Method for amplifying NK (Natural Killer) cells by combining anti-CD52 monoclonal antibody and cytokines and application

Abstract

The invention relates to the technical field of cellular immunotherapy, in particular to a method for amplifying NK cells through combination of an anti-CD52 monoclonal antibody and cell factors and application of the method, and the method comprises the following steps: (1) extracting mononuclear cells PBMCs from peripheral blood of a donor; (2) culturing the mononuclear cells together with an anti-CD52 monoclonal antibody, an anti-CD3 monoclonal antibody and a cytokine composition containing IL-2, IL-15 and IL-21, and performing multiplication culture for 14-21 days under the conditions of 37 + / -1 DEG C and 5 + / -0.5% CO2; (3) amplified cells are harvested, the purity of the CD3-CD56 + NK cells reaches 85% or above, and the number of the cells is increased by 1000 times or above of the number of the original NK cells; and (4) detecting the surface receptor expression and killing activity of the NK cells, and through the synergistic effect of the combination of the anti-CD52 monoclonal antibody and the specific cytokines, the amplification multiple of the NK cells can reach more than 1000 times.

Description

Technical Field

[0001] The present invention relates to the technical field of cell immunotherapy, and in particular to a method for efficiently amplifying natural killer (NK) cells by utilizing the combined action of anti-CD52 monoclonal antibodies and specific cytokines, and application of the prepared NK cells in the treatment of acute myeloid leukemia. Background Art

[0002] Natural Killer cells (NK cells) are an important type of immune effector cells that have the ability to recognize and kill tumor cells and virus-infected cells, and play a key role in tumor immune surveillance. In recent years, with the rapid development of tumor immunotherapy, NK cell therapy, as an effective adoptive immunotherapy, has shown significant application prospects in the treatment of hematological tumors, especially acute myeloid leukemia (AML).

[0003] In clinical applications, NK cell immunotherapy faces several major challenges: first, how to obtain a sufficient number of NK cells; second, how to maintain and improve the activity of NK cells; third, how to prolong the survival time of NK cells in the body; and finally, how to overcome the immune escape mechanism of tumor cells. To address the first challenge, researchers have developed a variety of NK cell in vitro expansion methods.

[0004] At present, conventional NK cell expansion method mainly contains several types. (Stimulation withK562 Cells Transfected with 4-1BBL and IL-15 Expands and Activates NaturalKiller (NK) Cells with Specific Cytotoxicity for Multiple Myeloma (MM) etc., Blood, November 2005, 3392) report the expansion method based on K562 feeder layer cells, stimulate NK cell proliferation by engineered K562 cells expressing 4-1BBL and membrane-bound IL-15, can obtain about 500-1000 times of expansion. However, the method uses leukemia cell line as feeder layer and has potential safety hazard, and needs complicated genetic engineering operation, is unfavorable for clinical standardization application.

[0005] Another commonly used method is the simple cytokine stimulation method. Ojo et al. (Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells, Scientific Reports, 2019, 9:14916) reported a method of stimulating peripheral blood mononuclear cells (PBMCs) using a combination of IL-2, IL-15, and IL-18. After 2 weeks of culture, the number of NK cells increased by about 294 times, and the purity increased from 7.67% to 70.2%. Although this method is simple to operate, the amplification multiple is relatively limited, and the batch-to-batch differences are large, making it difficult to meet clinical needs.

[0006] Masuyama et al. (Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies, Cytotherapy, 2016, 18:80-92) reported the anti-CD3 monoclonal antibody stimulation method, which activated T cells in PBMCs by anti-CD3 monoclonal antibodies and indirectly promoted the expansion of NK cells. Although this method is simple, the purity of NK cells is low, and it is often necessary to purify NK cells with steps such as magnetic bead sorting, which increases production costs and process complexity.

[0007] CD52 is a glycoprotein molecule present on the surface of various white blood cells. It is highly expressed on T cells and B cells, but expressed at a lower level on NK cell subsets. Hotta et al. (CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment , PLoS One, 2016) showed that only about 30% of liver NK cells and 70% of peripheral blood NK cells express CD52, suggesting that NK cells may be less sensitive to anti-CD52 antibody treatment. Alemtuzumab is a humanized monoclonal antibody targeting CD52, which is clinically used to treat chronic lymphocytic leukemia and multiple sclerosis.

[0008] Masuyama et al. (Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies, Cytotherapy, 2016, 18:80-90) first discovered in a study that co-stimulation with anti-CD3 and anti-CD52 monoclonal antibodies can promote NK cell expansion. However, this study mainly focused on the application of NK cells in the treatment of pancreatic cancer, lacked in-depth exploration of the NK cell expansion mechanism and optimization conditions, and did not explore the effect of cytokine combination optimization on NK cell expansion. The expansion efficiency needs to be improved.

[0009] On the other hand, in response to the problem of insufficient NK cell activity in the treatment of acute myeloid leukemia, recent studies have begun to focus on tumor cell pretreatment strategies. Fisher et al. (Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E, Blood, 2021, 138:2411) found that selinexor, a selective nuclear export protein (XPO1) inhibitor, can enhance NK cell activation and killing function by downregulating HLA-E expression on the surface of lymphoma cells. However, this study mainly focused on lymphoma models, and its application in the treatment of AML and its value in combination with NK cell therapy have not been fully studied.

[0010] In summary, the existing technologies face the following limitations in NK cell expansion: (1) insufficient expansion multiples or low purity; (2) complex genetic engineering is required or reliance on feeder cells with potential risks; (3) the production process is complex, costly, and difficult to standardize; (4) the problem of insufficient activity and poor persistence of NK cells in vivo is not effectively solved; (5) the immune escape mechanism of AML cells is not effectively addressed. Therefore, it is of great clinical significance and application value to develop a safe, efficient, and simple NK cell expansion method that can simultaneously solve the problem of insufficient activity of NK cells in the treatment of AML. Summary of the invention

[0011] In view of the problems existing in the prior art, the purpose of the present invention is to provide a safe, efficient and simple method for expanding NK cells, while solving the problem of insufficient activity of NK cells in the treatment of acute myeloid leukemia.

