease comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item BCX8G5) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of a cell population comprising inducible regulatory human T cells, wherein the cell population has a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity CTLA4 positivity
  • NT5E positivity NT5E positivity
  • ITGAE (CD103) positivity AREG positivity
  • CD172g positivity CD26 positivity
  • (Item BCX9a) The method of any one of the preceding claims, wherein the FoxP3 is stably expressed in the cell population.
  • (Item BCX9b) The method according to any one of the preceding items, wherein the FoxP3 is continuously expressed in the cell population for at least about 48 hours or more.
  • (Item BCX9c) The method according to any one of the preceding items, wherein the FoxP3 is stably expressed in the cell population at least from day 7 of culture onwards.
  • (Item BCX9d) The method according to any one of the preceding items, wherein the FoxP3 is expressed in the cell population even after freezing and thawing the cells.
  • (Item BCX10) The method according to any one of the preceding items, wherein the proportion of at least the CD172g positive and/or CD26 positive cells in the cell population is about 50% or more, respectively.
  • (Item BCX11) The method according to any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 60% or more.
  • (Item BCX12) The method according to any one of the preceding claims, wherein the proportion of the at least one characteristic in the cell population is about 80% or more.
  • (Item BCX12a) The method according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells in the cell population is about 50% or more.
  • (Item BCX12b) The method according to any one of the preceding items, wherein the proportion of stably strongly FoxP3 positive cells in the cell population is about 50% or more.
  • (Item BCX13) The method of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • (Item BCX14) The method of any one of the preceding items, wherein the induced regulatory human T cells are administered at about 10 8 to about 10 9 cells per administration, or at about 10 7 cells/kg.
  • the T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppression.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic
  • the present disclosure also provides: (Item BC1) A method for treating or preventing a T cell-related disease, comprising administering to a subject an effective amount of induced regulatory T cells having at least one characteristic selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, and AREG positive.
  • (Item BC2) The method according to the above-mentioned item, wherein the induced regulatory T cells have at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity.
  • (Item BC3) The method according to any one of the preceding items, wherein the induced regulatory T cells are at least CTLA4 positive.
  • (Item BC4) The method according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory T cells is demethylated.
  • (Item BC5) The method according to any one of the preceding items, wherein the induced regulatory T cells are CD4 positive or CD8 positive.
  • (Item BC6) The method of any one of the preceding claims, wherein the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.
  • the induced regulatory T cells are (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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;
  • (Item BC8) The method according to any one of the preceding items, comprising a T cell population in which about 50% or more of the cells are said induced regulatory T cells.
  • (Item BC9) The method according to any one of the preceding items, comprising a T cell population in which about 80% or more of the cells are said induced regulatory T cells.
  • (Item BC10) The method of any one of the preceding claims, wherein the T cell population is a regulatory T cell population.
  • (Item BC11) The method of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • (Item BC12) The method of any one of the preceding items, comprising administering the induced regulatory T cells at about 10 8 to about 10 9 cells per administration, or at about 10 7 cells/kg.
  • T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppression.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis,
  • the present disclosure also provides the following.
  • (Item CX14) Use of the inducible regulatory human T cell described in any one of the preceding items or the cell population described in any one of the preceding items for the manufacture of a medicament.
  • (Item CX15) Use of the inducible regulatory human T cell described in any one of the above items or the cell population described in any one of the above items for the production of a regenerative medical material or product.
  • (Item CCX1) Use of inducible regulatory human T cells having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive, for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1A) Use of NT5E-positive inducible human regulatory T cells for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1B) Use of ITGAE (CD103) positive inducible human regulatory T cells for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1C) Use of AREG-positive inducible human regulatory T cells for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1D) Use of inducible human regulatory T cells that are CD172g positive for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1E) Use of CD26-positive inducible human regulatory T cells for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1F) Use of CTLA4-positive inducible human regulatory T cells for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1G) Use of a stable FoxP3 positive inducible human regulatory T cell for the manufacture of a medicament for treating or preventing a T cell related disease.
  • (Item CCX1G1) Use of inducible regulatory human T cells having at least two characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive, for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1G2) Use of inducible regulatory human T cells having at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1G3) Use of inducible regulatory human T cells having at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1G4) Use of inducible regulatory human T cells having at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CCX1G5) Use of inducible human regulatory T cells having all of the following characteristics: CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive, for the manufacture of a medicine for treating or preventing a T cell-related disease.
  • (Item CCX2) The use according to any one of the preceding items, wherein the induced regulatory human T cells are at least CTLA4 positive and FoxP3 positive.
  • (Item CCX2a) 2. The use according to any one of the preceding claims, wherein the FoxP3 is stably expressed.
  • (Item CCX2b) The use according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item CCX2c) The use according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item CCX2d) The use according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item CCX3) The use according to any one of the preceding items, wherein the induced regulatory human T cells are at least CD172g positive and/or CD26 positive.
  • (Item CCX4) The use according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory human T cells is demethylated.
  • (Item CCX5) The use according to any one of the preceding items, wherein the induced regulatory human T cells are CD4 positive or CD8 positive.
  • the inducible human regulatory T cells are obtained or derived from human peripheral blood T cells or human tissue-derived T cells.
  • the induced regulatory human T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; (d) subjecting the cells obtained in step (c) to dormant culture in a medium containing IL-2 for at least about 1 to about 3 days.
  • (Item CCX8) The use according to any one of the preceding items, for the manufacture of a medicament for treating or preventing a T cell-related disease of a cell population comprising the induced regulatory human T cells, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item CCX8A) 1. Use of a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has an NT5E positive rate of about 50% or more.
  • (Item CCX8B) Use of a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has an ITGAE (CD103) positive rate of about 50% or more.
  • (Item CCX8C) Use of a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has an AREG positive rate of about 50% or more.
  • Item CCX8D 1.
  • a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a CD172g positive rate of about 50% or more.
  • (Item CCX8E) 1. Use of a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a CD26 positive rate of about 50% or more.
  • a pharmaceutical composition comprising a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a CTLA4 positive rate of about 50% or more.
  • (Item CCX8G) Use of a cell population comprising an inducible regulatory human T cell for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a stable FoxP3 positive rate of about 50% or more.
  • (Item CCX8G1) Use of a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a percentage of at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • (Item CCX8G2) 1.
  • a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • AREG positivity AREG positivity
  • CD172g positivity CD26 positivity
  • a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • AREG positivity CD172g positivity
  • CD26 positivity of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • AREG positivity CD172g positivity
  • CD26 positivity of about 50% or more.
  • a cell population comprising inducible regulatory human T cells for the manufacture of a medicament for treating or preventing a T cell-related disease, wherein the cell population has a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • CTLA4 positivity NT5E positivity
  • AREG positivity CD172g positivity
  • CD26 positivity of about 50% or more.
  • (Item CCX9b) The use according to any one of the preceding items, wherein the FoxP3 is continuously expressed for at least about 48 hours or more.
  • (Item CCX9c) The use according to any one of the preceding items, wherein the FoxP3 is stably expressed at least from day 7 of culture onwards.
  • (Item CCX9d) The use according to any one of the preceding items, wherein the FoxP3 is expressed even after freezing and thawing of the cells.
  • (Item CCX10) The use according to any one of the preceding items, wherein the proportion of at least the CD172g positive and/or CD26 positive cells in the cell population is about 50% or more, respectively.
  • (Item CCX11) The use according to any one of the preceding claims, wherein the proportion of said at least one characteristic in said cell population is about 60% or more.
  • (Item CCX12) The use according to any one of the preceding claims, wherein the proportion of said at least one characteristic in said cell population is about 80% or more.
  • (Item CCX12a) The use according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells in the cell population is about 50% or more.
  • (Item CCX12b) The use according to any one of the preceding items, wherein the proportion of stably strongly FoxP3 positive cells in the cell population is about 50% or more.
  • T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idi
  • the disease is selected from the group consisting of chronic hypertension, pemphigus, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune disease, Graves' disease, autoimmune liver disease, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell
  • the present disclosure also provides: (Item CC1) Use of an inducible regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity for the manufacture of a medicament for treating or preventing a T cell-related disease.
  • (Item CC2) The use according to the above item, wherein the induced regulatory T cells have at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity.
  • (Item CC3) The use according to any one of the preceding items, wherein the induced regulatory T cells are at least CTLA4 positive.
  • (Item CC4) The use according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene of the induced regulatory T cells is demethylated.
  • (Item CC5) The use according to any one of the preceding items, wherein the induced regulatory T cells are CD4 positive or CD8 positive.
  • (Item CC6) The use according to any one of the preceding claims, wherein the induced regulatory T cells are obtained or derived from human peripheral blood T cells or human tissue-derived T cells.
  • the induced regulatory T cells are (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) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;
  • (Item CC8) The use according to any one of the preceding items, comprising a T cell population in which about 50% or more of the cells are said induced regulatory T cells.
  • (Item CC9) The use according to any one of the preceding items, comprising a T cell population in which about 80% or more of the cells are said induced regulatory T cells.
  • (Item CC10) The use according to any one of the preceding claims, wherein the T cell population is a regulatory T cell population.
  • (Item CC11) The use of any one of the preceding claims, wherein about 90% or more of the cell population are T cells.
  • T cell-related disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM), idiopathic
  • the disease is selected from the group consisting of chronic hypertension, pemphigus, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune disease, Graves' disease, autoimmune liver disease, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell
  • the present disclosure can provide a method for inducing highly functional regulatory T cells in vitro from human peripheral T cells.
  • Regulatory T cells induced by the method of the present disclosure highly express genes associated with regulatory T cells and have strong suppressive capabilities. Therefore, as regulatory T cells, they can be used for the treatment and prevention of various immune diseases and inflammatory diseases such as autoimmune diseases.
