Application of combination of mitochondrial uncoupling agents BAM15 and CART in tumor treatment and preparation

Abstract

The invention provides application of combination of a mitochondrial uncoupling agent BAM15 and a mitochondrial uncoupling agent CART in tumor treatment and a preparation, and relates to the technical field of biology. The invention provides an application of a mitochondrial uncoupling agent BAM15 combined with CART in preparation of a medicine for treating tumors. According to the combined use scheme of the BAM15 and the CD7CAR-T cells, the targeted tumor killing effect of the CD7CAR-T can be enhanced, meanwhile, release of cell factors in the CD7CAR-T treatment process can be relieved, infiltration of inflammatory cells can be reduced, and the BAM15 and CD7CAR-T cells have high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application and preparation of a mitochondrial uncoupler BAM15 combined with CART in treating tumors. Background Art

[0002] Acute lymphoblastic leukemia / lymphocytic lymphoma (T-ALL / LBL) originates from hematopoietic stem and progenitor cells and is a group of heterogeneous, aggressive T-cell malignancies characterized by the proliferation and accumulation of primitive, immature lymphocytes with a T-cell phenotype. However, many T-ALL patients still have difficulty in achieving remission through chemotherapy or relapse after remission. For relapsed patients, treatment options are still limited, with a poor prognosis and relapse / refractory (R / R).

[0003] Chimeric antigen receptor (CAR) T cell therapy is one of the more mature cellular immunotherapies. Currently, many CART products targeting B-cell tumors, such as CD19CART and BCMA-CART, have shown a high complete remission rate, most of which are complete remissions with minimal residual disease (MRD) negative. CART based on antigen targets such as CD7 and CD5 has now overcome the barriers caused by the shared targets of tumor T cells and normal T cells, and technically realized the possibility of preparing CART targeting T-cell tumors. CD7CART, which is currently in clinical trials, has demonstrated effective anti-tumor effects. It provides a means of CART for the treatment of T-cell malignancies, so that more patients can benefit.

[0004] Although CAR-T cell therapy has made significant progress in hematological malignancies, it still faces many challenges. First, common side effects during treatment include cytokine release syndrome (CRS) and neurotoxicity. Second, treatment resistance and tumor antigen escape cause some patients to relapse after treatment. The preparation process of CAR-T cells is complicated and expensive, which limits its widespread application. In addition, CART has poor therapeutic effects on solid tumors, and the immunosuppressive effects of the tumor microenvironment further weaken the function of CAR-T cells. Individual differences among patients also lead to inconsistent treatment responses, and its long-term effects and durability still need to be verified.

[0005] In order to solve the serious problems of CRS and tumor immune escape in the reality of CART use, the current clinical methods first monitor the patient's vital signs and cytokine levels in real time, which can identify the signs of CRS early and intervene. When signs of CRS are found, drug intervention such as IL-6 antagonists (such as tocilizumab) and IL-6 receptor antagonists (such as siltuximab) have been shown to effectively relieve CRS symptoms. In addition, cell engineering optimization, such as designing a "safety switch" or regulatory CAR-T cells for CAR-T cells, can induce cell apoptosis through exogenous drugs or mechanisms when CRS occurs, reducing side effects. Combined immunomodulators such as immune checkpoint inhibitors and immunosuppressants (such as cyclosporine A) also help balance the immune response and reduce over-activation. And by regulating the tumor microenvironment, improving T cell function and inhibiting the recruitment of immunosuppressive cells, the occurrence of CRS can be further reduced. However, real-time monitoring in these methods causes patients to take multiple blood samples and occupy medical resources, and the use of drugs cannot be intervened in time, and even these methods cannot fight against the immune escape of tumor cells from CART. Therefore, it is necessary to find a method to both attenuate CRAT cytokine release and enhance CART targeted killing.

[0006] BAM15 is a mitochondrial uncoupler. Mitochondrial uncouplers are a class of chemicals that can disrupt the proton gradient of the inner mitochondrial membrane, thereby inhibiting ATP synthesis and causing metabolism to shift to heat generation. They uncouple oxidative phosphorylation from ATP synthesis, resulting in energy consumption to generate heat. Uncouplers have shown potential application value in many fields such as cancer immunotherapy, metabolic diseases, and neurodegenerative diseases.