[0012] To achieve the above object, the present invention adopts the following technical solution: A method for amplifying NK cells by combining anti-CD52 monoclonal antibody and cytokines, comprising the following steps: (1) Extracting mononuclear cells (PBMCs) from donor peripheral blood; (2) culturing the mononuclear cells with anti-CD52 monoclonal antibody, anti-CD3 monoclonal antibody and a cytokine composition comprising IL-2, IL-15 and IL-21 at 37±1°C and 5±0.5% CO 2 The culture was expanded for 14-21 days under the conditions; (3) harvesting the expanded cells, wherein the purity of CD3-CD56+ NK cells reaches more than 85%, and the cell number increases to more than 1000 times the original NK cell number; (4) Detecting the surface receptor expression and cytotoxic activity of the NK cells.

[0013] Preferably, the anti-CD52 monoclonal antibody is alemtuzumab, and the added amount is 5-15 μg / mL, and the anti-CD3 monoclonal antibody is OKT3 clone, and the added amount is 10-100 ng / mL. This combination can effectively stimulate NK cell proliferation and selectively inhibit excessive proliferation of T cells and B cells.

[0014] In one embodiment of the present invention, the concentration of IL-2 in the cytokine composition is 100-1000 IU / mL, the concentration of IL-15 is 10-50 ng / mL, and the concentration of IL-21 is 5-25 ng / mL. This cytokine combination can synergistically promote the proliferation and functional maturation of NK cells.

[0015] Preferably, the method further comprises the following steps: (a) supplementing the anti-CD52 monoclonal antibody on the 3rd to 4th day and the 7th to 8th day of cell expansion, with the supplement amount being 50% and 25% of the initial concentration, respectively; (b) supplementing the cytokine composition once every 3 to 4 days; (c) the cell density exceeds 2×10 6 cells / mL and maintain at 1-2×10 6 This phased addition strategy can maintain continuous stimulation of NK cell proliferation while avoiding the accumulation of inhibitory signals.

[0016] In some embodiments of the present invention, the culture is carried out in a serum-free medium, which is RPMI-1640 or OpTmizer™ T Cell Expansion SFM supplemented with 5-10% human serum albumin. The use of a serum-free culture system can improve batch consistency and reduce the risk of exogenous pathogens.

[0017] In addition, the method may further include a step of culturing under hypoxic conditions in Step (2), wherein the oxygen concentration is 5-7%, and 10 ng / mL IL-12 and 10 ng / mL IL-18 are added at the end of the culture for pretreatment for 6-24 hours to induce NK cells to produce memory-like properties. Hypoxic culture is closer to the physiological environment and can improve the survival rate and function of NK cells, while IL-12 / IL-18 pretreatment can induce NK cells to produce persistent functions similar to memory T cells.

[0018] The present invention also provides an NK cell preparation prepared by the above method, characterized in that the NK cell purity in the preparation is ≥85%, the CD3+T cell ratio is ≤5%, the viable cell rate is ≥80%, the killing rate of K562 standard target cells is ≥40% (E:T=5:1), and the NK cells express high levels of NKG2D and CD16 activation receptors. This high-purity and high-activity NK cell preparation can be directly used for clinical treatment.

[0019] At the same time, the present invention also provides a method for treating acute myeloid leukemia using the above NK cell preparation, comprising the following steps: (1) treating acute myeloid leukemia cells with 5-10 nM selinexor 6-24 hours before NK cell infusion; (2) intravenously infusing the NK cell preparation at a dose of 1-5×10 7 cells / kg body weight.

[0020] Preferably, selinexor enhances the recognition and killing ability of NK cells against leukemia cells by downregulating the expression of HLA-E on the surface of acute myeloid leukemia cells. HLA-E is an important immune checkpoint molecule that binds to the inhibitory receptor NKG2A on the surface of NK cells to inhibit the activation of NK cells.

[0021] In another embodiment of the present invention, the method further comprises the combined use of one or more of the following drugs: BCL-2 inhibitor Venetoclax, at a dose of 5-100 nM; demethylation drug azacitidine, at a dose of 0.1-1 μM; or anti-NKG2A antibody Monalizumab, at a dose of 1-10 μg / mL. These combined drug strategies can further enhance the therapeutic effect of NK cell therapy.

[0022] Compared with the prior art, the present invention has the following significant advantages: 1. High amplification efficiency: Through the synergistic effect of anti-CD52 monoclonal antibody and specific cytokine combination, the NK cell amplification multiple can reach more than 1000 times, which is significantly higher than the 294-fold amplification rate of cytokines used alone in the prior art.

[0023] 2. High purity: No NK cell sorting step is required, and the purity of NK cells obtained by direct amplification can reach more than 85%, which is higher than the purity level of 70.2% in the existing technology.

[0024] 3. Simple and safe process: No genetically engineered feeder cells are required, which avoids related safety risks, simplifies the production process and reduces production costs.

[0025] 4. Functional enhancement: Through hypoxic culture and IL-12 / IL-18 pretreatment, NK cells are induced to have persistent activity similar to memory characteristics.

[0026] 5. Synergistic effect: Combined with the strategy of pre-treating AML cells with selinexor, the inhibitory signal on NK cells is relieved by down-regulating HLA-E expression, significantly enhancing the killing ability of NK cells against AML cells.

[0027] 6. High potential for clinical transformation: Using clinical-grade reagents and simplified processes, it is easy to standardize production and clinical transformation applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a process flow chart of the NK cell expansion method of the present invention.

[0029] Figure 2 The figure is a growth curve of the number of NK cells after 14 days of expansion by the method of the present invention, comparing the expansion effects of different methods.

[0030] Figure 3 This is a flow cytometric analysis of the purity (CD3-CD56+) of NK cells after 14 days of expansion using the method of the present invention.

[0031] Figure 4 To expand the NK cell surface receptor expression analysis chart, display the expression levels of activation receptors such as NKG2D and CD16.

[0032] Figure 5 To amplify the cytotoxic activity of NK cells against K562 cells, the E:T ratios were 5:1, 2.5:1, and 1:1.

[0033] Figure 6 This is a flow cytometric analysis of the changes in HLA-E expression on the surface of AML cells before and after selinexor treatment.

[0034] Figure 7 This is a diagram showing the enhancement of NK cell cytotoxicity after pre-treatment of AML cells with selinexor.

[0035] Figure 8 This is a graph showing the effect of NK cell therapy in the humanized NSG mouse AML model, comparing the changes in tumor burden in the three groups treated with NK cells alone, selinexor alone, and combined therapy. DETAILED DESCRIPTION

[0036] Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail.It should be appreciated by those skilled in the art that these embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.Under the premise of not departing from the spirit and scope of the present invention, the present invention can have various changes and variations.

