Efficient multiplication culture method for memory NK cells

Abstract

The invention provides a multiplication culture method of high-purity memory NK cells, which comprises the following steps: in the initial stage of cell culture, adopting a CD6 antibody, an ADAM inhibitor, IL2, IL15 and IL18 as a starting signal combination; furthermore, a CD137 antibody, IL2 and IL21 are adopted as an amplification signal combination, IL2, IL15 and IL21 are supplemented regularly, and NK amplification is promoted. According to the invention, multiple factors and antibody combination are originally utilized, so that NK cell culture and / or NK cell amplification can be more efficiently started; the memory NK cell prepared by the method has the characteristics of high purity and high amplification multiple of NK, and has high killing ability.

Description

Technical Field

[0001] This invention relates to the field of cellular immunology, and more specifically to a method for culturing memory NK cells. Background Technology

[0002] NK cells (natural killer cells, NK) are innate lymphocytes with cytotoxic activity and immunomodulatory functions. They are characterized by their ability to exert immune responses without antigen sensitization, forming the body's first line of defense against viral infections and malignant tumors. Research over the past 20 years has shown that NK cells, upon activation by appropriate receptor signals, can also exhibit memory characteristics, significantly enhancing their survival and proliferative capacity. These receptor signals are divided into antigen-specific and non-specific types. The former includes the NKG2C receptor (antigen epitope signal presented by HLA-E), while the latter includes C family receptors and IL1 family receptors (activation signals provided by cytokines such as IL12 / 15 and IL18, respectively). In adoptive cell therapy for malignant tumors, enhanced memory NK cells are highly favored. Among them, cytokine-induced memory-like NK (CIML NK) has entered clinical trials for the treatment of hematological diseases, especially acute myeloid leukemia. Reported research indicates that intravenous infusion of these cells is safe and effective, with NK cell proliferation observed after infusion and their persistence in vivo for several months (Cubitt, Celia C et al. A novel fusion protein scaffold 18 / 12 / TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells. Molecular therapy oncolytics, vol. 24 585-596. 15 Feb. 2022; Tarannum, Mubin et al. “Innovative Strategies to Improve the Clinical Application of NK Cell-Based Immunotherapy.” Frontiers in immunology, vol. 13 859177. 25 Mar. 2022).Furthermore, studies on peritoneal xenograft models have demonstrated that CIML NK has a definite therapeutic effect on various solid tumors, including ovarian cancer (Uppendahl, Locke D et al. “Cytokine-induced memory-like natural killer cells have enhanced function, proliferation, and in vivo expansion against ovarian cancer cells.” Gynecologic oncology, vol.153,1:149-157, 2019; Wong, Pamela et al. “Flowcytometry-based ex vivo murine NK cell cytotoxicity assay.” STAR protocols, vol. 2,1 100262. 12 Jan. 2021; Foltz, Jennifer A et al. “Cytokines drive the formation of memory-like NK cell subsets via epigenetic rewiring and transcriptional regulation.” Science immunology, vol.9,96: eadk4893, 2024.). In addition, the therapeutic effects of this type of NK cell in hematogenous metastasis of solid tumors (such as killing circulating tumor cells, CTC) also have clinical potential.

[0003] In 2012, the University of Washington first reported the preparation method and function of CIML-NK (Romee, Rizwanet et al. “Cytokine activation induces human memory-like NK cells.” Blood, vol. 120, 24: 4751-60, 2012). Peripheral blood was collected and NK cells were isolated. After short-term activation with the addition of IL2 / 15 / 18 / 21, followed by long-term culture with low-concentration IL15, memory NK cells with enhanced anti-tumor function could be obtained. This protocol has now become the globally recognized classic preparation method for CIML-NK. However, the cost of preparing a single batch of cell preparation is too high, severely restricting its clinical development. Its limitations include: ① Extremely high blood collection requirements. NK cells only account for 5-15% of the total number of lymphocytes in peripheral blood, and typically 1000 cells are needed for a single NK clinical treatment. 9-10① Large quantities of peripheral blood mononuclear cells (PBMCs) are difficult to obtain from blood; ② Separate and purify 10 9-10 The cost of materials required for each PBMC is extremely high, making it unaffordable for most patients; ③ The preparation process is inefficient. The CIML-NK preparation process involves isolating and purifying NK cells from PBMCs, activating them with a combination of IL12 / 15 / 18 cytokines for 12-18 hours, then removing the cytokines and culturing for one week (only 1 ng / mL IL15 needs to be supplemented to maintain cell survival). Therefore, adding cytokines does not benefit the increase in cell number, and CIML-NK cannot be efficiently expanded in vitro. Obviously, if this type of memory NK cell is to be developed into a novel cell drug for industrialization, the problem of efficient expansion must first be solved.

[0004] Globally, NK cell culture has developed into two main systems: feeder cell culture and whole-cytokine culture. Feeder cell culture enables highly efficient in vitro NK cell expansion, typically using K562 tumor cells or autologous PBMCs as the feeder cells. In vitro culture for 2-3 weeks results in NK cell expansion exceeding 1000-fold. However, this method is limited by safety risks (such as residual tumor cells) and equipment constraints (requiring the construction of irradiation-compliant production facilities). Whole-cytokine culture relies on accessory cells to transmit signals and promote NK cell activation and expansion. However, it results in relatively low NK cell expansion, low purity of the expanded NK cells, and limited anti-tumor ability. Although optimizing the first signal can improve NK cell expansion and purity, there are few reports on whether the resulting NK cells possess memory characteristics.

[0005] For example, the Chinese patent, entitled "A Method for High-Efficiency Culture of Human NK Cells without Serum", patent publication number CN115478051 A, proposes an NK cell culture system that does not use feeder cells. The culture components required in the preparation process are serum-free culture medium containing FB23, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-γ, PMA, PHA and H3. After 14 days of in vitro culture, the NK cells expand more than 600 times and the purity reaches 89.62%.

[0006] Although this method can achieve a high NK cell expansion rate, the purity of the cells harvested after 14 days is less than 90%, with a residual T cell content of 9.51%. A high residual T cell content can cause serious side effects such as immune rejection in clinical applications. Therefore, the cells expanded using this method need to undergo further sorting or purification processes to remove T cells; otherwise, they are not suitable for allogeneic reinfusion. Furthermore, this patent does not describe the biological properties and functions of its NK cells, nor does it mention whether it possesses memory NK cell characteristics.

[0007] Therefore, in order to achieve efficient expansion of NK cells with immune memory characteristics in a non-trophoblast system, there is an urgent need in the field for a preparation method that can obtain high-purity NK cells with high expansion folds and memory NK function characteristics that can be directly applied in clinical practice. Summary of the Invention

[0008] To address one or more problems in the existing technology, this invention provides a novel method for the in vitro culture of memory NK cells with high purity and efficiency, thereby obtaining high-purity memory NK cells that can be applied clinically.