[0007] No technical solution for the combined use of BAM15 and CD7CAR-T cells has been found in the prior art. Summary of the invention

[0008] In order to solve the above problems, the present invention provides the use and preparation of mitochondrial uncoupler BAM15 in combination with CART in the treatment of tumors.

[0009] On the one hand, the present invention provides the use of the mitochondrial uncoupler BAM15 combined with CART in the preparation of drugs for treating tumors.

[0010] Specifically, the tumor includes solid tumors and non-solid tumors.

[0011] Furthermore, the tumors include acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, esophageal cancer, liver cancer, lung cancer, gastric cancer, intestinal cancer, breast cancer, prostate cancer, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, and melanoma.

[0012] On the other hand, the present invention provides the use of a mitochondrial uncoupler BAM15 in combination with CART in the preparation of a drug for improving the killing ability of CART cells.

[0013] On the other hand, the present invention provides the use of a mitochondrial uncoupler BAM15 in combination with CART in the preparation of a drug for treating cytokine release induced by CART therapy.

[0014] Specifically, in the above-mentioned drug, the cell amount of the CART cells is (1-5)×10 6 indivual;

[0015] Furthermore, the cell amount of the CART cells is 1.5×10 6 indivual.

[0016] Specifically, in the above-mentioned drugs, based on a 1:1 effector-target ratio of CART cells to tumor cells, the added concentration of the mitochondrial uncoupler BAM15 is 0.5 μM-5 μM;

[0017] Furthermore, based on a 1:1 effector-target ratio of CART cells to tumor cells, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-5 μM;

[0018] Furthermore, based on a CART cell to tumor cell effector-target ratio of 1:1, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-2.5 μM.

[0019] Specifically, in the above-mentioned drugs, based on a CART cell to tumor cell effector target ratio of 1:2, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-5 μM;

[0020] Furthermore, based on a CART cell to tumor cell effector-target ratio of 1:2, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-2.5 μM.

[0021] Specifically, the cytokines include but are not limited to IL-1β, IL-4, IL-6, IL-10, IL-12, IL-21, TNF-α, and IFN-γ-GZMB.

[0022] Specifically, the above-mentioned CART cells are CD19CART cells, BCMACART cells, CD5CART cells, CD20CART cells, CD22CART cells, CD23CART cells, CD30CART cells, CD33CART cells, CD37CART cells, CD38CART cells, CD43CART cells, and CD78CART cells.

[0023] Furthermore, the above-mentioned CART cells are CD7CART cells.

[0024] Specifically, the above-mentioned medicine may also include a pharmaceutically acceptable carrier.

[0025] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0026] Specifically, the dosage forms of the drug include oral preparations and injections.

[0027] Furthermore, the oral preparation includes tablets, capsules, granules and powders.

[0028] Furthermore, the injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intracavitary injection.

[0029] On the other hand, the present invention provides a combination therapeutic drug, which includes a mitochondrial uncoupler BAM15 and CART cells.

[0030] Specifically, the cell amount of the CART cells is (1-5)×10 6 indivual;

[0031] Furthermore, the cell amount of the CART cells is 1.5×10 6 indivual.

[0032] Specifically, based on the human dose, the amount of the CART cells is 5×10 5 / kg-5×10 6 / kg.

[0033] Specifically, based on a 1:1 effector-target ratio of CART cells to tumor cells, the added concentration of the mitochondrial uncoupler BAM15 is 0.5 μM-5 μM;

[0034] Furthermore, based on a 1:1 effector-target ratio of CART cells to tumor cells, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-5 μM;

[0035] Furthermore, based on a CART cell to tumor cell effector-target ratio of 1:1, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-2.5 μM.

[0036] Specifically, based on a CART cell to tumor cell effector-target ratio of 1:2, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-5 μM;

[0037] Furthermore, based on a CART cell to tumor cell effector-target ratio of 1:2, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-2.5 μM.

[0038] The beneficial effects of the present invention are:

[0039] The present invention provides a scheme for the combined use of BAM15 and CD7CAR-T cells, which can enhance the targeted tumor killing effect of CD7CAR-T, while reducing the release of cytokines during CD7CAR-T treatment and the infiltration of inflammatory cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the half inhibitory concentration of BAM15 on CCRF-CEM cells and Raji cells.

[0041] Figure 2 The figure shows the effect of different concentrations of BAM15 on CD7CAR-T targeted killing at different effector-target ratios.

[0042] Figure 3 This is a graph showing the effect of BAM15 on the release of cytokines when CD7CAR-T targets and kills tumor cells.

