Pharmaceutical application of CpG island binding protein KDM2B inhibitor

Abstract

The invention provides a pharmaceutical application of a CpG island binding protein KDM2B inhibitor, namely an application of the CpG island binding protein KDM2B inhibitor in preparation of drugs for preventing and / or treating cancers related to Treg activation. The invention also provides a pharmaceutical composition for preventing and / or treating cancers related to Treg activation, and the pharmaceutical composition comprises an effective amount of the CpG island binding protein KDM2B inhibitor for prevention and / or treatment, and pharmaceutically acceptable auxiliary materials. The invention clarifies the cytobiology basis of the effect of KDM2B on regulating and controlling the immunosuppression function in Treg, develops a new epigenetic regulation and control mechanism, and defines that KDM2B is used as a new target for reducing the immunosuppression activity of a tumor microenvironment and improving the treatment effect of an immune checkpoint inhibitor, thereby adding a new strategy for the treatment of malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the field of drugs for preventing and / or treating tumors. Specifically, the present invention relates to the use of CpG island binding protein KDM2B inhibitors for preventing and / or treating cancer, pharmaceutical compositions for preventing and / or treating cancer comprising CpG island binding protein KDM2B inhibitors, and the use of CpG island binding protein KDM2B inhibitors in the preparation of drugs for preventing and / or treating cancer. Background Art

[0002] During the malignant evolution of tumors, tumor cells not only use a variety of strategies to maintain growth and metastasis, but also use a variety of strategies to avoid being recognized and eliminated by the immune system. [1] Therefore, immune checkpoint therapy came into being. James Allison and Tasuku Honjo also won the 2018 Nobel Prize in Physiology or Medicine for this. However, for most tumors, these treatments often do not produce a "response". One of the important reasons is the presence of other immunosuppressive mechanisms in these tumors, such as the infiltration of regulatory T cells (Treg). Treg is a subpopulation of T cells that can inhibit autoreactive effector T cells and play an important role in immune homeostasis. [2，3] There are a large number of Tregs in the tumor microenvironment of both mice and humans, and they are currently considered to be the main obstacle to anti-tumor immunity and cancer immunotherapy. [4] Therefore, one of the approaches to improve the therapeutic effect of immune checkpoint inhibitors is to deplete Tregs or neutralize the immunosuppressive effects of Tregs, especially the latter. [5，6] .

[0003] Under normal conditions, most peripheral Tregs are in a resting or naive state, with low immunosuppressive capacity. However, in inflammatory and tumor environments, signals transmitted to cells through cytokine receptors and T cell antigen receptors (TCRs) can activate the inhibitory function of Tregs, turning resting Tregs (rTregs) into activated Tregs (aTregs). [7] In mice, rTreg cells are mainly found in lymphoid organs and can inhibit lymphoid tissue proliferation and the occurrence of autoimmune diseases. In inflammatory and tumor environments, "effect-memory-like" aTreg cells inhibit anti-tumor immunity. [1] .

[0004] Currently, a lot of research has been done on the regulatory factors of Treg activation and tumor suppression.

[0005] Considering the impact of epigenetic regulation on cell fate plasticity, scientists such as Stephen Baylin and Peter Jones have sought breakthroughs from chromatin regulatory proteins and discovered inhibitors of some key factors such as DNMT inhibitors. [8] , EZH2 inhibitors, etc. can reverse tumor immune escape [9-11] Bradley E. Bemstein, Shi Yang, Yan Qin and other epigenetic research teams found that the inactivation of histone methyltransferase SETDB1 and histone demethylases LSD1 and KDM5B can increase the immunogenicity of tumor cells and enhance the effect of tumor immunotherapy. [12-14] Therefore, the study of epigenetics combined with tumor immunity will bring hope to many refractory tumors.

[15] .

