Method for enhancing antitumor activity of NK (Natural Killer) cells

Abstract

The invention discloses a method for enhancing anti-tumor activity of NK cells, and relates to the technical field of anti-tumor treatment of NK cells, peripheral blood mononuclear cells are separated through a Ficoll density gradient centrifugation method, and CD56 + CD3-NK cells are purified after IL-2 amplification. Then, the NK cells are co-cultured with IL-21 (interleukin-21), DZNp (2, 3-phthalazinone) and resveratrol to promote immune activation, epigenetic regulation and antioxidation of the NK cells, so that the anti-tumor capability of the NK cells is remarkably enhanced. Through the synergistic effect of the IL-21, the DZNep and the resveratrol, the anti-tumor effect of the NK cells is maximized. IL-21 activates immune response, DZNep enhances cytotoxicity, resveratrol alleviates oxidation pressure, and IL-21, DZNep and resveratrol synergistically optimize immunocompetence and anti-tumor ability of NK cells and overcome a tumor immune escape mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell anti-tumor treatment, and in particular to a method for enhancing the anti-tumor activity of NK cells. Background Art

[0002] As an emerging cancer treatment, tumor immunotherapy has made significant clinical progress in recent years. Tumor immunotherapy has become an important means of treating various tumors by enhancing the body's immune system's ability to recognize and kill tumor cells, especially by adoptive immunotherapy to enhance the patient's immune ability. Natural killer cells (NK cells) are an important part of the innate immune system and have the ability to directly kill tumor cells and virus-infected cells, independent of prior antigen sensitization. NK cells recognize and attack abnormal tumor cells through specific immune receptors, such as NKG2D receptors. In recent years, researchers have achieved certain clinical efficacy by using cytokines to amplify and activate NK cells and adoptively immunotherapy them in clinical trials. However, due to the inhibitory effect of the tumor microenvironment, the function of NK cells in the peripheral blood of tumor patients is significantly reduced, which directly affects their effect in tumor immunotherapy. Therefore, how to effectively enhance the anti-tumor activity of NK cells has become a technical problem that needs to be solved in the field of tumor immunotherapy.

[0003] Existing related technologies mainly focus on activating and amplifying NK cells through cytokines or small molecule drugs to improve their anti-tumor ability. Current immune cell therapy methods mainly include the application of cytokines such as IL-2 and IL-15, as well as improving the tumor-killing ability of NK cells through genetic engineering. Although these technologies have improved the anti-tumor effect of NK cells to a certain extent, there are several obvious shortcomings. First, although the cytokine amplification method can increase the number of NK cells, the effect of enhancing their function is limited, especially in the tumor microenvironment, NK cells still find it difficult to effectively play their tumor-killing function. Secondly, although some small molecule drugs can promote the proliferation and activation of NK cells, the enhancement of cytotoxicity is not significant, and the treatment process is often accompanied by side effects, such as immune-related side effects such as cytokine storms. More importantly, NK cells often have an immune tolerance state in tumor patients, especially the interaction with the immune escape mechanism between tumor cells, which greatly reduces the immune surveillance function of NK cells. For example, tumor cells inhibit the function of NK cells by downregulating the expression of NKG2D ligands, thereby escaping immune surveillance. Although these existing technologies have improved the anti-tumor ability of NK cells to a certain extent, they have not fundamentally solved the fundamental problem of decreased NK cell function, especially how to restore or enhance the anti-tumor activity of NK cells by regulating epigenetic mechanisms.

[0004] Most of the current technologies focus on regulating the function of NK cells through the application of immune cytokines or other external stimuli. However, the existing technologies have failed to effectively solve the root cause of the decline in NK cell function. The functional inhibition of NK cells in tumor patients is largely caused by the immune escape mechanism in the tumor microenvironment. For example, tumor cells inhibit the anti-tumor effect of NK cells by downregulating the expression of NKG2D receptor ligands. In addition, some inhibitory receptors on the surface of NK cells, such as PD-1, may also play an important role in the process of tumor immune escape. The regulatory role of these epigenetic mechanisms is an area that most existing technologies have not yet covered. Therefore, how to regulate the immune function of NK cells through epigenetic means to overcome the escape of tumor cells from the immune system has become an important direction for improving the anti-tumor therapeutic effect of NK cells.