[0009] In a first aspect, the present invention provides a method for the efficient expansion and culture of memory NK cells, wherein CD6 antibody, ADAM inhibitor, IL2, IL15 and IL18 are used as a combination of initiation signals in the initial stage of cell culture.

[0010] In the above-mentioned efficient amplification and culture method, CD137 antibody, IL2, and IL21 amplification signal combination are also used during cell amplification and culture to promote NK cell amplification.

[0011] The above-mentioned efficient amplification and culture method includes the following steps:

[0012] (1) Collect peripheral blood, centrifuge to obtain peripheral blood mononuclear cells, and use magnetic beads to sort T-cell-free peripheral blood mononuclear cells (abbreviated as "dT-PBMC").

[0013] (2) Use CD6 antibody to coat the cell culture plate, and after inoculating the cells in step (1), add IL2, IL15, IL18 and ADAM inhibitor to activate dT-PBMC.

[0014] The above-mentioned efficient amplification and culture method also includes the following steps:

[0015] (3) On the third day, add 3-5% serum substitute to the NK medium, then add IL2, IL21 and CD137 antibody, and continue culturing;

[0016] (4) Every 2-3 days, replenish the NK medium containing 3-5% serum substitute, and supplement with IL2, IL21, and IL15. The above amplification culture method also includes the following steps:

[0017] (5) On day 14 of culture, samples were taken and counted to perform NK killing tests and other functional tests.

[0018] In the above-mentioned efficient amplification and culture method, in step (4), the cell culture container is adjusted according to the cell state and the color of the culture medium.

[0019] The aforementioned CD137 antibody is an IgG4 type CD137 antibody.

[0020] In a specific embodiment, the efficient amplification culture method of the present invention further includes the following steps:

[0021] (6) Continue to culture NK cells after 14 days, observe the cell status daily, supplement NK culture medium containing 3-5% serum substitute as appropriate, and add IL2, IL21 and IL15 cytokine combination.

[0022] Preferably, the efficient amplification culture method of the present invention includes the following steps:

[0023] (1) Collect peripheral blood, centrifuge to obtain peripheral blood mononuclear cells, and use magnetic beads to sort T-cell-free peripheral blood mononuclear cells;

[0024] (2) The cell culture plate was coated with CD6 antibody and the cells in step (1) were inoculated. IL2, IL15, IL18 and ADAM inhibitor were added to activate peripheral blood mononuclear cells.

[0025] (3) On the third day, add NK medium containing 3-5% serum substitute, then add 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 0.5-2 μg / mL CD137 antibody, and continue culturing;

[0026] (4) Every 2-3 days, adjust the cell culture container according to the cell status and the color of the culture medium, supplement the culture medium with NK medium containing 3-5% serum substitute, and add 500-1000 U / mL IL2, 10-50 ng / mL IL21, and 10-50 ng / mL IL15.

[0027] (5) On day 14 of culture, samples were taken and counted to perform NK killing tests and other functional tests.

[0028] The above-mentioned efficient amplification and culture method also includes the following steps:

[0029] (6) Continue to culture NK cells after 14 days, observe the cell status daily, supplement NK culture medium containing 3-5% serum substitute as appropriate, and add IL2, IL21 and IL15 cytokine combination.

[0030] The cell culture containers mentioned above are cell culture flasks or culture bags.

[0031] The aforementioned CD137 antibody is an IgG4 type CD137 antibody.

[0032] In step (2) above, the seeding density of peripheral blood mononuclear cells is 0.8-1.2 × 10⁻⁶. 6 cells / mL.

[0033] In step (1) above, PBMCs were obtained by gradient centrifugation using Ficoll separation solution, and T cells in PBMCs were removed using CD3 immune microspheres.

[0034] The amplification culture method of the present invention includes the following steps:

[0035] (1) After isolating peripheral blood PBMCs, T cells were removed by magnetic bead sorting to obtain culture initiation cells dT-PBMCs;

[0036] (2) Adjust the cell density to 0.8~1.2×10⁻⁶. 6 Cells / mL, seeded into cell culture plates, 1~2 mL / well;

[0037] (3) Start-up signal configuration: Pre-coat the cells with 1-100 ng / mL LCD6 antibody. After cell seeding, add 500-1000 U / mL IL2, 10-50 ng / mL IL15, 10-50 ng / mL IL18, and 1-8 µM ADAM inhibitor.

[0038] (4) On the third day of culture, add 1-2 mL of NK medium containing 3-5% serum substitute to each well, and then add 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 0.5-2 μg / mL CD137 antibody, and continue to culture.

[0039] (5) After culturing for 5-6 days, observe the cell status, transfer to T25 cell culture flasks in a timely manner, supplement NK medium containing 3-5% serum substitute, and add a combination of 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 10-50 ng / mL IL15 cytokines.

[0040] (6) On the 7th to 8th day of culture, observe the cell status, transfer to T75 cell culture flasks in a timely manner, supplement NK medium containing 3-5% serum substitute, and add a combination of 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 10-50 ng / mL IL15 cytokines.

[0041] (7) On the 9th to 10th day of culture, observe the cell status, transfer to a 300 mL culture bag when appropriate, supplement with NK medium containing 3-5% serum substitute, and add a combination of 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 10-50 ng / mL IL15 cytokines.

[0042] (8) On the 12th to 13th day of culture, observe the cell status, transfer to a 1000 mL culture bag when appropriate, supplement with NK medium containing 3-5% serum substitute, and add a combination of 500-1000 U / mL IL2, 10-50 ng / mL IL21 and 10-50 ng / mL IL15 cytokines.

[0043] (9) After culturing for 14 days, samples were taken and counted to perform NK killing tests and other functional tests.

[0044] If culture continues, observe cell status daily and supplement with NK medium containing 3-5% serum substitute as needed, along with a combination of cytokines: 500-1000 U / mL IL2, 10-50 ng / mL IL21, and 10-50 ng / mL IL15. The culture medium is from Beianji Biotechnology, the CD16 antibody and cytokines are from Tongli Haiyuan, the ADAM inhibitor is from MCE, the culture bag is from OriGen, and the CD137 antibody (Urelumab) is from MCE.

[0045] The innovation and novelty of this preparation method lies in:

[0046] (1) By adding an ADAM inhibitor, the downregulation of CD16 expression after NK activation is inhibited, thereby improving the transmission of activation signals and NK proliferation efficiency;

[0047] (2) The innovative use of CD6 antibody + ADAM inhibitor + IL2 + IL15 + IL18 initiation signal combination results in better proliferation effect;

[0048] (3) The CD137 antibody Urelumab + IL2 + IL21 amplification signal combination was used in an original way to promote NK amplification;

[0049] (4) The memory NK cells obtained by this novel preparation method have the characteristics of high NK purity and high amplification fold. Multiple batches of CD3-CD56+NK cells have a purity of >96%, an NK amplification fold of >500 times, and high killing ability.

