Pharmaceutical composition containing NK cells and application of pharmaceutical composition in cancer treatment

Abstract

The invention discloses a pharmaceutical composition for treating cancers and application thereof. The pharmaceutical composition comprises natural killer cells activated by in vitro amplification and a bispecific antibody specifically targeting to Glypican-3 and CD16A. The invention provides a specific amino acid sequence of the bispecific antibody and a high-purity sorting and large-scale amplification method of natural killer cells. In-vitro experiments prove that the composition has remarkable specific killing activity on Glypican-3 high-expression liver cancer cells and can effectively activate natural killer cells to secrete IFN-gamma and TNF-alpha. Animal in-vivo experiments further prove that the composition can effectively inhibit tumor growth, and meanwhile, the lifetime of tumor-bearing mice is remarkably prolonged. The invention provides a brand new and efficient immunotherapy strategy for Glypican-3 positive malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically relating to a pharmaceutical composition containing NK cells and its application in the treatment of cancer. Background Technology

[0002] Natural killer (NK) cells are core effector lymphocytes of the innate immune system, renowned for their ability to directly kill tumor cells and virus-infected cells without prior sensitization. This function is primarily achieved through the release of perforin and granzymes, as well as the expression of death ligands that induce apoptosis (such as FasL and TRAIL). Compared to T cell-mediated adaptive immunity, NK cells offer a faster onset of action and are less likely to induce graft-versus-host disease, making them a highly attractive "off-the-shelf" cell product in cancer immunotherapy.

[0003] However, despite the promising effects of NK cells in the treatment of hematological malignancies, their application in the treatment of solid tumors still faces several bottlenecks. First, solid tumors typically establish a highly immunosuppressive microenvironment, directly weakening the cytotoxic activity and proliferative capacity of NK cells by secreting immunosuppressive factors such as TGF-β, prostaglandin E2, and adenosine. Second, NK cells have limited persistence and proliferative capacity in vivo, often rapidly decaying after infusion, making it difficult to maintain a long-term anti-tumor effect. Third, NK cells have low homing efficiency to solid tumor sites; due to chemokine receptor-ligand mismatch, only a small number of infused NK cells successfully infiltrate the tumor tissue. Finally, solid tumor cells often lack sufficient stress ligands that can be recognized by the NK cell's natural recognition system (such as NKG2D ligand), resulting in ineffective NK cell activation.

[0004] To overcome these challenges, various strategies are being explored. Genetically engineered chimeric antigen receptor NK cells, such as CAR-NK cells targeting BCMA or CD19, have shown enhanced targeted killing capabilities and a lower risk of cytokine release syndrome in clinical trials. However, the preparation of CAR-NK cells still faces challenges such as unstable transduction efficiency, difficulty in amplification, silencing of CAR expression over time, and high preparation costs. Another widely used and relatively mature strategy is the use of bispecific or trispecific NK cell connectors. These connect NK cells to the vicinity of tumor cells by simultaneously binding to activating receptors (such as CD16) and tumor-associated antigens, effectively enhancing their antibody-dependent cell-mediated cytotoxicity. For example, trispecific NKCEs targeting CD5 have shown significant efficacy in preclinical models of T-cell malignancies. However, these protein molecules suffer from problems such as short in vivo half-life, potential induction of off-target toxicity (such as damage to normal tissues) associated with antibody-mediated cytotoxicity, and potential anti-drug antibody responses. In addition, although antibodies targeting solid tumor-associated antigens such as GPC3 have been developed, the effective combination of these antibodies with NK cell therapy still faces challenges such as tumor microenvironment suppression and NK cell function depletion.

[0005] In summary, there is an urgent need to develop a novel, highly targeted NK cell drug composition that can overcome the inhibitory effects of the tumor microenvironment. This composition should significantly enhance the ability of NK cells to recognize, infiltrate, activate, and kill specific solid tumors (such as Glypican-3 (GPC3) positive tumors), while ensuring good safety and in vivo persistence. The purpose of this invention is to provide such a composition based on the synergistic effect of NK cells and a novel bispecific antibody, and its applications. Summary of the Invention

[0006] The purpose of this invention is to provide a novel, synergistic pharmaceutical composition containing NK cells. This composition combines in vitro expanded highly active NK cells with a novel, high-affinity anti-GPC3 / CD16 bispecific antibody to achieve highly efficient and specific immunotherapy for GPC3-positive solid tumors (especially hepatocellular carcinoma), while avoiding the systemic toxic side effects that may result from the use of chemical cytotoxic drugs.

[0007] Therefore, this invention discloses a GPC3 / CD16A bispecific antibody, which comprises a signal peptide, an anti-GPC3 scFv, a (Gly4Ser)3 linker, an IgG4 Fc backbone, and an anti-CD16A scFv. The nucleotide sequence of the full-length gene of the GPC3 / CD16A bispecific antibody is shown in SEQ ID NO:5. The amino acid sequence of the anti-GPC3 scFv is shown in SEQ ID NO:1. The amino acid sequence of the anti-CD16A scFv is shown in SEQ ID NO:2. The amino acid sequence of the IgG4 Fc backbone is shown in SEQ ID NO:3. The amino acid sequence of the signal peptide is shown in SEQ ID NO:4. The amino acid sequence of the (Gly4Ser)3 linker is GGGGSGGGGSGGGGS.

[0008] In one aspect, the present invention also discloses a pharmaceutical composition for treating cancer, the composition comprising in vitro expanded and activated natural killer cells and the GPC3 / CD16A bispecific antibody. The cancer is a Glypican-3 positive tumor. The Glypican-3 positive tumor includes hepatocellular carcinoma, hepatoblastoma, melanoma, or ovarian cancer.

[0009] In one aspect, the present invention also discloses the use of the aforementioned GPC3 / CD16A bispecific antibody in the preparation of a drug for treating Glypican-3 positive tumors.

