Prophylactic and / or therapeutic agent for tumors

Abstract

A novel prophylactic / therapeutic agent for tumors is developed. Provided are: a prophylactic and / or therapeutic agent for tumors, said prophylactic and / or therapeutic agent containing an Ikzf1 [delta] E5 protein, an Ikzf1 [delta] E5 gene-related substance, or the like, which is a defective body in which some or all of the exon 5 in Ikzf1 is defective; and a method for screening the prophylactic and / or therapeutic agent for tumors.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This patent application claims priority and benefits under the Paris Convention based on Japanese Patent Application No. 2022-112660 (filed on July 13, 2022), and the contents described in the above application are incorporated herein by reference in their entirety.

[0003] The present invention relates to a preventive and / or therapeutic agent for tumors. Specifically, the present invention relates to a preventive and / or therapeutic agent for tumors containing Ikzf1ΔE5 protein or a substance related to Ikzf1ΔE5 gene, and a method for screening the same. Background Art

[0004] In recent years, research focusing on cancer immunotherapy has been ongoing. For example, ipilimumab, an immune checkpoint inhibitor that is an anti-CTLA-4 antibody, is a drug that enhances anti-tumor immunity by removing regulatory T cells (Treg cells).

[0005] IKAROS family zinc finger 1 (Ikzf1) is one of the five IKAROS family transcription factors found in mammals, and is also referred to as "IKAROS". Ikzf1 is a molecule related to chromatin remodeling that is expressed in the hematopoietic system of the fetus and adults. It is expressed in the hematopoietic system of the fetus and adults and acts as a regulatory factor for lymphocyte differentiation. Loss-of-function mutations and defects in Ikzf1 can be observed in B-lymphoblastic leukemia (B-ALL) and have been reported to be associated with poor prognosis. In addition, the association between Ikzf1 mutations in the germline and autoimmune diseases has been reported (Non-Patent Literature 1).

[0006] Non-patent document 2 describes that, by producing and analyzing mice in which B cells specifically express a defective exon 5 of Ikzf1, Ikzf is important for differentiation from preB cells, and that preB cells with suppressed differentiation produce B lymphoblastic leukemia (B-ALL). This indicates that Ikzf1 exon 5 is important in the differentiation of B cells and the occurrence of B-ALL. In addition, patent document 1 describes a composition and method containing T cells whose cytolytic activity is enhanced by suppressing the expression of Ikzf1 in cells.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: WO2014 / 138348

[0010] Non-patent literature

[0011] Non-patent document 1: Journal of Allergy and Clinical Immunology Volume 140, Issue 1, Pages 223-231 (2017)

[0012] Non-patent literature 2: Nat Immunol. 2014 Mar;15(3):294-304. doi: 10.1038 / ni.2821. Epub 2014 Feb 9. Summary of the invention

[0013] Problems to be solved by the invention

[0014] An object of the present invention is to develop a new preventive / therapeutic drug for tumors.

[0015] Methods used to solve problems

[0016] The present inventors have conducted intensive research to develop a new preventive or therapeutic method in immunotherapy, and as a result, have found that when a defective body in which exon 5 of Ikzf1 is partially or completely deleted is expressed in regulatory T cells (Treg cells) (hereinafter sometimes referred to as "Ikzf1ΔE5". In addition, a gene encoding Ikzf1ΔE5 is sometimes referred to as "Ikzf1ΔE5 gene", and a protein encoded by the Ikzf1ΔE5 gene is sometimes referred to as "Ikzf1ΔE5 protein".), the production of interferon γ from regulatory T cells is significantly enhanced, and tumor prevention and / or treatment becomes possible. The present inventors have further repeatedly conducted research based on the above findings, and have completed the present invention.

[0017] That is, the present invention includes the following aspects.

[0018] <One of the preventive and / or therapeutic agents for tumors> [1]

[0020] A preventive and / or therapeutic agent for tumors, comprising Ikzf1ΔE5 protein or Ikzf1ΔE5 gene-related substances. [2]

[0022] The preventive and / or therapeutic agent for tumors according to [1], which targets T cells. [3]

[0024] The tumor prevention and / or treatment agent according to [2], wherein the T cells are CD4-positive T cells. [4]

[0026] The tumor prevention and / or treatment agent according to [2], wherein the T cells are regulatory T cells and / or naive T cells. [5]

[0028] The agent for preventing and / or treating tumors according to any one of [1] to [4], wherein the Ikzf1ΔE5 gene-related substance is a nucleic acid molecule encoding the Ikzf1ΔE5 protein. [6]

[0030] The tumor prevention and / or treatment agent according to any one of [1] to [4], wherein the Ikzf1ΔE5 gene-related substance is a substance in which a part or all of exon 5 of the Ikzf1 gene is deleted. [7]

[0032] The tumor prevention and / or treatment agent according to [6], wherein the substance that causes a partial or complete deletion of exon 5 of the Ikzf1 gene is selected from the group consisting of an exon skipping agent targeting exon 5 of the Ikzf1 gene and a gene therapy vector such as CRISPR / Cas. [8]

[0034] The tumor prevention and / or treatment agent according to [7], wherein the exon skipping agent is a nucleic acid. [9]

[0036] The preventive and / or therapeutic agent for tumors according to [1], comprising T cells expressing Ikzf1ΔE5.

[10]

[0038] A method for preventing and / or treating tumors, comprising the step of administering the agent for preventing and / or treating tumors described in any one of [1] to [9].

[0039] The present invention further includes the following other aspects.

[0040] <Interferon gamma modulators> [twenty one]

[0042] An interferon gamma production regulator contains Ikzf1ΔE5 protein or Ikzf1ΔE5 gene-related substances. [twenty two]

[0044] The interferon-γ production regulator according to

[21] , which targets T cells. [twenty three]

[0046] The interferon-γ production regulator according to

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

[0048] The interferon-γ production regulator according to

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

[25]

[0050] The interferon-γ production regulator according to any one of

[21] to

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

[26]

[0052] The interferon-γ production regulator according to any one of

[21] to

[24] , wherein the Ikzf1ΔE5 gene-related substance is a substance in which exon 5 of the Ikzf1 gene is partially deleted.

[27]

[0054] The interferon-γ production regulator according to

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

[28]

[0056] The interferon-γ production regulator according to

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

[29]

[0058] The interferon-γ production regulator according to

[21] , comprising T cells expressing Ikzf1ΔE5.

[0059] <T cell modulators>

[31]

[0061] A T cell regulator contains Ikzf1ΔE5 protein or Ikzf1ΔE5 gene-related substances.

[32]

[0063] The T cell regulator according to

[31] targets T cells.

[33]

[0065] The T cell regulator according to

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

[34]

[0067] The T cell regulator according to

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

[35]

[0069] The T cell regulator according to any one of

[31] to

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

[36]

[0071] The T cell regulating agent according to any one of

[31] to

[34] , wherein the Ikzf1ΔE5 gene-related substance is a substance in which exon 5 of the Ikzf1 gene is partially deleted.

[37]

[0073] The T cell regulator according to

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

[38]

[0075] The T cell regulator according to

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

[39]

[0077] The T cell regulator according to

[31] , comprising T cells expressing Ikzf1ΔE5.

[0078] In addition, the present invention further includes the following other aspects.

[41]

[0080] A tumor prevention and / or treatment agent, comprising a substance that inhibits and / or promotes the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein, and / or an HDAC1 protein.

[42]

[0082] An interferon-γ production regulator comprises a substance that inhibits and / or promotes the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein.

[43]

[0084] A T cell regulator comprises a substance that inhibits the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein and / or a substance that promotes the formation of the complex.

[44]

[0086] The T cell regulator according to

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

[45]

[0088] The T cell regulator according to

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

[46]

[0090] The agent according to any one of

[41] to

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

[47]

[0092] The agent according to any one of

[41] to

[46] , wherein the Ikzf1 complex formation inhibitory substance contains a part or all of the exon 5 fragment of the Ikzf1 protein.

[48]

[0094] The agent according to any one of

[41] to

[47] , wherein the Ikzf1 complex formation inhibitory substance contains a part or all of a nucleic acid encoding an exon 5 fragment of the Ikzf1 protein.