[0006] KDM2B (Lysine Demethylase 2B) is a CpG island (CpG island: in mammals, CpG exists in two forms: one is dispersed in the DNA sequence; the other is highly aggregated, which is called a CpG island) binding protein with multiple epigenetic regulatory functions. Currently, a large number of studies have confirmed that KDM2B promotes tumor progression. KDM2B is highly expressed in a variety of solid tumors and hematological tumors. Cell and mouse genetic models have confirmed that KDM2B is an important epigenetic regulatory factor in tumor development: KDM2B knockdown will weaken the self-renewal ability of GBM glioblastoma (GBM) cancer stem cells (GSC) [16，17] Overexpression of KDM2B in hematopoietic stem cells induces myeloid leukemia or B lymphocytic leukemia

[18] Knockdown / knockout of KDM2B in diffuse large B-cell lymphoma, ovarian cancer, and pancreatic cancer cell lines reduces tumor formation ability [19-21] However, as a key factor that binds to almost all CpG islands without bias [22-24] How KDM2B coordinates the activation or silencing of target genes remains a mystery, and its functions in somatic cells and pathological conditions are even less understood. Currently, the mechanism by which KDM2B promotes tumor progression and the relationship between KDM2B and cancers associated with Treg activation are still unclear. Summary of the Invention

[0007] The technical solution of the present invention is proposed on the basis of the following research:

[0008] The inventors have found through research that KDM2B can promote Treg activation and transcriptional elongation of immunosuppressive-related genes. Inactivation of KDM2B in Treg will affect Treg activation and its immunosuppressive function, thereby improving the therapeutic effect of immune checkpoint inhibitors. Specifically, database analysis found that the expression level of KDM2B is related to the activation state of Treg and external antigen stimulation; the results of flow cytometry, RNA-seq, CUT&Tag and GSEA analysis also showed that KDM2B is indispensable for Treg activation and the expression and activation of immunosuppressive-related genes; further animal experiments suggest that the loss of KDM2B activity reduces the immunosuppressive effect of Treg in tumors, weakens tumor growth, and further increases the therapeutic effect of immune checkpoint inhibitors anti-PD-L1. Therefore, KDM2B is an important potential target for improving the therapeutic effect of immune checkpoint inhibitors.

[0009] Therefore, the present invention aims to provide a method for preventing and / or treating cancers associated with Treg activation, and a pharmaceutical composition for preventing and / or treating cancers comprising a CpG island binding protein KDM2B inhibitor.

[0010] In the present invention, "CpG island binding protein KDM2B inhibitors" include agents that reduce or inhibit the activity of the CpG island binding protein KDM2B or inactivate it by any means. For example, inactivation by Cre recombinase, siRNA or shRNA, and CRISPR proteins or variants thereof that delete the KDM2B gene, such as Cas9, dCas9, nCas9; Cas12, Cas13, and Cas14.

[0011] In one aspect, the present invention provides use of a CpG island binding protein KDM2B inhibitor in the preparation of a medicament for preventing and / or treating cancer associated with Treg activation.

[0012] According to some embodiments of the present invention, the cancer associated with Treg activation is selected from cancers in which tumor-infiltrating Treg activity or ratio is increased and associated with worse prognosis, such as one or more of colon cancer, melanoma and breast cancer.

[0013] According to some embodiments of the present invention, the CpG island binding protein KDM2B inhibitor is selected from one or more of an agent that causes KDM2B gene deletion (such as Cre recombinase), an agent that reduces or inhibits KDM2B expression (such as siRNA, shRNA or CRISPR / Cas), and an agent that reduces or inhibits KDM2B activity (such as an agent that inhibits the binding of KDM2B to chromatin).

[0014] According to some embodiments of the present invention, the agent for deleting the KDM2B gene is Cre recombinase.

[0015] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is selected from CRISPR protein or its variants, for example, one or more of Cas9, dCas9, nCas9, Cas12, Cas13 and Cas14.

[0016] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is shRNA, for example, shRNAs such as 5'-GAGGGTGGACTTCGGAGAAAT-3' (SEQ ID NO: 1) and 5'-CTGAACCACTGCAAGTCTATC-3' (SEQ ID NO: 2).

[0017] According to some embodiments of the present invention, the drug further comprises one or more other drugs for preventing and / or treating cancer; preferably, the other drugs for preventing and / or treating cancer are immune checkpoint inhibitors; more preferably, the immune checkpoint inhibitors are PD-1 antibodies and / or PD-L1 antibodies; further preferably, the PD-1 antibody or PD-L1 antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a chimeric antibody; particularly preferably, the antibody is a PD-L1 monoclonal antibody.