[0037] Example 1: Method for amplifying NK cells by combining anti-CD52 monoclonal antibody and cytokines 1.1 Materials and reagents In this example, we used the following materials and reagents: human peripheral blood was provided by healthy volunteers, approved by the ethics committee and with informed consent; density gradient centrifugation separation medium (Ficoll-Paque PLUS, GE Healthcare); anti-CD52 monoclonal antibody (alemtuzumab, Campath®, 10 mg / mL stock solution); anti-CD3 monoclonal antibody (OKT3 clone, BioLegend, 0.5 mg / mL); recombinant human IL-2 (PeproTech, activity ≥5×10 6 IU / mg); recombinant human IL-15 (PeproTech, activity ≥2×10 6 IU / mg); recombinant human IL-21 (PeproTech, activity ≥1×10 6 IU / mg); serum-free culture medium RPMI-1640 (Gibco); human serum albumin (HSA, 20% concentration, clinical grade); flow cytometry antibodies including anti-CD3-FITC, anti-CD56-PE, anti-CD16-APC, anti-NKG2D-PE-Cy7, anti-NKG2A-APC-Cy7 (all from BD Biosciences); K562 cell line (ATCC CCL-243).

[0038] 1.2 PBMC isolation 20 mL of peripheral blood was collected from healthy volunteers and anticoagulated with heparin. The blood was mixed evenly with an equal volume of PBS, and the diluted blood was gently added to a 50 mL centrifuge tube containing 15 mL Ficoll-Paque PLUS along the tube wall to form a distinct layer. Centrifuge at 400 g for 30 minutes at room temperature. After the centrifugation, the mononuclear cell layer was carefully aspirated into a new centrifuge tube, washed twice with PBS (300 g, 10 minutes), counted the cells and adjusted the concentration to 2 × 10 6 This step can obtain about 2-4×10 7 PBMCs, of which NK cells (CD3-CD56+) account for approximately 10-15%.

[0039] 1.3 NK cell expansion and culture The isolated PBMCs were resuspended in complete medium, which consisted of RPMI-1640 basal medium supplemented with 10% HSA, 2mM L-glutamine, and 1% penicillin / streptomycin. 10μg / mL alemtuzumab (anti-CD52 monoclonal antibody) and 50ng / mL OKT3 (anti-CD3 monoclonal antibody) were added to the medium, and a cytokine composition was added: 500IU / mL IL-2, 20ng / mL IL-15, and 10ng / mL IL-21. The cell suspension was aliquoted into the G-Rex10 cell culture system (Wilson Wolf), and 10mL of cell suspension (containing 2×10 7 The culture was placed in a 37°C, 5% CO2 incubator.

[0040] On the third day of culture, 5 μg / mL alemtuzumab was added; on the seventh day, 2.5 μg / mL alemtuzumab was added. At the same time, the cytokine mixture (IL-2, IL-15 and IL-21) was added every 3-4 days at the same concentration as the initial concentration. The cell density was monitored regularly and the cells were resuspended when the cell density exceeded 2×10 6 When the cells reached 1 × 10 / mL, dilute the cells to 1 × 10 6 Pieces / mL.

[0041] Cell counts and phenotypic analysis were performed on days 7, 14, and 21 of culture. Cell counts were performed using the trypan blue exclusion method to assess the number of viable cells, and phenotypic analysis was performed using flow cytometry to measure the proportion of CD3-CD56+ cells.

[0042] 1.4 Cell phenotype analysis Take a small amount of expanded cells (about 1×10 6 ), washed twice with PBS and resuspended in 100 μL FACS buffer (PBS containing 2% FBS and 0.1% NaN3). The following fluorescently labeled antibodies were added: anti-CD3-FITC, anti-CD56-PE, anti-CD16-APC, anti-NKG2D-PE-Cy7 and anti-NKG2A-APC-Cy7, and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with FACS buffer and finally resuspended in 300 μL FACS buffer. The cells were detected using a BD FACSCanto II flow cytometer and the data were analyzed using FlowJo software.

[0043] 1.5 NK cell cytotoxicity assay Flow cytometry was used to evaluate the killing activity of NK cells against K562 target cells. K562 cells were first labeled with 5 μM CFSE (Invitrogen), incubated at 37°C for 10 minutes, and then washed twice with complete medium. The labeled K562 cells were mixed with the expanded NK cells at different effector: target ratios (E:T = 5:1, 2.5:1, and 1:1) and co-cultured in 96-well U-bottom plates at 37°C for 4 hours. After the co-culture, 7-AAD (BD Biosciences) was added to label the dead cells, incubated at room temperature in the dark for 10 minutes, and then immediately subjected to flow cytometry detection. Calculate the specific lysis rate: Specific lysis rate (%) = [(CFSE+7-AAD+% spontaneous death in the experimental group %) / (100% spontaneous death %)] × 100%.

[0044] 1.6 Experimental Results After 14 days of culture, the number of NK cells increased from the initial approximately 3×10 6 (2×10 7 NK cells, which account for about 15% of PBMC, increased to about 3.2×10 9 The expansion multiple reached about 1067 times. Flow cytometry analysis showed that the proportion of CD3-CD56+ cells in the expanded cells was 87.5±3.2%, and the proportion of CD3+T cells was 4.2±1.3%, indicating that this method can efficiently expand high-purity NK cells without the need for additional purification steps.

[0045] Analysis of the expression of receptors on the surface of amplified NK cells showed that the positive expression rate of CD16 was 82.3±5.6%, and the positive expression rate of NKG2D was 93.5±2.8%, which were significantly higher than those of the initial NK cells without amplification (65.4±7.2% and 75.3±6.5%, respectively). At the same time, the expression of the inhibitory receptor NKG2A decreased slightly from the initial 52.7±8.4% to 42.3±7.9%. These results indicate that the amplified NK cells are in a good state of activation and have a high killing potential.

[0046] In the killing activity assay, the specific lysis rate of the expanded NK cells against K562 cells reached 61.8±5.3% when E:T was 5:1, which was significantly higher than that of the initial NK cells without expansion (38.2±4.7%). As the E:T ratio decreased, the killing rate decreased accordingly, and was 43.5±4.1% and 28.7±3.6% when E:T was 2.5:1 and 1:1, respectively. These results show that the NK cells expanded by the method of the present invention have strong killing activity.

