Methods of modulating proliferation of cells

Abstract

Disclosed are a method for producing a cell population in which regulatory T cells have proliferated, 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; a method for proliferating a cell population containing regulatory T cells, the method comprising (1A) culturing a cell population comprising 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 medicament comprising a cell population containing the regulatory T cells.

Description

Technical Field

[0001] This invention relates to a method for generating a cell population in which primary regulatory T cells (Tregs) have proliferated, a cell population containing regulatory T cells obtained by said method, and a drug comprising the cell population containing said regulatory T cells. Background Technology

[0002] In recent years, due to the immunosuppressive function of regulatory T cells (Tregs), research and development of cell therapies (Treg therapies) using regulatory T cells to treat various autoimmune diseases have made progress worldwide. Therefore, regulatory T cells are expected to be used to treat graft-versus-host disease (GvHD) and autoimmune diseases, as well as for the treatment and prevention of inflammatory and allergic diseases.

[0003] Regulatory T cells are broadly classified into two types: naturally occurring regulatory T cells (nTregs or thymic Tregs: tTregs) and inducible regulatory T cells (iTregs). nTregs are known to be naturally present in the thymus, while iTregs differentiate from naive T cells in peripheral blood through antigen stimulation and the induction of cytokines such as IL-2 and TGF-β. These regulatory T cells are further subdivided based on the types of biomarkers expressed within the cells.

[0004] When regulatory T cells are removed from living organisms, various organ-specific autoimmune diseases spontaneously arise. Transplantation of regulatory T cells in such cases can prevent the onset of these autoimmune diseases; therefore, regulatory T cells are considered to play a crucial role in maintaining peripheral immune tolerance. Subsequently, regulatory T cells have been shown to suppress not only autoimmunity but also most immune responses, such as inflammation induced by exogenous antigens, transplant rejection, infection immunity, allergic reactions, and tumor immunity. Currently, the transcription factor FOXP3 has also been identified as a key regulator of regulatory T cells.

[0005] FOXP3 is the master regulator of Tregs and is known to be constitutively expressed in Tregs, while constitutive expression of FOXP3 has not been observed in conventional T cells (Tconv). Tconv cells do not have inhibitory function and can also be referred to as effector T cells or inflammatory T cells. Methods for effectively proliferating primary Tregs while maintaining FOXP3 expression have been investigated, and compounds, cytokines, and combinations thereof with expression maintenance and cell proliferation activities have been reported. Examples of such compounds and cytokines include IL-2 (NPL 1 to 7), IL-4 (NPL 2, 8, and 9), and TNFR2 agonist antibodies (NPL 4, 5, 7, and 10).

[0006] Other examples of such compounds, cytokines, and combinations thereof include IL-3 (NPL 1), TGF-β (NPL 1, 3, 6, 8, and 11), etc.

[0007] Although the effects of IL-2, IL-4 and TNFR2 agonists on primary Tregs have been reported as described above, there are no reports indicating that a combination of IL-2, IL-4, IL-33 and TNFR2 antagonists (with the further addition of IL-3 or TGF-β) is particularly preferred.

[0008] Reference List Non-patent literature NPL 1: J Immunol (2011) 186: 2262-2272 NPL 2: Immunology. 2009 Jul; 127(3): 338-344 NPL 3: PLoS One., 11(2): e0148474. (2016) NPL 4: PLoS One., 11(5), e0156311 (2016) NPL 5: Front. Immunol. 9: 573 (2018) NPL 6: Clin. Exp. Immunol., 197(1): 52-63. (2019) NPL 7: Scientific Reports 3: 3153 (2013) NPL 8: Immunology (2009) 128: e670-e678 NPL 9: Front. Immunol. 8: 1508.doi: 10.3389 / fimmu.2017.01508 NPL 10: Sci. Signal. 13, eaba9600 (2020) NPL 11: J Exp Med (2003) 198(12): 1875-1886 Summary of the Invention

[0009] Technical issues The objective of this invention is to provide a method for generating a cell population in which regulatory T cells have proliferated, the method being able to efficiently proliferate primary regulatory T cells that are known to be difficult to proliferate, while maintaining the inhibitory function of the primary regulatory T cells.

[0010] Solution to the problem As a result of extensive research to achieve the above objectives, the inventors have discovered that by culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and TNFR2 agonists, regulatory T cells can proliferate efficiently while maintaining their inhibitory function.

[0011] This invention was accomplished through further research based on these findings, and provides the following methods for generating cell populations in which regulatory T cells have proliferated, cell populations containing regulatory T cells, drugs, etc.

[0012] [1] A method for creating a cell population in which regulatory T cells have proliferated, the method comprising: Step (1) Culture a population of cells containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33 and TNFR2 agonists.

[0013] [2] According to the method of [1], wherein step (1) is further performed in the presence of a TGF-βR agonist.

[0014] [3] According to the method of [1], wherein step (1) is further performed in the presence of IL-3.

[0015] [4] According to the method of [1], wherein step (1) is further performed in the presence of IL-3 and TGF-βR agonist.

[0016] [5] The method according to any one of [1] to [4], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

[0017] [6] The method according to [2], [4] or [5], wherein the TGF-βR agonist is TGF-β.

[0018] [7] The method according to any one of [1] to [6], wherein the culture in step (1) is further carried out in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists and CD28 agonists.

[0019] [7a] The method according to any one of [1] to [6], wherein the culture in step (1) is further cultured in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists and CD28 agonists, and then cultured in the absence of an agonist.

