NK cell and application thereof in preparation of antitumor drugs

Abstract

The invention provides a method for separating, culturing and activating NK (Natural Killer) cells and an application method of the NK cells in tumor immunity. The method comprises the following steps: separating mononuclear cells from peripheral blood of healthy adults, removing non-NK cell populations through a negative selection method, and enriching and purifying NK cells. Through stimulation of specific cytokines, NK cells are successfully amplified, and cytotoxicity of the NK cells is activated. By optimizing the preparation and activation method of the NK cells, the NK cells with efficient killing activity are obtained in vitro, and the tumor treatment effect is remarkably improved through combined use of the NK cells and the immune checkpoint inhibitor. In combination with the immune memory effect of the NK cells, the invention provides a new anti-tumor immunotherapy method which can be applied to preparation of new anti-tumor drugs and provides an innovative thought for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and oncology, and in particular to a method for preparing anti-tumor drugs by using natural killer (NK) cells and an application thereof in treating tumors. Background Art

[0002] Natural killer (NK) cells are an important part of the body's innate immune system and have a high ability to recognize and kill tumor cells. Unlike T cells, NK cells do not rely on prompts from antigen-presenting cells and can directly recognize and kill virus-infected cells, tumor cells, and cells in other pathological states. Therefore, NK cells play an important role in immune surveillance and anti-tumor immune responses. In recent years, NK cells have attracted widespread attention in tumor treatment as an immunotherapy strategy.

[0003] Although NK cells have potential anti-tumor capabilities, current NK cell immunotherapy still faces many challenges. First, in vitro expanded NK cells usually lose their activity during the expansion process and their functions are unstable. This problem may be related to the culture environment, the cytokines used, or the activation method. Secondly, the effect of NK cells is affected by the immunosuppressive microenvironment, especially in the tumor microenvironment, where tumor cells and immunosuppressive cells often inhibit the function of NK cells by secreting immunosuppressive factors. Therefore, how to enhance the killing activity of NK cells, improve their tolerance in the tumor microenvironment, and prolong their immune memory effect is a hot topic in current research.

[0004] At present, studies have attempted to enhance the anti-tumor efficacy of NK cells through the combined use of cytokines (such as IL-2, IL-15, IL-21, etc.), stimulation of tumor-specific antigens, and gene modification. In addition, the strategy of combining NK cells with chemotherapy drugs and immune checkpoint inhibitors has also made some progress in clinical trials. However, existing treatment methods still have problems such as immune escape, treatment resistance, and unsustainable treatment effects. Therefore, finding new methods to enhance the anti-tumor activity of NK cells and overcome tumor immune escape remains an important issue that needs to be urgently addressed in the field of immunotherapy.

[0005] Based on the above problems, the present invention proposes an optimized NK cell preparation method and explores its application in the preparation of anti-tumor drugs, in order to improve the clinical effect of tumor immunotherapy through a more effective NK cell treatment plan. Summary of the invention

[0006] The purpose of the present invention is to provide a new method for preparing NK cells and their use in preparing anti-tumor drugs. Specifically, the present invention optimizes the culture conditions, stimulation methods and cytokine combinations of NK cells to obtain NK cells with high-efficiency killing activity and long-term immune memory effect, and explores their potential in tumor treatment.

[0007] Therefore, the present invention discloses a method for isolating, culturing and activating NK cells, comprising the following steps:

[0008] (1) Isolation of NK cells: Mononuclear cells were isolated from the peripheral blood of healthy adults by Ficoll-Hypaque gradient centrifugation, and T cells, B cells, and monocytes were removed by negative selection to enrich the NK cell population;

[0009] (2) Cultivating NK cells: The sorted NK cells were inoculated into RPMI-1640 medium, and 10% fetal bovine serum and 1% penicillin / streptomycin were added. At the same time, 50 IU / mL IL-2 and 10 ng / mL IL-15 were added for proliferation. The cells were cultured for 14 days, and the medium was replaced every 3 days.

[0010] (3) Activation of NK cells: On the 7th day of culture, 10 ng / mL of IL-21 and 5 ng / mL of IL-12 were added to stimulate NK cells and enhance their cytotoxicity;

[0011] (4) Tumor antigen-induced activation: After cytokine activation on the 7th day, the cultured NK cells were collected and tumor antigen stimulation was added to the activated NK cells; and on the 10th day, anti-CD16 monoclonal antibodies were added to further enhance the killing ability of NK cells.

