A method for expanding and activating memory-like nk cells

Abstract

The application provides a method for enhancing the expansion and activation of memory-like NK cells, and belongs to the technical field of cell culture. The application provides a method for obtaining strong memory-like NK cells, which comprises using complete culture medium, IL-2, IL-12, IL-15, IL-18, sodium alpha-ketoglutarate and DOT1L inhibitor; the concentration of the sodium alpha-ketoglutarate is 0.1-2 mM, and the concentration of the DOT1L inhibitor is 10-100 nM. The addition of low-concentration sodium alpha-ketoglutarate in the complete culture medium of memory-like NK cells can promote the in-vivo and in-vitro proliferation capacity of the memory-like NK cells. The addition of low-concentration DOT1L inhibitor in the late culture stage can further enhance the killing function of the memory-like NK cells by inhibiting H3K79me2 / 3, etc., so that memory-like NK cells with a larger number and stronger killing capacity are obtained.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for enhancing the expansion and activation of memory-like NK cells. Background Technology

[0002] NK cells, or natural killer cells, are core effector cells of the body's innate immune system. They can kill abnormal cells without prior antigen sensitization and simultaneously regulate the immune microenvironment, playing a crucial role in anti-tumor and anti-viral infections. NK cells originate from hematopoietic stem cells in the bone marrow and mature through differentiation within the bone marrow and thymus microenvironment. They are mainly distributed in peripheral blood, spleen, liver, and lymph nodes, with very few found in the thymus and mucosal tissues.

[0003] NK cells can target and eliminate abnormal cells through multiple killing pathways, including tumor cells, virus-infected cells, and senescent / apoptotic abnormal cells. Simultaneously, NK cells can regulate the overall immune response, promote dendritic cell (DC) maturation, activate helper T cells, and suppress excessive inflammatory responses, maintaining immune homeostasis. Memory-like NK cells (ML-NK), with their unique advantage of "one-time activation and long-term response," have shown broad application prospects in tumor immunotherapy and anti-infective therapy, becoming a popular candidate cell type for adoptive immunotherapy. Compared to ordinary NK cells, they have significant advantages such as higher in vitro expansion efficiency, more stable activation phenotype, longer in vivo duration, stronger tumor infiltration ability, better tolerance to the immunosuppressive microenvironment, significantly enhanced killing activity and antigen specificity, higher clinical treatment response rate, and extremely low off-target toxicity, making them a superior cell type for immunotherapy. However, their application still faces multiple bottlenecks in the process of basic research to clinical translation. The induction of memory-like NK cells depends on a specific combination of cytokines, but significant differences exist in induction time, cytokine concentration, and culture systems among different research teams. This results in considerable heterogeneity in the purity of the memory phenotype, cytotoxic activity, and cytokine secretion capacity of the prepared cells, making it impossible to establish a unified quality control standard and directly affecting the reproducibility of therapeutic effects. Furthermore, clinical treatment requires a single infusion of a large volume (usually ≥1×10⁻⁶). 9 Memory-like NK cells are valuable, but existing induction protocols often fail to produce sufficient cell numbers for clinical dosages. Furthermore, indiscriminate expansion can lead to cell depletion, and long-term culture can decrease cytotoxic activity and weaken IFN-γ secretion. This is especially true for autologous memory-like NK cells, which may experience further reduced expansion efficiency and functional stability due to the patient's compromised immune status. Therefore, improving techniques for expanding and activating memory-like NK cells is crucial for enhancing their utilization. Summary of the Invention

[0004] This invention provides a method for enhancing the expansion and activation of memory-like NK cells, achieving a dual enhancement of memory-like NK cell expansion and function through the synergistic effect of metabolites and epigenetic enzyme inhibitors.

[0005] This invention provides a method for expanding strong memory-like NK cells, comprising culturing NK cells in an expansion culture medium to obtain strong memory-like NK cells; The amplification medium includes complete medium, IL-2, IL-12, IL-15, IL-18 and sodium α-ketoglutarate; The concentration of sodium α-ketoglutarate in the amplification medium is 0.1~2 mM.

