Pre-culturing method of nk cells and application thereof

Abstract

The present application relates to the technical field of biology, and particularly discloses a pre-culturing method of NK cells and application thereof. The pre-culturing method of NK cells comprises the step of pre-treating NK cells with an inducing agent; the active ingredient in the inducing agent comprises a MAPK inhibitor; and the MAPK inhibitor comprises a p38 MAPK inhibitor and / or a JNK inhibitor. The method of the present application enables NK cells to obtain tolerance to a tumor hypoxic microenvironment, enhances the expression of an activation marker, reduces the expression of an inhibition marker, and reduces the level of intracellular reactive oxygen species, thereby greatly improving the killing ability of the NK cells to tumor cells in a hypoxic environment compared with NK cells without pre-treatment, and providing a safe, simple and easy-to-convert clinical synergistic strategy for NK cell immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for pre-culturing NK cells and its application. Background Technology

[0002] Natural killer cells (NK cells) are an important component of the body's innate immune system. They have the ability to directly recognize and kill tumor cells and virus-infected cells without antigen pretreatment, and have broad application prospects in anti-tumor immunotherapy.

[0003] However, in solid tumors, the tumor microenvironment (TME) is characterized by widespread hypoxia, nutrient deficiency, and stress accumulation. Hypoxia is one of the typical features of the solid tumor microenvironment. In tumor tissue, due to abnormal angiogenesis and metabolic disorders, local oxygen concentrations decrease significantly, often falling below 1%-3%. This significantly weakens the cytotoxicity of natural killer (NK) cells. Numerous studies have shown that after NK cells enter a hypoxic tumor microenvironment, the hypoxic environment leads to metabolic disorders, decreased mitochondrial function, increased ROS accumulation, and reduced perforin / granzyme release. Their cytotoxic activity and proliferative capacity rapidly decline due to hypoxia exposure, resulting in severely limited killing function. This phenomenon greatly restricts the application of immune cells, including NK cells, in tumor therapy, drug screening, and clinical translation. Obtaining NK cells with hypoxia tolerance has significant scientific and clinical translational implications.

[0004] Currently, there are methods to enhance NK cell function under hypoxic conditions by culturing NK cells under normoxic conditions and then transferring them to hypoxic conditions for further culture. However, these methods offer limited improvement in NK cell cytotoxicity and often involve long culture cycles, complex procedures, and poor reproducibility, hindering their application in research or clinical settings. Furthermore, existing technologies disclose gene editing of NK cells to improve their hypoxia tolerance, but this alters the NK cell genome, increasing application safety risks. Therefore, a novel strategy is urgently needed to address the functional suppression of NK cells under hypoxic conditions. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for culturing NK cells that still exhibit ideal cytotoxic activity under hypoxic conditions.

[0006] This invention provides a method for pre-culturing NK cells, comprising the step of pre-treating NK cells with an inducer; the active ingredient in the inducer includes a MAPK inhibitor; the MAPK inhibitor includes a p38 MAPK inhibitor and / or a JNK inhibitor.

[0007] This invention provides a method for pretreating NK cells. By pretreating NK cells solely with a specific MAPK inhibitor, the metabolic adaptation program of NK cells can be initiated in advance. This allows NK cells to enhance the expression of activating markers and reduce the expression of inhibitory markers after entering the hypoxic tumor microenvironment, thereby reducing intracellular reactive oxygen species levels and restoring mitochondrial function. This enables NK cells to acquire tolerance to the hypoxic tumor microenvironment and significantly improves their ability to kill tumor cells in a hypoxic environment (oxygen concentration of 1-5%) without genetic modification of NK cells. This provides a safe, simple, and easily clinically applicable synergistic strategy for NK cell immunotherapy.

[0008] In the NK cell pre-culture method of the present invention, Preferably, the MAPK inhibitor is a p38 MAPK inhibitor and a JNK inhibitor.