[0020] [8] The method according to any one of [1] to [7a], wherein the regulatory T cell is CD25 + / FOXP3 + cell.

[0021] [9] According to the method of [8], wherein the CD25 + / FOXP3 + The cells are CD4 + .

[0022] [9a] The method according to any one of [1] to [7a], wherein the regulatory T cell is CD25 + / CD127 - cell.

[0023] [9b] The method according to any one of [1] to [9a], wherein the regulatory T cell is a Helios + cell.

[0024]

[10] A method for proliferating a cell population containing regulatory T cells, the method comprising: Step (1A) Culture a population of cells containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and TNFR2 agonists.

[0025] [10a] According to the method of

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

[0026] [10b] According to the method of

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

[0027] [10c] According to the method of

[10] , wherein step (1A) is further performed in the presence of IL-3 and TGF-βR agonist.

[0028] [10d] The method according to any one of

[10] to [10c], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.

[0029] [10e] The method according to [10a], [10c] or [10d], wherein the TGF-βR agonist is TGF-β.

[0030] [10f] The method according to any one of

[10] to [10e], wherein the culture in step (1A) is further carried out in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists and CD28 agonists.

[0031] [10f1] The method according to any one of

[10] to [10f], wherein the culture in step (1A) is further carried out in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists and CD28 agonists, and then cultured in the absence of an agonist.

[0032] [10g] The method according to any one of

[10] to [10f1], wherein the regulatory T cell is CD25 + / FOXP3 + cell.

[0033] [10h] The method according to [10g], wherein CD25 + / FOXP3 + The cells are CD4 + .

[0034] [10i] The method according to any one of

[10] to [10f1], wherein the regulatory T cell is CD25 + / CD127 - cell.

[0035] [10j] The method according to any one of

[10] to [10i], wherein the regulatory T cell is Helios + cell.

[0036] [10k] The method according to any one of [1] to [10j] comprises: Step (2) Introduce a foreign gene (especially a gene expressing a chimeric antigen receptor) into a cell population containing primary regulatory T cells or a cell population containing regulatory T cells obtained by any one of [1] to [10j].

[0037]

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

[0038]

[12] A drug comprising a cell population containing regulatory T cells as described in

[11] .

[0039]

[13] The drug described in

[12] is used to prevent and / or treat abnormally enhanced immune responses.

[0040]

[14] A method for preventing and / or treating abnormally enhanced immune responses, the method comprising administering a population of cells containing regulatory T cells as described in

[11] to a subject in need.

[0041]

[15] The cell population containing regulatory T cells as described in

[11] is used for prevention and / or treatment of abnormally enhanced immune responses.

[0042]

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

[11] in the manufacture of a medicament for the prevention and / or treatment of abnormally enhanced immune responses.

[0043] Advantages of the present invention According to the present invention, primary regulatory T cells can proliferate efficiently while maintaining the inhibitory function of primary regulatory T cells. Attached Figure Description

[0044] Figure 1 This is a graph showing the results obtained by isolating primary cultured Tregs from PBMCs using flow cytometry. The numerical values ​​in the punctuated graph represent Tregs (CD4+). + CD8α - CD25 + CD127 - The proportion of ).

[0045] Figure 2 This graph shows the change in cell number over time when primary cultured Treg cells were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. Optimized group: medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody; Control group: medium without IL-3, IL-4, IL-33, TGF-β1, or anti-TNFR2 antibody.

[0046] Figure 3 This is a graph showing the results obtained by examining the expression levels of proteins expressed in Tregs amplified in a culture medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. All scatter plots in the graph show CD3. + CD4 + CD8 - Population. The values ​​in the figure represent the number of Treg (CD3) cells in each cell population. + CD4 + CD8 - CD25 + FOXP3 + The proportion of ).

[0047] Figure 4The left graph shows the flow cytometry analysis results of cell populations expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibodies, co-cultured with allogeneic T cells derived from human PBMCs, and the right graph shows the target cell division inhibition rate (%). E:T indicates the ratio of Treg numbers to human T cell numbers.

[0048] Figure 5 This is a graph showing the change in cell number over time when primary cultured Tregs are expanded in a medium containing only IL-2, or in a medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody.

[0049] Figure 6 This is a graph showing the results obtained by detecting the expression levels of proteins expressed in Tregs amplified in a culture medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. All scatter plots in the graph show CD3. + CD4 + CD8 - Population. The values ​​in the figure represent the number of Treg (CD3) cells in each cell population. + CD4 + CD8 - CD25 + FOXP3 + The proportion of ).

[0050] Figure 7 This is a graph showing the inhibition rate (%) of target cell division after expanding the cell population in a medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from a medium containing IL-2, IL-3, IL-4, IL-33, and TGF-β1, and co-culturing anti-TNFR2 antibody with allogeneic T cells derived from human PBMCs. E:T indicates the ratio of Treg number to human T cell number.

[0051] Figure 8 This is a graph showing the change in cell number over time when primary cultured Treg cells are expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1 and anti-TNFR2 antibody, or in a medium obtained by removing IL-3, TGF-β1 and anti-TNFR2 antibody from the above-mentioned medium composition. Detailed Implementation

[0052] The embodiments of the present invention will be described in detail below.

