Transgenic NK cell and application thereof in cancer treatment

Abstract

The invention discloses a technical scheme for enhancing the antitumor activity of NK cells. The designed GPR34 antagonistic peptide-transmembrane fusion protein contains an antagonistic peptide mutant, a connecting peptide and a transmembrane domain, and can block tumor microenvironment inhibition signals. SgRNA is designed aiming at UBE2F gene exon3, UBE2F is knocked out through CRISPR / Cas9, and the sensitivity of NK cells to low-dose IL-15 is improved. The prepared transgenic NK cell is subjected to electrotransfection and three-dimensional culture, and is high in purity and remarkable in gene editing efficiency. The dual-targeting antibody is composed of anti-GPC3scFv, a connecting peptide and anti-MUC1VHH, and can target two tumor antigens at the same time. An in-vitro experiment shows that the enhanced NK cell (Combb-NK) has a high killing rate on double positive tumor cells, and the in-vivo tumor-bearing mouse median lifetime is prolonged to 68 days and is improved by 112% compared with a control group. Therefore, the technical scheme of the invention provides an innovative strategy for tumor immunotherapy, and has significant clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a transgenic NK cell and its application in treating cancer. Background Art

[0002] Natural killer cells (NK cells), as key effector cells of the innate immune system, have become a highly promising universal cell therapy option in the field of tumor immunotherapy due to their non-MHC-restricted killing properties and low risk of graft-versus-host disease (GvHD). NK cells recognize tumor cells through a dynamic balance between surface activating receptors (such as NKG2D and DNAM-1) and inhibitory receptors (such as the KIR family and NKG2A). When tumor cells downregulate the expression of MHC class I molecules, NK cell inhibitory signals are weakened, thereby activating killing mechanisms such as the perforin-granzyme pathway and the death receptor-mediated apoptosis pathway. At the same time, they can also kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and secrete cytokines such as IFN-γ and TNF-α to regulate the immune microenvironment.

[0003] However, NK cells face many technical bottlenecks in their clinical tumor treatment applications. In the treatment of solid tumors, conventional NK cells have difficulty penetrating the dense extracellular matrix barrier, with a local tumor infiltration rate of less than 5%. Their in vivo persistence is short, typically surviving no more than 7 days after infusion. Systemic administration of traditional cytokines (such as IL-15) can easily trigger severe toxic reactions such as cytokine storms. In the tumor microenvironment (TME), immunosuppressive factors (such as TGF-β, IL-10, and adenosine) and lipid metabolites (such as LysoPS) specifically inhibit the cytotoxic function of tissue-resident NK cells (ILC1s) by binding to the NK cell surface receptor GPR34. Furthermore, existing single-target chimeric antigen receptor (CAR) designs struggle to cope with tumor heterogeneity, and viral vector-mediated gene integration carries potential genomic safety risks.

[0004] To overcome these difficulties, researchers have explored various technologies. Chimeric antigen receptor-modified NK cell (CAR-NK) technology empowers NK cells with targeted killing capabilities, but lentiviral transduction carries the risk of insertional mutagenesis, and mRNA-LNP-mediated transient expression technology lasts less than 72 hours in vivo. Cytokine pre-activation strategies, while capable of enhancing NK cell activity, require continuous exogenous supply, which is costly and poses safety risks. Existing patented technologies primarily focus on classic immune checkpoints such as PD-1 / CTLA-4, but have limited application to novel metabolic immune regulatory targets such as GPR34.

[0005] Recent research has led to new directions. A 2024 study demonstrated that GPR34 is specifically and highly expressed in ILC1s, and its ligand, LysoPS, is significantly enriched in the stroma of solid tumors. Targeted knockout of GPR34 can enhance ILC1s' tumor infiltration and inhibit liver cancer progression. Cancer Cell also revealed that the UBE2F-ARIH2 pathway mediates IL-15R degradation, leading to NK cell functional inactivation. Targeted intervention can enhance NK cell sensitivity to IL-15 while avoiding systemic toxicity. However, a systematic approach combining novel metabolic immune target regulation, gene editing optimization, and multi-specific targeting strategies is currently lacking. There is an urgent need to develop novel NK cell engineering technologies for safe and effective tumor immunotherapy. Summary of the Invention

[0006] Based on the above problems, the purpose of the present invention is to provide a transgenic NK cell and its application in treating cancer.

[0007] Therefore, in one aspect, the present invention discloses a GPR34 antagonist peptide-transmembrane fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] The present invention also discloses an enhanced NK cell, wherein the enhanced NK cell is labeled as a Comb-NK cell, wherein the Comb-NK cell simultaneously comprises the following elements:

[0009] (1) expressing the GPR34 antagonist peptide-transmembrane fusion protein;

[0010] (2) expressing a UBE2F gene editing element, wherein the gene editing element comprises sgRNA1 and sgRNA2 targeting UBE2F exon3, wherein the sequences of sgRNA1 and sgRNA2 are shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively;

[0011] (3) A surface-coupled dual-targeting antibody comprising an anti-GPC3 scFv and an anti-MUC1 VHH, which are linked by GGGGSGGGGSGGGGS, wherein the nucleotide sequence encoding the dual-targeting antibody is shown in SEQ ID NO: 6; the amino acid sequence of the anti-GPC3 scFv is shown in SEQ ID NO: 4, and the amino acid sequence of the anti-MUC1 VHH is shown in SEQ ID NO: 5.

[0012] The present invention also discloses a method for preparing the enhanced NK cells, which comprises the following steps:

[0013] (1) pX330-UBE2F-sgRNA and pLVX-GPR34-antagonist were introduced into NK cells using electroporation editing technology, with the electroporation parameters being 1500V / 20ms×2 pulses;

[0014] (2) co-cultured and expanded with irradiated K562-mbIL-21 trophoblast cells in serum-free medium containing 1 ng / mL IL-15;

[0015] (3) The dual-targeting antibody is coupled to the surface of NK cells through the biotin-streptavidin system to enhance NK cells.

[0016] The present invention also discloses a pharmaceutical composition, which comprises the enhanced NK cells and a pharmaceutically acceptable cryoprotectant.

[0017] The present invention also discloses a method for enhancing NK cells in the preparation of GPC3 + and / or MUC1 + Application in solid tumor drugs, the solid tumors include liver cancer, breast cancer, and pancreatic cancer.

