Pharmaceutical composition for treating or preventing pemphigus

Abstract

The invention provides a medicine for treating or preventing pemphigus. More specifically, the present invention provides a pharmaceutical composition for treating or preventing pemphigus, containing, as an active ingredient, an induced regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating or preventing pemphigus, and more particularly, to a pharmaceutical composition comprising regulatory T cells specifically responsive to desmoglein 3 (Dsg3). Background Art

[0002] A key characteristic of CD25-positive CD4-positive regulatory T cells within the immune system is their specific expression of the transcription factor FoxP3. FoxP3 deficiency or mutation can impair the development and differentiation of regulatory T cells, as well as their suppressive function. In addition to FoxP3, regulatory T cells suppress the immune system through the expression of multiple genes, including CTLA4 and IL-10. Epigenetic states such as DNA demethylation are believed to contribute to the regulation of global gene expression in regulatory T cells, exemplified by the stable expression of FoxP3, and are associated with the phenotype of functional regulatory T cells. Summary of the Invention

[0003] Means of solving the problem

[0004] The inventors of the present invention have discovered for the first time that, when inducing regulatory T cells from human peripheral T cells, by inducing stable induced T cells from human peripheral T cells with stimulation using anti-CD3 antibodies, followed by resting culture, then stimulating again with anti-CD3 antibodies and culturing, and then resting culture again, regulatory T cells with high expression of inhibitory molecules and high inhibitory function can be induced. Furthermore, the inventors of the present invention have discovered that the induced regulatory T cells thus obtained have sufficient immunosuppressive activity to function as a drug for the treatment or prevention of pemphigus.

[0005] Therefore, the present invention provides the following.

[0006] (Item 1)

[0007] A pharmaceutical composition for treating or preventing pemphigus, comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity.

[0008] (Item 2)

[0009] A pharmaceutical composition for treating or preventing pemphigus, comprising NT5E-positive inducible regulatory T cells as an active ingredient.

[0010] (Item 3)

[0011] A pharmaceutical composition for treating or preventing pemphigus, comprising ITGAE (CD103)-positive inducible regulatory T cells as an active ingredient.

[0012] (Item 4)

[0013] A pharmaceutical composition for treating or preventing pemphigus, comprising AREG-positive inducible regulatory T cells as an active ingredient.

[0014] (Item 5)

[0015] A pharmaceutical composition for treating or preventing pemphigus, comprising CD172g-positive inducible regulatory T cells as an active ingredient.

[0016] (Item 6)

[0017] A pharmaceutical composition for treating or preventing pemphigus, comprising CD26-positive inducible regulatory T cells as an active ingredient.

[0018] (Item 7)

[0019] A pharmaceutical composition for treating or preventing pemphigus, comprising CTLA4-positive inducible regulatory T cells as an active ingredient.

[0020] (Item 8)

[0021] The pharmaceutical composition according to any one of the above items, wherein the inducible regulatory T cells are at least CTLA4-positive and FoxP3-positive.

[0022] (Item 9)

[0023] The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are at least CD172g positive and / or CD26 positive.

[0024] (Item 10)

[0025] The pharmaceutical composition according to any one of the above items, wherein the CNS2 site of the FOXP3 gene in the inducible regulatory T cells is demethylated.

[0026] (Item 11)

[0027] The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are CD4-positive or CD8-positive.

[0028] (Item 12)

[0029] The pharmaceutical composition according to any one of the above items, wherein the inducible regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

[0030] (Item 13)

[0031] The pharmaceutical composition according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps:

[0032] (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;

[0033] (b) a step of dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;

[0034] (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and

[0035] (d) a step of dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0036] (Item 14)

[0037] The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition contains a cell population comprising the inducible regulatory T cells as an active ingredient, and the proportion 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 in the cell population is approximately 50% or more.

[0038] (Item 15)

[0039] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the NT5E-positive ratio of the cell population is about 50% or more.

[0040] (Item 16)

[0041] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the ITGAE (CD103)-positive ratio of the cell population is about 50% or more.

[0042] (Item 17)

[0043] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the AREG-positive ratio of the cell population is about 50% or more.

[0044] (Item 18)

[0045] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CD172g-positive ratio of the cell population is about 50% or more.

[0046] (Item 19)

[0047] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CD26-positive ratio of the cell population is about 50% or more.

[0048] (Item 20)

[0049] A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CTLA4-positive ratio of the cell population is about 50% or more.

[0050] (Item 21)

[0051] The pharmaceutical composition according to any one of the above items, wherein the proportions of at least the CTLA4-positive cells and the FoxP3-positive cells in the cell population are each about 50% or greater.

[0052] (Item 22)

[0053] The pharmaceutical composition according to any one of the above items, wherein the proportion of at least the CD172g-positive and / or CD26-positive cells in the cell population is respectively about 50% or greater.

[0054] (Item 23)

[0055] The pharmaceutical composition according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 60% or more.

[0056] (Item 24)

[0057] The pharmaceutical composition according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 80% or more.

[0058] (Item 25)

[0059] The pharmaceutical composition according to any one of the above items, wherein the proportion of cells in the cell population that are strongly FoxP3 positive is about 50% or more.

[0060] (Item 26)

[0061] The pharmaceutical composition according to any one of the above items, wherein about 90% or more of the cell population are T cells.

[0062] (Item 27)

[0063] The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is administered at a dose of at least about 1×10 3 The inducible regulatory T cells are administered in an amount of 1:1.

[0064] (Item 28)

[0065] The pharmaceutical composition according to any one of the above items, wherein the pharmaceutical composition is administered by injection.

[0066] (Item 29)

[0067] The pharmaceutical composition according to any one of the above items, wherein after administration of the pharmaceutical composition to a patient, the pharmaceutical composition is additionally administered to a patient for whom the effect is ineffective or insufficient.

[0068] (Item 30)

[0069] The pharmaceutical composition according to any one of the above items, wherein the inducible regulatory T cells specifically respond to desmoglein 3 (Dsg3).

[0070] (Item 31)

[0071] A method for treating or preventing pemphigus, comprising administering the pharmaceutical composition according to any one of the above items to a subject.

[0072] (Item 32)

[0073] A pharmaceutical composition comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity, and specifically responding to desmoglein 3 (Dsg3).

[0074] (Project A1)

[0075] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of inducible regulatory T cells, wherein the inducible regulatory T cells have at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.

[0076] (Item A2)

[0077] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of NT5E-positive inducible regulatory T cells.

[0078] (Item A3)

[0079] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of ITGAE (CD103)-positive inducible regulatory T cells.

[0080] (Item A4)

[0081] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of AREG-positive inducible regulatory T cells.

[0082] (Item A5)

[0083] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of CD172g-positive inducible regulatory T cells.

[0084] (Item A6)

[0085] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of CD26-positive inducible regulatory T cells.

[0086] (Item A7)

[0087] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of CTLA4-positive inducible regulatory T cells.

[0088] (Item A8)

[0089] The method according to any one of the above items, wherein the inducible regulatory T cells are at least CTLA4-positive and FoxP3-positive.

[0090] (Item A9)

[0091] The method according to any one of the above items, wherein the induced regulatory T cells are at least CD172g positive and / or CD26 positive.

[0092] (Item A10)

[0093] The method according to any one of the above items, wherein the CNS2 site of the FOXP3 gene in the inducible regulatory T cells is demethylated.

[0094] (Item A11)

[0095] The method according to any one of the above items, wherein the inducible regulatory T cells are CD4-positive or CD8-positive.

[0096] (Item A12)

[0097] The method according to any one of the above items, wherein the inducible regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

[0098] (Item A13)

[0099] The method according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps:

[0100] (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;

[0101] (b) a step of dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;

[0102] (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and

[0103] (d) a step of dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0104] (Item A14)

[0105] The method according to any one of the above items, wherein the cell population containing the inducible regulatory T cells is contained as an effective ingredient, and the proportion 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 in the cell population is approximately 50% or more.

[0106] (Item A15)

[0107] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of NT5E positivity in the cell population is about 50% or more.

[0108] (Item A16)

[0109] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of ITGAE (CD103) positivity in the cell population is about 50% or more.

[0110] (Item A17)

[0111] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of AREG positivity in the cell population is about 50% or more.

[0112] (Item A18)

[0113] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of CD172g positivity in the cell population is about 50% or more.

[0114] (Item A19)

[0115] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of CD26 positivity in the cell population is about 50% or more.

[0116] (Item A20)

[0117] A method for treating or preventing pemphigus in a subject, comprising administering to the subject an effective amount of a cell population comprising inducible regulatory T cells, wherein the proportion of CTLA4 positivity in the cell population is about 50% or more.

[0118] (Item A21)

[0119] The method according to any one of the above items, wherein the proportion of at least the CTLA4-positive and FoxP3-positive cells in the cell population is respectively about 50% or greater.

[0120] (Item A22)

[0121] The method according to any one of the above items, wherein the proportion of at least the CD172g-positive and / or CD26-positive cells in the cell population is respectively about 50% or greater.

[0122] (Item A23)

[0123] The method according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 60% or more.

[0124] (Item A24)

[0125] The method according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 80% or more.

[0126] (Item A25)

[0127] The method according to any one of the above items, wherein the proportion of cells in the cell population that are strongly FoxP3 positive is about 50% or more.

[0128] (Item A26)

[0129] The method according to any one of the above items, wherein about 90% or more of the cell population are T cells.

[0130] (Item A27)

[0131] The method according to any one of the above items, wherein the amount of each administration is at least about 1×10 3 The inducible regulatory T cells are administered in an amount of 1:1.

[0132] (Item A28)

[0133] The method according to any one of the above items, wherein the pharmaceutical composition is administered by injection.

[0134] (Item A29)

[0135] The method according to any one of the above items, wherein after the pharmaceutical composition is administered to a patient, the pharmaceutical composition is additionally administered to a patient for whom the pharmaceutical composition has no effect or an insufficient effect.

[0136] (Item A30)

[0137] The method according to any one of the above items, wherein the inducible regulatory T cells specifically respond to desmoglein 3 (Dsg3).

[0138] (Item B1)

[0139] An inducible regulatory T cell for treating or preventing pemphigus, which has at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity and CD26 positivity.

[0140] (Item B2)

[0141] An inducible regulatory T cell for treating or preventing pemphigus, which is NT5E positive.

[0142] (Item B3)

[0143] An inducible regulatory T cell for treating or preventing pemphigus, which is ITGAE (CD103) positive.

[0144] (Item B4)

[0145] An inducible regulatory T cell for treating or preventing pemphigus, which is AREG-positive.

[0146] (Item B5)

[0147] An inducible regulatory T cell for treating or preventing pemphigus, which is CD172g positive.

[0148] (Item B6)

[0149] An inducible regulatory T cell for treating or preventing pemphigus, which is CD26 positive.

[0150] (Item B7)

[0151] An inducible regulatory T cell that is CTLA4 positive for treating or preventing pemphigus.

[0152] (Item B8)

[0153] The induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are at least CTLA4-positive and FoxP3-positive.

[0154] (Item B9)

[0155] The induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are at least CD172g positive and / or CD26 positive.

[0156] (Item B10)

[0157] The inducible regulatory T cells according to any one of the above items, wherein the CNS2 site of the FOXP3 gene in the inducible regulatory T cells is demethylated.

