Use of inhibitors targeting bcl-2 in the preparation of a medicament for modulating tumor immune escape

Abstract

This invention discloses the application of BCL-2-targeting inhibitors in the preparation of drugs for regulating tumor immune escape, relating to the field of biomedical technology. In immunocompetent C57BL / 6 mice and immunodeficient nude mouse tumor models, this invention demonstrated that veneclade significantly inhibited LLC and MC38 tumor growth in C57BL / 6 mice, while this effect was significantly weakened in nude mice, indicating that it exerts its anti-tumor effect dependent on a complete adaptive immune system. Flow cytometry analysis showed that veneclade effectively reduced the proportion of regulatory T cells, myeloid-derived suppressor cells, and macrophages in tumor tissue and spleen, while increasing the infiltration and killing function of CD8+ T cells, thereby reshaping the tumor immune microenvironment and inhibiting tumor immune escape. This invention reveals a novel mechanism by which veneclade reverses the tumor immunosuppressive environment, providing a scientific basis for its combination with immunotherapies such as PD-1 inhibitors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of BCL-2-targeting inhibitors in the preparation of drugs for regulating tumor immune escape. Background Technology

[0002] Malignant tumors have become a major disease threatening human health, with lung cancer and colorectal cancer being the most common solid tumors in clinical practice. During the occurrence and development of tumors, immune escape mechanisms enable tumor cells to evade the recognition and killing of the body's immune system, which is an important reason for the continuous growth, metastasis, and development of drug resistance in tumors.

[0003] Mounting evidence suggests that the regulatory network for tumor immune escape is extremely complex, involving not only immune checkpoint molecules but also metabolic reprogramming, epigenetic modifications, cytokine networks, and various immunosuppressive cell populations (such as regulatory T cells and myeloid-derived suppressor cells) within the tumor microenvironment. This implies that intervening in these processes to weaken immune escape may represent a novel strategy for cancer treatment.

[0004] ABT199 (Veneclax), a highly selective BCL-2 inhibitor, has been approved for the treatment of hematologic malignancies such as chronic lymphocytic leukemia. Its core mechanism of action is to directly induce apoptosis in BCL-2-dependent tumor cells.

[0005] Current technologies mainly focus on the direct cytotoxic effects of Veneclare. However, there is a lack of systematic research and clear mechanistic elucidation regarding whether and how it affects the tumor immune microenvironment of solid tumors. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide the application of a BCL-2-targeting inhibitor in the preparation of a drug for regulating tumor immune escape; and the application of Veneclare in combination with a PD-1 inhibitor in the preparation of an anti-tumor drug.

[0007] This invention selects C57BL / 6 mice and nude mice as experimental animals, and constructs a tumor-bearing mouse model subcutaneously on the back of the experimental animals; when the tumor volume of the tumor-bearing mice reaches 70-100 mm... 3In this study, tumor-bearing mice were randomly divided into two groups: a control group and an ABT199 monotherapy group. Subcutaneous tumor volume and body weight were measured every 3 days. Based on the measured data, tumor growth curves and survival curves were plotted to evaluate the anti-tumor effect of BCL-2 targeting. Lymphocytes were isolated from subcutaneous tumor tissue and spleen tissue of C57BL / 6 tumor-bearing mice, and the proportion of various immune cell subsets was analyzed using flow cytometry. This invention demonstrates that targeting BCL-2 can effectively reshape the tumor immune microenvironment, reduce the proportion of regulatory T cells, MDSCs, and macrophages, and increase cytotoxic T cell infiltration and function, thereby stimulating an effective systemic anti-tumor immune response and exerting a powerful anti-tumor effect.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides the use of an inhibitor targeting BCL-2 in the preparation of a drug for regulating tumor immune escape.

[0009] Furthermore, the regulation of tumor immune escape is achieved by reshaping the tumor immune microenvironment.

[0010] Furthermore, the remodeling of the tumor immune microenvironment includes reducing the proportion of immunosuppressive cells in tumor tissue and / or spleen tissue, wherein the immunosuppressive cells are selected from at least one of regulatory T cells, myeloid-derived suppressor cells, and macrophages.

[0011] Furthermore, the remodeling of the tumor immune microenvironment includes increasing the number of infiltrating CD8+ T cells and / or their killing function in tumor tissue.

[0012] Furthermore, the inhibitor targeting BCL-2 is Venecla.