[0053] The terms "comprise(s)" or "comprising" mean that the elements following the term are included, but not limited to. Therefore, the term indicates inclusion of the elements following the term, but does not exclude any other elements. The terms "consist(s) of / consisting of" mean that all elements following the term are included, and the subject matter is limited to those elements. Therefore, the term "consisting of" indicates that the listed elements are necessary or required, and that substantially no other elements are present. The terms "consist(s) essentially of / consisting essentially of" mean that any element following the term is included, and the subject matter is limited to other elements that do not affect the activity or function of those elements as specified in this disclosure. Therefore, the term "essentially of" indicates that the listed elements are necessary or required, but other elements are optional and may or may not be present depending on whether the other elements affect the activity or function of the listed elements.

[0054] As used herein, the term "culture" refers to the maintenance and / or proliferation of cells in an in vitro environment. The term "culture" means the maintenance and / or proliferation of cells outside of tissues or in vitro (e.g., in cell culture dishes or flasks).

[0055] As used herein, the phrase "culturing a cell population in the presence of a substance" refers to, for example, culturing a cell population in a culture medium containing said substance. Examples of such culturing include cultures performed in a culture medium in the presence of only said substance or in the presence of said substance in conjunction with another differentiation-inducing factor, etc. When said substance is added to the culture medium, it may be added directly to the culture medium or dissolved in a suitable solvent before use. Culture may also be performed using a substance immobilized on the surface of a substrate or carrier used for culture.

[0056] As used herein, the term "positive (+)" means that a protein or gene is expressed in a quantity detectable by methods known in the art. In the case of proteins expressed intracellularly and not present on the cell surface (e.g., transcription factors or their subunits), the protein of interest can be detected by expressing a reporter protein along with the protein and detecting the reporter protein. Genes can be detected using, for example, nucleic acid amplification methods and / or nucleic acid detection methods, such as RT-PCR, microarrays, biochips, or RNAseq.

[0057] As used herein, the term "negative (-)" means that the expression level of a protein or gene is below the detection limit of all or any of the known methods described above, or that the expression level is low. The detection limit for protein or gene expression may vary depending on the respective method. The expression level of a protein or gene (whether low or high) can be determined by comparison with results obtained from control cells measured under the same conditions. For example, the expression level of CD25 in a cell population can be determined by comparing the CD25 expression level in the cell population to the CD25 expression level in PBMC-derived Tconv (control: known to have low CD25 expression) using flow cytometry; low expression is determined when expression is observed to be comparable to that in control cells, and high expression is determined when expression is observed to be higher than that in the control.

[0058] As used herein, the term "marker" refers to a protein or gene specifically expressed on the cell surface, cytoplasm, or nucleus of a predetermined cell type. The marker is preferably a "cell surface marker." The term "cell surface marker" refers to a protein expressed on the cell surface, which can be labeled (stained) with a fluorescent substance and facilitates the detection, enrichment, and isolation of cells expressing the cell surface marker. A cell surface marker refers to a gene that is specifically expressed (positive marker) or non-specifically expressed (negative marker) in a predetermined cell type; specifically, it refers to a substance produced (positive marker) or not produced (negative marker) as mRNA by transcription (positive marker) or not produced (negative marker) by translation (positive marker) or not produced (negative marker) as protein.

[0059] These cell surface markers can be detected using immunoassays that use antibodies specific to the cell surface markers, such as ELISA, immunostaining, or flow cytometry.

[0060] As used herein, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter in a cell.

[0061] As used herein, the term "regulatory T cell" refers to a T cell that, upon receiving T cell receptor-mediated stimulation, has the ability to suppress the activation of effector T cells and is responsible for suppressing the immune response (immune tolerance). Regulatory T cells are typically CD25+ cells. + / FOXP3 + Cells or CD25 + / CD127 - Cells containing CD4 + or CD8 + Cells. In this invention, regulatory T cells can be CD4 cells. + Cells (i.e., CD4) + / CD25+ / FOXP3 + or CD4 + / CD25 + / CD127 - Or it could be CD8 + Cells (i.e., CD8) + / CD25 + / FOXP3 + or CD8 + / CD25 + / CD127 - ), and more preferably CD4 + Cells. Transcription factor FOXP3 is also known as CD4. + / CD25 + The main regulators of regulatory T cells. In addition, regulatory T cells can be positive for Helios, cytotoxic T-lymphocyte (associated) antigen 4 (CTLA4; CD152), CD39, and CD73, which are considered indicators of the inhibitory 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; T cell activation is inhibited when CTLA4 expressed on the surface of T cells binds to CD80 or CD86 on the surface of antigen-presenting cells. A mechanism is also known in which CD39 and CD73 hydrolyze ATP and AMP, respectively, to produce adenosine as the final product, and T cell activation is inhibited when adenosine acts on T cells. As used herein, in substances such as "CD25..." + / FOXP3 + The " / " in the notation means "and".

[0062] As used herein, the term "cell population" refers to two or more types of cells of the same or different types. The term "cell population" also refers to a large number of cells of the same or different types.

[0063] As used herein, the phrases “regulatory T cells have proliferated” and “proliferation of regulatory T cells” refer to a state in which the number (absolute number) of regulatory T cells in a cell population increases relative to the number in a pre-culture or control (e.g., cells obtained without the present invention), and mean, for example, an increase of at least 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 300, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 50000, or 100000 times relative to the number in a pre-culture or control. In some embodiments of the invention, a cell population is generated according to the invention such that the number (absolute number) of regulatory T cells in the cell population is increased by at least 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 300 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, 10000 times, 15000 times, 20000 times, 50000 times, or 100000 times.

[0064] From a therapeutic perspective, the various cells used in this invention are preferably cells that meet Good Manufacturing Practice (GMP) standards.