[0010] In one aspect, the present invention also discloses the use of the pharmaceutical composition described herein in the preparation of a drug for treating Glypican-3 positive tumors.

[0011] The pharmaceutical composition and preparation method of this invention have significant technical advantages, primarily in the high efficiency and activity stability of natural killer cell preparation. After optimized processing, the cells expand by 9841±32 times within 21 days, which is 9.8 times that of traditional methods, meeting the needs of large-scale clinical applications. After expansion, the cells highly express activation receptors such as NKG2D and NKp46, with a 95.2% NKG2D positivity rate and an 88.7% NKp46 positivity rate, while also exhibiting a 75.3% CD16 expression rate. They possess both natural killer and antibody-dependent cytotoxic functions, achieving a 25.3% lysis rate in HepG2 cells alone in vitro, demonstrating significantly superior activity compared to cells obtained using traditional expansion methods.

[0012] Secondly, the GPC3 / CD16A bispecific antibody exhibits excellent targeting and structural stability. The equilibrium dissociation constant for GPC3 is 2.0±0.2 nM, and for CD16A it is 5.7±0.5 nM, both superior to commercially available monoclonal antibodies, enabling efficient bridging of tumor cells and natural killer cells. Utilizing an IgG4 Fc backbone-binding (Gly4Ser)3 linker, its in vivo half-life is extended to 12 hours, a three-fold improvement over traditional scFv bispecific antibodies. Furthermore, this antibody specifically binds only to GPC3⁺ tumor cells and CD16A⁺ natural killer cells, exhibiting no binding ability to GPC3-negative normal cells, effectively avoiding off-target effects.

[0013] Furthermore, the synergistic killing effect of the composition is outstanding. In vitro experiments showed that the combination of natural killer cells and 10 μg / mL GPC3 / CD16A bispecific antibody achieved a specific lysis rate of 78.9% against HepG2 cells, which was 3.1 times higher than that of natural killer cells alone and 1.4 times higher than that of natural killer cells combined with 1 μg / mL bispecific antibody, and the effect was dose-dependent. The lysis rate against GPC3-low expression cells was less than 32%, and the lysis rate against GPC3-negative normal cells was less than 8%, demonstrating clear targeting. In in vivo experiments, the composition inhibited tumor growth in a HepG2 liver cancer xenograft model by 72.2%, which was 2.4 times that of the natural killer cell group alone and 1.7 times that of the bispecific antibody group alone. The median survival of the model mice exceeded 70 days, with a 100% survival rate on day 70, which was significantly better than the 45-day survival of the control group, and there were no symptoms such as tumor ulceration or cachexia. At the same time, the composition significantly increased the levels of IFN-γ and TNF-α secreted by natural killer cells, reaching 835.2 pg / mL and 488.6 pg / mL, respectively, which were 4.5 times and 3.8 times that of the natural killer cell group alone, and could further regulate the tumor microenvironment and enhance the anti-tumor immune circulation.

[0014] Furthermore, the composition showed good safety. During treatment, the experimental animals experienced a weight change rate of less than 3%, with no abnormal symptoms such as diarrhea, hair loss, or lethargy. Blood routine indicators, including white blood cell count, red blood cell count, hemoglobin, platelet count, and lymphocyte percentage, were all within the normal range, with no bone marrow suppression. Liver function indicators, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and kidney function indicators, such as blood urea nitrogen and serum creatinine, all met physiological reference values, with no organ damage.

[0015] Finally, this composition has high clinical application value, specifically targeting GPC3-positive cancers, especially liver cancer (GPC3 positivity rate 72%-85%), filling the gap in the efficacy of existing natural killer cell therapy for solid tumors; its preparation process is stable, the sorting and amplification of natural killer cells, and the expression and purification of bispecific antibodies can all be carried out on a large scale, the cost is controllable, and the safety meets clinical standards, showing broad translational prospects. Attached Figure Description

[0016] Figure 1 The results of SDS-PAGE (non-denaturing) detection of GPC3 / CD16A bispecific antibody (BsAb) are shown in Figure 1, where 1 represents GPC3 / CD16A bispecific antibody (BsAb). Detailed Implementation

[0017] 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.

[0018] 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.

[0019] Example 1: Isolation, activation and large-scale expansion of NK cells

[0020] I. Experimental Materials and Reagents

[0021] 1. Peripheral blood source: 3 healthy volunteers (aged 25-35, no history of infectious diseases, and signed informed consent forms), numbered Vol-1, Vol-2, and Vol-3;

[0022] 2. Reagents: Ficoll-Paque PLUS (density 1.077 g / mL, GE Healthcare); CD56 positive magnetic bead sorting kit (Miltenyi Biotec); NK cell culture medium (Gibco); human AB serum (Sigma, heat-inactivated 56℃ for 30 min); recombinant human IL-2 (PeproTech), IL-15 (PeproTech), IL-21 (PeproTech); flow cytometry antibodies: CD45-PE (BD), CD56-APC (BD), CD3-FITC (BD), CD16-PE-Cy7 (BD), NKG2D-PE (BD), NKp46-APC (BD), DNAM-1-PE-Cy5 (BioLegend); isotype control antibody (BD); trypan blue staining solution (Sigma).

[0023] 3. Instruments: Blood cell separator; biosafety cabinet; CO2 incubator; flow cytometer; hemocytometer, etc.

[0024] II. Experimental Procedure

[0025] 1. Peripheral blood collection and separation of PBMCs

[0026] (1) Peripheral blood collection: A blood cell separator was used to collect peripheral blood from 3 volunteers. 500 mL of peripheral blood was collected from each volunteer. The anticoagulant was ACD-A (1.5 mL of ACD-A was added for every 10 mL of blood). The blood was stored at 4°C after collection and processed within 2 hours.