  • the method disclosed herein can induce functional regulatory T cells in vitro from human peripheral T cells.
  • the regulatory T cells obtained by the method disclosed herein are functional (e.g., have high suppressive function) and are stable as regulatory T cells.
  • the method disclosed herein can induce highly functional regulatory T cells that stably express FoxP3, the master gene for regulatory T cells, from human peripheral T cells.
  • FIG. 1 shows a phenotype analysis (flow cytometry) of one embodiment of the induced regulatory T cells (also referred to as human highly functional stable iTreg (HSF iTreg) cells) of the present disclosure.
  • the induced regulatory T cells (HSF-iTreg in the figure) of the present disclosure were generated from human CD4 positive T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry. Comparison was made between cells stimulated with conventional nTreg (CD4 positive CD25 positive T cells) and conventional iTreg (Conventional iTreg: CD4 positive T cells stimulated with CD3/CD28 for 3 days in the presence of IL-2 and TGF- ⁇ 1).
  • FIG. 2 is a diagram showing epigenetic analysis (bisulfite method) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure.
  • the demethylation state of the FOXP3 CNS2 region of each cell in FIG. 1 was analyzed by the bisulfite method.
  • Figure 3 is a diagram showing an analysis of the suppressive ability (in vitro suppression assay) of one embodiment of the iTreg (HSF iTreg) cells disclosed herein. The in vitro suppressive ability of each cell in Figure 1 was analyzed.
  • FIG. 4 is a diagram showing gene expression pattern analysis (RNA-seq method) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. Comprehensive gene expression patterns were analyzed by RNA sequencing for each cell in FIG. 1 and FOXP3-negative cells (Tconv) obtained by stimulating CD4-positive T cells in the presence of IL-2.
  • Treg Suppression Inspector Miltenyi Biotec
  • FIG. 5 is a diagram showing CD103 expression (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure.
  • CD103 expression in FOXP3-positive cells was analyzed by flow cytometry for each of the cells in FIG. 6 is a diagram showing a phenotype analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure prepared from human CD8 positive T cells.
  • Regulatory T cells of the present disclosure were prepared from human CD8 positive T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry.
  • FIG. 7 shows CD25 expression and FOXP3 expression (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure.
  • the expression intensity of CD25 and FOXP3 was analyzed by flow cytometry using a BD FACSLyric flow cytometer.
  • 8 is a diagram showing a phenotypic analysis of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure.
  • the inducible regulatory T cells (HSF-iTreg) of the present disclosure were produced, and the expression of CD172g, CD26, etc. was analyzed.
  • Figure 9 shows an outline of a method for producing one embodiment of mouse induced regulatory T cells (also called mouse highly functional stable iTreg (HSF iTreg) cells) and the results of flow cytometry of the induced regulatory T cells.
  • Figure 9A shows an outline of the protocol for inducing iTreg cells from mouse CD4-positive naive T cells using various stimulation methods.
  • Figure 9B shows the results of flow cytometry analysis of FoxP3 and CD25 expression in the iTreg cells, activated T cells, and activated nTreg cells produced in Figure 9A.
  • FIG. 10 shows the results of analyzing FoxP3 expression by flow cytometry and the demethylation state of the Treg-specific demethylated region by the bisulfite method when SF-iTreg were produced from mouse CD4-positive naive T cells or effector T cells based on the production method according to one embodiment of the iTreg (SF-iTreg in the figure) of the present disclosure shown in FIG. 9 .
  • Fig. 11 shows the results of one embodiment in which the global gene expression pattern of each cell in Fig. 9 was analyzed by RNA sequencing.
  • Fig. 11A shows a PCA analysis plot
  • Fig. 11B shows a heat map of Treg-associated genes.
  • FIG. 12 shows the results of one embodiment in which the in vitro suppressive ability of each cell in Figure 9 was analyzed.
  • Mouse CD4-positive naive T cells were stained with Cell Trace Violet reagent, and mixed and cultured for 3 days in the presence of anti-mouse CD3 antibody + MHC-II positive cells or Dynabeads T-activator (Veritas) (5 ⁇ L/well) at a certain ratio with each cell in Figure 9, and the Cell Trace Violet intensity was analyzed by flow cytometry.
  • FIG. 13 shows the results of one embodiment in which the iTregs shown in the group not using a single stimulation CD28 antibody in FIG.
  • FIG. 14 shows the results of one embodiment in which a colitis model was created by transferring mouse CD4-positive naive T cells into RAG2-deficient mice, and the iTreg cells of the present disclosure were administered to analyze the therapeutic effect.
  • Fig. 14A shows the change in body weight (g)
  • Fig. 14B shows an HE stained image of colon tissue
  • Fig. 14C shows the analysis results of CD69 expression in lymph node T cells by flow cytometry.
  • FIG. 15 is a diagram showing the results of analysis of one embodiment of the iTreg of the present disclosure derived from a human Crohn's disease patient.
  • FIG. 15 is a diagram showing the results of analysis of one embodiment of the iTreg of the present disclosure derived from a human Crohn's disease patient.
  • FIG. 15A shows the results of flow cytometry analysis of the expression of FOXP3, CTLA4, and Helios in the inducible regulatory T cells of the present disclosure (HSF-iTreg in the figure) produced from CD4-positive T cells derived from a human Crohn's disease patient.
  • Conventional nTreg CD4-positive CD25-positive T cells
  • conventional iTreg Conventional iTreg: CD4-positive T cells stimulated with CD3/CD28 for 3 days in the presence of IL-2 and TGF- ⁇ 1 were compared.
  • FIG. 15B shows the results of analysis of the in vitro suppressive ability of each cell in FIG. 15A.
  • FIG. 16 is a diagram showing phenotypic analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure prepared from CD4-positive T cells derived from an SLE patient. Highly functional inducible regulatory T cells were prepared from CD4-positive T cells derived from an SLE patient, and the expression of FoxP3, CD4, and CTLA4 was analyzed by flow cytometry.
  • HSF iTreg iTreg
  • FIG. 17 is a diagram showing a phenotype analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure prepared from CD4-positive T cells derived from a rheumatoid arthritis patient. Highly functional inducible regulatory T cells were prepared from CD4-positive T cells derived from a rheumatoid arthritis patient, and the expression of FoxP3, CD4, and CTLA4 was analyzed by flow cytometry.
  • FIG. 18 is a diagram showing that in one embodiment of the present disclosure, induced regulatory human T cells in the cell population of the present disclosure stably express FOXP3 at all stages of stability testing, including the final product, after freeze-thawing, and after restimulation.
  • FIG. 18 is a diagram showing that in one embodiment of the present disclosure, induced regulatory human T cells in the cell population of the present disclosure stably express FOXP3 at all stages of stability testing, including the final product, after freeze-thawing, and after restim
  • FIG. 19 is a diagram showing the results of analyzing the stability of FOXP3 expression on Day 9 (after the second stimulation), Day 13 (final product), and Day 15 (re-stimulation of the final product; an example of a stability test) for the following medium specifications during production of inducible regulatory T cells in a cell population of the present disclosure: RPMI, RPMI with half the amount of glucose, and RPMI with 10-50% reduced glutamine.
  • FIG. 19 is a diagram showing the results of analyzing the stability of FOXP3 expression on Day 9 (after the second stimulation), Day 13 (final product), and Day 15 (re-stimulation of the final product; an example of a stability test) for the following medium specifications during production of inducible regulatory T cells in a cell population of the present disclosure: RPMI, RPMI with half the amount of glucose, and RPMI with 10-50% reduced glutamine.
  • FIG. 20 shows the results of analyzing the stability of FOXP3 expression in the induced regulatory T cells of the present disclosure after restimulation in a standard restimulation environment (RPMI+IL2 with CD3/CD28 stimulation) or with the addition of Th1 cytokines (IL-12, IFN- ⁇ ), Th2 cytokines (IL-4, IL-5), or Th17 cytokines (TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23) in one embodiment of the present disclosure.
  • Th1 cytokines IL-12, IFN- ⁇
  • Th2 cytokines IL-4, IL-5
  • Th17 cytokines TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23
  • FIG. 21 shows the results of flow cytometry analysis of FoxP3 and CTLA4 expression in the induced regulatory T cells of the present disclosure, which are derived from female T cells, for the final product of the induced regulatory T cells of the present disclosure, before restimulation and for the final product restimulated with a standard restimulation environment (RPMI+IL2 with CD3/CD28 stimulation) and with the addition of Th1 cytokines (IL-12, IFN- ⁇ ), Th2 cytokines (IL-4, IL-5), Th17 cytokines (TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23), and TNF- ⁇ , in one embodiment of the present disclosure, and the results of confirming the demethylation state by bisulfite sequencing, as well as the analysis of the stability of FOXP3 expression.
  • Th1 cytokines IL-12, IFN- ⁇
  • Th2 cytokines IL-4, IL-5
  • Th17 cytokines TGF- ⁇ , IL-6, IL-1 ⁇ , IL
  • FIG. 22 shows an analysis using an autoimmune hepatitis model induced by Treg removal through DTX administration.
  • S/F-iTregs were administered simultaneously with the induction, and the H&E stained liver images and serum ALT levels were analyzed 12 days later.
  • Figure 23 shows the results of an autoimmune colitis model induced by administering naive T cells to RAG-deficient mice. S/F-iTregs were administered at the same time as induction, and the body weight change over 6 weeks and the H&E stained image of the colon at the endpoint were analyzed.
  • gene names and their products are written in all capital letters, unlike the usual usage, it may refer to both the gene and the protein.
  • it may be used to distinguish between FOXP3 gene and FOXP3 protein, and when written as FoxP3, it refers to both the concept and the entity (whole) of the gene or protein.
