WO2023095802A1 - Ｔ細胞関連疾患を治療または予防するための医薬組成物 - Google Patents

Ｔ細胞関連疾患を治療または予防するための医薬組成物 Download PDF

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WO2023095802A1
WO2023095802A1 PCT/JP2022/043221 JP2022043221W WO2023095802A1 WO 2023095802 A1 WO2023095802 A1 WO 2023095802A1 JP 2022043221 W JP2022043221 W JP 2022043221W WO 2023095802 A1 WO2023095802 A1 WO 2023095802A1
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cells
pharmaceutical composition
positive
cell
inducible regulatory
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English (en)
French (fr)
Japanese (ja)
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統久 三上
志文 坂口
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Regcell Co Ltd
University of Osaka NUC
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Regcell Co Ltd
Osaka University NUC
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Priority to EP22898585.9A priority Critical patent/EP4438048A4/en
Priority to US18/713,036 priority patent/US20250352578A1/en
Priority to MX2024006370A priority patent/MX2024006370A/es
Priority to CN202280077889.6A priority patent/CN118591379A/zh
Priority to JP2023563705A priority patent/JPWO2023095802A1/ja
Priority to CA3238860A priority patent/CA3238860A1/en
Priority to KR1020247020426A priority patent/KR20240130083A/ko
Publication of WO2023095802A1 publication Critical patent/WO2023095802A1/ja
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    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
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    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)

Definitions

  • the present disclosure relates to pharmaceutical compositions for treating or preventing T cell-related diseases.
  • CD25+CD4+ regulatory T cells that are intrinsic to the immune system, they specifically express the transcription factor FoxP3. Functionality may be impaired. Regulatory T cells suppress the immune system by expressing various genes such as CTLA4 and IL-10 in addition to FoxP3. Epigenetic conditions such as DNA demethylation are thought to contribute to the global gene expression regulation of regulatory T cells, including stable expression of FoxP3. Correlated with cell phenotype.
  • the present inventors When inducing regulatory T cells from human peripheral T cells, the present inventors induced stable inducible T cells from human peripheral T cells by stimulation with an anti-CD3 antibody, followed by dormant culture, It was found for the first time that regulatory T cells with high expression of inhibitory molecules and high inhibitory function can be induced by culturing with anti-CD3 antibody stimulation again and then by performing dormant culture. The inventors have also found that the inducible regulatory T cells thus obtained have sufficient immunosuppressive potential to function as pharmaceuticals.
  • the present disclosure provides: (Item X1) A pharmaceutical composition for treating or preventing a T cell-related disease, at least selected from the group consisting of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive A pharmaceutical composition containing as an active ingredient an inducible regulatory human T cell having one characteristic.
  • (Item X1A) A pharmaceutical composition for treating or preventing a T-cell-related disease, the pharmaceutical composition comprising NT5E-positive inducible regulatory human T-cells as an active ingredient.
  • (Item X1F) A pharmaceutical composition for treating or preventing a T-cell-related disease, the pharmaceutical composition containing CTLA4-positive inducible regulatory human T-cells as an active ingredient.
  • (Item X1G1) A pharmaceutical composition for treating or preventing a T cell-related disease, having at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition containing as an active ingredient inducible regulatory human T cells having (Item X1G2) A pharmaceutical composition for treating or preventing a T cell-related disease, having at least three characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition containing as an active ingredient inducible regulatory human T cells having (Item X1G3) A pharmaceutical composition for treating or preventing a T cell-related disease, having at least four characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition containing as an active ingredient inducible regulatory human T cells having (Item X1G4) A pharmaceutical composition for treating or preventing a T cell-related disease, having at least five characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • a pharmaceutical composition containing as an active ingredient inducible regulatory human T cells having (Item X1G5) A pharmaceutical composition for treating or preventing a T-cell related disease, inducible regulatory human with all characteristics of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive and CD26-positive A pharmaceutical composition containing T cells as an active ingredient.
  • Item X2 The pharmaceutical composition according to any one of the preceding items, wherein the inducible regulatory human T cells are at least CTLA4-positive and FoxP3-positive.
  • Item X3 The pharmaceutical composition according to any one of the preceding items, wherein said inducible regulatory human T cells are at least CD172g-positive and/or CD26-positive.
  • the inducible regulatory human T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormant culture of the cells obtained in step (a) in 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) treating the cells obtained in step (c) with IL-2. dormant culture in culture medium for at least about 1 to about 3 days.
  • a cell population containing the inducible regulatory human T cells is contained as an active ingredient, and the cell population consists of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive.
  • Pharmaceutical composition which is the above.
  • a pharmaceutical composition for treating or preventing a T-cell-related disease comprising a cell population containing inducible regulatory human T cells as an active ingredient, wherein each of the cell populations has an ITGAE (CD103) positive percentage.
  • ITGAE CD103 positive percentage.
  • Item X8C A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein each of the cell populations has an AREG-positive rate of about 50%.
  • Pharmaceutical composition which is the above.
  • (Item X8D) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a CD172g positive rate of about 50%.
  • Pharmaceutical composition which is the above. (Item X8E) A pharmaceutical composition for treating or preventing a T cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, each of said cell populations having a CD26 positive rate of about 50%. Pharmaceutical composition which is the above.
  • (Item X8F) A pharmaceutical composition for treating or preventing a T-cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population has a CTLA4-positive rate of about 50%.
  • Pharmaceutical composition which is the above.
  • (Item X8G1) A pharmaceutical composition for treating or preventing a T-cell-related disease, comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive, NT5E-positive, ITGAE (CD103 )
  • a pharmaceutical composition for treating or preventing a T-cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive, NT5E-positive, ITGAE (CD103 )
  • a pharmaceutical composition wherein the proportion of at least three characteristics selected from the group consisting of positive, AREG positive, CD172g positive, and CD26 positive is about 50% or more, respectively.
  • a pharmaceutical composition for treating or preventing a T-cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive, NT5E-positive, ITGAE (CD103 )
  • a pharmaceutical composition for treating or preventing a T-cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive, NT5E-positive, ITGAE (CD103 )
  • a pharmaceutical composition wherein the proportion of at least five characteristics selected from the group consisting of positive, AREG positive, CD172g positive, and CD26 positive is about 50% or more, respectively.
  • a pharmaceutical composition for treating or preventing a T-cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive, NT5E-positive, ITGAE (CD103 )
  • T-cell associated disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects.
  • the autoimmune disease is Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymis inflammation, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (DCM), peripartum cardiomyopathy (DCM), peripart
  • the present disclosure also provides: (Item 1) A pharmaceutical composition for treating or preventing a T cell-related disease, wherein the inducible regulation has at least one characteristic selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive A pharmaceutical composition containing T cells as an active ingredient. (Item 2) The pharmaceutical composition according to any of the preceding items, wherein the inducible regulatory T cells have at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. (Item 3) The pharmaceutical composition according to any one of the above items, wherein the inducible regulatory T cells are at least CTLA4 positive.
