Daphne genkwa ester A preparation for promoting T cells to kill tumor cells and method thereof

Abstract

The invention discloses a daphne genkwa ester A preparation for promoting T cells to kill tumor cells and a method thereof. The daphne genkwa ester A preparation is prepared from daphne genkwa ester A with the concentration larger than 200 pg / ml. The preparation can promote T cells to kill tumor cells.

Description

Technical Field

[0001] The present application relates to the technical field of application of the natural molecule Yuanhuacine, and in particular to a Yuanhuacine preparation and method thereof for promoting T cells to kill tumor cells. Background Art

[0003] At present, tumor immunotherapy has become an important treatment method besides traditional treatment, and is even considered to be the most likely treatment method to conquer cancer. Among them, treatment with targeted PD-1 / PD-L1 and CTLA-4 immune checkpoint inhibitors has shown certain therapeutic effects in solid tumors, but the overall response rate of tumor patients is only 5%-25%, and new tumor immunotherapy molecules are urgently needed; in addition, adoptive T cell therapy has achieved remarkable success worldwide, especially CAR-T cell therapy, but its safety has always been controversial. For example, the FDA announced on November 28, 2023 that it would begin investigating the serious risk of secondary cancer after CAR-T treatment. The potential risks apply to all currently approved CAR-T therapies, including Yescarta and Tecartus from Kite Pharma under Gilead Sciences, Kymriah from Novartis and Breyanzi from Bristol Myers Squibb, Johnson & Johnson and Legend. Biotech's Carvykti; and soon on January 24, 2024, all BCMA and CD19-targeted CAR-T cell therapies were marked with the highest level of black box warning; since then, the risk of secondary cancer in cell therapies such as CAR-T, TCR-T, etc. that use viral vectors to introduce exogenous nucleic acid modifications has become the biggest obstacle to safety requirements, and there is an urgent need to explore new cell therapy methods.

[0004] Natural small molecule-regulated T lymphocyte immunotherapy has unique advantages over antibody drugs, such as protein preparations targeting PD-1 / PD-L1 and CTLA-4, or exogenous nucleic acid-modified cell therapy, such as CAR-T and TCR-T adoptive cell therapy.

[0005] Yuanhuacine A, also known as genkwa terpenes, is a diterpenoid compound extracted from the genkwa of the Thymelaeaceae plant, which has significant anti-cancer and abortion-inducing effects. The molecular formula of Yuanhuacine A is C37H44O10. For example, Wang Yujue, Shang Xinyue, Yao Guodong. Pharmacological effects and clinical applications of Yuanhuacine A [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2020, 25(10): 1188-1194. It was disclosed that "In 1986, Hall et al. found that Yuanhuacine A inhibits DNA synthesis as one of its anti-cancer mechanisms. Yuanhuacine A mainly acts on the S phase of cell division and inhibits DNA synthesis at multiple sites, thereby exerting an anti-tumor effect. In 1992, Zhang Dechang et al. found that Yuanhuacine A It is a specific antagonist of protein kinase C (PKC) receptor. Experiments have shown that dianthine selectively inhibits the activation of PKC by PdBu and shows a dose-effect relationship, but has no effect on PKC itself. In 2006, Zhang et al. conducted an in-depth study on the anticancer activity of dianthine and found that the inhibition of DNA topoisomerase I (DNAtopoI) may be another anti-tumor mechanism of daphne diterpenoids (including dianthine). In the topo I inhibitory activity experiment, compared with the positive control drug hydroxycamptothecin (IC50 = 48.0μmol / L), the IC50 of Yuanhuacine A was 40.0μmol / L, showing moderate inhibitory activity. Subsequent experiments also found that the daphne-type diterpenoid compounds are a DNA topo I inhibitor with a new structure. Therefore, Yuanhuacine A can inhibit DNA and protein synthesis by acting on multiple enzyme sites, while regulating the level of cell apoptosis genes, and inhibit tumor growth in multiple ways. This mechanism makes Yuanhuacine A perform well in various tumor inhibition tests. "However, existing studies have not proposed that Yuanhuacine A has the ability to promote T cells to kill tumor cells.

[0006] For example, CN201280035568.6 discloses that Yuanhuacine A can effectively increase the secretion of cytokines in Th1 immune cells and inhibit atopy in atopy mouse models. However, this use does not show that Yuanhuacine A has the ability to promote T cells to kill tumor cells.

