An NK cell and its use in the preparation of antitumor drugs

Abstract

This invention provides a method for isolating, culturing, activating, and applying NK cells in tumor immunology. The method includes isolating monocytes from peripheral blood of healthy adults, removing non-NK cell populations using negative selection, and enriching and purifying NK cells. Through stimulation with specific cytokines, NK cells are successfully expanded and their cytotoxicity is activated. This invention optimizes the preparation and activation methods of NK cells, obtaining NK cells with highly efficient cytotoxic activity in vitro, and significantly improving tumor treatment efficacy when used in combination with immune checkpoint inhibitors. Combining the immune memory effect of NK cells, this invention provides a novel anti-tumor immunotherapy method and can be applied to the preparation of new anti-tumor drugs, offering innovative ideas for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology and oncology, in particular to a method for preparing anti-tumor drugs using natural killer (NK) cells and its application in treating tumors. BACKGROUND

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

[0003] Despite the potential anti-tumor ability of NK cells, current NK cell immunotherapy still faces many challenges. First, in vitro expanded NK cells usually lose activity during expansion and are functionally unstable. This problem may be related to the culture environment, the use of cytokines or activation methods. Second, 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 of current research.

[0004] Currently, there have been attempts 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 genetic modification methods. In addition, strategies for combining NK cells with chemotherapy drugs and immune checkpoint inhibitors have also made some progress in clinical trials. However, existing treatment methods still have problems such as immune escape, treatment resistance, and non-persistent treatment effects. Therefore, finding new methods to enhance the anti-tumor activity of NK cells and overcome tumor immune escape is still an important issue to be addressed in the field of immunotherapy.

[0005] The present application is based on the above problems and 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 more effective NK cell treatment programs. SUMMARY

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

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

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

[0009] (2) Culturing NK cells: the sorted NK cells are inoculated in RPMI-1640 medium, 10% fetal bovine serum and 1% penicillin / streptomycin are added, and 50 IU / mL of IL-2 and 10 ng / mL of IL-15 are added for expansion, and the medium is replaced every 3 days for 14 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 are 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 are collected, and tumor antigen is added to stimulate the activated NK cells; and on the 10th day, anti-CD16 monoclonal antibody is added to further enhance the killing ability of NK cells.

[0012] Preferably, the tumor antigen of the present application is MC-38 colon cancer cells, wherein the MC-38 colon cancer cells are about 1 x 10 6 cells / mL and mixed with NK cells at a ratio of 1:1, the cell mixture is inoculated into a culture dish, complete RPMI-1640 medium is supplemented, and the cells are fully contacted 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 application are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

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

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

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

[0017] Preferably, the drug of the present application is injected with 1x10 6 NK cells and 20 mg / kg anti-PD-1 single-domain antibody at the same time every week.

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

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

[0020] The NK cell preparation method provided by the present application significantly improves the proliferation rate and activity of NK cells by optimizing the combination of cytokines and cell culture conditions. Through tumor immunotherapy experiments, the NK cells of the present application exhibit significant anti-tumor effects in mouse models, especially after combined with immune checkpoint inhibitors. In addition, the present application can also extract the secretions of NK cells to develop new anti-tumor drugs, providing a new treatment for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A schematic diagram of the preparation of a monoclonal antibody by mouse hybridoma cell technology.

[0022] Figure 2 A diagram of the SDS-PAGE detection results of the anti-PD-1 single-domain antibody, wherein 1 is the anti-PD-1 single-domain antibody.

[0023] Figure 3 A diagram of the western blot detection results of the anti-PD-1 single-domain antibody, wherein 1 is the PD-1 protein. DETAILED DESCRIPTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0025] Reagents, methods, and apparatuses of the present application are of a type as are commonly employed in the art, unless otherwise indicated. Reagents and materials used in the following examples were purchased, unless otherwise indicated.

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

[0027] 1. Sample collection: Collect peripheral blood (10 mL) from healthy adult, isolate mononuclear cells (PBMC) by Ficoll-Hypaque gradient centrifugation, which is not described here.

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

[0029] 2.1 Cell labeling

[0030] (1) Prepare antibody solution: Prepare antibody solution of anti-CD3, CD19 and CD14. The concentration of the antibody used is 1 μg / 10 6 cells.

[0031] (2) Antibody labeling: Add PBMC to the centrifuge tube containing PBS, the final concentration is 1 x 10 6 cells / mL. Add antibody of anti-CD3, CD19 and CD14, mix gently, reaction time is 15-20 minutes, 4°C. Avoid excessive incubation of antibody solution to avoid affecting the activity of cells.

