Application of homovanillic acid in enhancing anti-tumor function of CAR-T (Chimeric Antigen Receptor T) cells
By using hypervanillic acid to promote the enhancement of CAR-T cells' activity and inhibit depletion, the problem of CAR-T cells depletion in anti-tumor treatment was solved, and its killing ability to cancer cells was significantly improved.
Patent Information
- Application Number
- CN202510173390.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-04-29
AI Technical Summary
CAR-T cells are easily depleted during anti-tumor treatment, resulting in reduced proliferation and cytotoxicity, upregulation of inhibitory receptor expression, affecting the therapeutic effect.
The use of vanillic acid promotes the enhancement of CAR-T cells, inhibits their depletion, and improves their killing ability to cancer cells.
Hypervanillic acid significantly promotes the proliferation and killing ability of CAR-T cells in vitro, inhibits cell depletion, and improves the killing efficacy of B-cell acute lympholeukemia cells and other tumor cells.
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Figure CN119896657A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to the application of homovanillic acid in enhancing the anti-tumor function of CAR-T cells. Background Art
[0002] Homovanillic acid (HVA) is a metabolite of dopamine. The human adrenal gland releases catecholamine hormones, including adrenaline, norepinephrine and dopamine. These hormones circulate in the blood, and their metabolites are excreted through urine. Currently, the research on HVA mainly focuses on the field of neuroblastoma. This patent proposes for the first time that homovanillic acid can enhance the anti-tumor function of CAR-T cells.
[0003] Cellular immunotherapy is an innovative treatment method that aims to use the human immune system to eliminate cancer, among which CAR-T cell immunotherapy is revolutionary because it produces very effective and durable clinical responses. However, CAR-T cell exhaustion is a shortcoming in cellular immunotherapy, which is characterized by reduced proliferation and cytotoxicity and upregulated expression of inhibitory receptors, mainly induced by continuous stimulation of chronic antigens or tonic signals of CAR self-aggregation. Therefore, it is worth further studying to explore new strategies to improve CAR-T cell function through metabolite small molecule remodeling. Improving the anti-tumor function of CAR-T cells through small molecules has great potential in cancer treatment. Summary of the invention
[0004] In view of this, the present invention provides an application of homovanillic acid for enhancing the anti-tumor function of CAR-T cells.
[0005] In a first aspect, the present invention provides the effect of homovanillic acid on promoting the enhancement of CAR-T cell activity, wherein the CAR-T cells mainly include primary T cells and cell therapy products based on the transformation of the cells.
[0006] In some embodiments, homovanillic acid can promote in vitro killing of CAR-T cells.
[0007] In some embodiments, homovanillic acid can inhibit the exhaustion of CAR-T cells.
[0008] In a second aspect, the present invention provides that homovanillic acid enhances the ability of CAR-T cells to kill cancer cells, wherein the cancer cells include but are not limited to B cell acute lymphoblastic leukemia cells and other tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0010] Figure 1 Homovanillic acid promotes the proliferation of CAR-T cells in vitro; Figure 2 It indicates that homovanillic acid can inhibit CAR-T cell exhaustion; Figure 3 It indicates that homovanillic acid can promote the killing of B-ALL leukemia cells by CAR-T cells; DETAILED DESCRIPTION
[0011] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0012] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.
[0013] Example 1 Effect of homovanillic acid on the proliferation of CAR-T cells in vitro 1. Isolation of human peripheral blood mononuclear cells (PBMCs) and preparation of CAR-T cells (1) Take 15 ml of human peripheral blood lymphocyte separation medium and add it to a 50 ml conical bottom centrifuge tube. Take 15 ml of diluted cells and slowly add them along the wall of the test tube to an equal volume of lymphocyte separation medium. Centrifuge at 700 g for 20 min. (2) After centrifugation, take the middle white flocculent lymphocyte layer and transfer the lymphocytes to a new 50 ml centrifuge tube. Add PBS to a total volume of 40 ml and centrifuge at 400 g for 10 min to obtain PBMC cells.
[0014] (3) Every 10 7 Add 80 μl AutoMACS Running Buffer to 10 PBMC cells and mix well. 7 Each cell was added with 20 μl CD3 MicroBeads, human and resuspended, incubated at 4°C for 15 min, and CD3+ cells were sorted by magnetic rack.
[0015] (4) Resuspend the cell pellet in the prepared T cell-specific culture medium and transfer it to a culture flask or culture plate for culture. Add CD3 / CD28 Dynabeads at the same time. This is determined as the 0th day of CAR-T cell culture. After 24 hours, transfect CAR to obtain CAR-T cells.
[0016] 2. Cultivation of CAR-T cells (1) Prepare CAR-T cell activation medium: 913 mL of OptiVitroPT cell serum-free basal medium, 8 mL of T cell serum-free medium supplement components, and 1000 IU / mL of recombinant human IL-2 protein.
[0017] (2) The transfected CAR-T cells were divided into 2×10 6 The cells were inoculated at a density of 100 / ml in T medium and placed in a cell culture incubator at 37°C with 5% CO 2 .
[0018] (3) Subculture every 2-3 days, and control the subculture density at 1-2×10 6 The cells were cultured and expanded for about 12 days.
[0019] 3. Treatment of CAR-T cells with homovanillic acid (1) CAR-T cells were seeded into 6-well cell culture plates, with 2×10 cells per well. 6 cells, 2 mL of culture medium; (2) Add 500 nM homovanillic acid to each well and add an equal volume of DMSO to the control group and mix well.
