Preparation for improving anti-tumor effect of NKT cells, anti-tumor preparation and preparation method of anti-tumor preparation

Abstract

The invention provides a preparation for improving the anti-tumor effect of NKT cells, an anti-tumor preparation and a preparation method of the anti-tumor preparation, and belongs to the technical field of anti-tumor preparations. The research finds that (1, 6)-beta-D-glucan can enhance the killing ability of NKT cells to tumor cells and can effectively inhibit the growth of the tumor cells. The anti-tumor preparation provided by the invention contains (1, 6)-beta-D-glucan and NKT cells, and the killing power of the preparation on tumors is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor preparations, and in particular relates to a preparation for improving the anti-tumor effect of NKT cells, an anti-tumor preparation and a preparation method thereof. Background Art

[0002] Human peripheral blood contains white blood cells, red blood cells, platelets, and plasma. Among them, mononuclear cells (PBMC) are a part of white blood cells and belong to a type of immune cells. PBMC contains lymphocytes, monocytes, and a small number of dendritic cells. Lymphocytes contain T lymphocytes, B lymphocytes, and NK cells. T cells play a killing role after recognizing specific antigens on the surface of tumors. NK cells have natural killing activity and can directly kill virus-infected cells and tumor cells without prior sensitization. They have a broad spectrum of anti-tumor activity. B cells attack tumors by producing antibodies, which belongs to humoral immunity. T cells and NK cells are both immune cells.

[0003] NKT cells, also known as CIK (cytokine-induced killer) cells, are T cells with NK-like cells. They have shown certain efficacy in the treatment of tumor diseases. Currently, research on NKT cells mainly focuses on the culture system of NKT cells and the short-term transportation and preservation fluid of NKT cells. There are currently no reports on how to further enhance the anti-tumor effect of NKT cells. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the use of (1,6)-β-D-glucan in the preparation of a product for improving the anti-tumor effect of NKT cells. The use of (1,6)-β-D-glucan can enhance the killing ability of NKT cells against tumor cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Application of (1,6)-β-D-glucan in the preparation of products for improving the anti-tumor effect of NKT cells.

[0007] Preferably, the (1,6)-β-D-glucan increases the killing rate of NKT cells against tumor cells.

[0008] Another object of the present invention is to provide a preparation for improving the anti-tumor effect of NKT cells, wherein the preparation contains (1,6)-β-D-glucan.

[0009] Another object of the present invention is to provide an anti-tumor preparation, which contains, by volume, 1 to 2 parts of (1,6)-β-D-glucan solution, 25 to 50 parts of NKT cells, 1 to 5 parts of human albumin solution and 50 to 70 parts of 0.9% sodium chloride injection.

[0010] Preferably, the concentration of the (1,6)-β-D-glucan solution is 1 g / mL, the mass volume fraction of the human albumin solution is 20%, and the concentration of the NKT cells is 1×10 8 ~2×10 8 cells / mL.

[0011] Another object of the present invention is to provide a method for preparing an anti-tumor preparation, comprising the following steps: isolating mononuclear cells from peripheral blood, resuspending the mononuclear cells with a complete culture medium and then adding the complete culture medium for culturing, adding IFN-γ, CD3 antibody and CD28 antibody to the culture medium on the first day of culture, adding IL-2 to the culture medium on the 3rd, 5th, 7th, 10th and 12th days of culture, respectively, collecting the culture medium on the 14th day of culture and centrifuging, and collecting the precipitate to obtain NKT cells; resuspending the NKT cell precipitate with a human albumin solution and a 0.9% sodium chloride injection solution, and then adding a (1,6)-β-D-glucan solution to obtain the anti-tumor preparation.

[0012] Preferably, the final concentration of IFN-γ is 500-1500 U / mL, the final concentration of CD3 monoclonal antibody is 50-150 ng / mL, the final concentration of CD28 monoclonal antibody is 15-25 ng / mL, and the final concentration of IL-2 is 300-800 IU / mL.

