Application of arginine in preparation of kit for recovering NK cell function and treating leukemia

Abstract

The invention belongs to the technical field of biology, and discloses application of arginine in preparation of a kit for recovering NK cell functions and treating leukemia. The invention has the following beneficial effects: the inventor reports the application of supplementing arginine in the NK cells for treating acute myelogenous leukemia for the first time. Experimental results show that the ability of damaged bone marrow NK cells of AML patients to kill tumor cells can be recovered by supplementing arginine, and results of in-vivo experiments further show that the survival time of acute myelogenous leukemia model mice can be prolonged by recovering NK cell treatment cultured by arginine, and leukemia cell load is inhibited. Therefore, a certain experimental basis is provided for clinically treating the recurrent and refractory AML by using the arginine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of arginine in reversing the damaged anti-tumor function of NK cells in patients with relapsed leukemia after hematopoietic stem cell transplantation, and in treating acute myeloid leukemia in a mouse animal model. Background Technology

[0002] Acute myeloid leukemia (AML) is an aggressive malignant hematologic disorder with a high incidence, particularly among the elderly, and has been a challenge for hematologists for decades (Liu H. Emerging agents and regimens for AML. J Hematol Oncol. 2021 Mar 23; 14(1):49. doi:10.1186 / s13045-021-01062-w.). Allogeneic hematopoietic stem cell transplantation (alo-HSCT) is an effective treatment and the only option for most AML patients (Romero D. Sorafenib prevents AML relapse after allo-HSCT. Nat Rev Clin Oncol. 2020 Oct; 17(10):591. doi:10.1038 / s41571-020-00431-7.). Even with current treatments, relapse of the primary disease after transplantation remains frequent and is associated with poor prognosis. In particular, relapse becomes the main cause of death 100 days after allogeneic hematopoietic stem cell transplantation.

[0003] AML relapse is associated with the ability of AML cells to evade immune surveillance. Natural killer (NK) cells play a crucial role in cancer immune surveillance. Recent studies have shown that downregulation of NK cell killing of AML cells after allogeneic hematopoietic stem cell transplantation may help AML cells evade immune surveillance. NK cells are the first lymphocytes to be reconstituted, and in the first 100 days after transplantation, NK cells may account for 80% of peripheral blood lymphocytes (Limongello R, Marra A, Mancusi A, Bonato S, Hoxha E, Ruggeri L, Hui S, Velardi A, Pierini A. Novel ImmuneCell-Based Therapies to Eradicate High-Risk Acute Myeloid Leukemia. FrontImmunol. 2021 Aug 3; 12:695051. doi:10.3389 / fimmu.2021.695051.). NK cells kill leukemia cells by secreting granules containing cytolysis-related proteins (such as granzymes) and effector cytokines (such as interferon-γ (IFN-γ)). (Limongello R, Marra A, Mancusi A, Bonato S, Hoxha E, Ruggeri L, Hui S, Velardi A, Pierini A. Novel Immune Cell-Based Therapies to Eradicate High-Risk Acute Myeloid Leukemia. Front Immunol. 2021 Aug 3; 12:695051. doi:10.3389 / fimmu.2021.695051.) However, in the tumor microenvironment, the lack of arginine often leads to a decline in the anti-tumor capacity of immune cells, allowing tumor cells to evade NK cell-mediated cytotoxicity and immune surveillance.

[0004] Cell metabolism and nutrition, especially arginine, play an indispensable role in promoting T cell function, maintaining T cell survival and proliferation (Chapman NM, Boothby MR, Chi H. Metabolic coordination of T cellquiescence and activation. Nat Rev Immunol. 2020; 20(1):55-70. doi:10.1038 / s41577-019-0203-y). Arginine transport receptor CAT-1 (cationic amino acid transporter) enables peripheral arginine to be transported into cells to maintain their function. Low levels of arginine in the body are closely related to poor prognosis and low survival rate in patients with solid tumors (Peyraud F, Guégan JP, Bodet D, et al. Circulating L-arginine predicts the survival of cancer patients treated with immune checkpoint inhibitors. Ann Oncol. 2022; 33(10):1041-1051. doi:10.1016 / j.annonc.2022.07.001). However, it is still unclear whether arginine deficiency leads to NK cell dysfunction in AML disease. In addition, it has not been reported whether restoring arginine levels can enhance the effect of NK cell-dependent anti-leukemia therapy. Therefore, restoring arginine levels in the body to improve NK cell function is the focus of this study in the drug treatment of acute myeloid leukemia. It also has high prospects for basic research to clinical application. Summary of the Invention

[0005] In order to address the problems existing in the prior art, the present invention provides the use of arginine in the preparation of a kit for restoring NK cell function and treating leukemia.

