Application of Targeting BCL6 to regulation of Treg cells in preparation of antitumor drugs

Abstract

The invention relates to application of targeted BCL6 regulation and control of Treg cells in preparation of anti-tumor drugs and application of targeted BCL6 regulation and control of Treg cells combined with immune checkpoint blocking agents in preparation of anti-tumor drugs, a BCL6 targeting method comprises the step of using a BCL6 inhibitor, experiments prove that the methylation level of H3K4 of Treg cell histone of BCL6 knockout mice is remarkably reduced, and the methylation level of H3K4 is remarkably reduced. Compared with the prior art, the anti-PDL1 group and the combined group have the advantages that the anti-PDL1 group and the combined group have better tumor inhibition efficacy, the inhibition capability on CD8T cells is obviously reduced, after the BCL6 inhibitor is used, compared with a control group, the tumor volume is obviously reduced, a targeting BCL6 regulation and control Treg cell combined immune checkpoint blocking agent shows a synergistic effect, the tumor size can be obviously inhibited, and compared with the anti-PDL1 group and the combined group, the anti-PDL1 group and the combined group have better tumor inhibition efficacy, and the anti-PDL1 group and the combined group have obvious difference; according to the invention, the efficacy of tumor immunotherapy is effectively improved, and a new direction and inspiration are provided for related research in the field.

Description

Technical Field

[0001] The present invention belongs to the field of tumor biotherapy, and particularly relates to the application of targeting BCL6 to regulate Treg cells in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumors are diseases that seriously threaten human life and health. According to the statistics of the National Cancer Center and the International Agency for Research on Cancer (IARC, WHO) of the World Health Organization, the number of new cancer cases and death cases in China ranks first in the world. Moreover, in the past decade or so, the incidence and death of malignant tumors have been showing a continuous upward trend. The prevention and control situation is severe, which is a huge challenge for the implementation of the "Healthy China" strategy.

[0003] Under normal circumstances, the body's immune system mainly relies on CD8+ T cells to complete the specific recognition and direct killing of cancer cells. However, under specific circumstances, cancer cells escape immune surveillance through immune editing, form dominant clones and colonize locally, and finally establish an inhibitory tumor microenvironment (Tumor micro-environment, TME). There are various inhibitory immune cells in the TME, such as regulatory T cells (regulatory T cell, Treg), tumor-associated macrophages (tumorassociated macrophages, TAM), myeloid-derived suppressor cells (myeloid-derived suppressor cells, MDSCs), etc. These cells inhibit the anti-tumor function of tumor antigen-specific CD8+ T cells in different dimensions. The immunosuppressive factors and the continuously present tumor antigens jointly drive the functional exhaustion (Exhaustion) of CD8+ T cells, causing them to lose the ability to control tumors at the population level. The exhausted tumor-specific CD8+ T cells continuously and highly express a series of immune checkpoint molecules (Immune checkpoints), that is, co-inhibitory receptors (Co-inhibitory receptor), such as PD-1, TIM3, and CTLA-4. The binding of the co-inhibitory receptor to its corresponding ligand (such as PD-L1) activates downstream inhibitory signals, which is an important reason for the low effector function of exhausted CD8+ T cells. Blocking the inhibitory signals mediated by immune checkpoint molecules with monoclonal antibodies can, to a certain extent, reverse the exhaustion of CD8+ T cells, which constitutes the clinical application basis of immune checkpoint blockers (Immune Checkpoint Blockade, ICB) such as PD-1. Currently, the main immune strategy for CD8 T cell exhaustion is to block the expression of inhibitory receptors. The immune checkpoint blockade therapy represented by PD-1 / PD-L1 blocking antibodies has achieved good results in the treatment of malignant melanoma, non-small cell lung cancer, and renal cancer. However, the highest patient response rate does not exceed 30%. In addition, clinical studies have found that patients who respond in the initial stage will develop drug resistance or recurrence in the later stage, and only a very small number of patients (<5%) can be clinically cured. This suggests that using ICB alone cannot fundamentally change the exhausted state, and new strategies for improving immunosuppressive factors need to be explored starting from the microenvironment.