  • the inducible regulatory T cells are (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormant culture of the cells obtained in step (a) in 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) treating the cells obtained in step (c) with IL-2. dormant culture in culture medium for at least about 1 to about 3 days.
  • a pharmaceutical composition according to any one of the preceding items comprising a T cell population wherein about 50% or more of the cells in the T cell population are said inducible regulatory T cells.
  • a pharmaceutical composition according to any one of the preceding items comprising a T cell population wherein about 80% or more of the cells in the T cell population are said inducible regulatory T cells.
  • the pharmaceutical composition according to any one of the preceding items, wherein the T cell population is a regulatory T cell population.
  • the pharmaceutical composition according to any one of the preceding items, wherein about 90% or more of the cell population are T cells.
  • a pharmaceutical composition according to any one of the preceding items comprising the inducible regulatory T cells at a dose of about 10 8 to about 10 9 cells per dose, or about 10 7 cells/kg.
  • the T-cell associated disease comprises autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS that can be treated by immunosuppressive effects. .
  • the autoimmune disease is systemic lupus erythematosus, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and cardiomyopathy.
  • the present disclosure can provide a method for inducing highly functional regulatory T cells from human peripheral T cells in vitro.
  • Regulatory T cells induced by the method of the present disclosure highly express genes associated with regulatory T cells and have a strong suppressive ability. Therefore, they can be used as regulatory T cells for the treatment and prevention of various immune diseases and inflammatory diseases such as autoimmune diseases.
  • the methods of the present disclosure can induce functional regulatory T cells in vitro from human peripheral T cells.
  • Regulatory T cells obtained by the method of the present disclosure have a high suppressive function and are stable as regulatory T cells. That is, by the method of the present disclosure, highly functional regulatory T cells can be induced by stably expressing FoxP3, the master gene of regulatory T cells, from human peripheral T cells.
  • FIG. 1 shows an outline of a method for producing an embodiment of mouse-inducible regulatory T cells (also referred to as mouse high-functioning stable iTreg (HSF iTreg) cells) and results of flow cytometry on the inducible regulatory T cells.
  • FIG. 1A shows a schematic protocol for inducing iTreg cells from mouse CD4-positive naive T cells by various stimulation methods.
  • FIG. 1B shows the results of flow cytometry analysis of the expression of FoxP3 and CD25 in iTreg cells, activated T cells, and activated nTreg cells prepared in FIG. 1A.
  • FIG. 1 shows a schematic protocol for inducing iTreg cells from mouse CD4-positive naive T cells by various stimulation methods.
  • FIG. 1B shows the results of flow cytometry analysis of the expression of FoxP3 and CD25 in iTreg cells, activated T cells, and activated nTreg cells prepared in FIG. 1A.
  • FIG. 2 shows FoxP3 when SF-iTreg is produced from mouse CD4-positive naive T cells or effector T cells based on the production method according to one embodiment of the iTreg (SF-iTreg in the figure) of the present disclosure in FIG.
  • FIG. 2 shows the results of analysis of expression by flow cytometry and demethylation state of Treg-specific demethylated regions by bisulfite method.
  • FIG. 3 shows the result of one embodiment in which the global gene expression pattern for each cell in FIG. 1 was analyzed by RNA sequencing.
  • FIG. 3A shows a PCA analysis plot and
  • FIG. 3B shows a heatmap of Treg-associated genes.
  • FIG. 4 shows the results of one embodiment of analyzing the in vitro suppressive ability of each cell in FIG.
  • FIG. 5 is the results of one embodiment of analyzing Foxp3 expression of regulatory T cells transferred two weeks after administering the single-stimulation CD28 antibody-na ⁇ ve group of FIG. 1 and iTregs of the present disclosure to wild-type mice. .
  • FIG. 5 is the results of one embodiment of analyzing Foxp3 expression of regulatory T cells transferred two weeks after administering the single-stimulation CD28 antibody-na ⁇ ve group of FIG. 1 and iTregs of the present disclosure to wild-type mice. .
  • FIG. 6 shows the results of one embodiment in which mouse CD4-positive naive T cells were transferred to RAG2-deficient mice to create a colitis model, and iTreg cells of the present disclosure were administered to analyze the therapeutic effects.
  • FIG. 6A shows changes in body weight (g)
  • FIG. 6B shows HE-stained images of colon tissues
  • FIG. 6C shows the analysis results of CD69 expression in lymph node T cells by flow cytometry.
  • FIG. 7 shows the results of an analysis of one embodiment of iTregs of the present disclosure from human Crohn's disease patients.
  • FIG. 7A shows the results of generating inducible regulatory T cells (HSF-iTreg in the figure) of the present disclosure from CD4-positive T cells derived from human Crohn's disease patients, and analyzing the expression of FOXP3, CTLA4, and Helios by flow cytometry.
  • Normal nTreg CD4-positive CD25-positive T cells
  • conventional iTreg Conventional iTreg: CD4-positive T-cells stimulated with CD3/CD28 for 3 days in the presence of IL-2 and TGF- ⁇ 1 compared.
  • FIG. 7B is the analysis result of the in vitro suppressive ability of each cell in FIG. 7A.
  • FIG. 8 shows phenotypic analysis (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure generated from human CD8-positive T cells. Highly functional inducible regulatory T cells were generated from human CD8-positive T cells, and the expression of FOXP3, CTLA4 and Helios was analyzed by flow cytometry.
  • FIG. 9 shows CD25 and FOXP3 expression (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure.
  • the expression intensity of CD25 and FOXP3 was analyzed by flow cytometry using a BD FACSLyric flow cytometer.
  • FIG. 10 shows phenotypic analysis of one embodiment of iTreg (HSF iTreg) cells of the present disclosure.
  • Inducible regulatory T cells (HSF-iTreg) of the present disclosure were generated and analyzed for expression of CD172g, CD26 and others.
  • FIG. 11 shows phenotypic analysis (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure generated from SLE patient-derived CD4-positive T cells. Highly functional inducible regulatory T cells were prepared from SLE patient-derived CD4-positive T cells, and the expressions of FoxP3, CD4 and CTLA4 were analyzed by flow cytometry.
  • FIG. 12 shows phenotypic analysis (flow cytometry) of one embodiment of iTreg (HSF iTreg) cells of the present disclosure generated from rheumatoid arthritis patient-derived CD4-positive T cells. High-function-inducible regulatory T cells were prepared from rheumatoid arthritis patient-derived CD4-positive T cells, and the expressions of FoxP3, CD4, and CTLA4 were analyzed by flow cytometry.
  • both the gene and the protein may be indicated.
  • FOXP3 gene and FOXP3 protein are sometimes used properly, and the term FoxP3 refers to both the concept and the entity (whole) of the gene or protein.
  • regulatory T cells are T cells positive for FoxP3 expression. Also referred to herein as “Treg”. Tregs include endogenous regulatory T cells (Naturally Occurring Regulatory T cells: nTreg) and inducible regulatory T cells (Inducible Regulatory T cells: iTreg). Regulatory T cells can typically have a variety of functions, such as immunosuppressive functions.
