Agent for preventing and / or treating tumor

Abstract

To develop a new tumor preventative / therapeutic.　Provided are: an agent for preventing and / or treating a tumor, the agent containing, inter alia, an IKZF1ΔE5 protein, which is a deletion variant of IKZF1 in which part or all of exon 5 has been deleted, or an IKZF1ΔE5-gene-related substance; and a method for screening such agents.

Description

Preventive and / or therapeutic agent for tumors

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority under the Paris Convention to and the benefit of Japanese Patent Application No. 2022-112660 (filed July 13, 2022), the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a preventive and / or therapeutic agent for tumors. In particular, the present invention relates to a preventive and / or therapeutic agent for tumors comprising an Ikzf1ΔE5 protein or an Ikzf1ΔE5 gene-related substance, and a method for screening for the same.

[0003] In recent years, research has focused on cancer immunotherapy. For example, the immune checkpoint inhibitor ipilimumab, an anti-CTLA-4 antibody, is a drug that enhances antitumor immunity by eliminating regulatory T cells (Treg cells).

[0004] IKAROS family zinc finger 1 (Ikzf1), also known simply as "Ikaros," is one of five IKAROS family transcription factors found in mammals. Ikzf1 is a chromatin remodeling molecule expressed in the fetal and adult hematopoietic system, where it functions as a regulator of lymphocyte differentiation. Loss-of-function mutations and defects in Ikzf1 have been observed in B-acute lymphoblastic leukemia (B-ALL) and have been associated with poor prognosis. Germline Ikzf1 mutations have also been associated with autoimmune diseases (Non-Patent Document 1).

[0005] Non-Patent Document 2 describes the generation and analysis of mice expressing a B cell-specific mutant of Ikzf1 lacking exon 5, demonstrating that Ikzf is important in differentiation from preB cells and that B lymphoblastic leukemia (B-ALL) develops from differentiation-inhibited preB cells. This indicates that Ikzf1 Exon 5 is important in B cell differentiation and the development of B-ALL. Patent Document 1 also describes a composition and method comprising T cells that enhance cytolytic activity by inhibiting Ikzf1 expression in the cells.

[0006] WO2014 / 138348 publication

[0007] Journal of Allergy and Clinical Immunology Volume 140, Issue 1, Pages 223-231 (2017) Nat Immunol. 2014 Mar;15(3):294-304. doi: 10.1038 / ni.2821. Epub 2014 Feb 9.

[0008] An object of the present invention is to develop a new drug for preventing and treating tumors.

[0009] The present inventors have conducted extensive research with the aim of developing a new preventive or therapeutic method for immunotherapy, and as a result have found that expression of a deleted Ikzf1 mutant in which exon 5 is deleted in part or in whole (hereinafter, this may be referred to as "Ikzf1ΔE5"; the gene encoding Ikzf1ΔE5 may be referred to as the "Ikzf1ΔE5 gene" and the protein encoded by the Ikzf1ΔE5 gene may be referred to as the "Ikzf1ΔE5 protein") by partially or completely deleting exon 5 of Ikzf1 in regulatory T cells (Treg cells) significantly enhances interferon-γ production from regulatory T cells and enables the prevention and / or treatment of tumors. Based on this finding, the present inventors have conducted further studies and have completed the present invention.

[0010] That is, the present invention includes the following aspects. <Tumor preventive and / or therapeutic agent No. 1> [1] A tumor preventive and / or therapeutic agent comprising an Ikzf1ΔE5 protein or an Ikzf1ΔE5 gene-related substance. [2] The tumor preventive and / or therapeutic agent of [1], which targets T cells. [3] The tumor preventive and / or therapeutic agent of [2], wherein the T cells are CD4-positive T cells. [4] The tumor preventive and / or therapeutic agent of [2], wherein the T cells are regulatory T cells and / or naive T cells. [5] The tumor preventive and / or therapeutic agent of any of [1] to [4], wherein the Ikzf1ΔE5 gene-related substance is a nucleic acid molecule encoding the Ikzf1ΔE5 protein.

[0011] [6] The tumor preventive and / or therapeutic agent according to any one of [1] to [4], wherein the Ikzf1ΔE5 gene-related substance is a substance that deletes part or all of exon 5 of the Ikzf1 gene. [7] The tumor preventive and / or therapeutic agent according to [6], wherein the substance that deletes part or all of exon 5 of the Ikzf1 gene is selected from the group consisting of an exon skipping agent for exon 5 of the Ikzf1 gene and a gene therapy vector such as CRISPR / Cas. [8] The tumor preventive and / or therapeutic agent according to [7], wherein the exon skipping agent is a nucleic acid. [9] The tumor preventive and / or therapeutic agent according to [1], wherein the tumor includes T cells that express Ikzf1ΔE5.

[0012]

[10] A method for preventing and / or treating a tumor, comprising administering the agent for preventing and / or treating a tumor according to any one of [1] to [9].

[0013] The present invention further includes the following other embodiments. <Interferon gamma regulator>

[21] An interferon gamma production regulator comprising an Ikzf1ΔE5 protein or an Ikzf1ΔE5 gene-related substance.

[22] The interferon gamma production regulator of

[21] , which targets T cells.

[23] The interferon gamma production regulator of

[22] , wherein the T cells are CD4-positive T cells.

[24] The interferon gamma production regulator of

[22] , wherein the T cells are regulatory T cells and / or naive T cells.

[25] The interferon gamma production regulator of any of

[21] to

[24] , wherein the Ikzf1ΔE5 gene-related substance is a nucleic acid molecule encoding the Ikzf1ΔE5 protein.

[0014]

[26] The interferon-γ production regulator according to any one of

[21] to

[24] , wherein the Ikzf1ΔE5 gene-related substance is a substance that deletes exon 5 of the Ikzf1 gene.

[27] The interferon-γ production regulator according to

[26] , wherein the substance that deletes exon 5 of the Ikzf1 gene is selected from the group consisting of an exon skipping agent for exon 5 of the Ikzf1 gene and a gene therapy vector such as CRISPR / Cas.

[28] The interferon-γ production regulator according to

[27] , wherein the exon skipping agent is a nucleic acid.

[29] The interferon-γ production regulator according to

[21] , comprising T cells that express Ikzf1ΔE5.

[0015] <T cell regulator>

[31] A T cell regulator comprising an Ikzf1ΔE5 protein or an Ikzf1ΔE5 gene-related substance.

[32] The T cell regulator of

[31] , which targets T cells.

[33] The T cell regulator of

[32] , wherein the T cells are CD4-positive T cells.

[34] The T cell regulator of

[32] , wherein the T cells are regulatory T cells and / or naive T cells.

[35] The T cell regulator of any of

[31] to

[34] , wherein the Ikzf1ΔE5 gene-related substance is a nucleic acid molecule encoding the Ikzf1ΔE5 protein.

[0016]

[36] The T cell modulating agent according to any one of

[31] to

[34] , wherein the Ikzf1ΔE5 gene-related substance is a substance that deletes exon 5 of the Ikzf1 gene.

[37] The T cell modulating agent according to

[36] , wherein the substance that deletes exon 5 of the Ikzf1 gene is selected from the group consisting of an exon skipping agent for exon 5 of the Ikzf1 gene and a gene therapy vector such as CRISPR / Cas.

[38] The T cell modulating agent according to

[37] , wherein the exon skipping agent is a nucleic acid.

[39] The T cell modulating agent according to

[31] , comprising T cells that express Ikzf1ΔE5.

[0017] The present invention also includes the following additional embodiments.

[41] A tumor preventive and / or therapeutic agent comprising an inhibitor and / or promoter of the formation of an Ikzf1 complex containing Ikzf1 protein and Foxp3 protein, CHD4 protein, and / or HDAC1 protein.

[42] An interferon-γ production regulator comprising an inhibitor and / or promoter of the formation of an Ikzf1 complex containing Ikzf1 protein and Foxp3 protein, CHD4 protein, and / or HDAC1 protein.

[43] A T cell regulator comprising an inhibitor and / or promoter of the formation of an Ikzf1 complex containing Ikzf1 protein and Foxp3 protein, CHD4 protein, and / or HDAC1 protein.