[0027] (2) PBMC isolation: ① Dilute 500mL of peripheral blood with sterile PBS at a ratio of 1:1 (final volume 1000mL) and mix thoroughly; ② Add 15mL of Ficoll-Paque PLUS to a 50mL centrifuge tube, and slowly add 30mL of diluted blood along the tube wall (to avoid disrupting the density gradient), and prepare a total of 34 tubes; ③ Centrifuge at 400×g for 30min at 20℃ (centrifugation acceleration and deceleration are 0 to avoid layering disorder); ④ After centrifugation, the liquid is divided into 4 layers (from top to bottom: plasma layer, white membrane layer, Ficoll layer, red blood cell layer), carefully aspirate the white membrane layer (PBMCs) with a 10mL pipette to a new 50mL centrifuge tube, add 30mL of PBS to each tube, centrifuge at 300×g for 10min at 20℃, and discard the supernatant; ⑤ Repeat washing twice (resuspend in PBS, centrifuge at 300×g for 10min), and finally resuspend the PBMCs with 20mL of NK medium, and count the viability by trypan blue staining (the viability should be ≥95%).

[0028] 2. NK cell sorting (CD56) + (Positive sorting of magnetic beads)

[0029] (1) Pretreatment: Take the PBMCs suspension, centrifuge at 300×g for 10 min at 20℃, discard the supernatant, resuspend in sorting buffer (PBS + 0.5% BSA + 2mM EDTA), and adjust the cell concentration to 1×10⁻⁶. 8 cells / mL;

[0030] (2) Magnetic bead incubation: 10 μL of magnetic beads / 1×10 7 Add CD56 magnetic beads to the cells and incubate at 4°C in the dark for 30 min (mix gently once every 10 min).

[0031] (3) Preparation of the sorting column: Install the LS sorting column in the magnetic field, prewash the column with 3 mL of sorting buffer, and discard the eluent;

[0032] (4) Sorting: Add the incubated cell suspension to the sorting column and collect the effluent (CD56). - Cells (discard); after the liquid has completely flowed into the column, wash the column three times with 3 mL of sorting buffer, and collect the washing buffer (still CD56). - cell);

[0033] (5) Elution of positive cells: Remove the sorting column, remove it from the magnetic field, and elute CD56 cells with 5 mL of sorting buffer under pressure. + Transfer the cells to a new centrifuge tube, centrifuge at 20°C and 300×g for 10 min, discard the supernatant, and resuspend in NK medium;

[0034] (6) Purity detection: Take 200 μL of sorted cells, add CD45-PE (1:100), CD56-APC (1:100), and CD3-FITC (1:100) antibodies, stain at 4℃ in the dark for 30 min; add 2 mL PBS, centrifuge at 300×g for 10 min, discard the supernatant, and fix with 500 μL of 1% paraformaldehyde; detect CD45 purity by flow cytometry. + CD56 + CD3 - Cell percentage (NK cell purity), 1×10⁻⁶ cells per sample. 4 Each cell.

[0035] 3. NK cell activation and large-scale expansion

[0036] (1) Initial inoculation: The sorted NK cells were resuspended in NK-specific culture medium containing 10% human AB serum, 100 U / mL IL-2, 10 ng / mL IL-15, and 5 ng / mL IL-21, and the density was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL were inoculated into T75 culture flasks (25mL per flask) and incubated statically at 37°C in a 5% CO2 incubator (day 0).

[0037] (2) Medium change and density maintenance: On the 3rd day, gently pipette the cells (avoid vigorous shaking), take 100 μL of cell suspension, stain with trypan blue for counting, and adjust the density to 1×10⁻⁶. 6 cells / mL (fresh culture medium and cytokines were added to maintain the concentrations of IL-2, IL-15, and IL-21); on day 7, the cell density increased to 2 × 10⁶ cells / mL. 6 Transfer to a T175 culture flask (approximately 50 mL) and adjust the density to 1×10⁻⁶ cells / mL. 6 cells / mL; on day 10, after counting, cells were transferred to a cell culture bag with an initial volume of 200 mL and a density of 1×10⁶ cells / mL. 6 cells / mL; on days 14 and 17, fresh culture medium was added to 300 mL and 400 mL respectively, maintaining a density of 0.8-1.2 × 10⁶ cells / mL. 6 cells / mL, cell viability was measured before each medium change (viability ≥90%); final harvest: on day 21, cells were collected from the culture bag, centrifuged at 300×g for 10 min, resuspended in NK medium, and the total number of cells and viability were counted.

[0038] 4. Dynamic analysis of NK cell phenotype during expansion.

[0039] (1) Sampling time points: Day 0 (after sorting), Day 7, Day 14, and Day 21;

[0040] (2) Flow cytometry staining: 2 × 10⁻⁶ samples were taken at each time point. 5 Cells were divided into 6 tubes, and the following were added to each tube: ① isotype control (irrelevant antibodies labeled with PE, APC, FITC, PE-Cy7, and PE-Cy5); ② CD45-PE + CD56-APC + CD3-FITC; ③ CD16-PE-Cy7; ④ NKG2D-PE; ⑤ NKp46-APC; ⑥ DNAM-1-PE-Cy5. All antibodies were diluted 1:100. Cells were stained at 4°C in the dark for 30 min, washed twice with PBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry (1 × 10⁶ cells per tube). 4 (cells); the percentage of CD45 positive cells was analyzed using FlowJo 10.0 software. + CD56 + CD3 - The proportion of cells.

[0041] III. Experimental Results

[0042] 1. PBMC separation and NK cell sorting efficiency: An average of 8.9 × 10⁻⁶ NK cells were separated from 500 mL of peripheral blood from 3 volunteers. 8 PBMCs were obtained, with a viability of 97.2%; after CD56 magnetic bead positive sorting, NK cells (CD45) were also found. + CD56 + CD3 - The average purity was 98.5%, the viability was 97.2%, and each case yielded 13.1 × 10⁻⁶. 6 The initial NK cells showed stable sorting efficiency. Specific experimental results are shown in Table 1.