[0047] Example 2: Optimized NK cell expansion protocol Based on Example 1, we further optimized the NK cell expansion protocol by introducing hypoxic culture and terminal IL-12 / IL-18 pretreatment to improve the function and in vivo persistence of NK cells.

[0048] 2.1 Hypoxic culture conditions PBMCs were isolated and cultured as described in Example 1, except that the culture conditions were set at 37°C and 5% CO. 2 and 5% O 2 (Low oxygen). A three-gas incubator (Thermo Scientific) was used to control the oxygen concentration. The remaining culture steps were consistent with Example 1.

[0049] 2.2 IL-12 / IL-18 pretreatment On the 13th day of culture (one day before harvest), 10 ng / mL IL-12 and 10 ng / mL IL-18 were added to part of the culture system and incubated for 24 hours at 37° C. As a control, part of the culture system was retained without adding IL-12 / IL-18.

[0050] 2.3 NK cell function assessment In addition to the standard killing activity assay, the following functional assessments were performed: (1) Cytokine production: The expanded NK cells were co-cultured with K562 cells (E:T = 5:1) for 6 h, and the protein transport inhibitor GolgiStop (BD Biosciences) was added, followed by intracellular cytokine staining to detect the production of IFN-γ and TNF-α.

[0051] (2) Degranulation reaction: Anti-CD107a-PE-Cy5 antibody was added to the co-culture system to detect the expression of CD107a on the surface of NK cells, reflecting the degranulation situation.

[0052] (3) NK cell persistence: NK cells expanded under different culture conditions were labeled and infused into NSG mice. Blood was collected regularly to detect the proportion of human NK cells in peripheral blood and evaluate their in vivo persistence.

[0053] 2.4 Experimental Results Under hypoxic culture conditions, the NK cell expansion multiple reached about 1250 times in 14 days, slightly higher than the 1067 times of normal oxygen concentration culture. The purity of NK cells expanded under hypoxic conditions was about 89.3±2.7%, which was also slightly higher than normal oxygen concentration culture. The more significant difference was reflected in the function of NK cells: the killing rate of NK cells cultured under hypoxia against K562 (E:T = 5:1) was 68.5±4.6%, higher than the 61.8±5.3% of normal oxygen concentration culture.

[0054] IL-12 / IL-18 pretreatment further enhanced the function of NK cells. The pretreated NK cells showed a stronger ability to produce IFN-γ, with an intracellular staining positive rate of 76.5±5.2%, while that of the unpretreated group was 52.3±6.1%. At the same time, the CD107a expression of NK cells in the pretreated group was also higher, indicating a stronger degranulation reaction.

[0055] In the in vivo persistence experiment in mice, the detection rate of NK cells in the hypoxia + IL-12 / IL-18 pretreatment group 14 days after infusion was significantly higher than that in the conventional culture group, indicating that this optimized scheme can prolong the survival time of NK cells in vivo.

[0056] Example 3: Selinexor pretreatment of AML cells enhances NK cell killing effect This example aims to verify whether pre-treatment of AML cells with selinexor can enhance the killing effect of NK cells and explore the related mechanism.

[0057] 3.1 Materials and methods (1) AML cell lines: Three AML cell lines (ATCC) were used: THP-1, HL-60, and Kasumi-1. The cells were cultured in RPMI-1640 medium containing 10% FBS.

[0058] (2) Primary AML samples: Mononuclear cells were isolated from bone marrow samples of five newly diagnosed AML patients, with approval from the ethics committee and informed consent from the patients.

[0059] (3) Selinexor treatment: AML cell lines and primary AML cells were cultured at 5×10 5 Cells were inoculated at a density of 100 / mL, and different concentrations (5 nM, 10 nM and 20 nM) of selinexor (KPT-330, Selleck) were added and treated at 37°C for 12 hours. An equal volume of solvent (DMSO) was added to the control group.

[0060] (4) HLA-E expression analysis: After selinexor treatment, cells were collected and labeled with anti-HLA-E-PE antibody (clone 3D12, BD Biosciences), and HLA-E expression levels were detected by flow cytometry.

[0061] (5) NK cell killing experiment: NK cells expanded by the optimized scheme of Example 2 were used as effector cells, and AML cells pretreated with selinexor or DMSO were used as target cells. They were co-cultured at an E:T ratio of 5:1 for 4 hours, and the cell death rate was detected by flow cytometry.

[0062] 3.2 Experimental Results Treatment with selinexor significantly reduced the expression of HLA-E on the surface of AML cells. After treatment with 10 nM selinexor for 12 hours, the mean fluorescence intensity (MFI) of HLA-E on the surface of THP-1, HL-60, and Kasumi-1 cells decreased by 63.5 ± 5.2%, 58.7 ± 6.1%, and 67.2 ± 4.8%, respectively. A similar decrease in HLA-E expression was observed in primary AML samples, with an average decrease of 55.3 ± 8.7%.

[0063] In the NK cell killing experiment, compared with the DMSO control group, the sensitivity of THP-1, HL-60 and Kasumi-1 cells to NK cell killing was significantly increased after pretreatment with 10nM selinexor, and the killing rate increased by 2.1 times, 1.9 times and 2.3 times, respectively. The results of primary AML cells were similar, with 4 of the 5 samples showing a significant increase in sensitivity to NK cell killing (an average increase of about 1.8 times), and 1 case had no significant change.

[0064] Further mechanistic studies have shown that selinexor inhibits XPO1, leading to HLA-E nuclear accumulation and decreased surface expression, thereby reducing the contact of NK cell inhibitory receptor NKG2A and enhancing NK cell activation. At the same time, selinexor treatment also upregulated the expression of NKG2D ligands such as MICA / B and ULBP1-3 on the surface of AML cells, further promoting NK cell recognition and killing.

[0065] Example 4: In vivo validation of NK cells combined with selinexor in the treatment of AML This example aims to verify the in vivo efficacy of NK cells combined with selinexor in the treatment of AML, and further explore its mechanism of action and clinical application value.

[0066] 4.1 Establishment of humanized AML mouse model Select 6-8 week old NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ 40 (NSG) mice were randomly divided into 4 groups (n=10 / group) according to their body weight. 5×10 HL-60-luciferase cells (HL-60 cell line stably transfected with luciferase gene) were added. 6 On the 7th day after injection, the fluorescent signal was monitored using an in vivo imaging system (IVIS Spectrum, PerkinElmer) to confirm that the leukemia model was successfully established, and the groups were rebalanced according to the fluorescence intensity.