[0004] Treg cells are the most important immunosuppressive cell type in the TME. Treg cells account for 2%-5% of peripheral blood CD4+ T cells, while this proportion is as high as 10%-50% in the TME. The increase in the frequency of Treg cells and the increase in their ratio to effector T cells are associated with the progression of various types of tumors and poor patient prognosis. Currently, various strategies for targeting Treg cells alone or in combination with ICB therapy are undergoing preclinical / clinical trials. Unfortunately, the current efficacy of these strategies is not clear. On the one hand, it may eliminate effector T cells that also express this type of molecule. On the other hand, the deletion of systemic Treg cells may greatly increase the risk of immune-related adverse events (irAEs), especially autoimmune-related toxicity. Therefore, further deepening the understanding of Treg cell differentiation and function regulation is of great practical significance for developing safer Treg cell-targeted therapeutic strategies. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of targeting BCL6 to regulate Treg cells in the preparation of anti-tumor drugs.

[0006] The technical solution of the present invention is as follows:

[0007] The use of targeting BCL6 to regulate Treg cells in the preparation of anti-tumor drugs.

[0008] Further, the method of targeting BCL6 includes using a BCL6 inhibitor.

[0009] Further, the BCL6 inhibitor reduces the inhibitory effect of Treg cells on CD8+ T cells by reducing the histone H3K4 methylation level of Treg cells.

[0010] Further, the BCL6 inhibitor includes but is not limited to FX1.

[0011] Further, the administration method of the drug includes but is not limited to intraperitoneal injection.

[0012] Further, the drug further includes pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients include one or more of diluents, wetting agents, binders, disintegrants, lubricants, color, flavor and odor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local anesthetics, pH regulators, isotonic regulators and isosmotic regulators.

[0013] The use of targeting BCL6 to regulate Treg cells in combination with immune checkpoint blockers in the preparation of anti-tumor drugs.

[0014] Further, the immune checkpoint blocker includes, but is not limited to, one or more of PD1 antibodies and PDL1 antibodies.

[0015] Further, the method for targeting BCL6 includes using a BCL6 inhibitor.

[0016] Further, the BCL6 inhibitor includes, but is not limited to, FX1.

[0017] Further, the effective dose of the drug is 60 - 100 mg / kg, and the mass ratio of the BCL6 inhibitor to the immune checkpoint inhibitor is 70:0.15.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] 1. The present invention relates to the application of targeting BCL6 to regulate Treg cells in the preparation of anti-tumor drugs. The methylation level of histone H3K4 in Treg cells of BCL6 knockout mice is significantly reduced, and the inhibitory ability on CD8 T cells is significantly decreased; the expressions of Treg cell fate determination and function-related factors including Foxp3, CTLA4, CD25, and GITR in KO Treg cells are significantly lower than those in WT Treg cells. The proportion of central Treg cells in the draining lymph nodes of KO mice is significantly higher than that of WT mice, while the proportion of effector Treg cells is significantly lower than that of WT mice; moreover, the proportions and absolute numbers of TNF-α+CD8+, IFN-γ+CD8+, and CD107a / b+CD8+ T cells infiltrating the tongue of KO mice are significantly higher than those of WT mice; verification using the BCL6 inhibitor FX1 significantly inhibits tumor growth.

[0020] 2. The present invention also relates to the application of targeting BCL6 to regulate Treg cells in combination with an immune checkpoint blocker in the preparation of anti-tumor drugs. Compared with the control group, the combination of the BCL6 inhibitor and the immune checkpoint inhibitor shows a synergistic effect, can significantly inhibit tumor size, and has better tumor inhibitory efficacy than the group using anti-PDL1 alone, and there is a significant difference between the two; using the method of the present invention can accurately and effectively target tumor-infiltrating Treg cells, reduce immune-related side effects and show a significant synergistic effect with PD-1 / PD-L1 immune checkpoint blockade therapy. The invention effectively improves the efficacy of tumor immunotherapy and provides a new direction and inspiration for related research in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0022] Figure 1Experimental process of inducing oral mucosal carcinogenesis in mice by 4NQO, tongue lesion tissues, and comparison results of tongue lesion tissues between KO mice and WT mice in Example 2 of the present invention;

[0023] Figure 2 Analysis of the immune phenotypes of Treg cells in KO mice and WT mice in Example 3 of the present invention;