  • regulatory T cells are T cells that are positive for FoxP3 expression. In the present specification, they may also be referred to as “Tregs.” Tregs include naturally occupying regulatory T cells (nTregs) and inducible regulatory T cells (iTregs). Regulatory T cells can usually have various functions (e.g., immunosuppressive functions).
  • inducible regulatory T cells refers to regulatory T cells that are negative for IKZF2 (Helios) expression. iTregs are usually obtained by inducing differentiation from naive CD4 positive T cells, etc.
  • nTreg Naturally Occurring Regulatory T cells
  • IKZF2 Helios
  • CTLA4 CTLA4
  • peripheral T cells refers to T cells present outside the thymus gland, and can be obtained from peripheral blood, lymph nodes, and other tissues. When referring to “peripheral T cells” as used herein, it is sufficient that the cell group contains peripheral T cells, and it is not necessary for the T cells to be isolated. Cell fractions containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMC), may also be used.
  • PBMC peripheral blood mononuclear cells
  • flow cytometry refers to a technique for measuring the number of cells, individuals, and other biological particles suspended in a liquid, as well as the physical, chemical, and biological properties of each particle.
  • a device using this technique is called a “flow cytometer.”
  • the "positive” and “negative” of cell markers are determined by flow cytometry, as is commonly used in the field. More specifically, in flow cytometry, cells are lined up in a line and allowed to flow, and the number of cells is counted by a spectroscopic method.
  • cells labeled with fluorescence or luminescent enzymes are irradiated with laser light, and the fluorescence or luminescence signals emitted by the cells are detected by a detector such as a photodiode, thereby counting the number of target cells.
  • a detector such as a photodiode
  • the detection results from the detector can be input into a computer and a two-dimensional plot can be generated and displayed. This makes it easy to determine the presence or absence of target cells, as well as their number.
  • demethylation refers to the removal of methylation modifications from typically methylated adenine (e.g., m6A at position 6, m1A at position 1) and cytosine (e.g., m5C at position 5, m3C at position 3). Demethylation can be determined using methods known in the art, and can be measured, for example, using the bisulfite method.
  • a "cell population” refers to a population containing two or more cells, and may be, for example, a collection of cells in a planar form, or a cell mass formed by cells adhering to each other in a three-dimensional form. Furthermore, a "cell population” may be formed of a single type of cell, or may contain multiple types of cells.
  • stimulation means stimulation via TCR.
  • stimulation of T cells includes stimulation with anti-CD3 antibodies and complexes containing them, stimulation with peptides that bind to TCR and complexes containing peptides, stimulation with antigen-presenting cells, stimulation using anti-CD3 antibodies and antigen-presenting cells simultaneously, and stimulation using peptides or proteins and antigen-presenting cells simultaneously.
  • “dormant culture” refers to culturing cells in the absence of the above-mentioned stimuli.
  • stable means that the expression of a cell marker (e.g., FoxP3) is maintained in at least about 50% or more of the cells in a cell population for at least 48 hours or more at any time point under normal culture conditions (including after restimulation and after freeze-thawing), and preferably means that the expression continues at least from the 7th day of culture onwards.
  • a cell marker e.g., FoxP3
  • Whether or not a cell marker is stably expressed can be determined by a method well known in the field, such as flow cytometry.
  • the present disclosure relates to highly functional stable induced regulatory T cells (also referred to as highly functional stable iTregs, HSF iTregs) and uses thereof.
  • the present invention relates to medical uses of stable iTregs (also called stable iTregs or HSF iTregs).
  • an inducible regulatory human T cell that has at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising, as an active ingredient, an inducible regulatory human T cell that has at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.
  • the inducible regulatory T cell of the present disclosure may also have at least two characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, and AREG positive.
  • the induced regulatory T cells of the present disclosure may be at least CTLA4 positive.
  • the induced regulatory T cells of the present disclosure may be CD4 positive or CD8 positive.
  • the induced regulatory T cells of the present disclosure may be at least CTLA4 positive and FoxP3 positive. In one embodiment of the present disclosure, the induced regulatory T cells of the present disclosure may be at least CD172g positive and/or CD26 positive.
  • the present disclosure provides inducible regulatory human T cells that are CD172g positive.
  • the present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising inducible regulatory human T cells that are CD172g positive as an active ingredient.
  • CD172g is a tyrosine kinase-related protein that is involved in cell adhesion of neurons and the like (adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment), and therefore inducible regulatory human T cells that are CD172g positive, functions related to adhesion and the like are activated, and it is expected that the inducible regulatory human T cells will be highly effective against various diseases related thereto.
  • the present disclosure provides CD26-positive inducible regulatory human T cells.
  • the present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising CD26-positive inducible regulatory human T cells as an active ingredient.
  • CD26 is a gene also known as DPP4, and is related to glucose metabolism, insulin metabolism, and immune function (notably in infectious diseases), so CD26-positive inducible regulatory human T cells are expected to be highly effective against various diseases related to these.
  • the present disclosure provides NT5E-positive inducible regulatory human T cells.
  • the present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising NT5E-positive inducible regulatory human T cells as an active ingredient.
  • NT5E (CD73) is a cell membrane protein that has a catalytic activity of converting extracellular nucleotides into membrane-permeable nucleosides. The encoded protein is used as a determinant for lymphocyte differentiation. Since defects in this gene can cause calcification of joints and arteries, NT5E-positive inducible regulatory human T cells are expected to be highly effective against various diseases related to this.
  • the present disclosure provides ITGAE (CD103) positive inducible regulatory human T cells.
  • the present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising ITGAE (CD103) positive inducible regulatory human T cells as an active ingredient.
  • ITGAE (CD103) encodes an ⁇ integrin with an I domain, which undergoes post-translational cleavage in the extracellular domain to generate disulfide-linked heavy and light chains. This protein binds to ⁇ 7 integrin to form an E-cadherin-binding integrin known as human mucosal lymphocyte-1 antigen.
  • This protein is preferentially expressed on human intestinal intraepithelial lymphocytes (IELs) and may function as an accessory molecule for IEL activation in addition to its adhesive role. Therefore, ITGAE (CD103) positive inducible regulatory human T cells are expected to be highly effective against various diseases related to this.
  • the present disclosure provides an AREG-positive inducible regulatory human T cell.
  • the present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising an AREG-positive inducible regulatory human T cell as an active ingredient.
  • AREG is a member of the epidermal growth factor family and is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF- ⁇ ).
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor alpha
  • the protein interacts with the EGF/TGF- ⁇ receptor to promote the growth of normal epithelial cells and inhibit the growth of certain aggressive cancer cell lines. It also functions in the development of mammary glands, oocytes, and bone tissue.
  • the gene is associated with a skin phenotype resembling psoriasis, and with other pathological diseases, including various types of cancer and inflammatory conditions. Therefore, it is expected that the AREG-positive inducible regulatory human T cell will be highly effective against various diseases related thereto.
  • CTLA4-positive inducible regulatory human T cells The present disclosure also provides a pharmaceutical composition for treating or preventing a T cell-related disease, the pharmaceutical composition comprising CTLA4-positive inducible regulatory human T cells as an active ingredient.
  • CTLA4 belongs to the immunoglobulin superfamily and is a protein that transmits inhibitory signals to T cells.
  • Membrane-bound CTLA4 functions as a homodimer linked by disulfide bonds, and soluble CTLA4 functions as a monomer. Mutations in this gene are said to be associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases.
  • CTLA4-positive inducible regulatory human T cells will be highly effective against various diseases associated with these.
  • CTLA4-positive inducible regulatory human T cells are expected to be highly effective against autoimmune diseases such as pemphigus, autoimmune liver disease (primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), etc.), primary biliary cholangitis, and primary sclerosing cholangitis.
  • Regulatory T cells can be induced in vitro by culturing CD4-positive T cells in a medium containing IL2 and TGF ⁇ in the presence of anti-CD3 and anti-CD28 antibodies, and then culturing in a medium containing IL2 and TGF ⁇ .
  • a medium containing IL2 and TGF ⁇ a medium containing IL2 and TGF ⁇ .
  • FoxP3 expression in induced regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has not been confirmed.
  • the present disclosure can provide an inducible regulatory T cell that stably expresses FoxP3 and advantageously stably retains an immunosuppressive effect, and/or a pharmaceutical composition containing such an inducible regulatory T cell as an active ingredient, and such an inducible regulatory T cell can be produced, for example, by a method for producing an inducible regulatory T cell described elsewhere in this specification.
  • the present disclosure can provide an inducible regulatory T cell obtained by a method including the steps of: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.
  • the induced regulatory T cells produced in the present disclosure have an induced regulatory T cell-specific demethylation state.
  • the obtained regulatory T cells can be confirmed to be in a regulatory T cell-specific demethylation state, for example, by demethylation of the CNS2 site of the FoxP3 gene (FOXP3 (all italics)). Since such a demethylation state can be an indicator of a stable type, the induced regulatory T cells of the present disclosure can be shown to be stable induced regulatory T cells by confirming the demethylation state.
  • the immunosuppressive activity or immunosuppressive effect of the induced regulatory T cells of the present disclosure can be confirmed, for example, by measuring the Cell Trace Violet intensity in responder T cells.
  • the induced regulatory T cells of the present disclosure can stably provide immunosuppressive activity or immunosuppressive effect, for example, the induced regulatory T cells of the present disclosure can provide immunosuppressive activity or immunosuppressive effect for at least about two weeks.
  • the induced regulatory T cells of the present disclosure can have a higher immunosuppressive effect than conventional regulatory T cells (including induced and endogenous). Therefore, the induced regulatory T cells of the present disclosure can also be said to be functional or highly functional induced regulatory T cells.
