NK immune cell capable of enhancing activity and application of NK immune cell in cancer treatment

Abstract

The invention discloses an activity-enhanced NK immune cell and an application thereof in cancer treatment. The NK cell stably expresses an anti-PD-L1 / CD16 coupled bispecific antibody through a lentiviral vector, and the antibody is formed by connecting an anti-PD-L1 single-domain antibody and an anti-CD16 single-domain antibody in series through a flexible connecting peptide (G4S). The NK immune cell with enhanced activity has three functions at the same time: tumor cells are targeted through anti-PD-L1 sdAb, and a PD-1 / PD-L1 immunosuppression pathway is blocked; the ADCC effect of the NK cell itself is activated through the anti-CD16 sdAb; by virtue of the small molecular weight characteristic of the single-domain antibody, the penetrating ability to solid tumors is remarkably enhanced. An in-vitro experiment shows that the killing rate of the sdAb-NK cell on various PD-L1 positive tumor cells is greatly improved compared with that of an unmodified NK cell, and the tumor penetrating power is improved by 3.7 times. In-vivo animal experiments show that the tumor inhibition rate is up to 84.8%, no obvious toxicity is shown, and a new efficient and safe strategy is provided for immunotherapy of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of immune cell therapy technology, and particularly relates to an enhanced NK immune cell and its use in cancer treatment. Background Technology

[0002] Natural killer (NK) cells are core effector cells of the innate immune system, possessing the ability to rapidly recognize and eliminate malignant cells without causing graft-versus-host disease. Their function depends on the balance of activating / inhibitory receptor signaling and CD16-mediated antibody-dependent cytotoxicity (ADCC). These characteristics give NK cells unique advantages in adoptive cell therapy for hematologic malignancies (such as leukemia and lymphoma) and certain solid tumors.

[0003] However, traditional NK cell therapy faces a dual bottleneck in the treatment of solid tumors. On the one hand, the tumor microenvironment inhibits NK cell function through multiple mechanisms: highly expressed immune checkpoint ligands such as PD-L1 bind to NK cell surface receptors (such as PD-1), activating inhibitory signaling pathways such as SHP-1 / 2, weakening the release of cytotoxic granules and the production of effector factors; simultaneously, inhibitory factors such as TGF-β and adenosine further exacerbate NK cell depletion. On the other hand, solid tumors evade natural NK cell recognition by downregulating NKG2D ligands (such as MICA / B) or hydrolyzing their proteins; unmodified NK cells, lacking tumor-specific targeting capabilities, infiltrate insufficiently at the lesion site and may inadvertently damage normal tissues.

[0004] Existing technologies often suffer from limitations due to their singular function: CAR-NK cells targeting hematologic tumor antigens such as CD19 have limited effectiveness against solid tumors and lack integrated immune checkpoint blockade; while NK cells secreting PD-1 antibodies can block some inhibitory signals, they cannot enhance tumor targeting, and systemic antibody secretion may disrupt immune homeostasis; CAR-NK cells targeting solid tumor antigens (such as EGFR) are still easily inactivated in the immunosuppressive microenvironment. These single-function modification strategies are insufficient to overcome the complex defense system of solid tumors.

[0005] Therefore, developing an integrated strategy that simultaneously addresses the three major challenges of targeted recognition, immunosuppression, and in vivo persistence is crucial for improving the efficacy of NK cell therapy for solid tumors. The ideal approach should utilize multiple gene modifications to enhance tumor-specific targeting, block key immune checkpoints, and provide endogenous cytokine support, thereby achieving synergistic effects and overcoming the limitations of existing therapies. Summary of the Invention

[0006] To address the above technical problems, this invention provides an enhanced NK immune cell and its use in cancer treatment.

[0007] Therefore, this invention discloses, in one aspect, an anti-PD-L1 / CD16 conjugate bispecific antibody, the amino acid sequence of which is shown in SEQ ID NO:4. The nucleotide sequence of the gene encoding the anti-PD-L1 / CD16 conjugate bispecific antibody is shown in SEQ ID NO:3. This invention also discloses an expression vector, pCHO-sdAb-BsAb, which contains the aforementioned encoding gene. Furthermore, this invention discloses a CHO cell containing the aforementioned expression vector.

[0008] In one aspect, the present invention also discloses an enhanced NK immune cell, wherein the enhanced NK immune cell stably expresses the anti-PD-L1 / CD16 conjugate bispecific antibody via a lentiviral vector. The lentiviral vector has a backbone of pLVX-EF1α-IRES-ZsGreen1, comprising an expression cassette consisting of an EF1α promoter, an anti-PD-L1 / CD16 conjugate bispecific antibody encoding gene, and an IRES-ZsGreen1 reporter gene.

[0009] In one aspect, the present invention also discloses a pharmaceutical composition comprising the aforementioned enhanced NK immune cells and pharmaceutically acceptable excipients.

[0010] In one aspect, the present invention also discloses the application of the aforementioned anti-PD-L1 / CD16 conjugate bispecific antibody in the preparation of enhanced NK immune cells. The present invention further discloses the use of the aforementioned enhanced NK immune cells in the preparation of cancer therapeutic drugs, wherein the cancer is PD-L1 positive cancer; the PD-L1 positive cancer includes melanoma, non-small cell lung cancer, liver cancer, and colorectal cancer.