[0024] Figure 3 Analysis of the immune phenotypes of CD8 T cells in KO mice and WT mice in Example 3 of the present invention;

[0025] Figure 4 RNA-Seq data analysis and GO analysis of KO mice and WT mice in Example 4 of the present invention;

[0026] Figure 5 Results of the combination of BCL6 inhibitor and immune checkpoint inhibitor in Example 5 of the present invention. Detailed implementation manners

[0027] The present invention will be further described in detail below. It is necessary to point out here that the following specific implementation manners are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0028] The present invention generally and / or specifically describes the materials and test methods used in the experiments. For the test methods or testing methods involved, if there is no special description, they are all conventional methods; for the reagents or instruments used, if the manufacturer is not indicated, they are all commercially available conventional products and are prepared or used by conventional methods.

[0029] Reproduction and identification of conditional knockout mice in Example 1

[0030] Hybridize and breed knockout mice with conditional knockout of BCL6 in Treg cells

[0031] Mate female homozygous Foxp3 YFP-Cre gene-knockin mice and male BCL6 flox / flox gene-knockin mice to obtain the first-generation mice. After the first-generation mice are weaned at 4-6 weeks of age, they are numbered and marked, and their DNA is extracted for genotype identification. The genotypes of the female mice in this generation are all BCL6 flox / - Foxp3 Cre / - , and the genotypes of the male mice are all BCL6 flox / - Foxp3 Cre . Use the F1-generation mice for self-crossing to produce the F2-generation mice. Then, use the homozygous mice in the F2 generation, BCL6 flox / flox Foxp3 CreSelf-crossing was performed, and finally, stable homozygous mice (KO mice) were obtained, that is, the BCL6 gene was specifically knocked out in Foxp3+ cells (Treg cells) of homozygous mice.

[0032] Example 2 Construction of a multi-stage carcinogenesis model of mouse oral mucosa by 4NQO drinking water method

[0033] Using sterilized ddH 2 O Prepare a 2% stock solution of 4NQO powder (stored in the dark in a 4°C refrigerator). When constructing the tongue cancer model, dilute the stock solution to a concentration of 100 μg / ml, wrap the drinking water bottle with tin foil for light protection, and allow the experimental mice to drink freely for 16 consecutive weeks. Replace the 4NQO water once a week. After drinking for 16 weeks, replace the 4NQO water with sterile water until the end of the experiment ( Figure 1 A), cauliflower-like vegetations can be seen on the tongue surface of the mice, which are scattered squamous cell carcinoma tissues. The results showed that the number of lesions in the tongues of KO mice was less and the volume was smaller than that of WT mice, with significant differences ( Figure 1 B).

[0034] Example 3 Immunophenotypic analysis of Treg cells and CD8 T cells in a mouse tumor model by flow cytometry

[0035] 1. Tissue dissociation and preparation of single-cell suspension

[0036] Obtain the tongue lesion tissues and submandibular draining lymph nodes of mice. The tongue tissues were digested with a digestive solution containing 1 mg / mL type IV collagenase + 0.5 mg / mL type I collagenase + 0.1 mg / mL DNase I (dissolved in RPMI-1640 + 10% FBS) in a 37°C shaker at 150 rpm for 1 hour. After digestion, pour the liquid into a tissue culture dish, terminate the reaction with a large amount of sterile PBS, grind and filter with the tail end of a 5 ml labeled syringe, centrifuge at 1800 rpm for 6 minutes, discard the supernatant, and resuspend with PBS containing 2% fetal bovine serum, and mix well to prepare a single-cell suspension of tongue lesion tissues / subcutaneous tumor tissues. Grind the lymph nodes with a sterile frosted glass slide, pick the lymph nodes between the frosted surfaces of two glass slides and grind them, add sterile PBS, centrifuge at 1800 rpm for 6 minutes, discard the supernatant, and resuspend with PBS containing 2% fetal bovine serum, and mix well to prepare a single-cell suspension of lymph nodes. The single-cell suspension was used for the following flow cytometry to detect the immunophenotypes of Treg cells and T cells.