[0013] Furthermore, the drug is administered orally at a dose of 50-150 mg / kg.

[0014] Furthermore, the tumor is a solid tumor.

[0015] Furthermore, the solid tumor is lung cancer or colorectal cancer.

[0016] In a second aspect, the present invention provides the use of Veneclare in combination with a PD-1 inhibitor in the preparation of an antitumor drug, characterized in that the Veneclare is used to regulate tumor immune escape to enhance the antitumor efficacy of the PD-1 inhibitor, wherein the tumor is a solid tumor.

[0017] In a third aspect, the present invention also provides a method for screening candidate drugs that exert anti-tumor effects by regulating tumor immune escape, comprising the following steps: a) The candidate drug was administered to both immunocompetent tumor-bearing mouse models and immunodeficient tumor-bearing mouse models. b) Compare the antitumor effects of the candidate drug in the two models. If the antitumor effect of the candidate drug is significantly better in the immune-intact model than in the immune-deficient model, it suggests that the candidate drug relies on the adaptive immune system to exert its antitumor effect. c) Analyze tumor-infiltrating lymphocyte subsets in a healthy immune model after treatment with candidate drugs to assess the remodeling effect of candidate drugs on the tumor immune microenvironment.

[0018] Further, in step c), the assessment includes detecting changes in the proportion and function of at least one cell subset among regulatory T cells, myeloid-derived suppressor cells, macrophages, and CD8+ T cells.

[0019] The beneficial effects of this invention include at least the following: (1) This invention discloses for the first time a new use of Veneclax to exert anti-tumor effects by regulating tumor immune escape, breaking through the existing technology’s limited understanding that Veneclax is only used as a BCL-2 inhibitor to directly induce tumor cell apoptosis, thus expanding its new anti-tumor application scenarios.

[0020] (2) Through comparative experiments between C57BL / 6 mice and nude mouse tumor-bearing models, this invention first demonstrated that the anti-tumor effect of Venecra depends on the body's complete adaptive immune system, revealing that its mechanism of action is closely related to immune regulation, and providing an important basis for the precise use and indication selection of Venecra.

[0021] (3) This invention confirms that Veneclare can effectively reshape the tumor immune microenvironment, specifically by significantly reducing the proportion of immunosuppressive cells such as regulatory T cells, myeloid-derived suppressor cells and macrophages in tumor tissue and spleen, while increasing the number of infiltrating CD8+ T cells and their killing function, thereby reversing the tumor immune escape state at multiple targets and levels, overcoming the limitation of existing small molecule compounds that only act on a single target.

[0022] (4) This invention provides a scientific basis and experimental support for the combined use of Venecra and immune checkpoint inhibitors such as PD-1 inhibitors. It is expected to improve the response rate of immunotherapy, overcome primary and acquired drug resistance, and thus improve the overall prognosis of cancer patients through combined use. Attached Figure Description

[0023] Figure 1The antitumor effect of the BCL-2 inhibitor ABT199 in different animal models. (A) Schematic diagram of ABT199 administration method in mouse subcutaneous tumor model. Tumor growth curves (B), mouse survival curves (C), and single tumor growth curves (D) in the control group and ABT199 monotherapy group of the LLC subcutaneous tumor model of C57BL / 6 mice. Tumor growth curves (E), mouse survival curves (F), and single tumor growth curves (G) in the control group and ABT199 monotherapy group of the MC38 subcutaneous tumor model of C57BL / 6 mice. Tumor growth curves (H), mouse survival curves (I), and single tumor growth curves (J) in the control group and ABT199 monotherapy group of the LLC subcutaneous tumor model of HJ. Tumor growth curves (K), mouse survival curves (L), and single tumor growth curves (M) in the control group and ABT199 monotherapy group of the MC38 subcutaneous tumor model of nude mice.

[0024] Figure 2 The effects of the BCL-2 inhibitor ABT199 on the tumor immune microenvironment in mice. (A) The proportion of Tregs in tumors after ABT199 treatment. (B) The proportion of CD8-positive T cells to CD4+ T cells in the spleen after ABT199 treatment. The proportion of MDSCs in tumors (C) and spleen (D) after ABT199 treatment. The proportion of macrophages in tumors (E) and spleen (F) after ABT199 treatment. The proportion of B lymphocytes in tumors (G) and spleen (H) after ABT199 treatment. The proportion of dendritic cells in tumors (I) and spleen (J) after ABT199 treatment.

