Application of ononin in enhancing car-t cell anti-tumor function

Abstract

The application discloses application of ostruthin in enhancing CAR-T cell anti-tumor function, belongs to the technical field of biological medicine, and the ostruthin can promote cell proliferation, inhibit cell exhaustion, promote the secretion of cytokines related to killing cancer cells, and further enhance the anti-tumor function of CAR-T cells in vivo and in vitro as a cell enhancer of CAR-T cells; experimental results show that when ostruthin is combined with CAR-T cells for anti-tumor treatment, compared with ostruthin or CAR-T cells alone, the combination of the two can significantly inhibit the growth of solid tumors. The application provides a new technical means for improving the tumor treatment effect of CAR-T cell immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of rutin in enhancing the anti-tumor function of CAR-T cells. Background Technology

[0002] Cellular immunotherapy, as a cutting-edge treatment that utilizes the body's immune system to fight cancer, is gradually changing the landscape of cancer treatment. Among them, CAR-T (chimeric antigen receptor T) cell immunotherapy is groundbreaking, demonstrating remarkable efficacy in clinical treatment and bringing new hope to many cancer patients. However, CAR-T cell immunotherapy has some major limitations: First, the tumor microenvironment (TME) of solid tumors is complex and possesses strong immunosuppressive properties, making it difficult for CAR-T cells to effectively infiltrate and attack tumor cells; second, CAR-T cells lack persistence in vivo and are prone to exhaustion during long-term treatment, characterized by decreased proliferation and cytotoxicity, and upregulated expression of inhibitory receptors, mainly induced by continuous stimulation of chronic antigens or tetanic signals of CAR self-aggregation. Furthermore, CAR-T cell immunotherapy is accompanied by serious side effects, such as cytokine release syndrome and neurotoxicity, which limit its widespread application.

[0003] In recent years, natural compounds have attracted widespread attention due to their diverse biological activities and relatively low toxicity. In cancer treatment, many natural compounds have been found to possess multiple effects, including anti-tumor, antioxidant, and immunomodulatory properties. Therefore, exploring new strategies to improve CAR-T cell function through the remodeling of natural compounds is worthy of further research, as enhancing the anti-tumor function of CAR-T cells through natural compounds holds great potential in cancer treatment.

[0004] Orientin is a natural flavonoid compound belonging to the flavonoid C-glycoside family. It is widely found in various medicinal and edible plants, such as bamboo leaves. Phyllostachys spp. Golden lotus ( Trollius Chinensis Bge Passionflower ( Passiflora edulis Saint Basil Ocimum sanctumOrientin, as a flavonoid active ingredient, possesses good chemical stability and biological activity, participating in various physiological and pathological processes by regulating oxidative stress, inflammatory responses, and cell signal transduction. Studies have shown that orientin has significant effects in antioxidation, anti-inflammation, antitumor activity, and immunomodulation, and can influence key signaling pathways such as NF-κB, MAPK, and PI3K / AKT. In the field of tumor research, orientin has received increasing attention due to its potential roles in metabolic regulation, cell proliferation inhibition, and immune function enhancement. Furthermore, orientin also exhibits multiple biological functions, including cardiovascular protection, neuroprotection, and protection against radiation damage. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a strategy to improve CAR-T cell immunotherapy and enhance the anti-tumor function of CAR-T cells by using rutin, aiming to solve the technical problems of insufficient tumor treatment effect caused by immunosuppression and CAR-T cell depletion in CAR-T cell immunotherapy.

[0006] In a first aspect, the present invention provides the application of Orientin in the preparation of CAR-T cell enhancers.

[0007] Specifically, CAR-T cell enhancers are at least one of the following products: CAR-T cell enhancers that promote CAR-T cell proliferation; CAR-T cell enhancers that inhibit CAR-T cell exhaustion and apoptosis; CAR-T cell enhancers that enhance the ability of CAR-T cells to kill cancer cells.

[0008] The above applications include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2.

