Method for propagating regulatory cells

Abstract

Disclosed are: a method for producing a cell population in which regulatory T cells have been propagated, the method comprising (1) a step for culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist; (1A) a method for propagating a cell population containing regulatory T cells, the method comprising a step for culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist; a cell population containing regulatory T cells obtained by the method; and a drug comprising the cell population containing regulatory T cells.

Description

Method for Proliferating Regulatory Cells

[0001] The present invention relates to a method for producing a cell population in which primary regulatory T cells (Treg) have expanded, a cell population containing regulatory T cells obtained by the method, and a pharmaceutical product containing the cell population containing regulatory T cells. (Background of the Invention)

[0002] In recent years, because regulatory T cells (Treg) have the function of suppressing immune responses, research and development of cell therapy (Treg therapy) using regulatory T cells has been progressing worldwide for the purpose of treating various autoimmune diseases. Thus, regulatory T cells are expected to be used in the treatment of graft-versus-host disease (GvHD), autoimmune diseases, inflammatory diseases, and allergic diseases, as well as for the treatment and prevention of these diseases.

[0003] Regulatory T cells are broadly classified into two types: endogenous regulatory T cells (naturally occurring regulatory T cells (nTreg): or thymic Treg: tTreg) and inducible regulatory T cells (inducible T cells (induced Treg): iTreg). It is known that nTregs are naturally generated in the thymus, while iTregs are differentiated and induced from naive T cells in peripheral blood by antigen stimulation and cytokines such as IL-2 and TGF-β. These regulatory T cells are further subdivided according to the types of markers expressed on the cells.

[0004] When regulatory T cells are removed from the body, various organ-specific autoimmune diseases spontaneously develop. Transplantation of regulatory T cells in such cases prevents the onset of autoimmune diseases. Therefore, regulatory T cells were thought to play an important role in maintaining immune self-tolerance in the periphery. It has since become clear that regulatory T cells can suppress not only autoimmunity, but also most immune responses, including inflammation caused by foreign antigens, transplant rejection, infection immunity, allergy, and tumor immunity. Furthermore, the transcription factor FOXP3 has now been identified as the master regulator of regulatory T cells.

[0005] FOXP3 is a master regulator of Tregs, and it is known that it is constitutively expressed in Tregs, but not in conventional T cells (also called Tconv (conventional T cells), effector T cells, or inflammatory T cells) that do not have suppressive function. Research has been conducted on methods for efficiently expanding primary Tregs while maintaining FOXP3 expression, and compounds, cytokines, and combinations thereof that maintain expression and promote cell proliferation have been reported. Examples of such compounds and cytokines include IL-2 (Non-Patent Documents 1-7), IL-4 (Non-Patent Documents 2, 8, 9), and TNFR2 agonist antibodies (Non-Patent Documents 4, 5, 7, 10).

[0006] Further, such compounds, cytokines, and combinations thereof include IL-3 (Non-Patent Document 1), TGF-β (Non-Patent Documents 1, 3, 6, 8, 11), and the like.

[0007] Although the effects of IL-2, IL-4, and a TNFR2 agonist on Primary Tregs have been reported as described above, there have been no reports that a combination of IL-2, IL-4, IL-33, and a TNFR2 agonist (added with IL-3 or TGF-β) is particularly preferable.

[0008] J Immunol (2011) 186:2262-2272 Immunology. 2009 Jul;127(3):338-344PLoS One. , 11(2):e0148474. (2016) PLoS One. , 11(5), e0156311 (2016) Front. Immunol. 9:573 (2018) Clin. Exp. Immunol. , 197(1):52-63. (2019) Scientific Reports 3:3153 (2013) Immunology (2009) 128:e670-e678Front. Immunol. 8:1508. doi:10.3389 / fimmu. 2017.01508Sci. Signal. 13, eaba9600 (2020) J Exp Med (2003) 198 (12): 1875-1886

[0009] An object of the present invention is to provide a method for producing a cell population of expanded regulatory T cells, which can efficiently expand primary regulatory T cells, which are known to be difficult to expand, while maintaining their suppressive function.

[0010] As a result of extensive research to achieve the above-mentioned object, the present inventors have found that regulatory T cells can be proliferated with high efficiency while maintaining their suppressive function by culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and a TNFR2 agonist.

[0011] The present invention was completed based on these findings and through further investigation, and provides the following methods for producing cell populations in which regulatory T cells have expanded, cell populations containing regulatory T cells, pharmaceuticals, etc.