[0018] In another aspect, the present invention provides a method for preventing and / or treating cancer associated with Treg activation, comprising administering a preventively and / or therapeutically effective amount of a CpG island binding protein KDM2B inhibitor to a subject in need thereof.

[0019] According to some embodiments of the present invention, the cancer associated with Treg activation is selected from cancers in which tumor-infiltrating Treg activity or ratio is increased and associated with worse prognosis, such as one or more of colon cancer, melanoma and breast cancer.

[0020] According to some embodiments of the present invention, the CpG island binding protein KDM2B inhibitor is selected from one or more of an agent that causes KDM2B gene deletion (such as Cre recombinase), an agent that reduces or inhibits KDM2B expression (such as siRNA, shRNA or CRISPR / Cas), and an agent that reduces or inhibits KDM2B activity (such as an agent that inhibits the binding of KDM2B to chromatin).

[0021] According to some embodiments of the present invention, the agent for deleting the KDM2B gene is Cre recombinase.

[0022] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is selected from CRISPR protein or its variants, for example, one or more of Cas9, dCas9, nCas9, Cas12, Cas13 and Cas14.

[0023] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is shRNA, for example, shRNAs such as 5'-GAGGGTGGACTTCGGAGAAAT-3' (SEQ ID NO: 1) and 5'-CTGAACCACTGCAAGTCTATC-3' (SEQ ID NO: 2).

[0024] According to some embodiments of the present invention, it further comprises: administering to the subject one or more other drugs for preventing and / or treating cancer; preferably, the other drugs for preventing and / or treating cancer are immune checkpoint inhibitors; more preferably, the immune checkpoint inhibitors are PD-1 antibodies and / or PD-L1 antibodies; further preferably, the PD-1 antibody or PD-L1 antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a chimeric antibody; particularly preferably, the antibody is a PD-L1 monoclonal antibody.

[0025] In another aspect, the present invention provides a pharmaceutical composition for preventing and / or treating cancer associated with Treg activation, comprising a preventive and / or therapeutically effective amount of a CpG island binding protein KDM2B inhibitor and a pharmaceutically acceptable excipient.

[0026] According to some embodiments of the present invention, the cancer associated with Treg activation is selected from cancers in which tumor-infiltrating Treg activity or ratio is increased and associated with worse prognosis, such as one or more of colon cancer, melanoma and breast cancer.

[0027] According to some embodiments of the present invention, the CpG island binding protein KDM2B inhibitor is selected from one or more of an agent that causes KDM2B gene deletion (such as Cre recombinase), an agent that reduces or inhibits KDM2B expression (such as siRNA, shRNA or CRISPR / Cas), and an agent that inhibits KDM2B activity (such as an agent that inhibits the binding of KDM2B to chromatin).

[0028] According to some embodiments of the present invention, the agent for deleting the KDM2B gene is Cre recombinase.

[0029] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is selected from CRISPR protein or its variants, for example, one or more of Cas9, dCas9, nCas9, Cas12, Cas13 and Cas14.

[0030] According to some embodiments of the present invention, the agent that reduces or inhibits KDM2B expression is shRNA, for example, shRNAs such as 5'-GAGGGTGGACTTCGGAGAAAT-3' (SEQ ID NO: 1) and 5'-CTGAACCACTGCAAGTCTATC-3' (SEQ ID NO: 2).

[0031] According to some embodiments of the present invention, the pharmaceutical composition further comprises one or more other drugs for preventing and / or treating cancer; preferably, the other drugs for preventing and / or treating cancer are immune checkpoint inhibitors; more preferably, the immune checkpoint inhibitors are PD-1 antibodies and / or PD-L1 antibodies; further preferably, the PD-1 antibody or PD-L1 antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a chimeric antibody; particularly preferably, the antibody is a PD-L1 monoclonal antibody.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This study clarifies the cellular biological basis for KDM2B's regulation of immunosuppressive function in Treg cells. It also explores new regulatory mechanisms for KDM2B's involvement in gene activation and transcriptional elongation, demonstrating the crucial role of KDM2B in Treg activation and the expression of genes associated with immunosuppression. It also identifies KDM2B as a new target for reducing immunosuppressive activity in the tumor microenvironment and improving the therapeutic efficacy of immune checkpoint inhibitors, adding a novel strategy for the treatment of malignant tumors. Furthermore, according to the inventors' research, inhibition of KDM2B activity does not lead to a decrease in Treg numbers or induce severe autoimmune inflammatory responses, resulting in minimal side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0035] Figure 1 The expression of KDM2B in Tregs of different subtypes and tissues is shown.

