Method for preparing memory natural killer cells by using peripheral blood mononuclear cells and application of memory natural killer cells

Abstract

The invention relates to the technical field of biological medicine, in particular to a method for preparing memory natural killer cells by using peripheral blood mononuclear cells and application of the memory natural killer cells. According to the preparation method, the mononuclear cells are prepared by using peripheral blood, and a cytokine composition A and a cytokine composition B are added in the amplification process; the amplified cells containing the mNK cells are obtained. A detection result shows that the preparation method provided by the invention can activate and proliferate a large amount of NK cells in mononuclear cells, and the activated NK cells show memory-like characteristics of adaptive immunity, have stronger capability of killing cancerous cells, and can stimulate mNK cells to generate stronger expression of IFN-gamma and granzyme, so that the cancerous cells can be effectively killed. The two components cooperate to enable the mNK cell to play an anti-tumor role, and the mNK cell provided by the preparation method is expected to be applied to medicine preparation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for preparing memory natural killer cells by using peripheral blood mononuclear cells and the application of the memory natural killer cells. Background Art

[0002] Natural Killer (NK) cells are an important type of immune cell, not only related to anti-tumor, anti-viral infection and immune regulation. Since the killing activity of NK cells is not restricted by the histocompatibility complex (MHC) and does not rely on antibodies, it is called natural killer activity. The functional mechanisms of NK cells mainly include the following aspects: release of cytotoxic substances such as granzymes; secretion of cytokines such as interferon-γ (IFN-γ) to mediate immune regulation and immune clearance; expression of Fc receptors (CD16) to kill target cells through antibody-dependent cell-mediated cytotoxicity (ADCC) effects. In some cases, they also show memory-like characteristics of adaptive immunity. When these pre-activated NK cells are stimulated by antigens or cytokines again, they show enhanced IFN-γ secretion and degranulation, such as the upregulation of CD107a expression. In addition, mature NK cells also highly express surface proteins such as NKG2C. Based on the rapid response ability, unique recognition mechanism, powerful killing function and memory characteristics of NK cells, NK cells have become immunotherapy cells with important clinical application value.

[0003] At present, the in vitro expansion technology of NK cells can be divided into autologous peripheral blood mononuclear cells (PBMC), autologous pluripotent hematopoietic stem cells, allogeneic PBMC, umbilical cord blood and NK-92 cell lines according to the different sources of NK cells. Among them, patent CN118389428A discloses a method for culturing and expanding allogeneic NK cells, which is briefly described as follows: using human umbilical cord blood as the source of allogeneic NK cells, selecting RPMI-1640 culture medium in vitro, and adding a combination of 2-pyrimidone-B-nucleoside and falcarindiol by precise addition, and adding one of the cytokines interleukin-2, interleukin-12, interleukin-18, interleukin-15 or interleukin-21, the expansion rate and cytotoxicity of cultured NK cells can be enhanced, and high-quantity and high-quality NK cells can be obtained, providing strong support for NK cell immunotherapy. However, unlike PBMCs in peripheral blood, which are all differentiated and mature blood cells, the NK cells in this invention are derived from allogeneic umbilical cord blood, which contains more CD34 + Hematopoietic stem cell components have multidirectional differentiation potential, which on the one hand increases the cost of cytokines required in the process of induced differentiation; on the other hand, compared with autologous peripheral blood, the source of allogeneic blood is relatively difficult.

[0004] Therefore, how to obtain sufficient numbers of high-quality NK cells for clinical applications within a certain period of time is a technical problem that urgently needs to be solved. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a method for preparing memory Natural Killer (mNK) cells using peripheral blood mononuclear cells; the second object of the present invention is to provide an application of mNK cells.