[0005] Therefore, a new method is needed to effectively enhance the anti-tumor ability of NK cells, especially to overcome the immune escape mechanism in the tumor microenvironment and restore or enhance the immune surveillance function of NK cells. In particular, how to enhance the function of NK cells through epigenetic regulation is a technical challenge in current tumor immunotherapy. The research of the present invention aims to activate NK cells through epigenetic regulation, using specific small molecules, enhance their anti-tumor ability, and effectively overcome the tumor immune escape mechanism, and ultimately improve the efficacy of NK cell adoptive immunotherapy. Summary of the invention

[0006] In order to achieve the above-mentioned purpose of the invention and to solve the above-mentioned technical problems, the present invention provides a method for enhancing the anti-tumor activity of NK cells, the method comprising the following steps:

[0007] (a) Mononuclear cells were isolated from peripheral blood using Ficoll density gradient centrifugation and used to isolate NK cells;

[0008] (b) PBMCs were suspended in culture medium, the cell concentration was adjusted, 500 IU / mL of IL-2 was added to continue proliferation, and CD56 was purified. + CD3 - of NK cells.

[0009] (c) distributing the NK cells into a plurality of culture flasks, adding IL-21 (interleukin-21) solution into the culture flasks, and continuing to culture;

[0010] (d) DZNep (2,3-naphthylidene) and resveratrol were added to the culture medium, and NK cells were further cultured.

[0011] Preferably, the specific steps of step (a) are: slowly adding the collected peripheral blood sample to the Ficoll centrifuge solution, centrifuging at 1600 r / min for 30 minutes using a centrifuge. By gradient separation, the PBMC layer between the Ficoll layer and the plasma layer is collected;

[0012] The PBMCs were then carefully transferred from the Ficoll layer to a new centrifuge tube and washed with PBS buffer to remove plasma and unwanted cellular impurities, and were then prepared for NK cell isolation.

[0013] Preferably, in step (b), the cell density is adjusted to 1×10 6 -5×10 6 Pieces / mL.

[0014] Preferably, the X-VIVO 15 medium used in step (b) does not contain serum, the culture conditions are 37° C., 5% CO 2 , and the medium is replaced every 2-3 days during the culture process.

[0015] Preferably, in step (c), the final concentration of IL-21 in the culture medium is 20-50 ng / mL.

[0016] Preferably, the final concentration of DZNep in the culture medium is 1-5 μM, and the final concentration of resveratrol in the culture medium is 10-50 μM.

[0017] Preferably, the amplification time of step (b) is 12 days.

[0018] Preferably, the culturing time in step (c) is 24 hours.

[0019] Preferably, in step (d), the NK cells are cultured in an environment of 37° C. and 5% CO 2 for 24-72 hours.

[0020] The technical solution provided by the present invention brings beneficial effects:

[0021] Synergistic effect of immune activation and epigenetic regulation: The present invention effectively regulates the immune function of NK cells by combining IL-21, DZNep and resveratrol. As an immune enhancement factor, IL-21 can activate the proliferation and activity of NK cells and enhance their cytotoxicity; DZNep enhances the gene expression of NK cells through epigenetic regulation, increases the production of cytotoxic molecules, and further enhances the anti-tumor ability of NK cells. Resveratrol reduces cellular oxidative stress and protects NK cells from oxidative damage through antioxidant effects. These three complement each other and work together through different mechanisms to significantly enhance the immune activity of NK cells.