[44] The T cell regulator of

[43] , wherein the T cells are CD4-positive T cells.

[45] The T cell regulator of

[43] , wherein the T cells are regulatory T cells and / or naive T cells.

[46] The agent according to any one of

[41] to

[45] , wherein the Ikzf1 complex comprises an Ikzf1 protein and a Foxp3 protein.

[47] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation comprises a part or all of the exon 5 fragment of the Ikzf1 protein.

[48] The agent according to any one of

[41] to

[47] , wherein the inhibitor of Ikzf1 complex formation comprises a nucleic acid encoding a part or all of the exon 5 fragment of the Ikzf1 protein.

[49] The agent according to any one of

[41] to

[48] , wherein the inhibitor of Ikzf1 complex formation binds to a region comprising a part or all of the exon 5 fragment of the Ikzf1 protein.

[50] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation comprises a part or all of the FHD of the Foxp3 protein.

[51] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation comprises a nucleic acid encoding all or part of the FHD of Foxp3 protein.

[52] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation binds to a region comprising all or part of the FHD of Foxp3 protein.

[53] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation comprises a Foxp3 protein lacking all or part of the FHD.

[54] The agent according to any one of

[41] to

[46] , wherein the inhibitor of Ikzf1 complex formation comprises a nucleic acid encoding a Foxp3 protein lacking all or part of the FHD.

[0018]

[55] A method for preventing and / or treating a tumor, comprising administering the tumor preventive and / or therapeutic agent according to any one of

[41] and

[47] to

[54] .

[0019] <Screening method for a "regulator of Ikzf1 complex formation">

[61] A method for screening for a regulator of Ikzf1 complex formation comprising Ikzf1 protein, and Foxp3 protein, CHD4 protein, and / or HDAC1 protein, comprising the steps of: (a) contacting a candidate compound for a regulator of Ikzf1 complex formation with a system comprising Ikzf1 protein or a fragment thereof, and Foxp3 protein or a fragment thereof, CHD4 protein or a fragment thereof, and / or HDAC1 protein or a fragment thereof; (b) examining whether the candidate compound inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with HDAC1 protein or a fragment thereof; and (c) identifying the candidate compound as a regulator of Ikzf1 complex formation if it inhibits or promotes any of the associations.

[62] A method for screening for a modulator of Ikzf1 complex formation according to

[61] , comprising the steps of: (a) contacting T cells in vitro with a candidate compound for a modulator of Ikzf1 complex formation; (b) examining whether the candidate compound inhibits or promotes the association between Ikzf1 protein and Foxp3 protein, whether it inhibits or promotes the association between Ikzf1 protein and CHD4 protein, whether it inhibits or promotes the association between Ikzf1 protein and HDAC1 protein, whether it inhibits or promotes the association between Foxp3 protein and CHD4 protein, and / or whether it inhibits or promotes the association between Foxp3 protein and HDAC1 protein; and (c) identifying the candidate compound as a modulator of Ikzf1 complex formation if it inhibits or promotes any of the associations.

[63] A method for screening for a regulator of Ikzf1 complex formation according to

[61] , comprising the steps of: (a) contacting in vitro cultured cells or T cells in which Ikzf1 protein or a fragment thereof, Foxp3 protein or a fragment thereof, CHD4 protein or a fragment thereof, and / or HDAC1 protein or a fragment thereof have been forcibly expressed with a candidate compound for a regulator of Ikzf1 complex formation; (b) examining whether the candidate compound inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with HDAC1 protein or a fragment thereof; (c) If the candidate compound inhibits or promotes either of the associations, the candidate compound is identified as a regulator of Ikzf1 complex formation.

[64] A method for screening for a regulator of Ikzf1 complex formation according to

[61] , comprising the steps of: (a) contacting a purified Ikzf1 protein or a fragment thereof, a purified Foxp3 protein or a fragment thereof, a purified CHD4 protein or a fragment thereof, and / or a purified HDAC1 protein or a fragment thereof in vitro with a candidate compound for a regulator of Ikzf1 complex formation; (b) examining whether the candidate compound inhibits or promotes the association of the Ikzf1 protein or a fragment thereof with the Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of the Ikzf1 protein or a fragment thereof with the CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of the Ikzf1 protein or a fragment thereof with the HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of the Foxp3 protein or a fragment thereof with the CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of the Foxp3 protein or a fragment thereof with the HDAC1 protein or a fragment thereof; (c) If the candidate compound inhibits or promotes either association, it is identified as a substance that regulates Ikzf1 complex formation.

[65] The method of any of

[61] to

[64] , wherein the substance that regulates Ikzf1 complex formation is a substance that inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof.

[66] The method of any of

[61] to

[65] , wherein the substance that regulates Ikzf1 complex formation is a preventive and / or therapeutic agent for tumors, an agent that regulates interferon-γ production, or an agent that regulates T cells.

[0020] <Tumor preventive and / or therapeutic agent No. 2>

[71] A tumor preventive and / or therapeutic agent comprising an inhibitor of CHD4 protein activity.

[72] An interferon-γ production regulator comprising an inhibitor of CHD4 protein activity.

[73] A T cell regulator comprising an inhibitor of CHD4 protein activity.

[74] The T cell regulator of

[73] , wherein the T cells are CD4-positive T cells.

[75] The T cell regulator of

[73] , wherein the T cells are regulatory T cells and / or naive T cells.

[0021]

[76] A method for preventing and / or treating a tumor, comprising administering the tumor preventive and / or therapeutic agent according to

[71] . <Tumor preventive and / or therapeutic agent No. 3>

[81] A tumor preventive and / or therapeutic agent comprising an inhibitor of HDAC1 protein activity.

[82] An interferon-γ production regulator comprising an inhibitor of HDAC1 protein activity.

[83] A T cell regulator comprising an inhibitor of HDAC1 protein activity.

[84] The T cell regulator according to

[83] , wherein the T cells are CD4-positive T cells.

[85] The T cell regulator according to

[83] , wherein the T cells are regulatory T cells and / or naive T cells.

[0022]

[86] A method for preventing and / or treating a tumor, comprising administering the tumor preventive and / or therapeutic agent according to

[81] .

[0023] According to the present invention, a new agent for preventing and treating tumors can be provided.