[0009] Secondly, this invention provides the application of Orientin in the preparation of products that enhance the anti-tumor function of CAR-T cells. These products can be pharmaceuticals, health supplements, food, etc., and enhance the anti-tumor function of CAR-T cells through at least the following mechanisms: Promote CAR-T cell proliferation; Inhibit CAR-T cell exhaustion; Promote the secretion of CAR-T cells by cytokines that kill cancer cells; Enhance the ability of CAR-T cells to kill tumor cells.

[0010] In the above applications, CAR-T cells include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2; tumor cells include, but are not limited to, B-cell acute lymphoblastic leukemia cells and other tumor cells.

[0011] The experiments of this invention demonstrate that appropriate amounts of Orientin can promote CAR-T cell proliferation in vitro, promote the secretion of cytokines related to cancer cell killing in CAR-T cells, enhance the killing ability of CAR-T cells against tumor cells, and inhibit CAR-T cell depletion. Similarly, administering appropriate amounts of Orientin in combination with CAR-T cells to experimental animals significantly inhibits tumor development in vivo. Therefore, Orientin can enhance the anti-tumor function of CAR-T cells. Given its mechanism of action, when products using Orientin as the active ingredient are used to enhance the anti-tumor function of CAR-T cells, they can either be directly processed to obtain cell products with stronger anti-tumor capabilities, or they can be added separately after CAR-T cells have acted on tumor cells.

[0012] Thirdly, this invention provides the application of Orientin in combination with CAR-T cells in the preparation of anti-tumor drugs.

[0013] In the above applications, in addition to the two active ingredients, Orientin and CAR-T cells, the drug may also contain other active ingredients or pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients refer to pharmaceutical excipients widely used in the pharmaceutical manufacturing field, specifically one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, and flavoring agents.

[0014] In the above applications, CAR-T cells include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2; the tumor is preferably a solid tumor, including but not limited to ovarian cancer, lung cancer, colorectal cancer, and other solid tumors.

[0015] In the above applications, there are no specific restrictions on the method of administration of the obtained drug, which can be oral, subcutaneous injection, intravenous injection, intramuscular injection, etc.

[0016] Tumor treatment drugs prepared according to the above applications are also within the scope of protection of this invention.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to demonstrate that Orientin can enhance the anti-tumor function of CAR-T cells, effectively solving the problem of limited anti-tumor function caused by the loss of effector function and depletion of CAR-T cells in cell immunotherapy. Experiments show that when using CAR-T cells for anti-tumor treatment, administering an appropriate amount of Orientin can significantly inhibit tumor growth, and the two have a significant synergistic effect. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0019] Figure 1 This is the structural formula of Orientin used in this invention and a diagram illustrating the mechanism by which Orientin enhances the anti-tumor function of CAR-T cells. Figure 2 This is a graph showing the detection results of Orientin promoting the in vitro proliferation of CAR-T cells in Example 1 of the present invention; Figure 3 The image shows the detection results of different concentrations of Orientin promoting the killing of tumor cells by CAR-T cells for 24 hours in Example 2 of the present invention. A shows the result of Orientin promoting the killing of CD19 CAR-T cells, B shows the result of Orientin promoting the killing of MSLN CAR-T cells, and C shows the result of Orientin promoting the killing of CD30 CAR-T cells. Figure 4 The following graph shows the detection results of Orientin promoting CAR-T cell killing of tumor cells at different effector-target ratios in Example 2 of the present invention. A shows the result of Orientin promoting CD19 CAR-T cell killing, B shows the result of Orientin promoting MSLN CAR-T cell killing, and C shows the result of Orientin promoting CD30 CAR-T cell killing. Figure 5 This is a graph showing the detection results of Orientin promoting the killing of tumor cells by various CAR-T cells over 24 hours under different effector-to-target ratio gradients in Example 3 of the present invention. Figure 6This is a graph showing the detection results of Orientin promoting cytokine secretion in CAR-T cells in Example 4 of the present invention; Figure 7 This is a graph showing the detection results of Orientin inhibiting CAR-T cell exhaustion in Example 5 of the present invention; Figure 8 This is a schematic diagram of the animal experiment process in Embodiment 6 of the present invention; Figure 9 This is a graph showing the detection results of the combined in vivo effect of Orientin and MSLN CAR-T to inhibit tumor development in Example 6 of the present invention; Explanation of reference numerals in the attached figures: This means P ≤ 0.0001. This means P ≤ 0.001. This means P ≤ 0.01. P ≤ 0.05, ns indicates no significant difference. Detailed Implementation