[0158] (Item B11)

[0159] The induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are CD4-positive or CD8-positive.

[0160] (Item B12)

[0161] The induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

[0162] (Item B13)

[0163] The induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps:

[0164] (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;

[0165] (b) a step of dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;

[0166] (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and

[0167] (d) a step of dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0168] (Item B14)

[0169] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the proportion 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 in the cell population is greater than about 50%.

[0170] (Item B15)

[0171] A cell population for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the NT5E-positive ratio of the cell population is about 50% or more.

[0172] (Item B16)

[0173] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the ITGAE (CD103)-positive ratio of the cell population is about 50% or more.

[0174] (Item B17)

[0175] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the AREG-positive ratio of the cell population is about 50% or more.

[0176] (Item B18)

[0177] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the CD172g-positive ratio of the cell population is about 50% or more.

[0178] (Item B19)

[0179] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the CD26-positive ratio of the cell population is about 50% or more.

[0180] (Item B20)

[0181] A cell population for treating or preventing pemphigus, comprising inducible regulatory T cells, wherein the CTLA4-positive ratio of the cell population is about 50% or more.

[0182] (Item B21)

[0183] The cell population according to any one of the above items, wherein the proportions of at least the CTLA4-positive cells and the FoxP3-positive cells in the cell population are each about 50% or greater.

[0184] (Item B22)

[0185] The cell population according to any one of the above items, wherein at least the proportion of CD172g positivity and / or CD26 positivity in the cell population is respectively about 50% or greater.

[0186] (Item B23)

[0187] The cell population according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 60% or greater.

[0188] (Item B24)

[0189] The cell population according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 80% or more.

[0190] (Item B25)

[0191] The cell population according to any one of the above items, wherein the proportion of cells in the cell population that are strongly FoxP3 positive is about 50% or more.

[0192] (Item B26)

[0193] The cell population according to any one of the above items, wherein about 90% or more of the cell population are T cells.

[0194] (Item B27)

[0195] The cell or cell population according to any one of the above items, wherein the cell or cell population is administered at a rate of at least about 1×10 3 The inducible regulatory T cells are administered in an amount of 1:1.

[0196] (Item B28)

[0197] The cell or cell population according to any one of the above items, wherein the cell or cell population is administered by injection.

[0198] (Item B29)

[0199] The cell or cell population according to any one of the above items, wherein after administering the cell or cell population to a patient, the pharmaceutical composition is additionally administered to a patient for whom the effect is ineffective or insufficient.

[0200] (Item B30)

[0201] The cell or cell population according to any one of the above items, wherein the inducible regulatory T cells specifically respond to desmoglein 3 (Dsg3).

[0202] (Project C1)

[0203] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells have at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity.

[0204] (Project C2)

[0205] An induced regulatory T cell for use in producing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cell is NT5E positive.

[0206] (Item C3)

[0207] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells are ITGAE (CD103) positive.

[0208] (Item C4)

[0209] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells are AREG-positive.

[0210] (Item C5)

[0211] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells are CD172g positive.

[0212] (Item C6)

[0213] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells are CD26 positive.

[0214] (Item C7)

[0215] A use of induced regulatory T cells for manufacturing a medicament for treating or preventing pemphigus, wherein the induced regulatory T cells are CTLA4 positive.

[0216] (Item C8)

[0217] The use of induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are at least CTLA4-positive and FoxP3-positive.

[0218] (Item C9)

[0219] The use of induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are at least CD172g positive and / or CD26 positive.

[0220] (Item C10)

[0221] The use of the inducible regulatory T cells according to any one of the above items, wherein the CNS2 site of the FOXP3 gene in the inducible regulatory T cells is demethylated.

[0222] (Item C11)

[0223] The use of induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are CD4-positive or CD8-positive.

[0224] (Item C12)

[0225] The use of induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are obtained or induced from human peripheral blood T cells or human tissue-derived T cells.

[0226] (Item C13)

[0227] The use of the induced regulatory T cells according to any one of the above items, wherein the induced regulatory T cells are obtained by a method comprising the following steps:

[0228] (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;

[0229] (b) a step of dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days;

[0230] (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and

[0231] (d) a step of dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0232] (Item C14)

[0233] A use of a cell population for manufacturing a medicament for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the proportion 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 in the cell population is respectively greater than about 50%.

[0234] (Item C15)

[0235] A use of a cell population for producing a medicament for treating or preventing pemphigus, wherein the medicament comprises a cell population containing inducible regulatory T cells as an active ingredient, and the NT5E-positive ratio of the cell population is about 50% or more.

[0236] (Item C16)

[0237] A use of a cell population for manufacturing a medicament for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the proportion of ITGAE (CD103) positivity in the cell population is about 50% or more.

[0238] (Item C17)

[0239] A use of a cell population for manufacturing a drug for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the AREG-positive ratio of the cell population is about 50% or more.

[0240] (Item C18)

[0241] A use of a cell population for manufacturing a medicament for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the CD172g-positive ratio of the cell population is about 50% or more.

[0242] (Item C19)

[0243] A use of a cell population for manufacturing a medicament for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the CD26-positive ratio of the cell population is about 50% or more.

[0244] (Item C20)

[0245] A use of a cell population for manufacturing a medicament for treating or preventing pemphigus, wherein the cell population comprises inducible regulatory T cells, and the CTLA4-positive ratio of the cell population is about 50% or more.

[0246] (Item C21)

[0247] The use of the cell population according to any one of the above items, wherein the proportions of at least the CTLA4-positive cells and the FoxP3-positive cells in the cell population are each about 50% or greater.

[0248] (Item C22)

[0249] The use of the cell population according to any one of the above items, wherein the proportion of at least the CD172g-positive and / or CD26-positive cells in the cell population is respectively about 50% or greater.

[0250] (Item C23)

[0251] The use of a cell population according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 60% or greater.

[0252] (Item C24)

[0253] The use of a cell population according to any one of the above items, wherein the ratio of the at least one characteristic in the cell population is about 80% or more.

[0254] (Item C25)

[0255] The use of a cell population according to any one of the above items, wherein the proportion of cells in the cell population that are strongly FoxP3 positive is about 50% or more.

[0256] (Item C26)

[0257] The use of a cell population according to any one of the above items, wherein about 90% or more of the cell population are T cells.

[0258] (Item C27)

[0259] The use of cells or cell populations according to any one of the above items, wherein the cells or cell populations are administered at a rate of at least about 1×10 3 The inducible regulatory T cells are administered in an amount of 1:1.

[0260] (Item C28)

[0261] The use of a cell or a cell population according to any one of the above items, wherein the cell or cell population is administered by injection.

[0262] (Item C29)

[0263] The use of the cell or cell population according to any one of the above items, wherein after the cell or cell population is administered to a patient, the pharmaceutical composition is additionally administered to a patient for whom the pharmaceutical composition has no effect or has an insufficient effect.

[0264] (Item C30)

[0265] The use of a cell or cell population according to any one of the above items, wherein the inducible regulatory T cells specifically respond to desmoglein 3 (Dsg3).

[0266] In the present invention, one or more of the above features, in addition to the combinations explicitly stated, are also intended to be provided in further combinations. It should be noted that, after reading and understanding the following detailed description as needed, those skilled in the art will be able to recognize further embodiments and advantages of the present invention.

[0267] It should be noted that features, significant actions, and effects of the present invention other than those described above will become apparent to those skilled in the art by referring to the following embodiments of the present invention and the accompanying drawings.

[0268] Effects of the Invention

[0269] The regulatory T cells of the present invention can specifically react to desmoglein, an antigenic protein of pemphigus. Furthermore, the regulatory T cells of the present invention highly express genes associated with regulatory T cells and possess strong inhibitory capacity. Therefore, as regulatory T cells, they can be used to treat or prevent pemphigus, etc.

[0270] Furthermore, the regulatory T cells of the present invention can be obtained by inducing functional regulatory T cells in vitro from human peripheral T cells. The regulatory T cells of the present invention have high suppressive function and are stable as regulatory T cells. Specifically, the cells or cell populations of the present invention are capable of reacting specifically with desmogleins and, using human peripheral T cells as the starting material, stably express FoxP3, the master gene for regulatory T cells, and are therefore useful for treating or preventing pemphigus. BRIEF DESCRIPTION OF THE DRAWINGS

[0271] Figure 1 This is a schematic diagram showing an example of the production of Dsg3-specific S / F iTregs in one embodiment of the present invention.

[0272] Figure 2 Graphs showing the results of flow cytometry analysis and bisulfite analysis of Dsg3-specific S / F iTregs in one embodiment of the present invention.

[0273] Figure 3 This is a graph showing the immunosuppressive effect of Dsg3-specific S / F iTreg in one embodiment of the present invention.

[0274] Figure 4 This is a graph showing the dermatitis suppressive effect of Dsg3-specific S / F iTreg in one embodiment of the present invention.

[0275] Figure 5 This figure shows the volume-dependent inflammatory effect of Dsg3-specific S / F iTreg on dermatitis according to one embodiment of the present invention. Body weight indicates (change in) body weight, and Clinical score indicates clinical score.

[0276] Figure 6 This is a graph showing the effect of Dsg3-specific S / F iTreg in suppressing the production of inflammatory cytokines in one embodiment of the present invention.

[0277] Figure 7 This figure shows the long-term survival of Dsg3-specific S / F iTreg in dermatitis model mice according to one embodiment of the present invention.

[0278] Figure 8 This is a graph showing the inhibitory effect of Dsg3-specific S / F iTreg on a pemphigus model according to one embodiment of the present invention.

[0279] Figure 9 Graphs showing the results of flow cytometry analysis and bisulfite analysis of Dsg3-specific human S / F iTregs in one embodiment of the present invention.

[0280] Figure 10 This is a graph showing the immunosuppressive effect of Dsg3-specific human S / F iTregs in one embodiment of the present invention.

[0281] Figure 11 Schematic diagram of the treatment using Dsg3-specific S / F iTregs in Example 12. The number of Dsg3-specific B cells was measured in the spleen and lymph nodes using the ELISpot method.

[0282] Figure 12 The results of Example 12 are shown. The left panel shows the total B cell count, and the right panel shows the Dsg3-specific B cell count. sLN denotes sentinel lymph node, and Spleen denotes spleen.

[0283] Figure 13 Results show the titer of anti-Dsg3 antibodies elicited by Dsg3-specific S / F iTregs and the clinical severity in a pemphigus model. Dsg3-specific S / F iTregs suppress Dsg3-specific B cells, thereby inhibiting Dsg3 antibody production and clinical symptoms of pemphigus, and are therefore considered useful as antigen-specific treatments. DETAILED DESCRIPTION

[0284] Below, the best mode of the present invention will be represented and described. It should be understood that, throughout the scope of this specification, unless otherwise indicated, expressions in the singular also include the concept of their plural forms. Therefore, it should be understood that, unless otherwise indicated, articles in the singular (e.g., "a", "an", "the", etc. in English) also include the concept of their plural forms. In addition, it should be understood that, unless otherwise indicated, the terms used in this specification are used in the sense commonly used in the art. Therefore, unless otherwise defined, all terms and scientific and technical terms used in this specification have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In the event of inconsistency, this specification (including definitions) takes precedence.