[0036] Figure 2 Flow cytometry analysis showed that the loss of KDM2B affects the expression of Treg cells in the spleen in CD4 +The effects on the proportion, activation level, expression level of activation and immunosuppressive-related molecules, proliferation ability, and apoptosis level in T cells.

[0037] Figure 3 Flow cytometry analysis showed that the loss of KDM2B affects the expression of conventional CD4 + T cells (Tconv, conventional CD4 + T cells) and CD8 + HE staining was used to analyze the effect of KDM2B deficiency on the level of lymphoid infiltration in the small intestine, colon, lung, pancreas, liver, and kidney of mice.

[0038] Figure 4 The process of establishing a melanoma model using wild-type (WT) and cKO mice in Example 5 is shown.

[0039] Figure 5 The results show that the loss of KDM2B in Tregs affects the occurrence and development of tumors.

[0040] Figure 6 Shown are the effects of KDM2B deletion in Tregs on the mouse melanoma tumor microenvironment.

[0041] Figure 7 The process and dosing regimen for establishing a melanoma model in wild-type (WT) and cKO mice in Example 6 are shown.

[0042] Figure 8 The effect of KDM2B deletion in Tregs on the therapeutic efficacy of the immune checkpoint inhibitor anti-PD-L1 (anti-PD-L1) is shown. DETAILED DESCRIPTION

[0043] The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0044] The experimental methods used in the following examples, unless otherwise specified, are all conventional experimental methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from biochemical reagent sales companies.

[0045] Example 1: Expression of KDM2B in Tregs of different subtypes and tissues

[0046] This example uses published data (RNA-sequencing or SMART-sequencing) to analyze and compare the expression levels of KDM2B in activated regulatory T cells (aTreg) and resting regulatory T cells (rTreg) in mouse spleens, the expression levels of KDM2B in tumor-infiltrated Treg (TITR) and splenic Treg (Splenic Treg) in mouse melanoma and colorectal cancer models, and the expression levels of KDM2B in TITR and corresponding normal tissue Treg in colorectal cancer patients and breast cancer patients.

[0047] Analytical methods: Published data (derived from GEO databases GSE71309, GSE82008, GSE116347, GSE89225), as well as transcriptome sequencing (RNA-seq) and single-cell transcriptome sequencing (SMART-seq) were used to analyze: ① The difference in KDM2B expression levels in aTreg and rTreg in the spleen of wild-type mice (derived from GEO databases GSE71309, GSE82008); ② The mouse melanoma model (B16 mouse model, data ③ The difference in KDM2B expression levels in splenic Treg and tumor-infiltrating Treg in colorectal cancer models (data from GEO database GSE116347) and colorectal cancer models (MC38 mouse model and AM_CT26 mouse model, data from GEO database GSE116347); ③ The difference in KDM2B expression levels in normal tissue Treg and tumor-infiltrating Treg in colorectal cancer patients (data from GEO database GSE116347) and breast cancer patients (data from GEO database GSE89225).

[0048] The analysis results of this example are as follows Figure 1 shown. Specifically, Figure 1 The RNA-seq results in A showed that the expression level of KDM2B in aTreg of the spleen of wild-type mice (red) was significantly higher than that in rTreg (blue); Figure 1 SMART-seq results in B show that in mouse melanoma models and colorectal cancer models, the expression level of KDM2B in tumor-infiltrating Tregs (red) is significantly higher than the expression level of KDM2B in spleen Tregs (blue); Figure 1The SMART-seq results in C show that the expression level of KDM2B in tumor-infiltrating Tregs of colorectal cancer patients (red) is significantly higher than the expression level of KDM2B in normal colon tissue Tregs (blue); Figure 1 RNA-seq results in D show that KDM2B expression levels in tumor-infiltrating Treg cells (Treg-tumor) from breast cancer patients (red) are significantly higher than in Treg cells from normal breast parenchyma (Treg-NBP) (blue). These results indicate that KDM2B expression is upregulated after Treg activation, and that KDM2B expression levels in tumor-infiltrating Treg cells are higher than in Treg cells from normal tissue, suggesting that KDM2B plays an important role in Treg activation and immunosuppression.