[0006] In order to achieve the first purpose, the technical solution adopted by the present invention is: A method for preparing mNK cells using peripheral blood mononuclear cells comprises the following steps: S100, Preparation of mononuclear cell suspension using peripheral blood Ⅰ; S200, adjusting the cell density of mononuclear cell suspension I with a complete mixed culture medium to obtain mononuclear cell suspension II, performing initial activation culture on the cells in mononuclear cell suspension II with cytokine composition A, adding a complete mixed culture medium to obtain mononuclear cell suspension III containing initially activated natural killer cells; Among them, the components and contents of cytokine composition A are as follows: IL-12, 2-50 ng / mL; IL-18, 2-50 ng / mL; IL-15 and IL-15Ra complex, 1-100 ng / mL; S300, add cytokine composition B to mononuclear cell suspension III, and culture until the cell density is 0.8-5.0×10 6 / mL, and a mononuclear cell suspension containing memory natural killer cells was obtained; Among them, the components and contents of cytokine composition B are as follows: IL-2, 600-2000 IU / mL; IL-15 and IL-15Ra complex, 1-100 ng / mL; S400, collecting cells in mononuclear cell suspension IV, washing, and obtaining memory natural killer cells.

[0007] Further, step S100 includes the following steps: S110, centrifuging the peripheral blood into an upper layer and a lower layer, the upper layer being plasma and the lower layer being blood cells; S120. Dilute the lower layer of blood cells to obtain a blood cell suspension, add lymphocyte separation solution to the blood cell suspension, and prepare a mononuclear cell suspension I.

[0008] Further, in step S200, the completely mixed culture medium is prepared by the following steps: S210, mixing GT-T551 H3 medium and X-VIVO medium in a mass ratio of 1:9 to 9:1 to prepare a basic mixed medium; S220, adding the plasma obtained in step S110 to the basic mixed medium, mixing well, and obtaining a complete mixed medium; The amount of plasma added is 10% to 20% by volume.

[0009] Further, in step S200, the initial activation culture time is 12 to 24 hours; In step S300, the amplification culture time is 4 to 16 days.

[0010] Furthermore, the memory natural killer cells obtained in step S400 are subjected to antibody loading, and the antibody loading comprises the following steps: S410, adding a monoclonal antibody to a mononuclear cell suspension IV containing memory natural killer cells to obtain a mononuclear cell suspension V containing antibody-loaded memory natural killer (abbreviated as: AmNK) cells; Wherein, the monoclonal antibody is added at a concentration of 0.1 to 1 mg / ml; S420, collecting cells in the mononuclear cell suspension V, washing, and obtaining antibody-loaded memory natural killer cells.

[0011] In order to achieve the second purpose, the technical solution adopted by the present invention is: The application of mNK cells includes the application of mNK cells in the preparation of drugs, wherein the mNK cells are prepared by any of the above-mentioned methods for preparing mNK cells using peripheral blood mononuclear cells.

[0012] Furthermore, the drug also includes a monoclonal antibody, which binds to CD16 highly expressed on the mNK cells to form AmNK cells, and the AmNK cells are prepared by the above-mentioned method of preparing mNK cells using peripheral blood mononuclear cells.

[0013] Furthermore, the monoclonal antibody includes one or more of Herceptin, cetuximab, bevacizumab, ramucirumab, rituximab, ibritumomab tiuxetan, ofatumumab, alemtuzumab, pertuzumab, ipilimumab, nivolumab, atezolizumab, avelumab and durvalumab.

[0014] Furthermore, the medicine includes a medicine for cancer.

[0015] Furthermore, the cancer includes breast cancer and / or colon cancer.

[0016] Furthermore, the drug includes one or more of pharmaceutically acceptable solvents, excipients and carriers.

[0017] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a method for preparing mNK cells using peripheral blood mononuclear cells. The method uses the donor's autologous peripheral blood mononuclear cells as the source of mNK cells, directly activates and amplifies the mononuclear cells, and the NK cells are derived from mixed types of cells, without the need to sort the NK cells through a purification step.