[0012] Preferably, the tumor antigen of the present invention is MC-38 colon cancer cells, wherein the number of MC-38 colon cancer cells is about 1×10 6 The cells were mixed with NK cells at a ratio of 1:1, and the cell mixture was inoculated into a culture dish and supplemented with complete RPMI-1640 medium to ensure that the cells were fully in contact and co-cultured for 48 hours to evaluate the anti-tumor effect of NK cells.

[0013] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD16 monoclonal antibody of the present invention are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0014] Preferably, the final concentration of the anti-CD16 monoclonal antibody of the present invention is 10 μg / mL.

[0015] In one aspect, the present invention also discloses an anti-tumor drug, which comprises the NK cells prepared according to claim 1 and an anti-PD-1 single domain antibody.

[0016] Preferably, the VNAR sequence of the anti-PD-1 single domain antibody described in the present invention is as shown in SEQ ID NO.3.

[0017] Preferably, the drug of the present invention is injected 1×10 6 NK cells and 20mg / kg anti-PD-1 single domain antibody.

[0018] In one aspect, the present invention also discloses a use of the anti-CD16 monoclonal antibody in NK cell activation.

[0019] In one aspect, the present invention also discloses a use of the anti-PD-1 single domain antibody in the preparation of anti-tumor drugs.

[0020] The NK cell preparation method provided by the present invention significantly improves the proliferation rate and activity of NK cells by optimizing the cytokine combination and cell culture conditions. Through tumor immunotherapy experiments, it was verified that the NK cells of the present invention showed significant anti-tumor effects in mouse models, especially the therapeutic effect after combined with immune checkpoint inhibitors was more significant. In addition, the present invention can also develop new anti-tumor drugs by extracting the secretions of NK cells, providing a new treatment method for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation of monoclonal antibodies using mouse hybridoma cell technology.

[0022] Figure 2 Figure 1 is the SDS-PAGE test result of anti-PD-1 single domain antibody, where 1 is the anti-PD-1 single domain antibody.

[0023] Figure 3 Anti-PD-1 single domain antibody western blot test results, where 1 is PD-1 protein. DETAILED DESCRIPTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0025] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] Example 1: Isolation and culture of NK cells

[0027] 1. Sample collection: Peripheral blood (10 mL) was collected from healthy adults, and mononuclear cells (PBMC) were separated using conventional Ficoll-Hypaque gradient centrifugation method, which will not be described here.

[0028] 2. NK cell sorting: Use negative selection to remove T cells, B cells and monocytes and enrich the NK cell population. The specific process is as follows:

[0029] 2.1 Cell labeling

[0030] (1) Prepare antibody solution: Prepare antibody solutions against CD3, CD19 and CD14. The antibody concentration used is 1 μg / 10 6 cell.

[0031] (2) Antibody labeling: PBMCs were added to a centrifuge tube containing PBS at a final concentration of 1×10 6 cells / mL. Add antibodies against CD3, CD19 and CD14, mix gently, react for 15-20 minutes, and store at 4°C. Avoid over-incubation with antibody solution to avoid affecting cell activity.

[0032] (3) Washing: Add PBS for washing, discard the supernatant after centrifugation (500×g, 10 minutes), resuspend the cells, and mix gently.

[0033] 2.2 Magnetic separation

[0034] (1) Magnetic bead treatment: According to the kit instructions, add specific magnetic beads (anti-CD3, CD19 and CD14 antibody magnetic beads provided by Miltenyi Biotec). These magnetic beads bind to antibodies specifically to form complexes, thereby helping to remove T cells, B cells and monocytes. Usually 1 μL of magnetic beads / 10 6 Mix cells gently and incubate for 20 min at 4°C.

[0035] (2) Washing and centrifugation: After incubation, add the cell and magnetic bead mixture to a centrifuge tube containing PBS and centrifuge at 500×g for 10 minutes. Discard the supernatant and resuspend the cells to ensure that the cells are evenly distributed.

[0036] (3) Magnetic separation: Add the cell sample labeled with magnetic beads to Magnetic separation is performed in the sorting column to elute the NK cell population after negative selection to remove T cells, B cells and monocytes.

[0037] (4) Cell elution: Elute the cells (i.e., NK cell population) that are not adsorbed by the magnetic field in the separation column. Collect the eluate into a new centrifuge tube.