[0009] This invention has found that hypoxia can cause overactivation of p38 and JNK in the MAPK pathway, thereby triggering cellular oxidative stress. Therefore, inhibiting p38 and JNK during NK cell culture can enhance the killing function of NK cells under hypoxia.

[0010] The ERK pathway in MAPK inhibitors can promote cell proliferation, so it should have a positive effect on NK function under hypoxia. Therefore, ERK inhibitors not only cannot alleviate the functional loss of NK under hypoxia, but also have an inhibitory effect on NK under hypoxia.

[0011] In the NK cell pre-culture method of the present invention, the MAPK inhibitor is sesamin.

[0012] In the NK cell pre-culture method of the present invention, the pretreatment is carried out under normoxic conditions, wherein the oxygen concentration under normoxic conditions is 18%-22% (preferably 21%).

[0013] Preferably, in the pre-culture method of NK cells of the present invention, before the pretreatment, the NK cells are cultured to the logarithmic growth phase under normoxic conditions with an oxygen concentration of 18%-22% (preferably 21%).

[0014] In the pre-culture method of NK cells of the present invention, the concentration of the inducer during the pretreatment is 5 μM-30 μM, preferably 5 μM.

[0015] In the NK cell pre-culture method of the present invention, the pretreatment time is 24-72 hours, preferably 48 hours.

[0016] In this invention, NK cell culture can be carried out using NK cell culture media known in the art and conventional culture temperature (e.g., 37°C).

[0017] In the pre-culture method of NK cells of the present invention, the NK cells are selected from the NK92 cell line and / or iNK cells differentiated from induced pluripotent stem cells (iPSCs).

[0018] In this invention, the source of NK cells is not limited; they can be purchased or prepared using methods known in the art.

[0019] The present invention also provides a metabolically reconstructed NK cell, which is prepared by the above method; preferably, compared with untreated NK cells, the metabolically reconstructed NK cell has a higher level of mitochondrial oxidative phosphorylation under hypoxic conditions.

[0020] The NK cells of this invention exhibit ideal tumor-killing effects under hypoxic conditions.

[0021] NK cells treated with this invention exhibit the following characteristics in a hypoxic (1% O2) environment: ROS is reduced, cytotoxicity is enhanced, and the tumor cell killing ability is significantly improved (e.g., A549, H1299, and PC-9).

[0022] Without departing from the technical concept of this invention, the MAPK inhibitors described in this invention are not limited to sesamolin, but also include, but are not limited to, inhibitors of the JNK or p38 MAPK pathways, and their specific chemical structures and sources do not constitute a limitation of this invention.

[0023] The present invention also provides the application of the above-mentioned metabolically remodeled NK cells in the preparation of anti-solid tumor drugs.

[0024] In the application of this invention, the solid tumor is selected from respiratory system tumors, breast tumors, digestive system tumors, or reproductive system tumors; preferably, the solid tumor is a lung tumor.

[0025] In the application of this invention, the lung tumor is non-small cell lung cancer.

[0026] The NK cells pre-cultured according to this invention can be used for tumor immunotherapy.

[0027] The present invention also provides an antitumor drug comprising the above-mentioned metabolically remodeled NK cells, preferably wherein the tumor is non-small cell lung cancer.

[0028] Preferably, the drug of the present invention is an immune cell preparation.

[0029] The medicament of the present invention may also include pharmaceutically acceptable excipients. Its formulation is not limited, but is preferably an injectable form.

[0030] The drug of the present invention can be prepared using conventional methods and may contain a therapeutically effective amount of a pharmacologically active ingredient or may also contain one or more pharmaceutically acceptable carriers.

[0031] The unit content of the active ingredient contained in a single dose of the drug of the present invention does not necessarily need to constitute an effective amount, because the necessary effective amount can be achieved by administering multiple dose units.

[0032] The medicament of the present invention may also include other active ingredients with similar or different functions. The effective dosage of each active ingredient may vary depending on the ingredients used, the route of administration, and the severity of the condition being treated. Preferably, the total proportion of the various active ingredients administered in the combined formulation may be varied, for example, according to the needs of individual patients with different requirements.

