High affinity anti-tumor nk cell and preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to a high-affinity anti-tumor NK cell, its preparation method, and its application. This invention designs NK92 cells transfected with a haPD1 high-affinity chimeric conversion receptor, wherein the high-affinity chimeric conversion receptor includes the extracellular segment of haPD-1, the transmembrane segment of CD28, the intracellular segment of DAP10, and the intracellular segment of CD3ζ. This invention prepares haChR3-NK92 cells through the construction of a recombinant lentiviral vector, lentiviral packaging, and lentiviral transfection of NK92 cells. In this invention, haPD-1 serves as the extracellular recognition domain of the CAR structure, specifically binding to PD-L1 on tumor cells to achieve a stronger tumor-killing effect. The co-stimulatory molecule CD28 serves as the transmembrane region to transmit extracellular information into the cell, integrating the adaptor protein DAP10 of the NK cell surface activator receptor NKG2D into the cell, and further embedding the intracellular segment CD3ζ commonly used in CAR design to jointly promote NK cell activation. The preparation method of this invention can successfully construct haChR3-NK92 cells, achieve the expression of the target plasmid, and be applied in anti-tumor drug research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-affinity anti-tumor NK cell, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy is the fourth major cancer treatment after surgery, radiotherapy, and chemotherapy. Compared with other forms of tumor immunotherapy, adoptive cell therapy has more advantages and better efficacy. Among these, chimeric antigen receptor T-cell therapy (CAR-T) has developed most rapidly. CAR-T is effective in hematologic malignancies; however, its effectiveness in solid tumors is inferior, and it has potential side effects such as cytokine storms and neurotoxicity. In addition, because allogeneic T cells are prone to causing graft-versus-host disease (GVHD), patients generally can only use autologous T cells modified into CARs before reinfusion, making the clinical application of CAR-T complicated and expensive. Since natural killer cells (NK cells) in the innate immune system are natural anti-tumor cells, some researchers have begun to explore modifying NK cells using CAR structures (CAR-NK).

[0003] Compared to CAR-T therapy, NK cells, as effector cells, possess many superior characteristics. First, unlike T cells, they do not exhibit graft-versus-host disease, making them a promising candidate for universal cell therapy. Second, NK cells are not restricted by the major histocompatibility complex (MHC), resulting in stronger cytotoxicity. Third, the low-affinity CD16 molecule on the NK cell surface can mediate ADCC with the IgG antibody complex on the target cell surface. NK cells can also mediate apoptosis and release cytokines via the Fas / FasL pathway. Considering these advantages, chimeric antigen receptor NK (CAR-NK) cells modified from NK cells hold greater promise for future applications.

[0004] In the prior art, Chinese patent CN201911317011.1 discloses an anti-tumor NK cell, its preparation method, and its application. The chimeric antigen receptor includes an extracellular domain fragment of PD-1, an F2A peptide fragment, an extracellular region of the CD8a signal peptide, an extracellular variable region of a single-chain antibody that recognizes and binds to HER2 protein, a CD8α linker region, a transmembrane and intracellular co-stimulatory region of CD28, and a CD3ζ fragment for intracellular signal transmission. This traditional scFv cannot function properly because it exists extracellularly and cannot fold properly; therefore, enhanced affinity is needed to avoid this problem. Furthermore, domestic and international literature has confirmed… Combinations of CD8 transmembrane domain, 41BB intracellular domain, CD28 transmembrane domain, and DAP10 intracellular domain can enhance anti-tumor effects and improve in vivo survival time. However, it is necessary to select recombinant fragments with stronger combined effects and a stronger ability to differentiate into central memory phenotypes. In the process of NK cell modification research, it was found that there are many killer inhibitory receptors (KIRs) on the surface of NK cells. These receptors may be induced to express in the tumor microenvironment, thereby inhibiting the killing function of immune cells and causing immune cell dysfunction. These inhibitory receptors are called immunosuppressive checkpoints. For example, programmed death 1 (PD-1) can inhibit T cell activation and proliferation by binding to its ligand PD-L1, downregulate the secretion of T cell immunostimulatory cytokines, thereby inhibiting T cell immune response and promoting T cell apoptosis. Tumor cells can highly express PD-L1. After binding to PD-1 on the surface of T cells, it inhibits the killing activity of T cells and achieves immune escape. Summary of the Invention