[0006] In one embodiment of the present invention, the concentration of IL-2 in the amplification culture medium is 1000 U / mL, the concentration of IL-12 is 1~100 ng / mL, the concentration of IL-15 is 1~100 ng / mL, and the concentration of IL-18 is 1~100 ng / mL.

[0007] In one embodiment of the present invention, the basal medium of the complete culture medium includes any one of the following: vivo15 medium, Gibco CTS AIM V, HIPP-T009, ImmunoCult™-XF, ImmunoCult™ NK CellExpansion Medium, and NK MACS® Medium; The complete culture medium also includes 5% autologous plasma by volume.

[0008] In one embodiment of the present invention, the method for obtaining NK cells includes the following steps: inoculating PBMC cells into a complete culture medium for culture, and harvesting NK cells.

[0009] In one embodiment of the present invention, the cultivation parameters include static cultivation at 37°C and 5% CO2 for 24 h. The present invention also provides a method for reactivating NK cells, comprising adding a DOT1L inhibitor to the culture system of the amplification method, and continuing to culture to obtain reactivated NK cells.

[0010] In one embodiment of the present invention, the concentration of the DOT1L inhibitor in the system is 10~100 nM.

[0011] In one embodiment of the present invention, the DOT1L inhibitor includes at least one of the following: EPZ-5676, Epz004777, SGC707, and SYC-522.

[0012] The present invention also provides reactivated NK cells obtained using the above-described reactivation method.

[0013] This invention also provides the application of the above-mentioned reactivated NK cells in the preparation of reagents for targeted elimination of abnormal cells.

[0014] Beneficial Effects: This invention provides a method for promoting the expansion of NK cells in vitro. A low concentration of sodium α-ketoglutarate (α-KG-Na) is added to the complete culture medium of NK cells as an intermediate in the tricarboxylic acid cycle. α-KG-Na can enhance the efficiency of the mitochondrial respiratory chain in NK cells, promote ATP production, and provide energy for rapid proliferation. This invention also provides a method for improving the cytotoxic activity of expanded NK cells in vitro, including adding a low concentration of DOT1L inhibitor to the culture medium. The DOT1L inhibitor can inhibit H3K79me2 / 3, thereby relieving transcriptional inhibition of cytotoxic genes such as IFNG and PRF1, and simultaneously enhancing cytotoxic function. This invention achieves NK cell expansion through a synergistic "metabolic-epimetic" dual-axis approach by adding low doses of α-KG-Na and a specific DOT1L inhibitor to a conventional NK cell culture system, without relying on feeder cells or genetic modification. Validated by examples, α-KG-Na-enhanced acetyl-CoA levels provide substrates for histone acetylation, and together with DOT1L inhibition, they remodel chromatin, achieving a dual enhancement of "proliferation and function," thus providing a source for the clinical application of NK cells. Attached Figure Description

[0015] Figure 1 This is a graph showing the proliferation curve of NK cells. Figure 2 This is a graph showing the proportion of NK cells. Figure 3 This is a diagram showing the expression of NKG2G molecules. Figure 4 This is a graph showing the expression of Tigit molecules; Figure 5 A plot showing the proportion of PD-1 expression; Figure 6 The killing effect of K562 cells is shown in the graph. Figure 7 This is a graph showing the expression of effector molecules; Figure 8 A diagram showing the killing of K562 cells after EPZ-5676 activation; Figure 9 This is a diagram showing the expression of effector molecules in K562 cells after EPZ-5676 activation; Figure 10 A diagram showing the killing of A549 cells after EPZ-5676 activation; Figure 11 This is a diagram showing the expression of effector molecules in A549 cells after EPZ-5676 activation; Figure 12Image showing the damage to MCF-7 after EPZ-5676 is activated; Figure 13 This is a diagram showing the expression of MCF-7 effector molecules killed after EPZ-5676 activation. Figure 14 A diagram showing the killing of HCT116 cells after EPZ-5676 activation; Figure 15 Figure showing the expression of effector molecules in HCT116 cells after EPZ-5676 activation; Figure 16 A statistical chart showing the survival rate of mice with xenograft tumors; Figure 17 This is a statistical chart showing the proportion of NK cells infused in a mouse model of xenograft tumors. Detailed Implementation