[0033] The beneficial effects of this invention are at least as follows: 1. The method of this invention reveals for the first time that inhibiting the metabolic remodeling mediated by the p38 / JNK pathway can induce "hypoxia tolerance" in NK cells, thereby improving the metabolic stability, mitochondrial activity, and cytotoxic particle generation of NK cells.

[0034] 2. The method of this invention clarifies the differentiated regulatory strategy for different MAPK subtypes (inhibiting p38 / JNK and retaining ERK), which enhances the killing function without affecting the cell's proliferation potential.

[0035] 3. The method of the present invention has high safety. Functional enhancement can be achieved by short-term treatment with small molecule inducers without the need for gene modification, which significantly reduces the risk and cost of clinical translation and is suitable for ready-to-use NK cell products.

[0036] 3. The method of this invention is simple and widely applicable. Experiments have shown that this method has a significant synergistic effect on NK cells from various sources (such as NK92 and iNK) and various lung cancer cell lines, and has broad prospects for clinical application. Attached Figure Description

[0037] Figure 1 The results of mitochondrial ROS level detection in Example 2 are shown.

[0038] Figure 2 The results of the in vitro killing activity of NK92 cells against non-small cell lung cancer cells A549, H1299 and PC-9 in Example 2 are shown.

[0039] Figure 3 The results of in vitro killing activity of iNK cells against non-small cell lung cancer cells A549, H1299 and PC-9 in Example 2 are shown.

[0040] Figure 4This is a map showing the expression levels of NK92 cell function-related markers in the AH group and the hypoxia-treated MAPKi group in Example 2.

[0041] Figure 5 This chart shows the statistical results of expression levels of NK92 cell function-related markers in the AH group and the hypoxia-treated MAPKi group in Example 2. Blue bars represent the WT group, purple bars represent the AH group, and green bars represent the MAPKi+AH group.

[0042] Figure 6 This is a map showing the expression levels of iNK cell function-related markers in the AH group and the hypoxia-treated MAPKi group in Example 2.

[0043] Figure 7 The results show the statistical results of the expression levels of iNK cell function-related markers in the AH group and the hypoxia-treated MAPKi group in Example 2.

[0044] Figure 8 The results of granzyme B secretion detection in NK92 cells of the AH group and the hypoxia-treated MAPKi group in Example 2 are shown.

[0045] Figure 9 The results of granzyme B secretion detection in iNK cells of the AH group and the hypoxia-treated MAPKi group in Example 2 are shown.

[0046] Figure 10 The results of mitochondrial ROS detection in NK92 cells of the AH group and the hypoxia-treated MAPKi group in Example 2 are shown.

[0047] Figure 11 The results of Seahorse assays are for NK92 cells in the MAPKi group subjected to hypoxia treatment in Example 2.

[0048] Figure 12 The results of in vivo antitumor imaging of NK92 cells in the MAPKi group in Experiment Example 2 are shown.

[0049] Figure 13 The results show the changes in tumor volume after treatment with NK92 cells in the MAPKi group in Experiment Example 2. Detailed Implementation

[0050] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0052] The main experimental materials and methods involved in the specific embodiments of this invention are as follows: 1. MAPK inhibitor (MAPKi): Sesamolin (HY-N0809).

[0053] Sesamolin inhibits the MAPK cascade reaction by inhibiting JNK, p38 MAPKs and caspase-3 phosphorylation, but has no inhibitory effect on ERK.

[0054] 2. Cell lines A549, PC-9, and 293T cell lines were purchased from the American Type Culture Collection (ATCC), and NK92 cells were purchased from Procell Life Science & Technology.

[0055] The iNK cells described in this invention are natural killer cells derived from induced pluripotent stem cells (iPSC-derived NK cells, abbreviated as iNK). These iNK cells are obtained by in vitro directed differentiation of human induced pluripotent stem cells (iPSCs) using methods known in the art.