[0007] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] In view of the above technical problems, the present application provides a Yuanhuacine preparation and method for promoting T cells to kill tumor cells. The preparation can promote T cells to kill tumor cells.

[0009] The present application provides a Yuanhuacine A preparation for promoting T cells to kill tumor cells, comprising: Yuanhuacine A with a concentration greater than 200 pg / ml. The Yuanhuacine A preparation with this concentration can achieve the effect of promoting T cells to kill tumor cells. The substance is naturally derived and can reduce damage to the human body while achieving the killing of tumor cells.

[0010] Preferably, it comprises: Yuanhuacine A at a concentration of 200pg / ml to 200ng / ml. This concentration can promote the killing effect of T cells on various common tumor cells in the human body.

[0011] Preferably, the tumor cell is at least one of brain glioma cell T98G, lung cancer cell H1299, liver cancer cell MHCC97H, breast cancer cell BT549, colorectal cancer cell HCT116, pancreatic cancer cell BXPC-3, gastric cancer cell NUGC4, prostate cancer cell PC-3, melanoma cell B16F10, ovarian cancer cell SKOV3, cervical cancer cell Hela, osteosarcoma cell U2OS, renal cancer cell ACHN, leukemia cell MOLM13, and lymphoma cell U937. The tumor cells used are purchased from ATCC.

[0012] Preferably, Yuanhuacine A is added to the cell culture system until the final concentration of Yuanhuacine A is greater than 200 pg / ml.

[0013] Preferably, Yuanhuacine A is added to the cell culture system until the final concentration of Yuanhuacine A is 200 pg / ml to 200 ng / ml.

[0014] Preferably, it is used in the preparation of tumor treatment drugs. In the preparation of drugs for treating tumors, Yuanhuacine A can be added according to the aforementioned minimum efficacy standard, or the specific addition amount can be obtained by a technician in this field according to the actual drug administration form and human absorption efficiency through limited experimental tests. The drug can be various commonly used dosage forms, such as injections, tablets, capsules, oral liquids, granules, dry powder inhalers, etc.

[0015] Preferably, the tumor treatment drug includes: Yuanhuacine A preparation.

[0016] Another aspect of the present application provides a method for promoting T cells to kill tumor cells, comprising culturing at least T lymphocytes in a cell culture system to which the above-mentioned Yuanhuacine A preparation is added.

[0017] Preferably, the steps include:

[0018] T lymphocytes and tumor cells are mixed at a number ratio of 200 to 1:1, and then inoculated into a cell culture system, and Yuanhuacine A is added to the cell culture system at a final concentration of more than 200 pg / ml.

[0019] Preferably, the steps include:

[0020] The T lymphocytes cultured with the Yuanhuacine A preparation were mixed with tumor cells and then cultured.

[0021] The beneficial effects of this application include:

[0022] 1) The Yuanhuacine A preparation and method for promoting T cells to kill tumor cells provided in the present application, the natural molecule Yuanhuacine A can induce T lymphocytes to kill tumor cells and improve the effect of T lymphocytes killing tumor cells. By adding Yuanhuacine A, the proportion of T lymphocytes killing tumor cells can be increased from 0% to 10%-100% relative to the cell culture system without Yuanhuacine A, indicating that it has a significant effect of promoting T lymphocytes to kill tumor cells BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the test results of the cytotoxic activity of the natural molecule Yuanhuacine A provided in the examples of the present application on various types of tumor cells at a concentration of 2 μg / ml. The results show that it has almost no inhibitory effect on tumor cells under this concentration condition; the graph is a white light bright field imaging;

[0024] Figure 2 A bar graph of MTS cell viability analysis of the natural molecule Yuanhuacine provided in the examples of the present application at a concentration of 2 μg / ml in the treatment group and the control group;

[0025] Figure 3 The result diagram of the treatment group and the control group of the natural molecule Yuanhuacine A provided in the embodiment of the present application at a concentration of 200 pg / ml shows that the regulatory T lymphocytes effectively kill various types of tumor cells; the figure is formed by overlapping the green or red fluorescence channel and the white bright field MERGE;