[0013] Preferably, after collecting the culture medium on the 14th day of culture, centrifuge at 560-700 g for 7-9 min; collect the precipitate, add 0.9% sodium chloride injection for washing, and centrifuge at 260-430 g for 4-6 min. Repeat this process twice and collect the precipitate, which is the NKT cell.

[0014] Preferably, the culture is carried out at 37°C, 5% CO 2 cultured in an incubator.

[0015] Another object of the present invention is to provide the use of the anti-tumor preparation or the preparation method in the preparation of drugs for treating cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides the use of (1,6)-β-D-glucan in the preparation of a product that improves the anti-tumor effect of NKT cells. The present invention has found that (1,6)-β-D-glucan can enhance the killing ability of NKT cells on tumor cells and can effectively inhibit the growth of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is a statistical analysis chart of the killing ability of Example 1 and Comparative Example 1 on gastric cancer cells in Example 7. DETAILED DESCRIPTION

[0019] The present invention provides the use of (1,6)-β-D-glucan in the preparation of a product for improving the anti-tumor effect of NKT cells, wherein the (1,6)-β-D-glucan improves the killing ability of NKT cells on tumor cells.

[0020] The present invention also provides a preparation for improving the anti-tumor effect of NKT cells, the preparation containing (1,6)-β-D-glucan. The (1,6)-β-D-glucan in the present invention is purchased from Sigma Company and can effectively improve the killing ability of NKT cells against tumor cells.

[0021] The present invention also provides an anti-tumor preparation, which contains, by volume, 1 to 2 parts of (1,6)-β-D-glucan solution, 25 to 50 parts of NKT cells, 1 to 5 parts of human serum albumin solution and 50 to 70 parts of 0.9% sodium chloride injection; preferably, it contains 1 to 1.5 parts of (1,6)-β-D-glucan solution, 30 to 40 parts of NKT cells, 2 to 3 parts of human serum albumin solution and 60 to 68 parts of 0.9% sodium chloride injection; more preferably, it contains 1 part of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human serum albumin solution and 67 parts of 0.9% sodium chloride injection.

[0022] In the present invention, the concentration of the (1,6)-β-D-glucan solution is 1 g / mL, the mass volume fraction of the human albumin solution is 20%, and the concentration of the NKT cells is 1×10 8 ~2×10 8 cells / mL. In a specific embodiment of the present invention, the human albumin solution is purchased from Green Cross Co., Ltd. with a specification of 10g, 50mL, that is, the concentration of the human albumin solution is 20% (w / v). The concentration described in the present invention is the original concentration of the solution added when preparing the anti-tumor preparation. Taking the (1,6)-β-D-glucan solution as an example, in the anti-tumor preparation containing 1 part of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human albumin solution and 67 parts of 0.9% sodium chloride injection by volume, the original concentration of the added (1,6)-β-D-glucan solution is 1g / mL, and the final concentration of (1,6)-β-D-glucan in the above preparation is 10mg / mL.

[0023] The anti-tumor preparation provided by the invention has high ability to kill tumor cells.

[0024] The present invention also provides a method for preparing an anti-tumor preparation, comprising the following steps: isolating mononuclear cells from peripheral blood, resuspending the mononuclear cells in complete culture medium, and then supplementing the complete culture medium for culturing, wherein the culturing is carried out at 37°C and 5% CO 2 The cells are cultured in an incubator; IFN-γ, CD3 antibody and CD28 antibody are added to the culture medium on the first day of culture; IL-2 is added to the culture medium on the 3rd, 5th, 7th, 10th and 12th days of culture, respectively; the culture medium is collected and centrifuged on the 14th day of culture, and the precipitate is NKT cells; the NKT cell precipitate is resuspended with human albumin solution and 0.9% sodium chloride injection, and then (1,6)-β-D-glucan solution is added to obtain an anti-tumor preparation.