[0011] This invention, through synergistic innovation in bispecific antibody design, lentiviral vector optimization, and natural killer immune cell preparation, effectively overcomes the shortcomings of existing natural killer cell therapy technologies. Its core advantages are: the bispecific antibody employs a tandem structure of a single-domain antibody and a (G4S)3 linker peptide, with a molecular weight of only about 30 kDa, achieving a tumor penetration rate of 45.6%, which is 3.7 times that of unmodified natural killer cells, effectively penetrating the tumor stroma to reach the core region; simultaneously, the binding constant between the anti-PD-L1 single-domain antibody and PD-L1 reaches 3.5 × 10⁻⁶. -9 M, the binding constant of the anti-CD16 single-domain antibody to CD16 reaches 2.8 × 10⁻⁶. -9M, and none of them have cross-binding homologous proteins, ensuring target specificity and binding stability, simultaneously achieving tumor-targeted binding, PD-1 / PD-L1 pathway blockade, and CD16-mediated antibody-dependent cytotoxicity activation, solving the problem of single-function in existing protocols. The lentiviral vector deletes the puromycin resistance gene, directly selecting positive cells via ZsGreen1 fluorescence signal, avoiding drug damage to natural killer cells, maintaining cell viability above 97%; the recombinant vector achieves a positive clone rate of 75%, and the viral packaging titer reaches 3.7 × 10⁻⁶. 11 The TU / mL concentration meets the requirements for large-scale preparation, and the positive rate of bispecific antibody expression in enhanced natural killer immune cells exceeds 92%. The cells maintain stable expression after 14 days of in vitro culture without significant attenuation. In vitro functional assays show that these cells achieve a killing rate of 90.5% ± 4.2% against PD-L1-high expressing melanoma A375 cells, and a killing rate of 78.5% ± 3.5% against low-to-medium PD-L1 expressing colorectal cancer HCT116 cells, representing a 40%-65% improvement compared to unmodified natural killer cells. This makes them suitable for various PD-L1-positive solid tumors. In an A375 tumor-bearing nude mouse model, the tumor volume was only 198.5 ± 30.2 mm² after 28 days of administration. 3 The tumor inhibition rate reached 84.8%, significantly higher than the 48.6% of unmodified natural killer cells, and it effectively inhibited the continuous growth of tumor volume. Safety assessment showed that the cells had no adverse effects on the hematopoietic system of mice, with white blood cell count and platelet count within the normal reference range; no damage to liver and kidney function, with normal serum alanine aminotransferase and creatinine levels, and no inflammatory infiltration or tissue damage in liver, kidney, and lung tissues after HE staining; the mice maintained stable body weight, without lethargy, reduced appetite, or other abnormalities, demonstrating good in vivo tolerability. This provides a clinically translatable, highly effective, and safe treatment option for PD-L1 positive cancers such as melanoma, non-small cell lung cancer, liver cancer, and colorectal cancer. Attached Figure Description

[0012] Figure 1 SDS-PAGE detection results of PD-L1-sdAb-08, where 1 represents PD-L1-sdAb-08.

[0013] Figure 2 SDS-PAGE results of anti-PD-L1 / CD16 conjugate bispecific antibody (BsAb), where 1 represents BsAb.

[0014] Figure 3 Results of plasmid double enzyme digestion identification.

[0015] Figure 4 The effects of transduction were observed using a fluorescence microscope.

[0016] Figure 5Flow cytometry results of anti-PD-L1 / CD16 conjugate bispecific antibody expression.

[0017] Figure 6 Western blot analysis of anti-PD-L1 / CD16 conjugate bispecific antibody expression in sdAb-NK cell culture supernatant, where 1 is sdAb-NK cell culture supernatant. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0019] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0020] Example 1: Design, preparation and testing of anti-PD-L1 / CD16 conjugated bispecific antibodies

[0021] I. Experimental Materials

[0022] 1. Immunogens and reagents: Recombinant human PD-L1 protein, recombinant human CD16 protein, recombinant human PD-L2 protein, and recombinant human CD32 protein were all from Sinocare; phage display vector pCANTAB 5E, competent cells TG1, restriction endonucleases BamHI / XhoI, T4 DNA ligase; surface plasmon resonance (SPR) chip CM5, SDS-PAGE gel assay kit, high performance liquid chromatography column, etc.

[0023] 2. Laboratory animals: One 6-month-old female alpaca, weighing 65 kg, with no known infectious diseases.

[0024] II. Experimental Procedure

[0025] (I) Preparation and testing of anti-PD-L1 sdAb

[0026] 1. Alpaca immune and antiserum titer detection

[0027] (1) Immunization regimen: Using human PD-L1 recombinant protein as the immunogen, the "basic immunization + booster immunization" strategy was adopted: On day 0 (basic immunization), PD-L1 protein (0.5 mg / mL) was emulsified with Freund's complete adjuvant (Sigma) at a ratio of 1:1 and injected subcutaneously at multiple points on the back of the neck, with a total dose of 100 μg / animal; On days 14, 28 and 42 (booster immunization), PD-L1 protein (0.5 mg / mL) was emulsified with Freund's incomplete adjuvant at a ratio of 1:1, with the same dose as basic immunization; On day 56, 5 mL of blood was collected from the tail vein, the serum was separated and stored at -20℃.

[0028] (2) Potency assay (indirect ELISA): 100 μL of PD-L1 protein (2 μg / mL, coating buffer: 0.05 M carbonate buffer, pH 9.6) was added to each well of a 96-well plate and incubated at 4°C for 12 h; 200 μL of 5% skim milk-PBS was added to each well and incubated at 37°C for 2 h; serum was added at dilutions of 1:100, 1:1000, and 1:1000. 4 1:10 5 1:10 6 Serial dilutions were performed, 100 μL / well, incubated at 37°C for 1 h; HRP-labeled anti-alpaca IgG antibody (1:5000 dilution) 100 μL / well, incubated at 37°C for 1 h; TMB chromogenic solution 100 μL / well, reaction in the dark for 15 min, terminated with 2M H2SO4, and OD450 value was measured (OD450 ≥ 0.2 and twice that of the negative control was considered positive). Results showed that serum diluted 1:10... 5 OD450 = 1.82 ± 0.15 (n = 3), diluted 1:10 6 At OD450 = 0.35 ± 0.04, the potency was determined to be 1:10. 5 This meets the needs of document library construction.

[0029] 2. Construction of anti-PD-L1 sdAb phage display library

[0030] (1) Lymphocyte isolation and RNA extraction: Alpaca were slaughtered on day 56, and 5g of spleen tissue was aseptically taken. After grinding, lymphocytes were separated by centrifugation (2000rpm, 20min) using lymphocyte separation medium. Total RNA was extracted using Trizol reagent. The RNA purity was detected by Nanodrop: A260 / A280=1.92, A260 / A230=2.05. Electrophoresis showed that the 28S / 18S bands were clear and there was no degradation.

[0031] (2) cDNA synthesis and sdAb gene amplification: cDNA was synthesized using Oligo (dT) primers and M-MLV reverse transcriptase under the following conditions: 42℃ for 60 min and 70℃ for 15 min. The sdAb gene was amplified using alpaca sdAb-specific primers in a 50 μL reaction system (2×Taq Mix 25 μL, primers 1 μL each, cDNA 2 μL, ddH2O 21 μL). The following conditions were: 95℃ for 5 min, 95℃ for 30 s, 58℃ for 30 s, 72℃ for 40 s, 30 cycles, and 72℃ for 10 min. Agarose gel electrophoresis showed a single band of about 400 bp. After gel extraction and recovery, the concentration was measured to be 85 ng / μL.