[0024] Figure 1 (top) shows the structure of each Flag-Ikzf1 mutant. Figure 1 (bottom) is a photograph showing the results of Western blotting in which each Ikzf1 mutant and Myc-Foxp3 were expressed in HEK293T cells and immunoprecipitated with anti-Flag-M2 antibody. In the figure, α-Flag refers to anti-Flag-M2 antibody, and α-Myc refers to anti-c-Myc antibody. Figure 2 (left) shows the structure of each Foxp3 mutant. Figure 2 (right) is a photograph showing the results of Western blotting in which each Foxp3 mutant and Myc-Ikzf1 were expressed in HEK293T cells and immunoprecipitated with anti-Flag-M2 antibody. Figure 3 shows the structure of Ikzf1-expressing Treg cells (Foxp3 Cre ) and Ikzf1ΔE5-expressing Treg cells (Foxp3 Cre IKE5 f / f 4 shows the results of Western blotting in which endogenous Foxp3 or Ikzf1 was immunoprecipitated in Ikzf1-expressing Treg cells (Foxp3) under various conditions. Cre ) and Ikzf1ΔE5-expressing Treg cells (Foxp3 Cre IkE5 f / fFigure 5 shows the results of Western blotting of immunoprecipitation of HDAC1 with endogenous Foxp3 in human Treg cells. Figure 5 shows the results of Western blotting of immunoprecipitation with anti-Foxp3 antibody in human Treg cells. Figure 6 shows the percentage of IFN-γ-positive Treg cells after knockout of various Ikzfs and expression of Ikzf1ΔE5. Values ​​were calculated using Tukey's multiple comparison method after standard one-way ANOVA. * P<0.05, ** P<0.01, ***; P<0.001. Figure 7 shows the effect of Ikzf family knockout on IFN-γ expression in human Treg cells. Figure 8A shows the results of Western blotting confirming that pomalidomide and REF001329 are Ikzf family protein degraders in human Treg cells. Figure 8B is a graph showing the ratio of IFN-γ-positive Treg cells obtained by gating using flow cytometry. Figure 9 shows the flow cytometry results showing the differentiation effect into Treg cells when Ikzf1,3 protein knockdown using pomalidomide was performed on human naive T cells. Figure 10 shows the anti-tumor effect of Ikzf1ΔE5 Treg cells. Figure 10A shows the experimental procedure. Figure 10B shows the results of Foxp3eGFP-Cre-ERT2IkE5 Treg cells expressing Ikzf1ΔE5 specifically in Foxp3 cells. f / f Figure 10C is a graph showing that engraftment of B16FO cells was suppressed in mice expressing Ikzf1ΔE5 compared to Foxp3eGFP-Cre-ERT2. f / f 11 is a graph showing that the engraftment of MC38 cells was suppressed in mice treated with Foxp3 eGFP-Cre-ERT2 compared to Foxp3 eGFP-Cre-ERT2. Cre / + R26 RFP / + and Foxp3 Cre / + IkE5 f / f R26 RFP / +Spleens and lymph nodes were collected from mice (3-4 weeks old) and the percentage of exTreg cells was measured by FACS analysis. Foxp3-expressing Treg cells are positive for both RFP and YFP, while exTreg cells that have lost Foxp3 expression are detected as a cell population that is RFP-positive and YFP-negative. Figure 12 shows the results of FACS analysis of Foxp3. Cre Mouse (straight line) and Foxp3 Cre IkE5 f / f Normalized density plots of p300 and NFAT1 binding peaks around regions of increased Foxp3 binding in mouse CD4+YFP+ Treg cells (wavy lines). Normalized signal densities were plotted within a ±1 kb window centered around the Foxp3 binding site.

[0025] <Preventive and / or Therapeutic Agent for Tumors> In one embodiment, the present invention relates to a preventive and / or therapeutic agent for tumors, comprising an Ikzf1ΔE5 protein or an Ikzf1ΔE5 gene-related substance. Here, tumor refers to solid cancers, such as epithelial cell cancers such as lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, and malignant melanoma, non-epithelial cell cancers such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and leiomyosarcoma, and blood cancers such as leukemia, malignant lymphoma, and multiple myeloma.

[0026] In the present invention, "Ikzf1ΔE5 gene-related substances" includes not only nucleic acid molecules encoding the Ikzf1ΔE5 protein but also substances that cause partial or complete deletion of exon 5 of the Ikzf1 gene in target cells. Here, "a portion of exon 5 of the Ikzf1 gene" may refer to a fragment comprising at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the entire length of the portion. Preferably, "a portion of exon 5 of the Ikzf1 gene" refers to a portion comprising zinc finger domain 2 (ZF2) of Ikzf1, a portion comprising zinc finger domain 3 (ZF3) of Ikzf1, or a portion comprising both ZF2 and ZF3. Target cells include T cells, preferably CD4-positive T cells, and more preferably regulatory T cells or naive T cells.

[0027] In the present invention, when the Ikzf1ΔE5 gene-related substance is a substance that deletes part or all of exon 5 of the Ikzf1 gene, examples of such a substance include an exon skipping agent for exon 5 of the Ikzf1 gene and a gene therapy vector such as CRISPR / Cas. An exon skipping agent is a substance that acts within the nucleus of a cell to prevent the recognition (skipping) of a specific exon adjacent to a site where a specific pre-mRNA mutation has occurred, thereby joining adjacent exons and creating a target deletion mutant. In the present invention, the exon skipping agent is preferably a nucleic acid, more preferably an antisense nucleic acid.

[0028] In the present invention, gene therapy vectors such as CRISPR / Cas are not particularly limited as long as they are capable of deleting part or all of exon 5 of the Ikzf1 gene by genome editing, and for example, the technology described in WO2018 / 179578 can be applied.

[0029] In another aspect, the present invention relates to a preventive and / or therapeutic agent for tumors of the present invention, comprising T cells expressing Ikzf1ΔE5. Such an aspect can be applied to, for example, adoptive T cell therapy.

[0030] In another aspect, the present invention relates to a tumor prevention and / or treatment agent of the present invention, comprising an inhibitor of the formation of an Ikzf1 complex containing Ikzf1 protein, Foxp3 protein, CHD4 protein, and / or HDAC1 protein, or an inhibitor of the activity of CHD4 protein and / or HDAC1 protein. Such formation inhibitors can be screened by any known method. Preferably, such formation inhibitors inhibit the association of Ikzf1 protein with Foxp3 protein. Alternatively, such formation inhibitors may be proteins containing a partial or complete fragment of exon 5 of Ikzf1 protein, proteins containing a partial or complete FHD of Foxp3 protein, Foxp3 protein lacking a partial or complete FHD, or nucleic acids encoding such proteins. Here, the FHD in Foxp3 refers to the DNA-binding region (Forkhead domain) of Foxp3. In the present invention, FHD preferably refers to the C-terminal region of Foxp3 protein, beginning with residue 279, as set forth in SEQ ID NO: 4. A portion of the exon 5 fragment of the Ikzf1 protein may mean a fragment containing 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, 55% or more, 50% or more, 45% or more, 40% or more, 35% or more, 30% or more, 25% or more, 20% or more, 15% or more, 10% or more, or 5% or more of the total length of the portion, and preferably means a portion containing zinc finger domain 2 (ZF2) of Ikzf1, a portion containing zinc finger domain 3 (ZF3) of Ikzf1, or a portion containing both ZF2 and ZF3. A portion of the FHD of Foxp3 protein may refer to the full length of said portion, preferably a fragment comprising 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, 55% or more, 50% or more, 45% or more, 40% or more, 35% or more, 30% or more, 25% or more, 20% or more, 15% or more, 10% or more, or 5% or more of the C-terminal region from residue 279 onwards of the Foxp3 protein shown in SEQ ID NO:4.Furthermore, in this specification, a portion or all of the exon 5 fragment of the Ikzf1 protein encompasses peptides whose corresponding amino acid sequences are 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, or 55% or more identical, and a portion or all of the FHD of the Foxp3 protein encompasses peptides whose corresponding amino acid sequences are 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, 55% or more, or 50% or more identical.

[0031] Examples of inhibitors of CHD4 protein and / or HDAC1 protein activity include inhibitors of the chromatin remodeling activity, ATPase activity, DNA binding activity, and / or deacetylation activity of CHD4 protein and / or HDAC1 protein. Inhibitors of CHD4 protein and / or HDAC1 protein activity may be obtained by any known method, for example, by measuring chromatin remodeling activity using a "Mononucleosome disruption assay" (Nucleic Acids Res. 2001 Jun 15;29(12):2517-21.) and screening based on the measured activity.

[0032] In one embodiment, substances that bind to a region containing all or part of exon 5 of the Ikzf1 protein or a region containing all or part of the FHD of the Foxp3 protein can also be used as inhibitors of Ikzf1 complex formation. Examples of such substances include antibodies that have this region as an epitope. Antibodies that can be used as inhibitors of Ikzf1 complex formation can be produced by standard methods using, for example, a peptide or protein containing all or part of exon 5 of the Ikzf1 protein or all or part of the FHD of the Foxp3 protein as an antigen. Such antibodies can be administered directly as antibody formulations or in the form of gene therapy vectors such as mRNA or AAV.

[0033] The present invention is based on the discovery that inhibiting the formation of an Ikzf1 complex containing the Ikzf1 protein, Foxp3 protein, CHD4 protein, and / or HDAC1 protein in T cells reprograms the cells and enhances interferon-γ production. Therefore, the tumor preventive and / or therapeutic agents of the present invention encompass any and all embodiments in which the formation of an Ikzf1 complex containing the Ikzf1 protein, Foxp3 (e.g., FHD), CHD4 protein, and / or HDAC1 protein is inhibited.

