Tumor cell sensitive to killing effect of PD-1 inhibitor and preparation method thereof

Abstract

The invention provides a tumor cell sensitive to the killing effect of a PD-1 inhibitor and a preparation method of the tumor cell, and belongs to the technical field of tumor treatment. The preparation method of the tumor cells sensitive to the killing effect of the PD-1 inhibitor comprises the following steps: (1) culturing the tumor cells in a culture medium containing an inducer to obtain induced tumor cells; and (2) co-culturing the induced tumor cells and DC-CIK cells to obtain the tumor cells sensitive to the killing effect of the PD-1 inhibitor. Compared with the traditional tumor cells, the tumor cells prepared by the method disclosed by the invention have the advantage that the sensitivity to the killing effect of the PD-1 inhibitor can be obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to a tumor cell sensitive to the killing effect of a PD-1 inhibitor and a preparation method thereof. Background Art

[0002] Tumor immunotherapy is a treatment method that activates or enhances the body's own immune system to identify and attack tumor cells. In recent years, dendritic cells (DC) and cytokine-induced killer cells (CIK) combined with PD-1 inhibitors or the two drugs alone have shown good application prospects in tumor treatment.

[0003] CN106890330B discloses a composition of DC-CIK cells and anti-PD-1 antibodies and uses thereof. The patent involves the combined use of DC-CIK cells and anti-PD-1 antibodies (PD-1 inhibitors), specifically using PD-1 inhibitors as activators of DC-CIK cells. The method of use is to co-incubate DC-CIK cells with PD-1 inhibitors. During the incubation process, the PD-1 inhibitors bind to PD-1 on the surface of DC-CIK cells to prevent PD-1 on the surface of DC-CIK cells from binding to PD-L1 on the surface of tumor cells, thereby reducing the escape of tumor cells and improving the killing ability of DC-CIK cells against tumor cells.

[0004] However, current tumor cell therapy methods are not sensitive enough to the killing effects of DC-CIK and / or PD-1, and different tumor cells have different sensitivities to the killing effects of DC-CIK and / or PD-1 inhibitors, which limits their therapeutic effects. Summary of the invention

[0005] The purpose of the present invention is to provide a new method for treating tumor cells, which can significantly increase the sensitivity of tumor cells to the killing effect of PD-1 inhibitors.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors, comprising the following steps:

[0008] (1) culturing tumor cells in a culture medium containing an inducer to obtain induced tumor cells;

[0009] (2) Co-culturing the induced tumor cells with DC-CIK cells to obtain tumor cells that are sensitive to the killing effect of PD-1 inhibitors.

[0010] Preferably, the inducing agent in step (1) is IFN-γ or TNF-α.

[0011] Preferably, the dosage of IFN-γ is 8-12 μg / mL culture medium; the dosage of TNF-α is 8-12 μg / mL culture medium, and the culture medium is 1640 culture medium.

[0012] Preferably, the culture conditions in step (1) are culturing at 37±2°C for 24±2h.

[0013] Preferably, the ratio of the number of induced tumor cells to that of DC-CIK cells is 1:1.

[0014] Preferably, the co-culture condition is culturing at 37±2°C for 24±2h.

[0015] Preferably, the PD-1 inhibitor is Nivolumab, and the effective concentration is 3±0.3 μg / mL.

[0016] Preferably, the tumor cells are nasopharyngeal carcinoma cells or colon cancer cells.

[0017] The present invention also provides tumor cells that are sensitive to the killing effect of PD-1 inhibitors and are obtained by the preparation method.

[0018] Compared with traditional tumor cells, the tumor cells provided by the present invention can significantly improve the sensitivity to the killing effect of PD-1 inhibitors. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0020] Example 1

[0021] Preparation of DC-CIK cells: 50 mL of peripheral blood was collected from healthy donors, and further purified into mononuclear cells by lymphocyte separation fluid gradient centrifugation, and washed twice with serum-free medium to obtain PBMCs with a purity of more than 90%. 2E6 cells / mL PBMCs were transferred to a culture flask containing 1640 medium, and 10 μg / mL IFN-γ, 10 μg / mL CD3 antibody, and 10 μg / mL IL-2 were added for culture at 37°C for 14 days to induce differentiation into DC-CIK cells.