[0032] (3) Library construction and library capacity determination: The recovered sdAb gene was ligated with the pCANTAB5E vector digested with BamH I / Xho I (T4 ligase, reaction at 16℃ for 12h), and the ligation product was transformed into TG1 competent cells (electrotransformation: 2.5kV, 200Ω, 25μF); 10μL of the transformation solution was plated on LB-ampicillin plates and incubated at 37℃ for 12h. The colony count was 2.3×10⁻⁶. 3 One, estimated storage capacity = 2.3 × 10 3 ×(1000 / 10)=2.3×10 5 CFU was used to meet the screening requirements; the remaining transformation medium was used for large-scale culture to prepare a primary phage library.

[0033] 3. Anti-PD-L1 sdAb panning and positive clone identification

[0034] (1) Three rounds of screening: In the first round, PD-L1 protein (10 μg / mL) was coated onto immunotubes and incubated at 4°C for 12 h; after blocking, a primary phage library (1×10⁻⁶) was added. 11 PFU), incubated at 37℃ for 2 h; washed 10 times with PBST, eluted bound phages with 0.2M Glycine-HCl (pH 2.2), and the titer was measured after neutralization: 1.2 × 10⁻⁶. 5 PFU; In the second round, the PD-L1 protein concentration was reduced to 5 μg / mL, and the protein was washed 15 times with PBST. The titer after elution was 3.5 × 10⁻⁶. 7 PFU (enrichment factor ≈ 292-fold); in the third round, the PD-L1 protein concentration was reduced to 2 μg / mL, washed 20 times with PBST, and the titer after elution was 8.9 × 10⁻⁶. 9 PFU (enrichment factor ≈ 254 times).

[0035] (2) Screening of positive clones (phage ELISA): 30 colonies were randomly selected after the third round of screening. After inducing phage expression, the binding activity with PD-L1 was detected. The results showed that 22 clones had OD450≥1.0 (negative control OD450<0.1), with a positive rate of 73.3%. The clone with the highest OD value (numbered PD-L1-sdAb-08) was selected for sequencing, and the amino acid sequence was shown in SEQ ID NO:1.

[0036] 4. Prokaryotic expression and purification of PD-L1-sdAb-08

[0037] The PD-L1-sdAb-08 gene was inserted into the pET-28a vector (His tag), transformed into BL21 (DE3) competent cells, and sequenced to verify its correctness. Single colonies were picked and inoculated into 5 mL of LB-Kana medium, and cultured at 37°C with shaking for 12 h; then transferred to 500 mL of LB-Kana medium at a ratio of 1:100, and cultured at 37°C until OD600 = 0.6. IPTG (final concentration 0.5 mM) was added, and the cells were induced at 25°C for 16 h; the cells were collected by centrifugation (8000 rpm, 10 min, 4°C), sonicated (300 W power, 3 s on, 5 s off, 30 min total), and centrifuged (12000 rpm, 20 min, 4°C) to collect the supernatant. The supernatant was loaded onto a Ni-NTA column, equilibrated with PBS containing 20 mM imidazole, and eluted with PBS containing 500 mM imidazole. The eluent was further purified by Superdex 75 gel filtration chromatography, and the main peak was collected. After mixing, the mixture was filtered through a 220 nm filter membrane for sterilization and aliquoted. The BCA concentration was determined to be 1.2 mg / mL, and the result was analyzed by SDS-PAGE. Figure 1 A single band appears at approximately 15 kDa, with a purity of over 95%.

[0038] 5. Detection of anti-PD-L1 sdAb binding activity and specificity (SPR)

[0039] (1) Experimental steps: The CM5 chip was activated with EDC / NHS for 10 min. PD-L1 protein (5 μg / mL, pH 4.5 acetate buffer) was coupled to the chip surface at a coupling density of about 2000 RU. PD-L1-sdAb-08 was diluted with HBS-EP buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% Tween-20) to 0.1, 0.5, 1, 5, and 10 nM at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 300 s. The chip was regenerated with 10 mM Glycine-HCl (pH 2.0) at a flow rate of 30 μL / min for 30 s.

[0040] (2) Experimental results: Typical "rapid binding-slow dissociation" curves were observed at different concentrations. After fitting, the binding constant KD was found to be 3.5 × 10⁻⁶. -9 M (Table 1), compared to before mutation (KD=1.2×10 -8 The M) was increased by 3.4 times. When the PD-L2 protein was coupled to the chip, the binding signal of 10 nM PD-L1-sdAb-08 to PD-L2 was <50 RU (PD-L1 binding signal >1500 RU), indicating no cross-binding.

[0041] Table 1. SPR binding parameters of anti-PD-L1-sdAb-08 (n=3, x±s)

[0042]

[0043] (II) Preparation and testing of anti-CD16 sdAb

[0044] 1. Preparation of CD16-sdAb-06

[0045] An anti-CD16 sdAb was prepared according to the above-described method for preparing PD-L1-sdAb-08 and named CD16-sdAb-06. Its amino acid sequence is shown in SEQ ID NO:2.

[0046] 2. Detection of anti-CD16 sdAb binding activity and specificity

[0047] (1) Binding activity (SPR): CD16 protein (5 μg / mL, pH 4.0 acetate buffer) was coupled to a CM5 chip at a coupling density of 1800 RU; CD16-sdAb-06 was diluted to 0.1, 0.5, 1, 5, and 10 nM, with other parameters remaining the same; the results showed that KD = 2.8 × 10 -9 M (Table 2), slow dissociation rate (kd=0.8×10) -4 s -1 It exhibits good stability.

[0048] Table 2 SPR binding parameters of CD16-sdAb-06 (n=3, x±s)

[0049]

[0050] (2) Specific detection (ELISA): CD16 and CD32 proteins were coated on 96-well plates (2 μg / mL); the primary antibody was CD16-sdAb-06 (1 μg / mL) and the secondary antibody was HRP-anti-His antibody (1:5000); the results showed that the OD450 of the CD16 group was 2.15±0.12 and the OD450 of the CD32 group was 0.11±0.02 (n=3), with no cross-binding to CD32.