[49]

[0096] The agent according to any one of

[41] to

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

[50]

[0098] The agent according to any one of

[41] to

[46] , wherein the Ikzf1 complex formation inhibitory substance contains a part or all of FHD of Foxp3 protein.

[51]

[0100] The agent according to any one of

[41] to

[46] , wherein the substance that inhibits the formation of the Ikzf1 complex contains a part or all of a nucleic acid encoding FHD of the Foxp3 protein.

[52]

[0102] The agent according to any one of

[41] to

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

[53]

[0104] The agent according to any one of

[41] to

[46] , wherein the substance that inhibits the formation of the Ikzf1 complex contains a Foxp3 protein in which a part or all of FHD is defective.

[54]

[0106] The agent according to any one of

[41] to

[46] , wherein the substance inhibiting the formation of the Ikzf1 complex contains a nucleic acid encoding a Foxp3 protein in which a part or all of FHD is defective.

[55]

[0108] A method for preventing and / or treating tumors, comprising the step of administering the agent for preventing and / or treating tumors described in any one of

[41] and

[47] to

[54] .

[0109] <Method for screening "substances regulating the formation of Ikzf1 complex">

[61]

[0111] A method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, comprising the following steps:

[0112] (a) contacting a system containing 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 with a candidate compound of a substance that regulates the formation of an Ikzf1 complex;

[0113] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0114] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as an Ikzf1 complex formation modulator.

[62]

[0116] The method for screening a substance that regulates the formation of an Ikzf1 complex according to

[61] comprises the following steps:

[0117] (a) contacting T cells with a candidate compound that is a substance that regulates the formation of an Ikzf1 complex in vitro;

[0118] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein with Foxp3 protein, whether it inhibits or promotes the association of Ikzf1 protein with CHD4 protein, whether it inhibits or promotes the association of Ikzf1 protein with HDAC1 protein, whether it inhibits or promotes the association of Foxp3 protein with CHD4 protein, and / or whether it inhibits or promotes the association of Foxp3 protein with HDAC1 protein;

[0119] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[63]

[0121] The method for screening a substance that regulates the formation of an Ikzf1 complex according to

[61] comprises the following steps:

[0122] (a) contacting cultured cells or T cells forced to express 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 with a candidate compound of a substance that regulates the formation of an Ikzf1 complex in vitro;

[0123] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0124] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[64]

[0126] The method for screening a substance that regulates the formation of an Ikzf1 complex according to

[61] comprises the following steps:

[0127] (a) contacting purified Ikzf1 protein or a fragment thereof and purified Foxp3 protein or a fragment thereof, purified CHD4 protein or a fragment thereof and / or purified HDAC1 protein or a fragment thereof with a candidate compound of a substance that regulates the formation of a complex with Ikzf1 in vitro;

[0128] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0129] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[65]

[0131] The method according to any one of

[61] to

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

[66]

[0133] The method according to any one of

[61] to

[65] , wherein the substance regulating the formation of the Ikzf1 complex is a tumor preventive and / or therapeutic agent, an interferon-γ production regulator, or a T cell regulator.

[0134] <Tumor prevention and / or treatment agent 2>

[71]

[0136] A preventive and / or therapeutic agent for tumors, comprising a substance that inhibits the activity of CHD4 protein.

[72]

[0138] An interferon-γ production regulator comprising a substance that inhibits the activity of CHD4 protein.

[73]

[0140] A T cell regulator comprising a substance that inhibits the activity of a CHD4 protein.

[74]

[0142] The T cell regulator according to

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

[75]

[0144] The T cell regulator according to

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

[76]

[0146] A method for preventing and / or treating tumors, comprising the step of administering the agent for preventing and / or treating tumors described in

[71] .

[0147] <Tumor prevention and / or treatment agent 3>

[81]

[0149] A preventive and / or therapeutic agent for tumors, comprising a substance that inhibits the activity of HDAC1 protein.

[82]

[0151] An interferon-γ production regulator comprising an HDAC1 protein activity inhibitory substance.

[83]

[0153] A T cell regulator comprising an HDAC1 protein activity inhibitory substance.

[84]

[0155] The T cell regulator according to

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

[85]

[0157] The T cell regulator according to

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

[86]

[0159] A method for preventing and / or treating tumors, comprising the step of administering the agent for preventing and / or treating tumors described in

[81] .

[0160] Effects of the Invention

[0161] According to the present invention, a new preventive / therapeutic drug for tumors can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 : Figure 1 (Top) The structures of various Flag-Ikzf1 mutants are shown. 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 represents anti-Flag-M2 antibody, and α-Myc represents anti-c-Myc antibody.

[0163] Figure 2 : Figure 2 (Left) The structures of various Foxp3 mutants are shown. Figure 2 (Right) Photographs showing the results of Western blotting in which each Foxp3 mutant and Myc-Ikzf1 were expressed in HEK293T cells and immunoprecipitated using an anti-Flag-M2 antibody.

[0164] Figure 3 Figure 2 shows Treg cells expressing Ikzf1 (Foxp3 Cre ) and Treg cells expressing Ikzf1ΔE5 (Foxp3 Cre IKE5 f / f ) are photographs of Western blotting results in which endogenous Foxp3 or Ikzf1 was immunoprecipitated under various conditions.

[0165] Figure 4 Figure 2 shows Treg cells expressing Ikzf1 (Foxp3 Cre ) and Treg cells expressing Ikzf1ΔE5 (Foxp3 Cre IUsQ f / f ) is a photograph of the Western blot result in which HDAC1 and endogenous Foxp3 were immunoprecipitated.

[0166] Figure 5 The photographs show the results of Western blotting in which immunoprecipitation was performed using an anti-Foxp3 antibody in human Treg cells.

[0167] Figure 6 This graph compares the proportion of IFN-γ-positive Treg cells by knockdown of various Ikzfs and expression of Ikzf1ΔE5. The values ​​were calculated by Tukey's multiple comparison after conventional one-way ANOVA. P<0.05, P<0.01, ;P<0.001.

[0168] Figure 7 The graph shows the effect of knocking out the Ikzf family in human Treg cells on the expression of IFN-γ.

[0169] Figure 8 : Figure 8 A is a photograph showing the results of Western blotting confirming that pomalidomide and REF001329 are substances that degrade Ikzf family proteins in human Treg cells. Figure 8 B is a graph showing the ratio of IFN-γ-positive Treg cells gated by flow cytometry.

[0170] Fig. 9 The results of flow cytometry show the effect of knocking down Ikzf1,3 protein in human naive T cells using pomalidomide on differentiation into Treg cells.

[0171] Fig.10 The anti-tumor effect of Ikzf1ΔE5 Treg cells is shown. Fig.10 A shows the experimental steps. Fig.10 B is a graph showing that Foxp3eGFP-Cre-ERT2IkE5 specifically expresses Ikzf1ΔE5 in Foxp3 cells compared with Foxp3eGFP-Cre-ERT2. f / f Figure 3 shows that engraftment of B16FO cells is inhibited in mice. Fig.10 C is a graph showing the expression of Ikzf1ΔE5 in Foxp3eGFP-Cre-ERT2IkE5 compared with Foxp3eGFP-Cre-ERT2. f / f Figure 3 shows that the engraftment of MC38 cells is inhibited in mice.

[0172] Fig.11 It is shown that from Foxp3 Cre / + R26RFP / + and Foxp3 Cre / + IUsQ f / f R26 RFP / + The spleen and lymph nodes of mice (3-4 weeks old) were collected and the proportion of exTreg was determined by FACS analysis. Treg cells expressing Foxp3 were detected as a population of cells that were both RFP and YFP positive, and exTreg cells that lost Foxp3 expression were detected as a population of cells that were RFP positive and YFP negative.