[0026] Figure 4 A bar graph of MTS cell viability analysis of the natural molecule Yuanhuacine A provided in the examples of the present application at a concentration of 200 pg / ml in the treatment group and the control group; Figure 5 This is a graph showing the killing phenotype observation results of T lymphocytes in the treatment group and the control group treated with the natural molecule Yuanhuacine A provided in the embodiment of the present application at a concentration of 2 ng / ml on various tumor cells; the results show that T lymphocytes can adoptively kill various types of tumor cells; the graph is formed by overlapping the green or red fluorescence channel and the white light field MERGE;

[0027] Figure 6 This is a bar graph of MTS cell viability analysis of the treatment group and the control group when the natural molecule Yuanhuacine A provided in the examples of the present application is at a concentration of 2 ng / ml;

[0028] Figure 7This is a graph showing the observation results of the natural molecule Yuanhuacine A provided in the examples of the present application enhancing the secondary killing phenotype of the immune response of T lymphocytes to tumor antigens at a concentration of 2 ng / ml; the graph is formed by overlapping the green or red fluorescence channel and the white light field MERGE;

[0029] Figure 8 This is a bar graph of MTS cell viability analysis of the treatment group and the control group when the natural molecule Yuanhuacine A provided in the examples of the present application is at a concentration of 2 ng / ml. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0031] Example

[0032] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels. Yuanhuacine (CAS60195-70-2) was purchased from Chengdu Lemeitian, all tumor cells were purchased from ATCC, and T lymphocytes were separated from human peripheral blood according to existing methods; the detection methods used were all existing methods unless otherwise specified.

[0033] Example 1: Cytotoxic activity test of natural molecule Yuanhuacine A against various types of tumor cells at a concentration of 2 μg / ml

[0034] Brain glioma cells T98G, lung cancer cells H1299, liver cancer cells MHCC97H, breast cancer cells BT549, colorectal cancer cells HCT116, pancreatic cancer cells BXPC-3, gastric cancer cells NUGC4, prostate cancer cells PC-3, melanoma cells B16F10, ovarian cancer cells SKOV3, cervical cancer cells Hela, osteosarcoma cells U2OS, renal cancer cells ACHN, leukemia cells MOLM13, and lymphoma cells U937 were cultured in a medium of DMEM high glucose + 10% FBS. The tumor cells used in the following examples were all in this culture system.

[0035] When each cell line grows to the logarithmic growth phase, the cells are digested and counted, and cells are inoculated in a 24-well plate at 10,000 cells / well and 2 ml of cell culture medium / well. The natural molecule Yuanhuacine A is added to the cell culture system at a final concentration of 2 μg / ml. The control group (CON) is added with an equal volume of solvent (DMSO) and cultured in a 37°C cell culture incubator for 3 to 5 days. The test results are observed, and the cell viability is tested with MTS. MTS is diluted in the culture medium at 1:10, incubated in the cell culture incubator for 1 hour, and the absorbance is measured at 490 nm to calculate the cell viability.

[0036] The experimental results are as follows Figure 1 As shown, phenotypic observations showed that compared with the control group Con, the tumor cells of various lines were intact and not broken under the treatment of 2 μg / ml concentration of Yuanhuacine A, and MTS cell viability analysis (such as Figure 2 ) showed that the cell numbers in the two groups were similar, indicating that 2 μg / ml Yuanhuacine had no classical cytotoxic killing or inhibitory effect on the tested tumor cells.

[0037] Example 2: The natural molecule Yuanhuacine A at a concentration of 200 pg / ml regulates T lymphocytes to effectively kill various types of tumor cells

[0038] Glioma cells T98G, lung cancer cells H1299, liver cancer cells MHCC97H, breast cancer cells BT549, colorectal cancer cells HCT116, pancreatic cancer cells BXPC-3, gastric cancer cells NUGC4, prostate cancer cells PC-3, melanoma cells B16F10, ovarian cancer cells SKOV3, cervical cancer cells Hela, osteosarcoma cells U2OS, renal cancer cells ACHN, leukemia cells MOLM13, lymphoma cells U937 were cultured in vitro, and each tumor cell was fluorescently labeled with GFP or RFP for the convenience of observing the test results.