[0051] (III) Preparation of anti-PD-L1 / CD16 conjugate bispecific antibody

[0052] 1. Design and synthesis of single-chain bispecific antibody encoding genes

[0053] The nucleotide sequence of the anti-PD-L1 / CD16 conjugate bispecific antibody was tandemly linked according to the sequence of "PD-L1-sdAb-08 gene-(G4S)3 linker peptide gene-CD16-sdAb-06 gene" and optimized based on human codon preference. The optimized nucleotide sequence of the anti-PD-L1 / CD16 conjugate bispecific antibody is shown in SEQ ID NO:3. The sequencing results of the synthesized gene show that it matches the designed sequence 100% and there are no base deletions or mutations.

[0054] 2. Eukaryotic expression and purification of bispecific antibodies

[0055] (1) Insert the synthesized gene into the pCHO1.0 vector (containing CMV promoter and DHFR selection marker), verify the correctness of BamHI / XhoI double digestion, and construct the recombinant vector pCHO-sdAb-BsAb.

[0056] (2) CHO cell transfection and screening: pCHO-sdAb-BsAb was transfected into CHO-K1 cells using Lipofectamine 3000; 24 h after transfection, serum-free CHO medium (Gibco) was used, and the concentration of methotrexate (MTX) was gradually increased (from 0.1 μM to 1 μM) to screen for high expression clones; the antibody concentration in the culture supernatant of the clones was measured by ELISA, and the highest expression clone (numbered CHO-BsAb-12) was selected, with an expression level of 350 mg / L.

[0057] (3) Protein purification: The supernatant was purified by Protein A chromatography column. Elution conditions: 0.1M citrate buffer, pH 3.0; elution buffer was dialyzed with PBS (4℃, 24h, buffer changed 3 times). SDS-PAGE analysis showed that ( Figure 2 The result was a single band of approximately 30 kDa. The concentration was adjusted to 1 mg / mL and stored at -80°C (named BsAb, whose amino acid sequence is shown in SEQ ID NO:4).

[0058] (iv) Functional testing of bispecific antibodies (simultaneous binding ability of dual targets)

[0059] 1. Experimental procedure: In the first cycle, PD-L1 protein (2000 RU) was coupled to the CM5 chip, BsAb (5 nM) was injected, and the binding signal was recorded; in the second cycle, CD16 protein (5 nM) was injected onto the same chip surface, and the binding signal was recorded twice.

[0060] 2. Results: The binding signal of BsAb to PD-L1 in the first cycle was 1850 RU, and the binding signal of CD16 to BsAb in the second cycle was 920 RU, proving that BsAb can bind to PD-L1 and CD16 simultaneously, and the dual-target binding function is normal.

[0061] III. Experiment Summary

[0062] This embodiment successfully prepared an anti-PD-L1 / CD16 conjugated bispecific antibody (BsAb), whose core performance indicators met the design requirements: ① High dual-target binding activity (PD-L1 KD=3.5×10⁻⁶). -9 M, CD16 KD = 2.8 × 10 -9 M); ② High specificity (no PD-L2 or CD32 cross-binding); ③ Intact structure (purity 98.5%); ④ Can bind to PD-L1 and CD16 simultaneously, possessing the dual function of "targeting tumor cells (PD-L1) + activating NK cells (CD16)," laying the foundation for subsequent gene modification of NK cells and detection of anti-tumor activity.

[0063] Example 2: Preparation and testing of enhanced NK immune cells

[0064] I. Experimental Materials

[0065] 1. Vectors and plasmids: Lentiviral backbone pLVX-EF1α-IRES-ZsGreen1 (containing EF1α promoter and ZsGreen1 reporter gene), packaging plasmid pSPAX2 (providing gag / pol gene), and envelope plasmid pMD2.G (providing VSV-G envelope protein).

[0066] 2. Cell lines: 293T cells (adherent growth, lentivirus packaging), primary human NK cells (isolated from peripheral blood PBMCs of healthy volunteers), and four types of tumor cells (A375 melanoma cells, PD-L1 positive rate of 92% by flow cytometry; H1299 non-small cell lung cancer cells, PD-L1 positive rate of 68%; HepG2 liver cancer cells, PD-L1 positive rate of 55%; HCT116 colorectal cancer cells, PD-L1 positive rate of 42%).

[0067] 3. Reagents: RPMI 1640 medium (Gibco) containing L-glutamine and phenol red-free; fetal bovine serum (FBS, Gibco) heat-inactivated at 56°C for 30 min; penicillin and streptomycin-containing antibiotics; BamHI / XhoI restriction endonucleases with 10 U / μL activity and CutSmart Buffer; T4 DNA ligase with 400 U / μL activity and 10× ligation buffer; and Lipofectamine containing P3000 Enhancer. 3000 transfection reagents, 10 mg / mL sterile filtered polyglobulin, NK cell magnetic bead sorting kit with anti-human CD56 magnetic beads and a sorting purity ≥90%, flow cytometry antibodies including anti-human CD56-PE, anti-human CD16-FITC, anti-human PD-L1-APC, and rabbit anti-sdAb polyclonal antibody with a titer of 1:5000 (prepared in our laboratory), LDH release assay kit, CFSE dye in 10 mM stock solution and Cy3 dye in 5 mM stock solution, and Matrigel matrix gel with reduced growth factors at a concentration of 8 mg / mL.

[0068] II. Experimental Procedure

[0069] (I) Construction of lentiviral vectors

[0070] 1. Preparation of the single-chain bispecific antibody encoding gene: Based on the amino acid sequence of the bispecific antibody (SEQ ID NO. 4), the encoding gene (SEQ ID NO: 3) was designed according to human codon preference. The full-length gene was synthesized by Shanghai Sangon Biotech. A BamHI restriction site was added to the 5' end and an XhoI restriction site was added to the 3' end. Sequencing verification showed that it was 100% correct. The Nanodrop concentration was 250 ng / μL, A260 / A280 = 1.88, and there was no impurity contamination.

[0071] 2. Vector digestion and recovery

[0072] (1) The enzyme digestion reaction system is shown in Table 3.

[0073] Table 3 Enzyme digestion reaction system (50 μL)

[0074]

[0075] (2) Reaction conditions: 37℃ water bath for 2 hours, 65℃ for 20 minutes to inactivate enzyme activity;

[0076] (3) Vector fragment recovery: The enzyme digestion product was subjected to 1% agarose gel electrophoresis, and a vector backbone fragment of about 7.5kb was excised. The fragment was recovered using a DNA gel recovery kit, and the concentration was measured to be 80ng / μL with a recovery rate of 65%.

[0077] 3. Ligation of the target gene with the vector: Ligation was performed in a 16℃ metal bath for 16 hours to construct the recombinant vector pLVX-sdAb. The ligation system is shown in Table 4.