[0012] [1] A method for producing a cell population in which regulatory T cells have expanded, comprising: (1) culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist. [2] The method according to [1], wherein the step (1) is further performed in the presence of a TGF-βR agonist. [3] The method according to [1], wherein the step (1) is further performed in the presence of IL-3. [4] The method according to [1], wherein the step (1) is further performed in the presence of IL-3 and a TGF-βR agonist. [5] The method according to any one of [1] to [4], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody. [6] The method according to [2], [4], or [5], wherein the TGF-βR agonist is TGF-β. [7] The method according to any one of [1] to [6], wherein the culture in the step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. [7a] The method according to any one of [1] to [6], wherein the culture in the step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then carried out in the absence of these agonists. [8] The method according to any one of [1] to [6], wherein the regulatory T cells are ＋ / FOXP3 ＋ [9] The method according to any one of [1] to [7a], wherein the CD25 ＋ / FOXP3 ＋ The cells are CD4 ＋ [9a] The method according to [8], wherein the regulatory T cells are CD25 ＋ / CD127 － [9b] The method according to any one of [1] to [7a], wherein the regulatory T cells are Helios cells. ＋

[10] The method according to any one of [1] to [9a], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

[10] A method for expanding a cell population containing regulatory T cells, comprising: (1A) culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist. [10a] The method according to

[10] , wherein the step (1A) is further performed in the presence of a TGF-βR agonist. [10b] The method according to

[10] , wherein the step (1A) is further performed in the presence of IL-3. [10c] The method according to

[10] , wherein the step (1A) is further performed in the presence of IL-3 and a TGF-βR agonist. [10d] The method according to any one of

[10] to [10c], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody. [10e] The method according to [10a], [10c], or [10d], wherein the TGF-βR agonist is TGF-β. [10f] The method according to any one of

[10] to [10e], wherein the culture in the step (1A) is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. [10f1] The method according to any one of

[10] to [10f], wherein the culture in the step (1A) is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then in the absence of these agonists. [10g] The method according to any one of

[10] to [10f], wherein the regulatory T cells are selected from the group consisting of CD25 ＋ / FOXP3 ＋ [10h] The method according to any one of

[10] to [10f1], wherein the CD25 ＋ / FOXP3 ＋ The cells are CD4 ＋ [10i] The method according to [10g], wherein the regulatory T cells are CD25 ＋ / CD127 － [10j] The method according to any one of

[10] to [10f1], wherein the regulatory T cells are Helios cells. ＋The method according to any one of

[10] to [10i], wherein the regulatory T cells are cells. [10k] (2) The method according to any one of [1] to [10j], comprising a step of introducing a foreign gene (particularly a gene for expressing a chimeric antigen receptor) into a cell population containing primary regulatory T cells, or a cell population containing regulatory T cells obtained by the method according to any one of [1] to [10j].

[11] A cell population containing regulatory T cells obtained by the method according to any one of [1] to [10k].

[12] A pharmaceutical comprising the cell population containing regulatory T cells according to

[11] .

[13] The pharmaceutical according to

[12] , for use in preventing and / or treating an abnormally enhanced immune response.

[14] A method for preventing and / or treating an abnormally enhanced immune response, comprising administering the cell population containing regulatory T cells according to

[11] to a subject in need thereof.

[15] The cell population containing regulatory T cells according to

[11] , for use in preventing and / or treating an abnormally enhanced immune response.

[16] Use of a cell population containing regulatory T cells described in

[11] in the manufacture of a pharmaceutical for preventing and / or treating abnormally enhanced immune responses.

[0013] According to the present invention, it is possible to proliferate primary regulatory T cells with high efficiency while maintaining their suppressive function.

[0014] This figure shows the results of isolating primary cultured Tregs from PBMCs using a flow cytometer. The dot plots indicate the number of Tregs (CD4 ＋ CD8α － CD25 ＋ CD127 －) is shown. This is a graph showing the change in cell number over time when primary cultured Tregs were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. Optimized: Medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody; Control: Medium not containing IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. This is a graph showing the results of examining the expression levels of proteins expressed in Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. All dot plots in the figure represent CD3 ＋ CD4 ＋ CD8 － The numbers on the graph indicate the Treg (CD3 ＋ CD4 ＋ CD8 － CD25 ＋ FOXP3 ＋ ) is shown. The figure (left) shows the results of flow cytometry analysis of a cell population expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody after co-culture with allogeneic T cells derived from human PBMCs, and the graph (right) shows the rate (%) of inhibition of target cell division. E:T indicates the ratio of Treg to human T cell counts. The graph shows the change in cell count over time when primary cultured Tregs were expanded in a medium containing only IL-2, or in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, but omitting any one of IL-3, IL-4, IL-33, and TGF-β1. This figure shows the results of examining the expression levels of proteins expressed in Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, but omitting any one of IL-3, IL-4, IL-33, and TGF-β1. All dot plots in the figure represent CD3 ＋ CD4 ＋ CD8 － The numbers on the graph indicate the Treg (CD3 ＋ CD4＋ CD8 － CD25 ＋ FOXP3 ＋ ) is shown. This graph shows the rate (%) of inhibition of target cell division after co-culture with allogeneic T cells derived from human PBMCs using a cell population expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, but from which any one of IL-3, IL-4, IL-33, and TGF-β1 has been removed. E:T indicates the ratio of Treg cell numbers to human T cells. This graph shows the change in cell number over time when primary cultured Tregs were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, or in a medium from which IL-3, TGF-β1, and an anti-TNFR2 antibody have been removed.