[0018] The present invention activates and proliferates immune cells in mononuclear cells by adding cytokine composition A and cytokine composition B to the culture medium. The test results show that the culture and amplification system provided by the present invention can activate and proliferate a large number of NK cells in mononuclear cells, and the activated NK cells show memory-like characteristics of adaptive immunity, and have a stronger ability to kill cancerous cells. Such NK cells are called memory NK cells, or mNK cells for short. While activating and amplifying to obtain mNK cells, CD4+T cells in peripheral blood mononuclear cells will be activated to secrete IL-21 to form IL-21+CD4+T cells, which can not only participate in the proliferation and amplification of tumor-infiltrating lymphocytes and CD8+ memory T cells, but also stimulate mNK cells to produce IFN-γ and granzymes, which can better cooperate with mNK cells and CD8+T cells to exert anti-tumor effects after reaching the local tumor.

[0019] Therefore, the method for preparing mNK cells provided by the present invention can obtain activated cells of mNK cells and T cells, two types of synergistic anti-cancer cells, after one activation and amplification operation, using the donor's peripheral blood mononuclear cells as the source. Compared with other existing technologies, it can make full use of the effective cells in the donor's own blood, thereby achieving a greater therapeutic effect.

[0020] At the same time, in the preparation method provided by the present invention, monoclonal antibodies used in clinical tumor treatment can also be added to mNK cells. CD16 highly expressed on activated mNK cells can bind to antibodies. By utilizing the antibody's specific binding to tumors, mNK cells can quickly locate tumor tissues and exert a highly effective tumor cell-killing effect.

[0021] The mNK cell preparation method provided by the present invention is safe, simple to operate, and low in cost, and is suitable for large-scale production of mNK cells. It is expected that the preparation method provided by the present invention can be used to prepare mNK cells suitable for clinical applications.

[0022] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of mNK cells and AmNK cells provided in Test Example 4 of the present invention on the breast cancer cell line SKBR3.

[0024] Figure 2 This is a graph showing the effect of mNK cells and AmNK cells provided in Test Example 4 of the present invention on the breast cancer cell line T47D. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0026] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0027] Peripheral blood: provided by healthy volunteers.

[0028] Example 1 The preparation of mNK cells and AmNK cells includes the following steps: 1. Blood separation.

[0029] In a biosafety cabinet, pour about 50 ml of anti-coagulated peripheral blood into two 50 ml sterile centrifuge tubes and centrifuge at 3500 rpm for 5 minutes at room temperature. After centrifugation, take out the centrifuge tubes and you can see that the blood is divided into two layers, the upper layer is plasma and the lower layer is blood cells.

[0030] In a biosafety cabinet, use a sterile pipette to draw the upper plasma into a new sterile centrifuge tube, and keep the lower blood cells for later use. Place the plasma in a water bath, inactivate at 56°C for 30 minutes, centrifuge, and store at room temperature for later use.

[0031] 2. Extraction of lymphocytes.

[0032] In a light-proof environment, take out the lymphocyte separation solution (Ficoll ® PM 400), respectively, into 4 50ml sterile centrifuge tubes, 22.5ml per tube. Centrifuge the blood cells obtained in the above steps with PBS at 3000 rpm for 6 minutes at room temperature to obtain the lower layer of blood cells, dilute it to 50ml, and then evenly and slowly inject it into the upper layer of Ficoll liquid in the 4 centrifuge tubes. According to the instructions for use of lymphocyte separation solution, perform density gradient centrifugation using the Ficoll method, 1800 rpm, 20 minutes, room temperature, the acceleration of the rise is set to 9, and the acceleration of the fall is set to 0.

[0033] After centrifugation, use a pipette to transfer the mononuclear cell layer in the buffy coat to another 50 ml sterile centrifuge tube. Add phosphate buffered saline (PBS) to 50 ml, mix well, and count. The cell concentration is about 1.2 to 8.0 × 10 5The cells were centrifuged and washed once, and then mixed with 50 ml PBS. The prepared cell suspension is mononuclear cells, which are used for the activation and proliferation of mNK cells and AmNK cells.

[0034] 3. Induction, activation and expansion culture of mNK cells and AmNK cells.