[0018] The present invention blocks the inhibitory signal of the tumor microenvironment through GPR34 antagonist peptide-transmembrane fusion protein, thereby increasing the killing rate of NK cells against HepG2 from 38.2% to 89.5%, and reducing the tumor volume in vivo by 86%; UBE2F gene knockout reduces the IL-15 dosage to 1 / 10 of the traditional one, increases the proliferation rate in vitro by 5 times, and prolongs the median survival in vivo to 68 days; the dual-targeting antibody effectively overcomes tumor heterogeneity, and increases the killing rate of single-positive tumors by 2.78 times compared with traditional NK cells; gene editing and antibody coupling produce significant synergistic effects, with a synergistic index CI of 0.78-0.83, and the proportion of tumor-infiltrating NK cells increases by 13 times, CD69 + / NKG2D + The expression of activation markers such as GPC3 and MUC1 was significantly upregulated, comprehensively enhancing the anti-tumor activity and therapeutic effect of NK cells. This invention significantly improves the efficacy of NK cells against solid tumors through the triple synergistic mechanism of "releasing inhibition (GPR34 antagonism) + enhancing survival (UBE2F editing) + dual-target killing (anti-GPC3 / anti-MUC1)", providing an innovative solution for difficult-to-treat tumors such as liver cancer and breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 1 shows the SDS-PAGE test results of the dual-targeting antibody (anti-GPC3 / anti-MUC1), where 1 is the dual-targeting antibody (anti-GPC3 / anti-MUC1) with a molecular weight of approximately 28 kDa. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0022] Example 1: Design and preparation of GPR34 antagonist peptide-transmembrane fusion protein

[0023] In the field of tumor immunotherapy, the inhibition of NK cell function by the tumor microenvironment (TME) is one of the key factors affecting therapeutic efficacy. GPR34, as an important metabolic immunomodulatory target, specifically inhibits the cytotoxicity of tissue-resident NK cells (ILC1s) upon binding to its ligand LysoPS. Based on this, the present invention designed a GPR34 antagonist peptide-transmembrane fusion protein (SEQ ID NO: 1).

[0024] From the perspective of structural design, the fusion protein consists of three parts. The GPR34 antagonist peptide (mutant) at the front end is the core functional region, which is obtained by performing V6S / S8K mutations on the V6 and S8 sites of the wild-type antagonist peptide (MALWLLLPLALLLHAAPGRAQPSQKKKRKV 5 V 6 Y 7 S 8 P 9 K mutation is MALWLLLPLALLLHAAPGRAQPSQKKKRKV 5 S 6 Y 7 K 8 P 9 K), which increases its binding force to GPR34 by 3 times, and the dissociation constant KD is reduced from 4.5nM of the wild type to 1.2nM, which can more efficiently block the binding of LysoPS to GPR34. The middle connecting part (GGGGS) 3 serves as a flexible connecting peptide and adopts the classic glycine-serine repeat sequence. While ensuring the spatial independence of each functional domain, it maintains the structural stability of the fusion protein and avoids the impact of steric hindrance on the function. The terminal CD8α transmembrane domain gives the fusion protein transmembrane ability, ensuring that the GPR34 antagonist peptide can be accurately positioned on the surface of NK cells, and continuously plays a role in blocking the LysoPS-GPR34 signaling axis, thereby effectively relieving the inhibition of TME on NK cells.

[0025] Compared with existing technologies, the design of this fusion protein has significant characteristics. On the one hand, the binding force is greatly improved through targeted amino acid site mutations, which is different from traditional antagonistic peptides that only rely on natural sequences; on the other hand, the antagonistic function is integrated with the transmembrane function, so that NK cells can actively resist TME inhibition in the complex environment of the body. Compared with the simple addition of antagonistic peptides, it has obvious advantages in terms of duration and effectiveness of action. In actual applications, when transgenic NK cells carrying this fusion protein enter tumor tissues, they can quickly block GPR34-mediated inhibitory signals, activate the killing activity of NK cells, and enhance their ability to recognize and kill tumor cells, providing a new and effective strategy for cancer treatment.

[0026] The final GPR34 antagonist peptide-transmembrane fusion protein (SEQ ID NO: 1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The HPLC purity after synthesis was ≥95%, and the protein was stored at -80°C for future use.

[0027] Example 2: Design of UBE2F sgRNA

[0028] In the field of NK cell therapy, enhancing NK cell sensitivity to IL-15 and prolonging its survival in vivo is a key challenge. Recent studies have found that the UBE2F-ARIH2 pathway significantly impairs NK cell responsiveness to IL-15 by mediating the ubiquitination and degradation of IL-15Rα. To address this issue, the present invention designed two highly active sgRNAs targeting the exon 3 region of the UBE2F gene.

[0029] sgRNA1 and sgRNA2 (specific sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively) share a common 78-nt backbone (specific sequence: guuuuagagc uagaaauagc aaguuaaaauaaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggtgcuuuu). The resulting sgRNA1 and sgRNA2 were synthesized at Sangon Biotech (Shanghai) Co., Ltd. and stored at −80°C until use.

[0030] Through CRISPR / Cas9-mediated gene editing, these two sgRNAs were able to efficiently cleave the UBE2F gene locus, with cleavage efficiencies reaching 92.1% and 88.7%, respectively (verified by T7E1 digestion and Sanger sequencing). Precision editing of this region specifically inhibited UBE2F protein expression, blocking the ubiquitination and degradation pathway of IL-15Rα, thereby significantly increasing the expression level of IL-15Rα on the surface of NK cells. In in vitro experiments, UBE2F-knockout NK cells (UBE2F-KO NK) exhibited the following advantages:

[0031] (1) Cell proliferation: In the medium with an IL-15 concentration of 5 ng / mL, the proliferation rate of UBE2F-KO NK cells reached 215% after 72 hours, which was significantly higher than the 108% of wild-type NK cells.

[0032] (2) Cytokine secretion: After co-culture with tumor cells, the secretion of IFN-γ by UBE2F-KO NK cells increased by 3.2 times, and the secretion of TNF-α increased by 2.8 times.

[0033] (3) Cytotoxicity: The cytotoxicity rate against SK-BR-3 breast cancer cells (efficacy-target ratio 10:1) increased from 52.3% to 81.7%.

[0034] Therefore, the present invention uses bioinformatics algorithms to predict off-target effects on the entire genome, screens out highly specific targets in the exon3 region, and ensures editing specificity. Experimental data show that no cleavage activity was detected at the TOP10 potential off-target sites. Compared with the traditional IL-15 dosing regimen, the present invention regulates the stability of IL-15Rα from the source through gene editing, thereby increasing the responsiveness of NK cells to low-dose IL-15 (<10ng / mL) by more than 5 times. The edited NK cells can still maintain the expression levels of anti-apoptotic proteins (Bcl-2, Mcl-1) for more than 7 days without exogenous IL-15 supplementation, significantly prolonging their survival time in vivo.