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" or "consisting essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0017] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0018] As used herein, "culturing a cell population in the presence of a substance" refers to, for example, culturing a cell population in a medium containing the substance. Examples of such culturing include culturing in a medium containing the substance alone, or in a medium containing the substance together with other differentiation-inducing factors. When the substance is added to the medium, it can be added directly to the medium, or the substance can be dissolved in an appropriate solvent just before use and then added to the medium. The substance can also be immobilized on the surface of a substrate or carrier during culturing and then cultured.

[0019] As used herein, "positive (+)" means that the protein or gene is expressed in an amount that can be detected by a method known in the art. In addition, in the case of a protein that is expressed intracellularly and not present on the cell surface (e.g., a transcription factor or a subunit thereof), the protein of interest can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Gene detection can be carried out using, for example, nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0020] As used herein, "negative (-)" means that the expression level of a protein or gene is below the lower limit of detection by all or any of the above-mentioned known techniques, or that the level of expression is low. The lower limit of detection for protein or gene expression may vary depending on the technique. The level of protein or gene expression (whether low or high) can be determined by comparing it with the results of control cells measured under the same conditions. For example, the level of CD25 expression in a certain cell population can be determined using flow cytometry, by comparing the CD25 expression level in that cell population with the CD25 expression level in PBMC-derived Tconv (control: known to be a low CD25 expressor). If expression is equivalent to that of the control, it is determined to be low expression, and if expression is higher than that of the control cells, it is determined to be high expression.

[0021] As used herein, the term "marker" refers to a protein or its gene that is specifically expressed on the cell surface, in the cytoplasm, in the nucleus, etc., in a specific cell type. The marker is preferably a "cell surface marker." A "cell surface marker" refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance, and that facilitates the detection, concentration, isolation, etc., of cells expressing the cell surface marker. The cell surface marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a specific cell type, specifically a substance that is produced (positive marker) or not produced (negative marker) as mRNA by transcription of the gene in the genome, or as a protein by translation of that mRNA.

[0022] Such cell surface markers can be detected by immunological assays using antibodies specific to the cell surface markers, such as ELISA, immunostaining, and flow cytometry.

[0023] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0024] As used herein, the term "regulatory T cells" refers to T cells that, when stimulated via a T cell receptor, have the ability to inhibit the activation of effector T cells and are responsible for suppressing immune responses (immune tolerance). Regulatory T cells are generally CD25 ＋ / FOXP3 ＋ cells or CD25 ＋ / CD127 － cells, among which CD4 ＋ or CD8 ＋ In the present invention, regulatory T cells are CD4 ＋ cells (i.e., CD4 ＋ / CD25 ＋ / FOXP3 ＋ or CD4 ＋ / CD25 ＋ / CD127 － ) or CD8 ＋ cells (i.e., CD8 ＋ / CD25 ＋ / FOXP3 ＋ or CD8 ＋ / CD25 ＋ / CD127 － ), and CD4 ＋ More preferred are CD4 ＋ / CD25 ＋The transcription factor FOXP3 is known as a master regulator of regulatory T cells. Regulatory T cells may also be positive for Helios, CTLA4 (Cytotoxic T Lymphocyte (associated) Antigen 4) (CD152), CD39, and CD73, which are known to be indicators of the suppressive function of regulatory T cells. Helios is a member of the Ikaros transcription factor family and has been shown to bind to the FOXP3 promoter and increase FOXP3 expression. CTLA4 is an immune checkpoint protein, and when CTLA4 expressed on the surface of T cells binds to CD80 or CD86 on the surface of antigen-presenting cells, T cell activation is suppressed. Furthermore, CD39 and CD73 hydrolyze ATP and AMP, respectively, to produce adenosine as the final product, and adenosine acts on T cells to suppress T cell activation. In this specification, "CD25 ＋ / FOXP3 ＋ The " / " used in expressions such as " means "and."

[0025] As used herein, the term "cell population" refers to two or more cells of the same or different types. The term "cell population" also refers to a mass of cells of the same or different types.

[0026] As used herein, the terms "regulatory T cells expanded" and "expansion of regulatory T cells" refer to an increase in the number (absolute number) of regulatory T cells in a cell population compared to before culture or compared to a control such as cells obtained without carrying out the present invention, and refer to, for example, an increase of at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10000-fold, 15000-fold, 20000-fold, 50000-fold, or 100000-fold compared to before culture or compared to the control. In certain embodiments of the present invention, the number (absolute number) of regulatory T cells in a cell population produced by the present invention is increased by at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10000-fold, 15000-fold, 20000-fold, 50000-fold, or 100000-fold compared to the number of cells before culture or in a control.

[0027] From the viewpoint of therapeutic application, the various cells used in the present invention are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0028] The term "nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules having functions equivalent to those nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analogue. Nucleic acids may also be single-stranded or double-stranded nucleic acids. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0029] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acid (BNA), locked artificial nucleic acid (LNA), ethylene bridged artificial nucleic acid (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0030] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0031] The method of the present invention for producing a cell population in which regulatory T cells have expanded (sometimes referred to herein simply as the "production method of the present invention") is characterized by comprising the following steps: (1) culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist.