[0025] In the preparation method provided by the present invention, peripheral blood is collected, Ficoll liquid density gradient centrifugation is used, and mononuclear cells (PBMC) in the peripheral blood are separated using a lymphocyte high-efficiency centrifuge tube. The obtained PBMC is washed twice with physiological saline, and the PBMC is resuspended with complete medium, transferred into a culture bottle, and then supplemented with complete medium. The complete medium is a mixture of serum-free basal medium and plasma, or the complete medium is a mixture of serum-free basal medium and human serum substitute. When the complete medium is serum-free basal medium and plasma, the serum-free basal medium and plasma are mixed at a volume ratio of 50:1. When the complete medium is serum-free basal medium and human serum substitute, the serum-free basal medium and human serum substitute are mixed at a volume ratio of 20:1. In a specific embodiment of the present invention, the serum-free basal medium is GT-T551 medium purchased from TAKARA, and the human serum substitute is purchased from AventaCell BioMedical Corp.

[0026] In the present invention, the final concentration of IFN-γ is 500-1500U / mL, preferably 800-1200U / mL, and more preferably 1000U / mL; the final concentration of CD3 monoclonal antibody is 50-150ng / mL, preferably 80-120ng / mL, and more preferably 100ng / mL; the final concentration of CD28 monoclonal antibody is 15-25ng / mL, 18-22ng / mL, and more preferably 20ng / mL; the final concentration of IL-2 is 300-800IU / mL, preferably 400-600IU / mL, and more preferably 500IU / mL.

[0027] In the present invention, after the culture medium is collected on the 14th day of culture, centrifugation is first performed at a centrifugal force of 560-700g for 7-9 minutes, preferably 635g, and the centrifugation time is preferably 8 minutes; the precipitate is collected, added with 0.9% sodium chloride injection for washing, and then centrifuged at a centrifugal force of 260-430g for 4-6 minutes, preferably 400g, and the centrifugation time is preferably 5 minutes. After repeating twice, the collected precipitate is the NKT cell.

[0028] Another object of the present invention is to provide the use of the anti-tumor preparation or the preparation method in the preparation of drugs for treating cancer.

[0029] 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.

[0030] Example 1

[0031] An antitumor preparation, comprising, by volume, 1 part of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human albumin solution, and 67 parts of 0.9% sodium chloride injection; wherein the concentration of the (1,6)-β-D-glucan solution is 1 g / mL, the mass volume fraction of the human albumin solution is 20%, the concentration of the NKT cells is 1.67×10 8 The final concentration of (1,6)-β-D-glucan solution in the preparation was 10 mg / mL.

[0032] The preparation method is as follows: 80 mL of peripheral blood is collected, Ficoll liquid density gradient centrifugation is used, and mononuclear cells (PBMCs) in the peripheral blood are separated using a lymphocyte high-efficiency centrifuge tube, the obtained PBMCs are washed twice with normal saline, and the PBMCs are resuspended with 30 mL of complete culture medium, and then transferred into a T175 cell culture flask and 50 mL of complete culture medium is added, wherein the complete culture medium is a mixture of serum-free basal culture medium and human serum substitute in a volume ratio of 20:1.

[0033] at 37°C, 5% CO 2 The cells were cultured in an incubator. On the first day of culture, IFN-γ was added to the culture medium to a final concentration of 1000U / mL, CD3 monoclonal antibody to a final concentration of 100ng / mL, and CD28 monoclonal antibody to a final concentration of 20ng / mL. On the 3rd, 5th, 7th, 10th, and 12th days of culture, IL-2 was added to the culture medium to a final concentration of 500IU / mL, and the same amount of complete culture medium as the original bottle was supplemented. On the 7th day of culture, the cells in the culture flask were transferred to a 2L cell culture bag, and each culture bag was supplemented to 250mL of complete culture medium for continued culture.

[0034] On the 14th day of culture, the culture medium was collected and centrifuged at 635g for 8 minutes; the precipitate was collected, added to 0.9% sodium chloride injection for washing, and then centrifuged at 400g for 5 minutes. After repeating twice, the precipitate collected was the NKT cells; the NKT cell precipitate was resuspended with human albumin solution and 0.9% sodium chloride injection, and then the (1,6)-β-D-glucan solution was added to obtain the anti-tumor preparation.

[0035] Example 2

[0036] An anti-tumor preparation, the difference between this embodiment and embodiment 1 is that: by volume, it contains 1.5 parts of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human albumin solution and 66.5 parts of 0.9% sodium chloride injection. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 15 mg / mL.