[0022] Maximize the anti-tumor effect of NK cells: The concentration combination of IL-21, DZNep and resveratrol is carefully designed to coordinate immune activation, epigenetic regulation and antioxidant effects, maximizing the anti-tumor ability of NK cells. The appropriate concentration of IL-21 provides a strong immune enhancement effect, the epigenetic regulation of DZNep effectively enhances the cytotoxicity of NK cells, and resveratrol improves the function of NK cells by reducing oxidative stress, thereby achieving the best level of NK cell killing effect on tumor cells.

[0023] Reducing immune burden and optimizing anti-tumor activity: By adjusting the concentration combination of IL-21, DZNep and resveratrol, the present invention can avoid the problem of excessive immune burden and optimize the immune response and anti-tumor activity of NK cells.

[0024] Overcoming tumor immune escape mechanism: The technical solution of the present invention can effectively enhance the immune surveillance function of NK cells and overcome the immune escape mechanism in the tumor microenvironment by combining epigenetic regulation and immune activation. Experimental results show that the combined use of IL-21, DZNep and resveratrol not only enhances the immune activity of NK cells, but also enhances their ability to recognize and kill tumor cells, thereby effectively restoring the anti-tumor function of NK cells.

[0025] Comprehensive regulation of immune response and cytotoxicity: The combination provided by the present invention achieves the best balance between the immune response and cytotoxicity of NK cells by precisely regulating the concentrations of IL-21, DZNep and resveratrol. IL-21 promotes the immune activation of NK cells at a reasonable concentration, DZNep further increases cytotoxicity through epigenetic regulation, and resveratrol relieves oxidative stress in the immune response, ensuring that the anti-tumor activity of NK cells is not affected by adverse factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of the embodiments of the present invention on NK cell cytotoxicity, the target cells are MDA-MB-231, P < 0.05;

[0027] Figure 2 The NK cells were treated with different concentrations of the combination in the embodiment of the present invention to detect their killing effect on MDA-MB-231 target cells;

[0028] Figure 3 This is an embodiment of the present invention that down-regulates the expression of CD107a on NK cells. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. Of course, the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] A method for enhancing the anti-tumor activity of NK cells, comprising the following steps:

[0032] Step (a): Isolation of PBMCs by Ficoll density gradient centrifugation

[0033] Collection of peripheral blood samples: Peripheral blood was collected and stored in EDTA anticoagulant tubes.

[0034] Isolation of PBMCs: The collected peripheral blood samples were slowly added to Ficoll centrifuge fluid (Ficoll-Paque PLUS, GE Healthcare) to ensure a good interface between the blood sample and the Ficoll fluid.

[0035] Centrifugation conditions: Use a centrifuge at 1600 rpm for 30 minutes and set the centrifuge to room temperature.

[0036] Collect the PBMC layer: Collect the PBMC layer between the Ficoll layer and the plasma layer by gradient separation. Carefully transfer the PBMCs from the Ficoll layer to a new centrifuge tube.

[0037] Washing: PBMCs were washed twice with PBS buffer (calcium-free and magnesium-free) at 300 r / min for 5 minutes. After washing, the supernatant was removed to obtain clean PBMCs.

[0038] Step (b): Isolation and expansion of NK cells

[0039] Cell suspension: PBMCs were suspended in serum-free X-VIVO 15 medium and the cell concentration was adjusted to 2 × 10 6 Pieces / mL.

[0040] Add IL-2: Add 500 IU / mL of recombinant IL-2 (PeproTech) to the cell suspension to continue expanding NK cells.

[0041] Cell culture: Place cells in an incubator at 37°C and 5% CO2 for 12 days. Change the culture medium every 2-3 days to maintain an optimal cell growth environment.

[0042] Cell purification: using CD56 + CD3 -Purification of CD56 from PBMCs by antibody magnetic bead separation + CD3 - of NK cells.

[0043] Step (c): IL-21 treatment

[0044] Add IL-21: Distribute the purified NK cells into multiple culture flasks and add IL-21 (PeproTech) solution to each culture flask. The final concentration of IL-21 is 30 ng / mL.