[0006] Specifically, the present invention provides:

[0007] (1) Use of arginine in the preparation of kits for restoring NK cell function and treating leukemia.

[0008] (2) As described in (1), the kit contains NK cells.

[0009] (3) The use according to (1) or (2), wherein the leukemia is acute myeloid leukemia, preferably, the leukemia is relapsed acute myeloid leukemia.

[0010] (4) The use according to any one of (1)-(3), wherein the restoration of NK cell function is the restoration of the ability of damaged NK cells in leukemia patients to kill tumor cells.

[0011] (5) The use according to any one of (1)-(4), wherein the dosage of arginine used is 0.5-5mM, preferably 0.5-2mM, more preferably 1mM.

[0012] (6) A method for preparing NK cells with the ability to restore tumor cell killing ability, comprising: co-culturing NK cells with impaired tumor cell killing ability with arginine to prepare NK cells with the ability to restore tumor cell killing ability.

[0013] (7) According to the method of (6), the dosage of arginine used is 0.5-5mM, preferably 0.5-2mM, and more preferably 1mM.

[0014] (8) The method according to (6) or (7), wherein the NK cells with impaired tumor-killing ability are isolated from leukemia patients.

[0015] (9) According to the method described in (8), the leukemia is acute myeloid leukemia, preferably, the leukemia is relapsed acute myeloid leukemia.

[0016] The present invention has the following beneficial effects:

[0017] This invention reports for the first time the application of arginine supplementation in NK cell therapy for acute myeloid leukemia (AML). Experimental results show that arginine supplementation can restore the ability of damaged bone marrow NK cells to kill tumor cells in AML patients. In vivo experiments further demonstrate that NK cell therapy after restoring arginine culture can prolong the survival of AML model mice and inhibit leukemia cell burden. This provides experimental evidence for the clinical use of arginine in the treatment of relapsed / refractory AML. Attached Figure Description

[0018] Figure 1 This study showed the arginine levels in the bone marrow of patients with relapsed and non-relapsed acute myeloid leukemia after hematopoietic stem cell transplantation.

[0019] Figure 2 The study showed that arginine (L-arginine, Arg) deficiency leads to impaired antitumor function of human bone marrow NK cells. Figure 2 A represents a flow cytometry graph showing tumor cell apoptosis after co-culturing bone marrow NK cells and tumor cells under arginine-deficient and arginine-supplemented culture conditions. Figure 2B indicates that under arginine-deficient culture conditions, bone marrow NK cells have a low ability to kill tumor cells; when arginine is supplemented, the NK cell killing function can be restored.

[0020] Figure 3 Arginine showed that it restored the anti-tumor effector function of bone marrow NK cells derived from relapsed patients, among which... Figure 3 A represents a flow cytometry graph showing tumor cell apoptosis after co-culturing bone marrow NK cells with tumor cells in relapsed patients under standard culture conditions or under culture conditions supplemented with arginine. Figure 3 B indicates that the bone marrow NK cells of relapsed patients have a low ability to kill tumor cells; supplementing with arginine can reverse the tumor-killing ability of bone marrow NK cells in relapsed patients.

[0021] Figure 4 The results showed that arginine supplementation in a mouse model of acute myeloid leukemia could restore the anti-tumor capacity of NK cells impaired by arginine deficiency. Figure 4 A indicates that NK cells cultured under arginine-deficient conditions exhibit impaired leukemia treatment capabilities and vigorous tumor growth; however, NK cells cultured with supplemented arginine show significantly enhanced leukemia treatment capabilities and restricted tumor growth. Figure 4 B indicates that NK cells cultured under arginine-deficient conditions resulted in decreased survival in leukemia model mice after treatment; however, NK cells cultured with supplemented arginine significantly prolonged the survival of mice after treatment. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased from the market.