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0021] CAR-T cell immunotherapy offers new hope for cancer treatment. This therapy utilizes genetic engineering to transform T cells into CAR-T cells, which are then cultured in vitro to expand their numbers before being reinfused into the human body. CAR-T cells can specifically recognize tumor cells in the body and release a large number of effector factors through immune responses, thereby efficiently killing tumor cells and achieving the goal of treating tumors. However, the insufficient persistence of CAR-T cells in vivo limits the therapeutic effect of CAR-T cell immunotherapy. To address this technical problem, this invention provides a strategy for enhancing the in vivo anti-tumor function of CAR-T cells using orientin, offering a new approach to improving CAR-T cell immunotherapy.

[0022] like Figure 1As shown, Orientin can promote CAR-T cell proliferation, inhibit CAR-T cell depletion, promote the secretion of cytokines related to killing cancer cells by CAR-T cells, and enhance the killing ability of CAR-T cells against tumor cells, thereby achieving the goal of enhancing the anti-tumor function of CAR-T cells. Therefore, Orientin can be used to develop products as CAR-T cell enhancers and to improve the anti-tumor function of CAR-T cells.

[0023] In some embodiments of the present invention, the CAR-T cells are selected from CD19 CAR-T cells, CD20 CAR-T cells, CD22 CAR-T cells, BCMA CAR-T cells, CD30 CAR-T cells, CD70 CAR-T cells, CLDN18.2 CAR-T cells, MSLN CAR-T cells, GPC3 CAR-T cells, Epcam CAR-T cells, EGFRvIII CAR-T cells, and HER2 CAR-T cells, all of which have significant improvement effects.

[0024] Some embodiments of the present invention also employ Orientin and CAR-T cells in combination to treat mouse tumors, and experimental results show that the two have a significant synergistic effect in anti-tumor treatment and can significantly inhibit tumor development, and are expected to be developed into a combination drug.

[0025] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0026] Example 1 This example tested the effect of orientin on the in vitro proliferation of CAR-T cells. The specific experiment is as follows: (1) Isolation of human peripheral blood mononuclear cells (PBMCs) and preparation of CAR-T cells.

[0027] Add 15 ml of human peripheral blood lymphocyte separation medium to a 50 ml conical centrifuge tube; dilute the peripheral blood with PBS by 1:1, and slowly add 15 ml of the diluted peripheral blood along the tube wall to an equal volume of lymphocyte separation medium. Centrifuge at 700 g for 20 min; after centrifugation, collect the lymphocyte layer in the middle white membrane layer, transfer the lymphocytes to a new 50 ml centrifuge tube, add PBS to a total volume of 40 ml, centrifuge at 600 g for 5 min, and discard the supernatant. PBMC cells are then obtained.

[0028] After dispersing the cell clumps, add 30 ml of PBS, mix well, and sample for counting. Centrifuge at 400 g for 4 min, and aspirate the supernatant using a pipette; repeat every 5 × 10⁻⁶ cells. 7 Resuspend each PBMC cell in 90 μl of AutoMACS Running Buffer, add 20 μl of CD3MicroBeads, mix well, incubate at 4°C for 20 min, sort CD3+ cells into a new collection tube using a magnetic rack, resuspend in 30 ml of PBS for counting, centrifuge at 500 g for 4 min, and you will get CD3 positive T cells.

[0029] Resuspend the cell pellet in the prepared T-cell-specific culture medium, adjust the density to 5e6~1e7 / ml, add 1 / 100 of Enceed™ T cell Activation human, and transfer to a culture flask or plate. Simultaneously add CD3 / CD28 Dynabeads and incubate at 37°C with 5% CO2. This is designated as day 0 of CAR-T cell culture. After 24 hours, transfect the T cells with CD19 virus to obtain CD19 CAR-T cells.