[0034] In relation to this embodiment, the present invention relates to an interferon-γ production regulator comprising an Ikzf1ΔE5 protein, an Ikzf1ΔE5 gene-related substance, the above-mentioned Ikzf1 complex formation inhibitor, the above-mentioned Ikzf1 complex formation promoter (hereinafter, the above-mentioned Ikzf1 complex formation inhibitor and formation promoter may be collectively referred to as "Ikzf1 complex formation regulator"), or a substance that inhibits the activity of CHD4 protein and / or HDAC1 protein. In the present invention, modulation of interferon-γ production includes both increased and decreased production. Here, an increase in production may mean that interferon gamma production increases by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more after treatment with an interferon gamma production regulator, and a decrease in production may mean that interferon gamma production decreases by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more after treatment with an interferon gamma production regulator.

[0035] Interferon-γ is known to have antiviral, immunomodulatory, and antitumor effects, and interferon-γ-associated diseases include tumors, autoimmune diseases, inflammation, colitis, idiopathic pulmonary fibrosis (IPF), and the like. The present invention can be used for the prevention and / or treatment of these interferon-γ-associated diseases. Examples of tumors include those listed above. Examples of autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, lupus nephritis, and Crohn's disease.

[0036] As described above, the target cells of the present invention include T cells, preferably CD4+ T cells, and more preferably regulatory T cells or naive T cells. In this embodiment, the present invention relates to a T cell modulating agent comprising an Ikzf1ΔE5 protein, an Ikzf1ΔE5 gene-related substance, a modulator of the formation of the Ikzf1 complex, or an inhibitor of the activity of CHD4 protein and / or HDAC1 protein. In the present invention, the term "T cell modulating agent" refers to an agent that converts T cells, preferably CD4+ T cells, more preferably regulatory T cells or naive T cells, more preferably regulatory T cells, into T cells that produce interferon-γ; an agent that suppresses the conversion of T cells, preferably CD4+ T cells, more preferably regulatory T cells or naive T cells, more preferably regulatory T cells, into T cells that produce interferon-γ; an agent that converts T cells into T cells with reduced Foxp3 expression (exTreg); an agent that reduces regulatory T cells; or an agent that adjusts the ratio of regulatory T cells to exTreg.

[0037] As used herein, "treatment" refers to a method or process intended to (1) delay the onset of a tumor or other interferon-gamma-associated disorder; (2) slow or stop the progression, worsening, or progression of symptoms of a tumor or other interferon-gamma-associated disorder; (3) bring about the amelioration of symptoms of a tumor or other interferon-gamma-associated disorder; or (4) cure a tumor or other interferon-gamma-associated disorder. Treatment may be administered prior to the onset of a disease or condition as a preventative measure, or alternatively, treatment may be administered after the onset of a disease.

[0038] As used herein, "prevention" means preventing the onset of tumors and other interferon-γ related diseases.

[0039] In the present invention, the agent or pharmaceutical composition generally means a drug for treating or preventing a disease, or for testing or diagnosing a disease.

[0040] The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is set so that an appropriate volume within the specified range is obtained.

[0041] Sterile compositions for injection can be formulated according to conventional formulations using vehicles such as distilled water for injection.

[0042] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80(TM), HCO-50, etc.), may be used in combination.

[0043] Oily liquids include sesame oil and soybean oil, and may contain benzyl benzoate and / or benzyl alcohol as a solubilizer. Buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection solution is usually filled into suitable ampoules.

[0044] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, it can be in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

[0045] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a polypeptide can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be set, for example, in the range of 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these numerical values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but one skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0046] <Screening Method> In another embodiment, the present invention provides a method for screening for a regulator of the formation of an Ikzf1 complex containing an Ikzf1 protein, and a Foxp3 protein, a CHD4 protein, and / or a HDAC1 protein, the method comprising the following steps: (a) contacting a candidate compound for a regulator of the formation of an Ikzf1 complex with a system containing an Ikzf1 protein or a fragment thereof, and a Foxp3 protein or a fragment thereof, a CHD4 protein or a fragment thereof, and / or a HDAC1 protein or a fragment thereof; (b) examining whether the candidate compound inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with HDAC1 protein or a fragment thereof, and (c) identifying the candidate compound as a modulator of Ikzf1 complex formation if it inhibits or promotes any of the associations. Preferably, a modulator of Ikzf1 complex formation refers to a substance that inhibits or promotes the association of Ikzf1 protein with Foxp3 protein. Preferably, the substance that regulates the formation of the Ikzf1 complex includes a preventive and / or therapeutic agent for tumors, an interferon-γ production regulator, or a T cell regulator.

[0047] One aspect of this embodiment relates to a method for screening for a modulator of the formation of an Ikzf1 complex containing an Ikzf1 protein, and a Foxp3 protein, a CHD4 protein, and / or an HDAC1 protein, the method comprising the steps of: (a) contacting T cells in vitro with a candidate compound for a modulator of the formation of the Ikzf1 complex; (b) examining whether the candidate compound inhibits or promotes the association between the Ikzf1 protein and the Foxp3 protein, whether it inhibits or promotes the association between the Ikzf1 protein and the CHD4 protein, whether it inhibits or promotes the association between the Ikzf1 protein and the HDAC1 protein, whether it inhibits or promotes the association between the Foxp3 protein and the CHD4 protein, and / or whether it inhibits or promotes the association between the Foxp3 protein and the HDAC1 protein; and (c) identifying the candidate compound as a modulator of the formation of the Ikzf1 complex if it inhibits or promotes any of the associations.

[0048] In another aspect of this embodiment, there is provided a method for screening for a modulator of the formation of an Ikzf1 complex containing an Ikzf1 protein, and a Foxp3 protein, a CHD4 protein, and / or a HDAC1 protein, the method comprising the steps of: (a) contacting in vitro a cultured cell or a T cell that has been forced to express an Ikzf1 protein or a fragment thereof, and a Foxp3 protein or a fragment thereof, a CHD4 protein or a fragment thereof, and / or a HDAC1 protein or a fragment thereof, with a candidate compound that is a modulator of the formation of the Ikzf1 complex; (b) examining whether the candidate compound inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with HDAC1 protein or a fragment thereof; and (c) identifying the candidate compound as a regulator of Ikzf1 complex formation if it inhibits or promotes any of the associations.

[0049] In this embodiment, a fragment of Ikzf1 protein refers to a polypeptide containing a part of Ikzf1 protein (SEQ ID NO: 3), preferably a polypeptide containing part or all of the exon 5 fragment of Ikzf1 protein, and a fragment of Foxp3 protein refers to a polypeptide containing a part of Foxp3 protein (SEQ ID NO: 4), preferably a polypeptide containing the C-terminal region from residue 279 onwards of the Foxp3 protein shown in SEQ ID NO: 4.

[0050] In this embodiment, protein association can be assessed by any known method, such as NanoLuc (registered trademark) Binary Technology (NanoBiT), bioluminescence resonance energy transfer (BRET), chemically amplified luminescence proximity homogeneous assay (Alpha), and TR-FRET.

[0051] In this embodiment, the Ikzf1 complex not only promotes the expression of its target genes but also represses the transcription of those target genes by competing with coactivators such as the histone acetyltransferase (HAT) p300 and NFAT1 (nuclear factor of activated T cells 1), which regulates the transcription of the cytokine IL-2 in activated T cells, and this repression is relieved by inhibiting the formation of the Ikzf1 complex (Figure 12). Therefore, target genes or their corresponding promoters of the Ikzf1 complex, as well as target genes or their corresponding promoters of p300 or NFAT1, can be used as reporters to screen for substances that regulate the formation of the Ikzf1 complex.

[0052] Examples of target genes of the Ikzf1 complex include those described in J Biol Chem. 2001 Jul 20;276(29):27647-56. Examples of target genes of p300 include those described in J Biol Chem. 2001 Jul 20;276(29):27647-56. Examples of target genes of NFAT1 include those described in J Biol Chem. 2014 Sep 26;289(39):26752-26761. Furthermore, the corresponding promoter is used in the above screening in the form of a vector linked to a reporter gene. Examples of reporter genes include genes encoding known reporter proteins such as β-galactosidase, β-gluconidase, luciferase, green fluorescent protein (GFP), tdTomato, and cell surface proteins.