[0035] Example 3: Preparation of transgenic NK cells (TG-NK cells)

[0036] 1. For NK cell isolation from cord blood, the human NK cell line NK-92MI can also be used directly.

[0037] (1) Sample collection and pretreatment: 50-80 mL of fresh umbilical cord blood (meeting ethical requirements) was collected from healthy volunteers and placed in a sterile collection bag containing 10 U / mL sodium heparin, and then immediately subjected to density gradient centrifugation.

[0038] (2) Density gradient centrifugation for PBMC separation: Gently mix umbilical cord blood with an equal volume of PBS (containing 2% human AB serum) and add 15 mL of Lymphoprep separation buffer (density 1.077 g / mL) using a SepMate-50 centrifuge tube. Centrifuge at 1200 × g for 20 minutes (room temperature, no brake). Collect the interphase PBMCs and wash twice with PBS (300 × g, 10 minutes).

[0039] (3) Immunomagnetic bead separation of NK cells

[0040] The cells were sorted using the CliniMACS CD56 isolation kit and the cell concentration was adjusted to 1 × 10 8 / mL, add CD56 MicroBeads (20μL / 10 7 Cells were incubated at 4°C for 15 minutes, gently mixing twice. Positive isolation was performed using the CliniMACS Prodigy system.

[0041] Flow cytometry was used to detect the purity of the separation: BD FACSCanto II analyzer was used, and the detection antibody combination was CD56-APC and CD3-PerCP. The results showed that CD56 + CD3-cell purity was 96.8% ± 1.2% (n = 5 batches), in compliance with USP <1225> standard.

[0042] 2. Electroporation Editing

[0043] (1) Construction and verification of recombinant plasmids

[0044] pX330-UBE2F-sgRNA: sgRNA sequences targeting UBE2F exon 3 (SEQ ID NO: 2 and SEQ ID NO: 3) were cloned into the BbsI site of the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector. Sequencing verified the correct insertion sequence. Endotoxin levels were <0.1 EU / μg using the EndoFree Plasmid Maxi Kit.

[0045] pLVX-GPR34-antagonist: Synthesize a gene sequence encoding a GPR34 antagonist peptide-transmembrane fusion protein (SEQ ID NO: 1) with XhoI and EcoRI restriction sites at both ends. Clone into the multiple cloning site of the pLVX-IRES-ZsGreen1 vector (Clontech). Verify the vector construct by restriction enzyme mapping and sequencing.

[0046] (2) Optimization of electroporation parameters

[0047] The Neon Transfection System was used for condition optimization: voltage gradient experiment: 1200 V, 1350 V, 1500 V, 1650 V; pulse duration: 10 ms, 20 ms, 30 ms; pulse number: 1, 2, 3 times.

[0048] The optimal condition was determined to be 1500V / 20ms×2 pulses, at which the cell survival rate was 68.3%±4.5% and the transfection efficiency was 72.6%±3.8%.

[0049] (3) Electroporation procedure: Take 5×10 6 NK cells (self-isolated or human NK cell line NK-92MI) were resuspended in 100 μL R Buffer (Neon Transfection Kit). 10 μg of pX330-UBE2F-sgRNA and 8 μg of pLVX-GPR34-antagonist were added (DNA concentration ratio 1.25:1). Immediately after electroporation, cells were transferred to GT-T551 medium supplemented with 50 ng / mL IL-15 and incubated at 37°C for 2 hours before subsequent culture.

[0050] 3. In vitro expansion

[0051] (1) Optimization of serum-free culture medium

[0052] Basal medium: GT-T551 supplemented with 5% human AB serum (GemCell, tested for HIV, HBV, and HCV), 1 ng / mL rhIL-15 (PeproTech, endotoxin <0.01 EU / μg), 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine.

[0053] Osmolality was adjusted to 290-310 mOsm / kg.

[0054] (2) Preparation of trophoblast cells: K562-mbIL-21 cell lines were cultured in RPMI 1640 medium containing 10% FBS until the logarithmic growth phase. They were irradiated with a 137Cs γ-ray source at a dose rate of 1 Gy / min for a total dose of 100 Gy. After irradiation, the cells were washed three times with PBS and the cell concentration was adjusted to 1×10 6 / mL for future use.

[0055] (3) Establishment of three-dimensional culture system

[0056] Large-scale expansion using a G-Rex 10 incubator: seeding density 5 × 10 4 NK cells / mL; the trophoblast ratio was NK:K562=5:1 (effector-target ratio); the culture conditions were 37°C, 5% CO2, and 95% humidity.

[0057] Dynamic monitoring: Observe cell morphology daily under a microscope; monitor glucose (<200 mg / dL) and lactate (<20 mM) levels every 48 hours; replenish fresh culture medium according to cell density to maintain a cell concentration of 1-2 × 10 6 / mL.

[0058] 4. Phenotypic Identification

[0059] (1) Gene editing efficiency detection

[0060] T7E1 digestion method: Genomic DNA was extracted and the UBE2F target region was amplified by PCR (primer sequence: F-5'-GCTGACCTGGACAAGAAGGA-3'R-5'-CCAGCCATGTCCTCTTTCCA-3'). Amplification conditions were 95°C for 3 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles; 72°C for 5 minutes. After denaturation and annealing, 10 U of T7E1 enzyme was added and digested at 37°C for 20 minutes. Separation was performed by 2% agarose gel electrophoresis, and the grayscale of the bands was analyzed by ImageJ software. Knockout efficiency was calculated as follows: %KO = 100 × (1-(1-f) 0 · 5 ), where f = cut strip density / (cut strip density + uncut strip density).

[0061] Sequencing verification: Next-generation sequencing of PCR products at a depth of >10,000× revealed insertion / deletion (indel) frequencies of 87.9% and 85.2% for sgRNA1 and sgRNA2, respectively, with an average indel frequency of 86.6% ± 1.3% (n = 3).

[0062] (2) Protein expression detection

[0063] After washing the cells with FACS buffer (PBS containing 2% FBS), anti-His tag antibody-PE was added. The cells were incubated at 4°C in the dark for 30 minutes, washed, and loaded for analysis. Data were analyzed using FlowJo software, and isotype controls were established. The results showed that the positive expression rate of the GPR34 antagonist peptide was 76.3% ± 2.8% (n = 5).

[0064] (3) Cell phenotype analysis

[0065] The flow cytometry antibody combination was CD56-APC, CD3-PerCP, CD16-FITC, CD69-PE, and NKG2D-PE-Cy7. The results (n=5 batches) showed that CD56 + The purity of CD3- cells was 99.4% ± 0.3%; CD16 +The cell ratio was 96.2%±1.1%; the expression of activation marker CD69 was 32.7%±4.5%; and the expression of killer receptor NKG2D was 92.8%±2.3%.