[0043] Table 1 Results of PBMC isolation and NK cell sorting

[0044]

[0045] 2. NK cell proliferation capacity: After 21 days of culture, the total number of NK cells increased from the initial 13.1 × 10⁻⁶. 6 The number of units increased to 129233×10. 6The cells expanded by an average of 9841-fold, with only 0.3% difference between individuals (Vol-1: 9880-fold, Vol-2: 9826-fold, Vol-3: 9817-fold). Cell viability remained above 90% throughout the expansion process (average viability of 96.5% on day 21), and the growth curve exhibited typical exponential growth (rapid proliferation phase from day 3 to 14, with an average daily fold increase of 1.4-1.6-fold). See Table 2.

[0046] Table 2 Dynamics of NK cell expansion (total cell count and expansion fold)

[0047]

[0048] 3. Phenotypic stability: Throughout the expansion process, the positivity rate of CD56, a core marker of NK cells, remained above 98.5%, indicating high cell population homogeneity. The expression of activation receptors NKG2D, NKp46, and DNAM-1 gradually increased with culture time, reaching 95.2%, 88.7%, and 82.9% respectively on day 21, representing increases of 20.8%, 25.3%, and 25.8% compared to initial levels. CD16 (mediating antibody-dependent cytotoxicity, ADCC) expression increased from 63.2% to 75.3%, suggesting that the expanded NK cells possess the potential for both natural killer and ADCC functions. (See Table 3.)

[0049] Table 3. Phenotypic changes of NK cells during expansion (positive rate, %)

[0050]

[0051] IV. Summary

[0052] This embodiment establishes a highly efficient and stable protocol for the isolation and expansion of peripheral blood NK cells: high-viability PBMCs can be obtained through Ficoll density gradient centrifugation, and NK cells with a purity >98% can be rapidly obtained by CD56 magnetic bead sorting; using a special culture medium stimulated by IL-2+IL-15+IL-21, nearly 100,000 cells can be obtained within 21 days. 4 The cells are multiplied, and the expanded NK cells highly express activation receptors such as NKG2D and NKp46, maintain the functional expression of CD16, and have strong killing potential.

[0053] Example 2: Expression, purification, and functional validation of GPC3 / CD16A bispecific antibody (BsAb)

[0054] I. Design Background and Molecular Structure Details

[0055] The anti-GPC3 / anti-CD16A bispecific antibody (GPC3 / CD16A BsAb) designed in this embodiment aims to enhance the killing activity of NK cells against liver cancer through "tumor targeting-effect cell bridging". Its molecular structure design is based on the following scientific basis:

[0056] 1. Targeting tumor cells: GPC3 (phosphatidylinositol proteoglycan 3) is specifically highly expressed on the surface of liver cancer cells (positive rate 72%-85%), while it is hardly expressed in normal liver tissue. Therefore, anti-GPC3 single-chain antibody (scFv) was selected as the tumor targeting term.

[0057] 2. Targeting effector cells: CD16A (FcγRIIIa) is a key receptor on the surface of NK cells that mediates ADCC (antibody-dependent cytotoxicity). Anti-CD16A scFv can specifically bind to NK cells, thereby recruiting effector cells.

[0058] 3. Optimized backbone stability: The human IgG4 hinge region +CH2+CH3 domain is used as the linker backbone. The two scFv are connected to the backbone through a flexible linker ((Gly4Ser)3) to ensure the flexibility of dual-target binding.

[0059] II. Key amino acid sequences (SEQ ID NO:1-4)

[0060] 1. Anti-GPC3 scFv (SEQ ID NO:1), with the structure VH-(Gly4Ser)3-VL, is an antibody sequence obtained through screening and optimization in the early stage of this study. It consists of VH (1-118aa), VL (134-242aa), and a (Gly4Ser)3 linker (119-133aa) in the middle.

[0061] 2. Anti-CD16A scFv (SEQ ID NO:2), with the structure VH-(Gly4Ser)3-VL, was obtained through screening and optimization in the early stage of this study. The sequence consists of VH (1-118aa), VL (134-249aa), and the (Gly4Ser)3 linker (119-133aa) in the middle.

[0062] 3. IgG4 Fc backbone (SEQ ID NO:3): Contains S228P point mutation to enhance stability and maintain the spatial structure of the antibody Fc segment.

[0063] 4. Signal peptide (SEQ ID NO:4): Derived from human IgG1 signal peptide, it can efficiently guide recombinant proteins into the secretory pathway and be cleaved and removed during protein maturation.

[0064] III. Experimental Procedure

[0065] 1. Construction of BsAb expression plasmid

[0066] (1) Gene fragment assembly: The full-length gene “signal peptide (SEQ ID NO:4)-anti-GPC3 scFv (SEQ ID NO:1)-(Gly4Ser) 3-IgG4 Fc backbone (SEQ ID NO:3)-(Gly4Ser) 3-anti-CD16A scFv (SEQ ID NO:2)” (shown in SEQ ID NO:5) was synthesized, and HindIII (5' end) and XhoI (3' end) restriction sites were introduced at both ends, wherein the amino acid sequence of (Gly4Ser) 3 is GGGGSGGGGSGGGGS.

[0067] (2) Double digestion and ligation: The pcDNA3.4 vector and the full-length gene were digested with HindIII and XhoI respectively (37℃, 2h), and the vector backbone and the target fragment were recovered by agarose gel electrophoresis; T4 DNA ligase was used for ligation overnight at 16℃ (vector: fragment = 1:3, molar ratio).

[0068] (3) The ligation product was transformed into DH5α competent cells, three single clones were selected, plasmids were extracted and sequenced (covering the full-length gene), and positive plasmids with completely correct sequences were screened (named pcDNA3.4-GPC3 / CD16A).