  • inducible regulatory T cells refers to regulatory T cells negative for IKZF2 (Helios) expression. iTreg is usually obtained by inducing differentiation from naive CD4-positive T cells or the like.
  • Intrinsic regulatory T cells (Naturally Occurring Regulatory T cells, nTreg) refer to regulatory T cells that positively express IKZF2 (Helios) and CTLA4. Cells that normally exist in vivo.
  • peripheral T cells refer to T cells that exist outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues.
  • peripheral T cells it is sufficient that the cell group contains peripheral T cells, and the T cells need not be isolated.
  • Cell fractions containing various lymphocytes in addition to T cells such as peripheral blood mononuclear cells (PBMC) may also be used.
  • PBMC peripheral blood mononuclear cells
  • flow cytometry refers to a technique for measuring the number of cells, solids and other biological particles suspended in a liquid, and individual physical, chemical, and biological properties. Devices using this technique are called “flow cytometers.”
  • “positive” and “negative” of cell markers eg, FoxP3, CTLA4, Helios, CD103, etc.
  • flow cytometry cells are run in a line and the number of cells is counted by spectroscopic techniques.
  • the number of target cells is counted by irradiating cells labeled with a fluorescent or luminescent enzyme with laser light and detecting fluorescence or luminescence signals emitted from the cells with a detector such as a photodiode. It is also possible to import the detection results from the detector into a computer and generate and display a two-dimensional plot. This makes it possible to easily grasp the presence or absence of target cells, their number, and the like.
  • demethylation refers to typically methylated adenine (e.g., position 6; m6A, position 1; m1A) and cytosine (e.g., position 5; m5C, position 3; m3C). It refers to the removal of methylation modifications. Demethylation can be identified using techniques known in the art, and can be measured using, for example, the bisulfite method.
  • cell population refers to a population containing two or more cells. It may be a cell cluster composed of In addition, a “cell population” may be formed of a single type of cells, or may contain multiple types of cells.
  • stimulation means stimulation via TCR.
  • stimulation of T cells includes stimulation using an anti-CD3 antibody and a complex containing it, stimulation using a peptide that binds to TCR and a complex containing the peptide, stimulation using antigen-presenting cells, and anti-CD3 antibody. and antigen-presenting cells, and stimulation using peptides or proteins and antigen-presenting cells at the same time.
  • distal culture refers to culturing cells in the absence of the above stimuli.
  • the present disclosure relates to the medical use of highly functional stable inducible regulatory T cells (also referred to as highly functional stable iTreg, HSF iTreg).
  • highly functional stable inducible regulatory T cells also referred to as highly functional stable iTreg, HSF iTreg.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising: A pharmaceutical composition is provided that contains, as an active ingredient, inducible regulatory human T cells having at least one characteristic selected from the group consisting of:
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure exhibit at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure may be at least CTLA4 positive.
  • the inducible regulatory T cells in the pharmaceutical compositions of the present disclosure may be CD4-positive or CD8-positive.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure may be at least CTLA4-positive and FoxP3-positive. In one embodiment of the disclosure, the inducible regulatory T cells in the pharmaceutical composition of the disclosure may be at least CD172g positive and/or CD26 positive.
  • the present disclosure provides a pharmaceutical composition for treating or preventing T-cell-related diseases, the pharmaceutical composition containing CD172g-positive inducible regulatory human T cells as an active ingredient.
  • CD172g is a tyrosine kinase-related protein and is involved in cell adhesion of neurons (adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment). It is expected that functions related to adhesion and the like are activated, and that it is highly effective against various diseases related to this.
  • the present disclosure provides a pharmaceutical composition for treating or preventing T-cell-related diseases, the pharmaceutical composition containing CD26-positive inducible regulatory human T cells as an active ingredient.
  • CD26 is a gene also called DPP4, and is associated with glucose metabolism, insulin metabolism, and immune function (also prominent in infectious diseases). It is expected to be highly effective against the disease of
  • the present disclosure provides a pharmaceutical composition for treating or preventing a T-cell-related disease, the pharmaceutical composition containing NT5E-positive inducible regulatory human T cells as an active ingredient.
  • NT5E CD73
  • CD73 is a cell membrane protein that catalyzes the conversion of extracellular nucleotides into membrane permeable nucleosides.
  • the encoded protein is used as a determinant of lymphocyte differentiation. Defects in this gene can lead to calcification of joints and arteries, and thus NT5E-positive inducible regulatory human T cells are expected to be highly effective against a variety of related diseases. be.
  • the present disclosure provides a pharmaceutical composition for treating or preventing T-cell-related diseases, which contains ITGAE (CD103)-positive inducible regulatory human T cells as an active ingredient.
  • ITGAE (CD103) encodes an alpha integrin with an I domain that undergoes post-translational cleavage at the extracellular domain to produce disulfide-linked heavy and light chains.
  • This protein associates with ⁇ 7 integrin to form an E-cadherin binding integrin known as human mucosal lymphocyte-1 antigen.
  • This protein is preferentially expressed on human intestinal intraepithelial lymphocytes (IELs) and may function as an accessory molecule for IEL activation, in addition to its role in adhesion. Therefore, ITGAE (CD103)-positive inducible regulatory human T cells are expected to be highly effective against various diseases related thereto.
  • the present disclosure provides a pharmaceutical composition for treating or preventing T-cell-related diseases, the pharmaceutical composition containing AREG-positive inducible regulatory human T cells as an active ingredient.
  • AREG is a member of the epidermal growth factor family and is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). This protein interacts with the EGF/TGF- ⁇ receptor to promote normal epithelial cell growth and inhibit the growth of certain aggressive cancer cell lines. It also functions in the development of mammary glands, eggs and bone tissue. This gene has been associated with a psoriasis-like skin phenotype and has been associated with other pathological diseases, including various types of cancer and inflammatory conditions. Therefore, AREG-positive inducible regulatory human T cells are expected to be highly effective against various diseases related thereto.
  • CTLA4 belongs to the immunoglobulin superfamily and is a protein that transmits inhibitory signals to T cells.
  • Membrane-bound CTLA4 functions as a disulfide-linked homodimer, and soluble CTLA4 functions as a monomer. Mutations in this gene are said to be associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-related orbital disease, and other autoimmune diseases.
  • CTLA4-positive inducible regulatory human T cells are expected to be highly effective against various diseases related thereto.
  • CTLA4-positive inducible regulatory human T cells are also expected to be highly effective against autoimmune diseases such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis.
  • CD4-positive T cells were cultured in medium containing IL2 and TGF ⁇ in the presence of anti-CD3 antibody and anti-CD28 antibody, followed by culture in medium containing IL2 and TGF ⁇ .
  • the expression of FoxP3 in inducible regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has been confirmed. It has not been.
  • an inducible regulatory T cell that stably expresses FoxP3 and advantageously retains an immunosuppressive effect stably.
  • Such inducible regulatory T cells can be produced, for example, by methods for producing inducible regulatory T cells described elsewhere herein.