[0030] (2) Culture of CAR-T cells.

[0031] Prepare CAR-T cell activation medium (containing 913 mL of serum-free basal medium for OptiVitroPT cells, 8 mL of serum-free medium supplemented with T cells, and 1000 IU / mL of recombinant human IL-2 protein), and then place the transfected CAR-T cells at a ratio of 2 × 10⁻⁶ cells / mL. 6 CAR-T cells were seeded at a density of 100 cells / ml in CAR-T cell activation medium and cultured in a cell culture incubator at 37°C and 5% CO2. The cells were passaged every 2-3 days, with a passage density of 1-2 × 10⁻⁶ cells / ml. 6 Cells were expanded and cultured for about 12 days.

[0032] (3) Orientin treatment of CAR-T cells.

[0033] CAR-T cells were seeded into 6-well cell culture plates, 2 × 10⁶ cells per well. 6 Cells, 2 mL culture medium; add 10 μM Orientin to each well of the experimental group, and add an equal volume of DMSO to each well of the control group, mix well. Place in a cell culture incubator and count cells every 48 hours, then passage once.

[0034] The proliferation assay results of CAR-T cells after treatment with Orientin are as follows: Figure 2 As shown, this indicates that Orientin can significantly promote the in vitro proliferation of CAR-T cells.

[0035] Example 2 This case tested the effect of Orientin on the ability of CAR-T cells to kill tumor cells.

[0036] Taking CD19 CAR-T cells and Raji-Luci tumor cells as examples, the killing experiment is as follows: Raji-Luci tumor cell culture: cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO2, and 90% relative humidity, and passaged once every 2-3 days.

[0037] Raji-Luci tumor cells were aliquoted into 96-well plates, 20,000 cells per well, with 100 μL of culture medium per well. CD19 CAR-T cells were also aliquoted into 96-well plates, with 10,000, 4,000, 2,000, and 1,000 effective CD19 CAR-T cells added to each well according to effector-to-target ratios of 1:2, 1:5, 1:10, and 1:20, respectively, along with 100 μL of culture medium, and mixed thoroughly with the Raji-Luci tumor cells. 2 μM, 5 μM, 10 μM, or 20 μM of Orientin were added to the wells, while an equal volume of DMSO was added to the control group. The plates were incubated for 24 hours. 2.5 μL of sodium fluorescein was added, and the plates were incubated for 10 min. Fluorescence intensity was read using a microplate reader at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The number of surviving Raji-Luci tumor cells was calculated by comparing the fluorescence intensity ratio with the control group.

[0038] The results of the in vitro killing experiment of CD19 CAR-T cells against Raji-Luci tumor cells are as follows: Figure 3 As shown in Figure A, different concentrations of Orientin at an effector-to-target ratio of 1:5 significantly enhanced the killing ability of CD19 CAR-T cells against Raji-Luci tumor cells.

[0039] Referring to the experiments described above, this example also examined the effects of MSLN CAR-T cells and CD30 CAR-T cells on the killing ability of tumor cells. MSLN CAR-T cells and CD30 CAR-T cells were prepared according to Example 1 and existing techniques, and will not be described again here. Experimental results showed that higher concentrations of Orientin could also significantly promote the killing ability of MSLN CAR-T cells and CD30 CAR-T cells against Raji-Luci tumor cells. Figure 3 B and 3C).

[0040] in addition, Figure 4To compare the killing ability of CAR-T cells against Raji-Luci tumor cells under different effector-to-target ratios, the results showed that treatment with 10 μM Orientin could promote the killing ability of CAR-T cells against tumor cells under different effector-to-target ratios.

[0041] Example 3 Referring to Example 2, this example tested the effects of Orientin (10 μM) at different effector-to-target ratios on the killing ability of CD20 CAR-T cells, CD22 CAR-T cells, BCMA CAR-T cells, CD70 CAR-T cells, CLDN18.2 CAR-T cells, GPC3 CAR-T cells, Epcam CAR-T cells, CD20 CAR-T cells, EGFRvIII CAR-T cells, and HER2 CAR-T cells. The CAR-T cells were prepared according to Example 1 and existing techniques, and will not be described further here.