[0056] A549 and PC-9 cells were cultured in RPMI 1640 medium: RPMI 1640 (Thermo Fisher Scientific, catalog number 11875119) + 10% fetal bovine serum (ExCell, catalog number FSP500) + penicillin and streptomycin (Thermo Fisher Scientific, catalog number 15140122).

[0057] 293T cells were cultured in DMEM medium (DMEM; Thermo Fisher Scientific, catalog number 11995-065) + 10% fetal bovine serum (ExCell, catalog number FSP500) + penicillin and streptomycin (Thermo Fisher Scientific, catalog number 15140122). 293T cells were used to culture stable transgenic strains of A549-td-tomato-luc, H1299-td-tomato-luc, and PC-9-td-tomato-luc viruses.

[0058] NK92 cells were cultured in NK92 Cell Complete Medium (Procell Life Science & Technology, catalog number CM-0530).

[0059] The culture medium for iNK cells is an NK cell maintenance medium, which includes basal culture medium, serum or serum substitutes, glutamine or substitutes thereof, and may be supplemented as needed with cytokines that promote NK cell survival, proliferation and functional maintenance. Preferably, the culture medium contains interleukin-2 (IL-2), and may further contain IL-15, IL-7 or a combination thereof.

[0060] All cells were cultured in a humidified cell culture incubator at 37°C and 5% CO2, and mycoplasma testing was performed periodically.

[0061] 3. iNK cell differentiation In this embodiment of the invention, iPSCs are induced to differentiate in vitro using a stem cell differentiation kit. Through stages such as mesoderm induction, hematopoietic progenitor cell induction, and NK cell lineage commitment, they gradually differentiate into iNK cells with NK cell phenotypes and functions. During the differentiation process, the culture medium is replaced at stages and corresponding cytokines are supplemented to promote the generation and maturation of NK cells.

[0062] 4. Mice: Male and female M-NSG mice aged 6-8 weeks (catalog number NM-NSG-001) were purchased from Southern Model Biotechnology Co., Ltd. All male and female mice used in the experiment were bred and maintained at the Guangzhou National Laboratory, with a balanced sex ratio throughout the experiment. The age range of the mice throughout the experiment was 8-18 weeks. Animals were housed under specific pathogen (SPF) conditions, and were housed together with control group animals. At the end of the experiment, mice were euthanized by cervical dislocation after isoflurane anesthesia.

[0063] 5. Construction of the in vivo xenograft model (Cell Line-Derived Xenograft Mode): A549-Luc cells (A549 cells with bioluminescent tags, 5 × 10⁻⁶) 6Mice were cultured at 37 °C and 5% CO2, and then collected and resuspended in 100 μL of DPBS (Dulbecco's PBS, Thermo Fisher, #14190250) for subcutaneous tumor inoculation. Tumor burden in mice was assessed using in vivo bioluminescence imaging (BLI), and mice were divided into balanced groups based on BLI intensity. Seven days after tumor inoculation, mice were injected via tail vein with 200 μL of MAPKi group NK92 cells (1 × 10⁻⁶ cells) resuspended in DPBS. 7 Mice were injected with IL-2 every three days for three consecutive days to maintain NK92 cell activity. Simultaneously, to maintain NK92 cell activity in mice, 10,000 IU of IL-2 was injected every 2-3 days (the specific activity of the IL-2 used in this experiment was 20,000 IU / μg, and 1 IU is equivalent to 0.05 ng IL-2. Based on this, each injection of 10,000 IU IL-2 corresponds to approximately 0.5 μg of protein), for 21 days. Tumor burden was measured using digital calipers, and BLI imaging was performed on representative animals at key stages of tumor growth using the XenogenIVIS imaging system. Mouse weight and tumor size were recorded and measured every two days. Tumor volume was measured periodically using digital calipers and calculated using the following formula (unit: mm). 3 ): V = L × (W) 2 × 0.5. Where L is the longest diameter of the tumor, and W is the diameter perpendicular to L. Euthanasia was performed when the experiment ended (the tumor reached its maximum permissible volume or the mouse lost its ability to move).