[0035] The preparation process of complete mixed medium is as follows: Use commercially available GT-T551 H3 and X-VIVO at a mixing ratio of 1:9 to 9:1 to prepare a basic mixed medium, and add the autologous plasma obtained in step 1 at 10% volume and mix thoroughly. This is a complete mixed medium.

[0036] The mononuclear cell concentration was adjusted and the initial activation culture was performed as follows: The mononuclear cells suspended in PBS were centrifuged again at 1800 rpm for 20 minutes, the upper layer of PBS was discarded, and the cell suspension obtained above was adjusted with an appropriate volume of complete mixed culture medium to a cell density of (1-3) × 10 6 cells / mL, placing the cells in a T-225 culture flask, and adding cytokines and activation proteins according to the proportion of cytokine composition A; Among them, the cytokine composition A cytokine composition and its dosage: IL-12, 2-50 ng / mL; IL-18, 2-50 ng / mL; recombinant human interleukin-15 receptor α and interleukin-15 fusion protein complex, 1-100 ng / mL. Activation is carried out, and the process is controlled within 12-24 hours and placed at 37°C and 5% CO 2 Culture in an incubator.

[0037] The first expansion culture process is as follows: Initially activate the cells after 12 to 24 hours of culture by adding the same volume of mixed culture medium as on the first day of culture and adding cytokine composition B; The composition and content of the cytokine B cytokine composition are as follows: IL-2, 600-2000 IU / mL; recombinant human interleukin-15 receptor α and interleukin-15 fusion protein complex, 1-100 ng / mL, placed at 37°C and 5% CO 2 Culture in an incubator.

[0038] Supplement the culture medium and continue the expansion process as follows: Around the 4th to 16th day of culture, the cell growth status was observed. When the cell clones were larger, more in suspension, and the culture medium was light orange-yellow, the mixed culture medium containing cytokine composition B was added to adjust the final cell density to 0.8-5.0×10 6 / mL, and continue culturing.

[0039] 4. Cell harvesting.

[0040] After culturing until the day of recovery, the cells are collected, washed with injection-grade saline, and stored in a reinfusion bag to complete the preparation of mNK cells.

[0041] It can be used directly for treatment, or used to prepare mNK cell drugs, AmNK cells or AmNK cell drugs according to treatment needs.

[0042] 5. Preparation of AmNK.

[0043] Add monoclonal antibodies to the prepared mNK cell suspension at a concentration of 0.1-1 mg / ml and place at 37°C and 5% CO 2 The cells were incubated in an incubator for 30 minutes to obtain AmNK cells.

[0044] In the treatment of solid tumors, targeted antibody drugs involve multiple tumor-related signaling pathways and targets. The targeted antibody drugs included in AmNK cells include: the most common targeted drugs in the treatment of solid tumors are Herceptin (targeting epidermal growth factor receptor-2, i.e. EGFR-2 or HER-2 / neu), cetuximab (targeting epidermal growth factor receptor EGFR), bevacizumab (targeting vascular endothelial growth factor VEGF), in addition to ramucirumab targeting VEGF, rituximab, ibritumomab and ofatumumab targeting CD20, alemtuzumab targeting CD52, pertuzumab targeting HER2, ipilimumab targeting CTLA-4, nivolumab targeting PD-1, atezolizumab, avelumab and durvalumab targeting PD-L1, etc.

[0045] Structurally, the above antibodies all carry Fc fragments. A large number of studies have confirmed that the Fc fragment can be captured by CD16 on the surface of the cell membrane of NK cells, etc., so that NK cells can obtain antibody-dependent cell-mediated cytotoxicity (ADCC), improve the targeting of mNK cell killing, and thus greatly improve the killing efficiency.

[0046] The present invention provides a method for preparing memory natural killer cells using peripheral blood mononuclear cells. The obtained mNK cells express CD16 on the cell membrane after detection, which is the protein basis for capturing therapeutic antibodies through Fc fragments. When incubated with antibodies represented by Herceptin, the preparation method provided by the present invention can obtain higher killing activity of the obtained AmNK cells, which is achieved by effectively capturing antibodies through cell surface CD16.

