Application of 2-butanone acid in enhancing anti-tumor function of CAR-T cells

Abstract

The invention provides application of 2-butanone acid in enhancing the anti-tumor function of CAR-T cells, and relates to the technical field of medicines, in particular to 2-butanone acid serving as a CAR-T cell enhancer and application of 2-butanone acid serving as the CAR-T cell enhancer. When the compound is combined with a CAR-T cell treatment product for application, the anti-tumor effect can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the application of 2-ketobutyric acid in enhancing the anti-tumor function of CAR-T cells. Background Art

[0002] 2-Oxobutanoic acid, also known as α-ketoisovaleric acid, is an organic acid and an intermediate in the metabolism of branched-chain amino acids. It is a key intermediate in the metabolism of isoleucine and is involved in the transamination of amino acids into other metabolites. In cancer research, 2-oxobutanoic acid has received increasing attention due to its importance in metabolism and cell proliferation. This patent is the first to show that 2-oxobutanoic acid can enhance the anti-tumor function of CAR-T cells.

[0003] Acute lymphocytic leukemia (ALL) is a blood tumor formed by the malignant proliferation of lymphoid precursor cells that are blocked in the immature stage of differentiation. ALL can occur in the T-cell or B-cell precursors of lymphocytes, and is divided into T-ALL or B-ALL; among them, B-ALL accounts for the vast majority of ALL and seriously threatens human survival and health. In addition, the heterogeneity of clinical characteristics and prognosis is large, which makes its treatment effect poor, and it is urgent to explore new and effective clinical treatment options.

[0004] 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

[0005] The embodiments of the present invention provide the use of 2-ketobutyric acid in enhancing the anti-tumor function of CAR-T cells, so as to improve the anti-tumor efficacy.

[0006] In view of the above problems, the technical solution proposed by the present invention is:

[0007] In a first aspect, the present invention provides 2-ketobutyric acid in promoting the enhancement of CAR-T cell activity, wherein the CAR-T cells mainly include CD19 CAR-T cells and cell therapy products based on the cell transformation.

[0008] Application of 2-ketobutyric acid as a CAR-T cell enhancer.

[0009] Furthermore, 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

[0010] Furthermore, 2-ketobutyric acid can enhance the anti-tumor function of CAR-T cells.

[0011] Application of 2-ketobutyric acid in the preparation of adjuvants for CAR-T cell therapy.

[0012] Furthermore, 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

[0013] Furthermore, 2-ketobutyric acid can enhance the anti-tumor function of CAR-T cells.

[0014] In a second aspect, the present invention provides that 2-ketobutyric 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.

[0015] Application of 2-ketobutyric acid combined with CAR-T cells in the preparation of anti-tumor drugs.

[0016] Furthermore, 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

[0017] Furthermore, 2-ketobutyric acid can enhance the ability of CAR-T cells to kill cancer cells.

[0018] Furthermore, 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.

[0019] Compared with the prior art, the beneficial effect of the present invention is that the anti-tumor efficacy can be significantly improved when 2-ketobutyric acid is used in combination with a CAR-T cell therapy product.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1It indicates that 2-ketobutyric acid promotes the proliferation of CAR-T cells in vitro;

[0022] Figure 2 It indicates that 2-ketobutyric acid can inhibit CAR-T cell exhaustion;

[0023] Figure 3 It indicates that 2-ketobutyric acid can promote the killing of B-ALL leukemia cells by CAR-T cells. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] 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.

[0026] Embodiment 1:

[0027] Figure 1 The effect of 2-ketobutyric acid on the in vitro proliferation of CAR-T cells is shown, comprising the following steps:

[0028] 1. Human T cell sorting and activation

[0029] (1) Dilute the peripheral blood 1-fold with DMSO, mix well, add 20 mL of ficoll to the bottom of the tube, centrifuge at 700 g for 20 min, and extract the buffy coat cells into a new 50 mL tube;

[0030] (2) Disperse the cell pellet, add DMSO and centrifuge at 400 g for 5 min, discard the supernatant, and repeat this step;

[0031] (3) Disperse the cell clumps, add 30 mL of DMSO, mix well, take samples and count, centrifuge at 400 g for 4 min, and aspirate the supernatant with a pipette;

[0032] (4) Add 90 μL running buffer to every 5e7 PBMC cells, and add 20 ul CytoSinct TM CD3Nanobeads, shake the cells with a mixer and incubate at 4 degrees for 20 minutes;

[0033] (5) Place the separation magnetic rack in a clean bench for irradiation sterilization. Place the separation column on the magnetic rack 5 min before the end of the antibody incubation and add 5 mL of running buffer.

[0034] (6) After the running buffer has run out, resuspend the cells in 5 mL running buffer and gently blow up with a 5 mL pipette tip, add to the separation column, and after the liquid has run out, add 5 mL running buffer again. At this time, the CD3-positive T cells are in the adsorption column;

[0035] (7) Add 5 mL of Running Buffer to the separation column, remove it and align it with the collection tube, use the piston to blow the T cells into the collection tube, add 30 mL of DMSO to resuspend and count, and centrifuge at 500 g for 4 min;

[0036] (8) Add T cell culture medium, adjust the density to 5e6-1e7 / mL, and add 1 / 100 of Enced TM T cellActivation human, placed in an incubator, 37°C, 5% CO2;

[0037] (9) Transfection after 24 hours of activation to prepare CAR-T cells

[0038] 2. Cultivation of CAR-T cells

[0039] (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, 1000 IU / mL of recombinant human IL-2 protein, and zoledronic acid injection (working concentration 5 μM).