[0067] 4.2 Treatment options Mice were divided into the following four groups for treatment: (1) Control group: normal saline was injected intravenously twice a week for 4 weeks; (2) NK cell group: 1×10 NK cells amplified using the optimized protocol in Example 2 7 Intravenous injection, twice a week, for 4 weeks; (3) Selinexor group: Selinexor 5 mg / kg was administered orally 3 times a week for 4 weeks; (4) Combined treatment group: oral administration of selinexor 5 mg / kg (3 times a week), NK cells 1×10 7 Each animal was injected intravenously (twice a week) for 4 weeks. Selinexor was given 12 hours before NK cell infusion.

[0068] 4.3 Efficacy evaluation (1) Intravital imaging: Leukemia burden was monitored weekly using an intravital imaging system, and changes in tumor burden were quantitatively analyzed by bioluminescent signal intensity.

[0069] (2) Survival analysis: Record the survival status of each group of mice, draw survival curves, and perform statistical analysis.

[0070] (3) Bone marrow infiltration analysis: After 4 weeks of treatment, 5 mice were randomly selected from each group and euthanized. Bone marrow samples were obtained and the proportion of human CD45+CD33+ leukemia cells was detected by flow cytometry.

[0071] (4) In vivo dynamics of NK cells: NK cells were labeled with CellTrace Far Red before NK cell infusion. Peripheral blood samples were collected on days 1, 3, 7, 14, and 21 after infusion. Flow cytometry was used to detect the presence of labeled NK cells in peripheral blood to assess in vivo persistence.

[0072] (5) Organ pathology analysis: After euthanasia, liver, spleen, bone marrow and other tissues of some mice were obtained for pathological section examination to evaluate the leukemia infiltration and treatment effect.

[0073] 4.4 Experimental Results The results of in vivo imaging showed that compared with the control group, the leukemia burden in the NK cell group and the selinexor group was reduced to a certain extent, but the effect was limited. The leukemia burden in the combined treatment group was significantly reduced. By the fourth week of treatment, the average bioluminescence signal intensity in the combined treatment group was 82.5±6.3% lower than that in the control group, 65.7±7.2% lower than that in the NK cell alone treatment group, and 58.9±8.1% lower than that in the selinexor alone treatment group.

[0074] The results of survival analysis showed that the median survival of mice in the control group was 30.5 days, 40.5 days in the NK cell treatment group, 44.5 days in the selinexor treatment group, and 67.5 days in the combined treatment group. The survival time of the combined treatment group was significantly longer than that of the other three groups (P<0.01).

[0075] Bone marrow infiltration analysis showed that the proportion of human CD45+CD33+ leukemia cells in the bone marrow of the combined treatment group was 12.3±3.5%, which was significantly lower than that of the control group (75.6±8.2%), NK cell group (45.3±7.6%) and selinexor group (38.5±6.9%).

[0076] The in vivo dynamics study of NK cells found that the NK cells in the NK cell therapy group were almost undetectable on the 7th day after infusion, while the NK cells in the combined therapy group were still detectable on the 14th day after infusion, indicating that selinexor may prolong the survival time of NK cells in vivo by reducing the immunosuppressive effect of leukemia cells.

[0077] Organ pathological analysis also confirmed that the leukemia infiltration in the liver, spleen and other tissues of the combined treatment group was significantly reduced, and the tissue structure was well maintained, while the control group had a large number of leukemia cell infiltrations and tissue structure destruction.

[0078] Example 5: Optimization of combination drug strategy Based on Example 4, we further explored the optimized treatment strategy of NK cells and selinexor combined with other drugs to maximize the therapeutic effect.

[0079] 5.1 Materials and methods (1) In vitro combined drug experiment: THP-1 and HL-60 cell lines were used for pretreatment with the following drugs alone or in combination, followed by NK cell killing experiments: Selinexor (10nM) BCL-2 inhibitor Venetoclax (50nM) Hypomethylating drug azacitidine (0.5 μM) Anti-NKG2A antibody Monalizumab (5 μg / mL) (2) Combined treatment of AML mouse model: The NSG mouse AML model was established according to the method of Example 4, and the mice were divided into 8 groups (n=6 / group): Control group: normal saline NK cell alone group NK cell + selinexor group NK cell + Venetoclax group NK cell + azacitidine group NK cell + Monalizumab group NK cell + selinexor + venetoclax group NK cell + selinexor + monalizumab group 5.2 Experimental Results In vitro experimental results showed that compared with single pretreatment, the combined pretreatment of selinexor and venetoclax significantly enhanced the killing effect of NK cells on AML cells, and the killing rate was increased by about 40-50% compared with the pretreatment of selinexor alone. The combined use of selinexor and monalizumab also showed a synergistic effect, with the killing rate increased by about 35-45%. However, the effect of the combined use of selinexor and azacitidine was not significantly enhanced.

[0080] Mechanistic studies have shown that the combination of selinexor and venetoclax can simultaneously target the two survival pathways of XPO1 and BCL-2, increasing the sensitivity of AML cells to apoptosis. The combination of selinexor reducing HLA-E expression and monalizumab blocking NKG2A receptors can double-release the inhibitory signals of NK cells and significantly enhance their activation levels.

[0081] In the AML mouse model, the triple treatment group of NK cells + selinexor + venetoclax and the triple treatment group of NK cells + selinexor + monalizumab had the best therapeutic effects. The leukemia burden was reduced by 90.5±4.3% and 88.7±5.1% respectively compared with the control group, and the median survival was extended to 82.5 days and 79.5 days respectively.

[0082] Example 6: In vitro validation of primary AML patient samples In order to better evaluate the clinical translation potential of the method of the present invention, we used primary samples from AML patients for in vitro validation studies.

[0083] 6.1 Materials and methods (1) Patient samples: Bone marrow samples were collected from 15 patients with acute myeloid leukemia, including 8 newly diagnosed patients and 7 relapsed and refractory patients. All samples were obtained with informed consent from the patients and approved by the ethics committee.

[0084] (2) NK cell expansion: NK cells were expanded from the peripheral blood of healthy volunteers using the optimized protocol of Example 2.

[0085] (3) Primary AML pretreatment: Mononuclear cells were isolated from the patient's bone marrow and pretreated with 10 nM selinexor, 50 nM enetoclax, a combination of both, or DMSO (control) for 12 h.