[0025] Figure 3 Effects of the BCL-2 inhibitor ABT199 on the infiltration and function of tumor-infiltrating CD8+ T cells. (A) Number of CD8+ T cells in tumors after ABT199 treatment. (B) Expression of perforin and granulamycin B in CD8+ T cells in tumors after ABT199 treatment.

[0026] Figure 4 The tumor-suppressive effect of the BCL-2 inhibitor ABT199 in a patient-derived tumor explant model. (A) Schematic diagram of a patient-derived tumor explant model; (B) Number of cleaved-caspase-3 positive (apoptotic) cells after treatment of tumor explants with anti-CD3 / CD28 and ABT199. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] The following specific embodiments illustrate the solution proposed in this invention: Example 1 Step 1: Constructing a tumor-bearing mouse model: C57BL / 6 mice aged 6-8 weeks and weighing 20-22g and nude mice aged 6-8 weeks and weighing 20-22g were selected as experimental animals. A tumor-bearing mouse model was constructed subcutaneously on the back of the experimental animals. The tumor cells used were the mouse lung cancer cell line LLC and the mouse colon cancer cell line MC38. Step 2: Group processing: When the tumor volume in tumor-bearing mice reaches 70-100 mm 3 At that time, tumor-bearing mice were randomly divided into a control group and an ABT199 single-drug group, with no fewer than 5 mice in each group; Step 3: Physical Fitness Test The subcutaneous tumor volume of the tumor-bearing mice was measured every 3 days. The subcutaneous tumor volume was measured by measuring the longest and shortest diameters with calipers and calculated according to the formula: tumor volume = (length × width 2) / 2. Step 4: Record: Based on the measured data, mouse tumor growth curves and mouse survival curves were plotted to evaluate the anti-tumor effect. Step 5: Immune microenvironment analysis: Subcutaneous tumors and spleen tissues were isolated from C57BL / 6 tumor-bearing mice. Lymphocytes were purified by Fillcoll density gradient centrifugation, and the distribution of immune cell subsets in the subcutaneous tumors and spleen tissues was analyzed by flow cytometry. Step Six: Explant Model Validation: Using a patient-derived explant model of tumor, immunohistochemical staining with anti-Cleaved caspase-3 antibody was used to evaluate the effect of BCL-2 targeting combined with anti-PD-1 neutralizing antibody on the function of intratumoral effector T cells.

[0030] The detailed experimental method is as follows: 1. Establishment of tumor-bearing mouse model (1) Preparation of tumor cell suspension 1) Expand LLC and MC38 cells that are in good condition and in the logarithmic growth phase to a sufficient number of cells; 2) Cell passage method: Digest tumor cells into cell suspension and resuspend in PBS to remove complete culture medium components from the suspension; 3) Take 20 μl of cell suspension to calculate the cell concentration, and calculate the total number of cells according to 1*10^6 tumor cells per mouse. Then, use PBS to adjust the cell concentration to 5*10^6 / ml. 4) Place the tumor suspension in an ice box to ensure its cell viability and prepare for further tumor implantation.