[0049] Example 2: Effect of KDM2B inactivation on Treg

[0050] In this example, flox sites were inserted upstream and downstream of exon 13 of mouse KDM2B to construct KDM2B flox (CxxC) Mice. Subsequently, KDM2B flox(CxxC) Mice and Foxp3 Cre mice and obtained KDM2B flox(CxxC) Foxp3 Cre Mice, namely mice with Treg cell-specific KDM2B inactivation (conditional knockout mice, cKO mice). Flow cytometry analysis showed that the loss of KDM2B affected the CD4 Treg cell population in the spleen. + The effects on the proportion, activation level, expression level of activation and immunosuppressive-related molecules, proliferation ability, and apoptosis level in T cells.

[0051] Methods: WT control mice and KDM2B cKO mice of the same age and sex, 6-8 weeks old, were selected, spleens were removed, and flow cytometry analysis was performed. The pretreatment steps before analysis were as follows:

[0052] A. Mice were sacrificed by cervical dislocation, and spleens and lymph nodes were removed. The cells were placed on a 70 μm cell sieve pre-soaked in 3 ml of RPMI-1640 / DMEM / PBS containing 2% fetal bovine serum (FBS). The cells were thoroughly triturated using the plunger of a 5 ml syringe to obtain a single-cell suspension, which was then collected in a 15- or 50-ml centrifuge tube.

[0053] B. Centrifuge at 1500-1600 rpm for 6-7 minutes, discard the supernatant, add appropriate amount of red blood cell lysis buffer to resuspend the cells, and lyse on ice for 15 minutes;

[0054] C. Centrifuge at 1500-1600 rpm for 6-7 minutes, discard the supernatant, resuspend the cells in an appropriate amount of 2% FBS-PBS, and count the cells using a hemocytometer.

[0055] D. Centrifuge at 1500-1600 rpm for 6-7 minutes and discard the supernatant. The remaining liquid volume in the control tube should be approximately 50-100 μl.

[0056] E. Extracellular staining: Add antibodies (source: Biolegend, CD4-APC / Fire750, CD8-APC, CD44-PE / Cy7, CD62L-FITC, CD69-PE / Cy7, Nrp1-BV421), vortex to mix the cells, and stain at 4°C in the dark for 20-40 minutes.

[0057] F. Stimulate cell activation to secrete cytokines and terminate Golgi secretion: Prepare GolgiStop buffer (add 100 ng / ml phorbol myristate phosphate (PMA), 10 μg / ml ionomycin, and 100 μl BD GolgiStop per 100 ml of RPMI-1640 medium). TM Protein transport inhibitor (BD, Cat. No. 554724) was used to resuspend Tregs in buffer and culture in a cell culture incubator for 4.5-6 hours. Cells were collected by centrifugation and prepared for intracellular staining.

[0058] G. Intracellular staining: Mix Fixation / Permeabilizatioin Concentrate and Fixation / Permeabilizatioin Diluent (Invitrogen) at a ratio of 1:3, add 500 μl of the mixture to each centrifuge tube, vortex to mix, and fix the cells in the dark at 4°C for 40-50 minutes.

[0059] H. Dilute the Permeabilization Buffer (10×) with distilled water and add 2 ml of diluent to each tube after fixation.

[0060] Centrifuge at 1500-1600 rpm for 6-7 minutes and discard the supernatant. The remaining liquid volume in the control tube should be approximately 50-100 μl.

[0061] G. Add flow cytometry antibodies for intracellular staining (source: Biolegend, Foxp-PE, IL-17-perCP / Cy5.5, IFNγ-APC, Granzyme B-BV421, Ki67-BV605), vortex to mix the cells, and stain at 4°C in the dark for 40-50 minutes.

[0062] K. Add 3 ml of Permeabilization Buffer (1×), centrifuge at 1500-1600 rpm for 6-7 minutes, and discard the supernatant.