[0064] 6. Functional testing (degranulation and cytokine production) Effector cells (NK92 or iNK cells) were co-cultured with A549 tumor cells at an E:T ratio of 2:1. After 4 hours of co-culture, the supernatant NK cells were collected and labeled with the following antibodies: • PE-tagged anti-CD107a (1186062, clone H4A3, BioLegend); • APC-tagged anti-FasL (2922541, clone NOK-1, BioLegend); • PE-tagged anti-CD69 (3083391, clone W19310K, BioLegend). • PE-tagged anti-NKG2D (492961, clone 1D11, BioLegend). • APC-tagged anti-NKG2A (2888862, clone S19004C, BioLegend); • APC-tagged anti-NKp46 (2888734, clone 9E2, BioLegend); • PerCP / Cyanine5.5-tagged anti-T-bet (1595593, clone 4B10, BioLegend); • Brilliant Violet 421-tagged anti-TRAIL (3683318, clone RIK-2, BioLegend).

[0065] After incubation, cells were stained for viability using Live / Dead Fixable Aqua staining reagent (Thermo Fisher, L-34966) and fixed with 4% paraformaldehyde. Membrane permeation was then performed using Triton X-100, followed by intracellular staining with BV650-labeled anti-T-Bet antibody (RRID: AB_2563608, clone 4S.B3, BioLegend). Cell expression was analyzed using an Agilent NovoCyte™ flow cytometer.

[0066] 7. ROS detection To detect reactive oxygen species (ROS) levels, 500,000 purified NK cells were collected for each condition and stained with 5 μM MitoSOX Red Mitochondrial Superoxide Indicator (MCE, catalog no. HY-D1055). After staining, the cells were stored on ice and immediately analyzed using Agilent NovoCyte. TM The cells were analyzed using a flow cytometer.

[0067] 8. IncuCyte cytotoxicity assay A549, H1299, and PC-9 tumor cells stably expressing td-tomato and luciferase (A549-td-tomato-luc, H1299-td-tomato-luc, and PC-9-td-tomato-luc) were seeded in 96-well plates at 8000 cells per well and allowed to adhere overnight. The following day, effector cells NK92 or iNK cells (32000 cells / well) were added to each well, with an effector-to-target cell ratio of 4:1 (E:T = 4:1). The plates were placed in an IncuCyte live-cell imaging system, and fluorescence images of the three groups of tumor cells were acquired every 45 minutes. All experiments were independently repeated at least three times. The cytotoxicity of tumor cells was assessed by quantifying the red cell area (representing td-tomato signal).

[0068] 9. ELISA test (Granzyme B) NK cells and A549 cells under different treatment conditions were co-cultured at an E:T ratio of 4:1 for 4 h. The culture supernatant was collected, and cell debris was removed by centrifugation. Following the Granzyme B ELISA kit instructions, standards and samples were added to antibody-coated ELISA plates and incubated. After washing, enzyme-labeled secondary antibody was added, and incubation continued. After the colorimetric reaction was terminated, the absorbance was measured at 450 nm. The concentration of Granzyme B in the sample was calculated based on the standard curve.

[0069] 10. Seahorse detection (OCR) NK cells under different treatment conditions were seeded into Seahorse-specific assay plates and equilibrated according to the instrument's instructions. Oxygen consumption rate (OCR) was then measured using a cellular energy metabolism analysis kit. Oligomycin, FCCP, and Rotenone / Antimycin A were added sequentially during the assay to assess basal respiration, maximal respiration, and mitochondrial respiratory function parameters. Data were automatically calculated and output by the Seahorse analysis software.

[0070] 11. Preparation of hypoxic cells NK cells were subjected to hypoxia treatment using the Huayi Ningchuang Cell+100 intelligent cell preparation system, with an oxygen concentration of 1% (upper limit 3%, lower limit 0%) and a carbon dioxide concentration of 5%. The system automatically monitored and maintained a stable gas environment throughout the culture process to ensure consistent and reliable hypoxia treatment conditions.