[0022] After the nasopharyngeal carcinoma cell line C666-1 was revived, it was inoculated into 1640 medium and cultured at 37°C for 24 hours. The cell pellet was collected and resuspended in 1640 medium to adjust the cell concentration to 1E6 / mL.

[0023] The above nasopharyngeal carcinoma cells were inoculated into T25 culture flasks, and then IFN-γ was added at a final concentration of 10μg / mL, and cultured at 37°C for 24h to induce PD-L1 expression on the surface of tumor cells. At the same time, tumor cells without IFN-γ induction were used as controls, and the PD-L1 expression rate on the surface of tumor cells in the control group and the above experimental group was measured. The results showed that the PD-L1 expression rate on the surface of the control group was low, averaging about 20%. The PD-L1 expression rate on the surface of the experimental group increased significantly, averaging about 60%, indicating that the inducer successfully induced the expression of PD-L1 on the surface of tumor cells.

[0024] The induced tumor cells in the experimental group were co-cultured with DC-CIK cells in 1640 culture medium at 37°C for 24 hours at a cell number ratio of 1:1. Through cell-to-cell interactions, the sensitivity of tumor cells to PD-1 inhibitors was further enhanced, thereby obtaining nasopharyngeal carcinoma cells that were sensitive to the killing effects of PD-1 inhibitors.

[0025] Finally, PD-1 inhibitor Nivolumab was added to the co-culture system at 3 μg / mL and treated at 37°C for 24 hours (the control group received the same treatment). The sensitivity of the two tumor cells to the killing effect of PD-1 inhibitors was evaluated by indicators such as cell survival rate and apoptosis rate.

[0026] The cell survival rate of the control group (no DC-CIK cells were added subsequently) was higher, averaging about 90%.

[0027] The cell survival rate in the experimental group was significantly reduced, averaging about 30%.

[0028] The apoptosis rate of tumor cells in the control group was lower, averaging about 5%.

[0029] The apoptosis rate of tumor cells in the experimental group increased significantly, reaching an average of about 65%.

[0030] This shows that DC-CIK combined with PD-1 has a significant killing effect on tumor cells.

[0031] Repeatability verification

[0032] To verify the reliability of the experimental results, we conducted multiple repeated experiments. The results showed that the sensitivity of tumor cells was significantly improved in each experiment, and the trends of cell survival rate and apoptosis rate were consistent.

[0033] Example 2

[0034] After the colon cancer cell line SW480 was revived, it was inoculated into 1640 medium and cultured at 37°C for 24 hours. The cell pellet was collected and resuspended in 1640 medium to adjust the cell concentration to 1E5 cells / mL.

[0035] The other treatments were the same as those in Example 1, and colon cancer cells sensitive to the killing effect of PD-1 inhibitors were obtained.

[0036] It can be seen from the above examples that, compared with traditional tumor cells, the tumor cells prepared by the method of the present invention can significantly improve the sensitivity to the killing effect of PD-1 inhibitors, thereby improving the therapeutic efficacy.

[0037] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors, It is characterized in that The steps include: (1) culturing tumor cells in a culture medium containing an inducer to obtain induced tumor cells; (2) Co-culturing the induced tumor cells with DC-CIK cells to obtain tumor cells that are sensitive to the killing effect of PD-1 inhibitors.

2. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 1, It is characterized in that The inducing agent in step (1) is IFN-γ or TNF-α.

3. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 2, It is characterized in that The concentration of the IFN-γ is 8-12 μg / mL; the concentration of the TNF-α is 8-12 μg / mL, and the culture medium is 1640 culture medium.

4. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 3, It is characterized in that The culture conditions in step (1) are culturing at 37±2°C for 24±2h.

5. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 1, It is characterized in that The ratio of the number of induced tumor cells to that of DC-CIK cells is 1:

1.

6. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 5, It is characterized in that The co-culture condition is culturing at 37±2°C for 24±2h.

7. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 1, It is characterized in that The PD-1 inhibitor is Nivolumab, and the effective concentration is 3±0.3 μg / mL.

8. A method for preparing tumor cells that are sensitive to the killing effect of PD-1 inhibitors as claimed in claim 1, It is characterized in that The tumor cells are nasopharyngeal carcinoma cells or colon cancer cells.

9. A tumor cell sensitive to the killing effect of a PD-1 inhibitor obtained by the preparation method according to any one of claims 1 to 8.