[0285] Definitions of terms particularly used in this specification and / or basic technical contents are described below as appropriate.

[0286] In this specification, "about" refers to ±10% of the subsequent value. For example, "about 20" includes "18-22". A numerical range includes all values ​​between and including the values ​​at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" includes "18-33".

[0287] In this specification, when referring to gene names and their products, in contrast to conventional usage, when all capital letters are used, both the gene and the protein may be referred to. In some cases, the terms "FOXP3 gene" and "FOXP3 protein" may be used separately, for example, but when "FoxP3" is used, both the concept and the entity (the whole) of the gene or protein are referred to.

[0288] As used herein, "pemphigoid" refers to an autoimmune blistering disease in which lesions are found on the skin or mucous membranes. It is characterized by the formation of intraepidermal blisters caused by acantholysis, which is observed in histopathological studies due to the obstruction of intercellular adhesion in the epidermis. In immunopathological studies, autoantibodies directed against the surface of epidermal cell membranes are deposited in the skin tissue or observed in the circulating blood. It is known that the target antigen protein of pemphigus is desmoglein, a cadherin-type intercellular adhesion factor that plays an important role in intercellular adhesion in the epidermis.

[0289] Pemphigus is broadly classified into three types: pemphigus vulgaris, pemphigus foliaceus, and other types. Other types include paraneoplastic pemphigus, vegetative pemphigus, a subtype of pemphigus vulgaris, pemphigus erythematosus, a subtype of pemphigus foliaceus, pemphigus herpetiformis, and drug-induced pemphigus.

[0290] In this specification, "regulatory T cells" refer to T cells that are positive for FoxP3 expression. In this specification, they are sometimes also referred to as "Tregs". Tregs include endogenous regulatory T cells (Naturally Occurring Regulatory T cells: nTregs) and inducible regulatory T cells (Inducible Regulatory T cells: iTregs). Regulatory T cells can generally have various functions (such as immunosuppressive function).

[0291] In this specification, "inducible regulatory T cells (iTreg)" refers to regulatory T cells that are negative for IKZF2 (Helios) expression. iTregs are generally obtained by inducing differentiation from naive CD4-positive T cells.

[0292] In this specification, "nTregs (naturally occurring regulatory T cells)" refer to regulatory T cells that are positive for IKZF2 (Helios) and CTLA4 expression, and are generally cells present in a living body.

[0293] In this specification, "peripheral T cells" refer to T cells present outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues. In this specification, when referred to as "peripheral T cells", as long as the cell population contains peripheral T cells, it is not necessary to separate the T cells. In addition to T cells such as peripheral blood mononuclear cells (PBMC), cell fractions containing various lymphocytes can also be used.

[0294] In this specification, "flow cytometry" refers to a technique for measuring the number of particles, respective physical, chemical, and biological properties of cells, individuals, and other biological particles suspended in a liquid. The device using this technique is called a "flow cytometer." In the present invention, the "positive" and "negative" of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are determined by flow cytometry as commonly used in the art. More specifically, in flow cytometry, cells are arranged in a row and flowed, and the number of cells is counted by a spectroscopic method. For example, laser light is irradiated on cells labeled with fluorescence or luminescent enzymes, and the fluorescence or luminescent signals emitted thereby from the cells are detected by detectors such as photodiodes, thereby counting the number of target cells. In addition, the detection results of the detector can be imported into a computer to generate a two-dimensional graph for display. Thus, the presence or absence of target cells and their quantity, etc. can be easily grasped.

[0295] In this specification, "demethylation" refers to a methylation modification that removes a typically methylated adenine (e.g., position 6: m6A, position 1: m1A) or cytosine (e.g., position 5: m5C, position 3: m3C). Demethylation can be determined using methods known in the art, such as the bisulfite method.

[0296] As used herein, a "cell population" refers to a group of two or more cells, and may be, for example, a state in which cells aggregate together on a two-dimensional surface, or a cell mass formed by cells adhering to each other in three dimensions. Furthermore, a "cell population" may be composed of a single type of cell or may contain multiple types of cells.

[0297] In this specification, "stimulation" refers to stimulation via the TCR. For example, T cell stimulation includes stimulation using anti-CD3 antibodies and complexes containing the same, stimulation using peptides that bind to the TCR and complexes containing the peptides, stimulation using antigen-presenting cells, stimulation using both anti-CD3 antibodies and antigen-presenting cells, and stimulation using both peptides, proteins, and antigen-presenting cells.

[0298] In this specification, "dormant culture" means culturing cells in the absence of the above-mentioned stimulation.

[0299] (Preferred embodiment)

[0300] The preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is apparent that those skilled in the art can make appropriate changes within the scope of the present invention with reference to the description in this specification. In addition, the following embodiments of the present invention can be used alone or in combination.

[0301] The present invention relates to the pharmaceutical use of highly functional and stable inducible regulatory T cells (also referred to as highly functional and stable iTreg, HSFiTreg) for treating or preventing pemphigus.

[0302] In one aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. In another aspect of the present invention, a pharmaceutical composition is provided, comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive, and specifically reactive to desmoglein 3 (Dsg3).

[0303] Pemphigus is an autoimmune blistering disease characterized by lesions on the skin or mucous membranes. Pemphigus is characterized by the formation of intraepidermal blisters caused by acantholysis, a condition observed in skin histopathology, which disrupts intercellular adhesion in the epidermis. Immunopathologically, autoantibodies targeting the surface of epidermal cell membranes are deposited in the skin tissue or circulating in the blood. The target antigen protein of pemphigus is known to be desmoglein, a cadherin-type intercellular adhesion factor that plays an important role in intercellular adhesion in the epidermis.

[0304] Pemphigus is broadly classified into three types: pemphigus vulgaris, pemphigus foliaceus, and other types. Other types include paraneoplastic pemphigus, vegetative pemphigus, a subtype of pemphigus vulgaris, pemphigus erythematosus, a subtype of pemphigus foliaceus, pemphigus herpetiformis, and drug-induced pemphigus.

[0305] IgG autoantibodies found in pemphigus patients bind to desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). Desmoglein 1 and 3 are calcium-binding transmembrane glycoprotein components of desmosomes, the intercellular binding sites in stratified squamous epithelia. Desmoglein plays an important role in the adhesion of epidermal keratinocytes to epidermal keratinocytes. Pemphigus autoantibodies bind to desmoglein, hindering its adhesive function and causing epidermal cells to become fragmented, resulting in the formation of blisters in the epidermis.

[0306] Desmoglein 3 is a member of the desmoglein family and the cadherin superfamily of cell adhesion molecules. The precursor protein of desmoglein 3 is processed by proteolysis and converted into a mature glycoprotein. The gene encoding desmoglein 3 is located on chromosome 18 as a cluster along with other members of the desmoglein gene family.

[0307] In one embodiment of the present invention, the inducible regulatory T cells of the present invention can specifically respond to desmoglein 3 (Dsg3) to suppress immunity and inhibit skin inflammation.

[0308] In one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention may also have at least two characteristics selected from CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. Furthermore, in one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention may be at least CTLA4 positivity. Without intending to be limiting, the inducible regulatory T cells in the pharmaceutical composition of the present invention may be CD4 positivity or CD8 positivity.

[0309] In one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention may be at least CTLA4 positive and FoxP3 positive. In one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention may be at least CD172g positive and / or CD26 positive.

[0310] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, comprising CD172g-positive inducible regulatory T cells as an active ingredient. Since CD172g is a protein associated with tyrosine kinase and is involved in cell adhesion of neurons, etc. (cerebellar neuron adhesion, neurite outgrowth, and glial cell attachment), it is expected that CD172g-positive inducible regulatory T cells will activate functions related to adhesion, etc., and thus have a better effect on pemphigus.

[0311] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, comprising CD26-positive inducible regulatory T cells as an active ingredient. CD26, also known as DPP4, is a gene involved in glucose metabolism, insulin metabolism, and immune function (also significant in infectious diseases). Therefore, CD26-positive inducible regulatory T cells are expected to be more effective in treating pemphigus.

[0312] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, comprising NT5E-positive inducible regulatory T cells as an active ingredient. NT5E (CD73) is a cell membrane protein that catalyzes the conversion of extracellular nucleotides into membrane-permeable nucleosides. The encoded protein serves as a determinant of lymphocyte differentiation. Defects in this gene can lead to joint and arterial calcification.

[0313] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, which contains ITGAE (CD103)-positive inducible regulatory T cells as an active ingredient. ITGAE (CD103) encodes an α-integrin with an I domain, which undergoes post-translational cleavage in the extracellular domain to generate heavy and light chains bound by disulfide bonds. This protein binds to β7 integrin to form an E-cadherin-binding integrin known as human mucosal lymphocyte-1 antigen. This protein is preferentially expressed in human intestinal intraepithelial lymphocytes (IELs) and, in addition to its adhesion function, may also act as an auxiliary molecule for IEL activation. Therefore, it is expected that ITGAE (CD103)-positive inducible regulatory T cells will have a better effect on pemphigus.

[0314] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, comprising AREG-positive inducible regulatory T cells as an active ingredient. AREG is a member of the epidermal growth factor family and is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). This protein interacts with EGF / TGF-α receptors to promote the growth of normal epithelial cells and inhibit the growth of certain aggressive cancer cell lines. It also plays a role in the development of breast, egg, and bone tissue. This gene is associated with a skin phenotype similar to psoriasis and is also associated with other pathological conditions, including various cancers and inflammatory conditions.

[0315] The present invention provides a pharmaceutical composition for treating or preventing pemphigus, which contains CTLA4-positive inducible regulatory T cells as an active ingredient. CTLA4 belongs to the immunoglobulin superfamily and is a protein that transmits inhibitory signals to T cells. Membrane-bound CTLA4 functions as a disulfide-bonded homodimer, while soluble CTLA4 functions as a monomer. Mutations in this gene are believed to be associated with insulin-dependent diabetes mellitus, Basedow's disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-related orbitopathy, and other autoimmune diseases. Therefore, it is expected that CTLA4-positive inducible regulatory T cells will have a better effect on pemphigus.

[0316] FoxP3 expression in regulatory T cells can be induced in vitro by culturing CD4-positive T cells in a medium containing IL2 and TGFβ in the presence of anti-CD3 and anti-CD28 antibodies, followed by culturing in a medium containing IL2 and TGFβ. While several methods are known for inducing regulatory T cells from peripheral T cells, FoxP3 expression in induced regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has not been confirmed.

[0317] In recent years, a method for inducing DNA demethylation by adding ascorbic acid to the culture medium of induced regulatory T cells (Kasahara et al. Int. Immunol. (2017) 29 (10): 457-469) and a method without CD28 antibody stimulation (Mikami et al. Proc Natl Acad Sci US A. (2020) 117 (22): 12258-12268) has been developed. However, the expression of functional molecules in induced regulatory T cells produced by this method has not been confirmed, and sufficient immunosuppressive activity has not been obtained. There is also a clinical trial using induced regulatory T cells, which was conducted for GVHD, but the proportion of regulatory T cells in the cells used was low, and the effectiveness of preventing GVHD has not been confirmed so far (MacMillan et al., Blood Adv. (2021) 5 (5): 1425-1436).