[0042] Test results as follows Figure 5 As shown, the killing ability of all CAR-T cells was significantly improved under the action of an effective amount of Orientin, indicating that Orientin has universality in improving the anti-tumor ability of CAR-T cells.

[0043] Example 4 This example tested the effect of orientin co-incubation on cytokine secretion in CAR-T cells. The specific experiment is as follows: (1) In vitro co-incubation.

[0044] Raji-Luci cell culture: cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO2, and 90% relative humidity; passaged every 2-3 days.

[0045] CD19 CAR-T cells were counted and distributed into six-well plates, 2 × 10⁶ cells per well. 6 100 cells, 2 mL culture medium; count Raji-Luci cells and divide into 2 × 10⁶ cells per well in a 6-well plate. 5 Mix 1 mL of culture medium with 1 cell and CD19 CAR-T cells. Add Orientin (5 μM or 10 μM) to the wells, and add an equal volume of DMSO to the control group; incubate in a cell culture incubator for 48 h.

[0046] (2) Cell incubation with antibodies (cytokines).

[0047] ① Take 1×10 6 Centrifuge each cell to be tested at 300 g for 3 min and carefully discard the supernatant; ② Take 200 μL of T cell-specific culture medium, add 1% activating magnetic and 5 μg / μL BFA, and incubate in an incubator for 4 h. After 4 h, centrifuge to remove the supernatant, wash twice with PBS, and centrifuge at 400 g for 3 min. ③ After resuspending the cells in 100 μL PBS, divide them into two tubes, each containing 50 μL. Add 1 μL of FITC anti-human SCFV Antibody to each tube and incubate at 4℃ for 30 min. ④ After incubation, centrifuge at 300 g for 3 min and carefully discard the supernatant; ⑤ Cell fixation: 200 μL of fixative per well (to agitate the cells), incubate at room temperature in the dark for 30 min. After fixation, wash once with 200 μL of permeabilizing agent per well, centrifuge at 400 g for 4 min and discard the supernatant. ⑥ Membrane rupture: 200 μL / well of rupture agent, incubate at room temperature in the dark for 30 min, then centrifuge at 700 g for 4 min; ⑦ After the membrane is broken down, resuspend the cells in 50 μL of the membrane breaking agent, add 1 μL of CYTO antibody to each tube, and incubate the antibody at room temperature in the dark for 30 min. ⑧ Resuspend the cells in 1 ml PBS, centrifuge at 700 g for 4 min, carefully discard the supernatant, and resuspend the cells in 300 μL PBS; ⑨ Flow cytometry was used to detect cytokine secretion, and the signal intensity of CYTO-positive cells was analyzed to indicate the killing level of CAR-T cells.

[0048] Test results as follows Figure 6 As shown, Orientin can promote the secretion of cytokines such as GZMB, TNFα, and IFNr in CAR-T cells.

[0049] Example 5 This example tested the effect of orientin on CAR-T cell exhaustion. The specific experiment is as follows: (1) In vitro co-incubation.

[0050] Raji-Luci cell culture: cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S at 37°C, 5% CO2, and 90% relative humidity, and passaged every 2-3 days.

[0051] CD19 CAR-T cells were counted and distributed into six-well plates, 2 × 10⁶ cells per well. 6 100 cells, 2 mL culture medium; count Raji-Luci cells and divide into 2 × 10⁶ cells per well in a 6-well plate. 5Mix 10 cells, 1 mL of culture medium, and CD19 CAR-T cells. Add Orientin (5 μM or 10 μM) to the wells, and add an equal volume of DMSO to the control group; incubate in a cell culture incubator for 24 h.

[0052] (2) Cell incubation of antibodies.

[0053] ① Preparation of cell washing solution: Add 1% FBS to 1×PBS to make cell washing solution. Store at 4℃ and it can be used for one week.

