Application of cinnamyl alcohol in enhancing anti-tumor function of CAR-T cells

Abstract

The invention discloses an application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T (Chimeric Antigen Receptor T) cells. The invention specifically relates to cinnamyl alcohol as a CAR-T cell enhancer and application of cinnamyl alcohol in 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 application relates to the field of medicine, in particular to the application of Cinnamyl Alcohol in enhancing the anti-tumor function of CAR-T cells. BACKGROUND

[0002] Cinnamyl Alcohol (CA) is a naturally occurring aromatic compound found in various plant sources, including cinnamon leaves, Peruvian balsam, and hyacinth. Its unique sweet, balsamic, spicy, and cinnamon-like aroma and flavor make it a popular ingredient in the perfume and food industries, used in perfumes, flavor products, baked goods, and flavored beverages. In addition, it can also be used as a precursor for industrial applications to synthesize chemicals such as cinnamyl esters and taxol. Derivatives of cinnamyl alcohol have antioxidant, anti-inflammatory, hypolipidemic, antiviral, and antibacterial properties, and can therefore function as bioactive compounds. CA also shows potential medicinal properties beyond its industrial and culinary applications, including anti-inflammatory and antibacterial activity. Notably, it has been approved by the FDA for inclusion in the allergic patch test, a diagnostic tool designed to assist in the diagnosis of allergic contact dermatitis in individuals aged 6 years or older. In disease research, cinnamyl alcohol has received increasing attention due to its effects on preventing and treating high cholesterol, reducing blood sugar, lowering lipid levels, and lowering blood pressure. This patent is the first to show that cinnamyl alcohol can enhance the anti-tumor function of CAR-T cells.

[0003] Acute lymphoblastic leukemia (ALL) is a hematological tumor formed by the malignant proliferation of lymphoid precursor cells blocked at the immature stage of their differentiation. ALL can occur in T cell or B cell precursors of lymphocytes, thus classified as T-ALL or B-ALL; among them, B-ALL accounts for the vast majority of ALL, which seriously threatens human survival and health. The heterogeneity of clinical characteristics and prognosis makes the treatment effect poor, and there is an urgent need to explore new effective clinical treatment programs.

[0004] Cellular immunotherapy is an innovative treatment method aimed at using the human immune system to eliminate cancer, among which CAR-T cell immunotherapy is revolutionary as it produces very effective and durable clinical responses. However, CAR-T cell exhaustion is a shortcoming in cellular immunotherapy, characterized by decreased proliferation and cytotoxicity, up-regulated expression of inhibitory receptors, mainly induced by chronic antigenic stimulation or tonic signaling of CAR self-aggregation. Therefore, exploring new strategies to remodel and improve CAR-T cell function through metabolic small molecules is worthy of further study, and improving the anti-tumor function of CAR-T cells through metabolic molecules has great potential in cancer treatment. SUMMARY

[0005] The application provides application of cinnamyl alcohol in enhancing anti-tumor function of CAR-T cells, and solves the technical problems described above.

[0006] The application provides application of cinnamyl alcohol in enhancing anti-tumor function of CAR-T cells, and solves the technical problems described above.

[0007] Further, cinnamyl alcohol can promote in-vitro proliferation of CAR-T cells; and / or cinnamyl alcohol can inhibit exhaustion of CAR-T cells.

[0008] Further, cinnamyl alcohol can enhance anti-tumor function of CAR-T cells.

[0009] Further, cinnamyl alcohol can promote in-vitro proliferation of CAR-T cells; and / or cinnamyl alcohol can inhibit exhaustion of CAR-T cells.

[0010] Further, the CAR-T cells include but are not limited to CAR-T cell treatment products targeting CD19, CD20, CD22, BCMA, CLDN18.2, MSLN, GD2, CEA, EGFRvIII and HER2, etc.

[0011] The application provides application of cinnamyl alcohol in enhancing anti-tumor function of CAR-T cells.

[0012] In the first aspect, the application provides application of cinnamyl alcohol in promoting enhancement of activity of CAR-T cells, wherein the CAR-T cells mainly include CD19 CAR-T cells and cell treatment products based on the cells.

[0013] In some embodiments, cinnamyl alcohol can promote in-vitro proliferation of CAR-T cells.

[0014] In some embodiments, cinnamyl alcohol can inhibit exhaustion of CAR-T cells.

[0015] In the second aspect, the application provides application of cinnamyl alcohol in enhancing killing ability of CAR-T cells on cancer cells, wherein the cancer cells include but are not limited to B-cell acute lymphoblastic leukemia cells and other tumor cells.