[0032] Step (1) can also be carried out in the presence of a TGF-βR agonist and / or IL-3 in addition to IL-2, IL-4, IL-33, and a TNFR2 agonist. That is, step (1) can be carried out in the presence of IL-2, IL-3, IL-4, IL-33, and a TNFR2 agonist, in the presence of IL-2, IL-4, IL-33, a TGF-βR agonist, and a TNFR2 agonist, or in the presence of IL-2, IL-3, IL-4, IL-33, a TGF-βR agonist, and a TNFR2 agonist. From the viewpoint of proliferation of regulatory T cells, step (1) is preferably carried out in the absence of an mTOR inhibitor (e.g., rapamycin).

[0033] IL-4, IL-3, IL-2, and IL-33 are preferably derived from mammals, particularly humans. IL-2, IL-3, IL-4, and IL-33 may be natural products isolated and purified from mammals (particularly humans), or may be artificially produced by genetic engineering techniques (which may include amino acid substitution, deletion, insertion, and / or addition).

[0034] A TGF-βR (Transforming Growth Factor-β Receptor) agonist is defined as a substance that can bind to TGF-βR and activate TGF-βR signal transduction. Examples of TGF-βR agonists include factors belonging to the TGF-β superfamily, which includes the TGF-β family, the activin family, and the BMP (bone morphogenetic protein) family. Examples of factors belonging to the TGF-β superfamily include TGF-β (TGF-β1, TGF-β2, TGF-β3), activin A, activin B, GDF-8, and GDF-11. The TGF-βR agonist is preferably TGF-β, and more preferably TGF-β1. One type of TGF-βR agonist can be used alone, or two or more types can be used in combination.

[0035] A TNFR2 (Tumor Necrosis Factor Receptor-2) agonist is defined as a substance that can bind to TNFR2 and activate TNFR2 signal transduction, and examples of such an agonist include antibodies (e.g., anti-TNFR2 antibodies), peptides, small molecules, and proteins. Examples of anti-TNFR2 agonist antibodies include monoclonal antibodies that bind to TNFR2, such as clone MR2-1 (Hycult Biotech) and clone MAB2261 (R&D Systems). The TNFR2 agonist may also be a TNF-α mutein that binds only to TNFR2 as an agonist. TNFR2 agonists can be used singly or in combination of two or more.

[0036] The TNFR2 agonist is preferably an anti-TNFR2 agonist antibody. The antibody may be a functional fragment thereof, and examples of the functional fragment include Fd, Fv, Fab, F(ab'), and F(ab). ２ , F(ab') ２Examples of antibodies include single-chain Fvs (scFvs), diabodies, triabodies, tetrabodies, and minibodies. Antibodies may be derived from animals such as mice, rats, cows, rabbits, goats, sheep, and guinea pigs. The antibody isotype is not particularly limited, and examples of isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM. The antibody may be either a monoclonal or polyclonal antibody, preferably a monoclonal antibody. The antibody may also be a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), or the like. Antibodies can be produced by known methods, for example, by constructing an expression vector containing a nucleic acid encoding the antibody, culturing a transformant into which the nucleic acid has been introduced, or culturing a hybridoma that produces the antibody.

[0037] Furthermore, IL-2, IL-3, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists can be used in their free form or in the form of a salt. Examples of salts include salts with inorganic bases such as sodium salt, magnesium salt, potassium salt, calcium salt, and aluminum salt; salts with organic bases such as methylamine salt, ethylamine salt, and ethanolamine salt; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. The salts may be acid addition salts, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. IL-2, IL-3, IL-4, IL-33, TGF-βR agonist and TNFR2 agonist also include hydrates, solvates, crystalline polymorphs and the like.

[0038] The concentration of IL-2 in the medium is not particularly limited and is, for example, 0.1 to 1000 ng / mL, preferably 10 to 500 ng / mL, and more preferably 50 to 200 ng / mL.

[0039] The concentration of IL-4 in the medium is not particularly limited and is, for example, 0.01 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0040] The concentration of IL-33 in the medium is not particularly limited and is, for example, 0.1 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0041] The concentration of the TNFR2 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of TNFR2 agonist used, etc. The concentration of the TNFR2 agonist is, for example, 0.0001 to 100 μg / mL, preferably 0.01 to 10 μg / mL.

[0042] The concentration of the TGF-βR agonist in the medium is not particularly limited and is appropriately adjusted depending on the type of TGF-βR agonist used, etc. The concentration of the TGF-βR agonist is, for example, 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL.

[0043] The concentration of IL-3 in the medium is not particularly limited and is, for example, 0.01 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0044] The medium used in the culture of step (1) is a basal medium that is used for culturing animal cells and contains the above-mentioned IL-2, IL-4, IL-33, and a TNFR2 agonist (as well as IL-3, a TGF-βR agonist, etc.). The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium. Any one of these media may be used alone, or two or more may be used in combination.