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

[0033] 2.2 Magnetic sorting method

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

[0035] (2) Washing and centrifugation: After incubation, add the cell and magnetic bead mixture to the centrifuge tube containing PBS for centrifugation, 500 x g, 10 minutes, discard the supernatant. Resuspend the cells to ensure uniform distribution of the cells.

[0036] (3) Magnetic sorting: Add the magnetic bead-labeled cell sample to the Magnetic separation was performed in a sorting column, and the NK cell population after T cells, B cells and monocytes were removed by negative selection was eluted.

[0037] (4) Cell elution: The cells that were not attracted by the magnetic field in the sorting column (i.e. the NK cell population) were eluted. The eluate was collected in a new centrifuge tube.

[0038] (5) Cell washing: The sorted cells were washed once by adding PBS, centrifuged (500 x g, 10 min), the impurities were removed, and the cells were resuspended.

[0039] 2.3 Cell purity evaluation

[0040] (1) Cell counting and viability detection: The purified cells were counted using a cell counting plate. The cell viability was detected by trypan blue staining method to ensure that the sorted NK cells had high activity.

[0041] (2) Purity evaluation: Purity analysis was performed using flow cytometry to check the marker expression of NK cells. The standard NK cell marker is CD56 (expressed in most human NK cells), CD3 negative. The proportion of CD3-CD56+ NK cells is usually determined by flow cytometry.

[0042] Table 1 detection results

[0043]

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

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

[0046] Table 2 detection results during cell culture

[0047]

[0048] Example 2: Activation of NK cells

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

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

[0051] 3. Immune regulation: On day 10, anti-CD16 monoclonal antibodies (10 μg / mL final concentration) prepared by the present application or commercial anti-CD16 monoclonal antibodies (ab246222) were added to the co-cultured NK cell and MC-38 cell mixed culture system, and the culture dish was gently shaken to ensure uniform distribution of the antibodies. The culture dish was then placed in a 37°C, 5% CO2 incubator for further culture for 24 hours.

[0052] The results showed (Table 3) that the killing ability of activated NK cells was significantly improved, especially after the combined use of IL-21, IL-12 and anti-CD16 monoclonal antibodies, the killing rate could be increased, but the anti-CD16 monoclonal antibodies prepared by the present application had better stimulating effect than the commercial anti-CD16 monoclonal antibodies (ab246222), and the highest killing rate could reach 85%.

[0053] The anti-CD16 monoclonal antibodies prepared by the present application were prepared using conventional hybridoma cell technology, and the mode diagram is shown in Figure 1 The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0054] Table 3 Detection results of 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 x 10 4 cells / well) were inoculated in a 96-well plate and cultured for 24 hours to the logarithmic growth phase.

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

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

[0060] The results show (Table 4) that the activated NK cells have significant killing ability, especially after being combined with the anti-CD16 monoclonal antibody prepared in the application, the killing rate is significantly improved, reaching 85%.

[0061] Table 4: Results of LDH release kit detection

[0062]

[0063] Example 4: NK cell combined with immune checkpoint inhibitor treatment

[0064] 1. Mice model construction: 8-week-old C57BL / 6 mice were selected, subcutaneously injected with MC-38 colon cancer cells (5x10 6 cells / mouse) to establish a tumor model, and treatment was started when the tumor grew to 50mm 3 in size.

[0065] 2. Treatment regimen: divided into four groups, respectively blank group, NK cell alone treatment group, anti-PD-1 antibody alone treatment group (Keytruda), NK cell combined with anti-PD-1 antibody (Keytruda) treatment group, NK cell combined with anti-PD-1 single domain antibody treatment group.

[0066] (1) NK cell treatment group: 1x10 6 NK cells were injected per week, and treatment was continued for 3 weeks.

[0067] (2) PD-1 antibody treatment group: 20mg / kg anti-PD-1 antibody (Keytruda) was injected per week, and treatment was continued for 3 weeks.

[0068] (3) Combination treatment group 1: 1x10 6 NK cells and 20mg / kg anti-PD-1 antibody (Keytruda) were injected per week.

[0069] (3) Combination treatment group 2: 1x10 6 NK cells and 20mg / kg anti-PD-1 single domain antibody (prepared in the application) were injected per week.

[0070] 3. Tumor growth monitoring: the volume of mouse tumor was measured every two days, and the formula: tumor volume = length x width2 / 2 was used to evaluate the tumor growth.