[0086] (4) Killing experiment: The pretreated AML samples were co-cultured with expanded NK cells at an E:T ratio of 5:1 for 4 hours, and the mortality rate of CD33+ AML cells was detected by flow cytometry.

[0087] 6.2 Experimental Results Of the 15 AML samples, 13 (86.7%) showed a significant response to selinexor pretreatment, as evidenced by decreased HLA-E expression and increased NK cell killing sensitivity. Selinexor pretreatment increased the average NK cell killing rate of these samples from 28.3±7.5% to 56.7±9.2%, an increase of approximately 2-fold.

[0088] The combined pretreatment of selinexor and venetoclax further enhanced the killing effect of NK cells, with an average killing rate of 68.5±8.7%. In particular, the effect of combined pretreatment was more obvious in samples of relapsed and refractory patients, indicating that this strategy may be particularly beneficial for patients who are insensitive to traditional treatment.

[0089] It is worth noting that there is some heterogeneity in the response of different patient samples to selinexor pretreatment, which may be related to the patient's genetic background, disease type and treatment history. This suggests that in future clinical applications, it may be necessary to develop predictive biomarkers to identify the patient population most likely to benefit from this combination therapy.

[0090] Example 7: Preparation and quality control of NK cell preparation This example describes in detail the process flow and quality control standards for preparing clinical-grade NK cell preparations based on the method of the present invention.

[0091] 7.1 Preparation process (1) Obtaining starting materials: 50 mL of peripheral blood was collected from healthy donors and mononuclear cells were isolated using an FDA-approved cell separation device (such as Spectra Optia). The target number of mononuclear cells to be harvested was no less than 1×10 9 indivual.

[0092] (2) NK cell expansion: The expansion culture was performed according to the optimized scheme of Example 2, using clinical-grade reagents and aseptic operation. The expansion process was carried out in a cell factory or automated bioreactor system that complies with GMP standards.

[0093] (3) Harvesting and preparation: After 14 days of expansion, the cells were harvested, washed three times with PBS to remove the culture medium and cytokines, and finally resuspended in infusion solution (Plasmalyte-A containing 5% HSA) and the concentration was adjusted to 1-5×10 7 Pieces / mL.

[0094] (4) Cryopreservation (if necessary): Add freezing solution with a final concentration of 10% DMSO and 5% Trehalose, use a controlled rate freezing system, cool to -80°C at a rate of 1°C / min, and then transfer to a liquid nitrogen tank for long-term storage.

[0095] 7.2 Quality Control Standards (1) Identification criteria: NK cell (CD3-CD56+) ratio ≥ 85% T cell (CD3+) contamination ≤ 5% B cell (CD19+) contamination ≤ 2% Viable cell rate ≥80% (7-AAD negative) Killing rate against K562 ≥ 40% (E:T = 5:1) (2) Safety standards: Sterility: Negative bacterial and fungal cultures Mycoplasma: PCR test negative Endotoxin: <0.5EU / mL Virus detection: HIV, HBV, HCV and other nucleic acid tests are negative Tumorigenicity: normal karyotype, no obvious chromosomal abnormalities (3) Functional standards: Surface markers: CD16 and NKG2D expression ≥ 80% Cytokine production: IFN-γ secretion capacity ≥ 2000pg / mL / 10 6 Cells / 24 hours Batch consistency: Coefficient of variation between batches of key functional parameters ≤ 15% 7.3 Experimental Results According to the above process and quality control standards, we successfully prepared 5 batches of NK cell preparations, and all batches met the quality control requirements. The NK cell purity of the 5 batches of preparations was 87.5-93.2%, with an average of 90.3±2.3%; the viable cell rate was 85.3-92.1%, with an average of 88.7±2.8%; the killing rate against K562 was 58.3-68.7%, with an average of 63.5±4.2%. This shows that the method of the present invention can stably prepare high-quality NK cell preparations with good batch consistency.

[0096] The live cell rate of the cryopreserved preparation 24 hours after thawing was 83.5±3.7%, and the cytotoxic activity retention rate was 92.3±4.1%, indicating that the NK cell preparation of the present invention has good cryopreservation tolerance and is convenient for clinical application.

[0097] Example 8: Preclinical safety evaluation of NK cell therapy for AML This example evaluates the safety of the NK cell preparation prepared by the method of the present invention and the combined treatment regimen, providing a basis for future clinical trials.

[0098] 8.1 Materials and methods (1) In vitro safety evaluation: Normal hematopoietic cell toxicity: NK cells were co-cultured with normal donor bone marrow CD34+ cells to evaluate the effect on normal hematopoietic stem / progenitor cells. The survival rate and colony-forming ability of CD34+ cells were analyzed by flow cytometry.

[0099] Normal tissue cell toxicity: NK cells were co-cultured with normal human liver cell line L02, kidney epithelial cells HK-2, and lung epithelial cells BEAS-2B to evaluate the toxicity to normal tissue cells.

[0100] Cytokine release risk assessment: Measure the levels of proinflammatory cytokines (IFN-γ, TNF-α, IL-6, etc.) released by NK cells under in vitro activation to assess the potential risk of cytokine release syndrome.

[0101] (2) In vivo safety evaluation: Animal models: Two animal models are used: NSG mice and Cynomolgus monkeys (crab-eating monkeys) Dosage regimen: intravenous injection of NK cells (mice: 1×10 7 Monkey: 3×10 8 / kg), single or multiple administration; Safety indicators: vital signs monitoring, weight changes, hematological indicators, blood biochemical indicators, and histopathological analysis; Tissue distribution study: NK cells were labeled with CellTrace, and the tissue distribution and clearance of NK cells in vivo were evaluated through in vivo imaging and tissue section fluorescence detection.

[0102] (3) Safety evaluation of combined drug administration: Evaluate the safety of NK cells in combination with selinexor, venetoclax, or monalizumab in animal models; Focus on drug interactions and potential synergistic toxicities; 8.2 Experimental Results The results of in vitro safety evaluation showed that NK cells showed a slight inhibitory effect on normal hematopoietic stem / progenitor cells, but at an E:T ratio of 5:1, the survival rate of CD34+ cells remained above 85%. NK cells had low toxicity to normal tissue cells, and only showed a killing rate of about 15% on L02 cells at an E:T ratio of 10:1, and the killing rates on HK-2 and BEAS-2B cells were both less than 10%. This shows that NK cells have good specific recognition ability for tumor cells and have a low risk of toxicity to normal tissue cells.