[0043] Figure 4 Bioluminescence imaging of mice in each group.

[0044] Figure 5 Figure 2 shows the levels of cytokine release in mice in different treatment groups at different time points.

[0045] Figure 6 HE staining images of lung pathology of mice in each group.

[0046] Figure 7 HE staining images of liver pathology of mice in each group.

[0047] Figure 8 Schematic diagram of the gradient dilution of the standard. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0049] The manufacturer and product number of the experimental materials:

[0050] BAM15 was purchased from MCE Company with the catalog number HY-110284;

[0051] CD7CAR-T cells were prepared by Beijing Sanlikang Cell Technology Co., Ltd.

[0052] Example 1

[0053] (1) In vitro co-culture of mitochondrial uncouplers with tumor cell lines and CD7CART

[0054] 1. Preparation of separation buffer: PBS buffer containing 0.5% human albumin and 2mM EDTA, which requires 48.5ml PBS, 1.25ml 20% human albumin, and 0.2ml 0.5M EDTA. Refrigerate at 0-4℃ and ensure that it is still in a low temperature state when used to ensure cell activity.

[0055] 2. Dissolve BAM15 powder in DMSO to obtain a 10 mM concentration of BAM15.

[0056] 3. Take the CD7-overexpressing Raji cell line (CD7-Raji) in culture and add 1×10 4 Cells were plated in 24-well plates.

[0057] 4. Take the CD7CAR-T cells in culture or the frozen and revived CD7CAR-T cells, and add the corresponding number of CD7CAR-T cells to the cell culture plate at the ratio of effector to target of 2:1, 1:1, 1:2, and 1:4.

[0058] 5. 10mM BAM15 was gradiently diluted and added to the co-culture system of CD7CAR-T targeted killing of tumor cells, so that the concentration of BAM15 in the system was 5μM, 2μM, 1μM, 0.5μM, 0.2μM, and 0.1μM, respectively. After 48h of co-culture with the tumor cell line, the fluorescence value was detected by an ELISA instrument. The culture supernatant at each concentration gradient was collected before detection, marked, and frozen for subsequent cytokine detection.

[0059] (2) Experimental methods

[0060] This test was validated by in vitro cell experiments and in vivo mouse tumor models.

[0061] In vivo experiments were performed using 24 female NSG mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.), weighing 18g-22g, 6 mice in each group, and the mouse model experiment used BAM15 at a concentration of 1 mg / kg body weight. The NSG mice were injected with 1.5×10 6 CD7-Raji-luc cells were given on day 5: PBS treatment group 200ul / mouse, CD7CAR-T cells (1.5×10 6 ) treatment group, BAM15 (1 mg / kg) + CD7CAR-T cells (1.5×10 6 ) treatment group and BAM15 (1 mg / kg) alone treatment group. The amount of CD7CAR-T cells in the experiment was 1.5×10 6 indivual.

[0062] 2.1 In vitro cytokine detection

[0063] Experimental purpose: To verify the effect of BAM15 on cytokine release during CD7CART targeted killing

[0064] The experiment used Luminex200 for high-throughput liquid protein chip kit detection to detect multiple cytokines IFN-γ, TNF-α, IL-17, IL-6, IL-10, IL-21, IL-12, IL-15, and granzyme B.

[0065] a. Dilute 10× Wash Buffer (Wash Buffer) into 1× Wash Buffer (Wash Buffer) at a ratio of 1:9 by adding deionized water for use in subsequent steps.

[0066] b. Take out the standard, centrifuge at 2000g for 10s, add 50μL of universal detection buffer (Universal detection buffer) to the standard tube, mix gently for 30s and place on ice for 5-10min; mix the standards into 1 tube, add universal detection buffer (Universal detection buffer) to finally obtain 250μL of mixed standards.

[0067] c. Perform gradient dilution of the standard ( Figure 8), take out the PCR 8-tube strip from the kit to dilute the standard, add 200 μL of the mixed standard to the first tube as standard 1, add 150 μL of universal detection buffer to tubes 2-8 respectively, take 50 μL of the mixed standard from tube 1 and add it to tube 2, pipette up and down 10 times to mix, try to avoid the generation of bubbles, replace a new pipette tip, draw 50 μL of the diluted standard from tube 2 and transfer it to tube 3, pipette up and down 10 times to mix, transfer in sequence to complete the gradient dilution of the mixed standard; put it on ice for later use.