[0037] Example 3

[0038] An anti-tumor preparation, the difference between this embodiment and embodiment 1 is that: it contains 2 parts of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human albumin solution and 66 parts of 0.9% sodium chloride injection by volume. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 20 mg / mL.

[0039] Example 4

[0040] An anti-tumor preparation, the difference between this embodiment and embodiment 1 is that: by volume, it contains 1.5 parts of (1,6)-β-D-glucan solution, 35 parts of NKT cells, 2.5 parts of human albumin solution and 61 parts of 0.9% sodium chloride injection. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 15 mg / mL.

[0041] Example 5

[0042] An anti-tumor preparation, the difference between this embodiment and embodiment 1 is that: it contains 2 parts of (1,6)-β-D-glucan solution, 25 parts of NKT cells, 3 parts of human albumin solution and 70 parts of 0.9% sodium chloride injection by volume. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 20 mg / mL.

[0043] Example 6

[0044] An anti-tumor preparation, the difference between this embodiment and embodiment 1 is that: it contains 1 part of (1,6)-β-D-glucan solution, 45 parts of NKT cells, 4 parts of human albumin solution and 50 parts of 0.9% sodium chloride injection by volume. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 10 mg / mL.

[0045] Comparative Example 1

[0046] An anti-tumor preparation, the difference between this comparative example and Example 1 is that: it contains 30 parts of NKT cells, 2 parts of human albumin solution and 68 parts of 0.9% sodium chloride injection by volume.

[0047] Comparative Example 2

[0048] An antitumor preparation, the difference between this comparative example and Example 1 is that: it contains 1 part of (1,6)-β-D-glucan solution, 2 parts of human albumin solution and 97 parts of 0.9% sodium chloride injection by volume. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 10 mg / mL.

[0049] Comparative Example 3

[0050] An anti-tumor preparation, the difference between this comparative example and Example 1 is that: it contains 0.5 parts of (1,6)-β-D-glucan solution, 30 parts of NKT cells, 2 parts of human albumin solution and 67.5 parts of 0.9% sodium chloride injection by volume. The final concentration of the (1,6)-β-D-glucan solution in the preparation is 5 mg / mL.

[0051] Example 7

[0052] This example compares the ability of different preparations to kill tumor cells.

[0053] 1. Ability to kill gastric cancer cells.

[0054] The preparation of Example 1 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as preparation 1;

[0055] The preparation of comparative example 1 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as comparative preparation 1;

[0056] The preparation of comparative example 2 was diluted at the same dilution ratio as preparation 1 and recorded as comparative preparation 2.

[0057] The 96-well plate co-culture combined with CCK8 method was used to inoculate the NKT cells prepared in the embodiment of the present invention into a 96-well plate, with 2×10 5 cells as the effector cell group; gastric cancer SGC7901 cells (purchased from Beina Biotechnology Co., Ltd.) were inoculated in a 96-well plate, with 2×10 4 cells (concentration of 2×10 5 cells / mL, inoculated with 100 μL), and then the wells inoculated with gastric cancer SGC7901 cells were divided into 4 groups:

[0058] Target cell group: add 50 μL 0.9% sodium chloride injection;

[0059] Example 1: Add 50 μL of Preparation 1 to the well inoculated with gastric cancer SGC7901 cells;

[0060] Comparative Example 1: 50 μL of comparative preparation 1 was added to the wells inoculated with gastric cancer SGC7901 cells;

[0061] Comparative Example 2: 50 μL of comparative preparation 2 was added to the wells inoculated with gastric cancer SGC7901 cells.

[0062] Place the 96-well plate at 37 °C, 5% CO 2 Incubate in the incubator for 24 hours. After the incubation, take out the 96-well plate, add 10 μL cck8 reagent to each well, tap the well plate gently to mix, and put it into the incubator for 4 hours. After the incubation, add the stop solution to each well and mix to stop the color development reaction, and use a microplate reader to detect the absorbance OD value (wavelength 450nm). Record the OD value and calculate the killing rate. The killing rate calculation formula in Example 1 and Comparative Example 1 is as follows:

[0063] Killing rate = 1-[(OD 杀伤 -OD 效应 ) / OD 靶细胞 ]×100%.