[0038] (5) Cell washing: Wash the sorted cells once by adding PBS, centrifuging (500×g, 10 min), removing impurities, and resuspending the cells.

[0039] 2.3 Cell purity assessment

[0040] (1) Cell counting and activity detection: Count the purified cells using a cell counting plate. Detect cell activity using trypan blue staining to ensure that the separated NK cells have high activity.

[0041] (2) Purity assessment: Purity analysis is performed using flow cytometry to examine the expression of NK cell markers. The standard NK cell marker is CD56 (expressed in most human NK cells) and CD3 is negative. The CD3-CD56+ ratio of NK cells is usually measured by flow cytometry.

[0042] Table 1 Test results

[0043]

[0044] 3. NK cell expansion: The sorted NK cells were inoculated into RPMI-1640 medium, and 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin were added. IL-2 (50 IU / mL) and IL-15 (10 ng / mL) were added for expansion. The culture was carried out for 14 days, and the medium was replaced every 3 days.

[0045] The results showed (Table 2) that, through the stimulation of IL-2 and IL-15, NK cells successfully proliferated and maintained a high survival rate within 14 days, proving the effectiveness of the expansion protocol.

[0046] Table 2 Cell culture process test results

[0047]

[0048] Example 2: Activation of NK cells

[0049] 1. Cytokine activation: On the 7th day of culture, IL-21 (10 ng / mL) and IL-12 (5 ng / mL) were added to jointly stimulate NK cells and enhance their cytotoxicity.

[0050] 2. Tumor antigen activation: After cytokine activation on day 7, the cultured NK cells were collected and an equal amount of MC-38 colon cancer cells (about 1×10 6 / mL), and mixed with NK cells at a ratio of 1:1, and the cell mixture was inoculated into the culture dish, and the RPMI-1640 medium was supplemented to ensure that the cells were fully in contact and co-cultured for 48 hours. During the culture process, the pH value of the culture medium and the cell morphology were regularly checked to ensure that the cells were in good condition.

[0051] 3. Immunomodulation: On the 10th day, anti-CD16 monoclonal antibody (final concentration of 10 μg / mL, anti-CD16 monoclonal antibody prepared by the present invention or commercial anti-CD16 monoclonal antibody (ab246222)) was added to the co-cultured NK cells and MC-38 cells mixed culture system, the culture dish was gently shaken to ensure uniform distribution of the antibody, and the culture dish was continued to be placed in an incubator at 37°C, 5% CO2 for 24 hours.

[0052] The results show (Table 3) that the killing ability of activated NK cells is significantly improved, especially after the combined use of IL-21, IL-12 and anti-CD16 monoclonal antibody, the killing rate can be improved, but the anti-CD16 monoclonal antibody prepared by the present invention has a better stimulating effect than the commercial anti-CD16 monoclonal antibody (ab246222), and the killing rate can reach up to 85%.

[0053] The anti-CD16 monoclonal antibody prepared by the present invention is prepared using conventional hybridoma cell technology, and its schematic diagram is as follows: Figure 1 As shown, after detection and analysis, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0054] Table 3 Test results under different activation conditions

[0055]

[0056] Example 3: In vitro killing ability detection of NK cells

[0057] 1. Target cell inoculation: MC-38 mouse colon cancer cells (5×10 4 The cells were inoculated into 96-well plates and cultured for 24 hours until the logarithmic growth phase.

[0058] 2. Co-culture of NK cells and target cells: Co-culture the activated NK cells with MC-38 tumor cells at a ratio of 10:1 for 48 hours.

[0059] 3. LDH release method to detect cell killing activity: The LDH content in the co-culture supernatant was detected by LDH release kit (Promega) to evaluate the degree of cell lysis.

[0060] The results showed (Table 4) that the activated NK cells had significant killing ability, especially when combined with the anti-CD16 monoclonal antibody prepared by the present invention, the killing rate was significantly improved to 85%.

[0061] Table 4 LDH release kit test results

[0062]

[0063] Example 4: Combination therapy of NK cells and immune checkpoint inhibitors

[0064] 1. Construction of mouse model: 8-week-old C57BL / 6 mice were selected and subcutaneously injected with MC-38 colon cancer cells (5×10 6 cells / mouse), establish tumor model, and wait for tumor to grow to 50mm 3 Start treatment when

[0065] 2. Treatment plan: The patients were divided into four groups, namely, blank group, NK cell alone treatment group, anti-PD-1 antibody alone treatment group (Keytruda), NK cell and anti-PD-1 antibody (Keytruda) combined treatment group, and NK cell and anti-PD-1 single domain antibody combined treatment group.