[0031] (2) Implantation of subcutaneous tumors in mice 1) The skin of the planned tumor implantation area on the back of C57BL / 6 mice needs to be shaved off in advance with a razor (nude mice do not need to be shaved). Care should be taken to avoid shaving the skin during the operation. At the same time, ear tag clamps should be used to number the mice for subsequent operation records. 2) Mix the prepared tumor cell suspension by blowing or inverting, and draw up the cell suspension with a 1ml insulin injection. 3) Remove the prepared mouse, hold and fix the mouse to expose the skin at the tumor site, and disinfect the skin at the tumor site with iodine. 4) Insulin was injected subcutaneously into mice, 200 μl per mouse, to form a wheal; 5) Slowly rotate the needle to remove it, avoiding leakage; 6) Return the mouse to its cage and record the contents; 2. Grouping and treatment of tumor-bearing mice, and measurement of subcutaneous tumor volume in mice. (1) When the tumor volume reaches 70-100 mm 3 At that time, the tumor-bearing mice were randomly divided into groups to ensure that the initial differences in tumor volume were not statistically significant. (2) Divided into two groups: Control group: The same volume of sterile water was administered by gavage for 5 days, followed by a 2-day pause, for a total of no more than 2 cycles; Veneclare (ABT199) monotherapy group: ABT199 100mg / kg was administered by gavage for 5 days, followed by a 2-day pause, for a total of no more than 2 cycles; (3) After capturing the mouse, use a vernier caliper to measure the length and width of the subcutaneous tumor of the mouse. Calculate the tumor volume according to the formula = (length × width 2) / 2. Record the changes in tumor volume every 2-3 days. (4) When the tumor volume reaches 2000 mm 3 The mice were euthanized and the results were recorded. (5) Plot the mouse tumor growth curve and mouse survival curve based on the recorded data; 3. Analysis of the mouse immune microenvironment (1) Purification of subcutaneous tumor-infiltrating lymphocytes 1) Obtain mouse subcutaneous tumor tissue in an SPF-grade animal room. After removing necrotic tumor tissue and tumor capsule with sterile forceps and scissors, place the tissue into a centrifuge tube containing PBS buffer. 2) Place the tumor tissue block from the centrifuge tube into a culture dish containing 3 ml of 1×PBS buffer, and repeatedly cut it into pieces approximately 2 mm in size using a blade. 3 Fragments; 3) Add an appropriate amount of PBS buffer, aspirate the shredded tissue pieces and PBS buffer onto a 70µm sterile filter screen and grind them, then rinse and filter repeatedly, and collect the tissue filtrate into a 15ml centrifuge tube; 4) Centrifuge the above centrifuge tubes at 600g for 10 minutes in a horizontal low-speed centrifuge; 5) After centrifugation, remove the supernatant and resuspend the cell pellet in 4 ml of mouse lymphocyte separation medium. Then, while maintaining liquid separation, slowly add 2 ml of 1640 culture medium along the tube wall; 6) Set the centrifuge to slow deceleration mode, place the centrifuge tube in a horizontal low-speed centrifuge and centrifuge at 850g for 30 minutes. After centrifugation, mouse lymphocytes can be seen to be enriched in the white intermediate layer. 7) Carefully aspirate the intermediate layer containing mouse lymphocytes into a new centrifuge tube. Dilute with an appropriate volume of PBS buffer, mix well, discard some of the liquid for cell counting, and centrifuge the remaining cell suspension at 600g for 10 minutes. Then discard the supernatant, resuspend the cell pellet in 1640 complete culture medium or 1×PBS buffer, and use it for flow cytometry staining.

[0032] (2) Purification of spleen-infiltrating lymphocytes 1) Mouse spleens were obtained in an SPF-grade animal facility and ground in a six-well plate containing 3 ml of PBS; 2) Filter using a 70µm filter screen, transfer the lower filtrate to a 15ml centrifuge tube, centrifuge horizontally at 2000rpm for 5 min, and discard the supernatant; 3) Add 2 ml of erythrocyte lysis buffer to each tube, let stand at room temperature for 5 min, and add 12 ml of 1% FBS-PBS to stop erythrocyte lysis; 4) Centrifuge at 2000 rpm for 5 minutes; 5) Resuspend each group in 110 μl of PBS (take out 10 μl for counting), and use 100 μl per flow cytometry tube for flow cytometry staining.

[0033] (3) Flow cytometry staining of cell surface 1) Taking the staining of tumor-infiltrating lymphocytes as an example, after counting, take 5×10⁻⁶ cells. 5 Lymphocytes were suspended in PBS buffer and then centrifuged at 400g for 10 minutes in a low-temperature centrifuge. 2) Carefully aspirate the supernatant with a pipette, resuspend the cell pellet with 100 μl of PBS buffer, add 0.1 μl of Fixed Viability Dye eFluor™ 780 dye to each group, and stain at 4°C for 30 minutes or at room temperature for 15 minutes in the dark. 3) After staining for both live and dead dyes, add 400 μl of PBS buffer to a centrifuge tube, invert and mix to wash the live and dead dyes, and then centrifuge at 400g for 10 minutes in a low-temperature centrifuge. 4) Carefully aspirate the supernatant with a pipette, add an appropriate amount of flow cytometry surface antibody to PBS buffer, then take 100 μl of the above liquid and resuspend the cell pellet by pipetting, and stain at 4°C for 30 minutes or at room temperature for 15 minutes under the dark. 5) After surface staining, add 400 μl of PBS buffer to a centrifuge tube, invert to mix and wash the surface antibody by flow cytometry, and then centrifuge at 400g for 10 minutes in a low-temperature centrifuge. 6) Carefully aspirate the supernatant with a pipette, add 500 μl of PBS buffer, resuspend the cell pellet by pipetting, and then perform flow cytometry or subsequent intracellular flow cytometry staining.