[0318] In the present invention, the expression of FoxP3 is stable, and advantageously, a pharmaceutical composition containing induced regulatory T cells that stably maintain an immunosuppressive effect as an active ingredient can be provided. Such induced regulatory T cells can be produced, for example, by a method for producing induced regulatory T cells described elsewhere in the specification of this application. For example, the present invention can provide a pharmaceutical composition, wherein the induced regulatory T cells are obtained by a method comprising the following steps: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) culturing the cells obtained in step (a) in a culture medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) culturing the cells obtained in step (c) in a culture medium containing IL-2 for at least about 1 to about 3 days.

[0319] The inducible regulatory T cells in the pharmaceutical composition of the present invention have an inducible regulatory T cell-specific demethylation state. The resulting regulatory T cells are in a regulatory T cell-specific demethylation state, for example, can be confirmed by demethylation of the CNS2 site of the FoxP3 gene (FOXP3 (all in italics). Such a demethylation state can be an indicator of a stable type, so the inducible regulatory T cells in the pharmaceutical composition of the present invention can be shown as stable inducible regulatory T cells by confirming the demethylation state.

[0320] In this specification, the immunosuppressive activity or immunosuppressive effect of the inducible regulatory T cells in the pharmaceutical composition of the present invention can be confirmed by, for example, measuring the Cell Trace Violet intensity in the responding T cells. The inducible regulatory T cells in the pharmaceutical composition of the present invention can stably provide immunosuppressive activity or immunosuppressive effect. For example, the inducible regulatory T cells of the present invention can provide immunosuppressive activity or immunosuppressive effect in at least about 2 weeks. The inducible regulatory T cells in the pharmaceutical composition of the present invention can have a higher immunosuppressive effect than previous regulatory T cells (including inducibility and endogenous). Therefore, the inducible regulatory T cells in the pharmaceutical composition of the present invention can also be referred to as functional or highly functional inducible regulatory T cells.

[0321] In one embodiment, the inducible regulatory T cells in the pharmaceutical composition of the present invention can stably express FoxP3. Therefore, the inducible regulatory T cells in the pharmaceutical composition of the present invention can also be referred to as stable inducible regulatory T cells. The inducible regulatory T cells in the pharmaceutical composition of the present invention are highly functional and stable in one aspect and can be referred to as highly functional stable inducible regulatory T cells (HSF iTreg).

[0322] In one embodiment of the present invention, whether the marker in the inducible regulatory T cells of the present invention is positive can be carried out by measuring the positive rate using flow cytometry. For example, flow cytometry can be used for analysis, and it is determined whether it is positive according to the ratio of cells showing the antigen expression amount above the benchmark. According to the expression intensity of the cell surface marker, it can also be divided into negative, weak positive, medium positive, strong positive (weak positive, medium positive, and strong positive are collectively referred to as "positive") and the like. For example, according to the setting of the equipment, when the value of the fluorescence intensity median of each marker / the fluorescence intensity median of the negative control (using isotype control antibody staining) is less than 5, more than 5 and less than 10, more than 10 and less than 30, or more than 30, it can also be set to negative, weak positive, medium positive, and strong positive respectively. Such determination can be determined as described in (using flow cytometry positive rate determination), but the present invention is not limited thereto.

[0323] In one embodiment of the present invention, the expression intensity of the cell surface markers in the inducible regulatory T cells of the present invention, for example, according to the results of analysis by BD FACSLyric flow cytometer (BD Biosciences) using samples prepared as described in the examples of the present application, can be an expression intensity of 10 2 The above are weak positive, 10 3 Above is moderate positive, 10 4 Above is a strong positive, or 10 3 Above is a strong positive, below is a weak positive, or 10 2 A value above 10 is a strong positive, and a value below 10 is a weak positive. Such determination can be appropriately set according to the experimental conditions, experimental purpose, cell type, and device setting conditions, and the present invention is not limited thereto. In addition, when analysis is performed using a flow cytometer other than the BD FACSLyric flow cytometer, the expression intensity in other devices corresponding to the expression intensity when analyzed using the BD FACSLyric flow cytometer can also be calculated, and a value representing the expression intensity can be appropriately found according to the device used.

[0324] In one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention can be induced from any cells, but preferably can be induced from human peripheral blood T cells or T cells derived from human tissues.

[0325] In one embodiment of the present invention, the inducible regulatory T cells or cell populations thereof in the pharmaceutical composition of the present invention may be human cells, and may include inducible regulatory T cells or cell populations induced by a prescribed method using T cells obtained from human peripheral blood as materials. Therefore, in one embodiment of the invention, the inducible regulatory T cells in the pharmaceutical composition of the present invention preferably include inducible regulatory human T cells. The properties of human cells and mouse cells are significantly different, and even if the same cell surface markers are present, the cells cannot be considered to have the same properties. For example, in the field of inducible regulatory T cells, the applicable technologies are different in mice and humans. In the case of mice, antigen-unsensitized lymphocytes account for the majority, while in the case of humans, the degree of antigen sensitization and activation of T cells in peripheral blood vary. Therefore, in mice, although it is possible to directly produce highly functional and stable inducible regulatory T cells from T cells collected from mouse lymphoid tissue, this is difficult to do in humans, and cells cannot be considered to be the same simply based on the presence of cell surface markers.

[0326] In one embodiment of the present invention, the pharmaceutical composition of the present invention may comprise a T cell population, wherein about 50% or more of the cells in the T cell population are inducible regulatory T cells as described elsewhere in this specification. In one embodiment, with respect to the cell population of the present invention, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the T cells in the cell population may be inducible regulatory T cells as described elsewhere in this specification.

[0327] In one embodiment of the present invention, the T cells in the cell population of the present invention may be regulatory T cells. In this case, of the regulatory T cells in the cell population of the present invention, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more may be inducible regulatory T cells as described elsewhere in this application.

[0328] In one embodiment of the present invention, the cell population of the present invention may contain cells other than T cells, but preferably, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, or more than about 99% of the cell population of the present invention can be T cells, and preferably more than about 90% can be T cells.

[0329] In one aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the proportion of CTLA4 positivity and FoxP3 positivity of the cell population is approximately 50% or greater. In one embodiment, the proportion of CTLA4 positivity and FoxP3 positivity of the cell population in the pharmaceutical composition of the present invention may be approximately 60% or greater, approximately 65% ​​or greater, approximately 70% or greater, approximately 75% or greater, approximately 80% or greater, approximately 85% or greater, approximately 90% or greater, approximately 95% or greater, approximately 97% or greater, or approximately 99% or greater.

[0330] In one embodiment of the present invention, in the cell population in the pharmaceutical composition of the present invention, at least the proportion of CD172g positivity and / or CD26 positivity may be about 50% or more, respectively. In one embodiment, the proportion of CD172g positivity and / or CD26 positivity in the cell population in the pharmaceutical composition of the present invention may 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.

[0331] In one embodiment of the present invention, the proportion of cells in the pharmaceutical composition of the present invention that are strongly positive for FoxP3 may be about 50% or more. In one embodiment, the proportion of cells in the pharmaceutical composition of the present invention that are strongly positive for FoxP3 may be about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more. Without intending to be limiting, in the cell population in such a pharmaceutical composition, the expression intensity of the cell surface marker can be determined by measuring the positive rate using flow cytometry as described above.

[0332] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the cell population has a FoxP3-positive ratio of approximately 50% or greater. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may comprise, based on cell number, approximately 50% or greater, approximately 60% or greater, approximately 65% ​​or greater, approximately 70% or greater, approximately 75% or greater, approximately 80% or greater, approximately 85% or greater, approximately 90% or greater, approximately 95% or greater, approximately 97% or greater, or approximately 99% or greater of FoxP3-positive inducible regulatory T cells.

[0333] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CTLA4-positive proportion of the cell population is about 50% or more. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may comprise, based on cell number, 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 CTLA4-positive inducible regulatory T cells.

[0334] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the NT5E-positive ratio of the cell population is about 50% or more. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain, based on cell number, 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 NT5E-positive inducible regulatory T cells.

[0335] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the ITGAE (CD103)-positive ratio of the cell population is about 50% or more. Without intending to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may comprise, based on the number of cells, 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 ITGAE (CD103)-positive inducible regulatory T cells.

[0336] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the cell population has an AREG-positive ratio of approximately 50% or greater. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may comprise, based on cell number, approximately 50% or greater, approximately 60% or greater, approximately 65% ​​or greater, approximately 70% or greater, approximately 75% or greater, approximately 80% or greater, approximately 85% or greater, approximately 90% or greater, approximately 95% or greater, approximately 97% or greater, or approximately 99% or greater AREG-positive inducible regulatory T cells.

[0337] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, the pharmaceutical composition comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CD172g-positive ratio of the cell population is about 50% or more. Without intending to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may comprise, based on the number of cells, 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 T cells.

[0338] In another aspect of the present invention, a pharmaceutical composition for treating or preventing pemphigus is provided, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the cell population has a CD26-positive ratio of about 50% or more. While not intended to be limiting, in one embodiment, the cell population in such a pharmaceutical composition may contain, based on cell number, 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 T cells.

[0339] As described above, since the properties of human cells and mouse cells are significantly different, even if the induction conditions are the same, the induction ratios of specific cells in mouse cells and human cells are different. In the present invention, when induced by a prescribed induction method, a cell population with a positive ratio of a prescribed cell surface marker of approximately 50% or more can be obtained, and preferably a cell population with "CTLA4 positivity and FoxP3 positivity ratios of approximately 50% or more, respectively" can be obtained.

[0340] CTLA4 is a molecule localized within cells, at least not at a level that can be used as an indicator for concentration and purification. Furthermore, because FoxP3 is a transcription factor and a molecule present entirely within cells, it is completely undetectable on the surface and cannot be used for purification. In other words, CTLA4 and FoxP3 are markers expressed "inside" T cells, so sorting and concentration using CTLA4 and / or FoxP3 as indicators cannot yield a cell population with approximately 50% or more CTLA4-positive and / or FoxP3-positive cells. Therefore, using conventional methods to obtain a cell population with a CTLA4-positive and FoxP3-positive ratio of less than approximately 50%, respectively, and using such a cell population as a starting material and concentrating using CTLA4 and / or FoxP3 as indicators, it is impossible to obtain a cell population with approximately 50% or more of these cells, respectively.

[0341] In one embodiment of the present invention, the inducible regulatory T cells in the pharmaceutical composition primarily perform the immunosuppressive function of Tregs. By comprising at least half of a cell population, these cells can stably achieve a medically effective immunosuppressive effect, which is also crucial for both technical and medical outcomes. Specifically, by containing at least 50% of T cells that exert a medically effective immunosuppressive effect, a stable cell preparation can be provided. This effect is extremely important medically, not only in terms of quantity and value, but also in terms of quality, such as the viability of the preparation.