[0050] (5) Compared with PB-NK and CIML-NK, the NK memory cells prepared by this novel method have stronger anti-tumor activity, higher expression levels of metabolism-related markers, and have been verified to have memory characteristics from the aspects of epigenetics and energy metabolism reprogramming, and can be directly used in clinical practice. Attached Figure Description

[0051] Figure 1 CD6 antibody and IL18 synergistically promote NK cell activation and proliferation;

[0052] The bars in bar A, from left to right, represent: the group with IL18 added alone; the group with CD6 antibody added alone; and the group with both CD6 antibody and IL18 added.

[0053] Figure 2 IL15 promotes the expression of NK activating receptors CD25 and CD137;

[0054] Figure 3 ADAM inhibitors promote NK cell activation and proliferation;

[0055] Figure 4 IgG4 CD137 antibody combined with IL2 / IL21 cytokines promotes NK cell proliferation;

[0056] The left bar chart, from left to right, represents the IL21 group; the IL21+IgG4 CD137 antibody group; and the IL21+IgG1 CD137 antibody group. The right line, from top to bottom, represents the IL2 group; the IL2+IL21 group; the IL2+IgG4 CD137 antibody group; and the IL2+IL21+IgG4 CD137 antibody group.

[0057] Figure 5 Statistical chart of multiple batches of NK preparation data;

[0058] Figure 6 Compare the expression levels of biomarkers in NK cells amplified using different preparation methods;

[0059] The horizontal axis of the bar chart, from left to right, represents the PB-NK group; the Ex-NK group; and the CMBL-NK group. Figure 7 Compare the ability of NK cells amplified using different preparation methods to secrete granzyme B, perforin, and interferon;

[0060] The horizontal axis, from left to right, represents the PB-NK group; M-NK group; Ex-NK group; and CMBL-NK group.

[0061] Figure 8 NK cell toxicity assay results (image);

[0062] Figure 9 Pairwise differential expression analysis results of Ex-NK group, CIML-NK group, and PB-NK group and similarity analysis of all samples;

[0063] A. Comparison of pairwise differential expression analysis among the Ex-NK group, CIML-NK group, and PB-NK group;

[0064] B. Comparison chart of sample similarity analysis among Ex-NK group, CIML-NK group, and PB-NK group.

[0065] Figure 10Heatmap of average expression of NK cell surface receptors, function-related genes, and memory-related genes in each group;

[0066] A. Heatmap of average expression of NK cell surface receptor-related genes in the PB-NK group, CIML-NK group, and Ex-NK group (from left to right);

[0067] B. Heatmap of average expression of NK cell function-related genes in the PB-NK, CIML-NK, and Ex-NK groups (from left to right);

[0068] Heatmap of average expression of C.NK cell memory-related genes in the PB-NK, CIML-NK, and Ex-NK groups (from left to right).

[0069] Figure 11 Energy metabolism test results (image);

[0070] A. Comparison of real-time cellular energy metabolism data of the Ex-NK group, CIML-NK group, and PB-NK group (from bottom to top);

[0071] B. Statistical chart of basal glycolysis, maximum glycolysis and reserve glycolysis data for the PB-NK group, CIML-NK group and Ex-NK group (from left to right);

[0072] Statistical chart of OCR / ECAR (oxygen consumption rate / extracellular acidification rate) ratios for C.PB-NK, CIML-NK, and Ex-NK groups.

[0073] Figure 12 Statistical chart of IFNG-CNS1 demethylation detection results.

[0074] A. Statistical graph of DNA methylation status at six CpG sites in the CNS-1 region of the IFNG gene in cells of the PB-NK group, CIML-NK group, and Ex-NK group (from left to right);

[0075] B.Statistical graph of average DNA methylation in the CNS-1 region of the IFNG gene in cells of the PB-NK group, CIML-NK group, and Ex-NK group (from left to right).

[0076] Figure 13 Comparison of amplification after 14 days of in vitro culture using different combinations of cytokines.

[0077] The bar charts, from left to right, represent: control group without IL2 / IL21; IL2 alone; IL21 alone; and IL2 / IL21. Detailed Implementation

[0078] The present invention will now be described in more detail and specifically with reference to embodiments, but the following embodiments are not intended to limit the present invention.

[0079] In this example, the culture medium was from Beacon Biotech, the CD16 antibody and cytokines were from Tongli Haiyuan, the ADAM inhibitor was from MCE, the culture bag was from OriGen, and the CD137 antibody, i.e., Urelumab antibody, was from MCE. The preferred inoculation density of dT-PBMCs was 0.8-1.2 × 10⁻⁶. 6 cells / mL.

[0080] Example 1: Optimization Study of High-Efficiency Expansion and Culture Methods for NK Cells

[0081] (1) Preparation of dT-PBMC

[0082] Peripheral blood (PBMCs) were collected (50 mL) and separated by gradient centrifugation using Ficoll separation medium. T cells in the PBMCs were then depleted using CD3 immunoglobulins (ThermoFisher). The resulting dT-PBMCs were analyzed by flow cytometry, as shown in Table 1. The main cellular components of dT-PBMCs included CD3-CD56+ NK cells (35.03% ± 12.26%, n=9), with other cells including CD3-CD19+ B cells (13.31% ± 14.49%, n=9) and CD3-CD14+ monocytes (22.79% ± 10.64%, n=9). CD3... + The T cell residual rate was < 5% (1.30% ± 0.89%, n=9).

[0083] The specific implementation method is as follows: Peripheral blood mononuclear cells (PBMCs) are obtained from human whole blood: 50 mL of human peripheral blood is collected, and 5 mL of whole blood is slowly added to a 15 mL centrifuge tube containing 5 mL of lymphocyte separation medium (Ficoll separation medium) to form two interfaces. Centrifuge at 800g for 20 min. Gently remove the supernatant serum and collect the white membrane layer cells in the centrifuge tube. Add PBS solution to the centrifuge tube to a final volume of 40 mL and centrifuge at 500g for 8 min. Discard the supernatant and add PBS solution to the centrifuge tube to a final volume of 40 mL. Centrifuge at 500g for 5 min, repeating twice. Discard the supernatant, add 2 mL of PBS to resuspend the cells, sample and count them, and adjust the cell density to 1×10⁻⁶ cells with PBS. 7 cells / mL, at 25 μL / 1×10 7Calculate the required volume of CD3 magnetic beads based on the proportion of cells, add the corresponding volume of CD3 magnetic beads to the cell suspension, mix well, and incubate at room temperature for 30 minutes. After 30 minutes, place the centrifuge tube containing the cell suspension in a magnetic rack, let it stand for 5 minutes, and collect the supernatant to obtain dT-PBMCs.