[0069] 2. Transfection and expression of Expi293F cells

[0070] (1) Cell pretreatment: 24 hours before transfection, Expi293F cells were pretreated with 2×10⁻⁶ cells. 6 Cells / mL were seeded in 125mL shake flasks (final volume 30mL) and cultured at 37℃, 8% CO2, and 125rpm. The viability at transfection should be ≥96% (confirmed by trypan blue staining).

[0071] (2) Preparation of transfection complex: Take 15 μg pcDNA3.4-GPC3 / CD16A plasmid and dilute it to 1 mL with Opti-MEM; take another 45 μL PEI (1 mg / mL) and dilute it to 1 mL with Opti-MEM; after standing at room temperature for 5 min, mix the two solutions and incubate at room temperature for 20 min (to form plasmid-PEI complex; preliminary experiments show that the 1:3 ratio has the highest transfection efficiency).

[0072] (3) Culture and harvest: The complex was slowly added to the cell suspension and cultured at 37℃, 8% CO2, and 125 rpm. 18 h after transfection, 0.3 mL of ExpiFectamine 293 Enhancer was added. On day 6, the supernatant was collected, centrifuged at 300×g for 10 min (to remove cell debris), filtered through a 0.22 μm filter membrane, and stored at 4℃.

[0073] 3. BsAb purification (three-step method to ensure purity)

[0074] (1) Protein A affinity chromatography: ① Equilibration: Wash the column (5 mL) with equilibration buffer (20 mM Tris-HCl, pH 7.4) at a flow rate of 5 mL / min until A280 < 0.01; ② Loading: Load 30 mL of cell supernatant at a flow rate of 3 mL / min and collect the breakthrough fluid (A280 < 0.05, confirming complete loading); ③ Elution: Elute with elution buffer (0.1 M citric acid, pH 3.0), collect the elution peak (about 3-5 mL) with A280 > 0.1, and immediately neutralize to pH 7.0 with 1 M Tris-HCl (pH 8.0);

[0075] (2) Desalting and concentration: The solution was concentrated to 1 mL using an ultrafiltration tube (30 kDa cutoff, Millipore) at 4 °C, dialyzed overnight with PBS (pH 7.4) (PBS was changed 3 times), and the concentration was determined by the BCA method. After sterilization by passing the solution through a 220 nm filter membrane, the solution was aliquoted and stored at -80 °C for later use. Calculations showed that 1 L of cell supernatant yielded 184 ± 2.8 mg of BsAb, with a concentration of 3.2 ± 0.1 mg / mL.

[0076] 4. Quality Inspection

[0077] (1) SDS-PAGE analysis (non-reducing conditions): 10 μg protein + non-reducing buffer (without β-mercaptoethanol), 10% gel electrophoresis, Coomassie brilliant blue staining, results showed ( Figure 1 A single main band (containing glycosylation modification) appears at a molecular weight of approximately 90 kDa.

[0078] (2) Endotoxin detection: Endotoxin content was detected by the horseshoe crab reagent method. The detection result was 0.06±0.01EU / μg, which met the standard.

[0079] 5. Functional Verification

[0080] (1) SPR affinity assay: The CM5 chip was activated with 10 mM sodium acetate (pH 5.0) and coupled with GPC3 (density 1200 RU) and CD16A-Fc (density 1100 RU), respectively, with the blank channel serving as a control; BsAb was diluted with HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% Tween-20, pH 7.4) to 0.625, 1.25, 2.5, 5, and 10 nM, and flowed through the chip at a flow rate of 30 μL / min (binding for 60 s, dissociation for 120 s); the KD value was calculated using a 1:1 model fitted with Biacore T200 Evaluation software. The results showed that the SPR affinity (KD) was 2.0 ± 0.2 nM for GPC3 and 5.7 ± 0.5 nM for CD16A. This indicates that BsAb has high affinity for both GPC3 and CD16A, and its affinity is consistent with or better than that of commercially available monoclonal antibodies (the KD value of commercially available anti-GPC3 monoclonal antibody (ab216606) for GPC3 is 2.2±0.3 nM; the KD value of commercially available anti-CD16A monoclonal antibody (ab227665) for CD16A is 6.0±0.6 nM).

[0081] (2) Verification of dual cell binding: HepG2 cells (GPC3) + ) or NK-92 cells (CD16A) + ) with 1×10 4 Cells were seeded in 96-well plates, fixed with 4% paraformaldehyde for 15 min, and washed three times with PBS. The plates were then blocked with 5% skim milk at 37°C for 2 h. 0.5 μg / mL BsAb (negative control: human IgG4; positive control: anti-GPC3 monoclonal antibody + anti-CD16A monoclonal antibody, 0.5 μg / mL each) was added, and the plates were incubated at 37°C for 1 h. HRP-labeled goat anti-human IgG4 (1:5000) was added and incubated for 1 h. TMB was used for color development for 10 min, and the reaction was stopped with 2M H2SO4. OD450nm was measured. Results showed that the cell ELISA (OD450nm) values ​​were 1.81±0.13 for HepG2+BsAb, 1.74±0.11 for NK-92+BsAb, and 0.20±0.02 for the negative control. These results indicate that BsAb and GPC3... + HepG2 cells and CD16A + The binding capacity of NK-92 cells was significantly higher than that of the negative control (P<0.001) and comparable to that of the positive control (monoclonal antibody mixture), demonstrating that it can simultaneously bridge tumor cells and NK cells.

[0082] IV. Summary

[0083] This embodiment successfully prepared a GPC3 / CD16A bispecific antibody through precise molecular design and optimized experimental procedures. In its structure, anti-GPC3 scFv (SEQ ID NO:1) and anti-CD16A scFv (SEQ ID NO:2) are linked by a stable IgG4 backbone (SEQ ID NO:3). After efficient expression in Expi293F cells, the antibody was purified with Protein A to obtain a high-purity (>98.8%), low-endotoxin BsAb. Functional verification confirmed that this BsAb can bind to GPC3 with high affinity. + Tumor cells and CD16A + NK cells provide a key material for subsequent research on the synergistic killing of liver cancer cells by "BsAb+NK cells", and the preparation process is stable and controllable, with the potential for large-scale production.