[0023] As used herein, the term "treatment" has the meaning known in the art, including, for example, (1) preventing or delaying the onset of clinical symptoms of a condition, symptom, or illness in animals (especially mammals and especially humans) that may be susceptible to or prone to such condition, symptom, or illness but have not yet experienced or presented clinical or subclinical symptoms of such condition, symptom, or illness; (2) suppressing such condition, symptom, or illness (e.g., inhibiting, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms); and / or (3) alleviating symptoms (i.e., causing the resolution of at least one of the condition, symptom, or illness or its clinical or subclinical symptoms). The benefit to the treated patient is statistically significant or at least perceptible to the patient or physician.

[0024] As used herein, the term "impaired ability of NK cells to kill tumor cells" means a decrease in the ability of NK cells to kill tumor cells of at least 10%, for example, a decrease of at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, or at least 19%. Preferably, the decrease in the ability of NK cells to kill tumor cells of at least 20%, for example, a decrease of at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, or at least 29%. More preferably, the decrease in the ability of NK cells to kill tumor cells of at least 30%, for example, a decrease of at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, or at least 39%. More preferably, the ability of NK cells to kill tumor cells is reduced by at least 40%, for example, by at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, or at least 49%. More preferably, the ability of NK cells to kill tumor cells is reduced by at least 50%, for example, by at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, or at least 59%. More preferably, the ability of NK cells to kill tumor cells is reduced by at least 60%, for example, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, or at least 69%.

[0025] In some embodiments, the present invention relates to a method for preparing NK cells with the ability to restore tumor cell killing capacity, comprising: co-culturing NK cells with impaired tumor cell killing capacity with arginine to prepare NK cells with the ability to restore tumor cell killing capacity. In some embodiments, the co-culturing of NK cells with arginine includes the step of culturing NK cells in a cell culture medium containing arginine. In some embodiments, the cell culture medium may include any culture medium suitable for NK cell culture, such as RPMI 1640 medium. In some embodiments, the co-culturing includes supplementing a culture medium without amino acids (e.g., arginine) with arginine, and then culturing the NK cells. In some embodiments, the NK cells to be co-cultured include NK cells with impaired tumor cell killing capacity, which may be isolated from leukemia patients, such as patients with acute myeloid leukemia (e.g., relapsed acute myeloid leukemia).

[0026] laboratory animals

[0027] Female NOD / ShiLtJGpt-Prkdcem26Cd52IL-2rgem26Cd22 / Gpt(NCG) mice. Purchased from Jicui Pharmaceutical Co., Ltd. Housing conditions: SPF grade.

[0028] Experimental steps

[0029] Flow cytometry:

[0030] First, mononuclear cells were isolated from the bone marrow: Using the erythrocyte lysis method, 1X RBC Lysis Buffer (Biolegend, Cat#420302) was added to a 15 mL centrifuge tube at a ratio of 1 mL erythrocyte lysis buffer / 100 μL bone marrow sample for thorough lysis. After incubation at room temperature for 10 minutes, 3 mL of PBS (Phosphate buffer saline) was added to terminate the lysis, and the sample was centrifuged at 350 g for 10 minutes. The supernatant was discarded, and the sample was washed again with 3 mL of PBS and centrifuged at 350 g for 10 minutes. Bone marrow mononuclear cells were obtained.

[0031] Subsequently, NK cells were purified from bone marrow mononuclear cells using an NK cell isolation and purification kit (purchased from Miltenyi Biotec, catalog number #130-092-657) following the kit instructions. The cells were then purified at a concentration of 2.5 × 10⁻⁶ cells per cell line. 5Cells per well were added to 48-well cell culture plates and divided into different groups for treatment. Groups included: 1) Normal complete medium (RPMI 1640, HyClone, Cat#SH30809; with 10% fetal bovine serum); Group 2) Medium with amino acid deficiency (Meilunbio, Cat#MA0546; with 10% fetal bovine serum) (i.e., L-arginine deprivation + PBS group); Group 3) Medium with amino acid deficiency (Meilunbio, Cat#MA0546; with 10% fetal bovine serum), supplemented with arginine (Arg; concentration 1mM, Sigma, Cat#74-79-3). Cultured for 48 hours. NK cells were transferred from wells to 1.5 ml centrifuge tubes, centrifuged at 500g for 5 minutes, and the supernatant was discarded to obtain NK cells.