[0342] In one embodiment of the present invention, in the cell population in the pharmaceutical composition of the present invention, the inducible regulatory T cells may also have the characteristics of ITGAE (CD103) positive, NT5E positive and / or AREG positive. These cell markers are genes that play an important role in immunosuppressive function, and due to the high expression of these markers, after the cells are administered, better functional stability can be maintained compared with previous inducible regulatory T cells. For example, CD103 is important for the migration of regulatory T cells to the site of inflammation, NT5E (CD73) is important for the production of adenosine with immunosuppressive ability, and AREG is important for the regeneration of tissues by regulatory T cells. The cell population of the present invention can achieve the effect of maintaining functional stability by high expression of these markers.

[0343] In one aspect of the present invention, a pharmaceutical composition comprising the inducible regulatory T cells or cell populations of the present invention is provided. In addition, in another aspect, a regenerative medical material or product comprising the inducible regulatory T cells or cell populations of the present invention is provided. These drugs, regenerative medical materials or products can be used together with culture media or any other additives used in the art. As such a culture medium, a culture medium to which essential factors have been added 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. When provided as such a product, DMSO and the like may be included.

[0344] In one embodiment, the pharmaceutical composition of the present invention can be administered in a variety of dosages and routes, but is preferably administered by injection. In one embodiment, the pharmaceutical composition of the present invention can be administered in a dose of at least about 1×10 3 The manner in which the amount of each is administered comprises the inducible regulatory T cells described elsewhere in this specification.

[0345] In one embodiment, after the pharmaceutical composition of the present invention is administered to a patient, if there is no effect or insufficient effect, a further additional administration may be performed. Whether there is no effect or insufficient effect can be determined, for example, by the degree of inhibition of pemphigus symptoms as an indicator. In one embodiment, when additional administration is performed, the administration may be performed at least about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after the initial administration.

[0346] (Manufacturing Method)

[0347] The induced regulatory T cells contained in the pharmaceutical composition of the present invention are highly functional, stable induced regulatory T cells. The method for producing such a pharmaceutical composition will be described in detail below in this specification.

[0348] In one aspect of the present invention, a method for producing induced regulatory T cells is provided, comprising: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal culture medium for about 1 to about 5 days; (b) allowing the cells obtained in step (a) to rest and culture in a culture medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal culture medium for about 1 to about 5 days; and (d) allowing the cells obtained in step (c) to rest and culture in a culture medium containing IL-2 for at least about 1 to 3 days. In the present invention, the induced regulatory T cells in the pharmaceutical composition of the present invention can be produced using either CD4-positive T cells or CD8-positive T cells as raw materials. In one embodiment, the induced regulatory T cells in the pharmaceutical composition of the present invention can also be produced using a mixture of CD4-positive T cells and CD8-positive T cells as raw materials.

[0349] In one aspect of the present invention, a method for manufacturing a pharmaceutical composition for treating or preventing pemphigus is provided, wherein the pharmaceutical composition comprises as an active ingredient inducible regulatory 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, and the inducible regulatory T cells are obtained by a method comprising the following steps: (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal culture medium for about 1 to about 5 days; (b) subjecting the cells obtained in step (a) to dormant culture in a culture medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal culture medium for about 1 to about 5 days; and (d) subjecting the cells obtained in step (c) to dormant culture in a culture medium containing IL-2 for at least about 1 to 3 days.

[0350] In one embodiment, the method of the present invention may include the steps of culturing human peripheral T cells in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation, culturing them in a medium containing IL-2 in the absence of anti-CD3 antibodies, and culturing them again in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation, and regulatory T cells can be produced from human peripheral T cells (including CD4-positive T cells or CD8-positive T cells).

[0351] In one embodiment, T cell stimulation is stimulation for obtaining Tregs, and there is no particular limitation as long as it is stimulation via the TCR. For example, T cell stimulation may include stimulation using anti-CD3 antibodies and complexes containing them, stimulation using peptides that bind to TCRs and complexes containing peptides, stimulation using antigen-presenting cells, stimulation using both anti-CD3 antibodies and antigen-presenting cells, and stimulation using both peptides, proteins, and antigen-presenting cells. However, as long as the stimulation can obtain Tregs, the culture medium and conditions are not particularly limited.

[0352] In one embodiment, as dormant culture, as long as cells are cultured in a state without the aforementioned stimulation, the medium and conditions are not particularly limited.

[0353] As used herein, "anti-CD3 antibody stimulation" refers to specific stimulation of the CD3 receptor on cells. Examples of CD3 stimulation include anti-CD3 agonist antibodies. Commercially available research reagents can be used as anti-CD3 agonist antibodies, or they can be prepared using conventional methods. Anti-CD3 antibodies derived from animals such as mice, rabbits, goats, and cattle, as well as human antibodies, can be used.

[0354] In one embodiment, the method of the present invention, once iTregs are induced (stimulation of T cells to induce demethylation) (step a), the cells are allowed to recover by resting and culturing for 1-3 days by replacing the culture medium (step b), and then the T cells are stimulated again (step c) to induce demethylation, thereby obtaining functional and stable inducible regulatory T cells. It is common knowledge that T cells typically undergo apoptosis after two stimulations, but the method of the present invention has found that by performing "resting culture" and "second stimulation," functional and stable inducible regulatory T cells can be obtained without apoptosis.

[0355] That is, in the method of the present invention, functional and stable inducible regulatory T cells can be obtained by performing T cell stimulation, resting culture, and subsequent restimulation. Therefore, if the inducible regulatory T cells obtained in this way or a cell population containing such inducible regulatory T cells can achieve the desired effects of the present invention. Therefore, in one embodiment of the present invention, the type of culture medium or stimulus used for culture is not particularly limited, and functional and stable inducible regulatory T cells can be obtained by using a culture medium of any composition and any type of stimulus.

[0356] In one embodiment, the culture medium used in each step may further contain retinoic acid and / or ascorbic acid. Preferably, ascorbic acid may be contained in the culture medium. In one embodiment, a CDK8 inhibitor, a CDK19 inhibitor and / or a CDK8 / 19 inhibitor may be contained in the culture medium. Preferably, a CDK8 inhibitor, a CDK19 inhibitor and / or a CDK8 / 19 inhibitor may be contained in the culture medium.

[0357] In one embodiment of the present invention, the first basal medium and the second basal medium may each independently contain at least one, at least two, at least three, at least four, at least five, or all of the factors selected from anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors, and ascorbic acid. In other embodiments, the first basal medium and the second basal medium may each independently contain anti-CD3 antibodies, TGF-β1, IL-2, retinoic acid, CDK8 inhibitors, CDK19 inhibitors, CDK8 / 19 inhibitors, and ascorbic acid. The concentrations of these components may be conventional concentrations used in this field. In one embodiment, the concentration of the CDK8 inhibitor, CDK19 inhibitor and / or CDK8 / 19 inhibitor that can be used can be any suitable concentration that can be used in the art. For example, when SenexinA is used, it can be about 0.1 μM or more, about 0.5 μM or more, about 1 μM or more, about 2 μM or more, about 3 μM or more, about 4 μM or more, about 5 μM or more, about 6 μM or more, about 7 μM or more, about 8 μM or more, about 9 μM or more, about 10 μM or more, about 12 μM or more, about 14 μM or more, about 16 μM or more, about 18 μM or more, about 20 μM or more, etc., but is not limited to these concentrations. Those skilled in the art can make appropriate changes based on other culture medium compositions.

[0358] In one embodiment, regulatory T cells are manufactured from human peripheral T cells by the method of the present invention. Peripheral T cells include initial regulatory T cells, CD4-positive T cells, CD8-positive T cells, etc. Regulatory T cells can be induced from a culture containing a variety of T cells, or specific cells such as CD4-positive T cells and CD8-positive T cells can be isolated from these cells to induce regulatory T cells. In addition, regulatory T cells can also be induced based on the separation of T cells specific for a specific antigen. Therefore, the inducible regulatory T cells of the present invention include CD4-positive T cells and CD8-positive T cells. It should be noted that in the present specification, when "CD4-positive" or "CD4+", unless otherwise specified, it refers to a single positive cell that is CD4-positive and CD8-negative. In addition, in the manufacturing method of the present invention, any CD4-positive or CD8-positive T cell can be used as a raw material. After generating the inducible regulatory T cells, as long as no special operation is performed, the characteristics of CD4-positive or CD8-positive can be maintained.

[0359] In one embodiment, in the method of the present invention, the antibody can be added to the culture medium, or the antibody can be fixed on the inner wall of the culture container or on the surface of an insoluble carrier. The insoluble carrier is a component that can physically or chemically bind to the anti-CD3 antibody, and a carrier that is insoluble in aqueous solution can be used. As materials that can physically adsorb the anti-CD3 antibody, synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, or glass can be mentioned. The shape of the insoluble carrier is not particularly limited, and for example, a plate shape, a bead shape, a container shape, etc. can be used. The amount of anti-CD3 antibody varies depending on the titer and source of the antibody used, but it can be appropriately set to provide sufficient stimulation for inducing regulatory T cells.

[0360] In one embodiment, in the method of the present invention, a medium obtained by adding essential factors to a basal medium for animal cell culture can be used for cell culture. Examples of basal medium for animal cell culture that can be used in the method of the present invention include Iscove's modified Eagle's Medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), RPMI 1640 medium, Fischer's medium, mixed mediums thereof, and mediums in which some of the compositions are modified.

[0361] In the basal medium, serum (e.g., fetal bovine serum (FBS)) or serum-free can be contained. In the serum-free medium, as needed, one or more serum substitutes such as albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut serum substitute (KSR) (serum substitute during 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'-mercaptoglycerol, thioglycerol (Monothioglycerol) can be included. The basal medium may also contain one or more substances such as lipids (e.g., chemically defined lipid concentrates), amino acids, L-glutamine, GlutaMAX (ThermoFisher 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 their equivalents.

[0362] In one embodiment, the basal medium may be RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0363] In the method of the present invention, cells can be cultured under conventional animal cell culture conditions. The culture temperature is not limited to about 30-40°C, preferably about 37°C. Culture is preferably carried out in an atmosphere of air containing CO2, preferably with a CO2 concentration of about 2-5%.

[0364] In the methods of the present invention, the anti-CD3 antibody can be added directly to the culture medium, or it can be immobilized on the inner wall of the culture vessel or on the surface of an insoluble carrier. The amount of anti-CD3 antibody varies depending on the potency and source of the antibody used, but can be appropriately adjusted to provide sufficient stimulation for inducing regulatory T cells.

[0365] Examples of TGFβ used include TGFβ1, TGFβ2, and TGFβ3, with TGFβ1 being an example. The concentration of TGFβ is not particularly limited and can be determined appropriately by a person skilled in the art. When TGFβ1 or TGFβ3 is used as TGFβ, the concentration in the culture medium is not particularly limited and can be 0.25 to 25 ng / mL, for example, approximately 10 ng / mL.

[0366] The concentration of IL-2 in the culture medium used is not limited and may be about 5 U / mL to about 500 U / mL, for example, about 100 U / mL.

[0367] Retinoic acid and / or ascorbic acid may also be added to the culture medium used in the present invention. Preferably, ascorbic acid is included in the culture medium. The concentration of ascorbic acid is not limited and is about 1 to about 100 μg / mL, for example, about 10 μg / mL.