[0063] L. Add an appropriate amount of 2% FBS-PBS to resuspend the cells. Filter the cells using a 70 μm cell sieve and analyze them.

[0064] The analysis results of this example are as follows Figure 2 shown.

[0065] Figure 2 The results of the flow cytometry analysis are shown. Specifically, Figure 2 showed that KDM2B deficiency did not affect Treg expression in CD4 + The proportion of T cells (see Figure 2 A, of which CD4 + Foxp3 + cells are Treg cells), but it will reduce the activation level of Treg (see Figure 2 B, in which CD44 + CD62L - aTreg cells, CD44 - CD62L + The loss of KDM2B significantly downregulated the expression of CD44, CD69, and Nrp-1 in Treg cells, which are related to activation and immunosuppression (see Figure 2 C). Inactivation of KDM2B in Tregs slightly increases Treg proliferation (see Figure 2 E, Ki67 expression level indicates Treg proliferation ability), but does not affect Treg apoptosis (see Figure 2 D) These results indicate that KDM2B deficiency affects Treg activation and the expression of immunosuppressive genes.

[0066] Example 3: Effect of KDM2B inactivation in Treg on immune balance in mice

[0067] In this example, cKO mice (prepared as above) were used to analyze the effect of KDM2B deficiency on the expression of conventional CD4 + T cells (Tconv, conventional CD4 +T cells) and CD8 + The effect of KDM2B on T cell activity was analyzed by flow cytometry as above. HE staining was used to analyze the effect of KDM2B deficiency on the level of lymphoid infiltration in the small intestine, colon, lung, pancreas, liver, and kidney.

[0068] Analytical methods : ① Select 12 WT control mice and KDM2B cKO mice of the same age and sex between 6 and 8 weeks old, weigh the mice, and perform statistical analysis to see if there are significant differences. ② Select WT control mice and KDM2B cKO mice of the same age and sex between 6 and 8 weeks old, remove the spleen, and perform flow cytometry analysis. The pretreatment steps before analysis are the same as in Example 2. ③ Select WT control mice and KDM2B cKO mice of the same age and sex between 6 and 8 weeks old, take the small intestine, colon, pancreas, liver, lung, and kidney and fix them in fixative, then perform HE staining to evaluate the infiltration of lymphocytes in each organ.

[0069] The analysis results of this example are as follows Figure 3 shown.

[0070] Figure 3 A shows that the inactivation of KDM2B in Tregs did not affect the body weight of mice compared with wild-type mice. The inventors detected the infiltration of lymphocytes in the small intestine, colon, pancreas, liver, lungs, and kidneys. The results showed that only the level of lymphocyte infiltration in the kidneys increased compared with the control group, and the infiltration score increased (see Figure 3 B and C). Loss of KDM2B weakens the inhibitory effect of Treg on T cells, and CD4 + Tconv and CD8 + Increased activation of T cells (see Figure 3 D, among which CD44 + CD62L - For activated cells (activated), CD44 - CD62L + Cells are resting, but their proliferation capacity has not changed significantly (see Figure 3 E). In addition, CD4 + Tconv secretes increased levels of cytokines IFNγ (interferon gamma) and IL-17 (interleukin-17) (see Figure 3 F and G), CD8 + T cells secrete increased levels of cytokines IFNγ and Granzyme B (granzyme B) (see Figure 3H and I). Therefore, inhibition of KDM2B activity in Tregs reduces the immunosuppressive effect of Tregs and thus affects the immune balance of mice.

[0071] Example 4: Inactivation of KDM2B in Tregs slows the development and progression of melanoma in mice

[0072] Analytical methods WT control mice and KDM2B cKO mice of the same age and sex (prepared as above) were selected at 6-8 weeks of age and were subcutaneously implanted with tumors (2.5×10 5 Melanoma cells (B16-F10 / mouse), two weeks after tumor inoculation, tumor tissue was removed and tumor size was measured. The construction scheme of melanoma model mice is as follows Figure 4 shown.

[0073] The results show that the loss of KDM2B in Treg affects the occurrence and development of tumors. Figure 5 As shown. Figure 5 It can be seen that the tumor volume of cKO mice was significantly reduced compared with that of wild-type mice, indicating that the loss of KDM2B in Treg inhibits the occurrence and development of melanoma.