[0053] In another aspect of this embodiment, there is provided a method for screening for a modulator of the formation of an Ikzf1 complex comprising an Ikzf1 protein, and a Foxp3 protein, a CHD4 protein, and / or a HDAC1 protein, the method comprising the steps of: (a) contacting a purified Ikzf1 protein or a fragment thereof, a purified Foxp3 protein or a fragment thereof, a purified CHD4 protein or a fragment thereof, and / or a purified HDAC1 protein or a fragment thereof in vitro with a candidate compound for a modulator of the formation of the Ikzf1 complex; (b) examining whether the candidate compound inhibits or promotes the association of Ikzf1 protein or a fragment thereof with Foxp3 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with CHD4 protein or a fragment thereof, whether it inhibits or promotes the association of Ikzf1 protein or a fragment thereof with HDAC1 protein or a fragment thereof, whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with CHD4 protein or a fragment thereof, and / or whether it inhibits or promotes the association of Foxp3 protein or a fragment thereof with HDAC1 protein or a fragment thereof; and (c) identifying the candidate compound as a regulator of Ikzf1 complex formation if it inhibits or promotes any of the associations.

[0054] In this embodiment, the degree of association between purified proteins can be evaluated using any known method, such as immunoprecipitation, pull-down assay, Western blotting, NMR, surface plasmon resonance (SPR), gel shift assay, gel filtration, microscale thermophoresis (MST), etc. In general, it is preferable to perform the same assay system in the absence of a candidate compound, measure the interaction both in the presence and absence of the candidate compound, and compare the two to determine whether the candidate compound inhibits or promotes the association of the target proteins.

[0055] In this embodiment, a fragment of Ikzf1 protein refers to a polypeptide containing a part of Ikzf1 protein (SEQ ID NO: 3), preferably a polypeptide containing part or all of the exon 5 fragment of Ikzf1 protein, and a fragment of Foxp3 protein refers to a polypeptide containing a part of Foxp3 protein (SEQ ID NO: 4), preferably a polypeptide containing the C-terminal region from residue 279 onwards of the Foxp3 protein shown in SEQ ID NO: 4.

[0056] T cells used in the screening methods of the present invention may be prepared by techniques well known in the art, e.g., cells may be obtained by drawing blood or by biopsy from a patient or healthy individual, or may be purchased from an immunological and microbiological supplier, e.g., American Type Culture Collection, Manassas, VA.

[0057] The T cells used in the present invention can be cultured according to standard cell culture techniques. For example, cells are grown in a suitable container in a sterile environment at 37°C in an incubator containing humidified 95% air and 5% CO2. The container may contain agitated or static media. A variety of cell culture media may be used, including media containing undefined biological fluids (e.g., fetal bovine serum) as well as fully defined media, such as 293 SFM serum-free medium (Invitrogen Corp., Carlsbad, CA). Cell culture techniques are well known in the art, and established protocols are available for culturing a variety of cell types (see, for example, R.I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique," 2nd Edition, 1987, Alan R. Liss, Inc.).

[0058] In certain embodiments, the screening methods of the invention are carried out using cells contained in multiple wells of a multi-well assay plate. Such assay plates are commercially available, for example, from Strategene Corp. (La Jolla, CA) and Corning Inc. (Acton, MA), and include, for example, 48-well, 96-well, 384-well, and 1536-well plates.

[0059] "In vitro contact" refers to, for example, adding a candidate compound at a predetermined concentration to cells contained in multiple wells of a multi-well assay plate. The candidate compound is then examined for its activity in inhibiting the association of target proteins. The association inhibitory activity can be examined by any known method, such as an interaction test in which one of the target proteins is immunoprecipitated to determine whether the other protein is co-precipitated, or by using NanoBit or TR-FRET-related technologies.

[0060] The activity of CHD4 protein and / or HDAC1 protein may be measured by any known method. For example, chromatin remodeling activity can be measured by the "Mononucleosome disruption assay" (Nucleic Acids Res. 2001 Jun 15;29(12):2517-21.).

[0061] The present invention will be described in detail below with reference to examples. However, it should be noted that these examples do not limit the scope of the present invention and are merely illustrative.

[0062] The antibodies used in the examples are as follows: Table 1

[0063] Example 1: Analysis of Ikzf1-Foxp3 Association Foxp3 and Ikzf1 interact with each other as described in Nat Immunol. 2012 Oct; 13(10): 1010-9. Therefore, we narrowed down the regions required for the interaction between Foxp3 and Ikzf1 as follows.

[0064] Example 1-1: Identification of the Respective Regions of Foxp3 and Ikzf1 Involved in Foxp3-Ikzf1 Complex Formation First, we identified the Foxp3-binding region of Ikzf1. Specifically, HEK293T cells (human embryonic kidney cells) were transfected with expression vectors for Myc-Foxp3 and Flag-Ikzf1 mutants: Flag-Ikzf1 (Ik1), Flag-IkΔN, Flag-IkΔC, Flag-IkΔE4, Flag-IkΔE6, and Flag-IkΔE5, using FuGENE HD Transfection Reagent (Promega). Forty-eight hours after transfection, cells were harvested with immunoprecipitation lysis buffer (IP lysis buffer). Immunoprecipitation was performed using anti-Flag-M2 antibody overnight at 4°C, and cell lysates (Input) with equal protein amounts and immunoprecipitation samples (IP) were electrophoresed and analyzed by Western blotting using anti-Flag-M2 antibody or anti-c-Myc antibody.

[0065] The amino acid sequence of the DNA-binding protein Ikaros transcript variant 7 [Mus musculus] used to create the Flag-Ikzf1 mutant is as follows: (SEQ ID NO: 1)

[0066] The results are shown in Figure 1. This figure shows whether Foxp3 co-precipitates when each Ikzf1 mutant is immunoprecipitated with anti-Flag-M2 antibody. The ΔN and ΔE5 mutants do not co-precipitate Foxp3. This indicates that the region encoded by exon 5 of Ikzf1 interacts with Foxp3.

[0067] Next, we identified the Ikzf1-binding region in Foxp3. Specifically, we performed the same analysis as above using expression vectors for Myc-Ikzf1 and Flag-Foxp3 mutants: Flag-Foxp3, Flag-ΔNFoxp3, and Flag-ΔCFoxp3. The amino acid sequence of the forkhead box P3 [Mus musculus] used to construct the Flag-Foxp3 mutants is as follows: MPNPRPAKPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGSGGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPLVMVAPSGARLGPSPHLQALLQDRPHFMHQLSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEEPEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQQLELEKEKLGAMQAHLAGKMALAKAPSVASMDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRRHLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYATLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFEFRKKRSQRPNKCSNPCP (SEQ ID NO: 2)

[0068] The results are shown in Figure 2. This figure shows whether Ikzf1 co-precipitates when each Foxp3 mutant is immunoprecipitated with anti-Flag-M2 antibody. ΔCFoxp3 does not co-precipitate Ikzf1 (far right lane). This indicates that the FHD of Foxp3 interacts with Ikzf1.

[0069] Example 1-2: Foxp3 Cre IkE5 f / f Analysis of Ikzf1 complex using mouse-derived Treg cells Foxp3 Cre IkE5 f / f Mice, IkE5 f / f Mouse (Joshi et al Nat Immunol 2014) and Foxp3Cre These mice were obtained by crossing with transgenic mice (Rudensky et al., Immunity 2008), in which exon 5 (IkE5) of the Ikzf1 gene was deleted by homologous recombination in Foxp3-positive Treg cells, resulting in the induction of Ikzf1ΔE5 expression. Cre IkE5 f / f YFP prepared from mice + Treg cells (1×10 5 After 7 days of culture with TCR stimulation and IL-2 (1500 U / ml), the cells were lysed in immunoprecipitation cell lysis buffer. Immunoprecipitation was performed overnight at 4°C using anti-IgG or anti-Foxp3 antibodies (5 μg / sample). Cell lysates (input) with equal amounts of protein and immunoprecipitated samples (IP) were electrophoresed and analyzed by simple Western assay using anti-CHD4 antibody as the primary antibody. As a control, Foxp3 Cre Treg cells obtained in the same manner from transgenic mice were used.