[0103] Cytokine release assessment showed that NK cells release a certain amount of proinflammatory cytokines when activated, but the level is relatively low and is unlikely to cause severe cytokine release syndrome. Importantly, adding IL-12 / IL-18 pretreatment did not significantly increase this risk.

[0104] In the in vivo safety study of NSG mice, single or multiple NK cell infusions did not cause obvious toxic reactions. The mice's vital signs were stable, body weight changes were not significant, and hematological and biochemical indicators were within normal ranges. Histopathological analysis also did not reveal obvious organ damage or inflammatory reactions.

[0105] Tissue distribution studies have shown that infused NK cells are mainly distributed in the lungs, liver, and spleen, begin to appear in the bone marrow and lymph nodes after 24 hours, and are almost undetectable in the peripheral blood after 7-10 days. This distribution characteristic is consistent with the physiological migration characteristics of NK cells and reflects their normal clearance process in the body.

[0106] In the cynomolgus monkey model, high-dose NK cell infusion (3×10 8 The drug was administered at a dose of 100 mg / kg, resulting in transient fever and mild inflammatory response, but the symptoms resolved spontaneously within 24-48 hours. All biochemical and organ function indicators returned to normal after administration, and no long-term toxic effects were found.

[0107] The safety evaluation of combined administration showed that the combination of NK cells with selinexor, venetoclax or monalizumab did not produce obvious synergistic toxicity. It is worth noting that the combination of NK cells and selinexor may slightly increase the fluctuation of liver function indicators, but it is within the clinically acceptable range. It is recommended to strengthen liver function monitoring in future clinical trials.

[0108] The comprehensive evaluation results show that the NK cell preparation prepared by the method of the present invention has good safety characteristics, and the safety risks of using it alone or in combination with drugs such as selinexor are controllable, which supports the conduct of future clinical trials.

[0109] Example 9: Dose exploration of NK cell therapy regimen This example explores the optimal dose and administration regimen of NK cells and selinexor combined for the treatment of AML, providing a basis for the design of future clinical trials.

[0110] 9.1 Materials and methods (1) AML mouse model: NSG mouse AML model was established according to the method of Example 4 (2) NK cell dose gradient: The following NK cell dose groups were set (n=6 per group): Low dose group: 5×10 6 Piece / piece Medium dose group: 1×10 7 Piece / piece High dose group: 2×10 7 Piece / piece (3) Selinexor dose gradient: The following selinexor dose groups were set (n=6 in each group): Low dose group: 2.5 mg / kg Medium dose group: 5 mg / kg High dose group: 10 mg / kg (4) Exploration of dosing frequency: NK cell infusion: once a week, twice a week, or three times a week Selinexor dosing: 2 times per week, 3 times per week, or 4 times per week (5) Evaluation indicators: leukemia burden, survival, bone marrow infiltration rate, adverse reactions 9.2 Experimental Results The results of NK cell dose exploration showed that the medium dose (1×10 7 per animal) and high dose (2×10 7 The efficacy of the medium-dose group was similar to that of the low-dose group, which was significantly better than the low-dose group. Considering the complexity and cost of cell preparation, the medium-dose regimen has a better benefit ratio. In terms of dosing frequency, the effect of NK cell infusion twice a week is better than that of once a week, but the difference is not significant compared with 3 times a week, and the latter has a slightly higher risk of adverse reactions.

[0111] The results of selinexor dose exploration showed that 5 mg / kg (medium dose) is an ideal dose, balancing efficacy and safety. In terms of dosing frequency, 3 times a week is better than 2 times a week, but 4 times a week does not bring additional benefits, but increases the risk of toxicity.

[0112] Based on the above results, the recommended optimal dosing regimen is: NK cells 1×10 7 100 mg / kg, twice a week; 5 mg / kg of selinexor, 3 times a week. Under this regimen, the median survival of AML mice was extended to 2.2 times that of the control group, the bone marrow leukemia cell infiltration rate was reduced by about 85%, and the adverse reactions were controllable.

[0113] Example 10: NK cell culture process scale-up and automation This example demonstrates the process scale-up and automation process of the NK cell expansion method of the present invention, verifying its applicability in clinical-scale production.

[0114] 10.1 Materials and methods (1) Starting material: Leukopak leukocyte separation product from healthy donors, containing approximately 1×10 PBMC 10 indivual (2) Culture system: Pilot scale: WAVE Bioreactor 2 / 10 system (GE Healthcare), working volume 2 L Clinical scale: CliniMACS Prodigy cell processing system (Miltenyi Biotec), closed-loop automation platform (3) Culture parameters: Culture medium: CTS™ OpTmizer™ T Cell Expansion SFM supplemented with 5% HSA Cytokines: IL-2 (500 IU / mL), IL-15 (20 ng / mL), IL-21 (10 ng / mL) Antibodies: anti-CD52 monoclonal antibody (10 μg / mL), anti-CD3 monoclonal antibody (50 ng / mL) Culture conditions: 37°C, 5% CO 2 , 5% O 2 (Low Oxygen) Cultivation period: 14 days (4) Online monitoring parameters: pH, dissolved oxygen, glucose, lactate, cell density (5) Quality control: according to the standards of Example 7 10.2 Experimental Results Pilot scale culture (WAVE system): Starting PBMC number: 2×10 9 indivual Total number of NK cells after 14 days: 2.05×10 12 indivual NK cell purity: 88.5±2.7% Amplification multiple: about 1025 times Live cell rate: 89.7±2.3% Killing activity (E:T = 5:1): 62.3±4.1% Clinical scale culture (Prodigy system): Starting PBMC number: 5×10 9 indivual Total number of NK cells after 14 days: 4.85×10 12 indivual NK cell purity: 86.8±3.1% Amplification multiple: about 970 times Live cell rate: 87.5±2.5% Killing activity (E:T = 5:1): 60.7±4.5% The culture results of the two amplification systems were similar to those of the small-scale experiments, indicating that the method of the present invention has good scalability. As a fully automatic closed-loop platform, the Prodigy system can further reduce the risk of contamination and human operation errors, and is more suitable for the large-scale production of clinical-grade NK cell preparations.

[0115] Online monitoring data during the culture process showed that NK cell proliferation mainly occurred on the 4th to 10th day of culture, during which glucose consumption and lactate production rates increased significantly. Based on this feature, the culture medium supplementation strategy can be optimized, and the frequency of medium replacement can be increased on the 4th to 10th day to further improve the NK cell expansion efficiency and product quality.