[0047] Test Example 1 Evaluation of the proportion of each immune cell subset and cytokine secretion in mixed cells.

[0048] In this test example, the present invention evaluates the changes in the proportion of NK cell subsets in PBMC and mNK cells, as well as the expression of CD16 and NKG2C on the surface of NK cells before and after culture, and the secretion of intracellular granzyme Grzm-B and cytokine IFN-γ. Among them, CD16 is an Fc receptor, representing the ADCC killing efficacy of NK cells; NKG2C represents the proportion of cytokine-induced memory NK cells; granzyme Grzm-B and cytokine IFN-γ are also detection indicators for detecting the killing ability of NK cells, and cytokine IFN-γ also represents the immunomodulatory effect of NK cells. The results are shown in Table 1.

[0049] Table 1 Proportion of each immune cell subset and cytokine content in mixed cells The test group is the expanded cells containing mNK cells obtained by the preparation method provided in Example 1, MFI is the abbreviation of Mean Fluorescence Intensity; PBMC is the abbreviation of Peripheral Blood Mononuclear Cell; GrzmB is the granzyme.

[0050] The results show that after culturing cells using the method provided by the present invention, the proportion of NK cells increased significantly. After antibody labeling using flow cytometry, the proportion of NK cells in the mixed cells expanded from 8.7% in PBMC before culture to 44.7% in the final product. According to the preparation method provided by the present invention, more than 97% of the mNK cells obtained by culture are CD16+ and NKG2C+ NK cells, which indicates that the NK cells have strong antibody loading and memory functions. In addition, further detection of the expression of granzymes and IFN-γ showed that the proportion of cells expressing granzymes (Gram-B) in mNK cells in Example 1 was 100%, and the proportion of cells expressing IFN-γ in mNK cells was 26.2%, both of which were highly expressed, further confirming that mNK cells have strong killing ability.

[0051] The preparation method provided by the present invention can obtain the final amplified cells, in addition to mNK cells, CD3+CD56+ T cells, also known as NKT cells. It is amplified simultaneously with mNK by the amplification method in the technical invention from T cells in mononuclear cells. Therefore, after one activation and amplification operation, the preparation method provided by the present invention can simultaneously use the donor's peripheral blood mononuclear cells as the source to obtain activated cells of two types of synergistic anti-cancer cells, NK cells and T cells. Compared with other technologies, it can make more full use of the effective cells in the donor's autologous blood, so that it can obtain better therapeutic effects.

[0052] Test Example 2 Cytokine stimulation response detection before and after culture.

[0053] In this test example, 6 × 10 6 , dissolved in 6 ml serum-free medium to control the final cell concentration at 1 × 10 6 1 ul / ml of GolgiStop™ protein transport inhibitor was added to the above culture system, mixed by pipetting, and placed in 37°C, saturated humidity, 5% CO 2 After culturing in the incubator for 4 hours, the cells were taken out and the memory function of NK cells was detected by flow cytometry, that is, the secretion of cytokine IFN-γ and the expression of degranulated CD107a when encountering the stimulation factor again. The results are shown in Table 2.

[0054] Table 2 Response test results before and after cytokine stimulation The cells to be tested are respectively a negative control group and a test group; the negative control group is PBMC amplified cells without adding cell activation factors during the cell amplification process, and the test group is amplified cells containing mNK cells obtained by the method provided by the present invention.

[0055] The results showed that compared with the donor's own common PBMC without adding cytokines, the mNK cells activated by the preparation method provided by the present invention had a CD3 - CD56 + The proportion of NK in cells increased significantly; After the cultured mNK cells were stimulated by cytokine composition A, the CD107a expression and IFN-γ secretion were significantly higher than those in the group without cytokine stimulation, and the proportions reached were also significantly higher than those in the samples of ordinary PBMCs receiving cytokine stimulation. CD107a expression and IFN-γ secretion are both signs of NK cell differentiation and maturation, and have strong cancer cell killing function. This suggests that the mNK cells obtained by the preparation method provided by the present invention have good memory function.