[0065] The term "nucleic acid" can refer to any polymer molecule composed of nucleotides and molecules having the same function as nucleotides, and examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a polymer of a mixture of ribonucleotides and deoxyribonucleotides; and can also be a nucleotide polymer that includes nucleic acid derivatives. Nucleic acids can be single-stranded or double-stranded. Double-stranded nucleic acids also include those in which the second strand hybridizes with the first strand under stringent conditions.

[0066] Compared to RNA or DNA, nucleotide analogs can be any molecule that is modified by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve nuclease resistance or achieve stability, increase affinity for complementary nucleic acid strands, increase cell permeability, or make them visible. Nucleotide analogs can be naturally occurring or non-naturally occurring molecules, and examples include glycosidically modified nucleotide analogs (e.g., 2'-O-methylribose-substituted nucleotide analogs, 2'-O-propylribose-substituted nucleoside analogs, 2'-methoxyethoxyribose-substituted nucleic acid analogs, 2'-O-methoxyethylribose-substituted nucleotide analogs, 2'-O-[2-(guanidinium)ethyl]ribose-substituted nucleotide analogs, 2'-fluororibose-substituted nucleotide analogs, bridged nucleic acids (BNA), locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), peptide nucleic acids (PNA), oxypeptide nucleic acids (OPNA), and peptide ribonucleic acid (PRNA)), phosphodiester-modified nucleotide analogs (e.g., thiophosphate-substituted nucleotide analogs and N3'-P5' aminophosphate-substituted nucleic acid analogs), etc.

[0067] Nucleic acid derivatives can be any molecule obtained by adding another chemical substance to nucleic acids to improve nuclease resistance, achieve stability, enhance affinity for complementary nucleic acid chains, improve cell permeability, or achieve visualization compared to nucleic acids. Specific examples include derivatives with added 5'-polyamines, cholesterol, steroids, bile acids, vitamins, Cy5, Cy3, 6-FAM, or biotin.

[0068] The method of the present invention for generating a population of cells in which regulatory T cells have proliferated (also referred to herein as "the method of generation of the present invention") comprises the following steps.

[0069] (1) Culture a population of cells containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33 and TNFR2 agonists.

[0070] In addition to IL-2, IL-4, IL-33, and TNFR2 agonists, step (1) can also be performed in the presence of a TGF-βR agonist and / or IL-3. That is, step (1) can also be performed, for example, in the presence of IL-2, IL-3, IL-4, IL-33, and TNFR2 agonists; in the presence of IL-2, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists; or in the presence of IL-2, IL-3, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists. From the perspective of regulatory T cell proliferation, step (1) is preferably performed in the absence of mTOR inhibitors (e.g., rapamycin).

[0071] Interleukin (IL)-4, interleukin (IL)-3, interleukin (IL)-2, and interleukin (IL)-33 are preferably derived from mammals, and particularly preferably from humans. IL-2, IL-3, IL-4, and IL-33 may be naturally occurring products isolated and purified from mammals (especially humans), or artificially produced products through genetic engineering techniques (which may have undergone amino acid substitutions, deletions, insertions, and / or additions).

[0072] Transforming growth factor β receptor (TGF-βR) agonists are defined as substances capable of binding to TGF-βR and activating TGF-βR signaling. Examples of TGF-βR agonists include factors belonging to the TGF-β superfamily, which includes the TGF-β family, the activin family, and the bone morphogenetic protein (BMP) family. Examples of such factors belonging to the TGF-β superfamily include TGF-β (TGF-β1, TGF-β2, and TGF-β3), activin A, activin B, GDF-8, GDF-11, etc. TGF-βR agonists are preferably TGF-β, more preferably TGF-β1. A single type of TGF-βR agonist can be used alone, or in combination of two or more types of TGF-βR agonists.

[0073] Tumor necrosis factor receptor-2 (TNFR2) agonists are defined as substances capable of binding to TNFR2 and activating TNFR2 signaling, and are, for example, antibodies (e.g., anti-TNFR2 antibodies), peptides, low molecular weight compounds, or 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). TNFR2 agonists can also be TNF-α mutant proteins that bind only to TNFR2 as agonists. A single type of TNFR2 agonist can be used alone, or in combination of two or more types of TGF-βR agonists.

[0074] The TNFR2 agonist is preferably an anti-TNFR2 agonist antibody. The antibody can be a functional fragment, and examples of said functional fragments include Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), biantibodies, triantibodies, tetraantibodies, and microantibodies. Examples of antibodies include those derived from animals such as mice, rats, cattle, rabbits, goats, sheep, and guinea pigs. There are no particular limitations on the isotype of the antibody, and examples of isotypes include IgG (IgG1, IgG2, IgG3, and IgG4), IgA, IgD, IgE, and IgM. The antibody can be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody, and can also be a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), etc. For example, antibodies can be generated by constructing an expression vector containing a nucleic acid encoding the antibody, and culturing a transformant in which the nucleic acid has been introduced, culturing a hybridoma that produces the antibody, etc.

[0075] IL-2, IL-3, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists can be used in free form or in salt form. Examples of salts include salts formed with inorganic bases, such as sodium, magnesium, potassium, calcium, and aluminum salts; salts formed with organic bases, such as methylamine, ethylamine, and ethanolamine salts; salts formed with basic amino acids, such as lysine, ornithine, and arginine; and ammonium salts. The salts can be acid addition salts, and specific examples of such salts include acid addition salts formed with: inorganic 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 agonists and TNFR2 agonists also include hydrates, solvates, crystalline polymorphs, etc.