[0071] The results show (Table 5) that the tumor volume of the two combined treatment groups is significantly smaller than that of the single treatment group, and the tumor inhibition rate reaches 67.4% or more, indicating that the combination of NK cells and PD-1 antibodies has a synergistic anti-tumor effect, and the anti-PD-1 single-domain antibody prepared by the application has 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 antibody

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

[0076] (2) Cloning and sequence analysis: specific primers were designed to amplify the shark single-domain antibody (VNAR fragment), and 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 as follows: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and finally extension at 72°C for 5 minutes. The amplified VNAR fragment was cloned into the pGEM-T vector and transformed into DH5α E. coli competent cells. After selecting positive clones, the plasmid was extracted, sequenced and analyzed for VNAR sequence, and finally the VNAR sequence of the anti-PD-1 single-domain antibody (SEQ ID NO. 3) was obtained.

[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 *E. coli* BL21(DE3) competent cells. When the cells were cultured at 37°C until the OD600 reached 0.6, 1 mM 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 His-tag nickel affinity chromatography. The purified antibody was dialyzed to remove salts, then sterilized by passing through a 220 nm 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 using SDS-PAGE. The results showed that ( Figure 2 Its purity was high (>90%). Subsequently, Western blot was used to determine whether the antibody could specifically bind to the recombinant PD-1 protein. The results showed ( Figure 3 The single-domain antibody was found to specifically bind to the PD-1 protein, which has a molecular weight of approximately 17 kDa, validating its functionality. Finally, the expression level of this single-domain antibody was calculated to be above 0.5 g / L, demonstrating its high expression yield and suitability for large-scale production.

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

[0083] 1. Cytokine Analysis: The concentrations of cytokines (IFN-γ, TNF-α, IL-2, etc.) secreted by activated NK cells were detected using ELISA. After co-culturing NK cells with MC-38 tumor cells, the culture supernatant was collected, and the changes in the concentrations of cytokines such as IFN-γ, TNF-α, and IL-2 were detected.

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

[0085] The results (Table 6) showed 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. Detection results of cytokines and their inhibitory effects on tumors.

[0087]

[0088] Example 6: Immune Memory Effect of NK Cells

[0089] 1. Induction of immune memory: On day 14, NK cells were stimulated with tumor antigens to induce immune memory. NK cells were stimulated with antigens related to colon cancer cells from MC-38 mice and cultured for 48 hours.

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

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

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

[0093]

[0094] The technical solution of this invention successfully cultured and activated highly efficient NK cells, demonstrating significant tumor-killing capabilities in vitro and achieving significant therapeutic effects in mouse tumor models. The combined treatment strategy with anti-PD-1 single-domain antibodies further enhanced the anti-tumor efficacy of NK cells. Simultaneously, the immune memory effect of NK cells provided strong support for long-term anti-tumor therapy. These experimental results demonstrate the innovation of this invention and its potential in tumor immunotherapy.

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of isolating, culturing, activating NK cells, characterized in that, The method comprises the following steps: (1) Isolation of NK cells: mononuclear cells are isolated from peripheral blood of healthy adults by Ficoll-Hypaque gradient centrifugation, and T cells, B cells and mononuclear cells are removed by negative selection to enrich the NK cell population; (2) Culturing NK cells: the sorted NK cells are inoculated in RPMI-1640 medium, 10% fetal bovine serum and 1% penicillin / streptomycin are added, and 50 IU / mL of IL-2 and 10 ng / mL of IL-15 are added for expansion, and the medium is replaced every 3 days for 14 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 are added to stimulate NK cells and enhance their cytotoxicity; (4) Tumor antigen-induced activation: After cytokine activation on day 7, the cultured NK cells were collected, and tumor antigen stimulation was added to the activated NK cells; and on day 10, an anti-CD16 monoclonal antibody was added to further enhance the killing ability of the NK cells; the tumor antigen was MC-38 colon cancer cells, wherein the MC-38 colon cancer cells were about 1 x 10 6 6 / mL and mixed with the NK cells at a ratio of 1:1, the cell mixture was inoculated into a culture dish, supplemented with complete RPMI-1640 medium, and the cells were fully contacted and co-cultured for 48 hours to evaluate the anti-tumor effect of the NK cells; 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.

2. The method of claim 1, wherein, The final concentration of the anti-CD16 monoclonal antibody is 10 μg / mL.

3. An antitumor agent, characterized by comprising the compound of claim 1 or 2. The anti-tumor drug comprises the NK cells prepared in claim 1 and an anti-PD-1 single-domain antibody; the VNAR sequence of the anti-PD-1 single-domain antibody is shown in SEQ ID NO.

3.

4. The medicament according to claim 3, characterized in that, The drug is injected 1 x 10 6 NK cells and 20 mg / kg anti-PD-1 single-domain antibody simultaneously every week.

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