  • the induced regulatory T cells of the present disclosure can stably express FoxP3. Therefore, the induced regulatory T cells of the present disclosure can also be referred to as stable induced regulatory T cells. In one aspect, the induced regulatory T cells of the present disclosure are highly functional and stable, and can be referred to as highly functional stable induced regulatory T cells (HSF iTreg).
  • whether or not a marker in the induced regulatory T cells of the present disclosure is positive can be determined by measuring the positivity rate by flow cytometry.
  • the marker can be analyzed by a flow cytometer, and whether or not it is positive can be determined from the proportion of cells that show an antigen expression level equal to or higher than a standard.
  • it can also be classified as negative, weakly positive, moderately positive, strongly positive (weakly positive, moderately positive, and strongly positive are collectively referred to as "positive"), etc.
  • the values of the median fluorescence intensity of each marker/median fluorescence intensity of the negative control can be negative, weakly positive, moderately positive, and strongly positive, respectively, when they are less than 5, 5 to 10, 10 to 30, and 30 or more.
  • Such a determination can be made as exemplified in (measurement of the positivity rate by flow cytometry), but the present disclosure is not limited thereto.
  • the expression intensity of the cell surface marker in the induced regulatory T cells of the present disclosure may be, for example, based on the results of analysis using a sample prepared as described in the examples of the present application using a BD FACSLyric flow cytometer (BD Biosciences), and the expression intensity may be weakly positive when it is 10 2 or more, moderately positive when it is 10 3 or more, strongly positive when it is 10 4 or more, or strongly positive when it is 10 3 or more and weakly positive when it is 10 4 or more, or strongly positive when it is 10 3 or more and weakly positive when it is 10 2 or more and weakly positive when it is 10 2 or more.
  • the expression intensity in the other device corresponding to the expression intensity when analyzed using the BD FACSLyric flow cytometer can be calculated, and a value indicating the expression intensity can be appropriately found depending on the device used.
  • the inducible regulatory T cells produced in the present disclosure can be derived from any cells, but are preferably derived from human peripheral blood T cells or human tissue-derived T cells.
  • the inducible regulatory T cells or cell population of the present disclosure can be targeted at human cells, and can be inducible human regulatory T cells or cell populations thereof induced by a specified method using T cells obtained from human peripheral blood as material.
  • the properties of human cells and mouse cells are clearly different, and even if the same cell surface marker is present, the properties of the cells cannot be considered to be the same.
  • the techniques that can be applied to mice and humans are different, and while in mice, the majority of lymphocytes are antigen-na ⁇ ve, in humans, the T cells in peripheral blood have a variety of degrees of antigen sensitization and activation. Therefore, while in mice, highly functional stable inducible regulatory T cells can be directly produced from T cells collected from mouse lymphoid tissue, this is difficult in humans, and the cells cannot be considered to be similar simply based on the presence of a cell surface marker.
  • a cell population includes a cell population containing the above-described inducible regulatory human T cells, the cell population having a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having a CTLA4 positive and FoxP3 positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having a CTLA4 positive and FoxP3 positive rate of about 50% or more.
  • the cell population of the present disclosure can have a CTLA4 positive and FoxP3 positive rate of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.
  • the percentage of at least the CD172g positive and/or CD26 positive in the cell population of the present disclosure can be about 50% or more. In one embodiment, the percentage of CD172g positive and/or CD26 positive in the cell population of the present disclosure can be about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.
  • the percentage of strongly FoxP3 positive cells in the cell population of the present disclosure can be about 50% or more. In one embodiment, the percentage of strongly FoxP3 positive cells in the cell population of the present disclosure can be about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.
  • the expression intensity of the cell surface marker can be measured by measuring the positivity rate by flow cytometry as described above.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having a FoxP3 positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having a FoxP3 positive rate of about 50% or more.
  • such a cell population can contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of FoxP3 positive inducible regulatory human T cells based on cell number.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having a CTLA4 positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having a CTLA4 positive rate of about 50% or more.
  • such a cell population can contain CTLA4 positive inducible regulatory human T cells in an amount of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more based on the cell number.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having an NT5E positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having an NT5E positive rate of about 50% or more.
  • such a cell population can contain NT5E positive inducible regulatory human T cells in an amount of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more based on the cell number.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having an ITGAE (CD103) positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having an ITGAE (CD103) positive rate of about 50% or more.
  • such a cell population can contain ITGAE (CD103) positive inducible regulatory human T cells in an amount of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more based on the cell number.
  • ITGAE CD103
  • a cell population containing inducible regulatory human T cells is provided, the cell population having an AREG positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having an AREG positive rate of about 50% or more.
  • such a cell population can contain AREG positive inducible regulatory human T cells in an amount of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more based on the cell number.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having a CD172g positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having a CD172g positive rate of about 50% or more.
  • such a cell population can contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD172g positive inducible regulatory human T cells based on cell number.
  • a cell population containing inducible regulatory human T cells is provided, the cell population having a CD26 positive rate of about 50% or more.
  • a pharmaceutical composition for treating or preventing a T cell-related disease is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory human T cells as an active ingredient, the cell population having a CD26 positive rate of about 50% or more.
  • such a cell population can contain about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD26 positive inducible regulatory human T cells based on cell number.
  • a cell population in which the positive rate of a specific cell surface marker is about 50% or more can be obtained, and preferably a cell population in which "the positive rates of CTLA4 and FoxP3 are each about 50% or more.”
  • CTLA4 is a molecule that is localized within cells, and at least not at a level that can be used as an indicator for enrichment or purification.
  • FoxP3 is a transcription factor and is a molecule that is entirely present within cells, so it cannot be detected at all on the surface and cannot be used for purification.
  • CTLA4 and FoxP3 are markers that are expressed "inside" T cells, it is not possible to obtain a cell population in which CTLA4 and/or FoxP3 are about 50% or more positive by sorting and enriching using CTLA4 and/or FoxP3 as indicators.
  • the inducible regulatory human T cells of the present invention are primarily responsible for the immunosuppressive function of Tregs, and when these cells account for more than half of a cell population, a medically effective immunosuppressive effect can be stably achieved, which is important in terms of technical and medical effects.
  • a medically effective immunosuppressive effect can be stably achieved, which is important in terms of technical and medical effects.
  • a stable cell preparation can be provided, and this effect is an extremely important point medically, and is not merely a difference in quantity or numerical value, but is an important point in terms of quality as to whether the preparation can be established.
  • the induced regulatory human T cells can further have the characteristics of being ITGAE (CD103) positive, NT5E positive, and/or AREG positive.
  • ITGAE CD103
  • NT5E positive NT5E positive
  • AREG AREG
  • High expression of these markers allows the cell population of the present disclosure to achieve the effect of maintaining functional stability.
  • the T cells in the cell population of the present disclosure can be regulatory T cells.
  • the regulatory T cells in the cell population of the present disclosure can be inducible regulatory T cells as described elsewhere in this specification.
  • the cell population of the present disclosure may contain cells other than T cells, but preferably, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the cell population of the present disclosure may be T cells, and preferably, about 90% or more may be T cells.
  • the induced regulatory T cells in the cell population of the present disclosure can stably express FOXP3 even when cultured for a long period of time.
  • the induced regulatory T cells in the cell population of the present disclosure can express FOXP3 in at least about 50% or more of the cells in the cell population at any time point under normal culture conditions (including after restimulation and after freeze-thawing), for example, for at least about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, about 120 hours or more, about 132 hours or more, about 144 hours or more, about 156 hours or more, about 168 hours or more, about 180 hours or more.
  • the induced regulatory T cells in the cell population of the present disclosure can express FOXP3 at least from the 7th day of culture onwards.
  • the cells of the intermediate product on culture days 3, 4, 5, 6, 7, 8, 9, and 10 can stably express FOXP3.
  • the induced regulatory T cells in the cell population of the present disclosure are stably FOXP3-positive cells, and can stably express FOXP3 even when cryopreserved and thawed, and can stably express FOXP3 even when cells that may become the final product (e.g., cells on culture day 13) are frozen and thawed (see, for example, FIG.
  • the induced regulatory T cells in the cell population of the present disclosure can stably express FOXP3 even when cells that can be the final product (e.g., cells on day 13 of culture) are restimulated and cultured (including in a Th1 environment) regardless of whether they have been frozen and thawed.
  • cells that can be the final product e.g., cells on day 13 of culture
  • cells can stably express FOXP3 even on day 14 of culture (day 1 of stability test), day 15 of culture (day 2 of stability test), day 16 of culture (day 3 of stability test), day 17 of culture (day 4 of stability test), day 18 of culture (day 5 of stability test), etc.
  • the induced regulatory T cells in the cell population of the present disclosure can express FOXP3 for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days of culture. In other embodiments, the induced regulatory T cells in the cell population of the present disclosure can express FOXP3 even when frozen and thawed for at least about 1, 2, 3, 4, 5, or 6 months.
  • the stably FOXP3-positive cells can be stably used as a cell preparation.
  • the cell population or induced regulatory T cells of the present disclosure can stably express at least one marker selected from the group consisting of CTLA4, NT5E, ITGAE (CD103), AREG, CD172g, and CD26 in addition to FoxP3, and can preferably be stably CTLA4-positive cells.
  • compositions comprising the induced regulatory T cells or cell population of the present disclosure.
  • a regenerative medicine material or product comprising the induced regulatory T cells or cell population of the present disclosure is provided.
  • This pharmaceutical composition or regenerative medicine material or product can be used for autoimmune diseases, inflammatory diseases, and allergies that can be treated by immunosuppressive action.
  • These medicines, regenerative medicine materials or products can be used together with culture media and any other additives used in the field.