[0076] There are no particular limitations on the concentration of IL-2 in the culture medium, and it is, for example, 0.1 to 1000 ng / mL, preferably 10 to 500 ng / mL, and more preferably 50 to 200 ng / mL.

[0077] There are no particular restrictions on the concentration of IL-4 in the culture medium, and it is, for example, 0.01 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0078] There are no particular limitations on the concentration of IL-33 in the culture medium, and it is, for example, 0.1 to 100 ng / mL, preferably 10 to 100 ng / mL.

[0079] There are no particular restrictions on the concentration of TNFR2 agonist in the culture medium, and it should be adjusted appropriately according to the type of TNFR2 antagonist used. The concentration of TNFR2 agonist is, for example, 0.0001 to 100 μg / mL, preferably 0.01 to 10 μg / mL.

[0080] There are no particular restrictions on the concentration of TGF-βR agonist in the culture medium, and it should be adjusted appropriately according to the type of TGF-βR antagonist used. The concentration of TGF-βR agonist is, for example, 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL.

[0081] There are no particular restrictions on the concentration of IL-3 in the culture medium, and it is, for example, from 0.01 to 100 ng / mL, preferably from 10 to 100 ng / mL.

[0082] The culture medium used in step (1) includes the aforementioned IL-2, IL-4, IL-33, and TNFR2 agonists (as well as IL-3, TGF-βR agonists, etc.) found in the basal medium used for culturing animal cells. There are no particular limitations on the basal medium, as long as it is suitable 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, Medium 199, Eagle's MEM, αMEM, DMEM, Ham's medium, RPMI-1640, Fisher's medium, etc. Any of these media can be used alone, or two or more can be used in combination.

[0083] The culture medium may or may not contain serum. It may also contain serum substitutes (e.g., albumin, transferrin, knockout serum substitutes (KSR), fatty acids, insulin, collagen precursors, trace elements, 2'-mercaptoethanol, 3'-mercaptoglycerol, ITS supplements, or B27(TM) supplements). One type of serum substitute, or two or more types of serum substitutes, may be used.

[0084] Culture media 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, pyruvate, buffers, or inorganic salts. It is preferable to use culture media with a known composition (clearly defined chemical components) that do not contain substances of unknown composition (such as serum) because this reduces variability between large quantities of culture media and allows for the preparation of cells of stable quality.

[0085] The pH of the culture medium is typically 7.0 to 7.8, and preferably 7.2 to 7.6. Before use, the culture medium is preferably sterilized by methods such as filtration, ultraviolet irradiation, heat sterilization, or radiation irradiation to prevent contamination.

[0086] Culture is carried out with or without feeder cells. The production method of the present invention is ideally carried out in the absence of feeder cells because it prevents the introduction of substances of unknown composition and can stably produce regulatory T cells with uniform characteristics.

[0087] The cultivation conditions of the production method of the present invention are not particularly limited, and the cultivation temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C; and the CO2 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%.

[0088] The culture time is not particularly limited and can be appropriately determined by those skilled in the art when monitoring the number of regulatory T cells, etc., and is, for example, 7 days or longer, preferably 14 days or longer, more preferably 21 days or longer, and even more preferably 28 days or longer. There is no particular upper limit to the culture time, and it is, for example, 42 days or less, preferably 35 days or less, and more preferably 28 days or less. In the culture according to the invention, multiple passages can be performed as needed to obtain the required amount of regulatory T cells, and the culture medium can be added or changed. The culture according to the invention can be carried out using a known CO2 incubator. There are no particular limitations on the culture container, and it can be appropriately selected from culture plates, petri dishes, Piper dishes, flasks, bags, bottles, jars (culture vessels), bioreactors, etc. Containers bound to anti-CD3 antibodies can be used as culture containers.

[0089] In step (1), culture (cell stimulation) may be performed in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists, and CD28 agonists, and in the presence of IL-2, IL-4, IL-33, and TNFR2 agonists (with the possible addition of IL-3 and TGF-βR agonists). More preferably, culture (cell stimulation) may be initiated first in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists, and CD28 agonists, and then culture may be performed in the absence of these antibodies. Step (1) may be repeated (by passage culture) and may be performed by first initiating culture (cell stimulation) in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists, and CD28 agonists, and then culturing in the absence of these antibodies. The duration of each repeated culture is, for example, 7 to 28 days, and preferably 14 to 21 days. Specifically, an example includes culturing (stimulating) cells for approximately three days in the presence of at least one agonist selected from the group consisting of CD30 agonists, CD3 agonists, and CD28 agonists, followed by culturing in a medium without the agonist for approximately 4 to 11 days, and repeating this culture unit. There is no particular limitation on the number of culture repeats, and it can be appropriately determined by those skilled in the art while monitoring the number of immune cells, etc., of the present invention; for example, one or more, two or more, three or more, four or more, five or more, six or more, or seven or more. The upper limit for the number of culture repeats is, for example, 20.

[0090] There are no particular limitations on CD30 agonists, as long as they are molecules capable of transmitting signals from CD30 to the cell by specifically binding to CD30. Examples of CD30 agonists include anti-CD30 agonist antibodies or functional fragments of antibodies, CD30 ligands or functional fragments of ligands, etc. There are no particular limitations on the concentration of CD30 agonists in the culture medium, and they are adjusted appropriately according to the type of CD30 antagonist used. The concentration of CD30 agonists is, for example, 10 to 1000 ng / mL, preferably 100 to 500 ng / mL.