[0368] The culture medium used in the present invention may further include a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor. Any inhibitor may be used as the CDK8 inhibitor, the CDK19 inhibitor, and / or the CDK8 / 19 inhibitor, 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 a salt, hydrate, or solvate thereof, or U.S. Patent No. 8598344, WO2013 / 001310, WO2013 / 040153, WO2013 / 040154, or the like. 116786, WO2014 / 029726, WO2014 / 063778, WO2014 / 072435, WO2014 / 090692, WO2014 / 106606, WO2014 / 123900, WO2014 / 154723, WO2014 / 194245, WO2015 / 049325, WO2015 / 100420, WO2015 / 144290, WO2015 / 159937, WO2015 / 159938, WO2016 / 009076 or WO2018 / 139660, etc. As an example, Senexin A or AS2863619 is exemplified, but the present invention is not limited thereto. The concentration of the CDK8 inhibitor, CDK19 inhibitor and / or CDK8 / 19 inhibitor that can be used can be any suitable concentration used in the art, or any suitable concentration described in the above-mentioned literature. Those skilled in the art can make appropriate changes based on other culture medium compositions.

[0369] In the method of the present invention, regulatory T cells are induced by stimulating human peripheral T cells with anti-CD3 antibodies in a medium containing TGFβ and IL-2. Furthermore, after resting in a medium containing IL-2 but not anti-CD3 antibodies, the cells are stimulated again with anti-CD3 antibodies to induce regulatory T cells with high immunosuppressive function. The high immunosuppressive function of the resulting induced regulatory T cells can be confirmed by, for example, comprehensive gene expression analysis using RNA sequencing and in vitro cell proliferation inhibition assays.

[0370] In one embodiment of the present invention, the number of culture days in the step (a) of stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days can be appropriately set by those skilled in the art and is not particularly limited. For example, it can also be about 3 days.

[0371] In one embodiment, the number of days of culture in the dormant culture step of step (b) can be appropriately set by those skilled in the art and is not particularly limited. For example, it may be approximately 2 days.

[0372] In one embodiment, the number of days of culture in the culturing step in the second basal medium of step (c) can be appropriately set by those skilled in the art and is not particularly limited. For example, it may be approximately 3 days.

[0373] In addition, in one embodiment, the number of days of culture in the dormant culture step of step (d) can be appropriately set by those skilled in the art, is not particularly limited, and for example, can be about 2 days. In one embodiment, by culturing in the presence of IL-2 and without stimulation, regulatory T cells with induced regulatory T cell-specific demethylation state can be proliferated. The culture medium can further include ascorbic acid, and further cultured in a culture medium comprising IL-2, a stable regulatory T cell culture of the induced regulatory T cells can be obtained.

[0374] To isolate regulatory T cells from the resulting cell culture containing regulatory T cells, cells can be separated using conventional methods based on cell surface markers unique to regulatory T cells. For example, a FoxP3-positive fraction can be selected using a cell sorter. Alternatively, regulatory T cells specific for a specific antigen can be isolated as needed.

[0375] The induced regulatory T cells obtained by the method of the present invention are expected to be used to treat human inflammatory diseases, such as autoimmune diseases or allergies.

[0376] (Positive rate determined by flow cytometry)

[0377] In one embodiment, the positive rate determination using flow cytometry can be performed as follows.

[0378] Prepare

[0379] Fixation / permeabilization concentrate (hereinafter referred to as buffer) (eBio Science, 00-5123-43)

[0380] Fixation / permeabilization diluent (hereinafter referred to as diluent) (eBio Science, 00-5223-56)

[0381] Permeabilization buffer (10×) (eBio Science, 00-833-56)

[0382] FOXP3 monoclonal antibody (236A / E7), PE (hereinafter referred to as anti-FOXP3 antibody) (eBio Science, 12-4777-42)

[0383] Mouse IgG1κ isotype control (P3.6.2.8.1), PE (hereinafter referred to as PE control) (eBio Science)

[0384] BV421, mouse, anti-human, CD152 (hereinafter referred to as anti-CTLA4 antibody) (BD)

[0385] BV421 mouse IgG2a, kappa isotype control (hereinafter referred to as BV421 control) (BD)

[0386] CD4 monoclonal antibody (RPA-T4), APC (hereinafter referred to as anti-CD4 antibody) (eBio Science)

[0387] Mouse IgG1κ isotype control (P3.6.2.8.1), APC (hereinafter referred to as APC control) (eBio Science)

[0388] ·D-PBS(Nacalai Tesque, 14249-95)

[0389] FBS (HyClone, SH30084.03)

[0390] ·0.5mol / l-EDTA solution (pH 8.0) (Nacalai Tesque, 06894-14)

[0391] ·MilliQ water

[0392] Centrifuge

[0393] Safety cabinets

[0394] Micropipettes (P200, P1000)

[0395] 5 mL polystyrene tube (hereinafter referred to as special tube) (Falcon, 352008)

[0396] Nylon mesh (65μm) (Kyojin Riko Co., Ltd., Japan, PP-65N)

[0397] Reagent preparation

[0398] ※Fixation buffer (use 100μL per sample)

[0399] Mix the buffer and diluent in a ratio of 1:3.

[0400] ※Permeabilization buffer

[0401] Permeabilization buffer (10×) was diluted 10-fold with MilliQ water.

[0402] ※FACS buffer (when preparing 500 mL)

[0403] D-PBS 489 mL

[0404] FBS 10 mL (final concentration 2%)

[0405] 1 mL of 0.5 mol / l EDTA solution (final concentration 1 mM)

[0406] After preparation, the above reagents should be stored at 4°C or on ice.

[0407] method

[0408] (1) Add 500 μL of FACS buffer to a 1.5 mL tube.

[0409] (2) 1x10 6 The final product was added to the tube in (1) and gently suspended using a micropipette.

[0410] (3) Centrifuge at 500 × g and 4°C for 5 minutes.

[0411] (4) After removing the supernatant with an aspirator, add 100 μL of fixation buffer and pipette gently. → Be careful not to create bubbles.

[0412] (5) Place on ice in the dark for at least 30 minutes to allow for fixation. → 45 minutes is also acceptable.

[0413] (6) After fixation, add 1 mL of permeabilization buffer to the tube and gently pipette.

[0414] (7) Prepare new tubes of 1.5 ml according to the number of samples.

[0415] (8) Aliquot 500 μL of each sample for control and antibody staining.

[0416] (9) Centrifuge at 500 × g and 4°C for 5 minutes.

[0417] (10) During centrifugation, anti-FOXP3 antibody (stock concentration = 0.05 mg / ml), anti-CTLA4 antibody (Lot: 0030269 stock concentration = 0.2 mg / ml), and anti-CD4 antibody (stock concentration = 0.1 mg / ml) were diluted 100-fold with permeabilization buffer to prepare a solution (hereinafter referred to as the antibody preparation solution). For control samples, PE control (stock concentration = 0.1 mg / ml), BV421 control (stock concentration = 0.2 mg / ml), and APC control (stock concentration = 0.1 mg / ml) were adjusted to the same concentration as the corresponding pigment antibody (hereinafter referred to as the control solution).

[0418] *CTLA4 staining was not performed in the In-Process Control Test.

[0419] (11) After removing the supernatant with an aspirator, add 100 μL of the antibody preparation solution to the antibody-stained sample and 100 μL of the control solution to the control sample, and gently pipette. → Be careful not to create bubbles.

[0420] (12) Place on ice in the dark for 60 minutes to allow for fixation.

[0421] (13) Start the measuring device during staining.

[0422] (14) After staining, add 1 mL of FACS buffer and pipette gently.

[0423] (15) Centrifuge at 500 × g and 4°C for 5 minutes. → Remove the supernatant with an aspirator and repeat steps (14) and (15) for washing.

[0424] (16) After removing the supernatant with an aspirator, add 500 μL of FACS buffer and gently pipette.

[0425] (17) Use tweezers to place the nylon mesh into a dedicated tube and filter the suspension.

[0426] (18) Analyze using any flow cytometer. The number of cells recovered should be 10,000 or more. In calculating the target antigen positivity rate, a baseline is drawn such that the positivity rate of the control sample is less than 5%, and the proportion of cells expressing antigen above the baseline is regarded as the positivity rate.

[0427] Remark

[0428] The reagents used for fixation and staining can also be purchased as a set of three as the "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.: 00-5523).

[0429] Any device settings are acceptable as long as they do not deviate from a level that clearly indicates that normal measurement cannot be performed from a general, scientific perspective. Clear deviations include situations where various signals from the target cell population fall below the set fluorescence threshold, or where various signal values ​​from control samples or measurement samples fall below or exceed the instrument's normal measurement limits.

[0430] (Immunosuppressive activity assay)

[0431] The cells of the present invention may have immunosuppressive activity. Immunosuppressive activity can be measured by various methods.

[0432] In one embodiment, as described in the examples, it is possible to determine whether the immune response caused by the responding T cells is suppressed by measuring the cell proliferation. Preferably, in the present invention, it is desirable to use an in vivo model to confirm this. For example, tissue can be recovered from a model mouse to which the inducible regulatory T cells of the present invention have been administered for a certain period of time, and its activity can be determined based on whether immunosuppression is confirmed in the tissue. Confirmation of immunosuppression in the tissue can be achieved by various methods, for example, the presence or absence of immunosuppression can also be confirmed by staining analysis of the tissue.

[0433] (General Technology)

[0434] The molecular biology methods, biochemical methods and microbiological methods used in this specification are well known and commonly used in the art, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its third edition (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). ofMethods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al (1999).PCR Applications: Protocols for Functional Genomics, Academic Press, Experimental Medicine Special Issue "Gene Introduction & Expression Analysis Experimental Methods", Yangtusha, 1997, etc., the relevant parts (possibly all) of which are incorporated herein by reference.

[0435] DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes can be used, for example, by gene synthesis or fragment synthesis services provided by GeneArt, GenScript, Integrated DNA Technologies (IDT), and the like. In addition, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. Let al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press. Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., and the relevant parts of these are cited as reference in this specification.

[0436] In this specification, "or" is used when "at least one or more" of the items listed in the text can be used. The same applies to "or". When this specification explicitly states "within a range" of "two values", the two values ​​themselves are also included in the range.

[0437] References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as if each were specifically set forth.

[0438] The present invention has been described above by way of preferred embodiments for ease of understanding. The present invention will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the specific embodiments and examples described in this specification, but is defined solely by the claims.

[0439] Example

[0440] The inducible regulatory T cells of the present invention can be produced by using Figure 1 The model mice shown are described. Specifically, Dsg3-deficient mice were immunized with Dsg3 protein to generate Dsg3-reactive lymphocytes, which were then administered to Rag2-deficient mice to create a pemphigus model. Dsg3-specific TCRs were cloned from Dsg3-reactive T cells contained in these Dsg3-reactive lymphocytes, and purified T cells from these TCR transgenic mice (Dsg3H1 mice) were administered to Rag2-deficient mice to create a Dsg3-specific dermatitis model mouse.