[0005] The purpose of this invention is to use synthetic biology technology to design a novel high-affinity chimeric conversion receptor targeting PD-L1, transfect and modify NK92 cells, and use the haPD-1 extracellular domain to target and recognize and bind to PD-L1 ligands on the tumor surface, and transmit, activate and release activating signals through the CD28 transmembrane domain, DAP10 intracellular domain and CD3ζ intracellular domain, thereby enhancing the anti-tumor immune activity of NK92 cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A high-affinity anti-tumor NK cell is NK92 cells transfected with a haPD-1 high-affinity chimeric conversion receptor; the haPD-1 high-affinity chimeric conversion receptor includes the haPD-1 extracellular segment, CD28 transmembrane segment, DAP10 intracellular segment, and CD3ζ intracellular segment.

[0008] Specifically, the nucleotide sequence of the extracellular segment of haPD-1 is shown in SEQ ID No. 1.

[0009] Specifically, the nucleotide sequence of the CD28 transmembrane segment is shown in SEQ ID No. 2.

[0010] Specifically, the nucleotide sequence of the intracellular segment of DAP10 is shown in SEQ ID No. 3.

[0011] Specifically, the nucleotide sequence of the CD3ζ intracellular segment is shown in SEQ ID No. 4.

[0012] A method for preparing high-affinity anti-tumor NK cells includes the following steps:

[0013] Step 1: Construction of the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH;

[0014] The extracellular segment of haPD-1, the transmembrane segment of CD28, the intracellular segment of DAP10, and the intracellular segment of CD3ζ were tandemly synthesized using a whole-genome synthesis method to obtain the haPD1-CD28-DAP10-CD3ζ fragment. The haPD1-CD28-DAP10-CD3ζ fragment was subcloned into the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector to obtain the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH.

[0015] Step 2: Lentiviral packaging process;

[0016] The target plasmid haChR3 was extracted from the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH obtained in step one; then, the target plasmid haChR3, lentiviral packaging helper plasmid psPAX2, and lentiviral packaging helper plasmid PMD2.G were mixed in a ratio of 4:3:1 and transfected into 293T cells to produce a virus carrying the target plasmid; the viral supernatant harvested after transfection was concentrated using the PEG concentration method.

[0017] Step 3: Lentiviral transfection of NK92 cells;

[0018] By infecting NK92 cells with a concentrated viral suspension containing the target plasmid, a stable NK92 cell line expressing haChR3-pCDH can be obtained.

[0019] Specifically, the nucleotide sequence of the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH is shown in SEQ ID No. 8.

[0020] Specifically, the nucleotide sequence of the haPD1-CD28-DAP10-CD3ζ fragment is shown in SEQ ID No. 6; its amino acid sequence is shown in SEQ ID No. 5.

[0021] A high-affinity anti-tumor NK cell obtained by the above preparation method is applied to the research of anti-tumor cell drugs; in particular, it can kill SGC-7901 gastric cancer cells in vitro.

[0022] The beneficial effects of this invention are as follows:

[0023] The high-affinity anti-tumor NK cells obtained in this invention, namely haChR3-NK92 cells, utilize the extracellular domain fragment (24-170 aa) of haPD-1 (a high-affinity chimeric conversion receptor) to recognize antigens, the co-stimulatory region of the CD28 transmembrane segment (153-179 aa) and the DAP10 intracellular segment (70-93 aa), and the CD3ζ (52-164 aa) to transmit intracellular signals, thereby enhancing the anti-tumor immune activity of NK92 cells. Notably, haPD-1, as a mutant of wild-type PD-1, exhibits higher affinity for PD-L1 than wild-type PD-1. This invention facilitates better identification of targeted tumor cells. In particular, compared to traditional scFv, the high-affinity haPD-1 used in this invention has stronger affinity. Traditional scFv cannot function properly because it exists outside the cell and cannot fold properly, while the use of high-affinity haPD-1 can avoid this problem. Furthermore, the combined use of the CD28 transmembrane segment and the DAP10 intracellular segment helps to increase the secretion of cytokines, thereby enhancing the anti-tumor activity of CAR-NK cells and prolonging their existence time in vivo. CD3ζ transmits signals to the intracellular environment to exert anti-cancer effects, endowing CAR-NK cells with CAR-dependent killing ability.