[0173] Fig.12 It is Foxp3 Cre Mouse (straight) and Foxp3 Cre IUsQ f / f Normalized density plots of p300 and NFAT1 binding peaks around areas of increased Foxp3 binding in CD4+YFP+ Treg cells from mice (wavy lines). Normalized signal density is plotted within a window ±1 kb centered around the Foxp3 binding site. DETAILED DESCRIPTION

[0174] <Preventive and / or therapeutic agent for tumor>

[0175] The present invention relates to a preventive and / or therapeutic agent for tumors containing Ikzf1ΔE5 protein or a substance related to Ikzf1ΔE5 gene in one embodiment. Here, tumors refer to solid cancers, for example, epithelial cell cancers such as lung cancer, breast cancer, stomach cancer, colorectal cancer, liver cancer, and malignant melanoma, and non-epithelial cell cancers such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and leiomyosarcoma, and blood cancers such as leukemia, malignant lymphoma, and multiple myeloma.

[0176] In the present invention, the Ikzf1ΔE5 gene-related substance includes not only a nucleic acid molecule encoding the Ikzf1ΔE5 protein, but also a substance that causes a part or all of the exon 5 portion of the Ikzf1 gene to be deleted in a target cell. Here, the part of the exon 5 portion of the Ikzf1 gene may refer to 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 full length of the portion. Preferably, the part of the exon 5 portion of the Ikzf1 gene refers to a portion containing the zinc finger domain 2 (ZF2) of Ikzf1, a portion containing the zinc finger domain 3 (ZF3) of Ikzf1, or a portion containing both ZF2 and ZF3. Examples of target cells include T cells, preferably CD4-positive T cells, and more preferably regulatory T cells or naive T cells.

[0177] In the present invention, when the Ikzf1ΔE5 gene-related substance is a substance that causes a partial or complete deletion of exon 5 of the Ikzf1 gene, examples thereof include exon skipping agents and gene therapy vectors such as CRISPR / Cas for exon 5 of the Ikzf1 gene. An exon skipping agent refers to a substance that acts in the nucleus of a cell to cause a specific exon adjacent to a mutated position of a specific mRNA precursor to no longer be recognized (skipped), thereby connecting adjacent exons to each other to form a target deletion body. In the present invention, the exon skipping agent is preferably a nucleic acid, and more preferably an antisense nucleic acid.

[0178] In the present invention, gene therapy vectors such as CRISPR / Cas are not particularly limited as long as they can cause a partial or complete deletion of exon 5 of the Ikzf1 gene through genome editing. For example, the technology described in WO2018 / 179578 can be applied.

[0179] Another embodiment of the present invention relates to a tumor prevention and / or treatment agent of the present invention comprising T cells expressing Ikzf1ΔE5. This embodiment can be applied to adoptive T cell therapy, for example.

[0180] In another embodiment of the present invention, it relates to a tumor prevention and / or treatment agent of the present invention comprising a substance that inhibits the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, or a substance that inhibits the activity of a CHD4 protein and / or an HDAC1 protein. The formation inhibitory substance can be screened by any known method. Preferably, the formation inhibitory substance inhibits the association of the Ikzf1 protein with the Foxp3 protein. In addition, a protein containing part or all of the exon 5 fragment of the Ikzf1 protein, a protein containing part or all of the FHD of the Foxp3 protein, a Foxp3 protein in which a part or all of the FHD is missing, or a nucleic acid encoding them can be used as the formation inhibitory substance. Here, the FHD of Foxp3 refers to the DNA binding region (Forkhead domain) of Foxp3. In the present invention, FHD preferably refers to the C-terminal side region after residue number 279 of the Foxp3 protein shown in sequence number 4. A portion of the exon 5 fragment of the Ikzf1 protein may refer to 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 full length of the portion, and preferably refers to a portion containing the zinc finger domain 2 (ZF2) of Ikzf1, a portion containing the zinc finger domain 3 (ZF3) of Ikzf1, or a portion containing both ZF2 and ZF3. A portion of the FHD of the Foxp3 protein may refer to a fragment containing the full length of the portion, preferably 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the C-terminal region after residue number 279 of the Foxp3 protein shown in SEQ ID NO: 4. In addition, in the present specification, a part or all of the exon 5 fragment of the Ikzf1 protein includes a peptide that is 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 to the amino acid sequence of the corresponding part, and a part or all of the FHD of the Foxp3 protein includes a peptide that is 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 identical to the amino acid sequence of the corresponding part.

[0181] As the activity inhibitory substance of CHD4 protein and / or HDAC1 protein, for example, the inhibitory substance of chromatin remodeling activity, ATPase activity, DNA binding activity and / or deacetylation activity of CHD4 protein and / or HDAC1 protein can be listed. The activity inhibitory substance of CHD4 protein and / or HDAC1 protein can be obtained by any known method, for example, it can be obtained by measuring the chromatin remodeling activity by "Mononucleosome disruption assay" (Nucleic Acids Res. 2001 Jun15;29(12):2517-21.), and screening based on the measured activity.

[0182] In one embodiment, a substance that binds to a region containing part or all of the exon 5 portion of the Ikzf1 protein, or a substance that binds to a region containing part or all of the FHD of the Foxp3 protein can also be used as a substance that inhibits the formation of the Ikzf1 complex. Here, as the substance, an antibody having the region as an epitope can be exemplified. The antibody that can be used as a substance that inhibits the formation of the Ikzf1 complex can be prepared by a conventional method using, for example, a peptide or protein containing part or all of the exon 5 portion of the Ikzf1 protein, or part or all of the FHD of the Foxp3 protein as an antigen. The antibody can be directly administered in the form of an antibody preparation, or can be administered in the form of a gene therapy vector such as mRNA or AAV.

[0183] The present invention is based on the discovery that by inhibiting the formation of an Ikzf1 complex containing Ikzf1 protein and Foxp3 protein, CHD4 protein and / or HDAC1 protein in T cells, cell reprogramming and interferon-γ production are enhanced. Therefore, the tumor prevention and / or treatment agent of the present invention includes all aspects as long as it is an aspect that inhibits the formation of an Ikzf1 complex containing the Ikzf1 protein and Foxp3 (e.g., FHD) protein, CHD4 protein and / or HDAC1 protein.

[0184] In connection with the above-mentioned aspect, the present invention relates to an interferon-γ production regulator comprising an Ikzf1ΔE5 protein, an Ikzf1ΔE5 gene-related substance, a substance that inhibits the formation of the above-mentioned Ikzf1 complex, a substance that promotes the formation of the above-mentioned Ikzf1 complex (hereinafter, the above-mentioned Ikzf1 complex formation inhibitory substance and formation promoting substance are sometimes 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, the regulation of interferon-γ production includes both an increase in production and a decrease in production. Here, the increase in production may refer to an increase in interferon gamma production of 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 the decrease in production may refer to a decrease in interferon gamma production of 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.

[0185] It is known that interferon gamma has antiviral, immunomodulatory and antitumor effects. As interferon gamma related diseases, autoimmune diseases represented by tumors, inflammation, colitis, idiopathic pulmonary fibrosis (IPF), etc. can be listed. The present invention can be used for the prevention and / or treatment of these interferon gamma related diseases. As tumors here, the examples listed above can be exemplified. In addition, as autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, lupus nephritis, Crohn's disease, etc. can be exemplified.

[0186] As described above, the target cells of the present invention include T cells, preferably CD4-positive T cells, and more preferably regulatory T cells or naive T cells. In the above embodiment, the present invention relates to a T cell regulator containing Ikzf1ΔE5 protein, Ikzf1ΔE5 gene-related substances, the formation regulator of the above-mentioned Ikzf1 complex, or a substance that inhibits the activity of CHD4 protein and / or HDAC1 protein. In the present invention, the T cell regulator refers to: an agent that converts T cells, preferably CD4-positive T cells, more preferably regulatory T cells or naive T cells, and more preferably regulatory T cells into T cells that produce interferon γ; an agent that inhibits T cells, preferably CD4-positive T cells, more preferably regulatory T cells or naive T cells, and more preferably regulatory T cells from becoming T cells that produce interferon γ; an agent that converts T cells (exTreg) with reduced expression of Foxp3; an agent that reduces regulatory T cells; an agent that adjusts the ratio of regulatory T cells to exTreg.

