Dual-targeting enhanced anti-tumor nk cell and preparation method and application thereof

Abstract

The present application relates to a kind of dual-targeting enhanced anti-tumor NK cell and its preparation method, comprising: synthesis plasmid piece, the plasmid includes the gene fragment of the following amino acid expressed in series: PD-L1 nanobody, NKG2D extracellular segment, CD28 transmembrane segment and intracellular segment, costimulatory molecule 41BB, CD3 zeta, again the above-mentioned plasmid cloning construction is cloned to slow virus vector;By preparing slow virus to be infected NK92 cell, obtain the NK92 cell strain of stable expression above-mentioned plasmid gene.The present application is modified to NK92 cell, constructs a kind of multi-target, enhanced anti-tumor NK cell, the addition of PD-L1 nanobody strengthens the targeting of CAR-NK to tumor cell, NKG2D, CD28, 41BB, CD3 zeta reverses the negative effect of soluble MICA / B to NK cell while enhancing the activation of NK92 cell, obtain a new type of anti-tumor NK cell, to enhance its anti-tumor immune effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically relates to a dual-target enhanced anti-tumor NK cell, its preparation method, and its application. Background Technology

[0002] In recent years, tumor immunotherapy has shone brightly in cancer treatment due to its remarkable efficacy, especially CAR-T cells, which have demonstrated excellent targeting, killing power, and durability in the clinical treatment of hematologic malignancies. This has made it the most convincing breakthrough in the field of tumor immunotherapy, opening a new door of hope for conquering cancer. Despite continuous progress in CAR-T technology development, many challenges remain. One major obstacle is that while CAR-T cells possess a powerful ability to kill cancer cells, the excessive immune response they trigger can potentially threaten patients' lives. Furthermore, neither CAR-T nor TCR-T immune cell technologies have been effective in treating solid tumors to date, and treating solid tumors is precisely the main battleground in the fight against cancer. Therefore, overcoming the drawbacks of CAR-T technology and endowing immune cells with precise recognition and specific killing of solid tumor cells have become new breakthroughs and leaps urgently needed in immunotherapy.

[0003] NK cells are important immune cells in the body, accounting for approximately 10% of the total number of peripheral blood lymphocytes in healthy individuals. NK cells play a crucial role in the early immune response and immune surveillance during the development of solid tumors, serving as the body's first line of defense against tumors. The use of NK cells in clinical treatment has a long history. Compared to T cells, NK cells possess many unique immune characteristics and advantages: First, they exhibit non-specific tumor cell killing activity unrestricted by HLA, primarily through surface cytotoxic receptors that trigger the release of perforin and granzymes, producing cytotoxic factors and TNF-α, without producing IL-2 or other cytokines, thus avoiding lethal cytokine storms. Second, NK cells can also induce ADCC-specific killing by binding to antibody Fc fragments via CD16. Third, the killing effect of NK cells is continuous, moving from one target to another, killing up to 7-10 tumor cells, a continuous killing capability lacked by T cells. Fourth, because NK cells have a short lifespan and disappear quickly after exerting their effects, there is no need to consider the need for suicide genes, similar to CAR-T cells, to limit their long-term side effects, making them clearly superior to T cells. Due to these advantages, NK cells are effective and safe for patient treatment. Based on these advantages, this invention translates the design principles of CAR-T to NK cells, using NK92 instead of T cells as the host cell, making CAR-NK more safe and controllable than CAR-T.

[0004] Immune checkpoints are a class of immunosuppressive molecules that regulate immune cell activity through a series of co-inhibitory or co-stimulatory signaling pathways, preventing excessive immune activation and ensuring self-tolerance, thereby avoiding damage and destruction of normal tissues. Programmed death 1 (PD1) is one of the major inhibitory molecules. Its ligand 1 (PD-L1) is expressed in limited quantities in normal tissues, but its expression is significantly increased in most solid tumor cells and immunosuppressive cells. Tumors utilize the PD-1 / PD-L1 negative regulatory mechanism to escape the anti-tumor immune response. Currently, multiple preclinical studies and clinical trials have indicated that using PD-L1 or PD-1 blocking antibodies to enhance the anti-tumor immune response in cancer patients can serve as a novel tumor immunotherapy approach and can achieve sustained clinical responses. More significantly, research has found that constructing a chimeric co-stimulatory switch receptor using the extracellular segment of PD-1 and the transmembrane and intracellular segments of CD28, and introducing it into CAR-T cells targeting solid tumor antigens, has shown greater and better efficacy compared to current treatments using PD1 antibodies or CAR-T cells alone.