  • a culture medium a culture medium obtained by adding necessary factors to a basal culture medium for animal cell culture used 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.
  • it may contain DMSO, etc.
  • the pharmaceutical composition of the present disclosure can be used to treat or prevent a T cell-related disease, which is not limited to any disease that can be treated by immunosuppressive effects, but includes, for example, autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS.
  • the autoimmune disease includes, but is not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (PPCM),
  • autoimmune diseases include myocarditis, glaucoma, hypertension, pulmonary hypertension, malignant hypertension, pemphigus, Alzheimer's disease, systemic sclerosis, polymyositis, mixed connective tissue disease, antiphospholipid syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, crescentic glomerulonephritis, organ-specific autoimmune disease, Graves' disease, autoimmune liver disease, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), primary biliary cholangitis, autoimmune pancreatitis, Goodpasture's syndrome, autoimmune hemolytic anemia, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant
  • the autoimmune disease is preferably pemphigus, an autoimmune liver disease (primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), etc.), primary biliary cholangitis, or primary sclerosing cholangitis, more preferably pemphigus or autoimmune hepatitis, and even more preferably autoimmune hepatitis.
  • PSC primary sclerosing cholangitis
  • AIH autoimmune hepatitis
  • the pharmaceutical composition of the present disclosure can be administered in a variety of doses and is preferably administered by injection.
  • the pharmaceutical composition of the present disclosure can include about 10 to about 10 induced regulatory T cells, as described elsewhere herein, administered per dose, or about 10 cells /kg.
  • the pharmaceutical composition of the present disclosure can be administered to a patient in whom administration of the composition to the patient is ineffective or insufficient. Whether the composition is ineffective or insufficient can be determined, for example, by using as an indicator the degree of inflammation suppression in the target disease. In one embodiment, when an additional administration is performed, the composition can be administered at least about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after the first administration.
  • the present disclosure provides a method for producing the inducible regulatory T cells of the present disclosure.
  • the method of the present disclosure is a novel method capable of producing highly functional and stable inducible regulatory T cells, which is described in detail herein below.
  • a method for producing inducible regulatory T cells comprising the steps of (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days, (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.
  • the inducible regulatory T cells of the present disclosure can be produced using either CD4-positive T cells or CD8-positive T cells as a raw material, and in one embodiment, the inducible regulatory T cells of the present disclosure can also be produced using a mixed cell of CD4-positive T cells and CD8-positive T cells as a raw material.
  • the method disclosed herein can also be a method for producing regulatory T cells from human peripheral T cells (including CD4-positive T cells or CD8-positive T cells), comprising the steps of culturing human peripheral T cells in a medium containing TGF ⁇ and IL-2 in the presence of anti-CD3 antibody stimulation, culturing the cells in a medium containing IL-2 in the absence of anti-CD3 antibody, and culturing the cells again in a medium containing TGF ⁇ and IL-2 in the presence of anti-CD3 antibody stimulation.
  • human peripheral T cells including CD4-positive T cells or CD8-positive T cells
  • the stimulation of T cells refers to stimulation when obtaining Tregs, and is not particularly limited as long as it is stimulation via TCR.
  • stimulation of T cells can include stimulation with an anti-CD3 antibody or a complex containing the antibody, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation using an anti-CD3 antibody and antigen-presenting cells simultaneously, stimulation using a peptide or protein and antigen-presenting cells simultaneously, etc., but the medium and conditions are not particularly limited as long as Tregs can be obtained.
  • the dormant culture is not particularly limited in terms of the medium or conditions, so long as the cells are cultured in the absence of the above-mentioned stimuli.
  • anti-CD3 antibody stimulation means providing specific stimulation to the CD3 receptor on a cell.
  • An example of CD3 stimulation is an anti-CD3 agonist antibody.
  • the anti-CD3 agonist antibody may be a product commercially available as a research reagent, or may be prepared by a conventional method.
  • Anti-CD3 antibodies may be derived from animals such as mice, rabbits, goats, and cows, or from humans.
  • the method of the present disclosure involves first inducing iTregs (stimulating T cells to induce demethylation) (step a), then exchanging the medium and resting culture for 1-3 days to recover the cells (step b), and stimulating the T cells once more to induce demethylation (step c), thereby obtaining functional and stable inducible regulatory human T cells.
  • iTregs stimulating T cells to induce demethylation
  • step b exchanging the medium and resting culture for 1-3 days to recover the cells
  • step c stimulating the T cells once more to induce demethylation
  • functional and stable inducible regulatory human T cells can be obtained by performing T cell stimulation, dormant culture, and then re-stimulation, so that the desired effects of the present disclosure can be achieved with inducible regulatory human T cells obtained in this manner or a cell population containing such inducible regulatory human T cells. Therefore, in one embodiment of the present disclosure, the type of culture medium or stimulus used is not particularly limited, and functional and stable inducible regulatory human T cells can be obtained by using a medium of any composition and any type of stimulus.
  • the medium used in each step may further contain retinoic acid and/or ascorbic acid.
  • the medium may contain ascorbic acid.
  • the medium may further contain a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor.
  • the medium may contain a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor.
  • CDK8 inhibitor since the CDK8 inhibitor, the CDK19 inhibitor, and the CDK8/19 inhibitor may have overlapping functions, in this disclosure, they may be described as CDK8 inhibitors, CDK19 inhibitors, and CDK8/19 inhibitors, in which case at least one selected from the group consisting of CDK8 inhibitors, CDK19 inhibitors, and CDK8/19 inhibitors is selected.
  • the first basal medium and the second basal medium may each independently contain at least one, at least two, at least three, at least four, at least five, or all of the factors selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ (e.g., TGF- ⁇ 1, TGF- ⁇ 2, TGF ⁇ -3, etc.), IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8/19 inhibitors (at least one selected from the group consisting of CDK8 inhibitors, CDK19 inhibitors, and CDK8/19 inhibitors), and ascorbic acid.
  • TGF- ⁇ e.g., TGF- ⁇ 1, TGF- ⁇ 2, TGF ⁇ -3, etc.
  • IL-2 IL-2
  • retinoic acid retinoic acid
  • CDK8 inhibitors CDK19 inhibitors
  • CDK8/19 inhibitors at least one selected from the group consisting of CDK8 inhibitors, CDK19 inhibitors, and CDK8/19 inhibitors
  • ascorbic acid ascorbic
  • the first basal medium and the second basal medium may each independently contain an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8/19 inhibitors (at least one selected from the group consisting of CDK8 inhibitors, CDK19 inhibitors, and CDK8/19 inhibitors), and ascorbic acid.
  • the concentration of each of these components may be a normal concentration used in the field.
  • 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 the field.
  • the concentration can be about 0.1 ⁇ M or more, about 0.5 ⁇ M or more, about 1 ⁇ M or more, about 2 ⁇ M or more, about 3 ⁇ M or more, about 4 ⁇ M or more, about 5 ⁇ M or more, about 6 ⁇ M or more, about 7 ⁇ M or more, about 8 ⁇ M or more, about 9 ⁇ M or more, about 10 ⁇ M or more, about 12 ⁇ M or more, about 14 ⁇ M or more, about 16 ⁇ M or more, about 18 ⁇ M or more, about 20 ⁇ M or more, etc., but is not limited to these concentrations, and those skilled in the art can change them appropriately depending on other medium compositions.
  • the first basal medium used in the present disclosure comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor
  • the second basal medium comprises at least one selected from the group consisting of an anti-CD3 antibody, TGF- ⁇ , IL-2, retinoic acid, and ascorbic acid, and a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor.
  • At least one selected from the group consisting of a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor contained in the first basal medium comprises Senexin A or AS2863619.
  • at least one selected from the group consisting of a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8/19 inhibitor contained in the second basal medium comprises Senexin A or AS2863619.
  • the TGF- ⁇ contained in the first basal medium comprises TGF- ⁇ 1 and/or TGF- ⁇ 3.
  • the TGF- ⁇ contained in the second basal medium comprises TGF- ⁇ 1 and/or TGF- ⁇ 3.
  • the TGF- ⁇ contained in the first basal medium comprises TGF- ⁇ 1.
  • the TGF- ⁇ contained in the second basal medium comprises TGF- ⁇ 1.
  • the first basal medium comprises anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, and SelexinA.
  • the second basal medium contains anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, Selexin A, and ascorbic acid.
  • the first basal medium comprises anti-CD3 antibody, TGF- ⁇ 3, IL-2, retinoic acid, and SelexinA.
  • the second basal medium contains anti-CD3 antibody, TGF- ⁇ 3, IL-2, retinoic acid, Selexin A, and ascorbic acid.
  • the method of the present disclosure produces regulatory T cells from human peripheral T cells.
  • Peripheral T cells include naive regulatory T cells, CD4 positive T cells, CD8 positive T cells, etc.
  • Regulatory T cells may be induced from a culture containing multiple types of T cells, or specific cells such as CD4 positive T cells or CD8 positive T cells may be isolated from these cells and then the regulatory T cells may be induced.
  • Regulatory T cells may also be induced after isolating T cells specific to a specific antigen. Therefore, the induced regulatory T cells of the present disclosure include those that are CD4 positive and those that are CD8 positive.
  • the terms “CD4 positive” or “CD4+” refer to single positive cells that are CD4 positive and CD8 negative unless otherwise specified.
  • CD4 positive or CD8 positive T cells can be used as a raw material, and even after the induced regulatory T cells are generated, the characteristics of CD4 positive or CD8 positive can be maintained unless a special operation is performed.
  • the antibody in the method of the present disclosure, may be added to the culture medium, or may be immobilized on the inner wall of a culture vessel or the surface of an insoluble carrier.