[0187] In this specification, "treatment" refers to a method or process with the following goals: (1) delaying the onset of tumors or other interferon gamma-related diseases; (2) slowing down or stopping the progression, aggravation or deterioration of symptoms of tumors or other interferon gamma-related diseases; (3) alleviating the symptoms of tumors or other interferon gamma-related diseases; or (4) curing tumors or other interferon gamma-related diseases. Treatment can be implemented as a preventive measure before the onset of a disease or condition, or, alternatively, treatment can be implemented after the onset of the disease.

[0188] In the present specification, "prevention" refers to prevention of the onset of tumors and other interferon-γ-related diseases.

[0189] In the present invention, an agent or a pharmaceutical composition generally refers to an agent used for the treatment or prevention, or examination / diagnosis of a disease.

[0190] The pharmaceutical composition of the present invention can be formulated by methods well known to those skilled in the art. For example, it can be used non-orally in the form of an injection of a sterile solution or suspension agent with water or a pharmaceutically acceptable liquid. For example, it is considered that it can be appropriately combined with a pharmacologically acceptable carrier or medium, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, carrier, preservative, bonding agent, etc., and mixed and formulated with the unit dosage form required for the pharmaceutical implementation that is usually allowed. The amount of active ingredient in these preparations is set in a manner to obtain an appropriate volume of the indicated range.

[0191] Sterile compositions for injection can be formulated according to usual formulations using carriers such as distilled water for injection.

[0192] As aqueous solutions for injection, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary drugs (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be mentioned. Appropriate dissolution aids, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) can be used in combination.

[0193] As the oily liquid, sesame oil and soybean oil can be cited, and benzyl benzoate and / or benzyl alcohol as a dissolution aid can be used in combination. In addition, it can be matched with a buffer (such as phosphate buffer and sodium acetate buffer), a pain-relieving agent (such as procaine hydrochloride), a stabilizer (such as benzyl alcohol and phenol), and an antioxidant. The prepared injection is usually filled in an appropriate ampoule.

[0194] The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, it can be prepared into an injection dosage form, a nasal dosage form, a pulmonary dosage form, or a transdermal dosage form. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0195] The method of administration can be appropriately selected according to the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the polypeptide can be set to, for example, a range of 0.0001 mg to 1000 mg per kg of body weight each time. Alternatively, for example, the dosage can be set to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these numerical values. The dosage and method of administration vary according to the patient's weight, age, symptoms, etc., and those skilled in the art can set appropriate dosage and method of administration in consideration of these conditions.

[0196] <Screening method>

[0197] Another embodiment of the present invention relates to a method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, which comprises the following steps:

[0198] (a) contacting a system containing 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 with a candidate compound of a substance that regulates the formation of an Ikzf1 complex;

[0199] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0200] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[0201] Preferably, the substance regulating the formation of the Ikzf1 complex is a substance that inhibits or promotes the association of the Ikzf1 protein with the Foxp3 protein. In addition, the substance regulating the formation of the Ikzf1 complex preferably contains a tumor preventive and / or therapeutic agent, an interferon gamma production regulator, or a T cell regulator.

[0202] As one aspect of this embodiment, it relates to a method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, which comprises the following steps:

[0203] (a) contacting T cells with a candidate compound that is a substance that regulates the formation of an Ikzf1 complex in vitro;

[0204] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein with Foxp3 protein, whether it inhibits or promotes the association of Ikzf1 protein with CHD4 protein, whether it inhibits or promotes the association of Ikzf1 protein with HDAC1 protein, whether it inhibits or promotes the association of Foxp3 protein with CHD4 protein, and / or whether it inhibits or promotes the association of Foxp3 protein with HDAC1 protein;

[0205] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[0206] Another aspect of the present embodiment relates to a method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, comprising the following steps:

[0207] (a) contacting cultured cells or T cells forced to express 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 with a candidate compound of a substance that regulates the formation of an Ikzf1 complex in vitro;

[0208] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0209] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[0210] In the present method, a fragment of Ikzf1 protein refers to a polypeptide containing a portion of Ikzf1 protein (sequence number 3), preferably a polypeptide containing a portion or all of the exon 5 fragment of Ikzf1 protein, and a fragment of Foxp3 protein refers to a polypeptide containing a portion of Foxp3 protein (sequence number 4), preferably a polypeptide containing the C-terminal side region after residue number 279 of Foxp3 protein shown in sequence number 4.

[0211] In the present embodiment, protein association can be evaluated by any known method, and examples thereof include NanoLuc (registered trademark) Binary Technology (NanoBiT), bioluminescence resonance energy transfer (BRET), amplified chemiluminescence affinity homogeneous detection (Alpha), TR-FRET, and the like.

[0212] The Ikzf1 complex in this embodiment not only promotes the expression of its own target gene, but also inhibits the transcription of its target gene by competing with coactivators such as p300, a histone acetyltransferase (HAT), and NFAT1 (nuclear factor of activated T cells 1) responsible for the transcriptional control of the cytokine IL-2 in activated T cells. This inhibition can be relieved by inhibiting the formation of the Ikzf1 complex ( Fig.12 ). Therefore, the target gene of the Ikzf1 complex or the corresponding promoter, and the target gene of p300 and NFAT1 or the corresponding promoter can be used as a reporter gene for screening the substance regulating the formation of the Ikzf1 complex.

[0213] Here, as the target gene of Ikzf1 complex, J Biol Chem. 2001 Jul 20; 276 (29): genes recorded in 27647-56 can be cited, as the target gene of p300, J Biol Chem. 2001 Jul 20; 276 (29): genes recorded in 27647-56 can be cited, as the target gene of NFAT1, J Biol Chem. 2014 Sep 26; 289 (39): genes recorded in 26752-26761 can be cited. In addition, the corresponding promoter can be used for the above screening in the form of a vector combined with a reporter gene. Here, as the reporter gene, genes encoding known reporter proteins such as β-galactosidase, β-glucosidase, luciferase, green fluorescent protein (GFP), tdTomato, cell surface proteins, etc. can be cited.

[0214] Another aspect of the present embodiment relates to a method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein and a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, comprising the following steps:

[0215] (a) contacting purified Ikzf1 protein or a fragment thereof and purified Foxp3 protein or a fragment thereof, purified CHD4 protein or a fragment thereof and / or purified HDAC1 protein or a fragment thereof with a candidate compound of a substance that regulates the formation of a complex with Ikzf1 in vitro;

[0216] (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment;

[0217] (c) If the candidate compound inhibits or promotes any of the associations, it is identified as a substance that regulates the formation of the Ikzf1 complex.

[0218] In the present embodiment, the degree of association between purified proteins can be evaluated using any known method, and examples thereof include immunoprecipitation, pull-down assay, protein blotting, NMR, surface plasmon resonance (SPR), gel shift assay, gel filtration, microthermophoresis (MST), etc. In general, it is preferred to perform the same test system in the absence of the candidate compound, measure the interaction in the presence and absence of the candidate compound, and compare the two, thereby determining whether the candidate compound inhibits or promotes the association of the target protein.

[0219] In the present method, a fragment of Ikzf1 protein refers to a polypeptide containing a portion of Ikzf1 protein (sequence number 3), preferably a polypeptide containing a portion or all of the exon 5 fragment of Ikzf1 protein, and a fragment of Foxp3 protein refers to a polypeptide containing a portion of Foxp3 protein (sequence number 4), preferably a polypeptide containing the C-terminal side region after residue number 279 of Foxp3 protein shown in sequence number 4.

[0220] The T cells used in the screening method of the present invention can be prepared by techniques well known in the art, for example, cells can be obtained from patients or healthy individuals by blood sampling or biopsy, or can be purchased from immunology and microbiology suppliers, such as the American Type Culture Collection (Manassas, VA).

[0221] The T cells used in the present invention can be cultured according to standard cell culture techniques. For example, cells are placed in a suitable container, humidified and incubator containing 95% air-5% CO2, and propagated in a sterile environment at 37°C. The container may contain a stirred or stationary culture medium. Various cell culture fluids may be used, including culture medium containing unclear biological fluids (e.g., fetal bovine serum, etc.) and completely defined culture medium, such as 293 SFM serum-free culture medium (Invitrogen Corp., Carlsbad, CA). Cell culture techniques are well known in the art, and established protocols may be used for the culture of a variety of cell types (e.g., with reference to RI Freshney, "Culture of Animal Cells: A Manual of Basic Technique", 2nd Edition, 1987, Alan R. Liss, Inc.).