[0066] 5. Experimental Summary

[0067] (1) Gene editing efficiency: T7E1 enzyme digestion and deep sequencing confirmed that the UBE2F gene knockout efficiency reached 86.6% ± 1.3%. Flow cytometry confirmed that the positive expression rate of GPR34 antagonist peptide was 76.3% ± 2.8%.

[0068] 2. Cell expansion capacity: Under low-dose IL-15 (1 ng / mL) conditions, transgenic NK cells expanded 62.5 ± 5.8 times within 14 days (starting cells 5 × 10 6 The final cell count was 3.13±0.29×10 8 In contrast, unedited NK cells expanded only 11.7 ± 2.1-fold under the same conditions (p < 0.001).

[0069] 3. Phenotypic characteristics: The expanded cell population is highly purified to CD56 + CD3-NK cells (>99%) and CD16 co-expression rate >95% meet the NK cell criteria. High expression of activation marker CD69 and killer receptor NKG2D indicates that the cells are in a pre-activated state.

[0070] In summary, this example uses a dual-gene modification strategy to knock out the UBE2F gene using CRISPR / Cas9 to enhance the sensitivity of NK cells to low-dose IL-15, while simultaneously achieving membrane expression of the GPR34 antagonist peptide to block the immunosuppressive signals of the tumor microenvironment. Using cGMP-compliant immunomagnetic bead sorting and electroporation editing technology, a low-dose IL-15-dependent three-dimensional amplification system was established to achieve large-scale preparation of NK cells (the NK cells prepared in this way are transgenic NK cells, labeled TG-NK cells). Product consistency was ensured through multi-dimensional phenotypic identification of gene editing efficiency, protein expression, cell surface markers, etc. The prepared cells have high purity, high activity, and good proliferation ability, meeting application requirements, laying the foundation for subsequent functional verification and in vivo experiments, and have significant innovation and industrial application value.

[0071] Example 4: Design and preparation of dual-targeting antibodies (anti-GPC3 / anti-MUC1)

[0072] Tumor heterogeneity is one of the key challenges hindering NK cells from precisely killing tumors. Modification of a single target makes it difficult to achieve comprehensive coverage of complex tumor cell populations. The dual-targeted antibody (anti-GPC3 / anti-MUC1) designed in this invention significantly enhances the breadth of NK cell recognition and tumor killing efficiency by simultaneously targeting two tumor-specific antigens.

[0073] Anti-GPC3 scFv screening: Three rounds of biopanning were performed from a natural phage antibody library using recombinant GPC3 protein (ab316055) as the solid-phase antigen. The phage antibody library was incubated with a GPC3 protein-coated ELISA plate at 4°C for 2 hours. Non-specifically bound phage were washed thoroughly to remove them. Specifically bound phage were then eluted with glycine-HCl buffer (pH 2.2) and reinfected with logarithmically growing Escherichia coli TG1 for amplification. ELISA analysis identified the single-chain antibody clone with the highest affinity for GPC3. The amino acid sequence of the anti-GPC3 scFv (after humanization modification, shown in SEQ ID NO:4) was sequenced, and its equilibrium dissociation constant, KD, was determined to be 2.3 nM by SPR.

[0074] Anti-MUC1 VHH Screening: A shark immune phage display library was constructed, containing recombinant MUC1 protein (ab80082), and a similar biopanning process was used as described above. After the third round of panning, single clones were screened using indirect ELISA, and the clone with the highest OD value was selected. Specificity was further verified by competitive ELISA, ultimately determining the amino acid sequence of the anti-MUC1 VHH (SEQ ID NO: 5), with a KD value of 1.8 nM.

[0075] Gene synthesis and vector construction: GenScript was commissioned to synthesize the DNA sequences encoding the anti-GPC3 scFv (SEQ ID NO: 4), the (G4S)3 linker peptide (GGGGSGGGGSGGGGS), and the anti-MUC1 VHH (SEQ ID NO: 5) (i.e., the anti-GPC3 / anti-MUC1, which also contains the coding sequence for 6His at the C-terminus; the synthesized nucleotide sequence is shown in SEQ ID NO: 6). BamHI and EcoRI restriction sites were introduced at both ends, respectively. These DNA fragments were sequentially ligated into the pET-28a(+) expression vector, which had been treated with the same double enzymes, to construct the recombinant expression plasmid pET-28a-anti-GPC3 / anti-MUC1. Sequence accuracy was verified by Sanger sequencing.

[0076] Protein Expression: Transform the recombinant plasmid into competent E. coli BL21(DE3) cells. Pick a single colony and inoculate it into LB medium containing kanamycin (50 μg / mL). Cultivate with shaking at 37°C until the OD600 reaches 0.6-0.8. Add IPTG to a final concentration of 0.5 mM and induce expression at 18°C ​​for 16 hours. Harvest the cells by centrifugation, resuspend in PBS, and sonicate. The supernatant is then centrifuged and used for subsequent purification.

[0077] Protein purification: The lysate supernatant was purified using a Ni-NTA affinity chromatography column. The column was first equilibrated with a binding buffer containing 20 mM imidazole. After loading, the column was gradient eluted with an elution buffer containing 20-500 mM imidazole. The elution peak of the target protein was collected. The purified protein was dialyzed to remove imidazole and replaced with PBS buffer. The protein was then analyzed by SDS-PAGE ( Figure 1 The purity was identified by HPLC (as shown in Figure 5), and finally a high-purity (>95%) dual-targeting antibody (anti-GPC3 / anti-MUC1) was obtained. After filtering through a 220 nm filter membrane, it was stored at -80°C for future use.

[0078] The dual-targeting antibody designed and prepared above is mainly composed of three parts:

[0079] (1) Anti-GPC3 scFv (SEQ ID NO: 4): This single-chain variable region fragment is an anti-GPC3 (GPC3) fragment. Its amino acid sequence was screened and optimized using phage display technology. It can specifically recognize the GPC3 antigen, which is highly expressed on the surface of tumor cells such as liver cancer. This fragment binds to GPC3 on the surface of tumor cells, activating the NK cell killing signaling pathway and initiating an attack on tumor cells.

[0080] (2) (G4S)3 connecting peptide: As a flexible connecting element, its unique structure gives each functional domain of the antibody sufficient spatial freedom to avoid mutual interference, ensure the stability and flexibility of the dual-targeting antibody, and enable it to flexibly bind to the antigen in the complex tumor microenvironment.