[0084] Example 3: In vitro cytotoxicity experiment

[0085] I. Experimental Materials and Reagents

[0086] 1. Target cells: HepG2 human liver cancer cells (high expression of GPC3, positive rate 92%), Huh-7 (low expression of GPC3, positive rate 18%), and LO2 normal human liver cells (GPC3 negative) were all cultured in DMEM medium containing 10% FBS (37℃, 5% CO2).

[0087] 2. Effector cells: NK cells expanded in Example 1;

[0088] 3. Key reagents: Calcein-AM (2.5mM stock solution), GPC3 / CD16A BsAb (prepared in Example 2); IFN-γ ELISA kit, TNF-α ELISA kit;

[0089] II. Experimental Procedure

[0090] 1. Target cell preparation and Calcein-AM labeling: Target cells in logarithmic growth phase (HepG2, Huh-7, LO2) were digested with 0.25% trypsin, washed twice with PBS, and the concentration was adjusted to 1×10⁻⁶. 5 cells / mL; add Calcein-AM stock solution to a final concentration of 2.5 μM, incubate at 37 °C and 5% CO2 for 30 min (protected from light); after incubation, wash three times with DMEM containing 10% FBS (centrifuge at 300 × g for 5 min) to remove free fluorescent dye, and finally resuspend at 5 × 10⁻⁶ cells / mL. 4 cells / mL (100 μL / well, i.e., 5 × 10⁻⁶ cells / mL) 3 (cells / pores).

[0091] 2. Experimental grouping and sample loading (96-well black fluorescent plate, 6 replicates per group), as shown in Table 4.

[0092] Table 4 Experimental Groups

[0093]

[0094] 3. Cytotoxicity assay (Calcein-AM release method): 96-well plates were incubated at 37℃ in a 5% CO2 incubator for 4 h; after incubation, the plates were centrifuged at 300×g for 5 min, and 100 μL of the supernatant was transferred to a new 96-well fluorescent plate; fluorescence microplate reader was used for detection: excitation wavelength 485 nm, emission wavelength 535 nm, and the fluorescence intensity (FI) of each group was recorded; the specific lysis rate was calculated: specific lysis rate (%) = [(experimental group FI - spontaneous release group FI) / (maximum release group FI - spontaneous release group FI)] × 100%.

[0095] 4. Cytokine release assay (ELISA): A separate 24-well plate was prepared, and cells were seeded according to the above grouping and E:T = 5:1 (target cells 1 × 10⁶). 5 / well, NK cells 5×10 5 / well), final volume 1 mL; incubate at 37℃ and 5% CO2 for 24 h, then centrifuge at 300×g for 10 min and collect the supernatant; follow the ELISA kit instructions and calculate the cytokine concentration based on the standard curve.

[0096] III. The experimental results are shown in Tables 5 and 6.

[0097] 1. Significantly specific targeted killing effect.

[0098] (1) For HepG2 cells with high GPC3 expression: The specific lysis rate of Group C (the present invention) reached 78.9%, which was significantly higher than that of Group A (25.3%, p<0.001) and Group B (55.6%, p<0.01). Moreover, the higher the BsAb concentration (10 μg / mL vs 1 μg / mL), the more obvious the killing effect, which reflects the dose dependence.

[0099] (2) For Huh-7 cells with low GPC3 expression: the lysis rate of each group was <32%, and there was no statistically significant difference between Group C (31.5%) and Group A (22.7%) (p>0.05);

[0100] (3) For GPC3-negative LO2 cells: the lysis rate of each group was <8%, close to the spontaneous release level, proving that the composition is only for GPC3-positive cells and has no off-target toxicity.

[0101] 2. Enhanced NK cell activation effect: The IFN-γ release of Group C (835.2 pg / mL) was 4.5 times that of Group A (185.6 pg / mL), and the TNF-α release (488.6 pg / mL) was 3.8 times that of Group A (128.5 pg / mL) (p < 0.001 for both). In contrast, the cytokine concentration of Group D (BsAb only) was close to that of the blank control, indicating that BsAb itself does not induce cytokine release, but rather activates the immune function of NK cells by bridging NK cells and tumor cells.

[0102] 3. BsAb has no direct cytotoxicity: Group D (BsAb only) showed a lysis rate of <4% for all three target cell groups, which was not different from the spontaneous release group (p>0.05), proving that BsAb only plays a "target bridging" role and has no independent killing activity, and its safety is controllable.

[0103] Table 5. Specific lysis rate of different target cells for each group (%, x±s, n=6)

[0104]

[0105] Table 6. Cytokine release concentrations in each group (pg / mL, x±s, n=6)

[0106]

[0107] IV. Summary

[0108] This embodiment, through Calcein-AM release assay and ELISA verification, confirms that the drug composition "NK cells + 10 μg / mL GPC3 / CD16A BsAb" (Group C) has three core advantages: ① Significantly enhanced killing activity against GPC3-overexpressing hepatocellular carcinoma cells (HepG2) (lysis rate 78.9%), in a BsAb dose-dependent manner; ② Strict GPC3 targeting for killing, with no toxicity to normal hepatocytes (LO2); ③ Effectively activates NK cells to release IFN-γ and TNF-α, enhancing the immune response. These results lay the foundation for subsequent in vivo experiments and verify the synergistic mechanism of the NK cells in Example 1 and the BsAb in Example 2.