[0068] d. Wash the microspheres, take out the premixed 1× beads, vortex the microspheres for 30 seconds, add 50μL of premixed microspheres to each well of the 96-well plate, place the 96-well plate in the magnetic separation plate, make sure the well plate is firmly stuck, let it stand for 2 minutes, let the microspheres sink to the bottom, then quickly invert the magnetic plate and pour out the liquid in the well plate. Do not remove the 96-well plate from the magnetic separation plate during this process. Add 150μL of washing buffer to each well, let it stand for 30 seconds, then invert the magnetic plate and pour out the liquid in the well plate; in the inverted state, use a paper towel to absorb the residual liquid on the surface of the well plate.

[0069] e. Add samples for incubation. Add 50 μL of standard or sample to the corresponding wells, add 50 μL of universal detection buffer to the blank control, seal the well plate, shake and incubate at room temperature at 500 rpm for 30 minutes, and leave it at 4°C overnight. Take it out the next day and shake and incubate at room temperature at 500 rpm for 30 minutes.

[0070] f. Remove the incubation plate, place the 96-well plate in the magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid liquid splashing, remove the liquid in the well plate by inverting it, add 150μL of washing buffer to each well, let it stand for 30 seconds, and remove the liquid in the well plate by inverting it. Repeat the steps and wash 3 times in total. At the end of the last wash, absorb the residual liquid with a paper towel.

[0071] g. Add detection antibody, take out 1× detection antibody from the kit, add 25 μL 1× detection antibody to each well, seal the well plate with a new sealing film, take out the 96-well plate from the magnetic separation plate, and place it in a well plate shaker at 500 rpm at room temperature for 30 minutes.

[0072] h. Wash the incubation plate, place the 96-well plate in the magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid liquid splashing, remove the liquid in the well plate by inverting, add 150 μL of washing buffer to each well, let it stand for 30 seconds, remove the liquid in the well plate by inverting, wash 3 times in total, and absorb the residual liquid with a paper towel at the end of the last wash.

[0073] i. Add 50 μL of SA-PE to each well, seal the well plate with a new sealing film, remove the 96-well plate from the magnetic separation plate, and place it in a well plate shaker at 500 rpm at room temperature for 30 min.

[0074] j. Place the 96-well plate in a magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid splashing the liquid, and remove the liquid in the well plate by inverting it. Add 150 μL of washing buffer to each well, let it stand for 30 seconds, remove the liquid in the well plate by inverting it, and wash it 3 times in total. At the end of the last wash, absorb the residual liquid with a paper towel.

[0075] k. Add 120 μL of Reading Buffer to each well, seal the plate with a new sealing film, remove the 96-well plate from the magnetic separation plate, place it in a plate shaker at 500 rpm for 5 minutes at room temperature, gently remove the sealing film, and place it in the Luminex 200 instrument for reading. Use a five-parameter nonlinear regression method to fit the standard curve and calculate the concentration value of each cytokine.

[0076] 2.2 Animal experiments

[0077] Purpose of the experiment: To evaluate the synergistic effect of BAM15 on CAR-T targeted killing and explore its effect on cytokine release syndrome (CRS).

[0078] 2.2.1 Laboratory Animal Husbandry and Quarantine

[0079] 28 SPF female NOD / SCID IL2RGnull (NSG) mice (including 4 spare animals), 6-8 weeks old, weighing 18-22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (Animal Production License No.: SCXK (Beijing) 2024-0001). The experimental animals were received, quarantined in situ, adapted to the environment, and abnormal animals (if any) were handled in accordance with the "Rodent Reception and Quarantine" (SOPT002) of this institution. The quarantine period included at least 3 days on the day of receipt, and the animals were weighed on the day of receipt and at the end of the quarantine period.

[0080] The animals were kept in the A113 breeding room of the barrier facility of Beijing Boruishian Technology Co., Ltd. (License Number for the Use of Experimental Animals: SYXK (Beijing) 2022-0025), with ≤5 animals per cage, the temperature of the breeding room was 20℃-26℃, the humidity was 30%-70%, and they were fed with SPF-grade rat and mouse maintenance feed (Ke'ao Xieli (Tianjin) Feed Co., Ltd., Animal Feed Production License Number: SCXK (Tianjin) 2020-0004), free access to animal drinking water (121℃, 30 minutes autoclaved reverse osmosis water), and the bedding was autoclaved (121℃, 30 minutes) corn cob bedding (Beijing Ke'ao Xieli Feed Co., Ltd., Animal Bedding Production License Number: SCXK (Beijing) 2024-0010). The experimental animal breeding environment and facilities meet the relevant requirements of GB14925-2023.