[0084] Table 1. Preparation and flow cytometry data of dT-PBMCs

[0085] serial number <![CDATA[Number of viable dT-PBMC cells (×10 6 ).]]> NK T cell B cell Monocyte 001 6.24 28.22% 1.32% 45.75% 3.25% 002 12.65 36.98% 1.46% 9.50% 24.99% 003 22.40 44.25% 0.14% 10.17% 26.01% 004 4.54 32.61% 1.55% 1.62% 37.02% 005 9.05 43.16% 0.45% 0.88% 29.06% 006 7.19 20.34% 1.49% 9.57% 18.49% 007 8.34 32.63% 1.61% 11.50% 34.91% 008 13.70 57.89% 0.53% 3.46% 15.54% 009 14.00 19.18% 3.16% 27.33% 15.82% average value 10.90 35.03% 1.30% 13.31% 22.79% SD 5.46 12.26% 0.89% 14.49% 10.64%

[0086] (2) Optimization of startup signal configuration

[0087] The NK cell initiation effect was assessed using dye dilution assays. CFSE-labeled dT-PBMCs were cultured under different initiation signal conditions. Cells were collected on day 5 after activation, and CFSE concentrations in the CD56+ population were detected by flow cytometry. Low Cell proliferation was performed, and FlowJo software was used for proliferation fitting analysis. Cell proliferation after activation was assessed using the division index (DI) and proliferation index (PI). First, the effects of CD16 signaling and IL-18 cytokines on NK cell activation proliferation were compared. Figure 1 As shown, when CD6 antibody and IL18 cytokine were used in combination, the CFSE fluorescence expression level (MFI) was lower than that of the CD6 antibody group and the IL18 cytokine group, indicating that the cell proliferation in the combination group was higher than that in the CD6 antibody group and the IL18 group. Further flow cytometry analysis revealed that CD6 antibody was the main component promoting NK cell proliferation. After 5 days of culture, the proportion of progeny cells in the CD6 antibody group was 92.24%, while that in the IL18 group was only 22.16%, and that in the combination group was 94.33%, suggesting that CD6 antibody and IL18 have a synergistic effect in promoting NK cell proliferation. Statistical analysis showed that the mitotic index (DI) and proliferation index (PI) of the combination group were higher than those of CD16 antibody or IL18 alone, indicating that the average number of cell divisions and the proliferation intensity of dividing cells were better in the combination of CD6 antibody and IL18 than in the groups that only CD6 antibody or IL18 were added. The combination of the two is more conducive to NK cell activation and proliferation.

[0088] The specific implementation method is as follows: dT-PBMCs are labeled with 2.5 μM CFSE, and the cell density is adjusted to 1×10⁻⁶. 6Cells were cultured at 50 ng / mL and then added to 12-well plates in groups. Group 1 received only 50 ng / mL IL18. Group 2 pre-coated the plates with CD6 antibody (250 μL of 15 μg / mL CD6 antibody solution was added and the plates were incubated at 4°C for at least 12 hours). Group 3 pre-coated the plates with CD16 antibody and added 50 ng / mL IL18 after cell seeding. Cells were cultured at 37°C for 5 days in a 5% CO2 incubator and then collected. CFSE fluorescence expression was detected by flow cytometry, and the chromatograms were fitted and analyzed using the FlowJo proliferation analysis platform. The system generated the mitotic index (DI) and proliferation index (PI) data for each group.

[0089] We further explored the possibility of using IL15 to synergistically promote NK cell activation. For example... Figure 2 As shown, cells were divided into two groups during activation. One group was given CD6 antibody and IL18, while the other group was given the same concentration of CD6 antibody and IL18, plus 50 ng / mL IL15. On days 2, 3, and 4, samples were taken to detect the activation markers of NK cells, CD25 and CD137. The data showed that the expression levels of CD25 and CD137 in the CD16 / IL18 / IL15 group were higher than those in the CD16 / IL18 group for three consecutive days, suggesting that IL15 can promote the expression of CD25 and CD137. Therefore, we configured CD6 antibody, IL18, and IL15 as initiation signals.

[0090] The specific implementation method is as follows: The 12-well plates are pre-coated with CD6 antibody (250 μL of 15 μg / mL CD16 antibody solution is added to each well and incubated at 4°C for at least 12 hours), and the cell density of dT-PBMCs is adjusted to 1×10⁻⁶. 6 Cells were cultured at 37°C for 4 days in a 5% CO2 incubator, and samples were taken daily from day 2 onwards. The expression levels of activating receptors CD25 and CD137 were detected by flow cytometry. The cells were then divided into groups and added to 12-well plates. Group 1 received 50 ng / mL IL18, and Group 2 received 50 ng / mL IL18 and 50 ng / mL IL15.

[0091] (3) ADAM inhibitors maintain CD16 expression on the surface of NK cells

[0092] Following NK activation, CD16 protein detaches from the cell surface within a short period, leading to restricted activation of the CD16 signaling pathway. Selective inhibition of ADAM17 can reduce cytokine-induced CD16 shedding. Figure 3As shown, a two-week proliferation experiment revealed that the control group, which received CD6 antibody + IL18 + IL15, experienced a 30-fold increase in NK cell proliferation, while the experimental group, which received CD6 antibody + IL18 + IL15 and ADAM inhibitor, experienced a 174-fold increase in NK cell proliferation. The addition of ADAM inhibitor can increase the number of NK cells proliferating. Therefore, the optimal initiation signal configuration was determined to be CD6 antibody + IL18 + IL15 + ADAM inhibitor.

[0093] The specific implementation method is as follows: The 12-well plates are pre-coated with CD6 antibody (250 μL of 15 μg / mL CD6 antibody solution is added to each well and incubated at 4°C for at least 12 hours), and the cell density of dT-PBMCs is adjusted to 1×10⁻⁶. 6 Cells were cultured at 1 mL / well and then divided into two groups and added to 12-well plates. Group 1 was treated with 50 ng / mL IL18 and 50 ng / mL IL15, while Group 2 was treated with 50 ng / mL IL18, 50 ng / mL IL15, and an ADAM inhibitor. On day 3, both groups were simultaneously treated with 1 mL / well of complete culture medium, along with 500 U / mL IL2, 50 ng / mL IL21, and 1 μg / mL CD137 antibody. From day 5 onwards, the culture medium was replenished periodically according to the cell status, with 500 U / mL IL2, 50 ng / mL IL21, and 50 ng / mL IL15 added after each replenishment. Cells were cultured at 37°C in a 5% CO2 incubator throughout the process. On day 14, cells were collected for counting, and the purity of CD3-CD56+NK cells was detected by flow cytometry, and the NK cell expansion fold was calculated.