  • the present disclosure provides that inducible regulatory T cells: (a) stimulate CD4-positive T-cells or CD8-positive T-cells in peripheral blood in a first basal medium for about 1 to about 5 days; b) dormant culture of the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (d) dormant culture of the cells obtained in step (c) in medium containing IL-2 for at least about 1 to about 3 days. It is possible to provide a pharmaceutical composition obtained by
  • the inducible regulatory T cell in the pharmaceutical composition of the present disclosure has an inducible regulatory T cell-specific demethylation state. Whether the obtained regulatory T cells are in a regulatory T cell-specific demethylated state is confirmed, for example, by demethylating the CNS2 site of the FoxP3 gene (FOXP3 (all italics)). be able to. Since such a demethylation state can be an indicator of stable inducible regulatory T cells, confirming the demethylation state of the inducible regulatory T cells in the pharmaceutical composition of the present disclosure indicates stable inducible regulatory T cells. It can be shown that
  • the immunosuppressive activity or immunosuppressive effect of inducible regulatory T cells in the pharmaceutical composition of the present disclosure can be confirmed, for example, by measuring the Cell Trace Violet intensity in responder T cells.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can stably provide immunosuppressive activity or immunosuppressive effects, e.g., the inducible regulatory T cells of the present disclosure are immunosuppressive for at least about two weeks. It can provide activity or an immunosuppressive effect.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can have a higher immunosuppressive effect than conventional regulatory T cells (including inducible and endogenous). . Therefore, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can also be called functional or highly functional inducible regulatory T cells.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can stably express FoxP3. Therefore, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can also be referred to as stable inducible regulatory T cells. In one aspect, the inducible regulatory T cells in the pharmaceutical composition of the present disclosure are highly functional and stable, and can be referred to as highly functional stable inducible regulatory T cells (HSF iTreg).
  • whether or not the markers in the inducible regulatory T cells of the present disclosure are positive can be determined by measuring the positive rate by flow cytometry. For example, by analyzing with a flow cytometer, it is possible to determine whether or not the cells are positive based on the percentage of cells exhibiting an antigen expression level equal to or higher than the standard. It can also be classified as negative, weakly positive, moderately positive, strongly positive (weakly positive, moderately positive, and strongly positive are collectively referred to as "positive"), etc., depending on the expression intensity of the cell surface marker.
  • the median fluorescence intensity of each marker/median fluorescence intensity of the negative control is less than 5, 5 or more and less than 10, 10 or more and less than 30, or 30 or more.
  • Such a determination can be made as exemplified in (Positive rate measurement by flow cytometry), but the present disclosure is not limited thereto.
  • the expression intensity of cell surface markers in inducible regulatory T cells of the present disclosure is measured using a BD FACSLyric flow cytometer, e.g., using samples prepared as described in the Examples herein. (BD Biosciences), the expression intensity of 10 2 or more may be weak positive, 10 3 or more medium positive, 10 4 or more strong positive, or 10 3 or more strongly positive. The following may be considered weakly positive, or 10 2 or more may be considered strongly positive, and less than 10 2 may be considered weakly positive. Such determination can be appropriately set according to experimental conditions, experimental purposes, cell types, equipment setting conditions, and the like, and the present disclosure is not limited thereto.
  • the expression intensity in other instruments corresponding to the expression intensity when analyzed with the BD FACSLyric flow cytometer can be calculated, and the instrument to be used A value indicative of expression intensity can be found as appropriate.
  • the inducible regulatory T cells in the pharmaceutical composition of the present disclosure can be derived from any cell, but are preferably derived from human peripheral blood T cells or human tissue-derived T cells. can be obtained by
  • the inducible regulatory T cells or cell populations thereof in the pharmaceutical composition of the present disclosure can be human cells, and T cells obtained from human peripheral blood are used as materials. , can include inducible regulatory human T cells or cell populations thereof induced by a given procedure.
  • the properties of human cells and mouse cells are clearly different, and even if the same cell surface markers are present, the properties of the cells cannot be regarded as identical.
  • applicable techniques differ between mice and humans. T cells vary in the degree of antigen sensitization and the degree of activation. Therefore, in mice, highly functional stable inducible regulatory T cells can be directly generated from T cells collected from mouse lymphoid tissue, but in humans it is difficult to do so. cannot be regarded as similar cells based solely on the
  • the pharmaceutical composition of this disclosure provides 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 herein. can contain.
  • the cell populations of the present disclosure are about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can be inducible regulatory T cells as described elsewhere herein.
  • the T cells in the cell population of the present disclosure can be regulatory T cells.
  • 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, in the regulatory T cells of the cell populations of the present disclosure about 95% or more, about 97% or more, or about 99% or more can be inducible regulatory T cells as described elsewhere herein.
  • the cell population of the present disclosure may contain cells other than T cells, but preferably about 60% or more, about 65% or more, about 70% of the cell population of the present disclosure 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 T cells, preferably about 90% or more may be T cells.
  • a pharmaceutical composition for treating or preventing a T-cell-related disease containing as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is CTLA4-positive and FoxP3-positive percentages of about 50% or more, respectively.
  • the cell population in the pharmaceutical composition of the present disclosure has a CTLA4-positive and FoxP3-positive ratio of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, It can be about 85% or greater, about 90% or greater, about 95% or greater, about 97% or greater, or about 99% or greater.
  • the CD172g-positive and/or CD26-positive ratio can each be about 50% or more.
  • the cell population in the pharmaceutical composition of the present disclosure has a CD172g-positive and/or CD26-positive percentage of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% greater than or equal to about 85%, greater than or equal to about 90%, greater than or equal to about 95%, greater than or equal to about 97%, or greater than or equal to about 99%.
  • the ratio of strongly FoxP3-positive cells in the pharmaceutical composition of the present disclosure can be about 50% or more.
  • the cell population in the pharmaceutical composition of the present disclosure has a ratio of strongly FoxP3 positive of about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher. % or greater, about 90% or greater, about 95% or greater, about 97% or greater, or about 99% or greater.
  • the expression intensity of cell surface markers can be measured by flow cytometric positivity measurement as described above.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises FoxP3
  • a pharmaceutical composition having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises FoxP3-positive inducible regulatory human T cells at about 50% or more, about 60%, by cell number. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises CTLA4
  • a pharmaceutical composition is provided having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises CTLA4-positive inducible regulatory human T cells at about 50% or more, about 60%, based on cell number. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises NT5E
  • a pharmaceutical composition is provided having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises about 50% or more, about 60%, by cell number, NT5E-positive inducible regulatory human T cells. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population is ITGAE
  • a pharmaceutical composition having a (CD103) positive rate of about 50% or greater.