[0222] In a specific embodiment, the screening method of the present invention is carried out using cells contained in multiple wells of a multi-well test plate. Such test plates are commercially available, for example, from Strategene Corp. (La Jolla, CA) and Corning Inc. (Acton, MA), including, for example, 48-well, 96-well, 384-well, and 1536-well plates.

[0223] "In vitro contact" refers to, for example, adding a candidate compound adjusted to a predetermined concentration to cells contained in a plurality of wells of a multi-well test plate. Next, the candidate compound is investigated for its association inhibition activity with respect to the target proteins. Here, the association inhibition activity can be investigated using any known method, for example, an interaction test for investigating whether another protein co-precipitates when one of the target proteins is immunoprecipitated, NanoBit, or TR-FRET-related techniques.

[0224] The activity of CHD4 protein and / or HDAC1 protein can be measured using any known method. For example, the chromatin remodeling activity can be measured using the "mononucleosome disruption assay" (Nucleic Acids Res. 2001 Jun 15; 29(12): 2517-21).

[0225] Example

[0226] The present invention will be described in detail below by way of examples. However, it should be noted that these examples are not intended to limit the scope of the present invention but are merely illustrative.

[0227] The antibodies used in the Examples are as follows.

[0228]

[0229] Example 1

[0230] Ikzf1-Foxp3 association analysis

[0231] As described in Nat Immunol. 2012 Oct; 13(10): 1010-9., Foxp3 and Ikzf1 interact with each other. Therefore, the regions necessary for the interaction of Foxp3 and Ikzf1 were narrowed down as follows.

[0232] Example 1-1: Identification of the respective regions of Foxp3 and Ikzf1 involved in the formation of the Foxp3-Ikzf1 complex

[0233] First, the Foxp3-binding region in Ikzf1 was identified.

[0234] Specifically, Myc-Foxp3 and expression vectors of Flag-Ikzf1 mutants: Flag-Ikzf1 (Ik1), Flag-IkΔN, Flag-IkΔC, Flag-IkΔE4, Flag-IkΔE6, and Flag-IkΔE5 were introduced into HEK293T cells (human fetal kidney cells) using FuGENE HD Transfection Reagent (Promega). 48 hours after gene introduction, cells were recovered using immunoprecipitation cell lysis buffer (IP lysis buffer). In immunoprecipitation, anti-Flag-M2 antibody was used to react overnight at 4°C, and cell lysate (input) and immunoprecipitation sample (IP) with the same protein amount were subjected to electrophoresis, and Western blotting (Western Blot) was performed using anti-Flag-M2 antibody or anti-c-Myc antibody for analysis.

[0235] It should be noted that the amino acid sequence of the DNA binding protein Ikaros transcript variant 7 [mouse (Mus musculus)] used in the preparation of the Flag-Ikzf1 mutant is as follows:

[0236] (Serial No. 1)

[0237] The obtained results are shown in Figure 1 . Figure 1 This figure confirms whether Foxp3 is co-precipitated when each Ikzf1 mutant is immunoprecipitated with an anti-Flag-M2 antibody, and shows that Foxp3 is not co-precipitated in the case of the ΔN and ΔE5 mutants. This indicates that the region encoded by exon 5 of Ikzf1 interacts with Foxp3.

[0238] Then, the Ikzf1-binding region in Foxp3 was identified.

[0239] Specifically, analysis was performed in the same manner as above using expression vectors for Myc-Ikzf1 and Flag-Foxp3 mutants: Flag-Foxp3, Flag-ΔNFoxp3, and Flag-ΔCFoxp3.

[0240] It should be noted that the amino acid sequence of forkhead box P3 (mouse (Mus musculus)) used in the preparation of Flag-Foxp3 mutant is as follows:

[0241] MPNPRPAKPMAPSLALLGPSPGVLPSWKTAPKGSELLGTRGSGGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPLVMVAPSGARLGPSPHLQALLQDRPHFMHQLSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEEPEEFLKHC QADHLLDEKGKAQCLLQREVVQSLEQQLELEKEKLGAMQAHLAGKMALAKAPSVASMDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRRHLWGSHGNSSFPEFFH NMDYFKYHNMRPPFTYATLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFEFRKKRSQRPNKCSNPCP (serial number 2)

[0242] The obtained results are shown in Figure 2 . Figure 2 This is a diagram to confirm whether Ikzf1 is co-precipitated when each Foxp3 mutant is immunoprecipitated with anti-Flag-M2 antibody, and it is shown that Ikzf1 is not co-precipitated in the case of ΔCFoxp3 (rightmost lane). This indicates that the FHD of Foxp3 interacts with Ikzf1.

[0243] Example 1-2: Using the protein from Foxp3 Cre IUsQ f / f Analysis of Ikzf1 complex in mouse Treg cells

[0244] Foxp3 Cre IUsQ f / f Mouse is IkE5 f / f Mouse (Joshi et al Nat Immunol 2014) and Foxp3 Cre In mice obtained by mating with transgenic mice (Rudensky et al Immunity 2008), exon 5 (IkE5) of the Ikzf1 gene was deleted by homologous recombination in Foxp3-positive Treg cells, thereby inducing the expression of Ikzf1ΔE5. Cre IUsQ f / f YFP mouse preparation + Treg cells (1×105 ) were cultured for 7 days in the presence of TCR stimulation and IL-2 (1500 U / ml) and then lysed with immunoprecipitation cell lysis buffer. Immunoprecipitation was performed using anti-IgG or anti-Foxp3 antibodies (5 μg / sample) at 4°C overnight. Cell lysates (input) and immunoprecipitation samples (IP) with the same protein amount were electrophoresed and analyzed by simple Western blotting using anti-CHD4 antibody as the primary antibody. As a control, samples from Foxp3 Cre Transgenic mice were similarly provided with Treg cells.

[0245] The obtained results are shown in Figure 3 Results of immunoprecipitation of endogenous Foxp3 (α-Foxp3 lane), Treg cells expressing Ikzf1 (Foxp3 Cre ) showed co-precipitation of CHD4 (left), whereas Treg cells expressing Ikzf1ΔE5 (Foxp3 Cre IKE5 f / f ) did not co-precipitate CHD4 (right). On the other hand, in the results of immunoprecipitation of Ikzf1 (α-Ikzf1 lane), CHD4 co-precipitated in both Treg cells expressing Ikzf1 and Treg cells expressing Ikzf1ΔE5. This suggests that in the Ikzf1 complex, CHD4 acts together with Ikzf1 rather than Foxp3.

[0246] Example 1-3: Using the protein from Foxp3 Cre IUsQ f / f Analysis of Ikzf1 complex in mouse Treg cells

[0247] From Foxp3 Cre IUsQ f / f CD4+FYP+ purified from mouse spleen and lymph nodes Treg cells (1×10 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). During the culture, the culture medium (including the cultured cells) was divided into two equal parts on the 4th day, and a culture medium containing IL-2 (1500 U / ml) was added at 100 μl / well. After culture, the activated Treg cells were stimulated with Cell Stimulation Cocktail (eBioscience) at 37°C for 1 hour and lysed with Immunoprecipitation Cell Lysis Buffer (Thermo Fisher Scientific). After the cells were lysed, DynaBeads IgG magnetic beads (50 μl / sample) (Thermo Fisher Scientific) and 5 μg anti-IgG (Sigma-Aldrich) or 5 μg anti-Foxp3 (eBioscience) antibodies were added, and immunoprecipitation was carried out at 4°C overnight. After immunoprecipitation, DynaBeads were washed 5 times with washing buffer (1 ml / sample), and then suspended with sample buffer (25 μl / sample) to recover proteins. Equal amounts of proteins from whole cell lysate (input) or immunoprecipitate (IP) were analyzed by sample western blot. Cre Treg cells obtained from mice were similarly compared for changes in interaction with Foxp3 caused by induction of Ikzf1ΔE5 expression.

[0248] Western blotting of samples was performed using Jess Simple Western System (ProteinSimple) according to the manufacturer's standard protocol.