[0081] (3) anti-MUC1 VHH (SEQ ID NO: 5): A single-domain heavy chain antibody fragment against mucin 1 (MUC1) with high affinity and specificity for the MUC1 antigen. MUC1 is abnormally expressed in a variety of epithelial tumors (e.g., breast cancer and colorectal cancer). This fragment can accurately recognize and bind to MUC1 on the surface of tumor cells, further enhancing the targeting of NK cells to tumor cells.

[0082] In in vitro cell killing experiments, transgenic NK cells expressing dual-targeting antibodies demonstrated significant advantages: against the HepG2 liver cancer cell line expressing both GPC3 and MUC1, the killing rate reached 89.2% at an effector-target ratio of 10:1. This compares to 62.5% and 58.7% for transgenic NK cells expressing only anti-GPC3 or anti-MUC1 single-targeting antibodies, respectively, and a mere 35.6% for wild-type NK cells. After co-culture with tumor cells, transgenic NK cells expressing dual-targeting antibodies secreted 4.1-fold more IFN-γ and 3.8-fold more TNF-α than wild-type NK cells, effectively enhancing the anti-tumor immune microenvironment. These data demonstrate that the dual-targeting antibodies (anti-GPC3 / anti-MUC1) of the present invention can significantly enhance the targeted recognition and killing capabilities of transgenic NK cells against tumors, demonstrating their outstanding application value in cancer treatment.

[0083] Therefore, the present invention effectively solves the problem of tumor heterogeneity by simultaneously targeting GPC3 and MUC1, two antigens widely expressed in different tumor types and different cell subpopulations of the same tumor, and expands the killing range of NK cells, covering more tumor cells compared to single-target antibodies. After binding to tumor antigens, dual-target antibodies can synergistically activate multiple activating receptors on the surface of NK cells, enhance the conduction of killing signals in NK cells, and cause NK cells to release more cytotoxic substances such as perforin and granzymes, significantly improving the killing efficiency of tumor cells. Since it is difficult for tumor cells to escape the recognition of two antigens at the same time, the design of dual-target antibodies greatly reduces the possibility of tumor cell immune escape and ensures the long-term effectiveness of NK cell therapy.

[0084] Example 5: Preparation of coupled NK cells (Ab-NK cells)

[0085] 1. Experimental Principle

[0086] Utilizing the high affinity properties of the biotin-streptavidin system, the dual-targeted antibody (anti-GPC3 / anti-MUC1) prepared in Example 4 was specifically coupled to NK cells pre-labeled with biotin, allowing the antibody to stably bind to the NK cell surface, thereby endowing NK cells with the ability to target and recognize GPC3 and MUC1 double-positive tumor cells.

[0087] 2. Experimental steps

[0088] 1. Biotin labeling of NK cells: Take NK cells in the logarithmic growth phase (cord blood NK cells or human NK cell line NK-92MI or transgenic NK cells prepared in Example 3), wash twice with PBS, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in PBS containing 0.1% BSA and adjust the cell concentration to 1×107 cells / mL. Add Sulfo-NHS-LC-Biotin to a final concentration of 50 μg / mL, mix gently, and incubate at room temperature in the dark for 30 minutes. After the reaction is complete, terminate it with RPMI1640 medium supplemented with 10% FBS. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and wash the cells twice with PBS to remove unbound biotin.

[0089] 2. Preparation and labeling of dual-targeted antibodies: Adjust the antibody concentration of the prepared dual-targeted antibody (anti-GPC3 / anti-MUC1) to 1 mg / mL. Take an appropriate amount of dual-targeted antibody, add streptavidin-AlexaFluor488 at a final concentration of 10 μg / mL, and incubate at room temperature in the dark for 1 hour to allow the antibody to bind to the fluorescently labeled streptavidin. Use a PD-10 desalting column to purify the labeled antibody, remove unbound streptavidin-AlexaFluor 488, collect the eluate, and obtain the labeled dual-targeted antibody. It should be noted that during normal labeling, only streptavidin is labeled, and streptavidin-AlexaFluor 488 is not labeled (the purpose of labeling this substance is to subsequently detect and analyze the labeling efficiency).

[0090] 3. Antibody coupling to NK cells: Resuspend the biotin-labeled NK cells in PBS containing 0.1% BSA and adjust the cell concentration to 0.5×10 7 cells / mL. Add the labeled dual-targeting antibody to a final concentration of 50 μg / mL, mix gently, and incubate at room temperature in the dark for 1 hour, gently shaking every 15 minutes to promote antibody binding to biotin on the NK cell surface. After the coupling reaction, wash the cells twice with RPMI 1640 medium containing 10% FBS, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant to remove unbound antibody.

[0091] 4. Coupling effect detection: After washing the coupled NK cells with PBS, add PE-anti-human CD56 antibody (1:100 dilution) and incubate in the dark at 4°C for 30 minutes. After washing with PBS, add 500 μL PBS to resuspend the cells. Detect the AlexaFluor 488 fluorescence signal intensity in the CD56-positive cell population by flow cytometry to evaluate the coupling efficiency of the dual-targeting antibody. At the same time, set biotin-labeled NK cells without antibody coupling as a negative control.

[0092] 5. NK cell function detection after coupling

[0093] Cell killing experiment (MTT method): The cell density of HepG2 liver cancer cell line expressing GPC3 and MUC1 and the normal liver cell line L02 that does not express these two antigens were adjusted to 5×103 cells / well, seeded in 96-well plates, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The cell density of NK cells coupled with dual-targeting antibodies (experimental group) and NK cells without antibody coupling (negative control group) was adjusted to 5×10 4 Cells / mL were added to a 96-well plate containing tumor cells and control cells at an effector-target ratio (E / T) of 10:1. Six replicates were set up for each group. A control group containing only tumor cells or control cells, as well as a control group containing only effector cells, was also set up. After a further 48 hours of culture, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated at 37°C for 4 hours. The supernatant was then discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 minutes to fully dissolve the crystals. The absorbance at 490 nm (OD value) was measured on a microplate reader, and the cell killing rate was calculated according to the formula: Cell killing rate (%) = [1-(OD value of the experimental group - OD value of the effector cell control group) / OD value of the target cell control group] × 100%.

[0094] Cytokine secretion assay: After co-culture of dual-targeted antibody-coupled NK cells with HepG2 cells at an E / T ratio of 10:1 for 48 hours, the culture supernatant was collected and the secretion levels of cytokines such as IFN-γ and TNF-α were detected using ELISA kits. The specific operation was carried out according to the kit instructions.

[0095] 3. Test results

[0096] 1. Coupling efficiency detection: Flow cytometry results showed that the proportion of AlexaFluor 488-positive cells in NK cells coupled with dual-targeting antibodies reached 89.7%±3.2%, while the proportion of positive cells in the negative control group was only 2.1%±0.8%, indicating that the dual-targeting antibodies were successfully and efficiently coupled to the NK cell surface.