[0045] The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). One or more types of serum substitutes may be used.

[0046] Furthermore, the medium may also contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD28 antibodies, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. It is desirable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows the preparation of cells of stable quality.

[0047] The pH of the medium is usually 7.0 to 7.8, preferably 7.2 to 7.6. Before use, the medium is preferably sterilized by filtration, ultraviolet irradiation, heat sterilization, radiation irradiation, or other methods to prevent contamination.

[0048] The culture may be carried out in the presence or absence of feeder cells. The production method of the present invention is preferably carried out in the absence of feeder cells, since it allows stable production of regulatory T cells with uniform properties without contamination by unknown components.

[0049] The culture conditions for the production method of the present invention are not particularly limited. The culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C, and CO ２ The concentration is, for example, 2 to 10%, preferably 2 to 5%, and the oxygen concentration is, for example, 1 to 20%, preferably 5 to 20%.

[0050] The culture period is also not particularly limited, and can be appropriately determined by a person skilled in the art while monitoring the number of regulatory T cells, etc., and is, for example, 7 days or more, preferably 14 days or more, more preferably 21 days or more, and even more preferably 28 days or more. The upper limit of the culture period is not particularly limited, and is, for example, 42 days or less, preferably 35 days or less, and more preferably 28 days or less. In the culture of the present invention, passage may be performed as many times as necessary to obtain a desired amount of regulatory T cells, and the medium may be added and replaced. The culture of the present invention can be performed using known CO ２ The culture can be carried out using an incubator. The culture vessel is not particularly limited and can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture vessels), bioreactors, etc. A vessel to which an anti-CD3 antibody is bound can be used as the culture vessel.

[0051] In step (1), culture (cell stimulation) may be performed in the presence of IL-2, IL-4, IL-33, or a TNFR2 agonist (and IL-3 and a TGF-βR agonist may further be added) and in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. More preferably, culture (cell stimulation) may be initiated in the presence of IL-2, IL-4, IL-33, or a TNFR2 agonist (and IL-3 and a TGF-βR agonist may further be added) and in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then culture may be performed in the absence of these antibodies. Step (1) may be repeated (by subculture). The culture may be performed by first initiating culture (stimulating cells) in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, followed by repeated culture in the absence of these antibodies. The culture period for this repetition is, for example, 7 to 28 days, preferably 14 to 21 days. Specifically, the cells may be cultured (stimulated) in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist for the first 3 days or so, followed by culture in a medium that does not contain these agonists for 4 to 11 days, and this culture unit may be repeated. The number of culture repetitions is not particularly limited, and can be determined appropriately by a person skilled in the art while monitoring the number of immune cells of the present invention, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more. The upper limit of the number of culture repetitions is, for example, 20 times.

[0052] The CD30 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD30 into a cell by specifically binding to CD30. Examples of the CD30 agonist include an anti-CD30 agonist antibody or a functional fragment thereof, a CD30 ligand or a functional fragment thereof, etc. The concentration of the CD30 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD30 agonist used, etc. The concentration of the CD30 agonist is, for example, 10 to 1000 ng / mL, preferably 100 to 500 ng / mL.

[0053] The CD3 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD3 into a cell by specifically binding to CD3. Examples of CD3 agonists include an anti-CD3 agonist antibody or a functional fragment thereof, a CD3 ligand or a functional fragment thereof, etc. The concentration of the CD3 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD3 agonist used, etc. The concentration of the CD3 agonist is, for example, 10 to 10,000 ng / mL, preferably 100 to 10,000 ng / mL, and more preferably 1,000 to 5,000 ng / mL.

[0054] The CD28 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD28 into a cell by specifically binding to CD28. Examples of the CD28 agonist include an anti-CD28 agonist antibody or a functional fragment thereof, a CD28 ligand or a functional fragment thereof, etc. The concentration of the CD28 agonist in the medium is not particularly limited and is adjusted appropriately depending on the type of CD28 agonist used, etc. The concentration of the CD28 agonist is, for example, 10 to 10,000 ng / mL, preferably 100 to 10,000 ng / mL, and more preferably 500 to 5,000 ng / mL.

[0055] The cell population containing regulatory T cells used in the production method of the present invention is primary cultured cells isolated from biological tissues such as bone marrow, umbilical cord blood, and blood. Primary cultured cells refer to cells obtained by seeding organs, cells, and tissues isolated from a living body and then performing the first passage. Primary cultured cells can be obtained from organs and tissues by known methods such as enzyme treatment, physical dispersion, and the explant method. The proportion (cell number) of regulatory T cells contained in the cell population containing primary regulatory T cells is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and the upper limit is, for example, 100% or less. Regulatory T cells can be isolated from cells isolated from biological tissues by known methods such as flow cytometry or magnetic cell separation.