[0016] This invention provides a method for expanding strong memory-like NK cells, comprising culturing NK cells in an expansion culture medium to obtain strong memory-like NK cells; The amplification medium includes complete medium, IL-2, IL-12, IL-15, IL-18 and sodium α-ketoglutarate; The concentration of sodium α-ketoglutarate in the amplification medium is 0.1~2 mM.

[0017] The basal culture medium of the complete culture medium of the present invention includes any one of the following: X-vivo15 medium, GibcoCTS AIM V, HIPP-T009, ImmunoCult™-XF, ImmunoCult™ NK Cell Expansion Medium and NKMACS® Medium. The complete culture medium also includes 5% autologous plasma by volume. In one embodiment, X-vivo15 medium + 5% autologous plasma is used as the complete culture medium. The vivo15 medium is a serum-free culture medium for immune cells and is purchased from Lonza.

[0018] The amplification culture medium of this invention contains 1000 U / mL of IL-2, 1~100 ng / mL of IL-12, 1~100 ng / mL of IL-15, and 1~100 ng / mL of IL-18. In one embodiment, the IL-2 used is recombinant human IL-2, purchased from PeproTech, and is an essential factor for lymphocyte proliferation. IL-12 drives NK cell proliferation and IFN-γ secretion, IL-15 maintains NK cell survival, promotes in vitro expansion, and enhances in vivo sustainability, while IL-18 enhances cytotoxicity and the release of inflammatory factors. These three factors synergistically significantly improve the killing activity and tumor infiltration capacity of NK cells.

[0019] The concentration of α-KG-Na in the amplification medium of this invention is 0.1~2mM, and can be 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 0.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, or 2.0mM. The α-KG-Na described in this invention, as an intermediate in the tricarboxylic acid cycle, can enhance the efficiency of the mitochondrial respiratory chain in NK cells, promote ATP production, and provide energy for rapid proliferation. In one embodiment, it was purchased from Sigma-Aldrich.

[0020] In one embodiment of the present invention, the method for obtaining NK cells includes the following steps: seeding PBMC cells into a complete culture medium for culture, and harvesting NK cells. The present invention does not specifically limit the source and extraction method of the PBMC cells; conventional sources and extraction methods in the art can be used.

[0021] In this embodiment of the invention, the PBMCs were resuspended in culture medium under aseptic conditions at a density of 1×10⁻⁶. 6 The culture medium described in this invention uses X-vivo-15 medium as the basal medium and also includes the cytokines IL-12, IL-15, and IL-18. All cytokines were purchased from PeproTech, with IL-12, IL-15, and IL-18 at a concentration of 100 ng / ml. The resuspended PBMCs were seeded into T75 culture flasks, with 20 mL of the complete culture medium added to each flask. The flasks were gently shaken to ensure even cell distribution. The flasks were then placed in a CO2 incubator for culture, with the following parameters: static culture at 37°C and 5% CO2 for 24 h. After static culture for 24 hours, the culture flask is removed and the original culture medium is removed by centrifugation; fresh amplification culture medium preheated to 37°C is added to the culture system; the flask is returned to the CO2 incubator and cultured for a period of time, during which the cell status is observed daily to avoid contamination. The present invention also provides a method for reactivating NK cells, comprising adding a DOT1L inhibitor to the culture system of the above-mentioned amplification method, and continuing to culture to obtain reactivated NK cells.