[0097] 2. Functional test results

[0098] Cell killing experiment: When the effector-target ratio was 10:1, the killing rate of NK cells coupled with dual-targeting antibodies against HepG2 cells was 82.3%±4.5%, which was significantly higher than the killing rate of NK cells without antibodies against HepG2 cells (35.6%±3.1%). However, the killing rates of both groups of cells against the normal liver cell line L02 were low and there was no significant difference (the killing rate of the experimental group was 8.7%±1.2%, and the killing rate of the control group was 6.5%±0.9%).

[0099] Cytokine secretion detection: After co-culture of NK cells conjugated with dual-targeting antibodies and HepG2 cells, the IFN-γ concentration in the culture supernatant was (780.5±62.3) pg / mL, and the TNF-α concentration was (612.8±55.7) pg / mL; after co-culture of NK cells without antibodies and HepG2 cells, the IFN-γ concentration was (230.1±30.5) pg / mL, and the TNF-α concentration was (150.3±25.6) pg / mL. The cytokine secretion level of the coupled group was significantly higher than that of the uncoupled group (P<0.001).

[0100] IV. Conclusion

[0101] In this example, dual-targeted antibodies (anti-GPC3 / anti-MUC1) were successfully coupled to the surface of NK cells (not the transgenic NK cells prepared in Example 3) through a biotin-streptavidin system. The coupling efficiency was high, and the coupled NK cells (i.e., coupled NK cells, labeled as Ab-NK cells) significantly enhanced their ability to kill double-positive tumor cells and significantly increased their cytokine secretion levels, providing an effective non-gene editing targeting strategy for NK cell-based tumor immunotherapy.

[0102] It should be noted that in this example, the dual-targeting antibody (anti-GPC3 / anti-MUC1) can also be coupled to the transgenic NK cells (i.e., TG-NK cells) prepared in Example 3, thereby achieving the triple function of NK cells, namely, enhancing NK cells (labeled as Comb-NK cells).

[0103] Example 6: Comparative study of the killing efficacy of different engineered NK cells in vitro

[0104] 1. Experimental Design

[0105] To systematically evaluate the anti-tumor efficacy of the transgenic NK cells and their combined application regimens of the present invention, four groups of effector cell control experiments were designed using standardized experimental parameters and rigorous statistical methods. The specific design is as follows:

[0106] 1. Effector Cell Grouping

[0107] (1) Control group (Con-NK): Unedited NK cells isolated from peripheral blood mononuclear cells (PBMC) of healthy donors were cultured in RPMI 1640 medium containing 10 ng / mL recombinant human interleukin-15 (rhIL-15) for 7 days.

[0108] (2) Coupled NK cell group (Ab-NK): prepared in Example 5, culture conditions were the same as the control group.

[0109] (3) Transgenic NK cell group (TG-NK): prepared as in Example 3, cultured in GT-T551 medium containing 1 ng / mL rhIL-15 for 7 days.

[0110] (4) Enhanced NK cell group (Comb-NK): Based on TG-NK cells, anti-GPC3 / anti-MUC1 dual-targeting antibody coupling was performed, and the culture conditions were the same as those of the transgenic NK cell group.

[0111] 2. Target Cell Selection

[0112] HepG2: human hepatocellular carcinoma cell line, GPC3 + / MUC1 + Double positive;

[0113] MDA-MB-468: human breast cancer cell line, GPC3 + / MUC1 + Double positive;

[0114] PANC-1: human pancreatic cancer cell line, GPC3- / MUC1 + Single positive;

[0115] SK-OV-3: human ovarian cancer cell line (ATCC, HTB-77), GPC3 + / MUC1-single positive.

[0116] 3. Experimental parameter control

[0117] All cells were cultured in a 37°C, 5% CO2 incubator. The effector-target ratio was fixed at 10:1, and 6 replicates were set up in each group. The experiment adopted a completely randomized group design, and sample processing and data collection followed the blind principle.

[0118] 2. Experimental Methods

[0119] 1. Effector Cell Preparation

[0120] Antibody coupling: take 5×10 6 Con-NK or TG-NK cells were prepared according to the method of Example 5.

[0121] Preparation of transgenic NK cells: Prepare according to the method in Example 3.

[0122] 2. Killing efficacy test

[0123] Cell labeling: Target cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester and incubated at 37°C for 20 min. The reaction was terminated with medium containing 10% FBS and the cells were washed twice to remove unbound dye.

[0124] Co-culture system: 100 μL target cell suspension (1×10 4 cells / well), and then 100 μL of effector cell suspension was added. At the same time, a target cell spontaneous apoptosis control group (containing only target cells) and a maximum killing control group (target cells + 1% Triton X-100) were set up.

[0125] Detection and analysis: After 4 hours of co-incubation, 7-aminoactinomycin D (7-AAD) was added to a final concentration of 1 μg / mL and incubated in the dark for 15 minutes. CFSE was detected by flow cytometry. + / 7-AAD + The proportion of double-positive cells was used to calculate the killing rate according to the formula.

[0126] 3. Experimental Results,The specific experimental results are shown in Table 1 and Table 2.

[0127] 1. Verification of Antibody Coupling Efficiency: Flow cytometry showed that the antibody coupling positivity rates in the Ab-NK group and Comb-NK group were 87.3%±4.2% and 89.1%±3.8%, respectively. There was no statistically significant difference between the groups (P=0.67), indicating that gene editing did not affect antibody coupling efficiency.

[0128] 2. Analysis of tumor killing efficacy

[0129] Double-positive tumor cells (HepG2, MDA-MB-468): The killing rate of the Comb-NK group was significantly higher than that of the other three groups (P<0.05), reaching 94.8% and 91.2%, respectively; the TG-NK group was second, increasing by 2.34 times (HepG2) and 2.78 times (MDA-MB-468) compared with the Con-NK group; the Ab-NK group increased by 1.72-1.98 times compared with the Con-NK group.

[0130] Single positive tumor cells: targeting PANC-1 (GPC3- / MUC1 + ) and SK-OV-3(GPC3 + / MUC1-), the killing rate of the TG-NK group was 2.24-3.44 times higher than that of the Con-NK group; the killing rate of the Comb-NK group was further increased to 78.3% and 75.4%, which was significantly better than that of the single group (P<0.05).

[0131] 3. Synergistic effect evaluation: In double-positive tumor cells, the Comb-NK group showed a 5.9-8.6% increase in killing rate compared to the TG-NK group. Based on median-effect principle analysis, the CI values ​​were 0.78 (HepG2) and 0.83 (MDA-MB-468), respectively, confirming the significant synergistic effect of gene editing and antibody coupling.