[0005] Currently, using single-chain antibodies targeting PD1 as the extracellular segment of CAR structures has achieved good results in CAR-T cell therapy. However, in order for single-chain antibodies as extracellular segments to still exert their targeting effect without affecting the complete expression of the CAR structure, their original structure needs to be optimized. For example, the length and properties of the linker, whether it is rigid or flexible, whether the optimized scFv can correctly coil and fold, and whether it can recognize the corresponding antigen in the extracellular space all need to be tested, which will inevitably waste a lot of human and material resources.

[0006] The emergence of nanobodies (Nb) has precisely compensated for this disadvantage. Cloning the variable region of a naturally occurring heavy chain antibody (HCAs) found in the serum of camels, which lacks the light chain, yields a single-domain antibody composed only of the heavy chain variable region, called a VHH (variable domain of heavy chain of heavy-chain antibody), also known as a nanobody. Nanobodies consist of only one domain, are small in size (relative molecular mass of 15 kDa), and therefore do not require linker optimization. Furthermore, the camel VHH germline gene is highly homologous to the human VH3 family sequence, exhibiting low immunogenicity and good biocompatibility with humans. The VHH surface differs from human VH by only about 10 amino acids, making humanization modification easy. The CDR1 and CDR3 of nanobodies are longer than those of human antibody heavy chains (VH), which to some extent compensates for the reduced antigen-binding capacity caused by the loss of the light chain. Meanwhile, the CD3 of nanobodies has a convex structure, which is more compatible with the concave topology that antigenic epitopes often form, enabling them to bind to more concealed antigenic epitopes such as slit-shaped and pocket-shaped antigenic epitopes.

[0007] NKG2D molecules, as a prominent activating receptor on NK cells, can induce degranulation and cytokine production upon activation alone. Their ligands (NKG2DLs) are ubiquitous on the surface of infected or malignantly transformed cells, especially tumor cells. During tumor cell development, mechanisms such as the formation of soluble NKG2DLs (sNKG2DLs) have evolved to evade the immune system, resulting in immune escape. These shed NKG2DLs, such as MICA / B, mediate the endocytosis of NKG2D receptors, leading to a decrease or loss of NKG2D-dependent tumor immune surveillance. Furthermore, they hinder NK cell migration and recognition, promoting NK cell exhaustion. The shedding of antigens from the tumor surface makes single-target CAR-T / NK therapy difficult to achieve long-term effectiveness. Traditionally designed CARs only respond to tumor surface antigens, neglecting the impact of shed, soluble antigens on the tumor immune response. Summary of the Invention

[0008] This invention modifies NK92 cells to construct a multi-target, enhanced anti-tumor NK cell. The addition of PD-L1 nanobody enhances the targeting of CAR-NK to tumor cells. NKG2D, CD28, 41BB, and CD3ζ reverse the negative effects of soluble MICA / B on NK cells while enhancing the activation of NK92 cells, resulting in a novel anti-tumor NK cell to enhance its anti-tumor immune effect.

[0009] The technical solution adopted in this invention is as follows: A method for preparing dual-targeting enhanced anti-tumor NK cells includes the following steps: Step 1: Synthesis of the target plasmid sheet The target plasmid comprises gene fragments expressing the following amino acids in tandem: PD-L1 nanobody (1-129aa), NKG2D extracellular segment (82-216aa), CD28 transmembrane segment (153-179aa) and intracellular segment (180-220aa), co-stimulatory molecule 41BB (214-255aa), and CD3ζ (52-164aa). The sequences of the above amino acids are shown in SEQ ID NO. 1-6, and the corresponding expressed nucleotide sequences are shown in SEQ ID NO. 7-12. The target plasmid was cloned and constructed into a lentiviral vector; the lentiviral vector is preferably pCDH-CMV-MCS-P2A-copGFP-T2A-Puro.