[0054] ② Take 2×10 5 Centrifuge each cell at 300 g for 3 min, carefully discard the supernatant, resuspend the cells in 1 ml of cell washing buffer, divide into two tubes of 50 μL each, add 1 μL of PE anti-human PD1 Antibody and 1 μL of APC anti-human SCFV Antibody to each tube, and incubate at 4 °C for 30 min.

[0055] ③ After incubation, centrifuge at 300 g for 3 min, carefully discard the supernatant, resuspend the cells in 1 ml of cell washing buffer, centrifuge at 300 g for 3 min, carefully discard the supernatant, and resuspend the cells in 300 μL of cell washing buffer.

[0056] ④ Use flow cytometry to detect PE and APC.

[0057] ⑤ By analyzing the signal intensity of PD-1 in APC-positive cells, the level of CAR-T depletion can be indicated.

[0058] Test results as follows Figure 7 As shown, Orientin can inhibit CAR-T cell depletion when co-incubated with CAR-T cells in vitro.

[0059] Example 6 This study used animal experiments to test the effect of Orientin on tumor growth when used in combination with CAR-T cells in vivo.

[0060] Taking MSLN CAR-T cells and OVCAR8 tumors as examples, according to Figure 8 The procedure shown is for conducting animal experiments. The specific steps are as follows: ① Select 6-8 week old male NCG mice and inject them intraperitoneally with Orientin (10 mg / kg, 400 μL) on day 9. The control group was injected with PBS (with the same volume). ② Establishing a tumor model: On day-7, 1×10⁻⁶ tumor cells were subcutaneously implanted in the right back of experimental mice. 6 One mouse ovarian cancer cell line ovcar8; ③ After modeling, monitor tumor size. The tumor burden volume of the experimental mice is 100 mm². 3 MSLN CAR-T cells were injected into tumor-burdened mice via tail vein. ④ During the experiment, Orientin was administered intraperitoneally every two days, while the control group was given PBS with the same volume. ⑤ Measure the size of the tumor with calipers every 2 days to detect tumor growth (tumor volume calculation formula: L×W×W / 2); ⑥ Once the volume of a tumor in a single mouse exceeds 300 mm³, the mice will be euthanized according to animal welfare principles; the tumors will be removed, digested, and the expression of cytokines in tumor-infiltrating T cells will be detected by flow cytometry.

[0061] Test results as follows Figure 9 As shown, the ORI group received only Orientin, the MSLN CAR-T group received only PBS + MSLN CAR-T cells, and the ORI + MSLN CAR-T group received both Orientin and MSLN CAR-T cells. These results indicate that, compared to using Orientin or MSLN CAR-T cells alone, the combination of the two significantly inhibited tumor growth, and Orientin enhanced the anti-tumor ability of CAR-T cells in vivo.

[0062] In summary, Orientin can significantly promote the proliferation of CAR-T cells and their ability to kill tumor cells, while inhibiting cell exhaustion, thereby enhancing the anti-tumor function of CAR-T cells in vitro and in vivo. This is of great significance for the development and clinical application of CAR-T cell immunotherapy products.

[0063] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of rutin in the preparation of CAR-T cell enhancers.

2. The application according to claim 1, characterized in that, The CAR-T cell enhancer is at least one of the following products: CAR-T cell enhancers that promote CAR-T cell proliferation; CAR-T cell enhancers that inhibit CAR-T cell exhaustion and apoptosis; CAR-T cell enhancers that enhance the ability of CAR-T cells to kill cancer cells.

3. The application according to claim 1, characterized in that, The CAR-T cells include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2.

4. Application of rutin in the preparation of products that enhance the anti-tumor function of CAR-T cells.

5. The application according to claim 4, characterized in that, The products include pharmaceuticals, health supplements, and food.

6. The application according to claim 4, characterized in that, The CAR-T cells include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2.

7. Application of rutin combined with CAR-T cells in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 7, characterized in that, The CAR-T cells include, but are not limited to, CAR-T cells targeting CD19, CD20, CD22, BCMA, CD30, CD70, CLDN18.2, MSLN, GD2, GPC3, Epcam, EGFRvIII, or HER2.

10. A tumor treatment drug prepared according to any one of claims 7-9.

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