[0040] (2) The sorted T cells were divided into 2×10 6 The cells were inoculated at a density of 100 / mL in CAR-T cell activation medium and placed in a cell culture incubator at 37°C and 5% CO2.

[0041] (3) Subculture every 2-3 days, with a subculture density of 1-2×10 6 The cells were cultured and expanded for about 12 days.

[0042] 3.2-Ketobutyric Acid Treatment of CAR-T Cells

[0043] (1) Plate CAR-T cells in a 96-well flat-bottom plate, 10,000 cells / well;

[0044] (2) 10 μM 2-OXOBUTANOIC ACID was added to the experimental wells, and the corresponding DMSO was added to the control group, and the wells were placed in a carbon dioxide constant temperature incubator and incubated for 72 h;

[0045] (3) After 72 hours, 85 μL of culture medium was aspirated and discarded, 10 μL of cck8 solution was added to the wells and incubated in the incubator again for 2 to 3 hours. When the culture medium turned brown, 1% w / v SDS solution was added to terminate the reaction;

[0046] (4) The absorbance was measured at a wavelength of 450 nm using an ELISA reader. The efficiency of 2-OXOBUTANOICACID in promoting CAR-T cell proliferation was calculated by comparing the absorbance with that of the control group.

[0047] Embodiment 2

[0048] Figure 3 It is shown that 2-ketobutyric acid promotes CAR-T to kill cancer cells, including the following steps:

[0049] 1. CAR-T cell killing experiment in vitro

[0050] (1) Raji-luci cell culture: Cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO2, and 90% relative humidity. Subcultured once every 2-3 days.

[0051] (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;

[0052] (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;

[0053] (4) Add 10 μM 2-ketobutyric acid to the above wells, and add an equal volume of DMSO to the control group.

[0054] (5) Place in a cell culture incubator and incubate for 24 hours;

[0055] (6) Add 5 μL of fluorescein sodium salt and incubate in an incubator for 10 min;

[0056] (7) Using a microplate reader to read the Luciferace autoluminescence intensity;

[0057] (8) The number of surviving Raji-Luci cells was obtained by calculating the ratio of fluorescence intensity to that of the control group.

[0058] Embodiment 3

[0059] Figure 2 2-ketobutyric acid was shown to inhibit the exhaustion of CAR-T cells, comprising the following steps:

[0060] 1. In vitro Co-incubation

[0061] (1) Raji-luci cell culture: All T-ALL cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum and 1% P / S at 37°C, 5% CO2, and 90% relative humidity in an incubator. The cells were passaged every 2-3 days.

[0062] (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;

[0063] (3) Count the Raji-Luci cells and place them into six-well plates, with 2×10 cells per well. 5 cells, 1 mL of culture medium, and CAR-T mixed;

[0064] (4) Add 10 μM 2-ketobutyric 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;

[0065] 2. Cell Incubation with Antibodies

[0066] (1) Preparation of cell washing solution: Add 1% FBS to 1× DMSO to make the cell washing solution. Store at 4°C and use for one week.

[0067] (2) Take 2×105 cells to be tested, centrifuge at 300g for 3 minutes, carefully discard the supernatant, break the membrane and fix it, resuspend the cells with 1mL cell washing solution and divide them into two tubes, 50μL in each tube, add 1μL TOX Monoclonal Antibody (TXRX10), PE, eBioscience to each tube TM , incubate at 4°C for 30 min.

[0068] (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.

[0069] (4) Detect FITC and PE using flow cytometry.

[0070] (5) Finally, the signal intensity of PD-1 in FITC-positive cells was analyzed to indicate the exhaustion level of CAR-T.

[0071] The present invention provides the use of 2-ketobutyric acid in enhancing the anti-tumor function of CAR-T cells:

[0072] In a first aspect, the present invention provides 2-ketobutyric acid in promoting the enhancement of CAR-T cell activity, wherein the CAR-T cells mainly include CD19 CAR-T cells and cell therapy products based on the cell transformation.

[0073] In some embodiments, 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro.

[0074] In some embodiments, 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

[0075] In a second aspect, the present invention provides that 2-ketobutyric 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.

[0076] When 2-ketobutyric acid is used in combination with CAR-T cell therapy products, the anti-tumor efficacy can be significantly improved.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

Application of 1.2-butyric acid as a CAR-T cell enhancer.

2. The use according to claim 1, characterized in that: 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

3. The use according to claim 1, characterized in that: 2-Ketobutyric acid can enhance the anti-tumor function of CAR-T cells. Application of 4.2-ketobutyric acid in the preparation of adjuvants for CAR-T cell therapy.

5. The use according to claim 4, characterized in that: 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

6. The use according to claim 4, characterized in that: 2-Ketobutyric acid can enhance the anti-tumor function of CAR-T cells.

7. Application of 2-ketobutyric acid combined with CAR-T cells in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: 2-ketobutyric acid can promote the proliferation of CAR-T cells in vitro; and / or 2-ketobutyric acid can inhibit the exhaustion of CAR-T cells.

9. The use according to claim 7, characterized in that: 2-Ketobutyric 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.

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