  • the cell populations in such pharmaceutical compositions comprise ITGAE (CD103)-positive inducible regulatory human T cells at about 50% or more, based on cell number, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can contain.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises AREG
  • a pharmaceutical composition having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises about 50% or more, about 60%, by cell number, AREG-positive inducible regulatory human T cells. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises CD172g
  • a pharmaceutical composition is provided having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises about 50% or more, about 60%, by cell number, CD172g-positive inducible regulatory human T cells. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • a pharmaceutical composition for treating or preventing a T cell-related disease comprising as an active ingredient a cell population comprising inducible regulatory human T cells, wherein the cell population comprises CD26
  • a pharmaceutical composition having a positive rate of about 50% or greater.
  • the cell population in such pharmaceutical compositions comprises about 50% or more, about 60%, by cell number, CD26-positive inducible regulatory human T cells. about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more can.
  • the rate of induction of specific cells differs between mouse cells and human cells.
  • a predetermined induction method it is possible to obtain a cell population in which the positive percentage of a predetermined cell surface marker is about 50% or more. It is possible to obtain a cell population.
  • CTLA4 is a molecule that is localized in cells, and at least it is not at a level that can be used as an index for concentration or purification.
  • FoxP3 is a transcription factor and is a molecule that exists entirely within cells, so it cannot be detected on the surface at all and cannot be used for purification. That is, since CTLA4 and FoxP3 serve as markers expressed "inside" T cells, CTLA4 and/or FoxP3 are sorted and enriched using CTLA4 and/or FoxP3 as an index to obtain a cell population of about 50% or more that is CTLA4-positive and/or FoxP3-positive. It is not possible.
  • a cell population having a CTLA4-positive and FoxP3-positive ratio of less than about 50% is obtained according to a conventional method, and such a cell population is used as a raw material and concentrated using CTLA4 and / or FoxP3 as an indicator, and the cells are It is also not possible to obtain a cell population greater than about 50%.
  • the inducible regulatory human T cells in the pharmaceutical composition of the present invention are mainly responsible for the immunosuppressive function of Tregs, and these cells account for more than half of a certain cell population.
  • a medically effective immunosuppressive effect can be stably achieved, which is also important for technical and medical effects. That is, by containing 50% or more of T cells exhibiting a medically effective immunosuppressive effect, it is possible to provide a stable cell preparation. It is not only the difference in quantity and numerical value, but it is an important quality issue as to whether it can be established as a formulation.
  • the inducible regulatory human T cells in the cell population in the pharmaceutical composition of the present disclosure, can further be characterized as ITGAE (CD103)-positive, NT5E-positive, and/or AREG-positive.
  • ITGAE CD103
  • NT5E-positive NT5E-positive
  • AREG AREG-positive
  • compositions comprising the inducible regulatory T cells or cell populations of the present disclosure.
  • regenerative medicine materials or products are provided that include the inducible regulatory T cells or cell populations of the present disclosure.
  • This pharmaceutical composition, regenerative medicine material or product can be used for autoimmune diseases, inflammatory diseases, and allergies that can be treated by immunosuppressive action.
  • These medicines, regenerative medicine materials or products can be used together with media and any other additives used in the field.
  • a medium a medium obtained by adding necessary factors to a basal medium for animal cell culture used for cell culture can be used. Examples of such media are detailed elsewhere herein. Examples of components added to the medium are also detailed elsewhere herein.
  • it may contain DMSO and the like.
  • the pharmaceutical composition of the present disclosure can be used to treat or prevent a T-cell-related disease, any disease that can be treated by immunosuppressive effects.
  • T cell-related diseases include, but are not limited to, autoimmune diseases, infectious diseases, cancer, allergies, inflammatory diseases, and ALS.
  • autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I ), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis Fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, cardiomyopathy, dilated cardiomyopathy (
  • the autoimmune disease is preferably autoimmune hepatitis, primary biliary cholangitis, or primary sclerosing cholangitis, more preferably primary biliary cholangitis, or primary sclerosing cholangitis. and even more preferably primary sclerosing cholangitis.
  • the pharmaceutical composition of the present disclosure can be administered by various dosages and administration methods, but is preferably administered by injection.
  • the pharmaceutical composition of this disclosure contains about 10 8 to about 10 9 , or about 10 7 inducible regulatory T cells as described elsewhere herein per administration. /kg to be administered.
  • the pharmaceutical composition of the present disclosure can be additionally administered to a patient who is ineffective or inadequate when administered to the patient. Whether or not the effect is ineffective or insufficient can be determined using, for example, the degree of suppression of inflammation in the target disease as an indicator. In one embodiment, additional administrations, if any, can be administered at least about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after the initial administration.
  • the method of producing inducible regulatory T cells in the pharmaceutical composition of the present disclosure is a novel method capable of producing highly functional and stable inducible regulatory T cells, and is described herein below. I will explain in detail.
  • a method of producing inducible regulatory T cells comprising: (a) reducing CD4 positive T cells or CD8 positive T cells in peripheral blood from about 1 to about 5 cells in a first basal medium; (b) dormant culture of the cells obtained in step (a) in medium containing IL-2 for at least about 1 to about 3 days; (c) (d) resting the cells obtained in step (c) in medium containing IL-2 for at least about 1 to about 3 days; culturing.
  • inducible regulatory T cells in the pharmaceutical composition of the present disclosure can be produced using either CD4-positive T cells or CD8-positive T cells as raw materials.
  • the inducible regulatory T cells in the pharmaceutical composition can also be produced using mixed cells of CD4-positive T-cells and CD8-positive T-cells as raw materials.
  • the method of the present disclosure comprises culturing human peripheral T cells in medium containing TGF ⁇ and IL-2 in the presence of anti-CD3 antibody stimulation, IL-2 in the absence of anti-CD3 antibody culturing in medium, again in the presence of anti-CD3 antibody stimulation in medium containing TGF ⁇ and IL-2, from human peripheral T cells (including CD4-positive T-cells or CD8-positive T-cells) It can also be a method for producing T cells.
  • stimulation of T cells is stimulation for obtaining Treg, and is not particularly limited as long as it is stimulation via TCR.
  • stimulation of T cells includes stimulation using an anti-CD3 antibody and a complex containing it, stimulation using a peptide that binds to TCR and a complex containing the peptide, stimulation using antigen-presenting cells, and anti-CD3 antibody. and antigen-presenting cells at the same time, stimulation using peptides or proteins and antigen-presenting cells at the same time, etc., but the medium and conditions are not particularly limited as long as Tregs can be obtained.
  • the culture medium and conditions for dormant culture are not particularly limited as long as cells are cultured in the absence of the above stimuli.
  • anti-CD3 antibody stimulation means giving specific stimulation to CD3 receptors on cells.
  • Examples of CD3 stimulation include anti-CD3 agonist antibodies.
  • the anti-CD3 agonist antibody may be prepared using commercially available products as research reagents or by conventional methods.
  • Anti-CD3 antibodies can be derived from animals such as mice, rabbits, goats, and cows, and from humans.
  • step a once iTreg is induced (stimulation of T cells that causes demethylation induction) (step a), the medium is replaced and resting culture is performed for 1 to 3 days. (step b) and once again stimulating the T cells to induce demethylation (step c), resulting in functional and stable inducible regulatory human T cells Obtainable. Normally, it is common general knowledge that T cells exhibit apoptosis when stimulated twice. We have found to obtain functional and stable inducible regulatory human T cells.