  • the insoluble carrier may be a material capable of physically or chemically binding the anti-CD3 antibody and insoluble in an aqueous solution. Examples of materials capable of physically adsorbing the anti-CD3 antibody include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, and glass.
  • the shape of the insoluble carrier is not particularly limited, and may be, for example, a plate shape, a bead shape, or a container shape.
  • the amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide sufficient stimulation for the induction of regulatory T cells.
  • a medium in which necessary factors have been added to a basal medium for animal cell culture can be used for cell culture.
  • basal media for animal cell culture include Iscove's modified Eagle's Medium medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, RPMI1640 medium, Fischer's medium, and mixtures or media with partially modified compositions thereof.
  • Basal media may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free.
  • Serum-free media may optionally contain one or more serum substitutes, such as, for example, albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and monothioglycerol.
  • serum substitutes such as, for example, albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty
  • the basal medium may also contain one or more substances such as lipids (e.g., chemically defined lipid concentrate), amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), growth factors, antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.
  • lipids e.g., chemically defined lipid concentrate
  • amino acids amino acids
  • L-glutamine e.g., GlutaMAX (Thermo Fisher Scientific)
  • NEAA non-essential amino acids
  • vitamins e.g., nicotinamide, ascorbic acid
  • growth factors e.g., antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.
  • the basal medium is, for example, RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid).
  • the cells may be cultured under general animal cell culture conditions.
  • the culture temperature is, but is not limited to, about 30 to 40° C., preferably about 37° C.
  • the culture is preferably performed in an atmosphere of CO2- containing air, and the CO2 concentration is preferably about 2 to 5%.
  • the anti-CD3 antibody may be added directly to the culture medium, or may be immobilized on the inner wall of a culture vessel or on the surface of an insoluble carrier.
  • the amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide sufficient stimulation for the induction of regulatory T cells.
  • TGF ⁇ can be used by, for example, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3.
  • concentration of TGF ⁇ can be determined appropriately by those skilled in the art and is not particularly limited.
  • the concentration in the medium is not particularly limited, but may be 0.25 to 25 ng/mL, for example, about 10 ng/mL.
  • the concentration of IL-2 in the medium used is not limited, but can be about 5 U/mL to about 500 U/mL, for example, about 100 U/mL.
  • the medium used in the present disclosure may further contain retinoic acid and/or ascorbic acid.
  • the medium may contain ascorbic acid.
  • the concentration of ascorbic acid is not limited, but is about 1 to about 100 ⁇ g/mL, for example, about 10 ⁇ g/mL.
  • the medium used in the present disclosure may further contain a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor.
  • a CDK8 inhibitor, CDK19 inhibitor, and/or CDK8/19 inhibitor may be used, but 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. thereof, or the compounds disclosed in U.S.
  • concentrations of the CDK8 inhibitor, CDK19 inhibitor, and/or CDK8/19 inhibitor that can be used may be any suitable concentration that can be used in the art, or any suitable concentration described in the above literature, and can be appropriately changed by a person skilled in the art according to other medium compositions.
  • human peripheral T cells are stimulated with anti-CD3 antibody in a medium containing TGF ⁇ and IL-2 to induce regulatory T cells, and regulatory T cells with high immunosuppressive function can be induced by stimulating the cells again with anti-CD3 antibody after dormant culture in an IL-2-containing medium that does not contain anti-CD3 antibody.
  • the high immunosuppressive function of the obtained induced regulatory T cells can be confirmed, for example, by comprehensive gene expression analysis using RNA sequencing and in vitro cell proliferation inhibition tests.
  • the number of days of culture in the step (a) of stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days can be appropriately set by a person skilled in the art and is not particularly limited, but can be, for example, about 3 days.
  • the number of days for the dormant culture step in step (b) can be appropriately set by a person skilled in the art and is not particularly limited, but can be, for example, about 2 days.
  • the number of days for the culture step in the second basal medium in step (c) can be appropriately set by a person skilled in the art and is not particularly limited, but can be, for example, about 3 days.
  • the number of days for culture in the dormant culture step in step (d) can be appropriately set by those skilled in the art and is not particularly limited, but can be, for example, about 2 days.
  • regulatory T cells having an induced regulatory T cell-specific demethylation state can be expanded by culturing in an unstimulated state in the presence of IL-2.
  • the medium may further contain ascorbic acid, and a stable regulatory T cell culture of the induced regulatory T cells can be obtained by culturing in a medium further containing IL-2.
  • the cells can be isolated by a conventional method based on a cell surface marker specific to regulatory T cells, for example, by extracting a FoxP3-positive fraction using a cell sorter. If desired, regulatory T cells with specific antigen characteristics can also be isolated.
  • the inducible regulatory T cells obtained by the method disclosed herein are expected to be used to treat human inflammatory diseases, such as autoimmune diseases and allergies.
  • the positive rate measurement by flow cytometry can be performed as follows.
  • Preparation/Fixation/Permeabilization Concentrate (hereinafter referred to as Buffer) (eBio Science, 00-5123-43) ⁇ Fixation/Permeabilization Diluent (hereinafter referred to as Diluent) (eBio Science, 00-5223-56) ⁇ Permeabilization Buffer (10 ⁇ ) (eBio Science, 00-833-56) ⁇ FOXP3 Monoclonal Antibody (236A/E7), PE (hereinafter referred to as anti-FOXP3 antibody) (eBio Science, 12-4777-42) - Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE (hereinafter referred to as PE control) (eBio Science) ⁇ BV421,Mouse,Anti-Human,CD152 (hereinafter referred to as anti-CTLA4 antibody) (BD) - BV421 Mouse IgG2a, k Isotype Control (hereinafter referred to as BV421 control) (BD)
  • Fixation Buffer (use 100 ⁇ L per sample) Mix buffer and diluent in a 1:3 ratio. ⁇ Perm Buffer Dilute the Permeabilization Buffer (10x) 10 times with MilliQ water. *FACS Buffer (when preparing 500 mL) D-PBS 489mL FBS 10mL (final concentration 2%) 0.5mol/l EDTA solution 1mL (final concentration 1mM) After preparation, store the above reagents at 4°C or on ice.
  • Method (1) Add 500 ⁇ L of FACS Buffer to a 1.5 mL tube. (2) Add 1x106 cells of the final product to the tube (1) and gently suspend using a micropipette. (3) Centrifuge at 500 x g and 4°C for 5 minutes. (4) After removing the supernatant with an aspirator, add 100 ⁇ L of Fixation Buffer and gently pipette. ⁇ Be careful not to create bubbles. (5) Fix by leaving it on ice in the dark for at least 30 minutes. ⁇ 45 minutes is also acceptable. (6) After fixation, add 1 mL of Perm Buffer to the tube and gently pipette. (7) Prepare new 1.5 mL tubes for the number of samples.
  • the reagents used for fixation and staining can also be purchased as a set of three bottles called the "Foxp3/Transcription Factor Staining Buffer Set" (Cat.: 00-5523). - Any settings of the instrument are acceptable as long as they do not deviate to such an extent that it is clearly determined from a general and scientific point of view that normal measurements are not being performed. Clear deviations include cases where the various signals of the target cell population are below the set fluorescence threshold value, or where the various signal values of the control sample or the sample to be measured are below or above the limit of what can be measured normally by the instrument, etc.
  • the cells of the present disclosure may have immunosuppressive activity. Immunosuppressive activity can be measured in a variety of ways.
  • whether or not immune suppression is observed can be determined by measuring cell proliferation due to the immune response caused by responder T cells.
  • various kits can be used, including, but not limited to, a Treg immunosuppression assay (horizon discovery; https://www.horizondiscoverykk.com/products/research-services/immune-cell-based-assay/immune-suppression-assays/#Treg).
  • the immunosuppressive activity of the cells of the present disclosure may be enhanced compared to conventional iTregs.
  • tissues are collected from a colitis model mouse to which the inducible regulatory T cells of the present disclosure have been administered after a certain period of administration, and the activity can be measured based on whether or not immune suppression is confirmed in the tissue.
  • Immune suppression in the tissue can be confirmed by various methods, and for example, the presence or absence of immune suppression can be confirmed by staining analysis of the tissue.
  • Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annu al updates; Sninsky, J. J. et al. (1999).
  • glutamine-free RPMI1640 (containing 10% FCS (v/v), 60 ⁇ g/mL penicillin G, 100 ⁇ g/mL streptomycin, and 10 mM HEPES) was used as the basal medium. 30 to 300 mg/L of L-glutamine was added to the medium as necessary.
  • 5'-TTGGGTTAAGTTTGTTGTAGGATAG-3' was used for the forward side
  • 5'-ATCTAAACCCTATTATCACAACCCC-3' was used for the reverse side.
  • Foxp3-eGFP (eFox) reporter mice Ito et al. (2014) Science 346, 363-368) were used. Lymph node cells from Foxp3-eGFP (eFox) reporter mice were obtained, and CD4 + GFP + cells were sorted using FACSAriaII (BD) to obtain endogenous regulatory T cell (nTreg) fractions. CD4 + GFP - CD44 low CD62L high cells were sorted to obtain naive T cell fractions. Furthermore, CD4 + GFP - CD44 high CD62L low cell fractions were obtained and used as effector/memory T cells.
  • BD FACSAriaII
  • CD4-positive T cells were stimulated for 3 days with a basal medium containing anti-CD3 antibody (100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), and Senexin A (5 ⁇ M). Thereafter, the cells were cultured for rest 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 for rest for another 2 days.
  • a basal medium containing anti-CD3 antibody 100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 10 ng/ml
  • the cells were stimulated again for 2 days with a basal medium containing anti-CD3 antibody (100 ⁇ L of antibody at a concentration of 10 ⁇ g/ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), Senexin A (5 ⁇ M), and ascorbic acid (10 ⁇ g/ml).