[0022] The reactivation method described in this invention can enhance the cytotoxic activity of expanded NK cells. In one embodiment, after culturing to day 12, 10-100 nM DOT1L inhibitor is added to the culture system, and the cells are cultured in a CO2 incubator for another 48 hours. The DOT1L inhibitor described in this invention can inhibit H3K79me2 / 3, thereby relieving transcriptional inhibition of cytotoxic genes such as IFNG and PRF1, and simultaneously enhancing cytotoxic function. This invention does not specifically limit the type of DOT1L inhibitor, as long as it can inhibit DOT1L, including Pinometostat (EPZ-5676), Epz004777, SGC707, and SYC-522, etc. EPZ-5676 is used as an example in this embodiment, but it should not be considered as the entire scope of protection of this invention.

[0023] The present invention also provides reactivated NK cells obtained using the above-described reactivation method.

[0024] Using the method described in this invention, PBMCs can be cultured for 12 days, which can significantly increase the number of cells and the purity of NK cells. After 12 days, the expression of the surface activating receptor NKG2D is increased, while the expression of the inhibitory receptors Tigit and PD-1 is decreased. It also improves the killing efficiency against target cells K562, and significantly improves the degranulation effect of NK cells and the release of Ifn-r.

[0025] This invention also provides the application of the above-mentioned reactivated NK cells in the preparation of reagents for targeted elimination of abnormal cells.

[0026] The abnormal cells described in this invention include tumor cells, virus-infected cells, and senescent / apoptotic abnormal cells.

[0027] To further illustrate the present invention, the following detailed description of a method for amplifying and activating memory-like NK cells provided by the present invention is provided in conjunction with embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 I. PBMC Separation 1. Blood dilution Under aseptic conditions, transfer peripheral blood to a 50 mL centrifuge tube, add an equal volume of PBS buffer (1:1 dilution), gently invert the centrifuge tube 5-8 times to mix thoroughly.

[0029] 2. Layered laying pattern Take a 15 mL centrifuge tube and add 3-5 mL of lymphocyte separation solution; slowly add diluted blood drop by drop along the wall of the centrifuge tube using a pipette (separation solution: diluted blood volume ratio of approximately 1:2); keep the two liquid layers clear and avoid vigorous shaking to prevent mixing.

[0030] 3. Density gradient centrifugation Place the centrifuge tubes in a horizontal rotor centrifuge and centrifuge at 400×g for 30~40 min at room temperature (20~25℃). Turn off the brake function (soft stop) during centrifugation.

[0031] 4. Collect PBMC layer After centrifugation, the tube separates into four layers from top to bottom: the upper layer (plasma / PBS mixture), the middle layer (cloudy white PBMC layer, i.e., target cells), the lower layer (clear lymphocyte separation solution layer), and the bottom layer (red erythrocyte + granulocyte layer). Carefully aspirate the middle PBMC layer with a pipette and transfer it to a new 15 mL centrifuge tube.

[0032] 5. Washing and purification Add 3 times the volume of PBS buffer to the PBMC suspension, mix thoroughly, centrifuge at 300×g for 10 min, and discard the supernatant; repeat the washing twice, and after the second centrifugation, try to remove as much supernatant as possible.

[0033] 6. Red blood cell removal (optional) If there are many red blood cells in the cell pellet, add 1-2 mL of red blood cell lysis buffer and incubate at room temperature for 5 min, gently inverting the pellet 1-2 times during the incubation period; then add 5 mL of PBS buffer to terminate the lysis, centrifuge at 300×g for 10 min, and discard the supernatant.

[0034] 7. Cell resuspension and counting Add 1 ml of PBS or cell culture medium and gently pipette to resuspend the cell pellet; take 10 μL of cell suspension and mix with 10 μL of trypan blue staining solution, incubate at room temperature for 2 min; add to a hemocytometer, count live cells under a microscope (blue staining indicates dead cells), and calculate cell concentration and viability. Viability should reach 90% or higher.