[0039] T lymphocytes were isolated and cultured from human peripheral blood using magnetic bead negative separation technology. The kit used was Rosette Sep Human T Cell Enrichment Cocktail (#15061). The isolation steps were carried out strictly in accordance with the instructions of the kit.

[0040] The T cell in vitro culture medium consists of: RIPM 1640 + 10% FBS + 1% P / S (penicillin / streptomycin, Gibco, #15140-122) + 200U / ml IL-2 (Gibco, #PHC0026); tumor cells and T lymphocytes are counted separately, mixed according to the ratio of T cells: tumor cells = 100000:10000, and the resulting mixture is evenly inoculated in a 24-well plate; the natural molecule Yuanhuacine A is added to the above-mentioned T cell and tumor cell mixed culture system at a final concentration of 200pg / ml, mixed evenly, and an equal volume of solvent (DMSO) is added to the control group (CON), and the cells are placed in a 37°C cell culture incubator for culture for 3 to 5 days, and the test results are observed, and the proportion of surviving cells is tested with MTS, MTS is diluted in the culture medium at 1:10, incubated in the cell culture incubator for 1h, and the absorbance is measured at 490nm to calculate the cell viability.

[0041] The results are as follows Figure 3 , 4As shown, the T cells in the control group were loosely and randomly distributed around the tumor cells, without binding and killing; when the concentration of the natural molecule Yuanhuacine A in the culture system was 200pg / ml, the T cells in the treatment group aggregated around the tumor cells, tightly bound, and killed in groups, and the MTS cell viability test ( Figure 4 ), Figure 4 The results show that the proportion of surviving cells in the treatment group decreased significantly compared with that in the control group, which also proves that the proportion of surviving tumor cells after T cell killing under the treatment of Yuanhuacine A is seriously reduced. The results show that Yuanhuacine A can regulate T cells to kill tumors at 200pg / ml. This shows that Yuanhuacine A can promote the activity of T lymphocytes in killing tumor cells and achieve a better effect of killing tumor cells.

[0042] Example 3: The natural molecule Yuanhuacine A at a concentration of 2 ng / ml treated T lymphocytes to adoptively kill various types of tumor cells

[0043] T lymphocytes were seeded on a 24-well plate at 100,000 cells / well, and the natural molecule Yuanhuacine was added to the cell culture system described in Example 2 at a final concentration of 2 ng / ml, and the resulting culture system was added to the wells of the treatment group. The control group (CON) was added with an equal volume of solvent (DMSO) and placed in a 37°C cell culture incubator for 2 days.

[0044] T cells were collected by centrifugation and washed three times with PBS. T cells pretreated with Yuanhuacine A and DMSO control group were inoculated into the tumor cells in Example 1, respectively, and cultured in a 37°C cell culture incubator for 3-5 days. The killing effect was observed and the cell viability was tested with MTS.

[0045] The results are as follows Figures 5-6 As shown, the results show that the natural molecule Yuanhuacine A at a concentration of 2ng / ml can aggregate and bind to tumor cells in cultured T lymphocytes, thereby playing a role in killing tumor cells. The MTS cell viability test results ( Figure 6 ) showed that the killing effect of T lymphocytes on various tumor cells after treatment with Yuanhuacine A was significant compared with the blank group. This indicates that Yuanhuacine A can promote the activity of T lymphocytes in adoptively killing tumor cells, making the adoptive T cells achieve a better effect of killing tumor cells.

[0046] Example 4: The natural molecule Yuanhuacine A enhances the immune response of T lymphocytes to tumor antigens at a concentration of 2 ng / ml

[0047] The brain glioma cells T98G, lung cancer cells H1299, liver cancer cells MHCC97H, breast cancer cells BT549, colorectal cancer cells HCT116, pancreatic cancer cells BXPC-3, gastric cancer cells NUGC4, prostate cancer cells PC-3, melanoma cells B16F10, ovarian cancer cells SKOV3, cervical cancer cells Hela, osteosarcoma cells U2OS, renal cancer cells ACHN, leukemia cells MOLM13, lymphoma cells U937 were counted, 1*10^6 cells were taken, the cells were lysed by ultrasonication, and the supernatant was collected by centrifugation;

[0048] 10 μl of the supernatant of each tumor cell was taken as a tumor antigen and cultured with 1*10^5 T cells (RIPM 1640+10% FBS+1% P / S+200U / ml IL-2). The natural molecule Yuanhuacine A was added to the cell culture system at a final concentration of 2 ng / ml, and the configured culture system was added to a 24-well plate as a treatment group.