  • inducible regulatory human T cells can be obtained by performing T cell stimulation, dormant culture, and subsequent stimulation again.
  • Inducible regulatory human T cells thus obtained, or cell populations comprising such inducible regulatory human T cells can achieve the desired effects of the present disclosure. Therefore, in one embodiment of the present disclosure, the type of medium and stimulus for culture is not particularly limited, and by using a medium of any composition and any type of stimulus, it is possible to have functionality and stability. Certain inducible regulatory human T cells can be obtained.
  • the medium used in each step may further contain retinoic acid and/or ascorbic acid.
  • the medium may contain ascorbic acid.
  • the medium may further contain a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor.
  • the medium may contain a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor.
  • the first basal medium and the second basal medium are each independently anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, CDK8 inhibitor, CDK19 inhibitor , a CDK8/19 inhibitor, and ascorbic acid.
  • the first basal medium and the second basal medium each independently contain an anti-CD3 antibody, TGF- ⁇ 1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, CDK8/19 inhibitors and ascorbic acid can also be included.
  • the concentration contained as each of these components may be the usual concentration used in the relevant field.
  • the concentration of 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, e.g. is about 0.1 ⁇ M or more, about 0.5 ⁇ M or more, about 1 ⁇ M or more, about 2 ⁇ M or more, about 3 ⁇ M or more, about 4 ⁇ M or more, about 5 ⁇ M or more, about 6 ⁇ M or more, about 7 ⁇ M or more, about 8 ⁇ M or more, about 9 ⁇ M or more; It can be about 10 ⁇ M or more, about 12 ⁇ M or more, about 14 ⁇ M or more, about 16 ⁇ M or more, about 18 ⁇ M or more, about 20 ⁇ M or more, etc., but the concentration is not limited to these concentrations, and those skilled in the art can use other medium compositions as appropriate. can be changed accordingly.
  • the disclosed method produces regulatory T cells from human peripheral T cells.
  • Peripheral T cells include naive regulatory T cells, CD4 positive T cells, CD8 positive T cells, and the like. Even if regulatory T cells are induced from a culture containing multiple types of T cells, it is possible to induce regulatory T cells after isolating specific cells such as CD4-positive T cells and CD8-positive T cells from these cells. may Alternatively, regulatory T cells may be induced after isolating T cells specific to a particular antigen. Therefore, inducible regulatory T cells of the present disclosure include those that are CD4-positive and those that are CD8-positive.
  • CD4-positive or CD4+ refers to CD4-positive, CD8-negative, single-positive cells unless otherwise specified.
  • either CD4-positive or CD8-positive T cells can be used as raw materials, and even after the generation of inducible regulatory T cells, CD4-positive or CD8-positive T cells can be used unless special manipulations are performed. CD8-positive characteristics can be maintained.
  • the antibody in the method of the present disclosure, may be added to the medium or immobilized on the inner wall of the culture vessel or the surface of the insoluble carrier.
  • the insoluble carrier a member capable of physically or chemically binding the anti-CD3 antibody and insoluble in an aqueous solution can be used.
  • Materials capable of physically adsorbing the anti-CD3 antibody include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate and polypropylene, and glass.
  • the shape of the insoluble carrier is not particularly limited, and for example, a plate shape, a bead shape, a container shape, or the like can be adopted.
  • the amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide sufficient stimulation for the induction of regulatory T cells.
  • a medium obtained by adding necessary factors to a basal medium for animal cell culture can be used for culturing cells.
  • Animal cell culture basal media that can be used in the method of the present disclosure include, for example, Iscove's modified Eagle's Medium medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, RPMI 1640 medium, Fischer's medium, and mixed or partially modified media of these.
  • the basal medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free.
  • serum-free medium if necessary, for example, albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (serum replacement 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'-thiolglycerol, monothioglycerol, etc. It's okay.
  • BSA bovine serum albumin
  • KSR KnockOut Serum Replacement
  • the basal medium also contains lipids (eg, chemically defined lipid concentrate), amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins (eg, nicotinamide, ascorbic acid), growth factors , antibiotics (eg, penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.
  • lipids eg, chemically defined lipid concentrate
  • amino acids amino acids
  • L-glutamine e.g, GlutaMAX (Thermo Fisher Scientific)
  • NEAA non-essential amino acids
  • vitamins eg, nicotinamide, ascorbic acid
  • growth factors eg, antibiotics (eg, penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.
  • RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is exemplified as a basal medium.
  • cells may be cultured under general animal cell culture conditions.
  • the culture temperature is, but not limited to, about 30-40°C, preferably about 37°C.
  • Culturing is preferably carried out in an atmosphere of air containing CO 2 , the CO 2 concentration being preferably about 2-5%.
  • the anti-CD3 antibody may be added directly to the medium, or may be immobilized on the inner wall of the culture vessel or the surface of the insoluble carrier.
  • the amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide sufficient stimulation for the induction of regulatory T cells.
  • TGF ⁇ examples include TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3, such as TGF ⁇ 1.
  • concentration of TGF ⁇ is not particularly limited as long as it can be appropriately determined by those skilled in the art.
  • TGF ⁇ 1 or TGF ⁇ 3 is used as TGF ⁇ , the concentration in the medium is not particularly limited, but may be 0.25-25 ng/mL, for example about 10 ng/mL.
  • the concentration of IL-2 in the medium used is not limited, but can be from about 5 U/mL to about 500 U/mL, for example about 100 U/mL.
  • the medium used in the present disclosure may further contain retinoic acid and/or ascorbic acid.
  • the medium may contain ascorbic acid.
  • the concentration of ascorbic acid is not limited, but is from about 1 to about 100 ⁇ g/mL, such as about 10 ⁇ g/mL.
  • a CDK8 inhibitor, a CDK19 inhibitor, and/or a CDK8/19 inhibitor may be added to the medium used in the present disclosure.
  • Any CDK8 inhibitor, CDK19 inhibitor, and/or CDK8/19 inhibitor can be used, for example, 4-[1-(2-methyl-1H-benzimidazol-5-yl) -1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3- ⁇ 1-[1-(4-methoxyphenyl)piperidine-4- yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl ⁇ pyrazin-2-amine or salts, hydrates, solvates thereof, etc., or US Pat. No.
  • 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, and any suitable concentration described in the above literature. , and those skilled in the art can make appropriate changes according to other medium compositions.
  • regulatory T cells are induced by stimulating human peripheral T cells with anti-CD3 antibody in medium containing TGF ⁇ and IL-2, and anti-CD3 antibody-free IL-2-containing medium.
  • regulatory T cells with high immunosuppressive function can be induced. It can be confirmed that the obtained inducible regulatory T cells have a high immunosuppressive function, for example, by comprehensive gene expression analysis by RNA sequencing and in vitro cell growth suppression test.
  • the number of culture days in the step of (a) stimulating CD4-positive T cells or CD8-positive T cells in the peripheral blood with the first basal medium for about 1 to about 5 days can be determined by a person skilled in the art. It can be set as appropriate and is not particularly limited, but can be set to, for example, about three days.