[0056] Test Example 3 Detection of the killing efficiency of mNK cells on target cells K562.

[0057] Lactate dehydrogenase (LDH) is a relatively stable cytoplasmic enzyme that exists in the cytoplasm of normal cells. However, it can be released outside the cell when the cell is damaged or dead. LDH catalyzes lactate to form pyruvate, which is converted into a red formazan compound that can be detected by an enzyme-labeled instrument. At this time, the activity of LDH in the cell culture medium is proportional to the number of dead cells. By measuring it with an enzyme-labeled instrument and comparing it with the data of the target cell control well, the percentage of effector cells killing target cells can be calculated. This experimental method is simple and rapid to operate and can be applied to NK cell toxicity determination.

[0058] In this test example, the target cells used were NK cell sensitive cell line K562, and the cultured cells were used as effector cells. The killing experiments were performed using effector cell: target cell ratios of 40:1, 20:1, and 10:1, respectively. In addition, the effector cell spontaneous release well, target cell spontaneous release well, and culture medium background release group were set as controls. The killing efficiency calculation formula is as follows: ; According to the preparation method provided in Example 1, mNK cells were obtained. The test results are shown in Table 3, which show that mNK cells have significant K562 killing function.

[0059] Table 3 Detection results of the killing efficiency of mNK cells on K562 target cells

[0060] Test Example 4 Detection and comparison of the killing effects of mNK cells and AmNK cells on breast cancer tumor cell lines.

[0061] 1. Detection of target cell killing effect on breast cancer cell lines.

[0062] Breast cancer cell lines SKBR3 (HER2-positive cell line) and T47D (HER2-negative cell line) were used as target cells.

[0063] According to the preparation method provided in Example 1, mNK cells were prepared using donor peripheral blood PBMC.

[0064] AmNK cells incubated with Herceptin were prepared according to the preparation method provided in Example 1. Herceptin (humanized HER2 antibody) at a concentration of 0.1 mg / ml was added to the cell suspension containing the amplified mNK cells to prepare HER2-specific AmNK cells.

[0065] The specific process is as follows: Preparation of mNK cells: 6×10 6 18×10 cells are needed to kill three target cells. 6 cells; prepare antibody incubation solution: use GTT551 medium + 0.1mg / ml Herceptin, the amount of medium prepared is 9ml; resuspend 18×10 6 cells at 37°C with saturated humidity and 5% CO 2 Culture in the incubator for 0.5 hours; after half an hour, remove the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, discard the supernatant, collect the cells, and perform killing experiments.

[0066] The target cell killing experiment (LDH method) was used to detect the killing effect of mNK cells and AmNK cells. The operation process is as follows: According to the method provided in Example 1, mNK cells were prepared and AmNK cells incubated with Herceptin (humanized HER2 antibody, concentration of 0.1 mg / ml) were tested with target cells at a ratio of 40:1, 20:1 and 10:1. The killing efficiency was calculated as follows: ; The test results are shown in Table 4. The results show that the killing effect of AmNK cells is significantly higher than that of mNK cells for both HER2+ cell line SKBR3 and HER2+ cell line T47D. This result shows that AmNK cells have a more efficient killing effect with the assistance of antibody targeting.

[0067] Table 4 Detection results of the killing efficiency of mNK cells and AmNK cells on breast cancer cells

[0068] 2. Real-time cell analysis experiment of killing effect (RTCA method).

[0069] RTCA, or real-time label-free cell analysis, is a technique used to detect cell proliferation and cytotoxicity. It reflects cell growth and toxicity in real time by monitoring the physical changes of cells in cell culture plates. RTCA has the advantages of being fast, sensitive, and label-free, and is suitable for high-throughput screening and automated detection.

[0070] The present invention uses xCELLigence real-time cell analysis (RTCA) instrument to detect the killing effect of mNK cells and AmNK cells in real time.