[0045] Continue to culture: continue to culture the cells for 24 hours in an environment of 37°C and 5% CO2.

[0046] Step (d): Adding DZNep and resveratrol

[0047] Addition of DZNep: DZNep (2,3-naphthylidene, Selleck Chemicals) was added to the culture medium at a final concentration of 3 μM.

[0048] Addition of resveratrol: Resveratrol (Sigma-Aldrich) was added to the culture medium at the same time, with a final concentration of 30 μM.

[0049] Continue to culture: continue to culture NK cells in a 37°C, 5% CO2 environment for 24 hours.

[0050] Final culture conditions:

[0051] Temperature: 37℃

[0052] CO2 concentration: 5%

[0053] The culture flasks are sealed and placed in an incubator to ensure a constant temperature and stable gas environment.

[0054] Example 2

[0055] The preparation was carried out in the same manner as in Example 1, except that the final concentration of IL-21 was 50 ng / mL; the final concentration of DZNep was 1 μM; and the final concentration of resveratrol was 40 μM.

[0056] Example 3

[0057] The preparation was carried out in the same manner as in Example 1, except that the final concentration of IL-21 was 40 ng / mL; the final concentration of DZNep was 5 μM; and the final concentration of resveratrol was 10 μM.

[0058] Example 4

[0059] The preparation was carried out in the same manner as in Example 1, except that the final concentration of IL-21 was 20 ng / mL; the final concentration of DZNep was 3 μM; and the final concentration of resveratrol was 50 μM.

[0060] Comparative Example 1

[0061] The preparation was carried out in the same manner as in Example 1, except that step (d) was omitted and DZNep and resveratrol were not added.

[0062] Comparative Example 2

[0063] The preparation was carried out in the same manner as in Example 1, except that resveratrol was not added.

[0064] Comparative Example 3

[0065] The preparation was carried out in the same manner as in Example 1, except that DZNep was not added.

[0066] Comparative Example 4

[0067] The preparation was carried out in the same manner as in Example 1, except that, as a blank control, no IL-21, DZNep or resveratrol was added.

[0068] Experimental test:

[0069] 1. Cytotoxicity assay

[0070] The cytotoxicity of different effector-target ratios (5:1 -40:1) on target cells (MDA-MB-231) was detected by calcein release assay. The excitation and emission wavelengths of calcein are 490nm and 515nm, respectively.

[0071] Effector cells: Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3

[0072] Target cells: MDA-MB-231

[0073] Effector cells: target cells = 40:1, 20:1, 10:1, 5:1

[0074] 2. Streaming analysis

[0075] Detection of CD107a expression on NK cell surface by flow cytometry

[0076] pass Figure 1It can be seen that Example 1 shows the highest anti-tumor activity of NK cells. The three complement each other through immunomodulation, epigenetic regulation and antioxidant effects, significantly improving the proliferation, activation and killing of NK cells. The ability of tumor cells. Comparative Example 1 has low cytotoxicity and weak anti-tumor activity because it lacks the epigenetic regulation of DZNep and the antioxidant support of resveratrol, and only has the immune enhancement effect of IL-21. Comparative Example 2 retains the epigenetic regulation of DZNep, improves the immune function and cytotoxicity of NK cells, but due to the lack of antioxidant support of resveratrol, cells may be subject to greater oxidative stress in the immune response, and the anti-tumor activity is lower than that of Example 1. Although Comparative Example 3 retains the immune enhancement and antioxidant effects of resveratrol, it lacks the epigenetic regulation of DZNep, and cannot further enhance the immune activity of NK cells, resulting in an anti-tumor effect that is significantly lower than that of Example 1. Therefore, the combination of IL-21, DZNep and resveratrol has a synergistic effect in enhancing the anti-tumor activity of NK cells, and other schemes have a decreased anti-tumor effect when one of the components is missing.