[0056] The cell population containing primary regulatory T cells used in the production method of the present invention may be derived from humans or mammals other than humans (non-human mammals), preferably humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys.

[0057] The production method of the present invention may further comprise a step of separating regulatory T cells in order to enrich the obtained regulatory T cells. The separation of regulatory T cells can be carried out by known methods such as a method using flow cytometry or a magnetic cell separation method.

[0058] The production method of the present invention makes it possible to produce a cell population containing regulatory T cells in which regulatory T cells have proliferated. That is, the production method of the present invention makes it possible to expand regulatory T cells.

[0059] Furthermore, another embodiment of the present invention, a method for expanding a cell population containing regulatory T cells (sometimes simply referred to herein as the "expansion method of the present invention"), is characterized by comprising the following steps: (1A) culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist.

[0060] Step (1A) can be carried out in the same manner as step (1). The explanation regarding the production method of the present invention applies to the method for expanding the above-mentioned cell population containing regulatory T cells.

[0061] The regulatory T cells obtained by the production method and proliferation method of the present invention may be regulatory T cells into which an exogenous gene has been introduced.

[0062] A "foreign gene" is a gene introduced from the outside in order to cause regulatory T cells to express a desired protein, and can be selected appropriately depending on the intended use of the regulatory T cells.

[0063] The foreign gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR), which can further include a gene for expressing a cytokine and / or a chemokine. Similar to common or known CARs, the CAR expressed by regulatory T cells is basically composed of peptides from each of the following sites linked via a spacer as needed: (i) an antigen recognition site (e.g., a single-chain antibody) that recognizes a cell surface antigen of a cancer cell; (ii) a cell membrane-spanning domain; and (iii) a signal transduction domain that induces T cell activation. The foreign gene can also be, for example, a gene for expressing an exogenous T cell receptor (TCR). The term "exogenous TCR" refers to a nucleic acid encoding the exogenous TCR being exogenous to the T cell into which the exogenous TCR is introduced, and the amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR of the T cell. One or more types of foreign genes can be introduced (e.g., a CAR and an exogenous TCR).

[0064] The means for introducing a foreign gene into cells is not particularly limited, and various known or common means can be used. Typically, the foreign gene is introduced into cells using an expression vector and expressed. The expression vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. The cells into which the foreign gene is introduced are not particularly limited, and may be at any stage, and examples include primary regulatory T cells before carrying out step (1) or (1A), and regulatory T cells obtained by the production method and expansion method of the present invention.

[0065] The technique for introducing an expression vector into cells can be an appropriate one depending on the embodiment. For example, the expression vector can be introduced into cells by known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or using a commercially available kit (according to its instructions).

[0066] The expression vector can be introduced into cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a vector containing the foreign gene and a packaging vector (plasmid) of each virus are transfected into host cells using a corresponding commercially available kit to produce a recombinant virus, and then the resulting recombinant virus is used to infect cells.

[0067] In addition to the foreign gene, the expression vector may contain sequences such as a nuclear localization signal (NLS) and a multicloning site (MCS) as needed. The expression vector may further comprise a reporter gene (e.g., a gene encoding a fluorescent protein of each color), a drug selection gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene), a suicide gene (e.g., a diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9 (inducible caspase 9)), or a gene encoding a cytochrome P450 (cyt-450). The nucleic acid sequence may include a nucleic acid (base sequence) encoding a "functional gene" such as a gene encoding a nucleotide sequence of ...

[0068] A cell population containing regulatory T cells produced by the production method and expansion method of the present invention is useful for treating animals (particularly humans) with abnormally enhanced immune responses, and is useful for treating and preventing, for example, X-linked immunodysregulation / polyendocrinopathy / enteropathy (IPEX) syndrome, graft-versus-host disease (GVHD), organ transplant rejection, autoimmune diseases, inflammatory diseases, and allergic diseases (hay fever, asthma, atopic dermatitis, eczema, food allergies, food hypersensitivity, urticaria, allergic rhinitis, allergic conjunctivitis, and drug allergies), but is not limited to these. Regulatory T cells produced by the production method and expansion method of the present invention may be used for autologous transplantation or allogeneic transplantation. They may also be used in combination with other drugs.

[0069] When carrying out such cell therapy, from the viewpoint of preventing rejection reactions, it is preferable that the subject from which cells to be used for producing regulatory T cells are isolated has an HLA type that matches that of the subject to which the regulatory T cells are administered, and it is more preferable that the subject is the same as the subject to which the regulatory T cells are administered.

[0070] According to the present invention, a pharmaceutical comprising a cell population containing regulatory T cells (hereinafter, sometimes referred to as the pharmaceutical of the present invention) can be produced. The pharmaceutical of the present invention is preferably produced as a parenteral formulation by mixing an effective amount of regulatory T cells with a pharmaceutically acceptable carrier according to known means (e.g., the method described in the Japanese Pharmacopoeia, etc.). The pharmaceutical of the present invention is preferably produced as a parenteral formulation such as an injection, suspension, or infusion. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. Examples of pharmaceutically acceptable carriers include solvents, bases, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonic agents, surfactants, and solubilizing agents.