[0071] According to the culture method provided in Example 1, the mNK cells that have not been treated with antibodies are cultured, and the mNK cells are treated with HER2 antibodies to obtain AmNK cells; Wherein, the mNK cells include mNK1 cells, mNK2 cells and mNK3 cells, and the three types of mNK cells are obtained according to the method provided in Example 1; AmNK cells include AmNK1 cells, AmNK2 cells and AmNK3 cells, and the three types of AmNK cells are obtained according to the method provided in Example 1; mNK cells, AmNK2 cells and target cells were mixed and incubated at a ratio of 5:1, and the changes in the number of cells in the incubation system were detected in real time using the xCELLigence real-time cell analyzer. Figure 1 and Figure 2 As shown, the results show that, from the results of the cell number change curves of each group, both mNK cells and AmNK cells can quickly eliminate tumor cells in the incubation system, and the same elimination effect is observed for both the HER2-positive cell line SKBR3 and the HER2-negative cell line T47D. This shows that the mNK cells and AmNK cells amplified by the culture system provided by the present invention have tumor cell killing effects, and the killing effect of AmNK after antibody incubation is higher than that of the mNK cell group.

[0072] Test Example 5 Detection of the killing effect of mNK cells and AmNK cells on colon cancer cell lines.

[0073] Colon cancer cell lines SW620 (EGFR-positive cell line) and HCT116 (EGFR-negative cell line) were used as target cells.

[0074] According to the preparation method provided in Example 1, mNK cells were prepared.

[0075] AmNK cells incubated with nimotuzumab were prepared according to the preparation method provided in Example 1. Nimotuzumab (humanized EGFR antibody) at a concentration of 0.1 mg / ml was added to a cell suspension containing mNK cells to prepare EGFR-specific AmNK cells.

[0076] The process is as follows: Prepare mNK cells: 6 × 10 cells are required for each target cell. 6 18×10 cells are needed to kill three target cells. 6 cells; Prepare antibody incubation solution: use GTT551 medium + 0.1mg / ml nimotuzumab, the amount of medium prepared is 9ml; Resuspend 18×10 6 The cells were placed in a 37°C saturated humidity, 5% CO 2 Culture in the incubator for 0.5 hours; after half an hour, remove the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, discard the supernatant, collect the cells, and perform killing experiments.

[0077] The mNK cells prepared according to the method provided in Example 1 and the AmNK cells incubated with nimotuzumab (humanized EGFR antibody at a concentration of 0.1 mg / ml) were respectively tested with target cells at a ratio of 40:1, 20:1 and 10:1. The killing efficiency was calculated as follows: The results are shown in Table 5. The results show that the killing effect of AmNK is significantly higher than that of mNK for both the EGFR-positive cell line SW620 and the EGFR-negative cell line HCT116. This result shows that AmNK cells have a more efficient killing effect with the assistance of antibody targeting.

[0078] Table 5 Detection results of the killing efficiency of mNK cells and AmNK cells on colon cancer cells

[0079] In summary, the present invention provides a method for preparing memory natural killer cells using peripheral blood mononuclear cells. The provided NK cells have memory function and have more powerful killing and cytokine secretion functions on target cells.

[0080] At the same time, the present invention provides a method for preparing memory natural killer cells using peripheral blood mononuclear cells, wherein the activation-expansion scheme provided does not include trophoblast cells, thereby overcoming the disadvantage of low expansion capacity of peripheral blood NK cells and meeting the requirements of reinfusion in clinical work and practical applications.