[0034] (4) Intracellular flow cytometry staining 1) Prepare the fixative in advance. Mix the concentrated fixative and the diluent at a ratio of 1:3 according to the instructions. Store the prepared solution away from light. 2) After the flow cytometry staining of the cell surface is completed and rinsed, carefully aspirate the supernatant with a pipette, add 200-250 μl of fixative to each group, resuspend the cell pellet by pipetting, and then fix overnight at 4°C in the dark. 3) Pre-cool the centrifuge beforehand. After fixing the cells overnight, centrifuge at 400g for 10 minutes in a low-temperature centrifuge. 4) Prepare the cell disruption solution in advance by mixing 10x cell disruption solution concentrate with pure water at a ratio of 1:9. After centrifugation, carefully remove the fixative using a pipette. Add 500 μl of cell disruption solution to each group to rinse the cell pellet, and then centrifuge for 10 minutes at 400g. 5) Add an appropriate volume of flow cytometry antibody to the membrane permeation solution and mix well. Carefully aspirate the supernatant with a pipette. Add 100 µl of membrane permeation solution containing flow cytometry antibody to each group to resuspend the cell pellet. Then incubate at 4°C for 30-60 minutes in the dark. 6) After incubation, add 400µl of PBS buffer to each group, invert and mix to wash the intracellular antibody by flow cytometry, and then centrifuge at 400g for 10 minutes in a low-temperature centrifuge. 7) Carefully aspirate the supernatant with a pipette, resuspend the cell pellet with 500 μl of PBS buffer, and store at 4°C in the dark. Note that flow cytometry should be performed within 3 days after staining.

[0035] 4. Tumor explant models derived from lung cancer patients (1) Biopsy section of human lung cancer 1) Obtain tumors from non-small cell lung cancer patients in the operating room, remove non-tumor tissue, tumor capsule and necrotic tumor tissue, place the tumor tissue in centrifuge tubes containing DMEM complete culture medium and transport at low temperature; rinse the obtained fresh tumor tissue twice with sterile PBS in a laminar flow hood, and cut it into square tissue blocks with a blade for subsequent experiments. 2) Clean the vibratory microtome, pour in pre-cooled sterile PBS containing double antibiotics, and fill the surrounding area with ice to maintain a low temperature. 3) Set the speed of the vibratory microtome to 1, the amplitude to 2 mm, and the thickness to 300-400 μm. After attaching the blade, start slicing, and after completion, place the slices directly into a well plate containing pre-cooled sterile PBS.

[0036] 4) After disinfecting the well plate containing tissue sections with alcohol, place it in a laminar flow hood and rinse twice with sterile PBS containing antibiotics. Proceed to the next step of the experiment according to the experimental design.

[0037] (2) Culture and processing of lung cancer tissue samples 1) Rinse the tumor tissue twice with sterile PBS in a laminar flow hood, then cut the tumor tissue into pieces with a volume of approximately 5 mm using a sterile blade. 3 It can be cut into cubes or into 400μm thin slices using a vibratory slicer; 2) Take an appropriate amount of the separated tumor tissue block and put it into a 6-well or 12-well plate. Add 3 ml or 1 ml of DMEM complete culture medium to each well, and then add 5 µM ABT199 or 10 µg / ml of anti-PD-1 neutralizing antibody to each well. 3) Place the above-mentioned well plates in a cell culture incubator for culture, generally for 24-48 hours; 4) After culture, tumor-infiltrating lymphocytes were collected from the patient using the method described above and analyzed by flow cytometry. Simultaneously, thin slices of tumor tissue were fixed with 4% paraformaldehyde for sectioning and immunohistochemical staining.

[0038] 5. Immunohistochemical staining (1) Paraffin embedding of tissue 1) Fix the obtained subcutaneous tumor tissue or patient-derived lung cancer tissue in 4% paraformaldehyde at room temperature for 24-48 hours; 2) After fixation, the tissue was dehydrated stepwise from low to high concentration of alcohol, that is, it was soaked in 75%, 85%, 90%, and 95% alcohol solutions for 2 hours in sequence, then soaked in anhydrous ethanol in the first and second tanks for 40 minutes each, and finally soaked in xylene in the first tank for 10 minutes, xylene in the second tank for 8 minutes, wax in the first tank for 1 hour, and wax in the second tank for 1 hour until it became transparent. 3) The sample should be immersed in liquid paraffin with a melting point of 52-60℃ for at least 2 hours; 4) After the sample has solidified into a block in a -20℃ freezer, trim the wax block. 5) Section and spread the tissue, dry the sections in a slide dryer, mark them, and then proceed to the next step of the experiment.