[0066] (1) NK cell therapy group: 1×10 6 NK cells were treated for 3 weeks.

[0067] (2) PD-1 antibody treatment group: 20 mg / kg anti-PD-1 antibody (Keytruda) was injected weekly for 3 consecutive weeks.

[0068] (3) Combined treatment group 1: 1×10 6 NK cells and 20mg / kg anti-PD-1 antibody (Keytruda).

[0069] (3) Combined treatment group 2: 1×10 6 NK cells and 20 mg / kg of anti-PD-1 single domain antibody (prepared by the present invention).

[0070] 3. Tumor growth monitoring: The volume of mouse tumors was measured every two days, and the tumor growth was evaluated using the formula: tumor volume = length × width 2 / 2.

[0071] The results showed (Table 5) that the tumor volumes of the two combined treatment groups were significantly smaller than those of the single treatment groups, and the tumor inhibition rate reached 67.4% or more, indicating that the combined use of NK cells and PD-1 antibodies has a synergistic anti-tumor effect, and the anti-PD-1 single domain antibody prepared by the present invention has a better therapeutic effect than the anti-PD-1 antibody (Keytruda).

[0072] Table 5 Tumor growth monitoring results

[0073]

[0074] 4. Preparation and testing of anti-PD-1 single domain antibodies

[0075] (1) Immunization: Recombinant human PD-1 protein (ab174035) was emulsified with FCA to prepare a vaccine, ensuring that each milliliter of the vaccine contained 0.5 mg of PD-1 protein. The vaccine was subcutaneously injected into the shark at a dose of 0.5 mL. Two weeks after the first immunization, the second immunization was performed using the same dose of the vaccine emulsified with recombinant PD-1 protein and FIA. Two weeks later, the third immunization was performed, with the same dose and immunization method. Within seven days after the third immunization, shark peripheral blood was collected and lymphocytes were isolated. Total B cell RNA was extracted using the TRIzol method, and reverse transcription was performed according to the kit instructions to synthesize cDNA.

[0076] (2) Cloning and sequence analysis: Specific primers were designed to amplify shark single domain antibody (VNAR fragment). The primers used are as follows:

[0077] Upstream primer: 5'-gtcctggctcagggagagcgg t-3'

[0078] Downstream primer: 5'-ggtacggttggcctgttgagg-3'

[0079] The VNAR fragment was amplified by PCR, and the PCR conditions were: 94°C pre-denaturation for 5 minutes, followed by 35 cycles (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute), and finally extended at 72°C for 5 minutes. The amplified VNAR fragment was cloned into the pGEM-T vector and transformed into DH5α Escherichia coli competent cells. After selecting the positive clones, the plasmid was extracted, sequenced and analyzed for the VNAR sequence, and finally the VNAR sequence of the anti-PD-1 single domain antibody was obtained (SEQ ID NO.3).

[0080] (3) Expression and purification: The VNAR sequence of the anti-PD-1 single domain antibody was cloned into the pET28a vector and transformed into Escherichia coli BL21 (DE3) competent cells. When the cells were cultured at 37°C until OD600 reached 0.6, 1mM IPTG was added to induce protein expression. After 4 hours of culture, the cells were collected and soluble proteins were extracted using ultrasonic disruption. The anti-PD-1 single domain antibody was purified using nickel column affinity chromatography using the His-tag tag. The purified antibody was dialyzed to remove salts, sterilized through a 220nm filter membrane, and finally frozen at -80°C for subsequent use.

[0081] (4) Purity analysis and functional verification: The purity of the purified anti-PD-1 single domain antibody was analyzed by SDS-PAGE. The results showed that ( Figure 2 ) Its purity is high (>90%). Subsequently, Western blot was used to detect whether the antibody can specifically bind to the recombinant PD-1 protein. The results showed that ( Figure 3 ), the single-domain antibody was able to specifically bind to the PD-1 protein with a molecular weight of about 17kDa, verifying its functionality. Finally, it was calculated that the expression level of the single-domain antibody reached more than 0.5g / L, proving that it has a high expression yield and is suitable for large-scale production applications.