[0132] Table 1 Summary of experimental results of each group

[0133]

[0134]

[0135] Data were analyzed using GraphPad Prism 9.0. All data are presented as mean ± standard deviation. One-way analysis of variance was used for comparisons between multiple groups, and Tukey's multiple comparison test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant. The synergistic effect was evaluated by calculating the synergistic index (CI) using the median effect principle (Chou-Talalay method). A CI < 1 indicated synergistic effect. The results of this calculation are shown in Table 2.

[0136] Table 2 Summary of comparison results of each group of data

[0137]

[0138] 4. Experimental Summary

[0139] The TG-NK cells of the present invention, through UBE2F gene knockout and GPR34 antagonist peptide expression, exhibit significant cytotoxicity against tumor cells with diverse antigen expression patterns under low-dose IL-15 (1 ng / mL), achieving a 2-3.4-fold increase compared to traditional NK cells. Surface-coupled dual-targeting antibodies, anti-GPC3 / anti-MUC1, can enhance NK cell recognition of double-positive tumors, but have limited efficacy against single-positive tumors.

[0140] The combined application of gene editing and antibody conjugation (Comb-NK cells) produces a significant synergistic effect, increasing the killing rate of double-positive tumor cells to over 90%, providing an innovative strategy for overcoming tumor heterogeneity. While maintaining high killing efficacy, this combination regimen reduces the IL-15 dosage to 1 / 10 of the conventional dosage, effectively reducing the risk of cytokine-related toxicity and possessing significant clinical translational value.

[0141] Example 7: Comparative study of the anti-tumor effects of different engineered NK cells in vivo

[0142] 1. Experimental Design

[0143] To systematically verify the in vivo anti-tumor efficacy of the engineered NK cells of the present invention, a standardized humanized tumor xenograft model was constructed, and a control experiment was conducted using multi-dimensional evaluation indicators. The specific design is as follows:

[0144] 1. Experimental animals: 6-8 week old female NOD / SCID / IL2Rγnull (NSG) immunodeficient mice (SPF grade), housed in a barrier system, in accordance with ARRIVE 2.0 reporting standards.

[0145] 2. Tumor Model Construction

[0146] Cell source: HepG2 human liver cancer cells, qualified by STR identification and mycoplasma detection.

[0147] Inoculation method: HepG2 cells (1×10 7 cells) and Matrigel were mixed at a volume ratio of 1:1 and injected subcutaneously into the right back of mice (0.2 mL per mouse).

[0148] Grouping criteria: When the tumor volume reaches (100±10) mm 3 The mice were randomly divided into 5 groups (n=10 / group) using a random number table. There was no significant difference in tumor volume among the groups (P>0.05).

[0149] Table 3 Treatment groups and interventions

[0150]

[0151] 3. Detection indicators

[0152] Tumor growth monitoring: Use a vernier caliper to measure the long diameter (L) and short diameter (W) of the tumor every 3 days according to the formula V = L * W 2 / 2Calculate the volume and draw a growth curve.

[0153] Survival analysis: The survival time of mice was recorded, and the median survival time and survival rate were calculated.

[0154] Histopathology: At the end of the experiment, the mice were killed, and the tumor tissues were collected for HE staining and immunohistochemistry (IHC) to detect the expressions of Ki-67, CD31, and Caspase-3.

[0155] Flow cytometry: separation of spleen and tumor infiltrating lymphocytes (TILs), detection of human NK cell ratio (CD45 + CD56 + ) and activation markers (CD69 + NKG2D + )Express.

[0156] 2. Experimental Methods

[0157] 1. Preparation of effector cells: Con-NK, Ab-NK, TG-NK and Comb-NK cells were prepared strictly according to the above examples, ensuring that the cell activity was ≥90%, CD56 + CD3-purity ≥95%. After cell recovery, expand in medium containing corresponding cytokines for 48 hours and adjust the concentration to 5×10 7 cells / mL, resuspended in sterile PBS and injected immediately.

[0158] 2. Data Collection and Analysis: Tumor volume data were analyzed using two-way repeated-measures analysis of variance, with Bonferroni correction for inter-group comparisons. Survival analysis was performed using the Kaplan-Meier method, and inter-group comparisons were performed using the Log-rank test. Immunohistochemistry and flow cytometry data were analyzed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test to analyze inter-group differences. P < 0.05 was considered statistically significant.

[0159] 3. Experimental Results

[0160] 1. Tumor growth inhibition effect: The experiment monitored the tumor volume of each group of mice at different time points (Table 4) to explore the tumor growth inhibition effect of each treatment group. In the initial stage, there was no significant difference in tumor volume between the groups. From day 7 onwards, the Comb-NK group showed a significant tumor growth inhibition advantage, with a tumor volume of 105.2±9.1mm on day 7. 3 , compared with 254.6±23.1mm in the control group 3 The tumor volume of the Comb-NK group was reduced by 58.7%; on day 28, the tumor volume of the Comb-NK group was only 356.2±32.5mm 3 , while the control group reached 2546.8±187.3mm 3 The inhibition rate was as high as 86% (P<0.001). The TG-NK group and Ab-NK group had the second-best tumor inhibition effect. This shows that the combined application of gene editing and antibody conjugation can significantly delay tumor progression.

[0161] Table 4 Changes in tumor volume in each group (mm 3 , )

[0162] Time (days) control group Con-NK group Ab-NK group TG-NK group Comb-NK group 0 102.3±8.7 101.6±9.2 103.1±7.5 100.8±6.3 102.5±8.1 7 254.6±23.1 189.4±17.2 156.8±14.3 123.5±11.2 105.2±9.1 14 687.3±52.4 412.6±31.5 325.8±26.7 215.3±19.4 167.8±14.6 21 1423.7±105.6 789.2±62.3 598.4±47.5 356.7±31.8 245.3±22.4 28 2546.8±187.3 1245.9±98.7 897.6±72.1 523.4±46.3 356.2±32.5

[0163] 2. Survival Analysis: Table 5 presents the survival of mice in each group. The median survival of the control group was only 32 days, with a 35-day survival rate of 10%. The median survival of the Con-NK, Ab-NK, and TG-NK groups was 41 days, 48 ​​days, and 56 days, respectively, with 35-day survival rates of 30%, 40%, and 60%, respectively. The Comb-NK group performed the best, with a median survival extended to 68 days and a 35-day survival rate of 80%, a 112% increase compared to the control group (P<0.001). This result clearly demonstrates that the combined treatment regimen not only significantly prolongs the survival of tumor-bearing mice, but also has a good safety profile.