[0024] The preparation method of this invention can successfully construct high-affinity anti-tumor NK cells, namely haChR3-NK92 cells. At the mRNA level, haChR3-NK92 cells show normal haPD-1 expression; at the protein level, haPD-1 expression is also normal. In vitro toxicity tests show that haChR3-NK92 cells can kill cancer cells in vitro. This preparation method has a short process flow, is simple to operate, easy to implement, exhibits high expression stability, and is easily industrialized.

[0025] The high-affinity anti-tumor NK cells in this invention, namely haChR3-NK92 cells, can be used to prepare anti-tumor drugs. They can target tumor cells, enhance cytotoxicity, have low immunogenicity, are easily activated, and can be reinfused from other cells. The tumor killing effect of haChR3-NK92 cells is significantly improved, and the resulting anti-tumor drugs have good efficacy. Attached Figure Description

[0026] Figure 1 A schematic diagram of obtaining the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH by subcloning the haPD1-CD28-DAP10-CD3ζ fragment in the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector;

[0027] Figure 2 Fluorescence image (4X) of NK92 cells transfected with lentivirus 72 h later;

[0028] Figure 3 Fluorescence image (4X) of NK92 cells after lentivirus transfection with puromycin enrichment for 14 days.

[0029] Figure 4 To detect Flag and GFP expression in NK92 and haChR3-NK92 cells by flow cytometry;

[0030] Figure 5 To detect the expression of haPD-1 and GFP in NK92 and haChR3-NK92 cells by flow cytometry;

[0031] Figure 6 This is a schematic diagram showing the proliferation levels of NK92 and haChR3-NK92 cells.

[0032] Figure 7 The image shows the killing effect of NK92 and haChR3-NK92 cells on SCG-7901 tumor cells. Detailed Implementation

[0033] This invention utilizes the high expression of PD-L1 in tumor cells to find a high-affinity PD-1 (haPD-1) as the extracellular recognition domain of the CAR structure, which can replace the intracellular inhibitory domain with an activation domain to transmit positive activation signals inward; haPD-1 specifically binds to PD-L1 on tumor cells to achieve a stronger tumor killing effect.

[0034] In addition to the antigen-binding region, artificially constructing an NK cell activation pathway through gene modification requires the fusion of a transmembrane region and an intracellular signaling region. This invention uses the co-stimulatory molecule CD28 as the transmembrane region to transmit extracellular information to the intracellular region. The structure of the intracellular signaling region is crucial. Studies have shown that CARs designed using NK cell-related activation elements can better activate NK cells in vivo. Therefore, this invention integrates the adaptor protein DAP10 of the NK cell surface activator receptor NKG2D into the cell, and also embeds the intracellular segment CD3ζ commonly used in CAR design, to jointly promote NK cell activation.

[0035] In order to study anti-tumor cell drugs, this invention prepares high-affinity anti-tumor NK cells, namely haChR3-NK92 cells, through the following steps:

[0036] Step 1: Construction of the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH

[0037] The haPD1-CD28-DAP10-CD3ζ fragment (haChR3) was synthesized at Nanjing GenScript Biotech Co., Ltd. using a whole-genome synthesis method. The fragment contained a 435bp extracellular segment of haPD-1 (24-170 aa), an 81bp transmembrane segment of CD28 (153-179 aa), a 72bp intracellular segment of DAP10 (70-93 aa), and a 339bp intracellular segment of CD3ζ (52-164 aa). These fragments were then tandemly linked to obtain the haPD1-CD28-DAP10-CD3ζ fragment (haChR3). The haPD1-CD28-DAP10-CD3ζ fragment (haChR3) was subcloned into the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector. Figure 1 As shown, the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH (abbreviated as recombinant lentiviral vector haChR3-pCDH) was obtained, and the nucleotide sequence of haPD1-CD28-DAP10-CD3ζ-pCDH is shown in SEQ ID No. 8.