[0070] The results are shown in Figure 3. Endogenous Foxp3 was immunoprecipitated (α-Foxp3 lane), and Ikzf1-expressing Treg cells (Foxp3 Cre ) (left), whereas CHD4 was co-precipitated in Ikzf1ΔE5-expressing Treg cells (Foxp3 Cre IKE5 f / f ) did not co-precipitate CHD4 (right). On the other hand, when Ikzf1 was immunoprecipitated (α-Ikzf1 lane), CHD4 co-precipitated in both Ikzf1-expressing and Ikzf1ΔE5-expressing Treg cells. This indicates that CHD4 acts together with Ikzf1, but not Foxp3, in the Ikzf1 complex.

[0071] Examples 1-3: Foxp3 Cre IkE5 f / f Analysis of Ikzf1 complex using mouse-derived Treg cells Foxp3 Cre IkE5 f / f CD4+FYP+ Treg cells (1×10) purified from mouse spleens and lymph nodes 5) were cultured for 7 days with Dynabeads mouse CD3 / CD28 T cell stimulator (25 μl / ml) (Thermo Fisher Scientific) and IL-2 (1500 U / ml). On day 4 of culture, the culture medium (including the cultured cells) was divided into two portions, and 100 μl / well of culture medium containing IL-2 (1500 U / ml) was added. After culture, activated Treg cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 1 hour at 37°C and lysed in immunoprecipitation cell lysis buffer (Thermo Fisher Scientific). After cell lysis, immunoprecipitation was performed overnight at 4°C using DynaBeads IgG magnetic beads (50 μl / sample) (Thermo Fisher Scientific) and 5 μg anti-IgG (Sigma-Aldrich) or 5 μg anti-Foxp3 (eBioscience) antibodies. After immunoprecipitation, DynaBeads were washed five times with wash buffer (1 ml / sample) and then suspended in sample buffer (25 μl / sample) to recover proteins. Equal amounts of protein from the whole cell lysate (Input) or immunoprecipitate (IP) were analyzed by Simple Western assay. As a control, Foxp3 Cre We used Treg cells similarly isolated from mice to compare the changes in their interaction with Foxp3 following induction of Ikzf1ΔE5 expression. Simple Western assays were performed using the Jess Simple Western System (ProteinSimple) according to the manufacturer's standard procedures.

[0072] The results are shown in Figure 4. When endogenous Foxp3 was immunoprecipitated (α-Foxp3 lane), Ikzf1-expressing Treg cells (Foxp3 Cre ) CHD4 and HDAC1 were co-precipitated in Ikzf1ΔE5-expressing Treg cells (left), whereas Foxp3 Cre IkE5 f / f), CHD4 did not co-precipitate, but HDAC1 decreased (right). This suggests that, like CHD4, HDAC1 behaves with Ikzf1, but not Foxp3, in the Ikzf1 complex.

[0073] Example 1-4: Foxp3 and Ikzf1 interact in human Treg cells Human Treg cells (STEMCELL TECHNOLOGIES) were counted and then centrifuged (600 × g, 5 minutes). After removing the supernatant, the cell pellet was suspended in PBS and centrifuged (600 × g, 5 minutes). After centrifugation, the supernatant was removed, and 0.6 mL of RIPA buffer (Nacalai Tesque) was added to the human Treg cell pellet and allowed to stand (4°C, 10 minutes). The samples were then centrifuged (15,000 rpm, 10 min), and 300 μL of the supernatant was dispensed into 1.5 mL tubes. Each tube was mixed with 6 μg of anti-rat IgG2a antibody (eBioscience) or anti-Foxp3 antibody (eBioscience) and 20 μL of Protein G Dynabeads (Invitrogen) and rotated on a rotator (4°C, 180 min). After rotation, the samples were placed on a magnet, and the supernatant was removed without adsorbing the trapped Dynabeads. Fresh RIPA buffer was added, and this process was repeated three times. Then, 25 μL of sample buffer (Thermo) was added to the samples and heated (95°C, 5 min). The samples were electrophoresed by SDS-PAGE, and Foxp3 and Ikzf1 were detected by Western blotting.

[0074] The results are shown in Figure 5. Figure 5 shows that when Foxp3 is immunoprecipitated with anti-Foxp3 antibody, Ikzf1 is co-precipitated. This indicates that Ikzf1 and Foxp3 interact in human Tregs as well as in mouse Tregs. Furthermore, considering that human Ikzf1 and Foxp3 also contain exon 5 and FHD regions, respectively, it is highly likely that these regions also contribute to the interaction between Ikzf1 and Foxp3 in humans. The amino acid sequences of human Ikzf1, exon 5, and Foxp3 are as follows: Amino acid sequence of DNA-binding protein Ikaros transcript variant X5 [Homo sapience]: (SEQ ID NO: 3);

[0075] Human Ikzf1 Exon5 amino acid sequence: GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (SEQ ID NO: 10). Human Ikzf1 ZF2 amino acid sequence: QCNQCGASFTQKGNLLRHIKLHS (SEQ ID NO: 17). Human Ikzf1 ZF3 amino acid sequence: KCHLCNYACRRRDALTGHLRTHS (SEQ ID NO: 18).

[0076] Foxp3 [Homo sapience] amino acid sequence: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 4);

[0077] Example 2: Conversion of mice to IFN-γ-positive Treg cells by acting on Ikzf Example 2-1: Analysis of Ikzf1ΔE5 Treg cells Foxp3 Cre IkE5 f / f Cell suspensions were prepared from mouse spleens and lymph nodes. The cells were suspended in FACS buffer (PBS, 2% fetal bovine serum) containing an antibody against CD4 (BD Pharmingen) and stained for 15 minutes on ice. CD4 was then detected using a FACSAria II cytometer (BD). + YFP +Treg cells were sorted. To prepare samples for intracellular protein staining, Treg cells were stimulated for 4 hours in the presence of PMA / ionomycin and Brefeldin A. After washing, the cells were fixed with Foxp3-staining buffer (eBioscience) (45 minutes on ice). The fixed cells were washed with Perm buffer and stained with anti-IFN-γ antibody. Data were then acquired using a FACSCanto flow cytometer (BD) and analyzed using FlowJo (TreeStar). As a control, Foxp3 Cre Using Treg cells (expressing Ikzf1) similarly obtained from transgenic mice, we compared the change to IFN-γ-positive Treg cells due to induction of Ikzf1ΔE5 expression.

[0078] Example 2-2: Conversion of mouse Treg cells to IFN-γ-positive Treg cells by knockout of Ikzf family members. Guide RNAs (gRNAs) for the target genes used for knockout were designed using the IDT guide RNA design tool (bottom panel). Foxp3 Cre YFP from mice + Treg cells (1×10 5 ) were prepared and cultured for 7 days in the presence of TCR (Dynabeads) and IL-2 (1500 U / ml). 6) were suspended in P4 primary cell solution (P4 Primary Cell Solution; P4 Primary cell 4D-nucleofector X kit S) and transfected with Cas9 protein and gRNA using Amaxa 4D (DG137 program). After transfection, the cells were washed and cultured for 3 days in the presence of TCR (Dynabeads) and IL-2 (1500 U / ml). Cell surface and intracellular molecules were then stained. Control gRNA (INTEGRATED DNA TECHNOLOGIES) was used as a control. Using this method, we compared the transformation of Treg cells into IFN-γ-positive Treg cells among Treg cells without Ikzf family knockout, Treg cells with Ikzf1 knockout only, and Treg cells with Ikzf1 and Ikzf3 knockout.