[0032] At an effector cell:target cell ratio of 5:1, 5 × 10⁵ primitive tumor cells of the patient's myeloid leukemia were added to a 96-well plate. 4 Cells / well, along with the NK cells cultured above. Co-cultured at 37°C and 5% CO2 for 5 hours; then centrifuged at 500g for 5 minutes, discarded the supernatant, and collected the cells. The collected cells were first blocked with mouse serum for 15 minutes; then labeled with the anti-human flow cytometry antibodies CD45 (Biolegend, 304008), CD56 (Biolegend, 318328), and Annexin V (BD, 550474), and incubated at 4°C for 20 minutes; the cells were washed according to the manufacturer's instructions to remove non-specific antibodies. Five minutes before detection, 7AAD (BD, Cat#559925) detection antibody was added and mixed thoroughly with the cell suspension. Detection was then performed using an LSRII flow cytometer and analyzed using FlowJo software.

[0033] The inventors discovered that relapsed patients had reduced arginine levels, leading to weakened NK cell function. Therefore, to simulate the actual in vivo conditions, they used a culture medium lacking arginine during in vitro culture. In the third group, amino acids were supplemented to restore normal levels, and it was found that NK cells regained their function.

[0034] Mouse model construction and treatment

[0035] HL60 cells labeled with luciferase (Nanmo Biotechnology, catalog number: NM-B03-1) (5×10 5 The drug was injected into NCG mice via the tail vein (for 6 weeks). After one week, once tumor formation was confirmed, 2.5 × 10⁻⁶ human-derived drug was administered via the tail vein to the mice. 6NK cells (NK cells were isolated and purified from peripheral blood of healthy donors). Mice were intraperitoneally injected with 50,000 U IL-2 (purchased from Jiangsu Genscript Biotech) every two days. For the pretreatment of NK cells, the following groups were used: Group 1) cultured in normal culture medium; Group 2) cultured in L-arginine deprivation culture medium (purchased from Meilunbio); Group 3) cultured in L-arginine deprivation culture medium supplemented with arginine (1 mM, purchased from Sigma) for 48 hours.

[0036] When assessing the tumor burden of AML, D-Luciferin potassium salt (Sigma, 115144-35-9; dose 150 mg / kg) was administered intraperitoneally. Observation and statistical analysis were performed using a small animal imaging system (Perkin Elmer).

[0037] Data Analysis

[0038] Data were analyzed using GraphPad Prism 8. T-tests were used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0039] Example 1

[0040] First, the arginine level in bone marrow supernatant from patients with relapsed and non-relapsed acute myeloid leukemia after hematopoietic stem cell transplantation was measured using an ELISA (enzyme-linked immunosorbent assay). The kit information was: LSBio, catalog number LS-F13034. The test was performed according to the kit instructions.

[0041] Table 1: Arginine Levels in Bone Marrow (Each value represents the arginine concentration in a patient's bone marrow sample)

[0042]

[0043] Example 2

[0044] First, mononuclear cells were isolated from the bone marrow: Using the erythrocyte lysis method, 1X RBC Lysis Buffer (Biolegend, Cat#420302) was added to a 15 mL centrifuge tube at a ratio of 1 mL erythrocyte lysis buffer / 100 μL bone marrow sample for thorough lysis. After incubation at room temperature for 10 minutes, 3 mL of PBS (Phosphate buffer saline) was added to terminate the lysis, and the sample was centrifuged at 350 g for 10 minutes. The supernatant was discarded, and the sample was washed again with 3 mL of PBS and centrifuged at 350 g for 10 minutes. Bone marrow mononuclear cells were obtained.