[0081] 2.2.2 Test methods

[0082] 2.2.2.1 Tumor cell preparation and inoculation

[0083] Raji-Luc human Burkitt's lymphoma cells were cultured in RPMI-1640 medium (containing 10% FBS, 100U / mL penicillin, 100μg / mL streptomycin) in a 37°C, 5% CO2 incubator. The medium was usually changed every 2-3 days. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and fresh medium was added for subculture.

[0084] CD7-Raji-Luc cells in logarithmic growth phase, 1.5×10 6 The cells were washed, centrifuged and resuspended in PBS to a final cell concentration of 7.5 × 10 6 cells / mL, a total of 5.0 ml, slowly injected intravenously at 0.2 mL / animal (disinfected with alcohol cotton balls), press the injection site with dry cotton balls to stop bleeding after the injection, and then return the animal to the breeding cage for breeding.

[0085] 2.2.2.2 Animal grouping

[0086] The injection of CD7-Raji-Luc cells was recorded as Day 0. On Day 3, the fluorescence intensity of tumor cells was measured using a small animal in vivo imaging system. When the fluorescence signal reached 1×10 6 -1×10 7p / s, the mice were randomly divided into 4 groups according to the intensity of tumor fluorescence signal and combined with body weight, with 6 mice in each group, and the vehicle or test substance was given at the beginning; the average body weight difference between groups after the animals were grouped did not exceed ±20%. The groups were distinguished by animal cage cards, and the tail numbers were used to identify the animals during the quarantine period and the test period after grouping. After the grouping and the end of the test, the animals that did not enter the test were handled according to the relevant standard operating procedures, and the "Experimental Animal Transfer Record" was filled in. Animal grouping and dosage information are shown in Table 1.

[0087] Table 1 Animal groups and doses

[0088]

[0089] Note: PBS was injected intraperitoneally once, with a volume of 0.2 mL / mouse; CAR-T cells were injected intravenously once, with a volume of 0.2 mL / mouse; BAM15 was injected intraperitoneally multiple times, with a volume of 0.3 mL / mouse / time. IP means intraperitoneal injection, and IV means intravenous injection.

[0090] 2.2.2.3 Route, cycle and frequency of administration of test substance

[0091] Administration route and method: CAR-T cells are administered by intravenous injection, and BAM15 is administered by intraperitoneal injection.

[0092] Administration cycle and frequency: CAR-T cells were administered once; BAM15 was administered once every 3 days (in the first treatment of group G3, CAR-T cells were injected intravenously and then BAM15 was injected intraperitoneally), for a total of 5-7 times.

[0093] Dosage: See Table 1.

[0094] 2.2.2.4 Observation indicators and methods

[0095] 1) Clinical symptom observation

[0096] Observe at least once a day, record whether there are any abnormalities in the animal's appearance, nutrition, fur, skin, mucous membrane, mental behavior, head, breathing, digestion, urogenital, and other aspects. If there are any abnormalities, increase the number of observations. Record the animal's survival status every day until the end of the experiment, and draw an animal survival curve.

[0097] 2) Body weight measurement

[0098] Body weight was measured every 2 days.

[0099] 3) Temperature detection

[0100] The rectal temperature of the animals was measured before the first administration of CAR-T and 1, 2, 6, 12, and 24 hours after administration, and once a day for the rest of the time.

[0101] 4) Determination of tumor cell fluorescence signal intensity

[0102] Raji-Luc cells were inoculated on D3, 7, 11, 15, 19, and 23, and each animal was intraperitoneally injected with 0.2 mL of luciferase substrate (15 mg / mL). After isoflurane inhalation anesthesia, in vivo fluorescence imaging was performed using a small animal imaging system to measure the fluorescence signal intensity.