[0249] The obtained results are shown in Figure 4 Results of immunoprecipitation of endogenous Foxp3 (α-Foxp3 lane), Treg cells expressing Ikzf1 (Foxp3 Cre ), CHD4 and HDAC1 coprecipitated (left), whereas Treg cells expressing Ikzf1ΔE5 (Foxp3 Cre IUsQ f / f ), CHD4 was not co-precipitated, and HDAC1 was reduced (right). This suggests that, in the Ikzf1 complex, HDAC1, like CHD4, acts together with Ikzf1 rather than Foxp3.

[0250] Example 1-4: Foxp3 interacts with Ikzf1 in human Treg cells

[0251] After measuring the number of human Treg cells (STEMCELL TECHNOLOGIES), centrifugation was performed (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). After that, the sample was centrifuged (15000 rpm, 10 minutes), and the supernatant was dispensed into 1.5 mL tubes in 300 μL portions, and 6 μg of anti-rat IgG2a antibody (eBioscience) or anti-Foxp3 antibody (eBioscience) and 20 μL of ProteinG Dynabeads (ProteinG Dynabeads; Invitrogen) were added, and the tubes were rotated on a rotator (4°C, 180 minutes). After the rotation, the sample was placed on a magnet, the supernatant was removed so as not to aspirate the Dynabeads captured by the magnet, and new RIPA buffer was added. The above operation was repeated three times. Then, 25 μL of sample buffer (Thermo) was added to the sample and heated (95°C, 5 minutes). The sample was subjected to SDS-PAGE electrophoresis, and Foxp3 and Ikzf1 were detected by Western blotting.

[0252] The obtained results are shown in Figure 5 . Figure 5 It was shown that Ikzf1 co-precipitated when Foxp3 was immunoprecipitated with an anti-Foxp3 antibody. This means that Ikzf1 interacts with Foxp3 in human Tregs as well as in mouse Tregs. Furthermore, since human Ikzf1 and Foxp3 also have exon 5 and FHD regions, respectively, it is highly likely that these regions contribute to the interaction between Ikzf1 and Foxp3 in humans.

[0253] It should be noted that the amino acid sequences of human Ikzf1, exon 5, and Foxp3 are as follows.

[0254] Amino acid sequence of DNA binding protein Ikaros transcript variant X5 [Homo sapiens]:

[0255] (Serial No. 3);

[0256] Human Ikzf1 exon 5 amino acid sequence:

[0257] GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (sequence number 10).

[0258] Human Ikzf1 ZF2 amino acid sequence:

[0259] QCNQCGASFTQKGNLLRHIKLHS (serial number 17).

[0260] Human Ikzf1 ZF3 amino acid sequence:

[0261] KCHLCNYACRRRDALTGHLRTHS (serial number 18).

[0262] Amino acid sequence of Foxp3 [Homo sapiens]:

[0263] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQ ADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNM DYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (serial number 4).

[0264] Example 2

[0265] Changes to IFN-γ-positive Treg cells caused by the action of Ikzf in mice

[0266] Example 2-1: Analysis of Treg cells of Ikzf1ΔE5

[0267] From Foxp3 Cre IUsQ f / f Cell suspensions were prepared from the spleen and lymph nodes of mice. The cells were suspended in FACS buffer (PBS, 2% fetal bovine serum) containing an antibody against CD4 (BD Pharmingen) and stained on ice for 15 minutes. Afterwards, CD4 was sorted using a FACSAria II cytometer (BD). + YFP +Treg cells. When preparing samples for staining intracellular proteins, Treg cells were stimulated for 4 hours in the presence of PMA / Ionomycin and Brefeldin A. After washing the cells, they were fixed with Foxp3-staining buffer (Foxp3-staining buffer: eBioscience) (45 minutes, on ice). The fixed cells were washed with Perm buffer and stained with anti-IFN-γ antibody. After that, data were obtained with a FACSCanto flow cytometer (BD) and analyzed with FlowJo (TreeStar). As a control, the cells were isolated from Foxp3 Cre Treg cells (expressing Ikzf1) obtained in the same manner from transgenic mice were compared for changes to IFN-γ-positive Treg cells caused by induction of Ikzf1ΔE5 expression.

[0268] Example 2-2: Changes to IFN-γ-positive Treg cells caused by knockout of the Ikzf family in mouse Treg cells

[0269] The guide RNA (gRNA) for the target gene used in the knockout was designed using the IDT guide RNA design tool (lower paragraph). Cre Preparation of YFP mice + Treg cells (1×10 5 ) and cultured in the presence of TCR (Dynabeads) and IL-2 (1500 U / ml) for 7 days. 6 ) was suspended in P4 primary cell solution (P4 Primary Cell Solution; P4 Primary cell 4D-nucleofector X kit S), and Cas9 protein and gRNA were introduced into genes using Amaxa 4D (DG137 program). After gene introduction, the cells were washed and cultured for 3 days in the presence of TCR (Dynabeads) and IL-2 (1500U / ml). Afterwards, the cell surface and intracellular molecules were stained. As a control, a control gRNA (INTEGRATED DNA TECHNOLOGIES) was used. This method was used to compare the changes in IFN-γ positive Treg cells in Treg cells that did not knock out the Ikzf family, Treg cells that only knocked out Ikzf1, and Treg cells that knocked out Ikzf1 and 3.

[0270] Sequence of gRNA used in knockout

[0271] Ikzf1: AGAGCGATGCCACAACTACTTGG (SEQ ID NO: 5), and CAGAACTCCAAGAGTGATCGAGG (SEQ ID NO: 6)

[0272] Ikzf3: AGATGAACTGCGACGTGTGCGGG (SEQ ID NO. 7), and ATTATGAAGCCGGAGCCCATGGG (SEQ ID NO. 8), and CGACTATGAAAGCATTAAGCTGG (SEQ ID NO. 9)

[0273] Ikzf2: CGAAGGGGAACACGCCAATATGG (SEQ ID NO. 11), and GGGTAAAAGAAGCTCCGCACTGG (SEQ ID NO. 12), and TCAGTTTACCATTCGGAAGCCGG (SEQ ID NO. 13)

[0274] Ikzf4: AAGCTCAAGTGCGACGTCTGCGG (SEQ ID NO. 14), and CTTGTAGGGTTTGCCCACGGTGG (SEQ ID NO. 15), and TTATCCAGGAGCCGTTCATCCGG (SEQ ID NO. 16)

[0275] The results obtained in Examples 2-1 and 2-2 are summarized in Figure 6 (1)~(6) of. Figure 6 The effect of Ikzf on Treg cells → IFN-γ-positive Treg cells (IFN-γ production) was shown to be in the following order, and the rate of change to IFN-γ-positive Treg cells was significantly high in Treg cells expressing IkzfΔE5:

[0276] Ikzf1ΔE5 expression > Ikzfs (Ikzf1~4) KO > Ikzf1,3 KO > Ikzf1 KO.

[0277] Therefore, it was shown that, among Treg cells, the rate of change to IFN-γ-positive Treg cells was greater in the form of Ikzf1ΔE5 expression than in the form of Ikzf1 knockout, and further, compared with the form of Ikzf1 and 3, and Ikzf1 to 4 knockout, and the IFN-γ production capacity was improved.

[0278] Example 2-3: Changes to IFN-γ-positive Treg cells caused by overexpression of Ikzf1ΔE5 in mouse Treg cells

[0279] To produce retrovirus, HEK293T cells (2×10 5cells / well, 2 ml of culture medium) for 24 hours. Serum-free medium (Opti-MEM: Gibco) was dispensed into 1.5 ml tubes at 200 μl / well, and the retroviral vector (1 μg) integrated with Ikzf1ΔE5 was added together with the retroviral packaging vector pCL-Eco (1 μg). After that, HD (Promega) was added at 6 μl / tube, vortexed gently, and allowed to stand at room temperature for 20 minutes. After 20 minutes, serum-free medium containing the vector was added dropwise to the cultured HEK293T cells, and cultured for a further 2 days. After 2 days, the culture supernatant was recovered, filtered with a 0.45 μm filter, and used as a virus containing the target gene in the following experiments.