[0020] (3) Place the cells in a cell culture incubator and count and subculture them every 48 hours.
[0021] Example 2: Homovanillic acid promotes CAR-T to kill cancer cells 1. CAR-T cell killing experiment in vitro (1) Raji-luci cell culture: All T-ALL cells described above were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S at 37°C and 5% CO. 2 , cultured in an incubator at a relative humidity of 90%. Subcultured every 2-3 days.
[0022] (2) Count and distribute CAR-T cells into 96-well U-bottom plates, with 20,000 cells per well and 100 μl of culture medium; (3) Count the Raji-Luci cells and place them into a 96-well U-bottom plate, with 5,000 cells per well, 100 μL of culture medium, and mix with CAR-T cells; (4) Add 500 nM homovanillic acid to the above solution, and add an equal volume of DMSO to the control group.
[0023] (5) Place in an incubator and incubate for 24 hours; (6) Add 5 μL of fluorescein sodium salt and incubate in an incubator for 10 min; (7) Use a microplate reader to read the fluorescence intensity at an excitation wavelength of 495 and an emission wavelength of 525; (8) The number of surviving Raji-Luci cells was obtained by calculating the ratio of fluorescence intensity to that of the control group.
[0024] 2. CAR-T cell killing experiment in vitro (1) Raji-luci cell culture: RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S was cultured at 37°C and 5% CO. 2 , cultured in an incubator at a relative humidity of 90%. Subcultured every 2-3 days.
[0025] (2) Count and distribute CAR-T cells into 96-well U-bottom plates, with 20,000 cells per well and 100 μl of culture medium; (3) Count the Raji-Luci cells and place them into a 96-well U-bottom plate, with 5,000 cells per well, 100 μL of culture medium, and mix with CAR-T cells; (4) Add 500 nM homovanillic acid to the above solution, and add an equal volume of DMSO to the control group.
[0026] (5) Place in a cell culture incubator and incubate for 24 hours; (6) Add 5 μL of fluorescein sodium salt and incubate in an incubator for 10 min; (7) Use an ELISA reader to read the Luciferace autoluminescence intensity; (8) The number of surviving Raji-Luci cells was obtained by calculating the ratio of fluorescence intensity to that of the control group.
[0027] Example 3 Homovanillic acid inhibits CAR-T cell exhaustion 1. In vitro co-incubation (1) Raji-luci cell culture: All T-ALL cells described above were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S at 37°C and 5% CO. 2 , cultured in an incubator at a relative humidity of 90%. Subcultured every 2-3 days.
[0028] (2) Counting CAR-T cells was divided into six-well plates, with 2×10 cells per well. 6 2 mL of culture medium per cell; (3) Count the Raji-Luci cells and place them into six-well plates, 2×10 cells per well. 5 cells, 1 mL of culture medium, and CAR-T mixed; (4) Add 500 nM homovanillic acid to the above air, and add an equal volume of DMSO to the control group. Place in a cell culture incubator and incubate for 24 hours; 2. Incubate cells with antibodies (1) Preparation of cell washing solution: Add 1% FBS to 1× PBS to make the cell washing solution. Store at 4°C and use for one week.
[0029] (2) Take 2×10 5 Centrifuge the cells at 300 g for 3 minutes, discard the supernatant carefully, resuspend the cells with 1 ml of cell washing solution and divide into two tubes, 50 μL in each tube, add 1 μL of FITC anti-human PD1 Antibod and AF700 antihuman CD7 to each tube, and incubate at 4°C for 30 minutes.
[0030] (3) After incubation, centrifuge at 300g for 3 minutes, carefully discard the supernatant, resuspend the cells in 1 ml of cell washing solution, centrifuge at 300g for 3 minutes, carefully discard the supernatant, and resuspend the cells in 300 μL of cell washing solution.
[0031] (4) Detect FITC, PE and AF700 using flow cytometry.
[0032] (5) Finally, the signal intensity of PD-1 and Tim-3 of CD7-positive cells was analyzed to indicate the exhaustion level of CAR-T.
Claims
1. Application of homovanillic acid as a CAR-T cell enhancer.
2. The use according to claim 1, characterized in that: Homovanillic acid can promote the proliferation of CAR-T cells in vitro; and / or homovanillic acid can inhibit the exhaustion of CAR-T cells.
3. The use according to claim 1, characterized in that: Homovanillic acid can enhance the anti-tumor function of CAR-T cells.
4. Application of homovanillic acid in the preparation of adjuvants for CAR-T cell therapy.
5. The use according to claim 4, characterized in that: Homovanillic acid can promote the proliferation of CAR-T cells in vitro; and / or homovanillic acid can inhibit the exhaustion of CAR-T cells.
6. The use according to claim 4, characterized in that: Homovanillic acid can enhance the anti-tumor function of CAR-T cells.
7. Application of homovanillic acid combined with CAR-T cells in the preparation of anti-tumor drugs.
8. The use according to claim 7, characterized in that: Homovanillic acid can promote the proliferation of CAR-T cells in vitro; and / or homovanillic acid can inhibit the exhaustion of CAR-T cells.
9. The use according to claim 7, characterized in that: Homovanillic acid can enhance the ability of CAR-T cells to kill cancer cells.
10. The use according to claim 7, characterized in that: The CAR-T cells include but are not limited to CAR-T cell therapy products targeting CD19, CD20, CD22, BCMA, CLDN18.2, MSLN, GD2, CEA, EGFRvIII and HER2.