[0441] (Example 1: Preparation of Dsg3-specific iTregs)

[0442] Sorting CD4 from H1 mice + CD25 - CD62L hi CD44 lo TCRVb6 + Initial T cells were isolated and S / F iTregs were produced. The method is as follows. The purified T cells were stimulated for 3 days in a basal culture medium containing anti-CD3 antibody (100 μL of antibody at a concentration of 10 μg / mL was added to each well and allowed to stand at room temperature for 60 minutes to solidify in the container), hIL-2 (50 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), and AS2863619 (1 μM). The cells were then cultured in a dormant culture medium containing hIL-2 (100 U / ml) for 2 days. After 2 days, the culture medium was replaced in the same manner and the cells were cultured in a dormant culture medium for another 2 days. After 2 days, the cells were stimulated again in a basal medium containing anti-CD3 antibody (100 μL of antibody at a concentration of 10 μg / mL was added to each well and allowed to stand at room temperature for 60 minutes to solidify in the container), hIL-2 (100 U / ml), hTGFβ1 (2.5 ng / ml), retinoic acid (1 μM), AS2863619 (1 μM), and ascorbic acid (10 μg / mL) for 2 days. The cells were then cultured in a basal medium containing hIL-2 (100 U / ml) for 2 days. After 2 days, the medium was replaced in the same manner and the cells were cultured in a basal medium for another 2 days. The cells were analyzed by flow cytometry ( Figure 2Left) The expression of Foxp3 and CD25 in regulatory T cells obtained 2 days later was analyzed by bisulfite method ( Figure 2 Right) Analysis of the demethylation status of Treg-specific demethylation regions.

[0443] The demethylation of Foxp3 gene was 87.5%, which enabled the production of functionally stable Dsg3-specific iTreg.

[0444] (Example 2: Dsg3-specific immunosuppression in vitro)

[0445] CD4+ naive T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Thermo Scientific) using the following protocol. Cells suspended in PBS were plated at 1×10 6 Incubate 500 μl of 4°C RPMI medium at room temperature for 5 minutes. Incubate on ice for 20 minutes. Wash the cells once and transfer 4 × 10 4 labeled cells in the presence of 2 μM Dsg3H1 peptide and 4×10 3 CD11c + antigen-presenting cells and 2×10 3 4×10 3 1.3×10 4 4×10 4 WTS / FiTreg, H1 S / Fi iTreg, and WT nTreg were co-cultured. Cell proliferation was confirmed by flow cytometry after 72 hours with CFSE. Figure 3 As shown in the Figure 3, H1 S / F iTreg (Dsg3-specific S / F iTreg) exhibited a higher inhibitory effect on H1 T cells under Dsg3 peptide stimulation compared with WT (wild type) nTreg or WT (wild type) S / F iTreg.

[0446] (Example 3: Dsg3-specific immunosuppression in vivo: dermatitis model)

[0447] H1 naive T cells were added per 2 × 10 5 Dissolve in 200 μl PBS, from Rag2 - / - The Dsg3-specific dermatitis model was established by intravenous injection into the tail vein of mice. One week later, WT (wild type) S / F iTreg or H1 S / F iTreg were injected into the tail vein of mice at 2×10 4 Dissolve in 200 μl PBS, from Rag2 - / -The mice were injected intravenously into the tail vein to observe the therapeutic effect. iTregs were not administered to control mice. The therapeutic effect was evaluated weekly using the dermatitis score (see Table 1).

[0448] Dermatitis score

[0449] [Table 1]

[0450]

[0451] After 7 weeks, the mouse's palate was removed and fixed in 10% formaldehyde for 24 hours. The specimens were then dehydrated, defatted, and paraffin-infiltrated using an automatic embedding device. Paraffin blocks were prepared using a paraffin block maker. Thin sections of 4 μm thickness were cut using a microtome. The thinly sliced ​​specimens on glass slides were spread in a paraffin spreader set at 40°C and dried. The specimens were deparaffinized using xylene and then dexyleneized using pure ethanol. After washing with running water, the specimens were transferred to purified water. Hematoxylin solution* 1 Stain for 3 to 5 minutes. Stain 10 dips with 0.05% lithium carbonate and wash with running water for 3 minutes. *2 Stain. Dehydrate with anhydrous ethanol. Transparent with anhydrous xylene and seal with an automatic sealing device.

[0452] *1. Hematoxylin

[0453] Hematoxylin (Merck): 2g

[0454] Potassium alum (Sigma)*AIK(SO4)2·12H2O:50g

[0455] Sodium iodate (Wako) NaIO3: 0.4g

[0456] Glycerol (Sigma): 200ml

[0457] Purified water: 800ml

[0458] *2. Eosin treated with hydrochloric acid

[0459] Eosin Y (Merck): 10g

[0460] Concentrated hydrochloric acid (Sigma) (HCl): 10ml

[0461] Purified water: 1000ml

[0462] like Figure 4As shown, both WT (wild-type) S / F iTregs and H1 S / F iTregs suppressed the clinical symptoms of dermatitis. H1S / F iTregs were more effective in suppressing dermatitis than WT (wild-type) S / F iTregs. Comparing the number of lymphocytes infiltrating the pathological skin tissue revealed the same results: control mice had the highest number of infiltrating T cells, while mice administered H1S / F iTregs (Dsg3-specific S / F iTregs) had the lowest number of infiltrating T cells.

[0463] (Example 4: Dose dependence: dermatitis model)

[0464] H1 naive T cells were added per 2 × 10 5 2 × 10 5 Rag was administered intravenously via the tail vein 2- / - Mice were used to establish a Dsg3-specific dermatitis model. One week later, H1 S / F iTregs were administered, each containing 1×10 3 , 1×10 4 , 1×10 5 The treatment effect was observed by dermatitis scoring once a week. iTregs were not administered to the control mice. Figure 5 As shown, according to 1×10 5 Administration group<1×10 4 Administration group<1×10 3 The results showed that H1S / F iTreg can inhibit the symptoms of dermatitis in a dose-dependent manner.

[0465] (Example 5: Inhibitory Effects on Inflammatory Cell Count and IFNg and IL-17 Production: Dermatitis Model)

[0466] Flow cytometry analysis was performed in the Dsg3-specific dermatitis model to observe cytokine production. Skin lymph nodes were recovered, and a skin cell suspension was prepared from the dorsal skin of the mice. Hair was shaved with electric clippers, and subcutaneous tissue was mechanically removed with forceps. The remaining skin was minced with scissors and placed in 5 ml of RPMI containing 0.25 mg / ml Liberase TL research grade (Roche) and 1 g / ml DNase I (Sigma) in a 6-well plate and incubated at 37°C for 2 hours. The cells were dispersed, recovered, washed, and filtered through a 40 μm cell strainer (Falcon).

[0467] Add 100 μl of a solution containing anti-CD4 and anti-TCRVβ6 antibodies diluted 200-fold in PBS containing 2% FBS and stain on ice for 20 minutes. Add 100 μl of the BD Pharmingen Transcription Factor Buffer Set (BD) to the cells for fixation and incubate on ice for 30 minutes. Then, add 100 μl of a solution containing anti-IFN-g and anti-IL-17a antibodies diluted 200-fold in washing buffer and stain for 30 minutes. Flow cytometry analysis was performed.

[0468] like Figure 6 As shown, H1 S / F iTregs (Dsg3-specific S / F iTregs) had the lowest T cell counts and the highest suppression of inflammatory cytokines such as IFNγ and IL-17a.

[0469] (Example 6: Long-term survival in dermatitis model mice: 2 months later)

[0470] In the Dsg3-specific dermatitis model mice, 2×10 H1 S / F iTreg 5 Each of the antibodies was dissolved in 200 ml of PBS and injected intravenously through the tail vein. 56 days later, skin lymph nodes were harvested and 100 μl of a 200-fold dilution of anti-CD4 and anti-hCD2 antibodies in PBS containing 2% FBS was added. The cells were stained on ice for 20 minutes and analyzed by flow cytometry.

[0471] like Figure 7 As shown, CD4 + Foxp3 + cells, confirming the survival of H1 S / F iTreg in vivo for 56 days.

[0472] (Example 7: Inhibitory effect on pemphigus model)

[0473] The recombinant extracellular domain of mouse Dsg3 (mDsg3) was diluted with PBS-Ca (0.5 mM CaCl2, PBS) to a concentration of 100 μg / ml. On day 0, an emulsion of mDsg3 equal to Freund's complete adjuvant (SIGMA, F5881) was prepared with a syringe and 200 μl was administered subcutaneously. On days 7 and 14, an emulsion of mDsg3 equal to Freund's incomplete adjuvant (SIGMA, F5506) was prepared with a syringe and 200 μl was administered subcutaneously. On days 21 and 28, mDsg3 was diluted with PBS-Ca to 50 μg / ml and 200 μl was administered intraperitoneally. - / - Mice were immunized.

[0474] Dsg3 after immunization - / - The spleen and skin lymph nodes of mice were hemolyzed by ACK reaction for 1 minute and the cell fluid was recovered. 7 Cells from spleen and skin lymph nodes were suspended in 200 μl PBS and administered to Rag2- / - mice through the tail vein to create pemphigus model mice. 5 H1 S / FiTregs were administered to observe their symptom-suppressing effects in a pemphigus model. iTregs were not administered in the control group. Blood samples were collected weekly from the conjunctiva. Serum anti-Dsg3 IgG titers were measured by ELISA using plates coated with 5 μg / ml rDsg3 protein. Each serum sample was diluted 1000-fold with antibody diluent (a dilution prepared by adjusting 1% BSA + 0.05% Tween in PBS). Serum obtained from PV model mice was used as a positive control for anti-Dsg3 IgG. 50 μl of serum was added to the plate and incubated at room temperature for 1 hour. The plate was then washed, and 50 μl of a solution prepared by diluting anti-mouse IgG antibody 5000-fold with antibody diluent was added. The plate was washed at room temperature for 1 hour, and 100 μl of enzyme substrate solution was added and incubated at room temperature for 10 minutes. 100 μl of reaction stop solution was added, and the plate was allowed to stand at room temperature for 10 minutes. The absorbance at 450 nm was measured. The ELISA index (OD450 of the sample - OD450 of the negative control) / (OD450 of the positive control - OD450 of the negative control) was calculated using the positive and negative controls. Skin symptoms of pemphigus were evaluated weekly using a dermatitis score.

[0475] The results are shown in Figure 8 In. Figure 8 As shown, the H1 S / F iTreg-administered group had suppressed Dsg3 antibody production and clinical symptoms of pemphigus compared to the control group.

[0476] (Example 8: Production of highly functional stable iTregs from T cells derived from pemphigus patients)

[0477] As in Example 1, S / F iTregs were prepared from the peripheral blood of pemphigus patients. PBMCs were recovered from human blood by density gradient centrifugation and fractionated into CD14-positive and CD14-negative cells using beads conjugated to anti-CD14 antibodies. The CD14-negative cell fraction was then fractionated into CD4-positive and CD4-negative cells using beads conjugated to anti-CD4 antibodies. The resulting CD4-positive cells were stimulated for 3 days in a basal medium containing anti-hCD3 antibody (100 μL of 10 μg / mL antibody per well in a 96-well flat-bottom plate, 600 μL of 10 μg / mL antibody per well in a 24-well plate, and allowed to stand at room temperature for 60 minutes to solidify in the container), hIL-2 (50 U / mL), hTGFβ3 (10 ng / mL), AS2863619 (1 μM), and dupilumab (5 μg / mL). The cells were then cultured for two days in a basal medium supplemented with hIL-2 (100 U / ml) and AS2863619 (1 μM). After two days, the medium was replaced in the same manner, and the cells were cultured for another two days. Two days later, the cells were stimulated again for two days in a basal medium supplemented with anti-hCD3 antibody (100 μL of 10 μg / mL antibody was added to each well of a 96-well plate, and 600 μL of 10 μg / mL antibody was added to each well of a 24-well plate and allowed to stand at room temperature for 60 minutes to allow solid phase formation in the container), hIL-2 (50 U / ml), hTGFβ3 (10 ng / ml), AS2863619 (1 μM), dexamethasone (10 nM), and actosin (20 nM). The cells were then cultured for two days in a basal medium supplemented with hIL-2 (100 U / ml) and AS2863619 (1 μM). After two days, the medium was replaced in the same manner, and the cells were cultured for another two days. Flow cytometry ( Figure 9 Left) The expression of Foxp3 and CD4 in regulatory T cells obtained 2 days later was analyzed using the bisulfite method ( Figure 9 Right) Analysis of the demethylation status of Treg-specific demethylation regions.