[0082] Example 5: Effects of NK cell secreted factors on tumors

[0083] 1. Cytokine analysis: ELISA was used to detect the concentration of cytokines (IFN-γ, TNF-α, IL-2, etc.) secreted by activated NK cells. After co-culturing NK cells with MC-38 tumor cells, the culture supernatant was collected to detect changes in the concentration of cytokines such as IFN-γ, TNF-α, and IL-2.

[0084] 2. Effects of cytokines on tumors: The culture supernatant of NK cells was added to MC-38 mouse colon cancer cells to evaluate its inhibitory effect on tumor cells.

[0085] The results showed (Table 6) that the concentrations of IFN-γ and TNF-α in the supernatant of activated NK cells were significantly increased, and the tumor cell proliferation inhibition rate reached 60%, indicating that NK cells exert anti-tumor effects by secreting cytokines.

[0086] Table 6 Cytokines and their inhibitory effects on tumors

[0087]

[0088] Example 6: Immune memory effect of NK cells

[0089] 1. Immune memory induction: On the 14th day of NK cells, tumor antigens were used to stimulate the immune memory of NK cells. MC-38 mouse colon cancer cell-related antigens were used to stimulate NK cells and cultured for 48 hours.

[0090] 2. Long-term immune monitoring: Follow up the animals injected with NK cells and conduct tumor inoculation experiments 3 and 6 months after treatment to monitor their immune memory effects.

[0091] The results showed (Table 7) that the combined treatment group 2 had a better effect and still had an inhibition rate of 68.8% after 6 months.

[0092] Table 7 Follow-up test results after immunotherapy

[0093]

[0094] Through the technical solution of the present invention, highly efficient NK cells were successfully cultured and activated, showing significant tumor killing ability in vitro, and achieved significant therapeutic effects in mouse tumor models. The treatment strategy of combining anti-PD-1 single-domain antibodies further improved the anti-tumor efficacy of NK cells. At the same time, the immune memory effect of NK cells also provided a strong guarantee for long-term anti-tumor treatment. These experimental results demonstrate the innovativeness of the present invention and its potential in tumor immunotherapy.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for isolating, culturing and activating NK cells, characterized in that: The method comprises the following steps: (1) Isolation of NK cells: Mononuclear cells were isolated from the peripheral blood of healthy adults by Ficoll-Hypaque gradient centrifugation, and T cells, B cells, and monocytes were removed by negative selection to enrich the NK cell population; (2) Cultivating NK cells: The sorted NK cells were inoculated into RPMI-1640 medium, and 10% fetal bovine serum and 1% penicillin / streptomycin were added. At the same time, 50 IU / mL IL-2 and 10 ng / mL IL-15 were added for proliferation. The cells were cultured for 14 days, and the medium was replaced every 3 days. (3) Activation of NK cells: On the 7th day of culture, 10 ng / mL of IL-21 and 5 ng / mL of IL-12 were added to stimulate NK cells and enhance their cytotoxicity; (4) Tumor antigen-induced activation: After cytokine activation on the 7th day, the cultured NK cells were collected and tumor antigen stimulation was added to the activated NK cells; and on the 10th day, anti-CD16 monoclonal antibodies were added to further enhance the killing ability of NK cells.

2. The method according to claim 1, characterized in that The tumor antigen is MC-38 colon cancer cells, wherein the MC-38 colon cancer cells are about 1×10 6 The cells were mixed with NK cells at a ratio of 1:1, and the cell mixture was inoculated into a culture dish and supplemented with complete RPMI-1640 medium to ensure that the cells were fully in contact and co-cultured for 48 hours to evaluate the anti-tumor effect of NK cells.

3. The method according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD16 monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

4. The method according to claim 1, characterized in that The final concentration of the anti-CD16 monoclonal antibody is 10 μg / mL.

5. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the NK cells prepared according to claim 1 and an anti-PD-1 single domain antibody.

6. The drug according to claim 5, characterized in that The VNAR sequence of the anti-PD-1 single domain antibody is shown in SEQ ID NO.

3.

7. The drug according to claim 5, characterized in that The drug was injected 1×10 6 NK cells and 20mg / kg anti-PD-1 single domain antibody.

8. Use of the anti-CD16 monoclonal antibody according to claim 3 in NK cell activation.

9. Use of the anti-PD-1 single domain antibody as claimed in claim 6 in the preparation of anti-tumor drugs.

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