[0094] (4) Optimization of proliferation signal configuration

[0095] D1 signaling can initially activate NK cells and increase CD137 receptor expression. Therefore, a CD137 agonist antibody was added on day 3 of culture to enhance the activation of signaling pathways such as NF-κB and improve NK cell proliferation and functional activity. IL-21 belongs to the IL-2 subfamily of type I cytokines and activates the JAK-STAT signaling pathway. It has potential synergistic effects with CD137 signaling. For example, STAT3 signaling activated by IL-21 and NF-κB signaling activated by CD137 can jointly regulate anti-apoptotic protein Bcl-2 family members downstream, jointly promoting NK cell survival. Literature studies have shown that IgG1 monoclonal antibodies can directly upregulate the CD137 signaling pathway and promote NK cell activation (Lin W et al. Fc-dependent expression of CD137 on human NK cells: insights into "agonistic" effects of anti-CD137 monoclonal antibodies. Blood. 2008), while the IgG4 CD137 antibody Urelumab can also enhance its agonistic activity against NK cells in the presence of CD32A, CD32B, and CD64 (Leitner J et al. FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab. FrontImmunol. 2023). Therefore, we explored the activation effects of different CD137 antibody types (IgG1 or IgG4) on NK cells. Figure 4As shown, the average splitting index (DI) of the group that added IL21 and IgG4 type CD137 antibody (Urelumab) on the third day was 2.1, which was better than that of the IL21 group (DI=1.83) and the IL21 / IgG1 type CD137 antibody group (DI=2.0). This indicates that the IgG4 type antibody is more effective in activating NK than the IgG1 type, and the superposition of multiple signals is more conducive to promoting long-term NK amplification. In addition, several studies have demonstrated that IL2 activates various kinase pathways by binding to the heterotrimeric protein IL-2 receptor (IL-2R) expressed on NK cells, which can promote NK cell survival, activation, and enhance cytotoxicity in a short period of time (Warren HS. “NK cell proliferation and inflammation.” Immunol Cell Biol, 74(5):473-4801996; Liu X et al. PRDM1 decreases sensitivity of human NK cells to IL2-induced cell expansion by directly repressing CD25 (IL2RA). J Leukoc Biol, 109(5):901-914, 2021). Therefore, the optimal proliferation signal configuration for D3 was finally determined to be IgG4 type CD137 antibody + IL21 + IL2.

[0096] The specific implementation method is as follows: The 12-well plates were pre-coated with CD6 antibody (250 μL of 15 μg / mL CD16 antibody solution was added to each well and the plate was incubated at 4°C for at least 12 hours), dT-PBMCs were labeled with 2.5 μM CFSE, and the cell density was adjusted to 1 × 10⁻⁶ cells / well. 6 Cells were cultured at 37°C and 5% CO2 for 48 hours. Then, in groups, 1 mL of complete culture medium was added to each well. On day 1, both groups received 50 ng / mL IL18, 50 ng / mL IL15, and an ADAM inhibitor. On day 3, both groups received 1 mL / well of complete culture medium. Group 1 received only 50 ng / mL IL21, Group 2 received 50 ng / mL IL21 and 1 μg / mL IgG1 CD137 antibody, and Group 3 received 50 ng / mL IL21 and 1 μg / mL IgG4 CD137 antibody (Urelumab). After 5 days of culture at 37°C and 5% CO2, cells were collected, and CFSE fluorescence expression was detected by flow cytometry. The chromatograms were fitted and analyzed using the FlowJo proliferation analysis platform, and the system generated the mitotic index (DI) and proliferation index (PI) data for each group.

[0097] (5) Analysis of culture system and cell expansion stability

[0098] Through prior research and optimization of NK cell initiation and proliferation signals, an innovative NK cell expansion and culture protocol was developed, and multiple batches of NK cell cultures were conducted. Figure 5 High-purity NK cells (NK purity > 96.00%) can be obtained by collecting 50 mL of peripheral blood to separate dT-PBMCs and culturing them in vitro for 14-28 days. The NK cell amplification fold was approximately 230-fold on day 14, approximately 559-fold on day 21, and approximately 1382-fold on day 28. Tables 2 and 3 show the NK cell amplification data for two batches, respectively.

[0099] The NK cell expansion steps of this invention are as follows:

[0100] Preprocessing

[0101] 1. Antibody pre-coating: Add 250 μL of CD16 antibody solution with a concentration of 15 μg / mL to 10 wells of a 12-well plate and let stand overnight at 4°C.

[0102] 2. Preparation of complete culture medium: Add 50 mL of serum substitute and 10 mL of GlutaMAX additive (Gibco) to every 940 mL of Bepanthen HIPP 009 medium, mix well to prepare 1 L of NK complete culture medium. (Serve substitute content is 5%)

[0103] D1

[0104] 1. PBMCs were isolated from 50 mL of peripheral blood using a gradient centrifugation method (800×g for 20 minutes), and the cell density was adjusted to 1×10⁻⁶. 7 / mL, add 25 μL / 1×10⁻⁶ CD3 magnetic beads 7 After mixing the cells thoroughly in a 15ml centrifuge tube, incubate at room temperature for 30 minutes. Then, place the 15ml centrifuge tube on a magnetic rack and let it stand for 5 minutes. Collect the supernatant to obtain CD3- cells, which are dT-PBMCs.

[0105] 2. Adjust the dT-PBMC cell density to 1×10⁻⁶. 6 Cells / mL were seeded into pre-coated antibody-treated 12-well plates (the coating solution in the plates was removed beforehand). 2 mL of cell suspension was added to each well, followed by 500 U / mL IL-2, 50 ng / mL IL-15, 50 ng / mL IL-18, and 4 µM ADAM inhibitor.

[0106] 3. Incubate at 37℃ and 5% CO2 for 48 hours.

[0107] D3

[0108] 1. Add 1 mL of complete culture medium to each well.

[0109] 2. Add 500 U / mL IL2, 50 ng / mL IL21 and 1 μg / mL CD137 antibody to each well.

[0110] 3. After mixing thoroughly, incubate at 37℃ in a 5% CO2 incubator for 48-72 hours.

[0111] D5~6

[0112] 1. Observe the cell status daily. When the culture medium turns obviously yellow or obvious cell clusters are visible under the microscope, collect the cell suspension from each well.

[0113] 2. Transfer the cells to a T25 flask, add complete culture medium to 10 mL / flask, and add 500 U / mL IL2, 50 ng / mL IL21 and 50 ng / mL IL15.

[0114] 3. After mixing thoroughly, incubate at 37℃ in a 5% CO2 incubator for 48-72 hours.

[0115] D7~8

[0116] 1. Observe the cell status daily. When the culture medium turns obviously yellow, collect the cell suspension from each well.