[0091] There are no particular limitations on CD3 agonists, as long as they are molecules capable of transmitting signals from CD3 to the cell by specifically binding to CD3. Examples of CD3 agonists include anti-CD3 agonist antibodies or functional fragments of antibodies, CD3 ligands or functional fragments of ligands, etc. There are no particular limitations on the concentration of CD3 agonists in the culture medium, and they are adjusted appropriately according to the type of CD3 antagonist used. The concentration of CD3 agonists 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.

[0092] There are no particular limitations on CD28 agonists, as long as the CD28 agonist is a molecule capable of transmitting signals from CD28 to the cell by specifically binding to CD28. Examples of CD28 agonists include anti-CD28 agonist antibodies or functional fragments of antibodies, CD28 ligands or functional fragments of ligands, etc. There are no particular limitations on the concentration of the CD28 agonist in the culture medium, and it is adjusted appropriately according to the type of CD28 antagonist used. 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.

[0093] The cell population containing regulatory T cells used in the method of production of the present invention consists of primary cultured cells isolated from living tissues such as bone marrow, umbilical cord blood, and blood. The term "primary cultured cells" refers to cells obtained by inoculating organs, cells, and tissues isolated from living organisms and maintaining them to the first generation. Primary cultured cells can be obtained from organs and tissues by known methods, such as enzymatic treatment, physical dispersion, or explant methods. The proportion (number of cells) of regulatory T cells in the cell population containing 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 living tissues by known methods, such as using flow cytometry or magnetic cell separation methods.

[0094] The cell population containing primary regulatory T cells used in the method of production of the present invention can be derived from humans or mammals other than humans (non-human mammals), preferably from humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cattle, horses, sheep, and monkeys.

[0095] The method of production of the present invention may further include isolating regulatory T cells to enrich the obtained regulatory T cells. Regulatory T cells can be isolated using known methods such as flow cytometry or magnetic cell separation methods.

[0096] The method of this invention can produce a cell population containing regulatory T cells, wherein the regulatory T cells have proliferated. In other words, the method of this invention can be used to expand regulatory T cells.

[0097] According to another embodiment of the present invention, a method for proliferating a cell population containing regulatory T cells (also referred to herein as "the proliferation method of the present invention") includes the following steps.

[0098] (1A) Culture a population of cells containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and TNFR2 agonists.

[0099] Step (1A) can be performed in the same manner as step (1). The description relating to the method of generating cells of the present invention applies to the above-described method for proliferating a cell population containing regulatory T cells.

[0100] The regulatory T cells obtained by the generation and proliferation methods of the present invention can be regulatory T cells with introduced exogenous genes.

[0101] "Exogenous genes" are genes introduced from outside the body to enable regulatory T cells to express the proteins they require, and can be appropriately selected according to the intended use of the regulatory T cells.

[0102] The exogenous gene may be, for example, a gene for expressing a chimeric antigen receptor (CAR), and may also include genes for expressing cytokines and / or chemokines. CARs expressed by regulatory T cells are essentially configured in the same manner as conventional or known CARs by peptides optionally linking corresponding regions via spacer regions: (i) an antigen recognition region (e.g., a single-chain antibody) that recognizes cell surface antigens of cancer cells, (ii) a transmembrane region, and (iii) a signal transduction region that induces T cell activation. The exogenous gene may also be, for example, a gene for expressing an exogenous T cell receptor (TCR). The term “exogenous TCR” means that the TCR is exogenous to the T cell in which the nucleic acid encoding the exogenous TCR is introduced, and the amino acid sequence in the exogenous TCR may be the same as or different from the amino acid sequence of the endogenous T cell TCR. One type or two or more types of exogenous genes (e.g., CAR and exogenous TCR) may be introduced.

[0103] There are no particular limitations on the methods for introducing foreign genes into cells, and various known or commonly used methods can be employed. Typically, an expression vector is used to introduce and express the foreign gene into cells. The expression vector can be linear or circular, and can be a non-viral vector (such as a plasmid), a viral vector, or a transposon-based vector. There are no particular limitations on the cells into which the foreign gene is introduced, and they can be at any stage, including examples of primary regulatory T cells prior to step (1) or (1A) and regulatory T cells obtained by the generation or proliferation methods of the present invention.

[0104] The technique for introducing expression vectors into cells can be appropriately selected depending on the implementation plan. For example, expression vectors can be introduced into cells by known methods such as viral infection, calcium phosphate, lipid transfection, microinjection, or electroporation. Expression vectors can be prepared by known methods and, where appropriate, using commercially available kits (according to their instructions) in a form suitable for each technique.

[0105] Expression vectors can be introduced into cells via viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus vectors. When using these viral vectors, recombinant viruses can be generated by transfecting host cells with a vector containing the aforementioned exogenous gene and a packaging vector (plasmid) for each virus using appropriate commercially available kits. The resulting recombinant viruses can then be used to infect cells.

[0106] In addition to the exogenous genes mentioned above, expression vectors may also contain sequences, such as nuclear localization signals (NLS) or multiple cloning sites (MCS), as needed. Expression vectors may also include nucleic acids (nucleotide sequences) encoding “functional genes” such as reporter genes (e.g., genes encoding fluorescent proteins of various colors), drug selection genes (e.g., kanamycin resistance genes, ampicillin resistance genes, or puromycin resistance genes) or suicide genes (e.g., genes encoding diphtheria toxin A, herpes simplex virus 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, etc.).