[0049] The control group (CON) was added with an equal volume of solvent (DMSO).

[0050] The plates were placed in a 37°C cell culture incubator for 2 days. T cells were collected by centrifugation, washed 3 times with PBS, and then inoculated into a 24-well plate for further culture. After 24 hours, 1*10^4 of the above tumor cells were inoculated into a 24-well plate and cultured in a 37°C cell culture incubator for 3 to 5 days. The killing effect was observed, and cell viability was tested with MTS.

[0051] The results are as follows Figure 7 , 8 As shown, the T cells in the control group treated with DMSO did not respond to tumor antigens and could not kill tumor cells in the secondary killing test, while the T cells added with 2ng / ml concentration of Yuanhuacine A could respond to the added tumor antigens, so the T cells could kill tumor cells in the secondary killing test, and the MTS cell viability analysis ( Figure 8 ) showed that the cell viability of the treatment group decreased significantly compared with the control group, and also showed that T cells could kill tumor cells after adding Yuanhuacine A in the secondary killing test. The results showed that Yuanhuacine A could enhance the response of T cells to tumor antigens and subsequent tumor killing. This shows that Yuanhuacine A can promote the response of T lymphocytes to tumor antigens to achieve a better effect of killing tumor cells.

[0052] Example 5 Yuanhuacine A Preparation

[0053] Yuanhuacine A was added into acetic acid solvent to dissolve to prepare a preparation, and the amount of Yuanhuacine A added was 3ng / ml.

[0054] Example 6 Yuanhuacine A Preparation

[0055] The Yuanhuacine A micropowder is processed to obtain granules with a particle size of 1 to 5 microns, and then lactose is used as a carrier. The ratio of rough lactose to fine powder lactose is selected to be 10:1. The lactose mixture is then mixed with the Yuanhuacine A micropowder and evenly loaded using a vortex mixer to obtain a dry powder inhaler.

[0056] The amount of Yuanhuacine A added was 5 ng / ml.

[0057] Example 7

[0058] The difference from Example 2 is that the ratio of T cells to tumor cells is 200:1.

[0059] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Yuanhuacine A preparation for promoting T cells to kill tumor cells, characterized in that: include: The concentration of Yuanhuacine A is greater than 200pg / ml.

2. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 1, characterized in that: include: The concentration of Yuanhuacine A is 200pg / ml~200ng / ml.

3. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 1, characterized in that: The tumor cells are at least one of brain glioma cells T98G, lung cancer cells H1299, liver cancer cells MHCC97H, breast cancer cells BT549, colorectal cancer cells HCT116, pancreatic cancer cells BXPC-3, gastric cancer cells NUGC4, prostate cancer cells PC-3, melanoma cells B16F10, ovarian cancer cells SKOV3, cervical cancer cells Hela, osteosarcoma cells U2OS, renal cancer cells ACHN, leukemia cells MOLM13, and lymphoma cells U937.

4. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 1, characterized in that: Yuanhuacine A was added to the cell culture system until the final concentration of Yuanhuacine A was greater than 200 pg / ml.

5. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 1, characterized in that: Yuanhuacine A is added to the cell culture system until the final concentration of Yuanhuacine A is 200 pg / ml to 200 ng / ml.

6. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 1, characterized in that: Used in the preparation of tumor therapeutic drugs.

7. The Yuanhuacine A preparation for promoting T cells to kill tumor cells according to claim 6, characterized in that: Tumor treatment drugs include: Yuanhuacine A preparations.

8. A method for promoting T cells to kill tumor cells, characterized in that: At least T lymphocytes are cultured in a cell culture system to which the Yuanhuacine preparation according to any one of claims 1 to 7 is added.

9. The method according to claim 6, characterized in that The following steps are involved: After T lymphocytes and tumor cells are mixed at a number ratio of 200 to 1:1, they are inoculated into a cell culture system, and Yuanhuacide is added to the cell culture system at a final concentration of more than 200 pg / ml.

10. The method according to claim 6, characterized in that The following steps are involved: The T lymphocytes cultured with the Yuanhuacine A preparation were mixed with tumor cells and then cultured.

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