[0016] In the third aspect, the application provides application of cinnamyl alcohol in combination with CAR-T cells in treating solid tumors in vivo, which can significantly inhibit tumor growth and enhance anti-tumor immunity of CAR-T cells, wherein the solid tumor cells include but are not limited to mouse colorectal cancer cell lines and other tumor cells.

[0017] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 Cinnamyl alcohol promotes the in vitro proliferation or survival rate of CAR-T cells; Figure 2 Cinnamyl alcohol can inhibit the expression of PD1, a depletion molecule in CAR-T cells; Figure 3 Cinnamyl alcohol indicates that it can enhance the killing ability of CAR-T cells against B-ALL leukemia cells; Figure 4 This indicates that in vivo, cinnamyl alcohol, in combination with CAR-T, inhibits tumor development; Figure 5 This indicates that in vivo, cinnamyl alcohol can increase the secretion of cytokines related to killing in CAR-T cells; Figure 6 This indicates that in vivo, cinnamyl alcohol can inhibit the expression of PD1, a depleted molecule in CAR-T cells. Detailed Implementation

[0019] The following is in conjunction with the appendix Figures 1-6 The principles and features of the present invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. The advantages and features of the invention will become clearer from the following description and claims. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the invention.

[0020] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0021] 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 in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0023] Example 1: Effect of Cinnamyl Alcohol on In Vitro Proliferation of CAR-T Cells 1. Human T cell sorting and activation (1) Dilute peripheral blood with PBS by 1 time, mix well, add 20ml of ficoll to the bottom of the tube, centrifuge at 700g for 20min, and extract white membrane cells into a new 50ml tube. (2) Break up the cell clusters, fill with PBS 400g, centrifuge for 5 min, discard the supernatant, and repeat this step; (3) Break up the cell clusters, add 30 ml of PBS, mix well, take samples and count, centrifuge at 400g for 4 min, and remove the supernatant with a pipette; (4) Add 90 μL of running buffer and 20 μL of CytoSinct™ CD3 Nanobeads to every 5e7 PBMC cells. Mix the cells with a shaker and incubate at 4 degrees Celsius for 20 min. (5) Place the sorting magnetic rack in a clean bench for sterilization by irradiation. 5 minutes before the end of antibody incubation, place the sorting column on the magnetic rack and add 5 ml of running buffer; (6) After the running buffer has drained, resuspend the cells in 5 mL of running buffer and gently blow them up with a 5 mL pipette tip. Add the cells to the sorting column. After the liquid has drained, add another 5 mL of running buffer. At this time, the CD3 positive T cells are in the adsorption column. (7) Add 5ml of running buffer to the sorting column, remove it and align it with the collection tube. Use the piston to blow T cells into the collection tube, add 30ml of PBS to resuspend and count, and centrifuge at 500g for 4min. (8) Add T cell culture medium and adjust the density to 5e6~1e7 / ml. Add 1 / 100 of Enceed™ T cellActivation human and place in an incubator at 37℃ with 5% CO2. (9) Transfect CAR-T cells 24 hours after activation to prepare CAR-T cells. 2. Culture of CAR-T cells (1) Preparation of CAR-T cell activation medium: 913 mL of serum-free basal medium for OptiVitroPT cells, 8 mL of serum-free medium for T cells with added components, 1000 IU / mL of recombinant human IL-2 protein, and zoledronic acid injection (working concentration 5 μM).

[0024] (2) The sorted T cells were seeded into CAR-T cell activation medium at a density of 2×106 cells / ml and placed in a cell culture incubator at 37℃ and 5% CO2.

[0025] (3) Passage once every 2-3 days, with the passage density controlled at 1~2×106 cells, and expand culture for about 12 days.

[0026] 3. Cinnamyl alcohol treatment of CAR-T cells (1) CAR-T cells were plated in 96-well flat-bottom plates, 10,000 cells / well; (2) Add 10 μM CA to the experimental wells and add the corresponding DMSO to the control group. Incubate in a carbon dioxide constant temperature incubator for 72 h. (3) After 72 hours, take 85 μL of culture medium and discard it. Add 10 μL of cck8 solution to the well and incubate in the incubator for 2-3 hours. When the culture medium turns brown, add 1% w / v SDS solution to stop the reaction. (4) The absorbance was measured at a wavelength of 450 nm using an ELISA reader. The efficiency of CA in promoting CAR-T cell proliferation was calculated by comparing the absorbance with that of the control group.