[0071] Example 1 This embodiment provides a method for inducing NK cell tolerance to hypoxia in vitro, as detailed below: NK92 cells or fully differentiated iNK cells were cultured under normoxic conditions (21% O2) until the logarithmic growth phase. NK92 cells were cultured in NK92 Cell Complete Medium at 37°C, with half-changes every 48 hours. iNK cells were cultured in NK Cell Complete Medium at 37°C, with half-changes every 72 hours. Subsequently, NK cells were treated with 5 μM MAPKi for 48 hours (MAPKi group).

[0072] This embodiment further provides a method for detecting the performance of NK cells cultured by the above method under acute hypoxic conditions, specifically as follows: NK cells pretreated with the obtained MAPK inhibitor and NK cells not treated with the inhibitor were stimulated in 1% O2 for 36 hours to simulate the acute hypoxic inhibition faced by NK cells during their entry into the tumor microenvironment from the peripheral circulation, thus obtaining the hypoxic-treated MAPKi group (MAPKi+AH) and the AH group, respectively. NK cells cultured under normoxic conditions without inhibitor treatment were also used as the WT control group.

[0073] Example 2 To determine the optimal effective concentration of the MAPK inhibitor (MAPKi), this example first pretreated NK92 cells with different concentrations of MAPKi (5-30 μM) for 48 h, following the method described in Example 1. Cells were then cultured at 1% O2 for 36 h to simulate a hypoxic microenvironment, and mitochondrial ROS levels were detected by flow cytometry. The results are as follows: Figure 1 As shown, compared with the hypoxia control group (AH) without the use of inhibitors, all concentrations of MAPKi can reduce hypoxia-induced mitochondrial ROS levels. The lowest effective concentration of 5 μM with significant inhibitory effect was preferred for subsequent experiments.

[0074] This embodiment further examines the function of NK92 or iNK cells after hypoxia-tolerant induction culture and acute hypoxia treatment according to the method of Example 1, as follows: 1. To evaluate the effect of MAPK inhibitor pretreatment on NK cell effector function, the in vitro killing activity (E:T = 4:1) of MAPK inhibitor-pretreated NK92 cells and iNK cells against non-small cell lung cancer cells A549, H1299, and PC-9 was assessed using a live-cell real-time imaging system after 36 hours of treatment with 1% O2. Results for NK92 cells are shown below. Figure 2 As shown, the results of iNK cells are as follows: Figure 3As shown, the specific cytotoxicity of MAPKi group NK92 cells and iNK cells treated with 1% O2 for 36 hours was comparable to that of wild-type control group cultured under normoxic conditions (WT), but significantly higher than that of acute hypoxia control group (AH) treated directly under 1% O2 for 36 hours without MAPK inhibitor pretreatment. In multiple independent experiments, the killing efficiency of MAPKi group under hypoxia was approximately 3-5 times higher than that of AH group, indicating that hypoxia adaptation can significantly enhance the ability of NK cells to resist acute hypoxia inhibition.

[0075] 2. Functional markers were detected in NK92 cells and iNK cells from the AH group and MAPKi group after 36 hours of 1% oxygen treatment. Results for NK92 cells are shown below. Figure 4 and Figure 5 The results showed that, compared with the untreated group, the expression levels of activating markers (CD107a, FasL, NKG2D, NKp46, CD69, T-bet, TRAIL) were increased by approximately 2 to 3 times in the MAPK inhibitor pretreatment group, while the expression level of the inhibitory marker (NKG2A) was decreased by approximately 3 times. The results for iNK cells are shown below. Figure 6 and Figure 7 The study showed that, compared with the group without inhibitor pretreatment, the expression levels of activating markers (CD107a, FasL, NKG2D, NKp46, CD69) were significantly increased in the MAPK inhibitor pretreatment group, while the expression level of the inhibitory marker (NKG2A) was slightly decreased.