[0038] In the above content, the nucleotide sequence of the extracellular segment of haPD-1 is shown in SEQ ID No. 1, the nucleotide sequence of the transmembrane segment of CD28 is shown in SEQ ID No. 2, the nucleotide sequence of the intracellular segment of DAP10 is shown in SEQ ID No. 3, and the nucleotide sequence of the intracellular segment of CD3ζ is shown in SEQ ID No. 4; the amino acid sequence of the haPD1-CD28-DAP10-CD3ζ fragment (abbreviated as haChR3) is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6; the nucleotide sequence of pCDH-CMV-MCS-P2A-copGFP-T2A-Puro is shown in SEQ ID No. 7; and the nucleotide sequence of haPD1-CD28-DAP10-CD3ζ-pCDH is shown in SEQ ID No. 8.

[0039] Step 2: Lentiviral Packaging Process

[0040] (a) Extraction of haChR3 target plasmid

[0041] 1. Plasmid transformation

[0042] Remove *E. coli* TOP10 competent cells and add 1 μg of recombinant lentiviral vector haChR3-pCDH and lentiviral packaging helper plasmids psPAX2 (purchased from Addgene #12260) and PMD2.G (purchased from Addgene #12259) to each TOP10 competent cell. Mix well, incubate on ice for 30 min, heat shock in a 42℃ water bath for 90 s, and then incubate on ice for 2 min. Add 800 μL of antibiotic-free LB medium and incubate at 37℃ with shaking at 180 rpm for 1 h. Spread 100 μL of the bacterial culture onto LB solid medium (containing ampicillin (Amp) at a concentration of 50 μg / ml, added at a ratio of 1:1000) and incubate upside down at 37℃ for 12 h.

[0043] 2. Plasmid extraction

[0044] (1) Pick out single colonies from the above LB solid medium and inoculate them into 5 mL of fresh sterile LB liquid medium containing Amp (concentration of 50 μg / mL, inoculation ratio of 1:1000). Place them in a shaker at 37℃ and 220 rpm / min for 8 h.

[0045] (2) Take 200 μL of the above bacterial solution and inoculate it into 200 mL of fresh sterile LB medium containing Amp. Place it on a shaker and incubate at 37℃ and 220 rpm / min for 12 h until the OD600nm value reaches 1.5~2.0.

[0046] (3) Plasmids were extracted according to the OMEGA non-endotoxin plasmid large-scale extraction kit (purchased from OmegaBio-Tek, USA, product number D6926-03). The plasmid concentration and purity were measured using a Nanodrop micro spectrophotometer (purchased from Thermoelectric (Shanghai) Technology Instruments Co., Ltd., USA, model: NanoDrop2000), and then stored in a -20℃ freezer.

[0047] (ii) Lentiviral Packaging

[0048] First, culture 293T cells (purchased from ATCC, USA). When the cell density reaches 70%~80%, replace it with 15mL of 6% fresh DMEM medium (purchased from Hyclone, catalog number SH30022.01) containing 6% FBS (purchased from BI, catalog number 04-001-1ACS) and free of penicillin and streptomycin. Incubate in a cell culture incubator for 1 hour. Lentiviral packaging was performed using a calcium phosphate kit (purchased from Beyotime, catalog number C0508). The procedure was as follows: the target plasmid haChR3, helper plasmids pSPAX2 and PMD2.G were mixed in a ratio of 4:3:1 and added to the CaCl2 solution (provided in the kit). The mixture was then pipetted and mixed thoroughly. The mixture was then added dropwise to BBS solution, and the mixture was pipetted and mixed thoroughly to avoid the formation of white flocculent material. The mixture was incubated at room temperature for 20 min. The mixture was then added to 293T cells, gently shaken and mixed thoroughly. The cells were then incubated for another 12 h. The supernatant was discarded and replaced with 15 mL of fresh, preheated DMEM medium containing 10% FBS and 1% penicillin antibiotics. The viral supernatant was collected at 48 h, 72 h, and 96 h.

[0049] (iii) Lentiviral Concentration

[0050] Centrifuge the obtained viral supernatant at 3500g, 4℃ for 10 min, and filter through a 0.45μm sterile membrane to remove cell debris. Add fresh sterile PEG to the filtered viral supernatant and incubate at 4℃ for ≥24 h, inverting the container 1-2 times during this period. Centrifuge at 3000g, 4℃ for 30 min, discard the supernatant, resuspend the precipitate with an appropriate amount of PBS, aliquot, and store at -20℃ or -80℃, avoiding repeated freeze-thaw cycles.