[0010] Step 2: Infect NK92 cells with lentivirus to obtain NK92 cell lines that stably express the above-mentioned target plasmid gene.

[0011] Its specific preparation method includes the following steps: 1) Extraction of lentiviral packaging plasmids and target plasmids: The lentiviral packaging plasmids used are pSPAX2, PMD2G, and the target plasmids mentioned above; 2) Packaging of lentivirus: The target plasmid, pSPAX2, and PMD2G were transfected into 293T cells at a ratio of (3.5-4.5):(2.5-3.5):1 to produce a virus carrying the target plasmid; 3) Lentiviral concentration: The viral supernatant harvested after transfection (48-72h) is concentrated (using polyethylene glycol concentration method); 4) Lentiviral infection of NK92 cells: Explore the optimal MOI value of the corresponding virus, infect NK92 cells according to the MOI value, and obtain NK92 cell lines that stably express the target plasmid.

[0012] This invention employs whole-genome synthesis technology to obtain a fusion receptor, namely, a PD-L1 nanobody modified with NKG2D, a transmembrane segment (153-179 aa) and an intracellular segment (180-220 aa) of CD28, and co-stimulatory molecules 41BB (214-255 aa) and CD3ζ (52-164 aa). Utilizing this chimeric molecular module, on the one hand, the PD-L1 nanobody binds to PD-L1 on tumor cells, thus targeting tumor cells while avoiding the negative regulatory mechanism of PD-1 / PD-L1 immunity; on the other hand, NKG2D can bind to both MICA / B on the tumor surface and free soluble MICA / B in the tumor microenvironment. Through the intracellular linkage of CD28, 41BB, and CD3ζ, it can enhance the tumor-killing effect of NK92 cells and reverse the negative effects produced by the binding of soluble MICA / B to NKG2D.

[0013] Numerous experiments have demonstrated that the NN168ζ-NK92 cells of this invention can not only target tumor cells but also enhance the cytotoxic activity of NK92 cells. Compared with the targeting effect of CAR-NK against a specific antigen, NN168ζ-NK92 cells recognize tumor cells through the binding of PD-L1 nanobodies to PD1 and the NKG2D / MICA and MICB pathways. Since most tumors express PD-L1, MICA, and MICB, the targeting effect of NN168ζ-NK92 on tumor cells is more broad-spectrum, rather than being limited to one or a few specific antigen targets. Attached Figure Description

[0014] Figure 1 Schematic diagram of the recombinant lentiviral vector PDL1Nb-NKG2D-CD28-41BB-CD3ζ-pCDH; Figure 2 Fluorescence image of 293T cells 72 hours after plasmid transfection; Figure 3 Fluorescence image of NK92 72 h after lentiviral transfection; Figure 4 Flow cytometry was used to detect the expression of Flag and GFP in NK92 and NN168ζ-NK92 cells; Figure 5 qPCR was used to test the expression of nanobodies NKG2D-IgG4 and PDL1 nanobodies-Linker-NKG2D in NK92 and NN168ζ-NK92 cells. Figure 6 Schematic diagram of the proliferation levels of NK92 and NN168ζ-NK92 cells; Figure 7Killing of H1299 tumor cells by NK92 and NN168ζ-NK92 cells. Detailed Implementation

[0015] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, the experimental methods in the embodiments of the present invention are conventional methods.