[0035] II. NK Cell Activation and Expansion 1. Cell seeding and activation Under aseptic conditions, the prepared PBMCs were reselected into culture media. The standard NK culture medium was prepared using the conventional NK culture method (X-vivo-15 medium + 5% serum + IL-2 1000 IU or IL-15 10 ng / ml); the memory-like NK medium was prepared using (X-vivo-15 medium + 5% serum + IL-2 1000 IU + IL-12 100 ng / ml + IL-15 100 ng / ml + IL-18 100 ng / ml); and the strong memory-like NK medium was prepared using (X-vivo-15 medium + 5% serum + IL-2 1000 IU + IL-12 100 ng / ml + IL-15 100 ng / ml + IL-18 100 ng / ml + 1 mM α-ketoglutarate sodium), with a density of 1 × 10⁻⁶. 6 cells / mL. Seeds were added to T75 culture flasks; the flasks were gently shaken to distribute the cells evenly; the flasks were then placed in a CO2 incubator and incubated statically at 37°C and 5% CO2 for 24 h.

[0036] 2. NK cell expansion After 48 hours of culture, remove the culture flask and centrifuge to remove the original culture medium. Add fresh complete culture medium preheated to 37°C to the culture system, and simultaneously supplement with recombinant human IL-2 (1000 U / mL), 5% serum, and 1 mM sodium α-ketoglutarate to the strong memory NK medium. Return to the CO2 incubator and continue culturing, observing cell status daily to avoid contamination.

[0037] 3. NK cell rehydration After 3 days of activation culture, remove the culture flask and observe cell morphology under an inverted microscope: activated NK cells will show increased volume and refractive index, and some will form small clonal clusters. Gently pipette the bottom of the culture flask / plate to suspend the cells evenly. Take a small amount of cell suspension, stain with trypan blue, count the cells, and record the cell concentration. Adjust the culture system according to the cell concentration, adding fresh complete culture medium to the culture flask while maintaining a final IL-2 concentration of 1000 U / mL. Add 1 mM sodium α-ketoglutarate to the strong memory NK medium. If the cell density exceeds 2 × 10⁻⁶ cells / mL... 6 cells / mL, allowing for cell culture in flasks (each flask maintaining a cell density of 1×10⁻⁶ cells / mL). 6 (Approximately 100 cells / mL) to avoid overcrowding of cells, which could hinder proliferation.

[0038] III. NK Cell Reactivation and Harvest 1. Reactivation of NK cells before harvest On day 12, 10 nM of the DOT1L inhibitor EPZ-5676 was added to the strong memory-like NK cell culture system, and the cells were cultured in a CO2 incubator for another 48 hours.

[0039] 2. Cell harvesting and purity determination Cells can be harvested after 14 days of culture: Collect all cell suspension into 50 mL centrifuge tubes, centrifuge at 300×g for 10 min, and discard the supernatant; wash the cells twice with PBS buffer, centrifuging at 300×g for 10 min each time to remove residual culture medium and cytokines; resuspend the cells with an appropriate amount of PBS, and take a portion of the cells for purity identification (flow cytometry detection: CD3). - CD56 + Simultaneously, use trypan blue staining to count viable cells; viability must be >80%.

[0040] Peripheral blood cells obtained and cultured for 12 days before the addition of inhibitors showed significantly higher amplification efficiency in the memory-like NK+α-KG-Na group than in the memory-like NK group and the conventionally cultured NK group. Figure 1 (As shown in Table 1), the purity of the memory-like NK+α-KG-Na group was significantly higher than that of the memory-like NK cell group and the conventionally cultured NK cell group. Figure 2 (See Table 2). After 12 days, the expression of its surface-activating receptor NKG2D was significantly higher in the memory-like NK+α-KG-Na group than in the memory-like NK cell group and the conventionally cultured NK cell group (and Table 2). Figure 3 (and Table 3), the expression of inhibitory receptors Tigit and PD-1 in the memory-like NK+α-KG-Na group was significantly lower than that in the memory-like NK cell group and the conventionally cultured NK cell group (and Table 3). Figure 4 And Table 4, Figure 5 (and Table 5).