[0079] Sequences of gRNAs used for knockout Ikzf1: AGAGCGATGCCACAACTACTTGG (SEQ ID NO: 5) and CAGAACTCCAAGAGTGATCGAGG (SEQ ID NO: 6) Ikzf3: AGATGAACTGCGACGTGTGCGGG (SEQ ID NO: 7) and ATTATGAAGCCGGAGCCCATGGG (SEQ ID NO: 8) and CGACTATGAAAGCATTAAGCTGG (SEQ ID NO: 9) Ikzf2: CGAAGGGGAACACGCCAATATGG (SEQ ID NO: 11) and GGGTAAAAGAAGCTCCGCACTGG (SEQ ID NO: 12) and TCAGTTTACCATTCGGAAGCCGG (SEQ ID NO: 13) Ikzf4: AAGCTCAAGTGCGACGTCTGCGG (SEQ ID NO: 14) and CTTGTAGGGTTTGCCCACGGTGG (SEQ ID NO: 15) and TTATCCAGGAGCCGTTCATCCGG (SEQ ID NO: 16)

[0080] The results obtained in Examples 2-1 and 2-2 are summarized in Figures 6(1) to 6(6). Figure 6 shows that the effect of Ikzf on the conversion of Treg cells to IFN-γ-positive Treg cells (IFN-γ production) was in the following order, with IkzfΔE5-expressing Treg cells showing a significantly higher conversion rate to IFN-γ-positive Treg cells: Ikzf1ΔE5 expression > Ikzfs (Ikzf1-4) KO > Ikzf1,3 KO > Ikzf1 KO. Therefore, expression of Ikzf1ΔE5 in Treg cells resulted in a higher conversion rate to IFN-γ-positive Treg cells and enhanced IFN-γ production compared with knockout of Ikzf1, or knockout of Ikzf1 and 3, or Ikzf1-4.

[0081] Example 2-3: Conversion of mouse Treg cells into IFN-γ-positive Treg cells by overexpression of Ikzf1ΔE5 To prepare retrovirus, HEK293T cells (2 × 10 ５ The cells (cells / well, 2 ml medium) were cultured for 24 hours. Serum-free medium (Opti-MEM; Gibco) was dispensed into 1.5 ml tubes at 200 μl per well, and a retroviral vector (1 μg) incorporating Ikzf1ΔE5 was added along with the retroviral packaging vector pCL-Eco (1 μg). HD (Promega) was then added at 6 μl per tube, vortexed gently, and allowed to stand at room temperature for 20 minutes. After 20 minutes, the serum-free medium containing the vector was added dropwise to the cultured HEK293T cells and cultured for an additional 2 days. After 2 days, the culture supernatant was collected, filtered through a 0.45 μm filter, and used as the virus containing the target gene in the following experiments.

[0082] Foxp3 Cre YFP from mice + Treg cells were prepared and cultured (1 × 10 ) in the presence of IL-2 (100 U / ml) for 24–28 hours on a 96-well plate coated with anti-CD3 antibody (1 μg / ml) and anti-CD28 antibody (1 μg / ml). 5(100 μl / well). After incubation, polybrene (1.5 μl / ml) was added to the prepared retrovirus, and 200 μl / well of the retrovirus was slowly added to the culture wells. After retrovirus addition, viral infection was performed by centrifugation for 90 minutes at 32°C, 2500 rpm, with minimum acceleration and braking. After centrifugation, the cells were placed in a 37°C incubator for 30 minutes, after which the supernatant (240 μl) was slowly removed and 200 μl of culture medium was slowly added. After medium addition, the cells were centrifuged for 5 minutes at 32°C and 500 g. The supernatant (200 μl) was slowly removed and 200 μl of culture medium was slowly added. The cells were then cultured at 37°C for 4 days. On the second day of incubation, half of the culture medium (120 μl / well) was removed and the same amount of culture medium was added. After incubation, cell surface and intracellular molecules were stained. As a control, we used an empty retroviral vector containing no gene. Using this method, we compared the conversion of Ikzf1ΔE5-overexpressing Treg cells to IFN-γ-positive Treg cells.

[0083] The results are shown in Figures 6(7) and (8). These results demonstrate that overexpression of exogenous Ikzf1ΔE5 promotes the conversion to IFN-γ-positive Treg cells, similar to the expression of Ikzf1ΔE5 by homologous recombination, and that the degree of promotion is greater than that of Ikzf1 KO. Similar to the results in Examples 2-1 and 2-2, these results indicate that expression of Ikzf1ΔE5 in Treg cells leads to a greater conversion rate to IFN-γ-positive Treg cells and enhanced IFN-γ production than knockout of Ikzf1. Furthermore, because Ikzf1ΔE5 is an Ikzf1 mutant lacking the interaction domain with Foxp3, it competes with Ikzf1 to inhibit the association of Ikzf1 with Foxp3, thereby inhibiting the formation of the Ikzf1 complex and inducing the conversion to IFN-γ-positive Treg cells, suggesting that the induction is greater than that of Ikzf1 KO.

[0084] Example 3: Conversion of human Treg cells to IFN-γ-positive cells by acting on Ikzf Example 3-1: Conversion of human Treg cells to IFN-γ-positive Treg cells by knockout of Ikzf family EasySep Human CD4 + CD127lowCD25 + Human Treg cells were prepared using a Regulatory T Cell Isolation Kit (STEMCELL). Treg cells were cultured for 9 days in the presence of Dynabeads T-activator (2.5 μl / well, Thermo Fisher Scientific) and IL-2 (100 U / ml, Milteny Biotec). Cultured Treg cells were harvested and mixed with negative control gRNA or Ikzf1, 2, 3, and 4 gRNAs and Cas9 protein (Thermo Fisher Scientific), and then transfected into Treg cells via nucleofection. The next day, Treg cells were harvested and transfected at 1 × 10 4 Cells were seeded at 100 cells / well in a 96-well plate and cultured in the presence of 100 U / ml IL-2 and 2.5 μl / well of Dynabeads T-activator. After 3 days of culture, Brefeldin A, Monensin, and Cell Activation Cocktail (Biolegend) were added. After 5 hours of culture, cells were stained with anti-CD4 and anti-CD25 antibodies. After fixation, cells were stained for IFN-γ and the nuclear protein Foxp3, and the proportion of IFN-γ-expressing Treg cells was analyzed using a CytoFLEX LX (Beckman Coulter).

[0085] The results obtained are shown in Figure 7. These results demonstrate that knockout of all Ikzf1, 2, 3, and 4 induces the conversion of human Treg cells into IFN-γ-positive Treg cells.

[0086] Example 3-2: Conversion of human Treg cells into IFN-γ-positive Treg cells by treatment with Ikzf family degraders. Human CD4+ cells were prepared using the EasySep Human CD4+ T Cell Isolation Kit (STEMCELL). They were then stained with anti-CD4, anti-CD25, and anti-CD45RA antibodies, and Fr I (CD45RA+CD25+) and Fr II (CD45RA-CD25++) cells were sorted using MA900 (SONY). After confirming the purity of the sorted samples, 1 × 10 cells were cultured in the presence of Dynabeads T-activator (2.5 μl / well, Thermo Fisher Scientific), IL-2 (100 U / ml, Milteny Biotec), and pomalidomide (an Ikzf1,3 degrader) or REF001329 (an Ikzf2,4 degrader). 4 Cells were seeded at 1 cell / well in a 96-well plate and cultured for 9 days. The supernatant was then removed, and Brefeldin A, Monensin, and Cell Activation Cocktail (Biolegend) were added. After 5 hours of culture, the cells were stained with antibodies against CD4 and CD25. After fixation, the cells were stained for IFN-γ and the nuclear proteins Foxp3, Ikzf1, and Ikzf2. The samples were then analyzed using a CytoFLEX LX (Beckman Coulter).

[0087] The results are shown in Figure 8. Figure 8A shows that pomalidomide degrades Ikzf1,3, and REF001329 degrades Ikzf2,4. Figure 8B shows the ratio of IFN-γ-positive Treg cells, gated by flow cytometry. These results indicate that knockdown of Ikzf1,3 induces the conversion of Treg cells to IFN-γ-positive Treg cells. On the other hand, knockdown of Ikzf2,4 did not induce the conversion of Treg cells to IFN-γ-positive Treg cells.