[0016] Example 1 Construction of the recombinant lentiviral vector PD1-DAP10-41BB-pCDH: The following gene fragments were synthesized at Nanjing GenScript Biotech Co., Ltd. using a whole-genome synthesis method: 387 bp of PD-L1 nanobody (1-129aa), 405 bp of NKG2D extracellular segment (82-216aa), 81 bp of CD28 transmembrane segment (153-179aa) and 123 bp of CD28 intracellular segment (180-220aa), 126 bp of co-stimulatory molecule 41BB intracellular segment (214-255aa) and 339 bp of CD3ζ intracellular segment (52-164aa). These fragments were then tandemly synthesized to obtain the PDL1Nb-NKG2D-CD28-41BB-CD3ζ fragment. The PDL1Nb-NKG2D-CD28-41BB-CD3ζ fragment was then subcloned into the pCDH-CMV-MCS-P2A-CopGFP-T2A-Puro lentiviral vector to obtain the recombinant lentiviral vector NN168ζ-pCDH (e.g., Figure 1 (as shown in SEQ ID NO.13 and SEQ ID NO.14), where the nucleotide sequences of Gly4-Ser Linker and IgG4 Hinge are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0017] Example 2 Lentiviral packaging: 1. Extraction of lentiviral packaging plasmids and target plasmids (1) Plasmid transformation Take stbl3 competent cells (TransGenBiotech, catalog number CD521-01) and E. coli TOP10 competent cells; add 1 μg of the target plasmid (NN168ζ-pCDH) to the stbl3 competent cells and 1 μg of lentiviral packaging helper plasmids (pSPAX2, PMD2G) to the TOP10 competent cells respectively, mix thoroughly, incubate on ice for 30 min, immediately place in a 42℃ water bath for 90 s heat shock, then place on ice for 2 min, mix thoroughly. Then place in a 37℃ constant temperature shaking incubator and shake at 180 rpm for 1 h. Take 100 μl and spread it on LB solid medium (containing ampicillin, Amp, concentration of 50 μg / ml, added at a ratio of 1:1000), and incubate at 37℃ for 12 h.

[0018] (2) Plasmid extraction 1) Small shake: Pick single colonies from the above culture plates and inoculate them into 5 ml of fresh LB medium (containing Amp at a concentration of 50 μg / ml, added at a ratio of 1:1000), place them on a shaker at 37℃ and shake at 220 rpm / min for 12 h. 2) Shake vigorously. Take 200 μL of bacterial culture and inoculate it into 200 ml of LB medium (containing 200 μL of Amp). Place it on a shaker at 37℃ and shake at 220 rpm / min for 12-16 hours. 3) Plasmid extraction was performed according to the OMEGA endotoxin-free plasmid large-scale extraction kit (OmegaBio-Tek, USA, product code D6926-03). The plasmid concentration and purity were measured using a Nanodrop micro spectrophotometer (Thermoelectric (Shanghai) Technology Instruments Co., Ltd., USA, model: NanoDrop2000), and then stored at -20℃.

[0019] 2. Lentiviral packaging process When the 293T cell density reaches 70%-80%, replace 10ml of fresh DMEM medium (Hyclone, SH30022.01) containing 6% FBS (BI, catalog number 04-001-1ACS) without penicillin and streptomycin with 1 hour in advance, and continue to incubate in an incubator. After 1 hour, the virus is packaged. The plasmid was packaged using a calcium phosphate reagent kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0508). The packaging procedure was as follows: the target plasmid NN168ζ-pCDH, helper plasmids pSPAX2 and PMD2G were mixed in a 4:3:1 ratio. The mixed plasmid was added to CaCl2 solution (provided by the kit, product number C0508-2), and mixed by pipetting. The mixture was then added dropwise to BBS solution (provided by the kit, product number C0508-1), and mixed by pipetting. The mixture was incubated at room temperature for 30 min. Turbidity was observed, but no white precipitate formed. The mixture was then added to 293T cells, mixed, and incubated for 4-16 h. The supernatant was then removed, and the cells were gently washed once with PBS. The culture medium was replaced with 15 ml of fresh DMEM containing 10% FBS. Virus was collected at 48 h and 72 h. Fluorescence and white light images under a fluorescence microscope at 72 h (e.g., [images would be inserted here]). Figure 2 (As shown).

[0020] 3. Lentiviral Concentration Centrifuge the collected viral supernatant to remove cell debris, add polyethylene glycol (PEG), incubate for at least 12 hours, centrifuge at 4°C to collect viral particles, resuspend the concentrated viral particles in PBS, aliquot and store at -80°C to avoid repeated freeze-thaw cycles.

[0021] 4. Determination of lentivirus titer The lentivirus titers were determined using an abm qPCR lentivirus titer kit (catalog number LV900), and all obtained lentivirus titers were 1×10⁻⁶. 9 .