[0280] From Foxp3 Cre Preparation of YFP mice + Treg cells were cultured in 96-well plates coated with anti-CD3 antibody (1 μg / ml) and anti-CD28 antibody (1 μg / ml) in the presence of IL-2 (100 U / ml) for 24-28 hours (1×10 5 / well / 100μl). After culturing, add polybrene (1.5μl / ml) to the prepared retrovirus, and slowly add the retrovirus to the wells in culture at an amount of 200μl / well. After adding the retrovirus, centrifuge for 90 minutes at 32°C, 2500rpm, accelerator minimum, and decelerator minimum to carry out virus infection. After centrifugation, let it stand in an incubator at 37°C for 30 minutes, then slowly remove the supernatant (240μl), and slowly add 200μl of culture medium. After adding the culture medium, centrifuge for 5 minutes at 32°C and 500g, slowly remove the supernatant (200μl), slowly add 200μl of culture medium, and culture at 37°C for 4 days. During the culture, remove half of the culture solution (120μl / well) (including cultured cells) on the second day, and add an equal amount of culture medium.

[0281] After culture, cell surface and intracellular molecules were stained. As a control, an empty retroviral vector without any gene integration was used. By this method, the changes of Treg cells overexpressing Ikzf1ΔE5 to IFN-γ positive Treg cells were compared.

[0282] The results are shown in Figure 6(7), (8). The results here show that overexpression of exogenous Ikzf1ΔE5 promotes the change to IFN-γ positive Treg cells in the same way as the expression of Ikzf1ΔE5 based on homologous recombination, and the degree of change promotion is greater than that of Ikzf1 KO. The results here show that, similar to the results in Examples 2-1 and 2-2, in Treg cells, the expression of Ikzf1ΔE5 leads to a greater rate of change to IFN-γ positive Treg cells and an improved ability to produce IFN-γ compared to knocking out Ikzf1. Furthermore, Ikzf1ΔE5 is an Ikzf1 mutant that lacks the interaction region with Foxp3. Therefore, by competing with Ikzf1, it inhibits the association of Ikzf1 and Foxp3 and inhibits the formation of the Ikzf1 complex, thereby inducing the change to IFN-γ positive Treg cells, and its induction ability is greater than that of Ikzf1 KO.

[0283] Example 3

[0284] Changes to IFN-γ-positive Treg cells caused by the action of human Ikzf

[0285] Example 3-1: Changes to IFN-γ-positive Treg cells caused by knockout of the Ikzf family in human Treg cells

[0286] Using EasySep Human CD4 + CD127lowCD25 + Human Treg cells were prepared using the 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). The cultured Treg cells were recovered, and the negative control gRNA or gRNA of Ikzf1, 2, 3, and 4 was mixed with Cas9 protein (Thermo Fisher Scientific) and introduced into the Treg cells by nucleofection. The Treg cells were recovered the next day and 1×10 4Cells / well were inoculated in 96-well plates and cultured in the presence of 100 U / ml IL-2 and 2.5 μl / well Dynabeads T-activator. After 3 days of culture, Brefeldin A and Monensin, Cell Activation Cocktail (Biolegend) were added. After 5 hours of culture, staining was performed with anti-CD4 and anti-CD25 antibodies. After the cells were fixed, IFNγ and Foxp3, a nuclear protein, were stained, and the ratio of Treg cells expressing IFN-γ was analyzed using CytoFLEX LX (BeckmanCoulter).

[0287] The obtained results are shown in Figure 7 The results here show that knocking out all of Ikzf1, 2, 3, and 4 also induces a shift toward IFN-γ-positive Treg cells in human Tregs.

[0288] Example 3-2: Changes in human Treg cells to IFN-γ-positive Treg cells caused by treatment with Ikzf family-degrading substances

[0289] Using EasySep Human CD4+ Human CD4+ was prepared using a T cell isolation kit (STEMCELL). After staining with anti-CD4 antibody, anti-CD25 antibody, and anti-CD45RA antibody, Fr I (CD45RA+CD25+) and FrII (CD45RA-CD25++) were sorted using MA900 (SONY). After confirming the purity of the sorted samples, Dynabeads T-activator (2.5 μl / well, Thermo Fisher Scientific), IL-2 (100 U / ml, Milteny Biotec) and pomalidomide (Ikzf1,3 degrader) or REF001329 (Ikzf2,4 degrader) were used to increase the concentration of 1×10 4 Cells / well were seeded in 96-well plates and cultured for 9 days. After that, the supernatant was removed, and brefeldin A and monensin, a cell activation mixture (Biolegend) were added. After 5 hours of culture, the cells were stained with antibodies against CD4 and CD25, and after the cells were fixed, IFN-γ and nuclear proteins Foxp3, Ikzf1 and Ikzf2 were stained. The samples were then analyzed using CytoFLEX LX (Beckman Coulter).

[0290] The obtained results are shown in Figure 8 . Figure 8 A shows that Ikzf1,3 is degraded by pomalidomide, and Ikzf2,4 is degraded by REF001329. Figure 8 B is a graph showing the ratio of IFN-γ positive Treg cells by gating them using flow cytometry. These results show that knocking down Ikzf1 and 3 induces the change of Treg cells into IFNγ positive Treg cells. On the other hand, when Ikzf2 and 4 were knocked down, the change of Treg cells into IFNγ positive Treg cells was not induced.

[0291] Examples 3-1 and 3-2 show that Ikzf1 is likely to contribute to the transformation into IFN-γ-positive Treg cells in humans, and Examples 2-1 and 2-2 suggest that Ikzf1ΔE5 expression is also effective in humans as in mice.

[0292] Example 4

[0293] Effects of treatment with Ikzf family inhibitors on induction of iTreg cells from human naive T cells

[0294] Using human naive CD4+ Naive T cells were prepared from human PBMC using T Cell Isolation Kit II (Milteny Biotec), and inoculated on a plate coated with anti-CD3 antibody (8 μg / ml) using a medium containing TGF-β (10 ng / ml) and IL-2 (200 U / ml). Then, various concentrations of pomalidomide or REF001329 were added, and the cells were cultured for 10 days while performing medium exchange and passaging. After inoculation, cells were collected on the 4th, 6th, and 10th days, and the cells were stained using Human Regulatory T Cell Sorting Cocktail (BD) and anti-Foxp3 antibody, and the differentiated iTreg cells were analyzed using CytoFLEX LX (Beckman Coulter). In addition, for a portion of the sample on day 6, the amount of Ikzf family proteins was evaluated by Western blotting.

[0295] The obtained results are shown in Fig. 9 . Fig. 9 It was shown that knocking down Ikzf1 and 3 using pomalidomide in the knockdown of the Ikzf family inhibited differentiation into iTreg cells. Based on the results here, it is suggested from Example 2 that Ikzf1ΔE5 expression also acts on naive T cells and can affect differentiation into iTreg cells.

[0296] Example 5

[0297] Study on the anti-tumor effect of Ikzf1ΔE5 Treg cells in tumor-bearing mouse model

[0298] For Foxp3eGFP-Cre-ERT2 and Foxp3eGFP-Cre-ERT2IkE5 f / f Mice were subcutaneously transplanted with melanoma cell line B16FO or colorectal cancer cell line MC38, and tamoxifen was administered intraperitoneally on days 0, 1, and 3 after transplantation. Tumor diameter was measured every 2 days. Tumors and draining lymph nodes (dLNs) and non-draining lymph nodes (ndLNs) were sampled on day 18. Tumor-infiltrating lenkocytes (TILs) were prepared using a tumor dissociation kit and gentleMACS OCTO Dissociator according to the instructions.

[0299] The obtained results are shown in Fig.10 . Fig.10 A is a diagram showing the steps of this embodiment. Fig.10 B shows Foxp3eGFP-Cre-ERT2IkE5 expressing Ikzf1ΔE5 f / f In mice, the engraftment and proliferation of B16FO cells were inhibited compared with Foxp3eGFP-Cre-ERT2. Fig.10 C shows Foxp3eGFP-Cre-ERT2IkE5 expressing Ikzf1ΔE5 f / f In mice, the engraftment of MC38 cells was inhibited compared to Foxp3eGFP-Cre-ERT2. The results here indicate that Ikzf1ΔE5 is able to strongly inhibit tumor engraftment and proliferation, ie, can be used for the treatment and / or prevention of tumors.