[0103] 5) Cytokines

[0104] 2, 6, 12, and 24 hours after CAR-T administration, the animals were anesthetized by carbon dioxide inhalation, and blood was collected from the orbital venous plexus (for G1, blood was collected from 6 mice at one time at the 2nd hour, for G2, 3, and 4 groups, blood was collected from 4 mice in each group at the 2nd hour (the first 4 mice), and blood was collected from another 4 mice at 6 hours (the last 4 mice), the first 4 mice at 12 hours, and the last 4 mice at 24 hours. Blood was collected alternately in this way to ensure that no less than 50 μL of serum could be collected from each mouse), the serum was separated and stored at -80°C, and the number was recorded. After completion, the samples were used to determine the concentrations of IL-1, IL-4, IL-6, IL-10, IL-12p70, TNF-α, and IFN-γ using Luminex.

[0105] 6) Histopathology

[0106] The liver, kidney and lung tissues of mice were taken and fully fixed with 10% neutral formalin solution, and then HE histopathological staining analysis was performed.

[0107] 7) Handling of dead or dying animals

[0108] For animals in danger of death, record their status, symptoms and duration, and notify the veterinarian to determine whether to continue the experiment or perform euthanasia.

[0109] After measuring the body weight of dead or dying (euthanasia confirmed) animals, the experimenters will perform autopsies on the dead or dying animals, observe tissue and organ lesions with the naked eye, and fix and preserve organs that have changed in size, color, texture, etc. for histopathological examination.

[0110] When a necropsy cannot be performed in time, the dead animal should be refrigerated and necropsy should be performed as soon as possible.

[0111] 2.2.3 Experimental data processing and statistical analysis

[0112] The measurement data were expressed as mean ± standard deviation, and SPSS 19.0 or higher software was used for statistical analysis. One-way ANOVA was used to compare the measurement data. The variance homogeneity test was performed before analysis. When the variance was homogeneous, the LSD method was used for inter-group comparison analysis. When the variance was unequal, Dunnett's T3 post hoc multiple comparison was used. The statistical significance level of the two-sided test was set at p < 0.05. No statistical analysis was performed on the data collected when the animals were unplanned euthanasia and when the sample number (n) was less than 3.

[0113] 3 Pathological experiments

[0114] Experimental purpose: To verify the effect of BAM15 on the attenuation of CAR-T proliferation. Pathological examination experimental steps: Preparation of histopathological sections of mouse lungs and livers

[0115] a. The mice that died or were killed in the experiment were dissected, and the lung and liver tissues of the mice were removed, trimmed and fixed with formalin.

[0116] b. Fix mouse tissues with 10% neutral formalin for 12-24 hours to ensure that the tissues are completely fixed. Cut the fixed tissues into small pieces of 1-2 mm thick and dehydrate them in 85% ethanol for 3 min, 90% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol I for 10 min, and 100% ethanol II for 10 min, respectively, until the tissues are completely dehydrated.

[0117] c. Soak the tissue sections in xylene I for 10 minutes to remove the ethanol in the tissue and make the tissue transparent.

[0118] d. Immerse the transparent tissue in molten 60°C paraffin for 1 hour to ensure that the tissue is completely infiltrated with paraffin. Then take out the tissue soaked in wax and embed it in paraffin. After the paraffin cools down, use a microtome to cut the embedded tissue into 3μm thick slices. Put the cut paraffin slices in a water bath, wait for them to float, and transfer them to a slide to prepare for the subsequent operation steps.

[0119] e. Soak the tissue slices in xylene I again for 10 minutes, and soak the tissue slices in xylene I for 5 minutes to remove the paraffin. Hydrate the slices in 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, and 85% ethanol for 3 minutes to gradually restore the moisture of the tissue. Finally, rinse the slices with deionized water to remove ethanol and other residues to ensure that the slices are clean.

[0120] f. Place the slices in hematoxylin staining solution for 5 minutes until the tissue turns blue, rinse the slices with tap water for 3-5 minutes to remove excess dye, then place the slices in 1% hydrochloric acid alcohol for 10 seconds, and rinse with tap water for 1-3 minutes to promote pigment removal.

[0121] g. Eosin staining: soak the sections in acidified eosin ethanol solution for 5 seconds, and gently rinse the sections with tap water to remove excess dye.

[0122] h. Dehydrate the slices again by soaking in 95% ethanol for 5 min, 100% ethanol I for 5 min, and 100% ethanol II for 5 min, then soak in xylene I for 5 min and xylene II for 3 min to make the tissue transparent. Finally, add neutral gum to the coverslip and let the coverslip dry naturally for 1 h. Use LEICA DM3000 microscopic imaging system to observe and videotape.