[0109] Example 4: Evaluation of therapeutic effects in animals

[0110] I. Experimental Materials and Animals

[0111] 1. Tumor cells: Luciferase-labeled HepG2 cells (HepG2-luc): pGL4.51-luc2 vector was transfected with lentivirus, stable expression lines were selected with puromycin, and luciferase activity (RLU / cell > 1 × 10⁻⁶) was verified.4 );

[0112] 2. Experimental animals: 6-8 week old female NSG mice (immunodefective, without T / B / NK cells, suitable for xenotransplantation), n=32, purchased from Vital Rivers Beijing, and housed in an SPF-grade environment;

[0113] 3. Reagents: D-fluorescein potassium salt (prepared with 15 mg / mL physiological saline), GPC3 / CD16A BsAb (prepared in Example 2), NK cells (expanded in Example 1);

[0114] II. Experimental Procedure

[0115] 1. Establishment of a liver cancer xenograft model: Log-phase HepG2-luc cells were harvested, washed twice with PBS, and the concentration was adjusted to 5 × 10⁻⁶. 7 cells / mL; 100 μL of cell suspension (5 × 10⁻⁶ cells / mL) was subcutaneously injected into the right back of each mouse. 6 (cells / animal); observe tumor formation daily after inoculation, and measure tumor volume with calipers on day 10, selecting tumors with a volume of 80-100 mm. 3 The mice (a total of 24) were randomly divided into 4 groups (n=6 / group) according to the principle of "tumor volume balance". Day 10 was regarded as "day 0 of treatment".

[0116] 2. Grouping and Dosing Regimen

[0117] Table 7 Experimental Groups and Drug Administration

[0118]

[0119] 3. Monitoring Indicator Detection

[0120] (1) Dynamic monitoring of tumor volume: On days 0, 3, 6, 9, 12, 15, 18, and 21 of treatment, the long diameter (L) and short diameter (W) of the tumor were measured with calipers to an accuracy of 0.1 mm. The volume was calculated as follows: V = (L × W) 2 ) / 2. Calculate the tumor growth inhibition rate (TGI): TGI (%) = [1 - (mean volume on day 21 of the treatment group - mean volume on day 0 of the treatment group) / (mean volume on day 21 of the control group - mean volume on day 0 of the control group)] × 100%.

[0121] (2) Bioluminescence Imaging (BLI): On days 7, 14 and 21 of treatment, mice were injected intraperitoneally with potassium D-fluorescein (150 mg / kg, 100 μL / mouse) and incubated in the dark for 10 min (to allow the substrate to be fully distributed). After isoflurane anesthesia, the mice were placed in a live imaging system and exposed for 10 s. Bioluminescence images were acquired and the total number of photons in the tumor area (TotalFlux, unit: p / s) was analyzed using Living Image 4.7 software to reflect the tumor burden.

[0122] (3) Survival observation: Starting from day 0 of treatment, the mice’s condition (mental state, diet, tumor ulceration) was observed daily; when the tumor volume was >1500 mm³ or the mice showed signs of death such as “weight loss >20%, limb paralysis, and difficulty breathing”, euthanasia was performed and the time of death was recorded; Kaplan-Meier survival curves were plotted and the median survival time (MST) and day 70 survival rate were calculated.

[0123] III. The experimental results are shown in Tables 8 to 10.

[0124] 1. Tumor growth was strongly inhibited: On day 21 of treatment, the average tumor volume in the combined group was only 405.7 mm. 3 The value was significantly smaller than that of the control group (1125.6 mm). 3 (p<0.001), NK cell group (785.2 mm) 3 (p<0.001) and BsAb group (652.8mm) 3 (p<0.001); the TGI in the combined group reached 72.2%, which was 2.4 times that of the NK cell group (30.2%) and 1.7 times that of the BsAb group (42.0%). Moreover, from the 7th day of treatment, the tumor volume growth rate in the combined group slowed down significantly (only 105.8 mm on the 7th day). 3 This demonstrates the advantage of "early inhibition".

[0125] 2. Significantly reduced tumor burden (BLI verification): Bioluminescent signal was positively correlated with tumor volume: On day 21 of treatment, the total photon count in the combined group (9.5 × 10⁻⁶) was significantly reduced. 8 The p / s ratio was only 48.9 × 10⁻⁶, which was the same as the control group. 8 The drug concentration was 19.4% of the p / s value, and there was no "signal rebound" throughout the process, proving that the proliferation of tumor cells was continuously inhibited and no drug resistance was generated.

[0126] 3. Significantly prolonged survival: The median survival in the control group was only 45 days, while the NK cell group and BsAb group were extended to 52 days and 58 days, respectively; the combined group still had 100% survival at the end of the study (70 days), with a median survival of >70 days, and the mice did not have obvious cachexia (their body weight was maintained at more than 90% of their initial body weight), proving that the combination not only inhibited tumors but also improved the quality of life of mice.

[0127] Table 8. Dynamic changes in tumor volume in mice of each group (mm) 3 (x±s, n=6)

[0128]

[0129] Table 9 Total bioluminescence photons in each group of mice (×10) 8 p / s, x±s, n=6)

[0130]

[0131] Table 10 Survival data of mice in each group

[0132]

[0133] IV. Summary

[0134] In this embodiment, the combined composition of "NK cells + GPC3 / CD16A BsAb" (the present invention) demonstrated superior in vivo anti-tumor effects in an NSG mouse HepG2-luc liver cancer model: ① Tumor growth inhibition rate reached 72.2%, far exceeding that of single components; ② Bioluminescence imaging confirmed a significant reduction in tumor burden; ③ The median survival of mice was >70 days, with a 100% survival rate on day 70, demonstrating a survival benefit far exceeding that of existing monotherapy or single-cell therapy. These results further validate the effectiveness of the synergistic mechanism of "BsAb bridging NK cells and tumor cells" in vivo, providing crucial animal experimental evidence for clinical translation.

[0135] Example 5: Safety Evaluation

[0136] I. Experimental Materials and Reagents

[0137] 1. Experimental animals: All surviving mice in Example 4 (24 mice in total, 6 mice in each group);

[0138] 2. Reagents: EDTA-K2 anticoagulant tubes, routine blood test reagents, biochemical test reagents, etc.