[0022] Example 3 Lentiviral transfection of NK92 cells: Take 1-5×10 5 NK92 cells were seeded per well in 24-well plates, and concentrated NN168ζ virus solution (MOI=50-100) was added. Polyglobulin (Yisheng Biotechnology, catalog number 40804ES76) was added to each well to a final concentration of 8 μg / ml, mixed well, and incubated at 37℃. After 12-15 hours, the cells were centrifuged, the viral supernatant was removed, and fresh culture medium was added for further incubation. 72 hours after infection, GFP expression was observed under a fluorescence microscope to assess the infection effect (e.g., ...). Figure 3 (As shown). Infected NK92 cells can be used for subsequent experiments after 72 hours.

[0023] Example 4 Flow cytometry was used to detect the expression of target genes PD1 and NKG2D. NK92 cells, PCDH (empty vector), and NK92 cells were collected 72 hours after lentivirus infection. The cells were centrifuged at 300g for 5 minutes, and the cell count was 1×10⁻⁶. 6 The cells were washed 1-2 times with PBS containing 1% FBS (hereinafter referred to as wash buffer). APC-labeled mouse anti-Flag monoclonal antibody (Biolegend, catalog number 637307) was added to a 100 μL system, and the cells were incubated on ice in the dark for 30 min. The cells were then washed 2-3 times with wash buffer. Finally, the cells were resuspended in 400 μL of PBS and analyzed using a flow cytometer (BD FACSCantoII). The results are as follows: Figure 4 , Figure 5 As shown, the expression rate of Flag in NN168ζ-NK92 is approximately 90%.

[0024] Example 5 In vitro cytotoxicity assay: 1. Cell preparation H1299 lung cancer cells in the logarithmic growth phase after luciferase transfection were seeded into 96-well plates, ensuring 3 × 10⁶ cells per well. 4 Each cell type was divided into 3 subwells and incubated overnight in a 5% CO2, 37°C incubator.

[0025] 2. Experimental Design The experimental group was set up with two groups, and each group was further divided into three subgroups based on different effector cells (NN168ζ-NK92 cells and NK-92 cells), with effector-to-target ratios of 1:1, 5:1, and 10:1, respectively. Three control groups were also set up: target cell control wells, sample maximum enzyme activity control wells (target cell wells without effector cell treatment for subsequent lysis), and blank cell control wells without luciferase transfection. The total volume of each well was 100 μL.

[0026] 3. Luciferase assay This experiment uses ONE-Glo TM The Luciferase Assay System Promega was used 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 scheduled detection time, the 96-well plate was removed from the cell culture incubator, and Lysis Solution (10×) was added to the "Sample Maximum Enzyme Activity Control Well," at a volume equal to 10% of the original culture medium volume. After adding Lysis Solution, the plate was repeatedly pipetted and mixed thoroughly, and then the plate was 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.

[0027] 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, the modified NN168ζ-NK92 cells showed a 4-5 fold increase in killing efficiency against H1299 human lung cancer cells compared to the parental NK92 cells, indicating that the modified NN168ζ-NK92 cells have a significantly enhanced anti-tumor effect.

[0028] The above embodiments are part of the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing dual-targeted enhanced anti-tumor NK cells, characterized in that, Includes the following steps: Step 1: Synthesize the target plasmid fragment, which comprises gene fragments encoding the following amino acid sequences in tandem: PD-L1 nanobody, NKG2D extracellular segment, CD28 transmembrane and intracellular segments, co-stimulatory molecule 41BB, and CD3ζ. The amino acid sequences are shown in SEQ ID NO. 1-6, and the corresponding nucleotide sequences are shown in SEQ ID NO. 7-12. The PD-L1 nanobody and the NKG2D extracellular segment are linked using the nucleotide sequence shown in SEQ ID NO. 13, and the NKG2D extracellular segment and the CD28 transmembrane segment are linked using the nucleotide sequence shown in SEQ ID NO.

14. Then, clone or construct the above target plasmid fragment into a lentiviral vector. Step 2: Infect NK92 cells with lentivirus to obtain NK92 cell lines that stably express the above-mentioned target plasmid fragment.