[0077] from Figure 2 NK cells were treated with different concentration combinations to detect their killing effects on MDA-MB-231 target cells. It can be seen that in Example 1, the concentrations of IL-21, DZNep and resveratrol are coordinated with each other, maximizing the immune activation and epigenetic regulation effects of NK cells. The moderate concentration of IL-21 provides a good immune enhancement effect, DZNep regulates gene expression with its potent epigenetic regulation, enhances the cytotoxicity of NK cells, and resveratrol effectively relieves oxidative stress and enhances immune cell function. The synergistic effect of the three makes the anti-tumor activity of NK cells reach the strongest level. In Example 2, the concentration of IL-21 is high, which will cause immune burden, but the higher concentration of DZNep contributes to epigenetic regulation and enhances the immune function of NK cells. Therefore, despite the heavy immune burden, the overall anti-tumor effect is still strong. In Example 3, although the concentration of resveratrol is high, it provides a strong antioxidant effect and helps to relieve oxidative stress, but the low concentration of IL-21 causes the immune response to be not strong enough, which inhibits the overall immune activity of NK cells. Therefore, its anti-tumor effect is slightly inferior to Example 2. In Example 4, the concentrations of IL-21 and resveratrol are relatively high, which can enhance immune response and antioxidant capacity, but the concentration of DZNep is relatively low, lacking sufficient epigenetic regulation, and thus the overall anti-tumor effect is relatively weak.

[0078] Figure 3This indicates that the co-treatment of IL-21, DZNep and resveratrol can inhibit the degranulation effect of NK cells, thereby reducing their cytotoxic effects. The experimental results showed that the expression of CD107a on the surface of NK cells decreased, which means that the exocytosis of NK cells was interfered, thereby inhibiting their ability to kill tumor cells.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enhancing the anti-tumor activity of NK cells, characterized in that: The method comprises the following steps: (a) Mononuclear cells were isolated from peripheral blood using Ficoll density gradient centrifugation and used to isolate NK cells; (b) PBMCs were suspended in culture medium, the cell concentration was adjusted, 500 IU / mL of IL-2 was added to continue proliferation, and CD56 was purified. + CD3 - NK cells; (c) distributing the NK cells into a plurality of culture flasks, adding IL-21 (interleukin-21) solution into the culture flasks, and continuing to culture; (d) DZNep (2,3-naphthylidene) and resveratrol were added to the culture medium, and NK cells were further cultured.

2. The method according to claim 1, characterized in that The specific steps of step (a) are: The collected peripheral blood samples were slowly added to the Ficoll centrifuge solution, and centrifuged at 1600 r / min for 30 minutes. The PBMC layer between the Ficoll layer and the plasma layer was collected by gradient separation. The PBMCs were then carefully transferred from the Ficoll layer to a new centrifuge tube and washed with PBS buffer to remove plasma and unwanted cellular impurities, and were then prepared for NK cell isolation.

3. The method according to claim 1, characterized in that: In step (b), the cell density was adjusted to 1×10 6 -5×10 6 Pieces / mL.

4. The method according to claim 1, characterized in that: The X-VIVO 15 medium used in step (b) does not contain serum, and the culture conditions are 37° C. and 5% CO 2 . The medium is replaced every 2-3 days during the culture process.

5. The method according to claim 1, characterized in that In step (c), the final concentration of IL-21 in the culture medium is 20-50 ng / mL.

6. The method according to claim 1, characterized in that The final concentration of DZNep in the culture medium is 1-5 μM, and the final concentration of resveratrol in the culture medium is 10-50 μM.

7. The method according to claim 1, characterized in that The amplification time of step (b) is 12 days.

8. The method according to claim 1, characterized in that The culturing time in step (c) is 24 hours.

9. The method according to claim 1, characterized in that: In the step (d), the NK cells are cultured in an environment of 37° C. and 5% CO 2 for 24-72 hours.

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