[0064] In the wells of the killing group (Example 1 and Comparative Example 1), NKT cells and gastric cancer cells exist at the same time. The OD value of the killing group minus the OD value of the effector cell group is divided by the OD value of the gastric cancer cell group (target cell group) inoculated at the same time to obtain the percentage of gastric cancer cell survival rate.

[0065] The formula for calculating the killing rate in Comparative Example 2 is: killing rate = 1-(OD 对比例2 / OD 靶细胞 )×100%.

[0066] In this test, three holes were tested and the test was repeated three times. The results are shown in Table 1.

[0067] Table 1 The ability of different preparations to kill gastric cancer cells

[0068]

[0069]

[0070] The killing rates of Example 1 (experimental group) and Comparative Example 1 (control group) were calculated and then compared and statistically analyzed using T test. The results are as follows: Figure 1As shown, P value = 0.000240477, P < 0.01, which is a significant difference. It can be seen that the anti-tumor preparation provided by the present invention significantly enhances the ability of NKT to kill gastric cancer cells. In the anti-tumor preparation provided by the present invention, (1,6)-β-D-glucan and NKT cells work synergistically to effectively improve the killing effect on gastric cancer cells.

[0071] 2. Ability to kill leukemia cells.

[0072] The method in “1. Killing ability against gastric cancer cells” was adopted to replace gastric cancer SGC7901 cells with leukemia cells K562 (purchased from Beina Biotechnology Co., Ltd.) for testing. The results are shown in Table 2.

[0073] Table 2 The ability of different preparations to kill leukemia cells

[0074]

[0075] The kill rates of the comparative example 1 and the example 1 groups were calculated by the mean values ​​and then compared and statistically analyzed using a T test. The results are shown in Table 3.

[0076] Table 3 Significant difference

[0077] Serial number Group and Group P-value Is it significant 1 Comparative Example 1 VS. Example 1 0.0012 **

[0078] It can be seen that the anti-tumor preparation provided by the present invention significantly enhances the ability of NKT cells to kill leukemia cells. In the anti-tumor preparation provided by the present invention, (1,6)-β-D-glucan and NKT cells work synergistically to effectively enhance the killing effect on leukemia cells.

[0079] Example 8

[0080] This example compares the ability of different preparations to kill tumor cells.

[0081] The preparation of Example 1 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as preparation 1;

[0082] The preparation of Example 2 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as preparation 2;

[0083] The preparation of Example 3 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as preparation 3;

[0084] The preparation of comparative example 1 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as comparative preparation 1;

[0085] The preparation of comparative example 3 was diluted to a NKT cell concentration of 4×10 6 cells / mL, recorded as comparative preparation 3.

[0086] The 96-well plate co-culture combined with CCK8 method was used to inoculate the NKT cells prepared in the embodiment of the present invention into a 96-well plate, with 2×10 5 cells as the effector cell group; gastric cancer SGC7901 cells (purchased from Beina Biotechnology Co., Ltd.) were inoculated in a 96-well plate, with 2×10 4 cells (concentration of 2×10 5 cells / mL, inoculated with 100 μL), and then the wells inoculated with gastric cancer SGC7901 cells were divided into 6 groups:

[0087] Target cell group: add 50 μL 0.9% sodium chloride injection;

[0088] Example 1: Add 50 μL of Preparation 1 to the well inoculated with gastric cancer SGC7901 cells;

[0089] Example 2: Add 50 μL of Preparation 2 to the wells inoculated with gastric cancer SGC7901 cells;

[0090] Example 3: Add 50 μL of formulation 3 to the wells inoculated with gastric cancer SGC7901 cells;

[0091] Comparative Example 1: 50 μL of comparative preparation 1 was added to the wells inoculated with gastric cancer SGC7901 cells;

[0092] Comparative Example 3: 50 μL of comparative preparation 3 was added to the wells inoculated with gastric cancer SGC7901 cells.