[0116] The present invention provides an innovative NK cell expansion method, which can efficiently expand high-purity and high-activity NK cells through the synergistic effect of anti-CD52 monoclonal antibody and specific cytokine combination. This method has the following outstanding advantages: First, the amplification efficiency is significantly improved. The method of the present invention can achieve more than 1000-fold amplification of NK cells, which is significantly higher than the 294-fold amplification rate of cytokines alone in the prior art (Min et al., 2019) or the 500-1000-fold amplification rate of the K562 feeder layer method (Knorr et al., 2018). Importantly, this method does not require complex genetic engineering operations or potentially risky tumor cell feeder layers, and the process is simple, which is convenient for standardized and large-scale production.

[0117] Secondly, the product has high purity and does not require sorting. The purity of NK cells directly amplified by this method can reach more than 85%, without the need for additional magnetic bead sorting or flow sorting steps, which simplifies the production process and reduces production costs. This feature stems from the selective inhibitory effect of anti-CD52 monoclonal antibodies on T cells and B cells, while the effect on NK cells is relatively small, which is consistent with the finding reported by Hotta et al. (2016) that NK cell CD52 expression is lower than that of T / B cells.

[0118] Third, NK cell function is optimized and enhanced. Through hypoxic culture and IL-12 / IL-18 pretreatment, the NK cells amplified by this method express high levels of activation receptors (such as NKG2D and CD16) and show enhanced killing activity and cytokine production. More importantly, these NK cells have long-lasting functions similar to memory characteristics and prolong their survival time in vivo. This finding echoes the results reported by Romee et al. (2012) that IL-12 / 15 / 18 pretreatment induced NK cell memory-like characteristics.

[0119] Fourth, the innovative strategy of pre-treating AML cells with selinexor significantly enhanced the therapeutic effect of NK cells. This invention applied this strategy for the first time in the treatment of AML and found that selinexor significantly enhanced the recognition and killing ability of NK cells by downregulating HLA-E expression on the surface of AML cells and reducing the contact of NK cell inhibitory receptor NKG2A. This finding is consistent with the report of Fisher et al. (2021) in lymphoma models, but we further revealed its application value in the treatment of AML and explored its mechanism of action.

[0120] Fifth, the optimization and exploration of combined drug strategies provide more options for future clinical applications. The present invention verifies the triple therapy of NK cells and selinexor combined with venetoclax or monalizumab, showing a stronger synergistic anti-tumor effect. In particular, the triple therapy of NK cells + selinexor + venetoclax, by simultaneously targeting two key survival pathways, XPO1 and BCL-2, and enhancing NK cell activity, shows outstanding therapeutic potential in refractory relapsed AML models.

[0121] Sixth, the method of the present invention has good safety characteristics and clinical transformation potential. In vivo safety evaluation shows that the safety risk of NK cell preparations prepared by this method when used alone or in combination with drugs such as selinexor is controllable. Process amplification and automation studies also confirmed that this method is suitable for clinical scale production and has good scalability and batch consistency.

[0122] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for amplifying NK cells by combining anti-CD52 monoclonal antibody and cytokines, characterized in that: The method comprises the following steps: (1) Extracting mononuclear cells (PBMCs) from donor peripheral blood; (2) culturing the mononuclear cells with anti-CD52 monoclonal antibody, anti-CD3 monoclonal antibody and a cytokine composition comprising IL-2, IL-15 and IL-21, and expanding and culturing for 14-21 days at 37±1° C. and 5±0.5% CO2; (3) harvesting the expanded cells, wherein the purity of CD3-CD56+ NK cells reaches more than 85%, and the cell number increases to more than 1000 times the original NK cell number; (4) Detecting the surface receptor expression and cytotoxic activity of the NK cells.

2. The method according to claim 1, characterized in that The anti-CD52 monoclonal antibody is alemtuzumab, and the added amount is 5-15 μg / mL. The anti-CD3 monoclonal antibody is OKT3 clone, and the added amount is 10-100 ng / mL.

3. The method according to claim 1, characterized in that The concentration of IL-2 in the cytokine composition is 100-1000 IU / mL, the concentration of IL-15 is 10-50 ng / mL, and the concentration of IL-21 is 5-25 ng / mL.

4. The method according to claim 3, characterized in that: The method further comprises the following steps: (a) supplementing the anti-CD52 monoclonal antibody on days 3-4 and 7-8 of cell expansion, with the supplemented amounts being 50% and 25% of the initial concentration, respectively; (b) replenishing the cytokine composition once every 3-4 days; (c) Cell density exceeds 2×10 6 cells / mL and maintain at 1-2×10 6 cells / mL.

5. The method according to claim 1, characterized in that The culture is performed in a serum-free medium, which is RPMI-1640 or OpTmizer™ T Cell Expansion SFM supplemented with 5-10% human serum albumin.

6. The method according to claim 1, characterized in that The method further includes a step of culturing under hypoxic conditions in Step (2), wherein the oxygen concentration is 5-7%, and at the end of the culture period, 10 ng / mL IL-12 and 10 ng / mL IL-18 are added for pretreatment for 6-24 hours to induce NK cells to produce memory-like properties.

7. A NK cell preparation prepared by the method according to any one of claims 1 to 6, characterized in that: The NK cell purity in the preparation is ≥85%, the CD3+T cell ratio is ≤5%, the viable cell rate is ≥80%, the killing rate against K562 standard target cells is ≥40% (E:T=5:1), and the NK cells express high levels of NKG2D and CD16 activation receptors.

8. A method for treating acute myeloid leukemia using the NK cell preparation according to claim 7, comprising the following steps: (1) 6-24 hours before NK cell infusion, acute myeloid leukemia cells were treated with 5-10 nM selinexor; (2) Intravenous infusion of the NK cell preparation at a dose of 1-5×10 7 cells / kg body weight.

9. The method according to claim 8, characterized in that Selinexor downregulates HLA-E expression on the surface of acute myeloid leukemia cells, thereby enhancing the ability of NK cells to recognize and kill leukemia cells.

10. The method according to claim 8, characterized in that The method also includes the combined use of one or more of the following drugs: BCL-2 inhibitor Venetoclax, at a dose of 5-100 nM; demethylation drug Azacitidine, at a dose of 0.1-1 μM; or anti-NKG2A antibody Monalizumab, at a dose of 1-10 μg / mL.

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