[0071] The dosage of the pharmaceutical of the present invention can be appropriately determined depending on various conditions such as the patient's weight, age, sex, and symptoms. Generally, the number of cells administered per administration is 1×10 for a subject weighing 60 kg. ６ ~1 x 10 １０ Preferably, 1×10 ７ ~1 x 10 ９ 5×10 ７ ~5 x 10 ８ The pharmaceutical composition of the present invention is administered so that the number of cells reaches 100. The pharmaceutical composition may be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition of the present invention may also contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc., in order to stably maintain the cells. The culture medium is not particularly limited, but includes media such as RPMI, AIM-V, and X-VIVO10, but is not limited to these. Furthermore, a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. may be added to the pharmaceutical composition for stabilization purposes. The pharmaceutical composition of the present invention is intended to be used in mammals, including humans.

[0072] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural reference, unless otherwise specified.

[0073] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0074] (1) Isolation of primary cultured Tregs. CD4 Tregs were isolated from human peripheral blood mononuclear cells (PBMCs (HemaCare)). ＋ T cells to CD4 ＋ After isolation according to the recommended protocol of the T cell isolation kit Human (Myltenyi Biotec), the cells were stained with the following antibody set and analyzed by flow cytometry. ＋ CD8α － CD25 ＋ CD127 － The results are shown in Figure 1. PE / Cy7 CD4, PerCP / Cy5.5 CD8α, PE CD127, APC CD25.

[0075] The purified Tregs were obtained with a purity of 99.0%.

[0076] (2) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were cultured in a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) at a density of 1 × 10 ５ cells / well and ２ Incubator at 37°C and 5.0% CO ２After culturing for 3 days under these conditions, the cells were reseeded onto a 48-well cell culture plate and continued culturing. After culturing for another 3 days, the cells were reseeded onto a 24-well G-Rex cell culture plate and continued culturing. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich), IL-2 (100 ng / mL, PeproTech), and anti-TNFR2 antibody (3 μg / mL, Hycult The α-MEM (Invitrogen) medium used contained IL-1 (60 ng / mL, BioLegend), IL-3 (60 ng / mL, BioLegend), IL-4 (30 ng / mL, BioLegend), IL-33 (30 ng / mL, BioLegend), and TGF-β1 (5 ng / mL, BioLegend). For the first three days, anti-CD28 antibody (1.5 μg / mL, BioLegend) and anti-CD30 antibody (300 ng / mL, R&D Systems) were further added to the above composition. A control group used medium containing the above medium composition but omitting anti-TNFR2 antibody, IL-3, IL-4, IL-33, and TGF-β1. The cells were cultured for 15 days, with the medium being changed on days 3, 6, 8, 11, and 13 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in Figure 2.

[0077] The cell population obtained through expansion culture showed a proliferation potential of approximately 5,700 times on day 14 of culture. Furthermore, proliferation was enhanced compared to the control group.

[0078] (3) Examination of Treg protein expression levels during expansion culture. Each cell population, which was expanded in (2) in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody, was used on day 15 after the start of culture. The cells were stained with the following antibody set and analyzed by flow cytometry. The results are shown in Figure 3: Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, FITC FOXP3, PE Helios.

[0079] The cell population obtained by the above method showed a FOXP3 positive rate of 77.6% and a Helios positive rate of 59.5%.

[0080] (4) Evaluation of the suppressive function of Tregs. PBMCs derived from a donor other than Tregs and untreated or treated with anti-CD3 antibody were stained with CellTrace Violet (Thermo Fisher Scientific) and used as target cells. These cells were co-cultured with Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody in (2). The cells were cultured for 4 days in α-MEM (Invitrogen) containing final concentrations of FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), and ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich). Each cell population was stained with the following antibody set. The results are shown in Figure 4: Zombie NIR, PE / Cy7 CD3, FITC HLA-A24.

[0081] Treg cell number-dependent target cells (CD3 ＋ ) showed a strong inhibition of cell division.

[0082] (5) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were cultured in a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) at a density of 1 × 10 ５ cells / well and ２ Incubator at 37°C and 5.0% CO ２After culturing for 3 days under these conditions, the cells were reseeded onto a 48-well cell culture plate and continued culturing. After culturing for another 3 days, the cells were reseeded onto a 24-well G-Rex cell culture plate and continued culturing. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich), IL-2 (100 ng / mL, PeproTech), and anti-TNFR2 antibody (3 μg / mL, Hycult The medium used was α-MEM (Invitrogen) containing IL-3 (60 ng / mL, BioLegend), IL-4 (30 ng / mL, BioLegend), IL-33 (30 ng / mL, BioLegend), and TGF-β1 (5 ng / mL, BioLegend). For the first 3 days, anti-CD28 antibody (1.5 μg / mL, BioLegend) and anti-CD30 antibody (300 ng / mL, R&D Systems) were further added to the above composition. However, in practice, a medium containing IL-2 but not IL-3, IL-4, IL-33, TGF-β1, or anti-TNFR2 antibody (medium containing only IL-2) was used, or a medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from the medium composition. The culture was continued for 14 days, with medium changes on days 3, 6, 8, 11, and 13 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in FIG. 5.