[0037] 2. Immunophenotypic analysis of Treg cells

[0038] In this example, flow cytometry was used to detect the proportion and number of Treg cells (CD4+Foxp3+), and the proportion and number of functional subsets of Treg cells: central Treg cells (CD44-CD62L+) and effector Treg cells (CD44+CD62L-); the expression of molecules related to Treg cell fate determination and effector function (Foxp3, CD25, CTLA4, GITR, ICOS) was detected.

[0039] As can be seen from Figure 2 A and B, there was no significant difference in the proportion of Treg cells in the tongue lesion tissues and draining lymph nodes between KO mice and WT mice in this model; as can be seen from Figure 2 C and D, the expression of molecules related to Treg cell fate determination and effector function (Foxp3, CD25, CTLA4, GITR, ICOS) was significantly lower in KO Treg cells than in WT Treg cells, indicating that knocking out BCL6 could effectively inhibit Treg cell-related effector molecules; the functional subsets of Treg cells include central Treg cells (cTreg) and effector Treg cells (eTreg), and cTreg has lower inhibitory activity than eTreg. As can be seen from Figure 2 E, the proportion of cTreg in the draining lymph nodes of KO mice was significantly higher than that of WT mice, while the proportion of eTreg cells was significantly lower than that of WT mice, and the inhibitory activity of KO mice was lower than that of WT mice.

[0040] 3. Immunophenotype analysis of CD8 T cells

[0041] In this example, flow cytometry was used to detect the proportion, activation status of CD8 T cells, and the effector function of CD8 T cells.

[0042] As can be seen from Figure 3 A, the proportions of infiltrating CD4+T and CD8+T cells in the tongue lesion tissues of KO mice were significantly higher than those of WT mice; there was no difference in the proportion of infiltrating naive CD8+T cells in the tongue tissues of the two groups, the proportion of infiltrating central memory CD8+T cells in the tongue tissues of KO mice was lower than that of WT mice, and the proportion of effector memory CD8+T cells was significantly higher than that of WT mice ( Figure 3 B). In addition, the proportions and absolute numbers of TNF-α+CD8+, IFN-γ+CD8+ and CD107a / b+CD8+T cells infiltrating the tongue of KO mice were significantly higher than those of WT mice ( Figure 3 C).

[0043] Example 4 Flow sorting, high-throughput sequencing and bioinformatics analysis of Treg cells

[0044] 1. Cell sorting: According to the tumor progression, mice were sacrificed on the 16th - 18th day after tumor formation. Tumor tissues were collected and lymphocytes were extracted. Miltenyi separation magnetic beads were used to enrich CD4+ T cells. Then, flow antibodies CD4, CD25, CD44, and GITR were labeled, and Live / Dead dye was used to distinguish dead cells. Flow cytometry sorting was performed on BD FACS Aria III. The target cell population was Live / Dead–CD4+CD25+GITR+CXCR5-, which were the target Treg cells. After sorting, the purity of Treg cells was measured by flow cytometry and should be above 95%.

[0045] 2. RNA-Seq: The prepared RNA samples were reverse transcribed and library constructed using the Smart-Seq V4 kit from Takara, and the obtained samples were sent to the company for sequencing. The raw data obtained was processed for normalized data using the FeatureCounts software package, and differential gene analysis was performed using DESeq2 within the software package. Compared with WT Treg cells, 320 genes were upregulated in KO Treg cells, including Hspa1a, Hspa1b, Igfbp4, cd7, Il7r, etc. 534 genes were downregulated, including Foxp3, Mki67, Ccr6, histone H3K4 methyltransferase genes Setd1a, Setd1b, Kmt2a, Kmt2b, Kmt2c, Kmt2d, etc. ( Figure 4 A); Heatmap showed that Treg cell marker genes including Il2rα, Foxp3, Ikzf2, Malt1 were enriched in WT Treg cells; costimulatory molecules (Cd28, Icos, etc.) and activation-related genes (such as Nr4a1, Cd44, etc.) were also enriched in WT Treg cells; while resting-related genes such as Sell, Il7r, cd7, Klf2, S1pr1, etc. were enriched in KO Treg cells. In addition, some genes crucial for the inhibitory function of Treg cells such as Ctla4, Entpd1, Tigit, Lag3 were also enriched in WT Treg cells ( Figure 4 B). Further gene ontology (GO) analysis was performed on the differentially expressed genes of the two groups of Treg cells. The results showed that compared with WT Treg cells, the upregulated genes in KO Treg cells were enriched in pathways related to ribosome synthesis, mitochondrial gene expression, ATP biosynthesis, oxidative phosphorylation, etc. The downregulated genes in KO Treg cells were enriched in pathways related to histone modification, especially histone H3K4 methylation, small G protein-mediated signal transduction, lymphocyte activation, chromatin localization, etc. Figure 4C); Further RT-qPCR results showed that histone H3K4 methyltransferase genes Kmt2a, Kmt2c, and Kmt2d were significantly downregulated in KO Treg cells ( Figure 4 D).