[0078] Table 4. Connection reaction system (20 μL)

[0079]

[0080] 4. Transformation and Cloning Screening

[0081] (1) Transformation of competent cells: Take 5 μL of ligation product and add 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 45 s, incubate on ice for 2 min, add 900 μL of LB medium (antibiotic-free), and culture at 37℃ with shaking for 1 h;

[0082] (2) Cloning screening: Spread 100 μL of bacterial culture onto LB-amphetamine plates (amphetamine concentration 100 μg / mL), incubate at 37℃ for 12 h, pick 8 single clones and inoculate into 5 mL of LB-amphetamine medium, shake culture at 37℃ for 12 h, and extract plasmids using a kit.

[0083] (II) Identification of Lentiviral Vectors

[0084] 1. Enzyme digestion identification: Plasmids from 8 clones were digested with BamHI / XhoI according to the "Vector Enzyme Digestion Procedure", and detected by 1% agarose gel electrophoresis; the results showed that 6 clones simultaneously exhibited two bands, approximately 7.5kb (vector backbone) and approximately 800bp (target gene). Figure 3 The positive clone rate was 75%, and clone 3 (pLVX-sdAb-3) was selected for subsequent experiments.

[0085] 2. Sequencing Validation: pLVX-sdAb-3 was sent to Shanghai Sangon Biotech for Sanger sequencing. The results showed that the sequenced sequence matched the designed target gene sequence 100%, with no base insertions, deletions, or mutations, indicating successful vector construction. Nanodrop analysis showed that the pLVX-sdAb-3 concentration was 1.2 μg / μL, A260 / A280 = 1.89, and A260 / A230 = 2.01, meeting the requirements for lentiviral packaging.

[0086] (III) Packaging and titer determination of recombinant lentivirus

[0087] 1. Pretreatment of 293T cells

[0088] (1) Cell resuscitation and passage: The frozen 293T cells were resuscitated in DMEM medium containing 10% FBS and 1% penicillin and antibiotics, cultured at 37°C with 5% CO2, and passaged 2-3 times until the cells were stable (viability ≥95%).

[0089] (2) Optimization of seeding density: One day before transfection, 293T cells were seeded at a density of 5 × 10⁻⁶ cells / day. 6 Cells were inoculated into 10cm culture dishes (10mL of culture medium per dish). After 12 hours of culture, the cell confluence reached 70%-80%, which met the transfection requirements.

[0090] 2. Preparation of transfection system: Gently mix solution A and solution B, and incubate at room temperature for 15 min to form a DNA-liposome complex. See Tables 5 and 6 for the specific details of solutions A and B.

[0091] Table 5 Solution A (Plasmid Mixture)

[0092]

[0093] Table 6 Solution B (Transfection reagent mixture)

[0094]

[0095] 3. Transfection and virus collection: The complex was slowly added dropwise to a 293T cell culture dish, gently shaken, and incubated at 37°C with 5% CO2. 6 h after transfection, the culture medium containing the complex was discarded, and 10 mL of fresh DMEM (containing 10% FBS, antibiotic-free) was added, and the cells were incubated for another 6 h. The supernatant was collected at 48 h and 72 h after transfection, centrifuged at 3000 rpm for 10 min at 4°C to remove cell debris, and filtered through a 0.45 μm PVDF membrane to obtain the crude virus extract.

[0096] 4. Virus Concentration and Purity Detection: The crude virus extract was added to an SW32Ti centrifuge tube and centrifuged at 28,000 rpm for 2 hours at 4°C. The supernatant was discarded, and the virus pellet was resuspended in 1 mL of NK cell culture medium (containing IL-2 100 IU / mL and IL-15 50 IU / mL). The pellet was incubated at 4°C for 1 hour to allow the virus to fully dissolve, resulting in a concentrated virus solution. The total protein concentration in the concentrated solution was measured using a BCA protein concentration assay kit and found to be 0.8 mg / mL (low content of impurities, meeting the virus purity requirements).

[0097] 5. Titer determination: The concentrated virus solution was serially diluted 10-fold using DMEM medium (10... -1 Up to 10 -8 Add 100 μL of virus solution of each dilution to a 96-well plate, followed by inoculation of 2 × 10⁶ cells per well. 4293T cells were cultured in triplicate for each dilution. After 72 hours of incubation at 37°C and 5% CO2, cells were digested with trypsin and collected. The proportion of ZsGreen1-positive cells was determined by flow cytometry. Viral titer (TU / mL) was calculated using the following formula: Titer = (Proportion of positive cells × Total number of cells at inoculation × Dilution factor) / Virus volume. Results and calculations were compared within 10... -6 At the dilution, the percentage of ZsGreen1-positive cells was (18.5 ± 2.1)% (n=3). Substituting these values ​​into the formula, the titer was calculated as: titer = (0.185 × 2 × 10⁻⁶) / (n=3). 4 ×10 6 ) / 0.1mL = 3.7 × 10 11 TU / mL. Therefore, the titer of this concentrated virus solution is 3.7 × 10⁻⁶. 11 TU / mL. To visually demonstrate the transduction effect, observation was performed using a fluorescence microscope after 72 hours of culture. -2 A large number of cells in the dilution wells emitted green fluorescence. Figure 4 ).

[0098] (iv) Preparation and identification of enhanced active NK immune cells

[0099] 1. PBMC Isolation from Healthy Individuals: 50 mL of peripheral blood was collected from healthy volunteers (anticoagulated with EDTA-K2, anticoagulant ratio 1:9), stored at 4℃, and processed within 2 hours. In a 50 mL centrifuge tube, 20 mL of Ficoll-Paque Plus (density 1.077 g / mL) was added, and 25 mL of diluted peripheral blood (diluted 1:1 with PBS) was slowly added along the tube wall. The tube was centrifuged at 1500 rpm for 20 min at 20℃ (acceleration / deceleration set to minimum). After centrifugation, the solution separated into three layers. The middle white membrane layer (PBMCs) was transferred to a new centrifuge tube, 5 times the volume of PBS was added, and the tube was centrifuged at 1200 rpm for 10 min. The tube was washed twice and finally resuspended in RPMI 1640 medium. The results showed that 1.2 × 10⁶ PBMCs were isolated from 50 mL of peripheral blood. 7 The trypan blue staining viability was 98.5%.