[0045] Subsequently, using the magnetic-activated cell sorter (MACS) technology and an NK cell isolation and purification kit (purchased from Miltenyi Biotec, catalog number #130-092-657), NK cells were purified from bone marrow mononuclear cells according to the kit instructions, at a concentration of 2.5 × 10⁻⁶. 5 Cells per well were added to 48-well cell culture plates and divided into different groups for treatment. Groups included: 1) Normal complete medium (RPMI 1640, HyClone, Cat#SH30809; with 10% fetal bovine serum); Group 2) Medium with amino acid deficiency (Meilunbio, Cat#MA0546; with 10% fetal bovine serum) (i.e., L-arginine deprivation + PBS group); Group 3) Medium with amino acid deficiency (Meilunbio, Cat#MA0546; with 10% fetal bovine serum), supplemented with arginine (Arg; concentration 1mM, Sigma, Cat#74-79-3). Cultured for 48 hours. NK cells were transferred from wells to 1.5 ml centrifuge tubes, centrifuged at 500g for 5 minutes, and the supernatant was discarded to obtain NK cells.

[0046] At an effector cell:target cell ratio of 5:1, 5 × 10⁵ primitive tumor cells of the patient's myeloid leukemia were added to a 96-well plate. 4 Cells / well, along with the NK cells cultured above. Co-cultured at 37°C and 5% CO2 for 5 hours; then centrifuged at 500g for 5 minutes, discarded the supernatant, and collected the cells. The collected cells were first blocked with mouse serum for 15 minutes; then labeled with the anti-human flow cytometry antibodies CD45 (Biolegend, 304008), CD56 (Biolegend, 318328), and Annexin V (BD, 550474), and incubated at 4°C for 20 minutes; the cells were washed according to the manufacturer's instructions to remove non-specific antibodies. Five minutes before detection, 7AAD (BD, Cat#559925) detection antibody was added and mixed thoroughly with the cell suspension. Detection was then performed using an LSRII flow cytometer and analyzed with FlowJo software.

[0047] The expression of 7AAD and Annexin V in CD45-CD56- cells was detected, the proportion of 7AAD-positive leukemia blasts was calculated, and cytotoxicity was determined. It was found that arginine deficiency significantly reduced the killing function of NK cells against leukemia blasts; however, arginine supplementation significantly restored the tumor cell killing ability (A, B). n = 10 cases. Data analysis was performed using the one-way analysis of variance (ANOVA) test.

[0048] Table 2. Detection of NK cell tumor cell killing ability under different culture conditions.

[0049]

[0050] Example 3

[0051] First, bone marrow NK cells were isolated and purified from bone marrow mononuclear cells of AML patients who had relapsed after hematopoietic stem cell transplantation using the magnetic-activated cell sorter (MACS) technology and a kit (Miltenyi Biotec, Cat. #130-092-657). The steps were the same as described in Example 2.

[0052] NK cells were administered to relapsed patients at a rate of 2.5 × 10⁻⁶. 5 Cells / well were grouped and cultured in 48-well plates. 1) Cells were cultured in normal complete medium (RPMI 1640, HyClone, Cat#SH30809; with 10% fetal bovine serum added) for 48 hours; 2) Arginine (Arg; 1mM, Sigma, Cat#74-79-3) was added to the normal complete medium, and the cells were cultured for 48 hours. NK cells from each well were transferred to a 1.5 mL centrifuge tube, centrifuged at 500g for 5 minutes, and the supernatant was discarded to obtain NK cells.

[0053] Then, 5×10⁻⁶ ppm was added to the 96-well plate. 4 Leukemia blast cells (from the First Affiliated Hospital of University of Science and Technology of China) / well. The above NK cells and leukemia blast cells were co-cultured at an effector cell:target cell ratio of 5:1 for 5 hours. The expression of 7AAD and Annexin V in CD45-CD56- cells was detected, the proportion of 7AAD-positive leukemia blast cells was calculated, and cytotoxicity was calculated. It was found that the NK cells from the bone marrow of relapsed patients had lower killing function against leukemia blast cells; however, culturing with arginine supplementation significantly reversed the impaired NK cell killing ability against tumor cells (A, B). n = 10 cases. Paired t-tests were used for data analysis.