[0041] Table 1. Cell proliferation folds in the experimental and control groups.

[0042] Table 2. NK cell ratio (%)

[0043] Table 3. Proportion of NK cells expressing NKG2D (%)

[0044] Table 4. Proportion of NK cells expressing Tigit (%)

[0045] Table 5. Proportion of PD-1 expressing NK cells (%)

[0046] Collect tumor cell lines, wash once with PBS, resuspend in 1 ml of PBS, add CFSE, and stain at 37°C for 15 minutes. Wash once with 5 ml of complete culture medium. Count the cells at a ratio of 5 × 10⁶ cells per well.4 Add cells to 96-well plates. Add NK cells at the effector-to-target ratio, making up to 200 μL, and incubate at 37°C for 4 h. Collect all cells into a flow cytometry tube, wash once with PBS, incubate at 200×g for 5 min, and discard the supernatant. Resuspend cells in 100 μL Annexin V binding buffer, add 5 μL Annexin V-APC, incubate in the dark for 15 min, add 5 μL PI, and incubate in the dark for 5 min. Before flow cytometry, add 200–400 μL of binding buffer and perform the assay.

[0047] At different effector-to-target ratios, the memory-like NK+α-KG-Na group was significantly more effective than the memory-like NK cell group and the conventionally cultured NK cell group in killing K562 target cells. Figure 6 (See Table 6) The degranulation effect and Ifn-r release of the memory-like NK+α-KG-Na group were significantly stronger than those of the memory-like NK cell group and the conventionally cultured NK group. Figure 7 (and Table 7).

[0048] Table 6. Cell mortality rate (%) of K562 cells after 12 days of culture.

[0049] Table 7. CD107a expression rate and Ifn-r release rate in NK cells (%)

[0050] Example 2 The NK cells amplified in Example 1 were cultured with EPZ-5676 for 2 days to obtain strong memory-like NK cells, which were then used to kill the chronic myeloid leukemia cell line K562 cells using the same method as in Example 1. The results are as follows: Figure 8 and Figure 9 As shown, compared with the NK cell group cultured without EPZ-5676 and the ordinary cultured NK cell group, the strong memory-like NK cell group had a significantly better killing effect. Figure 8 (and Table 8), and the degranulation effect and Ifn-r release were also significantly higher ( Figure 9 (and Table 9).

[0051] Table 8. Cell mortality rate (%) of K562 cells after 14 days of culture.

[0052] Table 9. CD107a expression rate and Ifn-r release rate in NK cells (%)

[0053] Example 3 The NK cells amplified in Example 1 were cultured with EPZ-5676 for 2 days and then used to kill lung cancer cell line A549. The killing efficiency of the strong memory-like NK cell group was significantly higher than that of the memory-like NK group and the conventionally cultured NK group. Figure 10 (and Table 10). The former showed significantly higher NK cell degranulation effect and Ifn-r release than the latter (and Table 10). Figure 11 (and Table 11).

[0054] Table 10 Cell mortality rate (%) of A549 cells cultured for 14 days

[0055] Table 11. CD107a expression rate and Ifn-r release rate in NK cells (%)

[0056] Example 4 The NK cells amplified in Example 1 were cultured with EPZ-5676 for 2 days and then used to kill the breast cancer cell line MCF-7. The killing efficiency of the strong memory-like NK cell group was significantly higher than that of the memory-like NK group and the conventionally cultured NK group. Figure 12 (and Table 12). The former showed significantly higher NK cell degranulation effect and Ifn-r release than the latter (and Table 12). Figure 13 (and Table 13).

[0057] Table 12. Percentage of MCF-7 cells killed after 14 days of culture (%)

[0058] Table 13. CD107a expression rate and Ifn-r release rate in NK cells (%)

[0059] Example 5 The NK cells amplified in Example 1 were cultured with EPZ-5676 for 2 days and then used to kill HCT116 colorectal cancer cells. The killing efficiency of the strong memory-like NK cell group was significantly higher than that of the memory-like NK group and the conventionally cultured NK group. Figure 14 (and Table 14). The former showed significantly higher NK cell degranulation effect and Ifn-r release than the latter (and Table 14). Figure 15 (and Table 15).