[0117] 2. Transfer the cells into a T75 flask, add complete culture medium to 40 mL / flask, and add 500 U / mL IL2, 50 ng / mL IL21 and 50 ng / mL IL15.

[0118] 3. After mixing thoroughly, incubate at 37℃ in a 5% CO2 incubator for 48-72 hours.

[0119] D9

[0120] 1. Transfer cells from the T75 flask to an OriGen 300 mL cell culture bag, add complete culture medium to 300 mL, and add 500 U / mL IL2, 50 ng / mL IL21 and 50 ng / mL IL15.

[0121] 2. After mixing thoroughly, incubate at 37℃ in a 5% CO2 incubator for 72 hours.

[0122] D12

[0123] 1. Transfer cells from the OriGen 300 mL cell culture bag to the OriGen 1000 mL cell culture bag, add complete culture medium to 1000 mL, and add 500 U / mL IL2, 50 ng / mL IL21 and 50 ng / mL IL15.

[0124] 2. After mixing thoroughly, incubate at 37℃ in a 5% CO2 incubator for 72 hours.

[0125] D14

[0126] 1. Cells can be harvested on day 14 or cultured until day 21. If continued culture is required, replenish the medium every 3 days by adding 500 U / mL IL2, 50 ng / mL IL21, and 50 ng / mL IL15.

[0127] time Cell count Cell viability Cell expansion factor NK purity NK count NK amplification factor D1 6.24E+06 88.20% / 28.22% 1.76E+06 / D14 2.53E+08 84.80% 40.54 99.34% 2.51E+08 142.80 D21 9.63E+08 97.20% 154.33 99.34% 9.57E+08 543.55 D28 2.28E+09 98.80% 365.38 98.92% 2.26E+09 1281.46

[0128] Table 2 NK Culture Data for Batch NK001

[0129] time Cell count Cell viability Cell expansion factor NK purity NK count NK amplification factor D1 1.20E+07 94.10% / 30.31% 3.64E+06 / D14 4.74E+08 85.10% 39.50 97.23% 4.61E+08 126.71 D21 1.74E+09 74.70% 145.00 94.15% 1.64E+09 450.40 D28 5.68E+09 84.80% 473.33 95.00% 5.40E+09 1483.56

[0130] Table 3 NK cell culture data for batch NK002

[0131] Example 2: Phenotypic and functional detection of NK cells expanded using this novel method.

[0132] NK cells amplified using this novel method (Ex-NK) served as the experimental group, while two control groups were established: PBMC-derived NK cells (PB-NK) and CIML-NK cells prepared using the classic protocol (Romee, Rizwan et al. Cytokine activation induces human memory-like NK cells. Blood, 2012). PB-NK cells were purified from PBMCs using an NK sorting reagent (Miltenyi, Germany) and then cultured overnight with a low concentration of IL15 (1 ng / mL). CIML-NK cells were purified from PBMCs using an NK sorting kit (Miltenyi, Germany) and then activated overnight with a combination of IL12 (10 ng / mL), IL15 (100 ng / mL), and IL18 (50 ng / mL) cytokines. Flow cytometry was used to analyze the positive rate and fluorescence intensity of various markers. Cell surface markers were directly labeled with fluorescent antibodies, while cytoplasmic or nuclear markers were treated with a fixation and permeabilization reagent before fluorescent antibody labeling.

[0133] The specific implementation method is as follows: ① The Ex-NK group cell amplification method is the same as the NK cell amplification steps of the present invention.

[0134] ② Preparation of PB-NK cells: Collect 50 mL of human peripheral blood. Slowly add 5 mL of whole blood to each 15 mL centrifuge tube containing 5 mL of lymphocyte separation medium (Ficoll separation medium), forming two interfaces. Centrifuge at 800g for 20 min. Gently remove the supernatant serum and collect the white membrane layer cells in the centrifuge tube. Add PBS solution to the centrifuge tube to a final volume of 40 mL and centrifuge at 500g for 8 min. Discard the supernatant and add PBS solution to the centrifuge tube to a final volume of 40 mL. Centrifuge at 500g for 5 min, repeating twice. Discard the supernatant, add 2 mL of PBS to resuspend the cells, sample and count, and adjust the cell density to 1×10⁻⁶ cells with PBS. 7 NK cells were enriched using the Miltenyi NK Sorting Kit according to the manufacturer's instructions to obtain PB-NK cells, and the cell density was adjusted to 2 × 10⁶ cells / mL. 6 Add 1 ng / mL IL15 to the culture medium and incubate for 3-5 days. During this period, cells can be collected for subsequent detection as needed.

[0135] ③ CIML-NK cell preparation method: Collect 50 mL of human peripheral blood. The PBMC isolation and NK cell enrichment methods are the same as those for the PB-NK cell preparation method. Adjust the density of the enriched NK cells to 2 × 10⁻⁶. 6 Add 4 mL of IL-12, 100 ng / mL IL-15, and 50 ng / mL IL-18 to each well of a 6-well plate for short-term stimulation for 16-24 hours to prepare CIML-NK cells. After 16-24 hours, collect the cells, centrifuge at 500g for 5 minutes, discard the supernatant, and adjust the cell density to 2×10⁻⁶ cells / well. 6 Add 1 ng / mL IL15 and culture for 1-3 days. During this period, cells can be collected for subsequent detection as needed.

[0136] ④ M-NK cell preparation method: Collect 50 mL of human peripheral blood. The PBMC isolation and NK cell enrichment methods are the same as those for PB-NK cell preparation. Amplification and culture are performed using the Miltenyi NK Amplification Kit, and the amplification method is the same as the kit instructions.

[0137] (1) Identification of markers of high expression of Ex-NK such as CD25, CD71, GLUT1, SLC3A2, and EZH2.

[0138] like Figure 6As shown, flow cytometry was used to detect various cellular markers in PB-NK, CINML-NK, and Ex-NK cells, respectively. The results showed that the expression levels of the activating receptor CD25, memory-related markers CD71, GLUT1, SLC3A2, functional cytokine granzyme A, and epigenetic marker EZH2 in NK cells amplified by this new method (Ex-NK) were significantly better than those in PB-NK. In particular, the expression levels of memory-related markers GLUT1 and epigenetic marker EZH2, as well as the granzyme A secretion capacity, were superior to those in CINML-NK, suggesting that the NK cells amplified by this new method have the functional attributes of memory NK cells and have the potential to play a role in enhancing metabolic capacity, anti-tumor function, and epigenetic regulation.

[0139] (2) Identify that Ex-NK has the ability to secrete high levels of cytokines (IFNγ, Perforin, GZMB).