[0100] 2. NK cell magnetic bead sorting: Take 1×10 7Centrifuge PBMCs at 1200 rpm for 10 min, discard the supernatant, and resuspend in 300 μL buffer (PBS + 0.5% BSA + 2 mM EDTA). Add 100 μL of anti-human CD56 magnetic beads and incubate at 4°C in the dark for 15 min, gently mixing twice during incubation. Install the LS sorting column in a magnetic field, equilibrate the column with 3 mL of buffer, load the magnetic bead-cell mixture onto the column, wash the column three times with 3 mL of buffer, and collect the effluent (negative cells). Remove the column, quickly wash the column with 5 mL of buffer, and collect CD56+ cells (NK cells). The results showed that the number of NK cells after sorting was 2.4 × 10⁻⁶. 6 The recovery rate was 20% ± 3%, and the proportion of CD56+ cells detected by flow cytometry reached 91.5% ± 2.3%.

[0101] 3. NK cell activation culture: The sorted NK cells were cultured at a ratio of 1×10⁻⁶. 6 NK cells were seeded at 1 mL / well into 24-well plates, and 1 mL of NK cell-specific culture medium (containing 10% FBS, IL-2 100 IU / mL, and IL-15 50 IU / mL) was added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 h (after activation, the NK cells became larger and developed pseudopodia). After 24 h of incubation, the trypan blue staining viability was 97.2% ± 1.8%, and the plates were ready for viral infection.

[0102] 4. Viral infection of NK cells

[0103] (1) MOI preliminary experiment: three gradients were set up with MOI=5, 10 and 15, with 3 replicates for each gradient. The positive rate of ZsGreen1 was detected 72h after infection. The results showed that the positive rate was 42.3%±4.5% when MOI=5, 78.5%±3.2% when MOI=10 and 80.1%±2.9% when MOI=15. MOI=10 was selected for formal experiment (considering both positive rate and cytotoxicity).

[0104] (2) Formal infection: Add 1×10 to each well of the 24-well plate 6 One activated NK cell was added with recombinant lentivirus (MOI=10, at a dose of 1×10⁻⁶). 6 10 cells × 10 TU / cell = 1 × 10 7 TU, such as a viral fluid concentration of 1.2 × 10⁻⁶ 8 If the concentration of TU / mL is 83 μL, add 83 μL of virus solution and polybrene (final concentration 8 μg / mL), and mix gently. 24 h after infection, discard the virus-containing culture medium, add 1 mL of fresh NK cell culture medium, and continue culturing for 72 h.

[0105] 5. sdAb-NK cell screening and identification

[0106] (1) ZsGreen1 positive sorting: After culturing for 72 hours, the cells were digested and collected. ZsGreen1 positive cells were sorted by flow cytometry. The positive rate after sorting was 96.8%±1.5%. They were labeled as sdAb-NK cells and then cultured, cryopreserved or tested.

[0107] (2) Verification of bispecific antibody expression: Take 1×10 5 After sorting, 10 μL of anti-single-domain antibody-specific antibody (1:100 dilution) was added, and the cells were incubated at 4°C in the dark for 30 min. After washing twice with PBS, 5 μL of FITC-labeled goat anti-rabbit secondary antibody (1:200 dilution) was added, and the cells were incubated at 4°C in the dark for 20 min. After washing twice with PBS, the cells were analyzed by flow cytometry. The results showed that the positive rate of bispecific antibody expression reached 92.3% ± 2.1%. Figure 5 );

[0108] (3) Western blot verification: The supernatant of sdAb-NK cell culture was collected, subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, incubated with anti-single-domain antibody specific antibody (1:1000 dilution), and developed with HRP-labeled secondary antibody (1:5000 dilution). The results showed a band of about 30 kDa. Figure 6 This proves that the antibody was successfully expressed.

[0109] (v) In vitro functional testing

[0110] 1. In vitro antitumor activity assay

[0111] (1) Tumor cell pretreatment: A375, H1299, HepG2, and HCT116 cells were cultured in RPMI 1640 medium containing 10% FBS to the logarithmic growth phase, and the concentration was adjusted to 1×10⁻⁶ after digestion. 5 cells / mL;

[0112] (2) Inoculation and adhesion: Add 100 μL of tumor cell suspension (1×10⁻⁶) to each well of a 96-well plate. 4 Cells per well), cultured at 5% CO2 and 37℃ for 24 hours to ensure cell adhesion (adhesion rate ≥90%).

[0113] (3) Effector cell preparation: Unmodified NK cells and sdAb-NK cells were adjusted to a concentration of 5×10⁻⁶ cells using culture medium. 5 1 / mL (target-efficacy ratio 5:1);

[0114] (4) Grouping (Table 7) and co-culture conditions: 5% CO2, 37℃ for 4h;

[0115] Table 7 Experimental Grouping and Treatment

[0116]

[0117] (5) LDH detection: Aspirate 100 μL of supernatant into a new 96-well plate from each well, add 100 μL of LDH detection reagent, react at room temperature in the dark for 30 min, and measure the OD490 value with an enzyme-linked immunosorbent assay (ELISA) reader;

[0118] (6) Experimental results: Among the four types of tumor cells, sdAb-NK cells showed the best killing effect on A375 cells with high PD-L1 expression, with a killing rate of 90.5% ± 4.2%, which was 65.1% higher than that of unmodified NK cells. Even against HCT116 cells with low PD-L1 expression, the killing rate of sdAb-NK cells still reached 78.5% ± 3.5%, which was 77.6% higher than that of the control group. This proves that the bispecific antibody can activate ADCC effect by targeting CD16, which can make up for the effect of insufficient PD-L1 expression and achieve broad-spectrum anti-tumor activity. See Table 8 for details.

[0119] Table 8. Calculation results of OD490 values ​​and killing rates for each tumor cell line.

[0120]

[0121] 2. Tumor penetration ability detection

[0122] (1) Validation of tumor matrix model: 50 μL of Matrigel matrix gel (8 mg / mL) was laid in the upper chamber of Transwell (8 μm pore size), cured at 37℃ for 30 min, and then A375 cells (5 × 10⁶ cells) were seeded. 4 After culturing for 48 hours, the cells were observed under an inverted microscope to form a dense three-dimensional spherical structure (approximately 150-200 μm in diameter). Trypan blue staining showed a cell viability of 90% ± 3.5%, indicating successful model construction.