[0107] Cell populations containing regulatory T cells generated by the methods of production and proliferation of this invention can be used to treat animals (particularly humans) with abnormally enhanced immune responses, and can be used (but not limited to) to treat and prevent conditions such as X-linked polyendocrine enteropathy with immune dysregulation (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). Regulatory T cells generated by the methods of production and proliferation of this invention can be used for autologous or allogeneic transplantation. Immune cells can also be used in combination with another drug.

[0108] From the perspective of preventing rejection reactions when performing this cell therapy, the HLA type of the subject from whom the cells used to generate regulatory T cells are isolated is preferably matched with the HLA type of the subject who receives the regulatory T cells, and the subject from whom the cells are isolated is more preferably the same as the subject who receives the regulatory T cells.

[0109] According to the present invention, a drug comprising a cell population containing regulatory T cells (hereinafter sometimes referred to as "the drug of the present invention") can be produced. The drug of the present invention is preferably formulated as a parenteral formulation by known means (e.g., the method described in the Japanese Pharmacopoeia), for example, by mixing an effective amount of regulatory T cells with a pharmaceutically acceptable carrier. The drug of the present invention is preferably formulated as a parenteral formulation, such as an injectable formulation, a suspension, or an infusion formulation. Examples of parenteral administration methods include intravenous, intra-arterial, 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, solubilizers, etc.

[0110] The dosage of the drug of this invention can be appropriately determined according to various conditions, such as the patient's weight, age, sex, and symptoms, and the drug is typically administered to a subject weighing 60 kg, such that the cell count per administration is typically 1 × 10⁻⁶. 6 Up to 1×10 10 1 × 10 cells, preferably 1 × 10 7 Up to 1×10 9 5 × 10 cells, more preferably 5 × 10 7 Up to 5×10 8Cells. The drug can be administered once or multiple times. The drug of the present invention can be in known forms suitable for parenteral administration, such as injection or infusion adjuvants. The drug of the present invention may also contain physiological saline, phosphate-buffered saline (PBS), culture medium, etc., to stabilize and maintain cells. There are no particular limitations on the culture medium, and examples include, but are not limited to, RPMI, AIM-V, and X-VIVO10 media. For stability, pharmaceutically acceptable carriers (e.g., human serum albumin), preservatives, etc., may also be added to the drug. The drug of the present invention can be used in mammals, including humans.

[0111] As used in this specification and claims, unless the context specifically requires it, singular terms are intended to include plural forms, and plural terms are intended to include singular forms. Therefore, it should be understood that, unless otherwise specified, singular articles (e.g., “a,” “an,” or “the” in English) also include the corresponding plural concept.

[0112] Example The following embodiments are provided to describe the invention in more detail. However, the invention is by no means limited to these embodiments, etc.

[0113] (1) Isolation of Tregs from primary culture According to CD4 + The recommended protocol for the T-cell isolation kit (Miltenyi Biotec) is to isolate CD4 cells from human peripheral blood mononuclear cell fractions (PBMCs (HemaCare)). + T cells were stained using the following antibody group and analyzed by flow cytometry. Treg (CD4+) cells were also purified by flow cytometry. + CD8α - CD25 + CD127 - The results are shown in Figure 1 middle.

[0114] PE / Cy7 CD4, PerCP / Cy5.5 CD8α, PE CD127, APC CD25.

[0115] The purified Treg was obtained with a purity of 99.0%.

[0116] (2) Expansion culture of primary cultured Treg cells The primary cultured Tregs obtained in (1) were used at a rate of 1 × 10⁻⁶. 5Cells were seeded per well in 96-well cell culture plates pre-conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) and cultured for three days in a CO2 incubator at 37°C and 5.0% CO2. Cells were then re-seeded into 48-well cell culture plates for further culture. After a further three days of culture, cells were re-seeded into 24-well G-Rex cell culture plates for continued culture. α-MEM (Invitrogen) was used as the culture medium, which contained the following components 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), anti-TNFR2 antibody (3 μg / mL, Hycult Biotech), 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 additionally added to the above composition. As a control group, a culture medium obtained by removing anti-TNFR2 antibody, IL-3, IL-4, IL-33, and TGF-β1 from the above culture medium composition was used. Cells were cultured for 15 days, with the culture medium changed on days 3, 6, 8, 11, and 13 after the start of culture, and cell counts were measured to plot a line graph. Results are shown in... Figure 2 middle.

[0117] The cell population obtained through amplification culture exhibited approximately 5,700-fold proliferation capacity by day 14 of culture. Furthermore, the results showed enhanced proliferation compared to the control group.

[0118] (3) Study on protein expression levels in Tregs during amplification culture On day 15 after the start of culture, each cell population expanded in (2) in medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1 and anti-TNFR2 antibodies was stained with the following antibody set and analyzed by flow cytometry. Results are as follows: Figure 3shown. Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, FITCFOXP3, PE Helios.

[0119] The cell population obtained using the above method showed a 77.6% FOXP3 positive cell percentage. The Helios positive cell percentage was also 59.5%.

[0120] (4) Assessment of Treg inhibitory function PBMCs derived from donors different from the Treg donors, whether untreated or treated with anti-CD3 antibody and stained with CellTrace Violet (Thermo Fisher Scientific), were used as target cells and co-cultured with the Tregs amplified in (2) in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibodies. Cells were cultured for four 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), and ascorbic acid 2-phosphate (50 μg / mL, Sigma-Adrich). Each cell population was stained with the following antibody group. Results are shown in... Figure 4 middle.