[0074] Example 5: Inactivation of KDM2B in Tregs reduces their tumor suppressive ability

[0075] Analytical methods WT control mice and KDM2B cKO mice of the same age and sex (prepared as above) were selected at 6-8 weeks of age and were subcutaneously implanted with tumors (2.5×10 5 Melanoma cells (B16-F10 / mouse) were transplanted two weeks after implantation, and tumor tissue was removed for flow cytometry analysis of tumor-infiltrating T cells within the tumor. The pretreatment steps for tumor tissue prior to flow cytometry analysis were as follows:

[0076] A. Mice were sacrificed by cervical dislocation, and tumor tissue was removed and placed on a 70 μm cell sieve pre-soaked in 3 ml of RPMI-1640 / DMEM / PBS containing 2% FBS. The tissue was minced with scissors and collected in a 15- or 50-ml centrifuge tube. The tissue was then digested in digestion buffer (10% FBS, 0.1 mg / ml deoxyribonuclease I (DNase I), 1.5 mg / ml collagenase IV in RPMI-1640 medium) (37°C, 5% CO2, 1 hour, 200 rpm).

[0077] B. Filter through a 70 μm cell mesh to obtain a single-cell suspension;

[0078] C. Centrifuge at 1500-1600 rpm for 6-7 minutes, discard the supernatant, add an appropriate amount of red blood cell lysis buffer to resuspend the cells, and lyse on ice for 15 minutes;

[0079] D. Centrifuge at 1500-1600 rpm for 6-7 minutes, discard the supernatant, and resuspend the cells in 10-15 ml of 80% Percoll.

[0080] E. Pre-add 10-15 ml of 40% Percoll to a 50 ml centrifuge tube. Draw the cell suspension into a syringe. Insert the needle tip into the centrifuge tube containing 40% Percoll and slowly push the cell suspension in, taking care not to disrupt the interface of the solution.

[0081] F. Centrifuge at 325g for 23 minutes at room temperature. Carefully remove the tube and remove the white blood cells between the interfaces using a pipette. Remix.

[0082] G. Centrifuge at 1500-1600 rpm for 6-7 minutes, discard the supernatant, resuspend the cells in an appropriate amount of 2% FBS-PBS, and count the cells using a hemocytometer.

[0083] H. Flow cytometry antibody staining, the method is the same as before.

[0084] I. On-computer analysis.

[0085] The results are as follows Figure 6 Specifically, loss of KDM2B activity in Tregs did not affect the ratio of CD8+ T cells to CD4+ T cells within tumors (see Figure 6 A), nor does it affect the proportion of Treg in CD4+ T cells (see Figure 6 B). However, a significant decrease in the activation level of tumor-infiltrating Treg cells was observed, and the proportion of aTreg cells was significantly downregulated (see Figure 6 C), and the expression levels of CD44, CD69, and Nrp-1, which are related to activation and immunosuppression, were significantly decreased (see Figure 6 D), indicating a decrease in Treg activation and immunosuppression. Consistent with this, the activation level of tumor-infiltrating Tconv increased and the proportion of aTconv increased (see Figure 6 E), tumor-infiltrating Tconv secrete the cytokine IL-17 (see Figure 6 F), and CD8+ T cells secrete Granzyme B and IFNγ (see Figure 6 G and H) levels were significantly increased, indicating that the inactivation of KDM2B in Tregs affects the inhibitory effect of Tregs in tumors on Tconv and CD8+ T cells, thereby enhancing the activity of T cells and the ability to kill tumors.

[0086] Example 6 Inactivation of KDM2B in Treg enhances the therapeutic effect of immune checkpoint inhibitor anti-PD-L1 on tumors

[0087] Analytical methods: WT control mice and KDM2B cKO mice of the same age and sex (prepared as above) were selected at 6-8 weeks of age and were subcutaneously implanted with tumors (2.5×10 5 Melanoma cells (B16-F10 / mouse) were injected intraperitoneally with monoclonal antibody anti-PD-L1 (Selleck) starting 7 days after tumor initiation, 200 μg / mouse, once every three days, for a total of 4 injections. The size of the tumor was measured with a vernier caliper, and the tumor volume was calculated as follows: Volume = (length × width 2 ) / 2. The survival period of mice was recorded at the same time. The construction of melanoma model mice and the dosing regimen were as follows. Figure 7 shown.