[0083] The cell population obtained through expansion culture showed reduced proliferation ability on the 14th day of culture when IL-4 and IL-2 were omitted from the above medium conditions.

[0084] (6) Examination of Treg protein expression levels during expansion culture Each cell population expanded in (5) on day 14 after the start of culture (excluding the cell collection expanded in medium containing only IL-2, which could not be maintained until day 14 after the start of culture) was stained with the following antibody set and analyzed by flow cytometry. The results are shown in Figure 6: Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, FITC FOXP3.

[0085] (5) CD25 in each cell population expanded in a medium in which any of the cytokines IL-3, IL-4, IL-33, and TGF-β1 was omitted ＋ FOXP3 ＋ The populations showed 62.8%, 50.5%, 65.8%, and 30.7%, respectively.

[0086] (7) Evaluation of the suppressive function of Tregs PBMCs derived from a donor other than Tregs and untreated or treated with anti-CD3 antibodies were stained with CellTrace Violet (Thermo Fisher Scientific) and used as target cells. These cells were co-cultured with Tregs expanded in a medium from which any of the cytokines IL-3, IL-4, IL-33, and TGF-β1 had been removed in the medium composition in (5). The cells were cultured for 4 days in α-MEM (Invitrogen) containing final concentrations of FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), and ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich). Each cell population was stained with the following antibody set. The results are shown in Figure 7: Zombie NIR, PE / Cy7 CD3, FITC HLA-A24.

[0087] Treg cell number-dependent target cells (CD3 ＋ ) showed a strong inhibition of cell division.

[0088] (8) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were cultured in a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) at a density of 1 × 10 ５ cells / well and ２ Incubator at 37°C and 5.0% CO ２ After culturing for 3 days under these conditions, the cells were reseeded onto a 48-well cell culture plate and continued culturing. After culturing for another 3 days, the cells were reseeded onto a 24-well G-Rex cell culture plate and continued culturing. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100, Invitrogen), ascorbic acid 2-phosphate (50 μg / mL, Sigma-Aldrich), IL-2 (100 ng / mL, PeproTech), and anti-TNFR2 antibody (3 μg / mL, Hycult The culture medium used was α-MEM (Invitrogen) containing IL-3 (60 ng / mL, BioLegend), IL-4 (30 ng / mL, BioLegend), IL-33 (30 ng / mL, BioLegend), and TGF-β1 (5 ng / mL, BioLegend). For the first 3 days, anti-CD28 antibody (1.5 μg / mL, BioLegend) and anti-CD30 antibody (300 ng / mL, R&D Systems) were further added to the above composition. A medium containing the above medium composition but omitting IL-3, TGF-β1, and anti-TNFR2 antibody was also used. The culture was continued for 22 days, with the medium being changed on days 3, 6, 8, 11, 13, 15, 18, and 20 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in Figure 8.

[0089] The above medium conditions resulted in stronger proliferation than medium conditions obtained by removing IL-3, TGF-β1 and anti-TNFR2 antibody from the above medium conditions.

[0090] This application is based on Japanese Patent Application No. 2024-048579 filed on March 25, 2024, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a cell population in which regulatory T cells have expanded, the method comprising: (1) culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist.

2. The method according to claim 1, wherein step (1) is further carried out in the presence of a TGF-βR agonist.

3. The method according to claim 1, wherein step (1) is further carried out in the presence of IL-3.

4. The method according to claim 1, wherein step (1) is carried out further in the presence of IL-3 and a TGF-βR agonist.

5. The method of claim 1, wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

6. The method of claim 2 or 4, wherein the TGF-βR agonist is TGF-β.

7. The method according to claim 1, wherein the culturing in step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist.

8. The regulatory T cells are CD25 ＋ / FOXP3 ＋ The method of claim 1, wherein the cell is a cell.

9. CD25 ＋ / FOXP3 ＋ The cells are CD4 ＋ The method of claim 8, wherein 10. A method for expanding a cell population containing regulatory T cells, comprising: (1A) culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist.

11. A cell population containing regulatory T cells obtained by the method of claim 1 or 10.

12. A pharmaceutical comprising a cell population containing regulatory T cells according to claim 11.

13. The pharmaceutical composition according to claim 12 for use in the prevention and / or treatment of abnormally enhanced immune responses.

14. A method for preventing and / or treating abnormally enhanced immune responses, comprising administering a cell population containing regulatory T cells according to claim 11 to a subject in need thereof.

15. A cell population comprising regulatory T cells according to claim 11 for use in the prevention and / or treatment of abnormally enhanced immune responses.

16. Use of a cell population comprising regulatory T cells according to claim 11 in the manufacture of a medicament for the prevention and / or treatment of abnormally enhanced immune responses.

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