[0076] 3. Granulase B secretion was measured in NK92 cells from the AH and MAPKi groups after treatment with 1% O2 for 36 hours to evaluate the cytotoxic function of NK cells. The results are shown below. Figure 8 (NK92 cells) and Figure 9 (iNK cells). The results showed that, compared with the AH group without inhibitor pretreatment, the granzyme B secretion level in the MAPK inhibitor pretreatment group was significantly increased, suggesting that its cytotoxic function was restored or enhanced.

[0077] 4. Mitochondrial ROS levels were measured in NK92 cells from the AH and MAPKi groups after 36 hours of 1% oxygen treatment. Results are shown below. Figure 10 The results showed that, compared with the group without inhibitor pretreatment, the mitochondrial ROS in the MAPK inhibitor pretreatment group was reduced by about 5 times, indicating the restoration of mitochondrial function.

[0078] 5. Seahorse assays were performed on NK92 cells from the AH and MAPKi groups after 36 hours of 1% oxygen treatment to assess mitochondrial functional stability and oxidative phosphorylation levels. Results are shown below. Figure 11 The results showed that, compared with the untreated group, the MAPK inhibitor pretreatment group had significantly increased oxygen consumption rate (OCR) and related oxidative phosphorylation parameters, indicating a significant enhancement of mitochondrial function.

[0079] This embodiment further investigated the antitumor effect of NK92 cells cultured to tolerate hypoxia according to the method in Example 1 through in vivo experiments. In vivo imaging was used to monitor tumor growth in NSG mice. The specific method is described above, and the main procedure was as follows: 5 × 10⁶ A549-Luc cells were subcutaneously inoculated into each mouse. 6 Seven days later, NK92 cells were administered to either the untreated group (WT) or the MAPKi pretreated group, with each injection containing 1 × 10⁶ cells. 7 One animal per day, injected every three days, for a total of three injections. Representative animal imaging results during key tumor growth stages are shown below. Figure 12 The results of tumor size statistics are shown in [the original text]. Figure 13 (n=7) showed that at the end of the observation period, the average tumor volume of the MAPKi group mice was about one-third to one-quarter of that of the WT group, indicating that MAPKi pretreatment can effectively reduce the inhibitory effect of hypoxia in the tumor microenvironment on the killing function of NK92 cells.

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for pre-culturing NK cells, characterized in that, The method includes a step of pretreating NK cells with an inducer; the active ingredient in the inducer contains a MAPK inhibitor; the MAPK inhibitor includes a p38 MAPK inhibitor and / or a JNK inhibitor.

2. The method for pre-culturing NK cells according to claim 1, characterized in that, The MAPK inhibitors are p38MAPK inhibitors and JNK inhibitors.

3. The method for pre-culturing NK cells according to claim 2, characterized in that, The MAPK inhibitor is sesamolin.

4. The method according to any one of claims 1-3, characterized in that, The pretreatment is carried out under normal oxygen conditions, where the oxygen concentration is 18%-22%.

5. The method for pre-culturing NK cells according to any one of claims 1-4, characterized in that, During the pretreatment, the concentration of the inducing agent is 5-30 μM, preferably 5 μM; the pretreatment time is 24-72 hours, preferably 48 hours.

6. The method for pre-culturing NK cells according to any one of claims 1-5, characterized in that, The NK cells are selected from the NK92 cell line and / or iNK cells differentiated from induced pluripotent stem cells (iPSCs).

7. A metabolically remodeled NK cell, characterized in that, The metabolically remodeled NK cells are prepared by the method described in any one of claims 1-6; preferably, compared with untreated NK cells, the metabolically remodeled NK cells have a higher level of mitochondrial oxidative phosphorylation under hypoxic conditions.

8. The use of the metabolically remodeled NK cells according to claim 7 in the preparation of anti-solid tumor drugs.

9. The application according to claim 8, characterized in that, The solid tumor is selected from respiratory system tumors, breast tumors, digestive system tumors, or reproductive system tumors; preferably, the solid tumor is a lung tumor.

10. The application according to claim 9, characterized in that, The lung tumor in question is non-small cell lung cancer.

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