[0123] (2) Cell labeling efficiency: Unmodified NK cells were labeled with CFSE (final concentration 5 μM), and sdAb-NK cells were labeled with Cy3 (final concentration 3 μM). After incubation at 37℃ for 30 min, the labeling efficiency was detected by flow cytometry: CFSE labeling rate was 95.2% ± 2.1%, Cy3 labeling rate was 94.8% ± 2.3%, and the cell survival rate after labeling was > 90%, with no obvious toxicity.

[0124] (3) Penetration ability test results: Labeled NK cells (1×10⁻⁶) were added to the lower chamber. 5After culturing for 24 hours, the A375 tumor matrix in the upper chamber was collected and digested with trypsin into a single-cell suspension. The proportion of NK cells in the suspension was detected by flow cytometry (with labeled fluorescence as a positive signal): the proportion of unmodified NK cells penetrating into the tumor matrix was 12.3%±2.5%, and they were mainly distributed on the surface of the tumor matrix; the penetration proportion of sdAb-NK cells was 45.6%±3.8%, which was 3.7 times that of unmodified NK cells.

[0125] (4) Results analysis: The sdAb-NK cell penetration ability was significantly enhanced, mainly due to the single-domain antibody structure of the bispecific antibody: small molecule antibody can reduce the steric hindrance of tumor matrix (such as collagen and glycosaminoglycan), and at the same time, through the specific binding of anti-PD-L1 sdAb to PD-L1 on the surface of tumor cells, it enhances the migration and retention ability of NK cells in the matrix, laying the foundation for clearing tumor core cells.

[0126] III. Experiment Summary

[0127] This embodiment successfully prepared enhanced NK immune cells (sdAb-NK cells) through a complete process of "lentiviral vector construction - viral packaging - NK cell sorting and infection". The core advantages are as follows:

[0128] 1. Highly efficient vector construction: The lentiviral vector with the puromycin resistance gene deleted allows for direct screening via ZsGreen1, simplifying the process and avoiding drug damage to NK cells. The vector achieves a positive clone rate of 75%.

[0129] 2. High-quality NK cell preparation: NK cell purity reaches over 91.5% through magnetic bead sorting combined with activation culture, antibody expression rate exceeds 92% after viral infection, and viability remains above 97%.

[0130] 3. Significantly enhanced in vitro function: sdAb-NK cells not only increased the killing rate of various PD-L1 positive tumor cells by 40%-77.6%, but also showed the highest increase (77.6%) in HCT116 cells with low PD-L1 expression. This was attributed to the bispecific antibody activating ADCC through CD16, compensating for the effect of insufficient PD-L1 expression. In addition, due to the small molecule characteristics of the single-domain antibody, the tumor penetration ability was increased by 3.7 times. These results lay a solid foundation for subsequent in vivo anti-tumor experiments.

[0131] Example 3: In vivo antitumor activity detection and safety assessment

[0132] I. Experimental Materials

[0133] 1. Laboratory animals: 18 female BALB / c nude mice aged 6-8 weeks, weighing 18.2-21.8g, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; acclimatization feeding for 7 days.

[0134] 2. Cells and reagents: A375 melanoma cells (log phase, viability ≥95%), unmodified NK cells, sdAb-NK cells; Matrigel (growth factor reduced type, 8 mg / mL), physiological saline (0.9% NaCl), etc.

[0135] II. Experimental Procedure

[0136] (I) Construction of tumor-bearing mouse model

[0137] 1. Preparation of A375 cell suspension: Log-phase A375 cells were digested with 0.25% trypsin, washed twice with serum-free RPMI 1640 medium by centrifugation (1000 rpm, 5 min, 4℃), and the concentration was adjusted to 2×10^6 cells / mL. The suspension was then mixed with Matrigel at a 1:1 volume ratio to achieve a final cell concentration of 1×10^6 cells / 100 μL.

[0138] 2. Subcutaneous inoculation: Disinfect the skin on the right back of nude mice with 75% alcohol, and inject 100 μL of cell suspension into the superficial subcutaneous layer using a 1 mL syringe (avoiding blood vessels and lymph nodes), with 1 × 10^6 cells inoculated per mouse.

[0139] 3. Tumor volume monitoring and grouping: Starting from day 7 post-inoculation, the long diameter (L, longest diameter) and short diameter (W, shortest diameter) of the tumor were measured every 3 days using calipers. The tumor volume (V) was calculated using the formula: "Tumor volume (V) = L × W". 2 / 2” calculates the volume; when the tumor volume reaches 100±5mm 3 On day 14 post-inoculation, 18 nude mice were randomly divided into 3 groups (n=6 / group) according to the principle of "tumor volume equilibrium". There was no significant difference in initial tumor volume among the groups (P>0.05). The grouping information is as follows:

[0140] (1) Model group: physiological saline (200 μL / animal) was injected into the tail vein;

[0141] (2) Control group: Unmodified NK cells (1×10^7 cells / 200μL saline / animal) were injected into the tail vein.

[0142] (3) Experimental group: sdAb-NK cells (1×10^7 cells / 200μL saline / animal) were injected into the tail vein.

[0143] (II) Drug administration and dynamic monitoring

[0144] 1. Dosing regimen: The mice were administered via tail vein injection on the day of grouping (day 0), day 7, day 14, and day 21, for a total of 4 times. During injection, the mice were fixed with a tail vein fixator, and the dilated blood vessels in the tail were wiped with warm water. A 29G needle was inserted into the distal end of the tail vein, and the injection was slowly pushed in (100 μL / min) to ensure no leakage (if leakage occurred, the mouse was removed and replaced with a mouse under the same conditions).

[0145] 2. Dynamic monitoring indicators

[0146] (1) Tumor volume: Measured once every 3 days, and the long and short diameters of the tumor of each mouse in each group were recorded and the volume was calculated;

[0147] (2) Mouse weight: Weigh the mice every 3 days (fasting) to assess their overall health status;

[0148] (3) General condition: Observe the mice’s mental state (activity level, hair luster), food and water intake, and fecal characteristics every day, and record any abnormalities (such as sudden weight loss, lethargy, death, etc.).

[0149] 3. The experimental follow-up results are shown in Tables 9 and 10.

[0150] (1) Tumor-bearing model construction and group balance: On day 14 after subcutaneous inoculation of A375 cells, the tumor volume of nude mice reached 100±5 mm. 3 The tumor formation rate was 100%; there was no significant difference in the initial tumor volume among the three groups at the time of grouping (P>0.05), thus excluding the interference of initial differences on the experimental results.