[0051] Step 3: Lentiviral transfection of NK92 cells

[0052] 3.5 × 10⁵ NK92 cells / well were seeded into 24-well plates. Concentrated virus solution containing the target plasmid was added in a volume gradient. Polyglobulin (Yisheng Biotechnology, catalog number 40804ES76) was added to each well to a final concentration of 8 μg / mL. The plates were mixed and incubated at 37°C. After 12 hours, the viral supernatant was discarded, fresh culture medium was added, and the plates were incubated at 37°C for further culture. 72 hours after transfection, fluorescence expression was observed under a fluorescence microscope to detect the transfection effect. Figure 2 As shown, Figure 2 The magnification is 4×10, from Figure 2 As can be seen, the cells grow in clusters and are in good condition, with a small amount of cellular fluorescence observed. After selection with puromycin, the results were observed under a fluorescence microscope after 14 days, as shown... Figure 3 As shown, Figure 3 The magnification is 4×10, from Figure 3 As can be seen, the cells clustered together, were in good condition, and exhibited high fluorescence expression. Therefore, after successfully constructing haChR3-NK92 cells, fluorescence microscopy was used to detect the fluorescence expression of stably transfected NK92 cells. Five days later, the transfected NK92 cells were used for subsequent experiments.

[0053] Experiment 1

[0054] The expression of haPD-1, Flag, and GFP in NK92 cells and haChR3-NK92 cells was detected by flow cytometry, as follows:

[0055] Three × 10⁶ haChR3-NK92 cells and NK92 cells, enriched with puromycin for 14 days, were centrifuged at 300g for 5 min. The supernatant was discarded, and the cells were washed twice with PBS containing 1% FBS (hereinafter referred to as wash buffer). APC-labeled mouse anti-Flag monoclonal antibody (Biolegend, catalog number 637307) and APC-labeled mouse anti-human PD1 monoclonal antibody (Invitrogen, catalog number 17-9969-42) were added to a 100 μL system. The cells were incubated on ice in the dark for 30 min, and washed twice with wash buffer. The cells were resuspended in 400 μL of wash buffer and analyzed using a flow cytometer (BD FACSCantoII). The results are as follows: Figure 4 , Figure 5 As shown.

[0056] Depend on Figure 4 It can be seen that cells in the third quadrant are negative for both Flag and GFP. Because they contain the target gene, cells in the first quadrant show a Flag expression rate of 99.2% in haChR3-NK92. Figure 5It can be seen that PD-1 and GFP are negative in the third quadrant; due to the presence of the target gene, the expression rate of PD-1 in haChR3-NK92 cells in the first quadrant is 98.9%.

[0057] Experiment 2

[0058] The proliferation of haChR3-NK92 cells and NK92 cells was detected using CCK8 assay, as follows:

[0059] HaChR3-NK92 and NK92 cells were harvested at 1×10⁴ cells per well, centrifuged at 300 g for 5 min, and resuspended in 100 μL of culture medium per well. Four sub-wells were prepared for each cell type, and the cells were evenly seeded into three wells. At 24 h, 48 h, and 72 h, one well was removed, and 10 μL of CCK8 assay solution was added to each well. Simultaneously, 100 μL of culture medium was added to the other untested wells, with the assay solution being added progressively. The cells were then incubated at 37°C for 2–3 h, and the OD values ​​of each group were measured using a microplate reader. The CCK8 proliferation assay was used to detect whether there was a difference in the proliferation rate between haChR3-NK92 and NK92 cells, with three assay time points.

[0060] The results are as follows Figure 6 As shown, there was no significant difference in the proliferation rate of haChR3-NK92 and NK92 cells at the three detection time points of 24 h, 48 h, and 72 h, indicating that the modification of NK92 cells still maintains the original proliferation capacity of NK92 cells.

[0061] Experiment 3

[0062] In vitro cytotoxicity experiments were conducted using the constructed haChR3-NK92 cells, as follows:

[0063] 1. Cell preparation

[0064] SGC-7901 gastric cancer cells in the logarithmic growth phase after luciferase transfection were seeded into 96-well plates, ensuring 1×10⁴ cells per well. Three accessory wells were set up for each cell type. The plates were incubated overnight in a 5% CO₂ incubator at 37°C.

[0065] 2. Experimental Design

[0066] The experiment was set up in 4 groups: the target cell maximum lysis group, the target cell blank group, and the effector cell (NK92 cells, haChR3-NK92 cells) killing experimental group. Each group had three auxiliary wells with a total volume of 100 μL per well and an effector-to-target ratio of 5:1.