  • the cells were then cultured for 2 days in a basal medium containing hIL-2 (100 U/ml). After 2 days, the medium was replaced in the same manner, and the cells were cultured for another 2 days in a basal medium.
  • the expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after 2 days was analyzed by flow cytometry (FIG. 1).
  • Conventional induced regulatory T cells have insufficient expression of functional molecules (for example, CTLA4 in this case), and the efficiency of FoxP3 induction is also low.
  • endogenous regulatory T cells nTreg
  • the highly functional induced regulatory T cells obtained by the production method of the present application are Helios negative, and it can be confirmed that they are a population containing many cells expressing FoxP3 and CTLA4.
  • Helios is a marker for regulatory T cells endogenous to the body, and being Helios negative indicates that they are induced regulatory T cells.
  • Inducible regulatory T cells obtained by the conventional method are, for example, CTLA4 negative, so at least in terms of being CTLA4 positive, it can be seen that the obtained induced regulatory T cells are induced regulatory T cells newly induced in vitro.
  • the induced regulatory T cells obtained in the above examples are different from endogenous nTregs and are newly induced regulatory T cells in vitro, but they have been shown to have characteristics not found in existing induced regulatory T cells, such as high induction efficiency, high CTLA4 expression, and demethylation of the FOXP3 CNS2 region.
  • Example 2 In vitro suppressive activity of regulatory T cells Regulatory T cells were obtained using the same experimental system as in Example 1.
  • Cell Trace Violet-labeled responder T cells (5x10 4 ) and the prepared regulatory T cells (5x10 4 ) were mixed and cultured for 3 days in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 ⁇ L/well), and the Cell Trace Violet intensity was analyzed by flow cytometry.
  • Treg Suppression Inspector Miltenyi Biotec
  • the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak.
  • the regulatory T cells obtained by the preparation method of the present disclosure strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a higher suppressive function than existing regulatory T cell populations (FIG. 3).
  • Example 3 Comprehensive gene expression analysis of regulatory T cells
  • the gene expression pattern of the regulatory T cells prepared by the method of Example 1 was confirmed by RNA sequencing ( Figure 4).
  • RNA sequencing was performed using a known method (Kitagawa et al. (2017) Nat. Immunol. 18, 173-183).
  • High expression of genes of FOXP3, IL-2RA (CD25), and major inhibitory molecules of Treg (CTLA4, etc.) was confirmed in the induced regulatory T cells and nTreg cells obtained by the preparation method of the present disclosure, compared to existing induced regulatory T cells and activated Tconv cells.
  • IKZF2 a marker of nTreg cells
  • the induced regulatory T cells obtained by the preparation method of the present disclosure have high expression of several inhibitory molecules such as ENTPD1, NT5E, and AREG, compared to other regulatory T cell populations. Additionally, high expression of adhesion molecules such as ITGAE and chemokine receptors such as CCR4 was confirmed ( Figure 4).
  • the induced regulatory T cells obtained by the production method of the present application had higher CD103 expression than other regulatory T cell populations ( Figure 5).
  • the induced regulatory T cells obtained by the production method of the present application are expected to exhibit higher suppressive ability than existing regulatory T cells.
  • CD8-positive T cells were stimulated for 3 days with a basal medium containing anti-CD3 antibody (100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), and Senexin A (5 ⁇ M). Thereafter, the cells were cultured for rest 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 for rest for another 2 days.
  • a basal medium containing anti-CD3 antibody 100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 10 ng/ml
  • the cells were stimulated again for 2 days with a basal medium containing anti-CD3 antibody (100 ⁇ L of antibody at a concentration of 10 ⁇ g/ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), Senexin A (5 ⁇ M), and ascorbic acid (10 ⁇ g/ml).
  • the cells were then cultured for 2 days in a basal medium containing hIL-2 (100 U/ml). After 2 days, the medium was replaced in the same manner, and the cells were cultured for another 2 days in a basal medium.
  • the expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after 2 days was analyzed by flow cytometry ( FIG. 6 ).
  • the inducible regulatory T cells of the present disclosure which were created from CD8-positive T cells, were also confirmed to be Helios-negative and to be a population containing a large number of cells expressing FoxP3 and CTLA4.
  • Example 5 Production of inducible regulatory T cells from human peripheral T cells Using human peripheral T cells, induced regulatory T cells or cell populations thereof are obtained in the same manner as in Example 1. A pharmaceutical containing the induced regulatory T cells or cell populations thereof and a carrier is manufactured. This pharmaceutical contains about 2 x 105 cells as the induced regulatory T cells or cell populations thereof, and its effectiveness can be confirmed based on the degree of inflammation in tissues collected one month after administration to a patient with an inflammatory disease.
  • Example 6 Formulation Example Inducible regulatory T cells or a cell population thereof are obtained in the same manner as in Example 1. A pharmaceutical comprising the inducible regulatory T cells or a cell population thereof and a carrier is produced.
  • Example 7 Expression intensity analysis
  • the expression intensities of CD25 and FOXP3 in the cell population of induced regulatory T cells produced in Example 1 were analyzed by flow cytometry using a BD FACSLyric flow cytometer. The results are shown in Figure 7. It was confirmed that a high proportion of the cell population of FOXP3 Hi inducible regulatory T cells was present in all cells tested.
  • Example 8 Antibody panel analysis of inducible regulatory T cells
  • Example 8 Antibody panel analysis of inducible regulatory T cells
  • the induced regulatory T cells produced in Example 1 were stained using BioLegend's LEGENDScreen Human PE Kit, and antibodies reacting with the induced regulatory T cells of the present disclosure were analyzed by flow cytometry. A portion of the results (those in which clear and significant expression was confirmed) are shown in Tables 1 to 4.
  • Tables 1 to 4 When stained with an antibody panel containing just under 400 types, the positive/negative characteristics of the cell surface markers in the induced regulatory T cells of the present disclosure could be confirmed by at least 185 types of surface marker molecules.
  • Example 9 Analysis of cell surface markers in induced regulatory T cells Characteristic results from the antibody screening carried out in Example 8 are shown in Figure 8. It was found that the induced regulatory T cells of the present disclosure were positive for specific cell surface markers such as CD172g and CD26.
  • Example 10 Method for producing highly functional stable regulatory T cells in mice
  • Mouse CD4-positive T cells were stimulated for 3 days with a basal medium containing anti-CD3 antibody (100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (2.5 ng/ml), retinoic acid (1 ⁇ M), and Senexin A (5 ⁇ M). Thereafter, the cells were cultured for rest 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 for rest for another 2 days.
  • a basal medium containing anti-CD3 antibody 100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container
  • hIL-2 100 U/
  • the cells were stimulated again for 2 days with a basal medium containing anti-CD3 antibody (100 ⁇ L of an antibody at a concentration of 10 ⁇ g/ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (2.5 ng/ml), retinoic acid (1 ⁇ M), Senexin A (5 ⁇ M), and ascorbic acid (10 ⁇ g/ml).
  • the cells were then cultured for 2 days in a basal medium containing hIL-2 (100 U/ml). After 2 days, the medium was replaced in the same manner, and the cells were cultured for another 2 days in a basal medium.
  • the expression of FoxP3 and CD25 in the regulatory T cells obtained after 2 days was analyzed by flow cytometry ( FIG. 9 ), the demethylation state of the Treg-specific demethylated region was analyzed by the bisulfite method ( FIG. 10 ), and the comprehensive gene expression pattern was analyzed by RNA-seq ( FIG. 11 ).
  • Example 11 In vitro suppressive activity of regulatory T cells Regulatory T cells were obtained using the same experimental system as in Example 1.
  • the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak. It was confirmed that the regulatory T cells obtained by the preparation method of the present application strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a higher suppressive function than existing regulatory T cell populations ( FIG. 12 ).
  • Example 12 In vivo stability of regulatory T cells Regulatory T cells were prepared from Thy1.2/eFox reporter mice by the method of Example 1, and Foxp3-positive cells (2 ⁇ 10 5 cells) were separated by FACS and administered to Thy1.1/wild-type mice via the tail vein. After 2 weeks, the transferred Thy1.2 cells were collected from lymph nodes and Foxp3 expression was analyzed ( FIG. 13 ). Regulatory T cells induced without CD28 stimulation and subjected to stabilization culture were confirmed to stably express Foxp3 even after 2 weeks in vivo.
  • Example 13 Inhibitory effect on colitis model
  • a colitis model was prepared by administering 2x10 5 wild-type mouse-derived CD45RB-highly expressing CD4-positive naive T cells to RAG-deficient mice. After one week, 2x10 5 highly functional stable iTreg cells prepared by the method of Example 1 were administered, and the change in body weight was measured over time (FIG. 14A). Then, one month after administration, colon tissue and lymph nodes were collected, and HE staining analysis of colon tissue (FIG. 14B) and CD69 expression analysis in lymph node T cells (FIG. 14C: flow cytometry method) were performed. As a result, it was confirmed that administration of highly functional stable iTreg suppressed colitis. In addition, the therapeutic effect was confirmed at a dose of 2x10 5 per mouse (about 20 g), and the efficacy was confirmed at a dose of 1x10 7 /kg.
  • Example 14 Production of regulatory T cells from human peripheral T cells CD4-positive T cells derived from a patient with Crohn's disease were stimulated for 3 days with a basal medium containing an anti-CD3 antibody (100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), and Senexin A (5 ⁇ M). Thereafter, the cells were cultured for rest in a basal medium containing hIL-2 (100 U/ml) for 2 days.
  • a basal medium containing an anti-CD3 antibody 100 ⁇ L per well of an antibody at a concentration of 10 ⁇ g/ml was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 10 ng/ml
  • the medium was replaced in the same manner, and the cells were cultured for rest for another 2 days.