[0039] (2) Immunohistochemical staining 1) Select tissue sections according to the experimental design and place them in the slide rack. Set the drying temperature to 75℃, then place the slide rack in the drying oven and continue drying for at least 60 minutes. 2) Remove the slide rack from the drying oven and dewax the tissue sections in the following order and time: soak twice in xylene for 15 minutes each time; soak twice in anhydrous ethanol for 5 minutes each time; soak twice in 95% ethanol for 5 minutes each time; and finally soak in 75% ethanol for 5 minutes. 3) Gently rinse the tissue section with tap water for 1 minute, being careful to avoid direct water flow onto the tissue; 4) During the rinsing process, prepare the antigen retrieval solution. Dilute 20×EDTA antigen retrieval solution (pH=9.0) with pure water to a 1× concentration, and perform antigen retrieval treatment in an antigen retrieval pot, ensuring that the tissue sections are completely immersed in the antigen retrieval solution; 5) Carefully remove the slide rack from the antigen retrieval pot and cool it to room temperature in tap water; 6) Shake off the liquid on the slide, draw circles around the tissue with an immunohistochemistry pen, then add 3% hydrogen peroxide solution to cover the tissue, and incubate in a humidified box at room temperature for 10 minutes in the dark to reduce non-specific staining; 7) Shake off the hydrogen peroxide solution on the slides, wash with PBS buffer on a shaker for 3-5 minutes, and repeat this step 3 times; 8) Prepare a 5% BSA solution in advance during the washing process. Then, shake off the liquid on the slides, add the 5% BSA solution dropwise onto the tissue, and incubate in a humidified chamber at room temperature for 30-60 minutes in the dark. 9) Dilute the primary antibody solution of the corresponding concentration with 5% BSA, take an appropriate volume to cover the tissue section, place the section in a humidified chamber, and incubate overnight at 4°C; 10) After the primary antibody has fully bound to the tissue, allow the humidified chamber to warm to room temperature for 30 minutes in preparation for subsequent experiments; 11) Place the slides into a slide holder and wash them on a shaker for 5 minutes with PBS buffer. Repeat this step 3 times. 12) During the washing process, prepare the secondary antibody solution in advance, add an appropriate volume of the secondary antibody solution to cover the tissue, and place it in a humidified chamber to incubate at room temperature in the dark for 30-60 minutes; 13) Shake off the liquid on the slide and wash with PBS buffer on a shaker for 5 minutes. Repeat this step 3 times. 14) During the washing process, prepare the DAB developing solution in advance. It should be prepared fresh and used immediately, and stored away from light. After adding the DAB developing solution to cover the tissue, control the development time carefully, generally 3-5 minutes. Then, place the section in tap water to stop the development process. 15) Next, rinse the slide with tap water for 1 minute, being careful to avoid direct water flow onto the tissue; 16) Immerse the sections in hematoxylin for 5-10 minutes, then rinse with tap water for 1 minute, being careful to avoid direct water flow onto the tissue; 17) Immerse the slices in 1% hydrochloric acid alcohol for 3-4 seconds, turning them from side to side during the immersion process, then rinse with tap water for 1 minute, while preheating warm water. 18) Place the slices in warm water for 30 seconds to allow them to re-blue. 19) Perform dehydration treatment in the following order and time: soak in 75% ethanol for 5 minutes, soak in 95% ethanol for 5 minutes, soak in anhydrous ethanol for 5 minutes, soak in anhydrous ethanol for 5 minutes, soak in xylene for 15 minutes, soak in xylene for 15 minutes; 20) After dehydration, place the slicing rack and slices in a fume hood to air dry for at least 2 hours; 21) After the sections are fully air-dried, mount them with neutral resin and coverslips. After mounting, let them stand at room temperature for 1 day, then store them in a cool place or under an optical microscope for observation, photography, and storage. Then, use software to analyze the immunohistochemical results.