[0081] In addition, compared with ordinary NK cells, the AmNK cells prepared in this product after incubation with tumor-specific antibodies have acquired tumor antigen-specific targeting recognition capabilities and have stronger killing functions. Compared with CAR-NK technology, the NK cell culture technology in this product does not involve gene transfection methods, simplifies the expansion and culture process, greatly reduces the expansion and culture costs, and is easy to use on a large scale in clinical practice.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing memory natural killer cells using peripheral blood mononuclear cells, characterized in that: The steps include: S100, Preparation of mononuclear cell suspension using peripheral blood Ⅰ; S200, adjusting the cell density of mononuclear cell suspension I with a complete mixed culture medium to obtain mononuclear cell suspension II, performing initial activation culture on the cells in mononuclear cell suspension II with cytokine composition A, adding a complete mixed culture medium to obtain mononuclear cell suspension III containing initially activated natural killer cells; Among them, the components and contents of cytokine composition A are as follows: IL-12, 2-50 ng / mL; IL-18, 2-50 ng / mL; IL-15 and IL-15Ra complex, 1-100 ng / mL; S300, add cytokine composition B to mononuclear cell suspension III, and culture until the cell density is 0.8-5.0×10 6 / mL, and a mononuclear cell suspension containing memory natural killer cells was obtained; Among them, the components and contents of cytokine composition B are as follows: IL-2, 600-2000 IU / mL; IL-15 and IL-15Ra complex, 1-100 ng / mL; S400, collecting cells in mononuclear cell suspension IV, washing, and obtaining memory natural killer cells.

2. The method for preparing memory natural killer cells using peripheral blood mononuclear cells according to claim 1, characterized in that: Step S100 includes the following steps: S110, centrifuging the peripheral blood into an upper layer and a lower layer, the upper layer being plasma and the lower layer being blood cells; S120. Dilute the lower layer of blood cells to obtain a blood cell suspension, add lymphocyte separation solution to the blood cell suspension, and prepare a mononuclear cell suspension I.

3. The method for preparing memory natural killer cells using peripheral blood mononuclear cells according to claim 2, characterized in that: In step S200, the completely mixed culture medium is prepared by the following steps: S210, mixing GT-T551 H3 medium and X-VIVO medium in a mass ratio of 1:9 to 9:1 to prepare a basic mixed medium; S220, adding the plasma obtained in step S110 to the basic mixed medium, mixing well, and obtaining a complete mixed medium; The amount of plasma added is 10% to 20% by volume.

4. The method for preparing memory natural killer cells using peripheral blood mononuclear cells according to claim 1, characterized in that: In step S200, the initial activation culture time is 12 to 24 hours; in step S300, the expansion culture time is 4 to 16 days.

5. The method for preparing memory natural killer cells using peripheral blood mononuclear cells according to claim 1, characterized in that: The memory natural killer cells obtained in step S400 are subjected to antibody loading, wherein the antibody loading comprises the following steps: S410, adding a monoclonal antibody to a mononuclear cell suspension IV containing memory natural killer cells to obtain a mononuclear cell suspension V containing antibody-loaded memory natural killer cells; Wherein, the monoclonal antibody is added at a concentration of 0.1 to 1 mg / ml; S420, collecting cells in the mononuclear cell suspension V, washing, and obtaining antibody-loaded memory natural killer cells.

6. The use of memory natural killer cells, characterized in that: The application includes the application of memory natural killer cells in the preparation of medicines, wherein the memory natural killer cells are prepared by the method for preparing memory natural killer cells by using peripheral blood mononuclear cells as claimed in any one of claims 1 to 4.

7. The use of memory natural killer cells according to claim 6, characterized in that: The drug also includes a monoclonal antibody, which binds to CD16 on the memory natural killer cells to form antibody-loaded memory natural killer cells. The antibody-loaded memory natural killer cells are prepared by the method for preparing memory natural killer cells using peripheral blood mononuclear cells as described in claim 5.

8. The use of memory natural killer cells according to claim 7, characterized in that: The monoclonal antibody includes one or more of Herceptin, cetuximab, bevacizumab, ramucirumab, rituximab, ibritumomab tiuxetan, ofatumumab, alemtuzumab, pertuzumab, ipilimumab, nivolumab, atezolizumab, avelumab and durvalumab.

9. The use of memory natural killer cells according to claim 6, characterized in that: The drugs include drugs for cancer.

10. The use of memory natural killer cells according to claim 6, characterized in that: The drug includes one or more of pharmaceutically acceptable solvents, excipients and carriers.

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