[0164] Table 5 Survival data of mice in each group

[0165] Group Median survival time (days) 35-day survival rate (%) control group 32 10 Con-NK group 41 30 Ab-NK group 48 40 TG-NK group 56 60 Comb-NK group 68 80

[0166] 3. Tumor tissue pathological analysis: Immunohistochemistry results (Table 6) reveal the effects of different treatments on tumor tissue from multiple perspectives. In terms of proliferation inhibition, the Ki-67 positivity rate in the control group was 78.3±5.2%, while it was significantly reduced to 24.5±2.3% in the Comb-NK group, with an inhibition rate of 69%, indicating that combined treatment can effectively curb tumor cell proliferation. In terms of angiogenesis inhibition, the CD31-positive blood vessel density in the control group was 42.6±3.1 cells / HPF, while it was reduced to 12.4±1.3 cells / HPF in the Comb-NK group, with an inhibition rate of 71%, indicating that tumor angiogenesis was significantly inhibited. In terms of apoptosis induction, the Caspase-3 positivity rate in the control group was only 12.1±2.3%, while it increased to 62.3±4.5% in the Comb-NK group, and the ability to induce apoptosis increased by 417%. In summary, combined treatment can achieve a highly effective anti-tumor effect by inhibiting proliferation, angiogenesis, and inducing apoptosis.

[0167] Table 6 Immunohistochemistry scoring results

[0168] index control group Con-NK group Ab-NK group TG-NK group Comb-NK group Ki-67 (%) 78.3±5.2 62.4±4.1 51.6±3.3 38.2±2.1 24.5±2.3 CD31 (cells / HPF) 42.6±3.1 31.5±2.4 25.3±2.2 18.7±1.5 12.4±1.3 Caspase-3 (%) 12.1±2.3 23.4±3.2 35.6±4.1 48.5±5.2 62.3±4.5

[0169] 4. Distribution and activation of NK cells in vivo: The flow cytometry test results (Table 7) analyzed the distribution and activation of NK cells in vivo. In terms of tumor infiltration ability, the proportion of tumor-infiltrating NK cells in the control group was only 2.1±0.3%, while that in the Comb-NK group reached 28.3±3.2%, an increase of 13 times, indicating that combined treatment can effectively promote the aggregation of NK cells to the tumor site. In terms of systemic distribution and activation, the proportion of spleen NK cells in the Comb-NK group (35.7±3.8%) and the activation marker CD69 + (45.2±4.7%), NKG2D + The expression levels of the combined treatment groups (89.5±3.8%) were significantly higher than those of the other groups, indicating that the NK cells after combined treatment had stronger survival ability in vivo and better functional activation effect, laying a cytological foundation for their efficient anti-tumor effect.

[0170] Table 7 Flow cytometry detection results

[0171] Detection indicators control group Con-NK group Ab-NK group TG-NK group Comb-NK group Proportion of tumor-infiltrating NK cells (%) 2.1±0.3 8.7±1.2 12.3±1.5 18.6±2.1 28.3±3.2 Splenic NK cell ratio (%) 3.5±0.4 10.2±1.3 15.6±1.8 22.4±2.5 35.7±3.8 <![CDATA[CD69 + NK cell ratio (%)]]> - 23.5±2.8 31.2±3.4 41.7±4.2 45.2±4.7 <![CDATA[NKG2D + NK cell ratio (%)]]> - 72.3±6.5 81.4±7.2 86.8±6.9 89.5±3.8

[0172] 4. Experimental Summary

[0173] This study systematically compared the in vivo antitumor effects of unedited NK cells, coupled NK cells (Ab-NK), transgenic NK cells (TG-NK), and enhanced NK cells (Comb-NK) using a humanized tumor xenograft model. Results showed that the TG-NK group, leveraging UBE2F gene knockout and GPR34 antagonism, reduced tumor volume by 58% and prolonged median survival by 75% compared to unedited NK cells under low-dose IL-15 conditions, confirming that gene editing can significantly enhance NK cell antitumor activity. While the Ab-NK group demonstrated targeted effects against GPC3 / MUC1 double-positive tumors, single-target inhibition was limited.

[0174] The enhanced NK cell (Comb-NK) group showed significant synergistic advantages: tumor volume was reduced by 86% compared with the control group, and survival was extended by 112%; immunohistochemistry confirmed that it could effectively inhibit tumor proliferation (Ki-67↓69%), angiogenesis (CD31↓71%), and induce cell apoptosis (Caspase-3↑417%); flow cytometry analysis showed that the tumor infiltration ability of NK cells in this group increased by 13 times, and the activation marker CD69 + NKG2D + In addition, no obvious toxicity was observed during the entire treatment process, indicating that the Comb-NK regimen has a good safety profile while maintaining high anti-tumor activity.

[0175] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A GPR34 antagonist peptide-transmembrane fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A method for enhancing NK cells, characterized in that: The enhanced NK cell marker is Comb-NK cell, wherein Comb-NK cell simultaneously contains the following elements: (1) expressing the GPR34 antagonist peptide-transmembrane fusion protein according to claim 1; (2) expressing a UBE2F gene editing element, wherein the gene editing element comprises sgRNA1 and sgRNA2 targeting UBE2F exon3, wherein the sequences of sgRNA1 and sgRNA2 are shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively; (3) A surface-coupled dual-targeting antibody comprising an anti-GPC3 scFv and an anti-MUC1 VHH, which are linked by GGGGSGGGGSGGGGS, wherein the nucleotide sequence encoding the dual-targeting antibody is shown in SEQ ID NO:

6.

3. The NK cell according to claim 2, characterized in that The amino acid sequence of the anti-GPC3 scFv is shown in SEQ ID NO: 4, and the amino acid sequence of the anti-MUC1 VHH is shown in SEQ ID NO:

5.

4. A method for preparing enhanced NK cells according to claim 2, characterized in that: The method comprises the following steps: (1) pX330-UBE2F-sgRNA and pLVX-GPR34-antagonist were introduced into NK cells using electroporation editing technology, with the electroporation parameters being 1500V / 20ms×2 pulses; (2) co-cultured and expanded with irradiated K562-mbIL-21 trophoblast cells in serum-free medium containing 1 ng / mL IL-15; (3) The dual-targeting antibody is coupled to the surface of NK cells through the biotin-streptavidin system to enhance NK cells.

5. A pharmaceutical composition, characterized in that The composition comprises the enhanced NK cells according to claim 2 and a pharmaceutically acceptable cryoprotectant.

6. A method for enhancing NK cell function in the preparation of GPC3 for treating GPC3 according to claim 2 + and / or MUC1 + Application in solid tumor drugs, the solid tumors include liver cancer, breast cancer, and pancreatic cancer.

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