[0140] The Ex-NK cells amplified using this novel method were used as the experimental group. Three control groups were also set up: PB-NK, CIML-NK, and M-NK cells amplified using a commercially available NK amplification kit. The NK cells from each group were co-cultured with K562 cells (E:T=1:1) for 24 hours, and the supernatant was collected. Cytokine secretion capacity, including interferon-gamma (IFNγ), perforin, and granzyme B (GZMB), was detected using an ELISA kit. The results are as follows: Figure 7 As shown:

[0141] The results of the GZMB ELISA kit showed that after co-culturing with target cells for 24 hours, the GZMB secretion capacity of NK cells in peripheral blood was 2.45±0.99 ng / mL. After amplification with commercial NK kits, the NK cells were 43.21±17.23 ng / mL, CIML-NK cells were 118.19±32.06 ng / mL, and Ex-NK cells were 303.92±98.42 ng / mL. The NK cells obtained by this process had the highest GZMB secretion capacity.

[0142] The results of the Perforin ELISA kit showed that after co-culturing with target cells for 24 hours, the Perforin secretion capacity of NK cells in peripheral blood was 1.34±1.19 ng / mL, CIML-NK was 3.42±0.51 ng / mL, and after amplification with a commercial NK kit, NK was 1.93±1.09 ng / mL and Ex-NK was 6.24±2.07 ng / mL. The NK cells obtained by this process had the highest Perforin secretion capacity.

[0143] The results of the IFNγ ELISA kit showed that after 24 hours of co-culture with target cells, the IFNγ secretion capacity of NK cells in peripheral blood was 54.28±57.23 pg / mL. After amplification with a commercial NK kit, the IFNγ secretion capacity of NK cells was 969.01±922.46 pg / mL, CIML-NK cells were 33776.63±28108.44 pg / mL, and Ex-NK cells were 33138.89±35590.36 pg / mL. CIML-NK cells had the highest secretion capacity, followed by Ex-NK cells, while the secretion level of NK cells from peripheral blood was the lowest.

[0144] In summary, Ex-NK cells showed increased secretion of all cytokines compared to PB-NK cells, with Perforin and GZMB secretion levels being superior to those of the positive control cell CIML-NK, consistent with the characteristics of memory-NK cells.

[0145] (3) The Ex-NK was identified as having a strong target cell killing ability.

[0146] K562 cells were labeled with Sartorius Cytolight fluorescent dye and co-cultured with NK cells at different effector-to-target ratios for 4 hours. Flow cytometry was then used to detect the FITC+K562 PI+ positivity rate. The killing rate was calculated using the formula: (Experimental group K562 PI+% - K562 control group PI+%) / (100% - K562 control group PI+%).

[0147] Data from multiple batches of cytotoxicity experiments were compiled and analyzed, such as... Figure 8 As shown, Ex-NK cells have a killing effect on K562 target cells. The killing rate is >50.0% when E:T=5:1 and >70.0% when E:T=20:1. There are significant differences among the effector-target ratio groups, suggesting that NK cells have the ability to efficiently kill K562 target cells.

[0148] (4) Bioinformatics analysis

[0149] We performed bulk mRNA sequencing to determine the transcriptional landscape of ExNK and CIML-NK cells relative to PB-NK cells. Differential expression analysis was performed on the three bulk RNA-seq sequencing data. Significantly differentially expressed genes were screened based on |log2FC| > 1 and padjust < 0.05, and the data were visualized using volcano plots. Figure 9 As shown in Figure A, the amplified Ex-NK and CIML-NK showed high similarity, while both differed significantly from PB-NK, with considerable overlap in upregulated genes. Correlation analysis was performed on all samples (expression matrices of all genes), and the correlation coefficients were displayed using corplot, as shown below. Figure 9As shown in B, the analysis results confirm that the correlation between Ex-NK and CIML-NK is significantly higher than that between the two and PB-NK.

[0150] Further, heatmaps of biomarker expression were created. Mean values ​​were applied to the samples within each of the Ex-NK, CIML-NK, and PB-NK groups, and the rows were visualized. For example... Figure 10 As shown, the gene expression levels of surface markers CCR5, CXCR3, CCR1, CXCR6 and KLRC1, functional markers GZMB, TNFSF10, GZMA, FASLG and IFNG, and memory markers SLCTA5, SLC1A5, KLRC1 and SLCZA1 in the Ex-NK group were superior to those in PB-NK and CIML-NK. Ex-NK cells were superior to or approximately equal to CIML-NK in several specific aspects, and were also superior to PB-NK.

[0151] Based on this, we can conclude that the expanded Ex-NK cells are similar to the positive control CIML-NK in terms of overall transcriptome expression level, meaning they may be consistent with CIML-NK in terms of proliferation, function, and memory.

[0152] (5) Energy metabolism reprogramming

[0153] To assess the metabolic levels of Ex-NK cells, we used the Seahorse Energy Metabolism Analyzer to evaluate mitochondrial oxidative phosphorylation and glycolytic function in NK cells by measuring cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Figure 11 As shown, Ex-NK and CIML-NK cells exhibited significantly upregulated glucose metabolism compared to PB-NK cells, manifested as stronger glycolysis, glycolytic capacity, and glycolytic reserve. Figure 11 -B). However, there was no significant difference in mitochondrial oxidative phosphorylation function among the three. The OCR:ECAR ratio is often used to detect changes in cellular energy use; a lower ratio indicates that the cell is more inclined to use glucose metabolism for energy. Figure 11 -C). Consistent with previous results, CIML-NK has a lower OCR:ECAR ratio. Similarly, Ex-NK exhibits an even lower OCR:ECAR ratio than CIML-NK.

[0154] (6) Epigenetic remodeling

[0155] The epigenetic structure of the IFNG gene plays a crucial regulatory role in its transcription. In naive NK cells, the IFNG gene promoter is open, while the CNS-1 region is closed. Therefore, demethylation of the CpG site in the CNS-1 region significantly promotes IFNγ secretion and is an important marker of CIML-NK cells. Figure 12 As shown in -A, the detection results revealed that six important CpG sites in the CNS-1 region of the IFNG gene in Ex-NK cells showed significant ultra-low methylation, which was significantly lower than that in CIML-NK and PB-NK. Figure 12 -B indicates the average methylation level of these 6 CpG sites, which is as high as 76.7% in PB-NK, 54.8% in CIML-NK, and only 6% in Ex-NK. This means that the IFNG gene is in a more open state in Ex-NK cells and may have more sustained IFNγ secretion energy.

[0156] (6) Verify the effect of IL2 / IL21 cytokines on NK cell amplification.

[0157] Based on bioinformatics analysis, we believe that IL-2 family cytokines and the potent JAK-STAT signaling pathway play a key role in the effective expansion of NK cells. Figure 13 As shown, we cultured cells in vitro for 14 days using different combinations of cytokines. The results showed that the average fold increases of the four groups—IL-2- / IL-21-, IL-2+ / IL-21-, IL-2- / IL-21+, and IL-2+ / IL-21+—were 17-fold, 52-fold, 45-fold, and 128-fold, respectively. These data ultimately confirmed that IL-2 and IL-21 play important roles in activating the JAK-STAT signaling pathway and promoting NK cell proliferation, and that their combined use produces a strong synergistic effect.