[0478] (Example 9: Inhibitory Effect on Highly Functional Stable iTregs from Pemphigus Patients)

[0479] As in Example 2, Dsg3-specific immunosuppression of highly functional stable iTreg from pemphigus patients was confirmed. Human PBMC was suspended in a 5 μM CFSE solution adjusted with MACS buffer, and iTreg was suspended in a 1 μM Far Red solution adjusted with MACS buffer, and each was placed in the dark for 20 minutes for staining. Then, centrifuged at 500 × g for 5 minutes, the precipitate was suspended in a 5% human albumin solution, and placed on ice for 5 minutes. Then, centrifuged at 500 × g for 5 minutes, the supernatant was discarded, and PBMC or iTreg were suspended at 10 6 Cells were suspended in a culture medium at a final concentration of 0.5% in PRIME-XVT Cell Expansion XSFM serum-free medium. 100 μL (10 5 ) PBMC. S / F iTregs were shaken and mixed at a ratio of 0:1 to 1:8 relative to PBMCs, and culture medium was added to the wells with insufficient capacity to make the amount of culture medium in each well consistent, and hIL-2 was added at a final concentration of 10U / ml. In addition, 5μL of beads bound to anti-CD2, CD3, and CD28 antibodies were added to 200μL of culture medium for stimulation and cultured at 37°C for 4 days. The CD4 in the cells was then stained with PE-Cy7 and analyzed by FACS. Ultra iTregs were removed by gating on far-red negative cells, and the proportion of divided CD4-positive cells was analyzed from the PE-Cy7-positive and CFSE-positive cell populations.

[0480] The results are as follows Figure 10 The addition of beads bound to anti-CD2, CD3, and CD28 antibodies significantly increased the proportion of dividing CD4-positive cells, but the addition of iTregs reduced the proportion of dividing CD4-positive cells in a cell-number dependent manner, demonstrating the high inhibitory effect of iTregs on cell division.

[0481] (Example 10: Clinical trial plan for the treatment of human pemphigus)

[0482] As a single intravenous injection of highly functional, stable iTregs from pemphigus patients (3×10) in patients with steroid-resistant pemphigus vulgaris and pemphigus foliaceus 7 / patient (weighing more than 50kg) or 1×10 8 A Phase I clinical trial of this drug was planned with single-center, open-label, dose-escalation single-dose trial to evaluate safety and efficacy.

[0483] The cells constituting this product are activated T cells contained in human PBMCs obtained from patients through apheresis, converted into iTregs, and then administered to the patients.

[0484] The primary evaluation criteria in this trial were safety (type, frequency, and severity of adverse events) after 24 weeks of iTreg administration. Secondary evaluations were blinded to changes in the Pemphigus Disease Area Index (PDAI, International Pemphigus Severity Scale) clinical symptom score from baseline to 24 weeks of iTreg administration, as well as changes in desmoglein 3 and 1 antibody titers from baseline to 24 weeks of iTreg administration.

[0485] (Example 11: Treatment of human pemphigus)

[0486] In patients with pemphigus, there is a situation where symptoms often recur during the process of reducing steroids. At this time, high-functioning stable iTreg from the patient can be administered intravenously while temporarily increasing steroids or without increasing steroids. When symptoms worsen, the clinical symptom score pemphigus disease area index (PDAI) increases. By administering high-functioning stable iTreg, the steroid dosage can be reduced to below the dosage at the time of relapse without an increase in PDAI. In addition, by administering high-functioning stable iTreg, a reduction in desmoglein 3 or 1 antibody titer can be expected. In the case where the antibody titer decreases with administration, the antibody titer can also be monitored on a monthly basis to evaluate the continued effectiveness of the administered high-functioning stable iTreg. At this point, as the antibody titer rises again in the future, at the stage where the PDAI rises again and is confirmed, high-functioning stable iTreg is administered again, and the patient's symptoms can be alleviated again.

[0487] (Example 12: Treatment using Dsg3-specific S / F iTreg)

[0488] In this example, treatment with Dsg3-specific S / F iTregs was investigated.

[0489] (Biochemical test)

[0490] 1) Method

[0491] Lymphocytes from the spleen and lymph nodes of Dsg3KO mice immunized with recombinant Dsg3 protein were adjusted and administered to Rag2KO mice via the tail vein at a dose of 1.5×10⁷ cells per mouse to establish a pemphigus model. One week later, 1.0×10⁷ Dsg3H1-specific S / F iTreg cells were administered. No cells were administered to the control group. N = 5 in each treatment and control group.

[0492] Total B cell counts were measured in spleens and lymph nodes 6 weeks after pemphigus model mice were induced. The cells were then stained for B220 and analyzed by FACS.

[0493] The number of Dsg3-specific B cells was measured by ELISpot in the same spleen and lymph nodes as above ( Figure 11 ).

[0494] 2) Results

[0495] Compared with the control group, treatment with Dsg3-specific S / F iTregs did not affect the total number of B cells, but significantly reduced the number of Dsg3-specific B cells ( Figure 12 ).

[0496] 3) Inspection

[0497] Dsg3-specific S / Fi Tregs suppressed B cells in a Dsg3-specific manner.

[0498] (Clinical score)

[0499] 1) Method

[0500] Peripheral blood was collected from the eyes of mice treated as described above once a week, and the anti-Dsg3 antibody titer was measured by ELISA. Clinical evaluation was performed weekly using a dermatitis score to assess systemic skin symptoms and the extent of hair loss.

[0501] 2) Results

[0502] Dsg3-specific S / FiTregs suppressed anti-Dsg3 antibodies and clinical symptoms.

[0503] 3) Inspection

[0504] Dsg3-specific S / F iTregs suppress B cells in a Dsg3-specific manner, thereby inhibiting Dsg3 antibody production and clinical symptoms of pemphigus, and are therefore considered useful as antigen-specific treatments ( Figure 13 ).

[0505] (Note)

[0506] As described above, although the present invention has been illustrated using the preferred embodiments of the present invention, it should be understood that the scope of the present invention should be interpreted only by the scope of the claims. It should be understood that the patents, patent applications and other documents cited in this specification are cited as references to this specification, just as their contents themselves are specifically recorded in this specification. This application is an application claiming priority to Special Application No. 2023-17733 filed with the Japan Patent Office on February 8, 2023, and its contents are cited as references in this application as needed.

[0507] [Industrial Applicability]

[0508] The cells or cell populations of the present invention can be used to treat or prevent pemphigus. In addition, the method for producing the pharmaceutical composition of the present invention can stably induce highly functional regulatory T cells from peripheral T cells, and its application in the medical field is expected.

Claims

1. A pharmaceutical composition for treating or preventing pemphigus, comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive and CD26-positive.

2. A pharmaceutical composition for treating or preventing pemphigus, comprising NT5E-positive inducible regulatory T cells as an active ingredient.

3. A pharmaceutical composition for treating or preventing pemphigus, comprising ITGAE (CD103)-positive inducible regulatory T cells as an active ingredient.

4. A pharmaceutical composition for treating or preventing pemphigus, comprising AREG-positive inducible regulatory T cells as an active ingredient.

5. A pharmaceutical composition for treating or preventing pemphigus, comprising CD172g-positive inducible regulatory T cells as an active ingredient.

6. A pharmaceutical composition for treating or preventing pemphigus, comprising CD26-positive inducible regulatory T cells as an active ingredient.

7. A pharmaceutical composition for treating or preventing pemphigus, comprising CTLA4-positive inducible regulatory T cells as an active ingredient.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein The induced regulatory T cells are at least CTLA4 positive and FoxP3 positive.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein The induced regulatory T cells are at least CD172g positive and / or CD26 positive.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein The CNS2 site of the FOXP3 gene in the inducible regulatory T cells is demethylated.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein The inducible regulatory T cells are CD4 positive or CD8 positive.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein The inducible regulatory T cells are obtained or induced from human peripheral blood T cells or T cells from human tissues.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein The induced regulatory T cells are obtained by a method comprising the following steps: (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) a step of 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) a step of dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein The pharmaceutical composition contains a cell population comprising the induced regulatory T cells as an active ingredient, and the proportion 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 in the cell population is approximately 50% or more.

15. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the NT5E-positive ratio of the cell population is about 50% or more.

16. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the proportion of ITGAE (CD103) positivity in the cell population is about 50% or more.

17. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the AREG-positive ratio of the cell population is about 50% or more.

18. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the proportion of CD172g positivity in the cell population is about 50% or more.

19. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the proportion of CD26 positivity in the cell population is about 50% or more.

20. A pharmaceutical composition for treating or preventing pemphigus, comprising a cell population containing inducible regulatory T cells as an active ingredient, wherein the CTLA4-positive ratio of the cell population is about 50% or more.

21. The pharmaceutical composition according to any one of claims 14 to 20, wherein In the cell population, at least the proportions of CTLA4-positive and FoxP3-positive cells are respectively about 50% or more.

22. The pharmaceutical composition according to any one of claims 14 to 21, wherein In the cell population, at least the proportion of CD172g-positive and / or CD26-positive cells is respectively about 50% or more.

23. The pharmaceutical composition according to any one of claims 14 to 22, wherein The proportion of the at least one characteristic in the cell population is greater than about 60%.

24. The pharmaceutical composition according to any one of claims 14 to 23, wherein The proportion of the at least one characteristic in the cell population is greater than about 80%.

25. The pharmaceutical composition according to any one of claims 14 to 24, wherein The proportion of FoxP3-strongly positive cells in the cell population is about 50% or more.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein About 90% or more of the cell population are T cells.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein The pharmaceutical composition is administered at a dose of at least about 1×10 3 The inducible regulatory T cells are administered in an amount of 1:

1.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein The pharmaceutical composition is administered by injection.

29. The pharmaceutical composition according to any one of claims 1 to 28, wherein After the pharmaceutical composition is administered to a patient, the pharmaceutical composition is additionally administered to a patient for whom the effect is not effective or the effect is insufficient.

30. The pharmaceutical composition according to any one of claims 1 to 29, wherein The induced regulatory T cells specifically respond to desmoglein 3 (Dsg3).

31. A pharmaceutical composition comprising as an active ingredient inducible regulatory T cells having at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive and CD26-positive and specifically responding to desmoglein 3 (Dsg3).

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