[0088] The results are as follows Figure 8 Compared with wild-type mice, the tumor volume of cKO mice was significantly reduced ( Figure 8 A), the survival of mice was significantly prolonged in the mid-term ( Figure 8 B), indicating that the loss of KDM2B in Treg can further significantly enhance the therapeutic effect of the immune checkpoint inhibitor anti-PD-L1 on mouse melanoma.

[0089] Although the present invention has been described to a certain extent, it is obvious that appropriate changes can be made to various conditions without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which include equivalent replacements of each factor described.

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Claims

1. Use of a CpG island binding protein KDM2B inhibitor in the preparation of a medicament for preventing and / or treating cancers associated with Treg activation.

2. The use according to claim 1, characterized in that The cancer associated with Treg activation is selected from cancers in which increased activity or proportion of tumor-infiltrating Tregs is associated with a worse prognosis, such as one or more of colon cancer, melanoma, and breast cancer.

3. The use according to claim 1 or 2, characterized in that The CpG island binding protein KDM2B inhibitor is selected from one or more of an agent that causes KDM2B gene deletion, an agent that reduces or inhibits KDM2B expression, and an agent that reduces or inhibits KDM2B activity.

4. The use according to claim 3, characterized in that The reagent for deleting the KDM2B gene is Cre recombinase; Preferably, the agent that reduces or inhibits KDM2B expression is selected from CRISPR proteins or variants thereof, for example, one or more of Cas9, dCas9, nCas9, Cas12, Cas13 and Cas14; Preferably, the agent that reduces or inhibits KDM2B expression is shRNA, for example, shRNAs such as 5'-GAGGGTGGACTTCGGAGAAAT-3' (SEQ ID NO: 1) and 5'-CTGAACCACTGCAAGTCTATC-3' (SEQ ID NO: 2).

5. The use according to any one of claims 1 to 4, characterized in that The drug further comprises one or more other drugs for preventing and / or treating cancer; preferably, the other drugs for preventing and / or treating cancer are immune checkpoint inhibitors; more preferably, the immune checkpoint inhibitors are PD-1 antibodies and / or PD-L1 antibodies; further preferably, the PD-1 antibody or PD-L1 antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a chimeric antibody; particularly preferably, the antibody is a PD-L1 monoclonal antibody.

6. A pharmaceutical composition for preventing and / or treating cancer associated with Treg activation, characterized in that: The pharmaceutical composition comprises a preventive and / or therapeutically effective amount of a CpG island binding protein KDM2B inhibitor and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that The cancer associated with Treg activation is selected from cancers in which increased activity or proportion of tumor-infiltrating Tregs is associated with a worse prognosis, such as one or more of colon cancer, melanoma, and breast cancer.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that The CpG island binding protein KDM2B inhibitor is selected from one or more of an agent that causes KDM2B gene deletion, an agent that reduces or inhibits KDM2B expression, and an agent that reduces or inhibits KDM2B activity.

9. The pharmaceutical composition according to claim 10, characterized in that The reagent for deleting the KDM2B gene is Cre recombinase; Preferably, the agent that reduces or inhibits KDM2B expression is selected from CRISPR proteins or variants thereof, for example, one or more of Cas9, dCas9, nCas9, Cas12, Cas13 and Cas14; Preferably, the agent that reduces or inhibits KDM2B expression is shRNA, for example, shRNAs such as 5'-GAGGGTGGACTTCGGAGAAAT-3' (SEQ ID NO: 1) and 5'-CTGAACCACTGCAAGTCTATC-3' (SEQ ID NO: 2).

10. The pharmaceutical composition according to any one of claims 6 to 9, characterized in that The drug further comprises one or more other drugs for preventing and / or treating cancer; preferably, the other drugs for preventing and / or treating cancer are immune checkpoint inhibitors; more preferably, the immune checkpoint inhibitors are PD-1 antibodies and / or PD-L1 antibodies; further preferably, the PD-1 antibody or PD-L1 antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a chimeric antibody; particularly preferably, the antibody is a PD-L1 monoclonal antibody.

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