[0067] 3. Luciferase assay

[0068] This experiment used the ONE-Glo™ Luciferase Assay System Promega for detection. Effector cells and target cells were incubated together for 12 hours in a 5% CO2 incubator at 37°C. One hour before the predetermined detection time, the 96-well plate was removed from the cell culture incubator, and Lysis Solution (10×) was added to the control wells with the maximum enzyme activity of the sample, at a volume of 10% of the original culture medium. After adding Lysis Solution, the plates were repeatedly pipetted and mixed thoroughly before being returned to the incubator for further incubation. After the co-incubation time was reached, the detection solution was added, and the cells were detected using a microplate reader.

[0069] Cytotoxicity (%) = (RLU min – RLU sample) / (RLU min – RLU max) × 100. Results are as follows: Figure 7 As shown, at an effector-to-target ratio of 5:1, parental NK92 cells killed approximately 18% of SGC-7901 cells, while haChR3-NK92 cells killed approximately 89%, representing a 4-fold increase in killing power. This indicates that the modified haChR3-NK92 cells exhibited significantly increased cytotoxicity.

Claims

1. A high-affinity anti-tumor NK cell, characterized in that, It is NK92 cells transfected with a high-affinity chimeric conversion receptor for haPD-1; the high-affinity chimeric conversion receptor for haPD-1 includes an extracellular segment of haPD-1, a transmembrane segment of CD28, an intracellular segment of DAP10, and an intracellular segment of CD3ζ; the nucleotide sequence of the extracellular segment of haPD-1 is shown in SEQ ID No. 1, the nucleotide sequence of the transmembrane segment of CD28 is shown in SEQ ID No. 2, the nucleotide sequence of the intracellular segment of DAP10 is shown in SEQ ID No. 3, and the nucleotide sequence of the intracellular segment of CD3ζ is shown in SEQ ID No.

4.

2. A method for preparing high-affinity anti-tumor NK cells, characterized in that, Includes the following steps: Step 1: Construction of the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH; The extracellular segment of haPD-1, the transmembrane segment of CD28, the intracellular segment of DAP10, and the intracellular segment of CD3ζ were tandemly synthesized using a whole-genome synthesis method to obtain the haPD1-CD28-DAP10-CD3ζ fragment. The nucleotide sequence of the extracellular segment of haPD-1 is shown in SEQ ID No. 1, the nucleotide sequence of the transmembrane segment of CD28 is shown in SEQ ID No. 2, the nucleotide sequence of the intracellular segment of DAP10 is shown in SEQ ID No. 3, and the nucleotide sequence of the intracellular segment of CD3ζ is shown in SEQ ID No.

4. The haPD1-CD28-DAP10-CD3ζ fragment was subcloned into the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector to obtain the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH. Step 2: Lentiviral packaging process; The target plasmid was extracted from the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH obtained in step one; then the target plasmid, lentiviral packaging helper plasmid psPAX2, and lentiviral packaging helper plasmid PMD2.G were mixed in a ratio of 4:3:1 and transfected into 293T cells to produce a virus carrying the target plasmid; the viral supernatant harvested after transfection was concentrated using the PEG concentration method. Step 3: Lentiviral transfection of NK92 cells; The concentrated viral suspension containing the target plasmid was used to infect NK92 cells.

3. The method for preparing high-affinity anti-tumor NK cells according to claim 2, characterized in that, The nucleotide sequence of the recombinant lentiviral vector haPD1-CD28-DAP10-CD3ζ-pCDH is shown in SEQ ID No.

8.

4. The method for preparing high-affinity anti-tumor NK cells according to claim 2, characterized in that, The nucleotide sequence of the haPD1-CD28-DAP10-CD3ζ fragment is shown in SEQ ID No. 6; its amino acid sequence is shown in SEQ ID No.

5.

5. A high-affinity anti-tumor NK cell according to claim 1, or a high-affinity anti-tumor NK cell prepared according to claims 2-4, characterized in that, It is used in the research of anti-tumor cell drugs.

6. The high-affinity anti-tumor NK cell according to claim 5, characterized in that, It was used to kill SGC-7901 gastric cancer cells in vitro.

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