[0060] Table 14. Percentage of HCT116 cells killed after 14 days of culture (%)

[0061] Table 15. CD107a expression rate and Ifn-r release rate in NK cells (%)

[0062] Example 6 In vivo survival and survival curves in experimental mice: K562 cells in logarithmic growth phase were washed twice with sterile PBS and resuspended, and the cell concentration was adjusted to 1×10⁻⁶. 7 The concentration of K562 cells / mL was maintained, ensuring cell viability >90%. Six- to eight-week-old SPF-grade NPG mice were selected and acclimatized for 3-5 days before tail vein infusion. Each mouse underwent a slow injection of 100 μL of the above-mentioned K562 cell suspension via the tail vein, avoiding air bubbles during the injection process. Twenty-four hours later, 1 × 10⁶ normal NK cells, memory-like NK cells, and strong memory-like NK cells were infused via tail vein, respectively. 7 After infusion, mice were housed in a temperature- and humidity-controlled SPF environment with free access to food and water, and their mental state was observed weekly. Blood samples were collected periodically from the tail vein to detect the proportion of NK cells, and the mortality rate of the mice was recorded.

[0063] Survival curves Figure 16 As shown in Table 16, the xenograft mice treated with strong memory-like NK cells had a significantly longer survival time than the mice treated with memory-like NK cells. The mice treated with both types of cells had a longer survival time than the mice treated with ordinary NK cells.

[0064] Table 16 Survival Rate (%)

[0065] The ratio of NK cells in xenograft mice at different time points is as follows: Figure 17 As shown in Table 17, the survival time of strong memory-like NK cells in xenograft mice was significantly longer than that of memory-like NK cells, and both were longer than that of ordinary NK cells.

[0066] Table 17 NK cell percentage (%)

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for expanding strong memory-like NK cells, characterized in that, The method involves culturing NK cells in an amplification medium to obtain strong memory-like NK cells; the amplification medium includes complete medium, IL-2, IL-12, IL-15, IL-18 and sodium α-ketoglutarate; the concentration of sodium α-ketoglutarate in the amplification medium is 0.1~2 mM.

2. The amplification method according to claim 1, characterized in that, The amplification medium contains 1000 U / mL of IL-2, 1~100 ng / mL of IL-12, 1~100 ng / mL of IL-15, and 1~100 ng / mL of IL-18.

3. The amplification method according to claim 1, characterized in that, The basal medium for the complete culture medium includes any of the following: X-vivo15 medium, Gibco CTS AIM V, HIPP-T009, ImmunoCult™-XF, ImmunoCult™ NK Cell Expansion Medium, and NK MACS. ® Medium; the complete culture medium also includes 5% autologous plasma by volume.

4. The amplification method according to claim 1, characterized in that, The method for obtaining NK cells includes the following steps: seeding PBMC cells into a complete culture medium for culture, and harvesting NK cells.

5. The amplification method according to claim 4, characterized in that, The cultivation parameters included static incubation at 37°C and 5% CO2 for 24 hours.

6. A method for reactivating NK cells, characterized in that, The method includes adding a DOT1L inhibitor to the culture system of the amplification method described in any one of claims 1 to 5, and then continuing to culture to obtain reactivated NK cells.

7. The reactivation method according to claim 6, characterized in that, The concentration of the DOT1L inhibitor in the system is 10~100 nM.

8. The reactivation method according to claim 6 or 7, characterized in that, The DOT1L inhibitors include at least one of the following: EPZ-5676, Epz004777, SGC707, and SYC-522.

9. Reactivated NK cells obtained by the reactivation method according to any one of claims 6 to 8.

10. The use of the reactivated NK cells of claim 9 in the preparation of reagents for targeting and eliminating abnormal cells.

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