  • the cells were stimulated again for 2 days with a basal medium containing anti-CD3 antibody (100 ⁇ L of antibody at a concentration of 10 ⁇ g/ml was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U/ml), hTGF ⁇ 1 (10 ng/ml), retinoic acid (1 ⁇ M), Senexin A (5 ⁇ M), and ascorbic acid (10 ⁇ g/ml).
  • the cells were then cultured for 2 days in a basal medium containing hIL-2 (100 U/ml).
  • Conventional induced regulatory T cells have insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained using the method of the present application have been confirmed to be a population containing a large number of cells expressing FoxP3 and CTLA4.
  • Example 15 In vitro suppressive activity of regulatory T cells Regulatory T cells were obtained using the same experimental system as in Example 10. Cell Trace Violet-labeled responder T cells (5 ⁇ 10 4 ) and the prepared regulatory T cells (5 ⁇ 10 4 ) were mixed and cultured for 3 days in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 ⁇ L/well), and the Cell Trace Violet intensity was analyzed by flow cytometry. When responder T cells cause an immune response, the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak. It was confirmed that the regulatory T cells obtained by the preparation method of the present application strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a higher suppressive function than existing regulatory T cell populations.
  • Treg Suppression Inspector Miltenyi Biotec
  • Example 16 Production of regulatory T cells from T cells derived from a patient with systemic lupus erythematosus (SLE) Using CD4-positive T cells derived from an SLE patient, regulatory T cells were obtained in the same experimental system as in Example 1. The expression of FoxP3, CD4, and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry ( FIG. 16 ).
  • Conventional induced regulatory T cells have insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained by the disclosed method of production can be confirmed to be a population containing many cells expressing FoxP3 and CTLA4.
  • Example 17 Production of regulatory T cells from T cells derived from rheumatoid arthritis (RA) patients
  • Regulatory T cells were obtained using the same experimental system as in Example 1, using CD4-positive T cells derived from RA patients.
  • the expression of FoxP3, CD4, and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry ( Figure 17).
  • Conventional induced regulatory T cells have insufficient expression of functional molecules (CTLA4 in this case) and low FoxP3 induction efficiency, but the highly functional induced regulatory T cells obtained by the method of the present application have been confirmed to be a population containing many cells expressing FoxP3 and CTLA4.
  • Example 18 FOXP3 stability evaluation
  • FOXP3 was stably expressed consistently from Day 7 of culture (Day 7) in the in-process control test through Day 13 (final product), Day 15 (Day 2 of stability test), and Day 17 (Day 4 of stability test).
  • the stability of FOXP3 expression was analyzed on Day 9 (after the second stimulation), Day 13 (final product), and Day 15 (re-stimulation of the final product; an example of a stability test) for the cases where RPMI was used, and where the amount of glucose was reduced by half from RPMI and the amount of glutamine was reduced by 10-50% from RPMI ( Figure 19).
  • the inducible regulatory T cells of the present disclosure can be produced at a constant rate, and the inducible regulatory T cells consistently exhibit stable regulatory T cell properties from the time the cell properties were determined (Day 9).
  • Example 19 Evaluation of FOXP3 stability after freeze-thaw It was confirmed that the induced regulatory T cells in the cell population of the present disclosure were stable FOXP3-positive cells even after freezing and thawing.
  • the induced regulatory T cells in the cell population of the present disclosure were frozen and thawed after 7.5 months (freezing date: 2021.10.16, thawing date: 2022.06.06), and FACS analysis was performed after leaving them for a certain period of time.
  • the freezing solution used was Kymriah composition (1 x 10 8 cells/freezing bag).
  • dextran injection solution 40 mL was used, and the cells were left at 4 ° C. or room temperature, and the cells were sampled and analyzed after 0 hours, 1 hour, 2 hours, and 3 hours. The results are shown in Table 5.
  • the induced regulatory T cells in the cell population disclosed herein stably express FOXP3 even when frozen and stored for more than six months, demonstrating that cell storage for more than six months is possible. Furthermore, after thawing, the condition of the cells was good when stored at 4°C for up to two hours. Even at room temperature, no significant changes were observed when stored for up to two hours.
  • Example 20 Production of regulatory T cells from female derived T cells and FOXP3 stability assessment
  • the final product of the inducible regulatory T cells of the present disclosure was analyzed by flow cytometry for expression of FoxP3 and CTLA4 in the obtained regulatory T cells before restimulation and after restimulation in a normal restimulation environment (RPMI+IL2 with CD3/CD28 stimulation) with the addition of Th1 cytokines (IL-12, IFN- ⁇ ), Th2 cytokines (IL-4, IL-5), Th17 cytokines (TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23) and TNF- ⁇ (FIG. 21).
  • the demethylation state of each was confirmed by bisulfite sequencing (FIG. 21).
  • CTLA4 was further increased in cells restimulated by adding Th1 cytokines (IL-12, IFN- ⁇ ), Th2 cytokines (IL-4, IL-5), Th17 cytokines (TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23) and TNF- ⁇ to a normal restimulation environment (RPMI+IL2 and CD3/CD28 stimulation) compared to those before restimulation.
  • Th1 cytokines IL-12, IFN- ⁇
  • Th2 cytokines IL-4, IL-5
  • Th17 cytokines TGF- ⁇ , IL-6, IL-1 ⁇ , IL-283
  • TNF- ⁇ TNF- ⁇
  • RPMI+IL2 and CD3/CD28 stimulation a normal restimulation environment
  • the stability of FOXP3 expression in the resulting induced regulatory T cells was analyzed for the final product of the induced regulatory T cells disclosed herein before restimulation and when restimulated in a normal restimulation environment (RPMI + IL2 with CD3/CD28 stimulation) with the addition of Th1 cytokines (IL-12, IFN- ⁇ ), Th2 cytokines (IL-4, IL-5), Th17 cytokines (TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23), and TNF- ⁇ (Figure 21).
  • Th1 cytokines IL-12, IFN- ⁇
  • Th2 cytokines IL-4, IL-5
  • Th17 cytokines TGF- ⁇ , IL-6, IL-1 ⁇ , IL-23
  • TNF- ⁇ Figure 21
  • Example 21 Demonstration of effect on autoimmune liver disease
  • PSC primary sclerosing cholangitis
  • AIH autoimmune hepatitis
  • the present disclosure is a cell preparation that is expected to suppress the overall immune response, particularly with T cells. Therefore, the transliteration command will conduct experiments targeting autoimmune liver disease (AILD) as a development target with the aim of confirming the effect on T cell-mediated autoimmune diseases in general.
  • autoimmune liver disease a development target with the aim of confirming the effect on T cell-mediated autoimmune diseases in general.
  • AILD mainly includes primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH), and since PSC is often accompanied by inflammatory bowel diseases such as ulcerative colitis, it is considered important to comprehensively control T cell-mediated autoimmune responses. Therefore, we believe that control of T cell-mediated autoimmune responses by regulatory T cells is highly likely to be effective in controlling the pathology of the disease, and we are currently conducting non-clinical development.
  • safety will be assessed through general toxicity tests, and the risk of tumorigenesis will be evaluated through growth characteristic analysis tests.
  • Efficacy will be assessed through in vitro growth inhibition tests using human T cells, and in vivo allograft tests using mouse cells.
  • ⁇ Growth characteristic analysis test> A growth characteristic analysis test of the formulation of the present disclosure was carried out, and it was confirmed that the formulation did not show uncontrollable growth characteristics by long-term culture, i.e., it did not have tumorigenicity.
  • extended culture was carried out using two lots of the final product, and the number of days until proliferation stopped was 26 and 22 days with stimulation, respectively, and 16 and 20 days without stimulation, respectively. It was shown that proliferation stopped within 30 days even under conditions using stimuli that promote T cell proliferation.
  • the cells that make up the formulation used in this example are expected to disappear eventually after administration into the body, and it can be determined that there is no risk of tumorigenicity.
  • Inflammatory cytokines IL-4, IL-6, IL-17, IFN ⁇ , TNF ⁇
  • Anti-inflammatory cytokine IL-10
  • S/F-iTreg or source cells peripheral blood mononuclear cells: PBMC
  • PBMC peripheral blood mononuclear cells
  • S/F-iTregs for non-clinical use were stored at -80°C for more than 6 months, but the provisional specifications were maintained for cell count, viability, CD4+FOXP3+ cell rate, and CTLA4+ rate, and no differences were observed in the results compared to those at month 0.
  • ⁇ T cell proliferation inhibition test A general cell proliferation test to evaluate the control ability of S/F-iTreg using healthy subjects confirmed that the product exhibited autologous T cell proliferation inhibition ability compared to the control group ( Figure 3B). In this example, the effect on the target disease will be confirmed using T cells derived from AILD patients.
  • ⁇ Mouse colitis model Since some of the targeted autoimmune liver diseases are accompanied by inflammatory bowel disease symptoms, we are conducting POC using a colitis model induced by transferring CD45RB-positive naive T cells derived from BALB/c mice to RAG2-deficient mice, with the possibility of including comprehensive improvement of autoimmune symptoms as an evaluation item. To date, we have confirmed the suppressive effect of S/F-iTreg cells in the colitis model.
  • the inducible regulatory T cells and/or cell populations disclosed herein can be used to treat or prevent various immune disorders and inflammatory diseases such as autoimmune diseases. Furthermore, the method for producing the inducible regulatory T cells and/or cell populations disclosed herein makes it possible to stably induce highly functional regulatory T cells from peripheral T cells, which is expected to be applied in the medical field.
  • SEQ ID NO: 1 Forward primer used in the examples
  • SEQ ID NO: 2 Reverse primer used in the examples

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