[0027] Example 2: Cinnamyl alcohol promotes CAR-T cell killing of cancer cells 1. In vitro killing experiment of CAR-T cells (1) Raji-luci cell culture: cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% P / S at 37 ℃, 5% CO2 and 90% relative humidity. passaged once every 2-3 days.

[0028] (2) Count the CAR-T cells and separate them into 96-well U-plates, with 20,000 cells per well and 100 μL of culture medium per well; (3) Count Raji-Luci cells and separate them into 96-well U-plates, 5000 cells per well, 100 μL of culture medium, and mix with CAR-T; (4) Add 10 μM cinnamyl alcohol to the above wells, and add an equal volume of DMSO to the control group.

[0029] (5) Incubate in a cell culture incubator for 24 hours; (6) Add 5 μL of sodium fluorescein and incubate in an incubator for 10 min; (7) Use an ELISA reader to read the autoluminescence intensity of Luciferace; (8) The cell viability of Raji-Luci was obtained by calculating the ratio of fluorescence intensity to that of the control group.

[0030] Example 3: Cinnamyl alcohol inhibits CAR-T cell depletion 1. In vitro co-incubation (1) Raji-luci cell culture: cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% P / S at 37°C, 5% CO2 and 90% relative humidity. passaged once every 2-3 days.

[0031] (2) Count the CAR-T cells and divide them into six-well plates, 2×106 cells per well, and 2 mL of culture medium; (3) Count the Raji-Luci cells and divide them into six-well plates, 2 × 10⁵ cells per well, and mix them with CAR-T cells; (4) Add 10 μM cinnamyl alcohol to the above wells, and add an equal volume of DMSO to the control group. Incubate in a cell culture incubator for 24 hours. 2. Cell incubation of antibodies (1) Preparation of cell washing solution: Add 1% FBS to 1×PBS to make cell washing solution. It can be stored at 4℃ and used for one week.

[0032] (2) Take 2×105 cells to be tested, 300g, centrifuge for 3 minutes, carefully discard the supernatant, resuspend the cells with 1ml of cell washing solution, divide into two tubes, 50 μL in each tube, add 1μL of PE anti-human PD1 Antibody to each tube, and incubate at 4℃ for 30 minutes.

[0033] (3) After incubation, centrifuge at 300g for 3 minutes, carefully discard the supernatant, resuspend the cells in 1ml 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.

[0034] (4) FITC and PE were detected using flow cytometry.

[0035] (5) Finally, the depletion level of CAR-T was indicated by analyzing the signal intensity of PD-1 in FITC-positive cells.

[0036] Example: Tetrathioamine pyrophosphate, when used in combination with CAR-T cells in vivo, significantly inhibited tumor growth and enhanced the anti-tumor immunity of CAR-T cells. 1. Animal experiments (1) Select 6-8 week old male C57BL / 6J experimental mice and inject CA intraperitoneally on day-3 and day-1; (2) Construction of tumor model: Day0 1*E5 mouse colon cancer cells Mc38 were subcutaneously implanted in the right back of experimental mice; (3) After modeling, monitor the tumor size. When the tumor burden volume of the experimental mice reaches 100 mm3, CAR-T cells are injected intravenously into the tumor-bearing mice. (4) During the experiment, CA was administered intraperitoneally every two days, while the volume of PBS remained constant; (5) Measure the size of the tumor with calipers every 2 days to detect tumor growth (tumor volume calculation formula: L*W*W / 2); 2. Flow cytometry detection After the tumor cells were minced and digested, they were processed according to the experimental steps of cell incubation with antibodies in Example 3 above, and the expression of CAR-T and killing-related cytokines was detected by instrument.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. The application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T cells, characterized in that, This includes the application of cinnamyl alcohol as a CAR-T cell enhancer.

2. The application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T cells according to claim 1, characterized in that, Cinnamyl alcohol can promote the in vitro proliferation of CAR-T cells; and / or cinnamyl alcohol can inhibit the depletion of CAR-T cells.

3. The application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T cells according to claim 1, characterized in that, Cinnamyl alcohol can enhance the anti-tumor function of CAR-T cells.

4. The application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T cells according to claim 3, characterized in that, Cinnamyl alcohol can promote the in vitro proliferation of CAR-T cells; and / or cinnamyl alcohol can inhibit the depletion of CAR-T cells.

5. The application of cinnamyl alcohol in enhancing the anti-tumor function of CAR-T cells according to claim 4, 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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