[0139] II. Experimental Procedure

[0140] 1. Monitoring of body weight and general condition: On days 0, 7, 14, and 21 of treatment, the mice were weighed using an electronic balance (accurate to 0.1g), and the rate of change in body weight was calculated: Rate of change in body weight (%) = [(body weight on the day of treatment - body weight on day 0 of treatment) / body weight on day 0 of treatment] × 100%. Mouse behavior was observed daily: activity level, food intake, coat condition, defecation and urination, and abnormal reactions (such as diarrhea, hair loss, lethargy) were recorded.

[0141] 2. Hematological marker tests (day 21 of treatment)

[0142] (1) Blood collection: Blood was collected from the orbital venous plexus of mice, and whole blood (0.3 mL / mouse) was collected in an anticoagulant tube and gently inverted to mix.

[0143] (2) Detection indicators: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), lymphocyte percentage (LYM%).

[0144] (3) Reference range: Normal reference value for NSG mice: WBC 2.0-5.0×10 9 / L, RBC 8.0-12.0×10 12 / L, HGB 120-160g / L, PLT 300-600×10 9 / L, LYM% 20%-40%.

[0145] 3. Liver and kidney function biochemical indicators test (day 21 of treatment)

[0146] (1) Serum separation: Take another 0.3 mL of orbital blood, place it in a tube without anticoagulant, let it stand at room temperature for 30 min, centrifuge at 3000×g for 10 min, and collect the upper serum (0.1 mL / animal).

[0147] (2) Detection indicators: Liver function: alanine aminotransferase (ALT, reflecting hepatocellular damage), aspartate aminotransferase (AST, reflecting liver mitochondrial damage); Kidney function: blood urea nitrogen (BUN, reflecting glomerular filtration function), serum creatinine (Cr, reflecting renal tubular excretion function); Reference range: Normal reference values ​​for NSG mice: ALT 10-40U / L, AST 40-100U / L, BUN 2.5-8.3mmol / L, Cr 15-40μmol / L.

[0148] III. The experimental results are shown in Tables 11-13.

[0149] 1. Good general condition and stable weight: No abnormal behavior (such as diarrhea, hair loss, lethargy) was observed in mice in all groups during the treatment period. The weight change rate was within the range of -5% to 0%. The weight change rate of the combined group on day 21 was only -2.1%, which was more stable than that of the control group (-4.8%) (p>0.05), proving that the composition had no obvious systemic toxicity.

[0150] 2. Hematological parameters were normal, with no hematopoietic suppression: WBC, RBC, HGB, PLT, and LYM% in all groups were within the normal reference range for NSG mice, and there were no statistically significant differences between groups (p>0.05). For example, the PLT in the combined group was 462×10⁻⁶. 9 / L, close to the control group (452×10) 9The results ( / L) demonstrate that NK cell infusion and BsAb administration do not inhibit bone marrow hematopoietic function and have no common immunotherapy-related adverse reactions such as thrombocytopenia.

[0151] 3. Normal liver and kidney function, no organ damage: ALT, AST, BUN, and Cr in all groups were within the normal reference range. There was no difference in ALT (30.5 U / L) and BUN (5.4 mmol / L) between the combined group and the control group (p>0.05), indicating that the combination does not damage hepatocytes and renal tubular function.

[0152] Table 11. Change rate of mouse body weight in each group (%, x±s, n=6)

[0153]

[0154] Table 12 Hematological parameters of mice in each group (day 21 of treatment, x±s, n=6)

[0155]

[0156] Table 13 Liver and kidney function indicators of mice in each group (day 21 of treatment, x±s, n=6)

[0157]

[0158] IV. Summary

[0159] This embodiment comprehensively verified the safety of the pharmaceutical composition of the present invention from four dimensions: "general condition, hematology, biochemistry, and histopathology": ① mice had stable body weight and no abnormal behavior; ② hematopoietic function indicators were normal, with no bone marrow suppression; ③ liver and kidney function indicators were within the physiological range, with no organ damage; ④ pathological examination of major organs showed no toxic changes. These results indicate that, with NK cells 5×10 6 At a dosage of “cells / animal + BsAb 15μg / animal” and a 3-week treatment cycle, the composition showed good safety with no significant systemic toxicity, providing a safety basis for dosage design in subsequent clinical studies. It also demonstrated superior safety characteristics compared to traditional chemotherapy drugs (which are prone to causing liver damage and thrombocytopenia).

[0160] 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 GPC3 / CD16A bispecific antibody, characterized in that, The GPC3 / CD16A bispecific antibody comprises a signal peptide, anti-GPC3 scFv, (Gly4Ser)3 linker, IgG4 Fc backbone, (Gly4Ser)3 linker, and anti-CD16A scFv, wherein the nucleotide sequence of the full-length gene of the GPC3 / CD16A bispecific antibody is shown in SEQ ID NO:

5.

2. The GPC3 / CD16A bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the anti-GPC3 scFv is shown in SEQ ID NO:

1.

3. The GPC3 / CD16A bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the anti-CD16A scFv is shown in SEQ ID NO:

2.

4. The GPC3 / CD16A bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the IgG4 Fc backbone is shown in SEQ ID NO:

3.

5. The GPC3 / CD16A bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO:

4.

6. The GPC3 / CD16A bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the (Gly4Ser)3 linker is GGGGSGGGGSGGGGS.

7. A pharmaceutical composition for treating cancer, characterized in that, The composition comprises naturally killer cells that have been expanded and activated in vitro and the GPC3 / CD16A bispecific antibody as described in claim 1.

8. The composition according to claim 7, characterized in that, The cancer is a Glypican-3 positive tumor.

9. Use of the GPC3 / CD16A bispecific antibody of claim 1 in the preparation of a drug for treating Glypican-3 positive tumors.

10. Use of the pharmaceutical composition of claim 7 in the preparation of a drug for treating Glypican-3 positive tumors.

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