2. The preparation method according to claim 1, characterized in that: Step two also includes the following steps: (1) Extraction of lentiviral packaging plasmids and target plasmid fragments: The lentiviral packaging plasmids used were pSPAX2, PMD2G and the target plasmid fragments; (2) Packaging of lentivirus: The target plasmid fragment, pSPAX2, and PMD2G are transfected into 293T cells at a ratio of (3.5-4.5):(2.5-3.5):1 to produce a virus carrying the target plasmid fragment; (3) Lentiviral concentration: The viral supernatant harvested after transfection is concentrated; (4) Lentiviral infection of NK92 cells: Explore the optimal MOI value of the corresponding virus, infect NK92 cells according to the MOI value, and obtain NK92 cell lines that stably express the target plasmid fragment.

3. The preparation method according to claim 1, characterized in that: The target plasmid fragment was synthesized using a whole-genome synthesis process.

4. The preparation method according to claim 1, characterized in that: The lentiviral vector is pCDH-CMV-MCS-P2A-copGFP-T2A-Puro.

5. The preparation method according to claim 2, characterized in that: The extraction of the lentivirus packaging plasmid and the target plasmid fragment includes the following steps: (1) Plasmid transformation: Take out stbl3 competent cells and E. coli TOP10 competent cells; add the target plasmid fragment to stbl3 competent cells, and add lentiviral packaging helper plasmids pSPAX2 and PMD2G to TOP10 competent cells respectively. Mix thoroughly, incubate on ice, immediately place in a 40-45℃ water bath for heat shock, then place on ice and mix thoroughly; then place in a 35-40℃ constant temperature shaking incubator for shaking culture, and then take an appropriate amount and spread it on LB solid medium and incubate in a 35-40℃ constant temperature incubator. (2) Plasmid extraction: ①Small shake: Pick single colonies from the above LB solid medium, inoculate them into fresh LB medium, and place them on a shaker at 35-40℃ for constant temperature shaking; ② Shake vigorously, take an appropriate amount of bacterial solution and inoculate it into LB medium, place it in a shaker at 35-40℃ and shake it at a constant temperature; ③ Extract plasmids according to the requirements of the extraction kit, measure the plasmid concentration and purity, and store them in a refrigerator.

6. The preparation method according to claim 2, characterized in that: The packaging of the lentivirus specifically includes the following steps: When the 293T cell density reaches 70-80%, replace the medium with 10 ml of fresh DMEM containing 6% FBS and free of penicillin and streptomycin, and continue culturing in an incubator. Then, use a calcium phosphate kit to package the virus. Mix the target plasmid fragment, helper plasmid pSPAX2, and PMD2G in a 4:3:1 ratio. Add the mixed plasmid to CaCl2 solution and mix by pipetting. Add the mixed solution dropwise to BBS solution and mix by pipetting. Incubate at room temperature until turbidity is observed and no white precipitate is formed. Add the above mixed solution to the 293T cells, mix well, and continue culturing in an incubator for 4-16 h. Then, remove the supernatant, wash with PBS buffer, replace with 15 ml of fresh DMEM medium containing 10% FBS, and collect the virus at 48-72 h.

7. The preparation method according to claim 2, characterized in that: The lentivirus concentration specifically includes the following steps: The collected viral supernatant was centrifuged to remove cell debris, polyethylene glycol was added, and the mixture was incubated for at least 12 hours. The virus particles were then collected by centrifugation at 4°C. The concentrated viral particles were resuspended in PBS and aliquoted for storage.

8. The preparation method according to claim 2, characterized in that: The lentivirus infection of NK92 cells specifically includes the following steps: Take 1×10 5 -5×10 5 NK92 cells were seeded per well in 24-well plates, and concentrated virus solution was added. The MOI was 50-100. Polybrene was added to each well to a final concentration of 6-10 μg / ml. The mixture was stirred and incubated at 35-40℃. After 12-15 hours, the cells were centrifuged to remove the viral supernatant. Fresh culture medium was then added and the cells were incubated for another 72 hours.

9. Dual-targeting enhanced anti-tumor NK cells obtained by the preparation method according to any one of claims 1-8.

10. The use of the dual-targeting enhanced anti-tumor NK cells as described in claim 9 in the preparation of anti-lung cancer immunotherapy drugs.

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