[0088] Examples 3-1 and 3-2 showed that Ikzf1 is highly likely to contribute to the conversion to IFN-γ-positive Treg cells in humans as well, and in light of Examples 2-1 and 2-2, it was suggested that Ikzf1ΔE5 expression is effective in humans as well as in mice.

[0089] Example 4: Effect of Ikzf family degrading agent treatment on iTreg cell induction from human naive T cells. Naive T cells were prepared from human PBMCs using the Naive CD4+ T Cell Isolation Kit II, human (Milteny Biotec). They were seeded onto plates coated with anti-CD3 antibody (8 μg / ml) in medium containing TGF-β (10 ng / ml) and IL-2 (200 U / ml). Various concentrations of pomalidomide or REF001329 were then added, and the cells were cultured for 10 days, with medium changes and passaging. Cells were harvested on days 4, 6, and 10 after seeding and stained with Human Regulatory T Cell Sorting Cocktail (BD) and anti-Foxp3 antibody. Differentiated iTreg cells were analyzed using a CytoFLEX LX (Beckman Coulter). In addition, the amount of Ikzf family proteins was assessed by Western blot in a portion of the day 6 sample.

[0090] The results are shown in Figure 9. Figure 9 shows that knockdown of Ikzf family members, including Ikzf1 and Ikzf3, using pomalidomide suppressed differentiation into iTreg cells. These results, in light of Example 2, suggest that Ikzf1ΔE5 expression may also affect naive T cells and influence their differentiation into iTreg cells.

[0091] Example 5: Examination of the antitumor effect of Ikzf1ΔE5 Treg cells in a tumor-bearing mouse model Foxp3eGFP-Cre-ERT2 and Foxp3eGFP-Cre-ERT2IkE5 f / fMice were subcutaneously implanted with the melanoma cell line B16F0 or the colon cancer cell line MC38, and tamoxifen was administered intraperitoneally on days 0, 1, and 3 after implantation. Tumor diameter was measured every 2 days. On day 18, tumors and draining lymph nodes (dLNs) and non-draining lymph nodes (ndLNs) were collected. Tumor-infiltrating lymphocytes (TILs) were prepared using a Tumor Dissociation Kit and a gentleMACS OCTO Dissociator according to the manufacturer's protocol.

[0092] The results are shown in Figure 10. Figure 10A shows the procedure of this example. Figure 10B shows the Foxp3eGFP-Cre-ERT2IkE5 expressing Ikzf1ΔE5. f / f In mice, the engraftment and proliferation of B16FO cells was suppressed compared to Foxp3eGFP-Cre-ERT2. Figure 10C shows that Foxp3eGFP-Cre-ERT2IkE5 expressing Ikzf1ΔE5 was suppressed compared to Foxp3eGFP-Cre-ERT2. f / f In mice, Ikzf1ΔE5 significantly suppressed tumor growth and survival in MC38 cells compared with Foxp3eGFP-Cre-ERT2. These results demonstrate that Ikzf1ΔE5 can strongly suppress tumor growth and survival, and thus may be useful for tumor treatment and / or prevention.

[0093] Example 6 To evaluate the effect of Exon 5 deletion of Ikzf1 on the stability of Treg cells in vivo, Foxp3 Cre IkE5 f / f Mouse Rosa26 RFP By crossing these mice with reporter mice, we created fate-mapping mice (Foxp3 Cre / + IkE5 f / f R26 RFP / + ) was created. Rosa26 RFPThe reporter mouse is a genetically engineered mouse in which a STOP cassette flanked by loxP sites is inserted into the ROSA26 locus before the DNA sequence encoding red fluorescent protein (RFP). Upon Cre expression, the STOP cassette is removed, resulting in RFP expression. Cre / + IkE5 f / f R26 RFP / + In + mice, Foxp3-expressing Treg cells are positive for both RFP and YFP, and T cells that once expressed Foxp3 as Tregs but then lost Foxp3 expression (hereinafter sometimes referred to as "exTregs") can be detected by FACS analysis as a cell population that is RFP-positive and YFP-negative. Therefore, using this system, we investigated the relationship between Foxp3 and Treg cells. Cre / + R26 RFP / + and Foxp3 Cre / + IkE5 f / f R26 RFP / + Spleens and lymph nodes were collected from mice (3-4 weeks old) and the proportion of exTregs was measured by FACS analysis. Cre / + IkE5 f / f R26 RFP / + The proportion of exTregs in mice was significantly increased (Fig. 11), suggesting that deletion of Exon 5 of Ikzf1 impairs the stability of Tregs and induces their transformation into exTregs.

[0094] Example 7 Analysis of p300 and NFAT1 target gene expression in Ikzf1ΔE5-expressing Treg cells Foxp3 Cre IkE5 f / fCD4+FYP+ Treg cells purified from mouse spleens and lymph nodes were stimulated with Cell Stimulation Cocktail (eBioscience) for 1 hour at 37°C. Stimulated Treg cells were then treated with 1% formaldehyde for 30 minutes at room temperature. After nuclear extraction, DNA was fragmented by sonication using a Digital Sonifier (Branson). The cells were then incubated overnight at 4°C with 5 μg of anti-Foxp3 (Abcam), anti-p300 (Abcam), or anti-NFAT1 (Abcam) antibodies pre-immunized with 100 μl of DynaBeads IgG magnetic beads (Thermo Fisher Scientific). After incubation, the DynaBeads were washed four times with RIPA buffer (1 ml / sample) and once with TE-NaCl buffer (1 ml / sample), and DNA was extracted with 100 μl of elution buffer per sample. After extraction, the DNA was reverse-crosslinked at 65°C and 1100 rpm for 24 hours, and then purified using the ChIP DNA Clean & Concentrator (Zymo Research) (40 μl / sample). The purified ChIP DNA was fragmented using a Covaris Focused-ultrasonicator S220 (Covaris) and then used to prepare libraries. Libraries were prepared using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) according to the manufacturer's instructions and sequenced using the IonS5 sequencer system (Thermo Fisher Scientific). Foxp3 was used as a control. Cre Using Treg cells similarly obtained from mice, we quantified changes in the genomic binding of p300 and NFAT1 around the genomic region where Foxp3 binding was increased by induction of Ikzf1ΔE5 expression and compared the two.

[0095] Data analysis began with fastQC, which checked the quality of the sequencing reads and confirmed that the average Phred score was ≥ 20. The sequencing reads were then trimmed using fastx_trimmer from the Fastx_tool kit, and mapped to the mouse genome mm10 using Bowtie2 with default settings. Peak calling was performed using MACS2. ChIP-seq peaks were defined using FindPeaks with a size of 500 base pairs, a minimum peak distance of 500 base pairs, default FDR, and local filtering disabled. Shared and unique regions of Foxp3 binding between wild-type and Ikzf1ΔE5-expressing Treg cells were identified using HOMER (getDifferentialPeaks) with default parameters. Normalized density plots of p300 and NFAT1 binding peaks around the Foxp3 binding site were calculated using annotatePeaks from the Homer package.

[0096] Figure 12 shows normalized density plots of p300 and NFAT1 binding peaks around the Foxp3-increased binding region. Combined with Figure 4, this indicates that in Ikzf1ΔE5-expressing Treg cells, the histone acetyltransferase (HAT) p300 interacts with Foxp3, resulting in enhanced binding to the Foxp3-increased binding region. These results suggest that Foxp3 may form an inhibitory complex with Ikzf1, potentially competing with the active Foxp3-p300 complex for regulating gene transcription. Figure 12 also demonstrates that NFAT1 binding to the Foxp3-increased binding region is indeed increased in Ikzf1ΔE5 Treg cells, suggesting that NFAT1 binding to the Foxp3-increased binding region is inhibited by the Foxp3-Ikzf1 complex. These results suggest that the Foxp3-Ikzf1 complex induces a closed chromatin structure via the nucleosome-remodeling deacetylase (NuRD) complex in an Ikzf1-dependent manner, thereby competing with coactivators such as p300 and NFAT1 and thereby suppressing the expression of its target genes.

[0097] INDUSTRIAL APPLICABILITY The present invention provides a technology relating to a novel method for treating and preventing tumors and the like, and has extremely high industrial applicability.