[0040] 6. Statistical Analysis Methods Statistical analyses were performed using GraphPad Prism 8.0 (San Diego, USA). All results are expressed as mean ± standard deviation (SD). Differences were compared using independent t-tests (Student's test), one-way ANOVA, or two-way ANOVA, depending on the specific circumstances. The Kaplan-Meier method was used for animal survival analysis. A p-value < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0041] 7. Experimental Results C57BL / 6 mice are immunocompetent mice, while nude mice are adaptively immunodeficient mice lacking a thymus. To preliminarily explore the anti-tumor efficacy of BCL-2 targeting, this example used LLC (mouse lung cancer cell line) and MC38 (mouse colorectal cancer cell line) tumor cell lines to construct tumor-bearing mouse models in C57BL / 6 mice and nude mice, respectively. Patients were grouped and treated according to control and BCL-2-targeted single-drug groups (veneclade, ABT199), and treatment data were recorded. Figure 1 A). The results showed that in the C57BL / 6 tumor-bearing mouse model, ABT199 monotherapy significantly inhibited the growth of LLC and MC38 tumors (A). Figure 1 B-1G), and this anti-tumor effect was significantly inhibited in a nude mouse tumor-bearing model ( Figure 1 H-1M). The above results demonstrate that the drug ABT199, which targets BCL-2, can exert an effective anti-tumor effect by relying on the body's intact adaptive immune system.

[0042] Table 1. Mouse tumor volume growth data 1

[0043] Table 2. Data on the growth trend of mouse tumor volume 2

[0044] To further explore the mechanism, this invention analyzed the immune microenvironment of mice in different groups of the C57BL / 6 MC38 tumor-bearing model. Analysis using lymphocyte isolation and flow cytometry revealed that Treg cells in the subcutaneous tumor tissue and spleen tissue of tumor-bearing mice treated with ABT199 (…) Figure 2 A, 2B), MDSCs ( Figure 2 C, 2D), macrophages ( Figure 2 The proportions of E and 2F cells were significantly reduced, while those of B cells ( Figure 2 G, 2H), DC cells ( Figure 2 The proportions of I and 2J showed no significant change. These results demonstrate that the drug ABT199, which targets BCL-2, can effectively reduce the distribution of immunosuppression-related cells and reshape the immune microenvironment.

[0045] To further verify the mechanism, this invention further analyzed the infiltrative number and function of CD8+ T cells in subcutaneous tumor tissue of the C57BL / 6 mouse MC38 tumor-bearing model. Immunohistochemical staining results of CD8+ in the subcutaneous tumor tissue suggested that ABT199 significantly promoted the infiltrative number of CD8+ T cells (…). Figure 3 A), flow cytometry results indicated that ABT199 can enhance the killing function of tumor-infiltrating CD8+ T cells (A), Figure 3 B).

[0046] Furthermore, this invention utilizes tumor tissue from untreated lung cancer patients to construct a patient-derived explant model. Figure 4 A), under in vitro TCR stimulation, the expression level of cleaved caspase-3 in tissues treated with ABT199 was analyzed to assess tumor apoptosis. Figure 4 (B) to demonstrate the effect of ABT199 in promoting anti-tumor immunity.

[0047] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0048] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of BCL-2-targeting inhibitors in the preparation of drugs for regulating tumor immune escape.

2. The application according to claim 1, characterized in that, The regulation of tumor immune escape is achieved by reshaping the tumor immune microenvironment.

3. The application according to claim 2, characterized in that, The remodeling of the tumor immune microenvironment includes reducing the proportion of immunosuppressive cells in tumor tissue and / or spleen tissue, wherein the immunosuppressive cells are selected from at least one of regulatory T cells, myeloid-derived suppressor cells, and macrophages.

4. The application according to claim 2, characterized in that, The remodeling of the tumor immune microenvironment includes increasing the number of infiltrating CD8+ T cells and / or their killing function in tumor tissue.

5. The application according to claim 1, characterized in that, The inhibitor targeting BCL-2 is Veneclare.

6. The application according to claim 5, characterized in that, The drug is administered orally at a dose of 50-150 mg / kg.

7. The application according to any one of claims 1-6, characterized in that, The tumor is a solid tumor.

8. The application according to claim 7, characterized in that, The solid tumor is either lung cancer or colorectal cancer.

9. The application of veneclade in combination with a PD-1 inhibitor in the preparation of antitumor drugs, characterized in that, The Veneclare is used to regulate tumor immune escape to enhance the anti-tumor efficacy of PD-1 inhibitors, wherein the tumor is a solid tumor.

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