[0093] Place the 96-well plate at 37 °C, 5% CO 2 Incubator for 24 hours, take out the 96-well plate after the culture, add 10 μL cck8 reagent to each well, tap the well plate gently to mix, and put it into the incubator for 4 hours. After the incubation, add the stop solution to each well and mix to stop the color reaction, and use a microplate reader to detect the absorbance OD value (wavelength 450nm). Record the OD value, calculate the killing rate, and the calculation formula is shown in Example 7. The results are shown in Table 4. This test was carried out simultaneously with the "1. Killing ability of gastric cancer cells" test in Example 7, so the test results of the target cell group, effector cell group, comparative example 1 group and example 1 group are the same as the results in Table 1.

[0094] Table 4 The ability of different preparations to kill tumor cells

[0095]

[0096]

[0097] The killing rates of Examples 1 to 3, Comparative Examples 1 and 3 were calculated by means of the T test and compared with the statistical analysis. The results are shown in Table 5.

[0098] Table 5 Significant difference

[0099] Serial number Group and Group P-value Is it significant 1 Comparative Example 1 VS. Comparative Example 3 0.3341 ns 2 Comparative Example 1 VS. Example 1 <0.0001 **** 3 Comparative Example 1 VS. Example 2 0.0022 ** 4 Comparative Example 1 VS. Example 3 0.0049 **

[0100] It can be seen that the anti-tumor preparation provided by the present invention contains 10 to 20 mg / mL of (1,6)-β-D-glucan, which effectively improves the ability of NKT to kill gastric cancer cells.

[0101] 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. Application of (1,6)-β-D-glucan in the preparation of products for enhancing the anti-tumor effect of NKT cells.

2. The use according to claim 1, characterized in that: The (1,6)-β-D-glucan increases the killing rate of NKT cells on tumor cells.

3. A preparation for improving the anti-tumor effect of NKT cells, characterized in that: The preparation contains (1,6)-β-D-glucan.

4. An anti-tumor preparation, characterized in that: The anti-tumor preparation contains 1-2 parts of (1,6)-β-D-glucan solution, 25-50 parts of NKT cells, 1-5 parts of human albumin solution and 50-70 parts of 0.9% sodium chloride injection by volume.

5. The anti-tumor preparation according to claim 4, characterized in that The concentration of the (1,6)-β-D-glucan solution is 1 g / mL, the mass volume fraction of the human albumin solution is 20%, and the concentration of the NKT cells is 1×10 8 ~2×10 8 cells / mL.

6. A method for preparing an antitumor preparation, characterized in that: The steps include: The mononuclear cells in the peripheral blood are separated, and the mononuclear cells are resuspended in a complete medium and then supplemented with the complete medium for culture. On the first day of culture, IFN-γ, CD3 monoclonal antibody and CD28 monoclonal antibody are added to the culture medium. On the 3rd, 5th, 7th, 10th and 12th days of culture, IL-2 is added to the culture medium respectively. On the 14th day of culture, the culture medium is collected and centrifuged, and the collected precipitate is NKT cells; the NKT cell precipitate is resuspended in a human albumin solution and a 0.9% sodium chloride injection solution, and then a (1,6)-β-D-glucan solution is added to obtain an anti-tumor preparation.

7. The preparation method according to claim 6, characterized in that: The final concentration of IFN-γ is 500-1500 U / mL, the final concentration of CD3 monoclonal antibody is 50-150 ng / mL, the final concentration of CD28 monoclonal antibody is 15-25 ng / mL, and the final concentration of IL-2 is 300-800 IU / mL.

8. The preparation method according to claim 6, characterized in that: On the 14th day of culture, the culture medium was collected and centrifuged at 560-700 g for 7-9 min. The precipitate was collected, washed with 0.9% sodium chloride injection, and then centrifuged at 260-430 g for 4-6 min. This was repeated twice and the precipitate was collected, which was the NKT cells.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The culture was carried out in an incubator at 37° C. and 5% CO 2 .

10. Use of the antitumor preparation according to claim 4 or 5 or the preparation method according to any one of claims 6 to 9 in the preparation of a drug for treating cancer.

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