  • the number of days of culture in the dormant culture step of step (b) can be appropriately set by a person skilled in the art, and is not particularly limited, but can be set to, for example, about two days.
  • the number of days of culture in the second basal medium of step (c) can be appropriately set by a person skilled in the art, and is not particularly limited, but is, for example, about 3 days. can also
  • the number of days of culture in the dormant culture step of step (d) can be appropriately set by a person skilled in the art, and is not particularly limited, but can be set to, for example, about two days.
  • regulatory T cells with an induced regulatory T cell-specific demethylation state can be expanded by culturing in the presence of IL-2 under unstimulated conditions.
  • the medium may further comprise ascorbic acid, and stable regulatory T cell cultures of induced regulatory T cells can be obtained by culturing in a medium further comprising IL-2.
  • regulatory T cells In order to isolate regulatory T cells from the obtained cell culture containing regulatory T cells, cells may be isolated by conventional methods based on cell surface markers specific to regulatory T cells, for example, using a cell sorter. A FoxP3-positive fraction may be taken out by . Also, regulatory T cells with specific antigenic properties may be isolated if desired.
  • the inducible regulatory T cells obtained by the method of the present invention are expected to be used for the treatment of human inflammatory diseases, such as autoimmune diseases and allergies.
  • Positive rate determination by flow cytometry can be performed as follows.
  • Preparation/Fixation/Permeabilization Concentrate (hereinafter referred to as Buffer) (eBio Science, 00-5123-43) ⁇ Fixation/Permeabilization Diluent (eBio Science, 00-5223-56) ⁇ Permeabilization Buffer (10X) (eBio Science, 00-833-56) ⁇ FOXP3 Monoclonal Antibody (236A/E7), PE (hereafter, anti-FOXP3 antibody) (eBio Science, 12-4777-42) ⁇ Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE (hereinafter referred to as PE control) (eBio Science) ⁇ BV421, Mouse, Anti-Human, CD152 (hereinafter referred to as anti-CTLA4 antibody) (BD) ⁇ BV421 Mouse IgG2a, k Isotype Control (BV421 Control) (BD) ⁇ CD4 Monoclonal Antibody (RPA-T4), APC (hereafter, anti
  • Reagent preparation *Fixation Buffer (100 ⁇ L used per sample) Mix Buffer and Diluent in a 1:3 ratio. * Perm buffer Dilute Permeabilization Buffer (10x) 10-fold with MilliQ water. *FACS Buffer (for 500 mL preparation) 489 mL of D-PBS 10 mL FBS (final concentration 2%) 0.5 mol/l EDTA solution 1 mL (final concentration 1 mM) Store the above reagents at 4°C or on ice after preparation.
  • Method (1) Add 500 ⁇ L of FACS Buffer to a 1.5 mL tube. (2) Add 1 ⁇ 10 6 of the final product to the tube of (1) and gently suspend with a micropipette. (3) Centrifuge at 500 xg, 4°C for 5 minutes. (4) After removing the supernatant with an aspirator, add 100 ⁇ L of Fixation Buffer and gently pipette. ⁇ Be careful not to make bubbles. (5) Fix by standing on ice for 30 minutes or more in the dark. ⁇ 45 minutes is fine. (6) After fixation, add 1 mL of Perm Buffer to the tube and pipette gently. (7) Prepare new 1.5 mL tubes for the number of samples.
  • Remarks ⁇ Reagents used for fixation and staining can be purchased as a set of 3 as "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.: 00-5523). ⁇ There is no problem with the settings of the equipment, as long as they do not deviate from a general/scientific point of view to the extent that it can be clearly determined that normal measurements cannot be performed. Significant deviations are when the signals of the cell population of interest are below the set fluorescence threshold value, when the signal values of the control or measured samples are below or above the instrument's normal measurement limits, and so on.
  • Cells of the disclosure may have immunosuppressive activity. Immunosuppressive activity can be measured by various techniques.
  • suppression can be determined by measuring cell proliferation caused by an immune reaction caused by responder T cells.
  • the present disclosure is demonstrated using an in vivo model.
  • tissues are collected after a certain period of administration from colitis model mice administered with the inducible regulatory T cells of the present disclosure, and immunosuppression occurs in the tissues. Its activity can be measured by whether it is confirmed or not. Confirmation of immunosuppression in tissues can be achieved by various techniques. For example, the presence or absence of immunosuppression can also be confirmed by tissue staining analysis.
  • Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.; M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.; M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Volume Experimental Medicine "Gene Introduction & Expression Analysis Experimental Method” Yodosha, 1997, etc., and these are the relevant parts (may be all) of this specification. is incorporated by reference.
  • glutamine-free RPMI1640 (containing 10% FCS (v/v), 60 ⁇ g/mL penicillin G, 100 ⁇ g/mL streptomycin, 10 mM HEPES) was used as the basal medium. 30 to 300 mg/L of L-glutamine was added to the medium as needed.
  • Genomic DNA was obtained from induced cells, treated with the MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures), and examined for demethylation of genes characteristic of regulatory T cells. Analysis of demethylation of each gene used known methods and primers (Floess et al. (2007) PloS Biology Volume 5, Issue 2, e38 and Ohkura et al. (2012) Immunity 37(5) 785 -799).
  • 5'-TTGGGTTAAGTTTGTTGTAGGATAG-3' (SEQ ID NO: 1) was used on the forward side and 5'-ATCTAAACCCTATTATCACAACCCC-3' (SEQ ID NO: 2) was used on the reverse side.
  • Foxp3-eGFP (eFox) reporter mice Ito et al. (2014) Science 346, 363-368) were used.
  • Foxp3-eGFP (eFox) reporter mouse lymph node cells were obtained and the endogenous regulatory T cell (nTreg) fraction was obtained by sorting CD4 + GFP + cells by FACSAriaII (BD).
  • CD4 + GFP ⁇ CD44 low CD62L high cells were sorted to obtain the naive T cell fraction.
  • a CD4 + GFP ⁇ CD44 high CD62L low cell fraction was obtained and used as effector/memory T cells.
  • Human CD4 + T cells were isolated from PBMC of Crohn's disease patients. Purified from concentrated PBMC using CliniMACS CD4 GMP MicroBeads according to the manufacturer's protocol.
  • Example 1 Method for producing mouse highly functional stable regulatory T cells
  • Mouse CD4-positive T cells were treated with an anti-CD3 antibody (100 ⁇ L of 10 ⁇ g/ml antibody was added per well and allowed to stand at room temperature for 60 minutes to solidify the container), hIL-2 (100 U/ml), hTGF ⁇ 1. (2.5 ng/ml), retinoic acid (1 ⁇ M), SenexinA (5 ⁇ M) in basal medium for 3 days. After that, the cells were cultured in a basal medium containing hIL-2 (100 U/ml) for 2 days in a dormant state. Two days later, the medium was replaced in the same manner, and rest culture was carried out for another two days.