[0123] 4. In vivo cytokine release assay

[0124] Experimental purpose: In vivo verification of the effect of BAM15 on cytokine release during CD7CART targeted killing

[0125] The experiment used Luminex200 for high-throughput liquid protein chip kit detection to detect multiple cytokines IFN-γ, TNF-α, IL-1, IL-4, IL-6, IL-10, and IL-12.

[0126] a. Dilute 10× Wash Buffer by adding deionized water at a ratio of 1:9 to 1× Wash Buffer for use in subsequent steps.

[0127] b. Take out the standard, centrifuge at 2000g for 10s, add 50μL of universal detection buffer to the standard tube, mix gently for 30s and place on ice for 5-10min; mix the standards into 1 tube, add universal detection buffer, and finally obtain 250μL of mixed standards.

[0128] c. Perform gradient dilution of the standard ( Figure 8 ), take out the PCR 8-tube strip from the kit to dilute the standard, add 200 μL of the mixed standard to the first tube as standard 1, add 150 μL of universal detection buffer to tubes 2-8 respectively, take 50 μL of the mixed standard from tube 1 and add it to tube 2, pipette up and down 10 times to mix, try to avoid the generation of bubbles, replace a new pipette tip, draw 50 μL of the diluted standard from tube 2 and transfer it to tube 3, pipette up and down 10 times to mix, transfer in sequence to complete the gradient dilution of the mixed standard; put it on ice for later use.

[0129] d. Wash the microspheres, take out the premixed 1×Beads, vortex the microspheres for 30 seconds, add 50μL of premixed microspheres to each well of the 96-well plate, place the 96-well plate in the magnetic separation plate, make sure the well plate is firmly stuck, let it stand for 2 minutes, let the microspheres sink to the bottom, then quickly invert the magnetic plate and pour out the liquid in the well plate. Do not remove the 96-well plate from the magnetic separation plate during this process. Add 150μL of washing buffer to each well, let it stand for 30 seconds, then invert the magnetic plate and pour out the liquid in the well plate; in the inverted state, use a paper towel to absorb the residual liquid on the surface of the well plate.

[0130] e. Add samples for incubation. Add 50 μL of standard or sample to the corresponding wells, add 50 μL of universal detection buffer to the blank control, seal the well plate, shake and incubate at room temperature at 500 rpm for 30 minutes, and leave it at 4°C overnight. Take it out the next day and shake and incubate at room temperature at 500 rpm for 30 minutes.

[0131] f. Remove the incubation plate, place the 96-well plate in the magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid liquid splashing, remove the liquid in the well plate by inverting it, add 150μL of washing buffer to each well, let it stand for 30 seconds, and remove the liquid in the well plate by inverting it. Repeat the steps and wash 3 times in total. At the end of the last wash, absorb the residual liquid with a paper towel.

[0132] g. Add detection antibody, take out 1× detection antibody from the kit, add 25 μL 1× detection antibody to each well, seal the well plate with a new sealing film, take out the 96-well plate from the magnetic separation plate, and place it in a well plate shaker at 500 rpm at room temperature for 30 minutes.

[0133] h. Wash the incubation plate, place the 96-well plate in the magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid liquid splashing, remove the liquid in the well plate by inverting, add 150 μL of washing buffer to each well, let it stand for 30 seconds, remove the liquid in the well plate by inverting, wash 3 times in total, and absorb the residual liquid with a paper towel at the end of the last wash.

[0134] i. Add 50 μL of SA-PE to each well, seal the well plate with a new sealing film, remove the 96-well plate from the magnetic separation plate, and place it in a well plate shaker at 500 rpm at room temperature for 30 min.

[0135] j. Place the 96-well plate in a magnetic separation plate, let it stand for 2 minutes, gently remove the sealing film to avoid splashing the liquid, and remove the liquid in the well plate by inverting it. Add 150 μL of washing buffer to each well, let it stand for 30 seconds, remove the liquid in the well plate by inverting it, and wash it 3 times in total. At the end of the last wash, absorb the residual liquid with a paper towel.

[0136] k. Add 120 μL of Reading Buffer to each well, seal the plate with a new sealing film, remove the 96-well plate from the magnetic separation plate, place it in a plate shaker at 500 rpm for 5 minutes at room temperature, gently remove the sealing film, and place it in the Luminex 200 instrument for reading. Use a five-parameter nonlinear regression method to fit the standard curve and calculate the concentration value of each cytokine.