[0300] Example 6

[0301] To evaluate the effect of Ikzf1 exon 5 deletion on the stability of Treg cells in vivo, Foxp3 Cre IUsQ f / f Mouse and Rosa26 RFP Fate-mapped Foxp3-expressing mice were prepared by mating reporter gene mice (Foxp3 Cre / + IUsQ f / f R26 RFP / + ). Rosa26 RFPThe reporter gene mouse is a genetically modified mouse in which a STOP box sandwiched by loxP sites is inserted into the ROSA26 locus in front of the DNA sequence encoding red fluorescent protein (RFP). With the expression of Cre, the STOP box falls off, thereby expressing RFP. Cre / + IUsQ f / f R26 RFP / + In mice, Treg cells expressing Foxp3 are both RFP and YFP positive, and T cells that once expressed Foxp3 as Treg but then lost Foxp3 expression (hereinafter sometimes referred to as "exTreg") can be detected by FACS analysis as a cell population that is RFP positive and YFP negative. Cre / + R26 RFP / + and Foxp3 Cre / + IUsQ f / f R26 RFP / + The spleen and lymph nodes of mice (3-4 weeks old) were collected and the proportion of exTreg was determined by FACS analysis. The results showed that Foxp3 Cre / + IUsQ f / f R26 RFP / + The proportion of exTreg in mice increased significantly ( Fig.11 ). This suggests that the loss of exon 5 of Ikzf1 impairs the stability of Tregs and induces the change from Tregs to exTregs.

[0302] Example 7

[0303] Analysis of target gene expression of p300 and NFAT1 in Treg cells expressing Ikzf1ΔE5

[0304] From Foxp3 Cre IUsQ f / f CD4+FYP+ purified from mouse spleen and lymph nodes Treg cells were stimulated with a cell stimulation cocktail (eBioscience) at 37°C for 1 hour. After that, the stimulated Treg cells were treated with 1% paraformaldehyde at room temperature for 30 minutes. After nuclear extraction, DNA was fragmented by ultrasonic treatment using a digital sonifier (Digital Sonifier) ​​(Branson), and incubated overnight at 4°C with 5 μg of anti-Foxp3 (Abcam) antibody, anti-p300 (Abcam) antibody, or anti-NFAT1 (Abcam) antibody that had been reacted with 100 μl DynaBeads IgG magnetic beads (Thermo Fisher Scientific). After incubation, DynaBeads were washed 4 times with RIPA buffer (1 ml / sample), washed once with TE-NaCl buffer (1 ml / sample), and DNA was extracted with 100 μl / sample of elution buffer. After extraction, reverse crosslinking was performed at 65°C and 1100 rpm for 24 hours, and DNA was purified using ChIP DNA Clean & Concentrator (Zymo Research) (40 μl / sample). The purified ChIP DNA was fragmented using Covaris Focused-ultrasonicator S220 (Covaris) to prepare the library. The library was prepared using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) according to the manufacturer's instructions, and the sequence was determined using the IonS5 sequencer system (Thermo Fisher Scientific). As a control, a 50-well clone from Foxp3 Cre Treg cells obtained from mice in the same manner were quantified for changes in genomic binding of p300 and NFAT1 around the genomic region where Foxp3 binding increased due to induction of Ikzf1ΔE5 expression, and the two were compared.

[0305] In data analysis, fastQC was first used to confirm the quality of the sequence reads, and the average Phred score was confirmed to be above 20. After that, the sequencing reads were trimmed using fastx_trimmer of Fastx_tool kit, and the sequencing reads were mapped to the mouse genome mm10 with default settings using Bowtie2. Peak identification was implemented using MACS2. ChIP-seq peaks were defined using FindPeaks, with the size set to 500 base pairs, the minimum distance between peaks set to 500 base pairs, the FDR set to default, and the local filter set to off (OFF). Foxp3 binding was identified using HOMER (getDifferentialPeaks) with default parameters in areas common to wild-type Treg cells and Treg cells expressing Ikzf1ΔE5, and in areas unique to each cell. The normalized density map of the binding peaks of p300 and NFAT1 around the Foxp3 binding site was calculated using annotatePeaks of the Homer software package.

[0306] The normalized density plots of p300 and NFAT1 binding peaks around the Foxp3 binding increased region are shown in Fig.12 . Also combined with Figure 4 It was found that in Treg cells expressing Ikzf1ΔE5, p300, a histone acetyltransferase (HAT), interacted with Foxp3 and enhanced its binding to the Foxp3 binding-enhanced region. These results suggest the possibility that Foxp3 forms an inhibitory complex with Ikzf1 and competes with the active Foxp3-p300 complex in the control of gene transcription. Fig.12 The fact that NFAT1 binding to the Foxp3 binding-enhanced region was indeed increased in Ikzf1ΔE5Treg cells suggests that the binding of NFAT1 to the Foxp3 binding-enhanced region is inhibited in the Foxp3-Ikzf1 complex. This suggests that the Foxp3-Ikzf1 complex induces a closed chromatin structure through the nucleosome remodeling deacetylase (NuRD) complex in a manner dependent on Ikzf1, thereby competing with coactivators such as p300 and NFAT1, resulting in the inhibition of target gene expression.

[0307] Industrial Applicability

[0308] According to the present invention, a technology related to a new treatment / prevention method for tumors and the like can be provided, and the industrial applicability is extremely high.

Claims

A preventive and / or therapeutic agent for tumors, comprising Ikzf1ΔE5 protein or a substance related to the Ikzf1ΔE5 gene.

2. The preventive and / or therapeutic agent for tumors according to claim 1, which targets T cells.

3. The tumor prevention and / or treatment agent according to claim 2, wherein: The T cells are CD4 positive T cells.

4. The preventive and / or therapeutic agent for tumors according to claim 2, wherein: The T cells are regulatory T cells and / or naive T cells.

5. The preventive and / or therapeutic agent for tumors according to any one of claims 1 to 4, wherein The Ikzf1ΔE5 gene-related substance is a nucleic acid molecule encoding the Ikzf1ΔE5 protein.

6. The preventive and / or therapeutic agent for tumors according to any one of claims 1 to 4, wherein The Ikzf1ΔE5 gene-related substance is a substance in which a part or all of exon 5 of the Ikzf1 gene is deleted.

7. The preventive and / or therapeutic agent for tumors according to claim 6, wherein: The substance that causes a partial or complete deletion 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 prevention and / or treatment agent according to claim 7, wherein: Exon skipping agents are nucleic acids.

9. The preventive and / or therapeutic agent for tumors according to claim 1, wherein: Containing T cells expressing Ikzf1ΔE5.

10. A method for screening a substance that regulates the formation of an Ikzf1 complex containing an Ikzf1 protein, a Foxp3 protein, a CHD4 protein and / or an HDAC1 protein, comprising the following steps: (a) contacting a system containing 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 with a candidate compound of a substance that regulates the formation of an Ikzf1 complex; (b) investigating whether the candidate compound inhibits or promotes the association of Ikzf1 protein or its fragment with Foxp3 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with CHD4 protein or its fragment, whether it inhibits or promotes the association of Ikzf1 protein or its fragment with HDAC1 protein or its fragment, whether it inhibits or promotes the association of Foxp3 protein or its fragment with CHD4 protein or its fragment, and / or whether it inhibits or promotes the association of Foxp3 protein or its fragment with HDAC1 protein or its fragment; (c) If the candidate compound inhibits or promotes either association, it is identified as a substance that modulates the formation of the Ikzf1 complex.

11. The method according to claim 10, wherein: The substance that regulates the formation of the Ikzf1 complex is a substance that inhibits or promotes the association between the Ikzf1 protein or a fragment thereof and the Foxp3 protein or a fragment thereof.

12. The method according to claim 10 or 11, wherein: The substance that regulates the formation of the Ikzf1 complex is a preventive and / or therapeutic agent for tumors, an interferon-γ production regulator, or a T cell regulator.

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