  • anti-CD3 antibody 100 ⁇ L of 10 ⁇ g/ml concentration antibody was added per well and allowed to stand at room temperature for 60 minutes to immobilize it on the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 2. 5 ng/ml
  • retinoic acid 1 ⁇ M
  • SenexinA 5 ⁇ M
  • ascorbic acid 10 ⁇ g/ml
  • Example 2 In vitro suppressive activity of regulatory T cells
  • Regulatory T cells were obtained by the same experimental system as in Example 1.
  • Cell Trace Violet-labeled responder T cells (5 ⁇ 10 4 ), prepared regulatory T cells (5 ⁇ 10 4 ), anti-CD3 antibody (1 ⁇ g/ml) + antigen presenting cells (MHC-II positive cells: 1 ⁇ 10 4 ) or in the presence of Dynabeads T-activator (Veritas) (5 ⁇ L/well) for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry.
  • the responder T cells generate an immune response, the Cell Trace Violet intensity exhibits multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains a single strong peak.
  • the regulatory T cells obtained by the production method of the present application strongly suppress the immune reaction of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a higher suppressive function than the existing regulatory T cell population (Fig. 4).
  • Example 3 In vivo stability of regulatory T cells
  • Regulatory T cells were prepared from Thy1.2/eFox reporter mice by the method of Example 1, Foxp3-positive cells (2 ⁇ 10 5 cells) were isolated by FACS, and administered to Thy1.1/wild-type mice through the tail vein. . Two weeks later, the transferred Thy1.2 cells were collected from the lymph nodes and analyzed for Foxp3 expression (Fig. 5). It was confirmed that regulatory T cells induced without CD28 stimulation and subjected to stabilizing culture stably expressed Foxp3 even after 2 weeks in vivo.
  • Example 4 Suppressive effect on colitis model
  • a colitis model was prepared by administering 2 ⁇ 10 5 CD4-positive naive T cells derived from wild-type mice with high expression of CD45RB to RAG-deficient mice.
  • 2 ⁇ 10 5 highly functional stable iTreg cells produced by the method of Example 1 were administered, and changes in body weight were measured over time (FIG. 6A).
  • colon tissues and lymph nodes were collected, and HE staining analysis of colon tissues (Fig. 6B) and CD69 expression analysis in lymph node T cells (Fig. 6C: flow cytometry method) were performed.
  • Fig. 6B HE staining analysis of colon tissues
  • Fig. 6C flow cytometry method
  • Example 5 Production of regulatory T cells from human peripheral T cells
  • anti-CD3 antibody 100 ⁇ L per well of 10 ⁇ g / ml antibody was added and allowed to stand at room temperature for 60 minutes to immobilize the container
  • hIL-2 100 U / ml
  • hTGF ⁇ 1 10 ng/ml
  • retinoic acid 1 ⁇ M
  • SenexinA 5 ⁇ M
  • anti-CD3 antibody 100 ⁇ L per well of 10 ⁇ g/ml concentration antibody was added and allowed to stand at room temperature for 60 minutes to solidify the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 10 ng/ ml
  • retinoic acid 1 ⁇ M
  • SenexinA 5 ⁇ M
  • ascorbic acid 10 ⁇ g/ml
  • Example 6 In vitro suppressive activity of regulatory T cells
  • Regulatory T cells were obtained by the same experimental system as in Example 1.
  • Cell Trace Violet-labeled responder T cells (5 ⁇ 10 4 ) and prepared regulatory T cells (5 ⁇ 10 4 ) were mixed-cultured for 3 days in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 ⁇ L/well), Cell Trace Violet intensity was analyzed by flow cytometry.
  • Treg Suppression Inspector Miltenyi Biotec
  • Example 7 Production of regulatory T cells from CD8-positive human peripheral T cells
  • CD8-positive T cells anti-CD3 antibody (100 ⁇ L per well of 10 ⁇ g / ml concentration of antibody was added and allowed to stand at room temperature for 60 minutes to immobilize the container), hIL-2 (100 U / ml), hTGF ⁇ 1 ( 10 ng/ml), retinoic acid (1 ⁇ M), SenexinA (5 ⁇ M) in basal medium for 3 days. After that, the cells were cultured in a basal medium containing hIL-2 (100 U/ml) for 2 days in a dormant state. Two days later, the medium was replaced in the same manner, and rest culture was carried out for another two days.
  • anti-CD3 antibody 100 ⁇ L per well of 10 ⁇ g/ml concentration antibody was added and allowed to stand at room temperature for 60 minutes to solidify the container
  • hIL-2 100 U/ml
  • hTGF ⁇ 1 10 ng/ ml
  • retinoic acid 1 ⁇ M
  • SenexinA 5 ⁇ M
  • ascorbic acid 10 ⁇ g/ml
  • Example 8 Expression intensity analysis
  • the cell population of inducible regulatory T cells produced in Example 1 was analyzed for the expression intensity of CD25 and FOXP3 by flow cytometry using a BD FACSLyric flow cytometer. The results are shown in FIG. A high proportion of FOXP3 Hi -inducible regulatory T cell populations could be observed in all cells tested.
  • Example 9 Antibody panel analysis of inducible regulatory T cells
  • BioLegend's LEGENDScreen Human PE Kit was used to stain the inducible regulatory T cells produced in Example 1, and antibodies reactive with the inducible regulatory T cells of the present disclosure were analyzed by flow cytometry. Some of the results (for which clear and significant expression could be confirmed) are shown in Tables 1-4. Staining with an antibody panel comprising just under 400, at least 185 surface marker molecules were able to confirm the positive/negative character of the cell surface markers in the inducible regulatory T cells of the present disclosure.
  • Example 10 Cell surface marker analysis in inducible regulatory T cells
  • Characteristic results of the antibody screening performed in Example 9 are shown in FIG.
  • Inducible regulatory T cells of the present disclosure were found to be positive for characteristic cell surface markers such as CD172g and CD26.
  • Example 11 Production of regulatory T cells from systemic lupus erythematosus (SLE) patient-derived T cells
  • SLE systemic lupus erythematosus
  • Regulatory T cells were obtained in the same experimental system as in Example 1 using SLE patient-derived CD4-positive T cells.
  • Expression of FoxP3, CD4 and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry (Fig. 11).
  • Example 12 Production of regulatory T cells from rheumatoid arthritis (RA) patient-derived T cells
  • Regulatory T cells were obtained in the same experimental system as in Example 1 using RA patient-derived CD4-positive T cells.
  • Expression of FoxP3, CD4 and CTLA4 in the obtained regulatory T cells was analyzed by flow cytometry (Fig. 12).
  • the cell populations of the present disclosure can be used for the treatment and prevention of various immune diseases and inflammatory diseases such as autoimmune diseases.
  • the method for producing a cell population of the present disclosure enables stable induction of highly functional regulatory T cells from peripheral T cells, and is expected to be applied to the medical field.
  • SEQ ID NO: 1 Forward primer used in Examples
  • SEQ ID NO: 2 Reverse primer used in Examples

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