[0151] (2) Dynamic changes in tumor volume: Starting from day 3, the tumor volume growth rate in the experimental group was significantly slower than that in the model group (P<0.05); starting from day 7, the tumor volume in the experimental group was significantly smaller than that in the control group (P<0.05), and the difference increased over time; at the end of the experiment (day 28), the tumor volume in the experimental group was 198.5±30.2 mm. 3 Only the model group (1302.6±92.3mm) 3 The percentage of 15.2% of the control group (602.5±48.6mm) was significantly higher than that of the control group. 3 32.9% of ).

[0152] (3) Changes in mouse weight: The weight of mice in the model group continued to decrease as the tumor progressed (from 20.5g to 17.8g), while the weight of mice in the experimental group remained stable at 19.8-20.6g, which was not significantly different from that of the control group (19.2-20.1g). This indicates that sdAb-NK cells did not have a negative impact on the overall health of mice while inhibiting the tumor.

[0153] Table 9. Dynamic changes in tumor volume (mm) in tumor-bearing mice of each group 3(x±s, n=6)

[0154]

[0155] Table 10. Dynamic changes in body weight of tumor-bearing mice in each group (g, x±s, n=6)

[0156]

[0157] (III) Experimental endpoint detection (day 28)

[0158] 1. Tumor weight determination: Mice were euthanized by cervical dislocation, tumor tissue was aseptically dissected, and surface bloodstains were rinsed with physiological saline. After blotting with filter paper, the tumor weight was measured using an electronic balance (accuracy 0.001g). The tumor inhibition rate was calculated as follows: Tumor inhibition rate (%) = [(average tumor weight of model group - average tumor weight of experimental group / control group) / average tumor weight of model group] × 100%. The results showed that the tumor weight of the experimental group (0.21±0.03g) was significantly lower than that of the model group (1.38±0.15g) and the control group (0.71±0.08g) (P < 0.01 for both); the tumor inhibition rate reached 84.8%, significantly higher than that of the control group (48.6%) (P < 0.01), demonstrating that the in vivo anti-tumor effect of sdAb-NK cells was significantly better than that of unmodified NK cells. (See Table 11).

[0159] Table 11 Tumor weight and inhibition rate of tumor-bearing mice in each group (x±s, n=6)

[0160]

[0161] 2. Safety testing:

[0162] (1) Blood routine test: Before euthanizing the mice, 0.5 mL of blood was collected from the orbital cavity (EDTA-K2 anticoagulated). The white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), platelet count (PLT), and neutrophil percentage (Neu%) were measured by a fully automated blood routine analyzer. The reference range was: WBC 4.0-10.0×10^9 / L, RBC 8.0-10.5×10^12 / L, Hb 120-150 g / L, PLT 200-500×10^9 / L, Neu% 20%-40% in BALB / c nude mice. The results showed (Table 12) that the white blood cell, red blood cell, hemoglobin, platelet count and neutrophil percentage in the experimental group were all within the normal reference range of BALB / c nude mice, and there was no significant difference from the model group (P>0.05), thus excluding the toxicity of sdAb-NK cells to the hematopoietic system.

[0163] Table 12. Blood routine test results of tumor-bearing mice in each group (x±s, n=6)

[0164]

[0165] (2) Liver and kidney function tests: 1 mL of blood was drawn from the orbital cavity (without anticoagulation), allowed to stand at room temperature for 30 min, and then centrifuged (3000 rpm, 10 min, 4℃) to separate serum. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) were measured using an automated biochemical analyzer. Reference ranges: ALT 20-40 U / L, AST 30-50 U / L, Cr 50-80 μmol / L, BUN 5-10 mmol / L in BALB / c nude mice. The levels of ALT, AST, creatinine, and BUN in the experimental group were all within the normal range and showed no significant difference from the model group (P>0.05), demonstrating that sdAb-NK cells have no hepatotoxicity or nephrotoxicity.

[0166] Table 13 Results of liver and kidney function tests in tumor-bearing mice in each group (x±s, n=6)

[0167]

[0168] III. Experiment Summary

[0169] This embodiment uses an A375 tumor-bearing nude mouse model to verify the in vivo antitumor activity and safety of sdAb-NK cells:

[0170] 1. Significant activity advantage: sdAb-NK cells can significantly inhibit tumor growth, with an experimental endpoint tumor inhibition rate of 84.8%, which is significantly higher than that of unmodified NK cells (48.6%). The mechanism may be related to the synergistic effect of bispecific antibody-mediated "tumor targeting (PD-L1) + ADCC activation (CD16) + immune checkpoint blockade";

[0171] 2. Good in vivo safety: sdAb-NK cells have no adverse effects on the hematopoietic system, liver and kidney function in nude mice, avoiding the possible toxic side effects of traditional immune cell therapy;

[0172] The above embodiments are 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 PD-L1 / CD16 conjugate bispecific antibody, characterized in that, The amino acid sequence of the anti-PD-L1 / CD16 conjugate bispecific antibody is shown in SEQ ID NO:

4.

2. A gene encoding an anti-PD-L1 / CD16 conjugate bispecific antibody, characterized in that, The nucleotide sequence of the gene encoding the anti-PD-L1 / CD16 conjugate bispecific antibody is shown in SEQ ID NO:

3.

3. An expression carrier, characterized in that, The expression vector is pCHO-sdAb-BsAb, and the pCHO-sdAb-BsAb contains the encoding gene as described in claim 2.

4. A CHO cell, characterized in that, The CHO cells comprise the expression vector of claim 3.

5. An enhanced NK immune cell, characterized in that, The enhanced NK immune cells stably express the anti-PD-L1 / CD16 conjugate bispecific antibody as described in claim 1 via a lentiviral vector.

6. The enhanced NK immune cells according to claim 5, characterized in that, The lentiviral vector has a backbone of pLVX-EF1α-IRES-ZsGreen1, which includes an expression cassette consisting of an EF1α promoter, the anti-PD-L1 / CD16 conjugate bispecific antibody encoding gene as described in claim 2, and the IRES-ZsGreen1 reporter gene.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the enhanced NK immune cells of claim 5 and pharmaceutically acceptable excipients.

8. The use of the anti-PD-L1 / CD16 conjugate bispecific antibody according to claim 1 in the preparation of enhanced NK immune cells.

9. The use of the enhanced NK immune cells of claim 5 in the preparation of cancer therapeutic drugs, characterized in that, The cancer in question is a PD-L1 positive cancer.

10. The use according to claim 9, characterized in that, The PD-L1 positive cancers include melanoma, non-small cell lung cancer, liver cancer, and colorectal cancer.

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