[0121] Zombie NIR, PE / Cy7 CD3, FITC HLA-A24.

[0122] Target cells (CD3) + Splitting is strongly suppressed in a manner that depends on the number of Tregs.

[0123] (5) Expansion culture of primary cultured Treg cells The primary cultured Tregs obtained in (1) were used at a rate of 1 × 10⁻⁶. 5Cells were seeded per well in 96-well cell culture plates pre-conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) and cultured for three days in a CO2 incubator at 37°C and 5.0% CO2. Cells were then re-seeded into 48-well cell culture plates for further culture. After a further three days of culture, cells were re-seeded into 24-well G-Rex cell culture plates for continued culture. α-MEM (Invitrogen) was used as the culture medium, which contained the following components 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), anti-TNFR2 antibody (3 μg / mL, Hycult Biotech), 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 additionally added to the above composition. However, in practice, the above culture medium components are used in a medium containing IL-2 but without IL-3, IL-4, IL-33, TGF-β1, or anti-TNFR2 antibody (a medium containing only IL-2), or a medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from the above culture medium composition. Cells were cultured for 14 days, with the medium changed on days 3, 6, 8, 11, and 13 after the start of culture, and cell counts were measured to plot a line graph. The results are shown in... Figure 5 middle.

[0124] On day 14 of culture, the cell populations obtained by expansion culture showed reduced proliferation capacity under conditions where IL-4 and IL-2 were removed from the above culture medium.

[0125] (6) Study on protein expression levels in Tregs during amplification culture On day 14 after the start of culture, each cell population expanded in (5) (excluding cell populations expanded in IL-2-only medium, which could not be maintained until day 14 after the start of culture) was stained with the following antibody group and analyzed by flow cytometry. Results are as follows Figure 6 As shown. Zombie NIR, BV510 CD3, BV421 CD4, PE / Cy7 CD8β, APC CD25, FITC FOXP3.

[0126] CD25 cells in the corresponding cell population expanded in a culture medium obtained by removing any one of the cytokines IL-3, IL-4, IL-33, and TGF-β1 from the culture medium composition in (5) + FOXP3 + The percentages for the groups were 62.8%, 50.5%, 65.8%, and 30.7%, respectively.

[0127] (7) Assessment of Treg inhibitory function PBMCs derived from donors different from the Treg donors, whether untreated or treated with anti-CD3 antibody and stained with CellTrace Violet (Thermo Fisher Scientific), were used as target cells and co-cultured with the Tregs amplified in (5) in a medium obtained by removing any of the cytokines IL-3, IL-4, IL-33, and TGF-β1 from the culture medium composition. Cells were cultured for four days in α-MEM (Invitrogen) containing the final concentrations of FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1 / 100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1 / 100), and ascorbic acid 2-phosphate (50 μg / mL, Sigma-Adrich). Each cell population was stained with the following antibody group. Results are shown in... Figure 7 middle.

[0128] Zombie NIR, PE / Cy7 CD3, FITC HLA-A24.

[0129] Target cells (CD3) + Splitting is strongly suppressed in a manner that depends on the number of Tregs.

[0130] (8) Expansion culture of primary cultured Treg cells The primary cultured Tregs obtained in (1) were used at a rate of 1 × 10⁻⁶. 5Cells were seeded per well in 96-well cell culture plates pre-conjugated with anti-CD3 antibody (3 μg / mL, eBioscience) and cultured for three days in a CO2 incubator at 37°C and 5.0% CO2. Cells were then re-seeded into 48-well cell culture plates for further culture. After a further three days of culture, cells were re-seeded into 24-well G-Rex cell culture plates for continued culture. α-MEM (Invitrogen) was used as the culture medium, which contained the following components 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), anti-TNFR2 antibody (3 μg / mL, Hycult Biotech), 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 additionally added to the above composition. A culture medium obtained by removing IL-3, TGF-β1, and anti-TNFR2 antibodies from the above culture medium composition was also used. Cells were cultured for 22 days, with the culture medium changed at days 3, 6, 8, 11, 13, 15, 18, and 20 after the start of culture, and cell counts were measured to plot a line graph. Results are shown in... Figure 8 middle.

[0131] The results showed that the cell proliferation capacity under the above culture conditions was stronger than that under the culture conditions obtained after removing IL-3, TGF-β1 and anti-TNFR2 antibody from the above culture conditions.

[0132] This application is based on Japanese Patent Application No. 2024-048579, filed in Japan on March 25, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A method for creating a cell population in which regulatory T cells have proliferated, comprising: Step (1) Culture a population of cells containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33 and TNFR2 agonists.

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

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

4. The method according to claim 1, wherein, Step (1) is further performed in the presence of IL-3 and TGF-βR agonists.

5. The method according to claim 1, wherein, The TNFR2 agonist is an anti-TNFR2 agonist antibody.

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

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

8. The method according to claim 1, wherein, The regulatory T cells are CD25. + / FOXP3 + cell.

9. The method according to claim 8, wherein, The CD25 + / FOXP3 + The cells are CD4 + .

10. A method for proliferating a cell population containing regulatory T cells, the method comprising: Step (1A) Culture a population of cells containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and TNFR2 agonists.

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

12. A drug comprising a cell population containing regulatory T cells as described in claim 11.

13. The medicament according to claim 12, used for the prevention and / or treatment of abnormally enhanced immune responses.

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

15. The cell population containing regulatory T cells according to claim 11, for the prevention and / or treatment of abnormally enhanced immune responses.

16. Use of the cell population containing 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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