[0054] Table 3. Arginine supplementation enhances the ability of bone marrow NK cells to kill tumor cells in relapsed patients.

[0055]

[0056] Example 4

[0057] HL60 cells carrying luciferase (Nanmo Biotechnology, catalog number: NM-B03-1) were transferred via tail vein into NCG mice, 5 × 10⁶ cells per cell line. 5 / 200 µL PBS / mouse, construct AML model.

[0058] NK cells derived from human peripheral blood were pretreated in groups: Group 1) cultured in normal complete medium (RPMI 1640, HyClone, Cat#SH30809; with 10% fetal bovine serum); Group 2) cultured in amino acid-deficient medium (Meilunbio, Cat#MA0546; with 10% fetal bovine serum) (i.e., L-arginine deprivation + PBS group); Group 3) cultured in amino acid-deficient medium (Meilunbio, Cat#MA0546; with 10% fetal bovine serum), supplemented with arginine (Arg; concentration 1 mM, Sigma, Cat#74-79-3). NK cells were collected after 48 hours.

[0059] One week after the AML model was constructed, the aforementioned NK cells were transfused via the tail vein into the affected groups for treatment, at a dose of 2.5 × 10⁻⁶. 6 NK cells / 200 μL PBS / mouse. Mice were intraperitoneally injected with 50,000 U IL-2 (purchased from Jiangsu Genscript Pharmaceutical Co., Ltd.) every two days. Every 7 days, tumor burden was observed and statistically analyzed using a small animal imaging system (Perkin Elmer) via intraperitoneal injection of D-fluorescein potassium salt (Sigma, Cat#115144-35-9; dose 150 mg / kg).

[0060] The study found that NK cell therapy in mice cultured under arginine-deficient conditions significantly increased tumor burden; however, upon restoration of arginine-supplemented culture, NK cells significantly inhibited tumor growth, and mice exhibited longer survival (A, B). Figure (A) uses one-way ANOVA. Figure (B) uses log-rank Mantel-Cox statistical analysis. "1" represents mouse survival; "0" represents mouse death.

[0061] Table 4. Detection of fluorescence values ​​of HL60 cells in mice at different time points.

[0062]

[0063] The numerical values ​​represent the fluorescence intensity values ​​of each mouse at each detection time point.

[0064] Table 5 Survival period detection of different groups of mice

[0065]

[0066] "0" represents death; "1" represents survival.

[0067] in conclusion

[0068] Arginine can significantly restore the anti-tumor capacity of bone marrow NK cells in relapsed patients. In both in vitro and in vivo treatment, it can significantly reverse the inhibitory effect of arginine deficiency on NK cell anti-tumor function, reduce tumor burden in mice, and prolong survival. These results suggest that arginine supplementation can effectively assist in the treatment of acute myeloid leukemia.

Claims

1. Use of arginine in the preparation of kits for restoring NK cell function and treating leukemia.

2. The use according to claim 1, wherein the kit comprises NK cells.

3. The use according to claim 1 or 2, wherein the leukemia is acute myeloid leukemia, preferably, the leukemia is relapsed acute myeloid leukemia.

4. The use according to any one of claims 1-3, wherein the restoration of NK cell function is the restoration of the ability of damaged NK cells in leukemia patients to kill tumor cells.

5. The use according to any one of claims 1-4, wherein the dosage of arginine is 0.5-5 mM, preferably 0.5-2 mM, and more preferably 1 mM.

6. A method for preparing NK cells with the ability to restore the killing ability of tumor cells, comprising: NK cells with impaired tumor-killing ability were co-cultured with arginine to prepare NK cells with restored tumor-killing ability.

7. The method according to claim 6, wherein the dosage of arginine is 0.5-5 mM, preferably 0.5-2 mM, and more preferably 1 mM.

8. The method according to claim 6 or 7, wherein the NK cells with impaired tumor-killing ability are isolated from leukemia patients.

9. The method according to claim 8, wherein the leukemia is acute myeloid leukemia, preferably, the leukemia is relapsed acute myeloid leukemia.

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