[0046] Example 5: BCL6 inhibitor FX1 combined with PD-1 / PD-L1 antibody treatment experiment

[0047] 1. Mouse Oral Squamous Cell Carcinoma Subcutaneous Tumor Model

[0048] In this example, 6-8 week old mice were subcutaneously inoculated with 1×10 6 SCC7 cells were used to closely observe the tumor growth of mice after inoculation. After the tumor was measurable, the longest diameter (a) and shortest diameter (b) of the tumor were measured with a vernier caliper and recorded. The measurement was performed once a day. The tumor volume was calculated using the formula: V = a × b 2 / 2, draw the mouse tumor growth curve.

[0049] 2. BCL6 inhibitor FX1 treatment experiment

[0050] Prepare the required experimental concentration of FX1 according to the manufacturer's instructions. Construct a subcutaneous tumor model of oral cancer in KO mice and WT mice according to the above subcutaneous tumor model construction method. When the mouse tumor grows to about 5×5mm in size, the mice are randomly divided into groups. The FX1 treatment group is intraperitoneally injected with the experimental dose of FX1 once a day for 8 consecutive days, and the control group is intraperitoneally injected with an equal volume of solvent. Monitor tumor growth and draw a tumor growth curve.

[0051] 2. BCL6 inhibitor FX1 combined with PD-1 / PD-L1 antibody treatment experiment

[0052] FX1 was configured and a subcutaneous tumor model of head and neck squamous cell carcinoma in mice was constructed according to the above method. When the mouse tumor grew to a size of about 5×5 mm, the KO mice and WT mice were randomly divided into 4 groups: control group, anti-PDL1 group, FX1 group, and FX1+anti-PDL1 combined treatment group; the FX1 group and the control group were treated as above; the anti-PDL1 group was administered once on the 2nd, 4th, 6th, and 8th days of the FX1 administration cycle, with each mouse intraperitoneally injected with 150 μg of anti-PDL1 each time; the FX1+anti-PDL1 combined treatment group was administered with the addition of anti-PDL1 on the basis of the FX1 group ( Figure 5A). Monitor tumor growth and plot the tumor growth curve. The results showed that compared with the control group, the tumor growth of mice in the FX1 group, anti-PDL1 group, and combination therapy group was significantly slowed down. The tumor growth in the combination therapy group was significantly slower than that in the anti-PDL1 group, indicating that FX1 could enhance the efficacy of anti-PDL1. The tumor growth in the combination therapy group was also slower than that in the FX1 group, although there was no statistical difference. Figure 5 B). At the observation endpoint, the tumor weight in the combination therapy group was the lightest and that in the control group was the heaviest. The combination therapy group had better tumor control efficacy than anti-PDL1 alone. Figure 5 C).

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the specification of the present invention.

Claims

1. Application of targeting BCL6 to regulate Treg cells in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: Methods targeting BCL6 include the use of BCL6 inhibitors.

3. The use according to claim 2, characterized in that: The BCL6 inhibitor reduces the inhibitory effect of Treg cells on CD8+T cells by reducing the methylation level of histone H3K4 in Treg cells.

4. The use according to claim 2, characterized in that: The BCL6 inhibitors include but are not limited to FX1.

5. The use according to claim 4, characterized in that: The administration method of the drug includes but is not limited to intraperitoneal injection.

6. The use according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. Targeting BCL6 to regulate Treg cells and combining them with immune checkpoint blockers in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The immune checkpoint blockers include but are not limited to one or more of PD1 antibodies and PDL1 antibodies.

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