[0137] (3) Experimental results

[0138] 1) BAM15 was co-cultured with cell lines in vitro to detect the half-inhibitory concentration of BAM15 on tumor cell lines. The half-inhibitory concentration of BAM15 on CCRF-CEM was 1.101 μM, and the half-inhibitory concentration on CD7Raji cells was 1.976 μM ( Figure 1 ).

[0139] 2) Compared with the BAM15 monotherapy group and the CD7CAR-T cell monotherapy group, when the effect-target ratio was 1:1, 2.5μM and 1μM BAM15 could significantly enhance the killing effect of CAR-T (2.5μM, BAM15 vs BAM15+CD7CAR-T, ****P<0.0001, 1μM, BAM15 vs BAM15+CD7CAR-T, ****P<0.0001). When the effect-target ratio was 1:2, 2.5μM and 1μM BAM15 could also significantly enhance the killing effect of CD7CAR-T (2.5μM, BAM15 vs BAM15+CD7CAR-T, ****P<0.0001, 1μM, BAM15 vs BAM15+CD7CAR-T, ****P<0.0001) ( Figure 2 ).

[0140] 3) The cytokine release when CD7CAR-T cells were killed by 1μM BAM15 with a CD7Raji effector-target ratio of 1:1 was observed. The results showed that compared with the CD7CAR-T cell killing group without BAM15, the cytokines IL-6 (***P<0.001), IL-10 (***P<0.001), IL-21 (****P<0.0001), TNFα (****P<0.0001) and granzyme B (GZMB) (****P<0.0001) were significantly reduced ( Figure 3 ).

[0141] 4) The experimental results showed that the tumor burden of mice in the PBS treatment group and the BAM15 treatment group was not controlled, while the growth of tumors in the CD7CAR-T cell treatment group and the BAM15+CD7CAR-T cell combined treatment group was effectively inhibited ( Figure 4 ).

[0142] 5) Cytokine detection found that the release levels of IL-1β, IL-4, and IL-6 in the BAM15+CD7CAR-T cell group were lower than those in the CD7CAR-T cell treatment group at 2 hours after treatment (2h: CD7CAR-T vs. BAM15+CD7CAR-T, N=5, *P<0.05, **P<0.01); between 6-12 hours after treatment, the release levels of IL-6, IL-10, IL-12, TNF-α, and IFN-γ were reduced (6-12hours: CD7CAR-T vs. BAM15+CD7CAR-T, N=5, *P<0.05, **P<0.01) Figure 5 ). The results showed that BAM15 could reduce cytokine release in response to CD7CAR-T cell therapy.

[0143] 6) Among the four treatment groups, the CD7CAR-T cell treatment group induced inflammatory cell infiltration in the lungs and liver, and the addition of BAM15 could reduce the infiltration of inflammatory cells ( Figure 6-Figure 7 ).

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of mitochondrial uncoupler BAM15 combined with CART in the preparation of drugs for treating tumors.

2. The application of mitochondrial uncoupler BAM15 combined with CART in the preparation of drugs to enhance the killing ability of CART cells.

3. Application of mitochondrial uncoupler BAM15 combined with CART in the preparation of drugs for treating cytokine release induced by CART therapy.

4. The use according to any one of claims 1 to 3, characterized in that: In the drug, the amount of CART cells is (1-5)×10 6 indivual.

5. The use according to any one of claims 1 to 3, characterized in that: In the drug, based on a CART cell to tumor cell effector-target ratio of 1:1, the added concentration of the mitochondrial uncoupler BAM15 is 0.5 μM-5 μM.

6. The use according to any one of claims 1 to 3, characterized in that: In the drug, based on a CART cell to tumor cell effector target ratio of 1:2, the added concentration of the mitochondrial uncoupler BAM15 is 1 μM-5 μM.

7. The use according to any one of claims 1 to 3, characterized in that: The CART cells are CD19CART cells, BCMACART cells, CD5CART cells, CD20CART cells, CD22CART cells, CD23CART cells, CD30CART cells, CD33CART cells, CD37CART cells, CD38CART cells, CD43CART cells or CD78CART cells.

8. The use according to claim 7, characterized in that: The CART cells are CD7CART cells.

9. The use according to any one of claims 1 to 3, characterized in that: The medicine may also include a pharmaceutically acceptable carrier.

10. A combined therapeutic drug, characterized in that: The combined therapeutic drug includes the mitochondrial uncoupler BAM15 and CART cells.

Images