[0158] The specific implementation method is as follows: The 12-well plates are pre-coated with CD16 antibody (250 μL of 15 μg / mL CD16 antibody solution is added to each well and the plate is incubated at 4°C for at least 12 hours), and the dT-PBMC cell density is adjusted to 1×10⁻⁶. 6Cells / mL were seeded into pre-coated antibody-treated 12-well plates (the coating solution in the wells was removed beforehand). 2 mL of cell suspension was added to each well. Group 1 (IL2-IL21-) and Group 3 (IL2-IL21+) were treated with 50 ng / mL IL15, 50 ng / mL IL18 and 4 µM ADAM inhibitor. Group 2 (IL2+IL21-) and Group 4 (IL2+IL21+) were treated with 500 U / mL IL2, 50 ng / mL IL15, 50 ng / mL IL18 and 4 µM ADAM inhibitor. After culturing the cells at 37°C and 5% CO2 for 48 hours, on day 3, each group was supplemented with 1 mL of complete culture medium per well. Group 1 was supplemented with only 1 μg / mL CD137 antibody Urelumab, Group 2 was supplemented with 500 U / mL IL2 and 1 μg / mL CD137 antibody Urelumab, Group 3 was supplemented with 50 ng / mL IL21 and 1 μg / mL CD137 antibody Urelumab, and Group 4 was supplemented with 500 U / mL IL2, 50 ng / mL IL21 and 1 μg / mL CD137 antibody Urelumab. From day 5 onwards, fluid was replenished periodically according to cell status. After each replenishment, different cytokines were added to each group: Group 1 received only 50 ng / mL IL15; Group 2 received 500 U / mL IL2 and 50 ng / mL IL15; Group 3 received 50 ng / mL IL21 and 50 ng / mL IL15; and Group 4 received 500 U / mL IL2, 50 ng / mL IL21, and 50 ng / mL IL15. Cells were cultured at 37°C in a 5% CO2 incubator throughout the process. On day 14, cells were collected for counting, and the purity of CD3-CD56+NK cells was detected by flow cytometry, and the fold increase of NK cells was calculated.

[0159] The NK cell culture system proposed in this invention does not use feeder cells. The required culture components are CD16 antibody, ADAM inhibitor, CD137 antibody, IL2 / IL15 / IL18 / IL21, serum substitute, and serum-free culture medium. After 14 days of in vitro culture, NK cells expand more than 100-fold with a purity exceeding 98%. Although the expansion fold is lower than that of CN 115478051 A, this method yields high-purity NK cells that can be directly used for allogeneic infusion with high safety and strong practicality. A series of functional, metabolic, and epigenetic studies further demonstrate that the expanded NK cells produced by this method are memory NK cells with stronger anti-tumor activity and longer in vivo survival capacity, possessing significant application value. See [link to relevant documentation]. Figure 5 .

[0160] Furthermore, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that various modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the scope and essence of the present invention.

Claims

1. A method for efficiently expanding memory NK cells, characterized by, In the initial stage of cell culture, CD6 antibody, ADAM inhibitor, IL2, IL15, and IL18 are used as the starting signal combination.

2. The method for efficient expansion and culture of memory NK cells as described in claim 1, characterized in that, During the cell expansion culture process, CD137 antibody, IL2, and IL21 expansion signal combination are used to promote NK expansion.

3. The high-efficiency expansion culture method according to claim 1, wherein The method comprises the following steps: (1) collecting peripheral blood, centrifuging to obtain peripheral blood mononuclear cells, and using magnetic bead sorting to obtain T cell-depleted peripheral blood mononuclear cells; (2) using CD6 antibody to coat a cell culture plate, inoculating the cells of step (1), and adding IL2, IL15, IL18, and ADAM inhibitor to activate the peripheral blood mononuclear cells.

4. The high-efficiency expansion culture method according to claim 3, wherein The method further comprises the following steps: (3) on the third day, adding 3-5% serum substitute NK culture medium, and then adding IL2, IL21, and CD137 antibody for continuous culture; (4) every 2-3 days, supplementing the NK culture medium containing 3-5% serum substitute, and supplementing the addition of IL2, IL21, and IL15.

5. The high-efficiency expansion culture method according to claim 4, wherein The method further comprises the following steps: (5) on the 14th day of culture, sampling and counting, and performing NK killing detection and other function detection.

6. The high-efficiency expansion culture method according to claim 5, wherein During the culture process of step (4), the cell culture container is adjusted according to the cell state and the culture medium color; and the CD137 antibody is an IgG4 type CD137 antibody.

7. The high efficiency expansion culture method of any one of claims 3-6, wherein, The method further comprises the following steps: (6) continuing to culture the NK cells after 14 days, observing the cell state every day, supplementing the NK culture medium containing 3-5% serum substitute in time, and adding IL2, IL21, and IL15 cytokine combination.

8. The high-efficiency expansion culture method according to claim 3, wherein The method comprises the following steps: (1) collecting peripheral blood, centrifuging to obtain peripheral blood mononuclear cells, and using magnetic bead sorting to obtain T cell-depleted peripheral blood mononuclear cells; (2) using CD6 antibody to coat a cell culture plate, inoculating the cells of step (1), and adding IL2, IL15, IL18, and ADAM inhibitor to activate the peripheral blood mononuclear cells; (3) on the third day, adding 3-5% serum substitute NK culture medium, and then adding 500-1000 U / mL IL2, 10-50 ng / mL IL21, and 0.5-2 μg / mL CD137 antibody for continuous culture; (4) every 2-3 days, adjusting the cell culture container according to the cell state and the culture medium color, supplementing the NK culture medium containing 3-5% serum substitute, and supplementing the addition of 500-1000 U / mL IL2, 10-50 ng / mL IL21, and 10-50 ng / mL IL15; (5) on the 14th day of culture, sampling and counting, and performing NK killing detection and other function detection.

9. The expansion culture method according to claim 7, wherein The method comprises the following steps: (6) continuing to culture the NK cells after 14 days, observing the cell state every day, supplementing the NK culture medium containing 3-5% serum substitute in time, and adding IL2, IL21, and IL15 cytokine combination.

10. The high-efficiency expansion culture method according to claim 6 or 7, wherein The cell culture container is a cell culture bottle or a culture bag; and the CD137 antibody is an IgG4 type CD137 antibody.

11. The high-efficiency expansion culture method according to claim 8, wherein The peripheral blood mononuclear cell seeding density in step (2) is 0.8-1.2 x 10 6 cells / mL.

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