Preparation method of NK cell and application of NK cell in tumor immunotherapy

Abstract

The invention relates to a fusion protein and application thereof in enhancing the immune function of natural killer (NK) cells. The fusion protein comprises a CD16a signal peptide, an IL-15Ralpha functional region, a flexible connecting peptide, an IL-15 active core sequence and a high-affinity mutant CD16a transmembrane structural domain, and can be effectively positioned on a cell membrane and maintain the functional activity of IL-15. The fusion protein is introduced into an NK cell through a lentiviral vector, the obtained genetic engineering NK cell stably expresses the fusion protein in a low-oxygen environment, and the cell proliferation capacity, the ADCC function and the tumor killing activity are remarkably improved. In-vitro experiments show that the engineering NK cell shows an excellent killing effect in a breast cancer PDX model, and especially the killing rate reaches up to 97.1% under the action of a combined bispecific antibody. The invention provides a novel and effective molecular tool for cellular immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and particularly relates to a method for preparing NK cells and its application in tumor immunotherapy. Background Art

[0002] Natural Killer (NK) cells are important effector cells of the innate immune system and can effectively eliminate virus-infected cells and tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and non-MHC-dependent direct killing mechanisms. Therefore, NK cell-based immunotherapy is regarded as an important direction for anti-tumor treatment. However, current NK cell therapies still face multiple key bottlenecks in clinical applications, limiting their wide application and efficacy.

[0003] First, the downregulation of CD16 expression seriously affects the ADCC function of NK cells. Although IL-2 stimulation commonly used in in vitro expansion can promote proliferation, it significantly reduces the expression of CD16 (FcγRIIIa). Research shows that after culturing with IL-2 for 7 days, the positive proportion of CD16 in NK cells drops suddenly from >90% to <30%, directly weakening its ability to mediate antibody-dependent killing.

[0004] Second, the inhibitory effect of the solid tumor immune microenvironment is significant. Tumor tissues are often in a hypoxic state (O2 < 1%) and are rich in immunosuppressive factors such as TGF-β (>50 ng / mL) and metabolites such as adenosine. These factors can significantly weaken the cytotoxicity of NK cells. Clinical data show that the expression of granzyme B in tumor-infiltrating NK cells of solid tumor patients decreases by about 78% compared with that in peripheral blood.

[0005] Third, insufficient targeting is also a current treatment difficulty. Traditional NK cell therapies mostly rely on non-specific antibodies to mediate ADCC. However, solid tumors generally lack highly expressed and specific target antigens, and the antibody permeability is limited. For example, the objective remission rate of trastuzumab in the treatment of HER2-positive breast cancer is only 12 - 24%.

[0006] Although existing improvement strategies can improve the efficacy to a certain extent, there are still obvious limitations. For example, IL-15 can extend the survival period of NK cells but is prone to induce severe cytokine storms; CAR-NK enhances targeting, but the antigen heterogeneity of solid tumors brings the risk of immune escape; high-affinity CD16 variants can enhance ADCC, but there is no significant improvement in the immunosuppressive microenvironment.

[0007] Therefore, there is an urgent need to develop a new NK cell treatment strategy that integrates targeted activation, metabolic adaptation, and microenvironment regulation to achieve more efficient and persistent immune clearance of solid tumors. Summary of the Invention

[0008] The present invention provides a novel fusion protein and its application in the preparation of genetically engineered NK cells, aiming to enhance the membrane localization ability, endogenous IL-15 signal transduction ability, and CD16-mediated ADCC function of NK cells, thereby enhancing their anti-tumor activity.

[0009] Therefore, on the one hand, the present invention discloses a fusion protein, and the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1.

[0010] Preferably, the nucleotide sequence of the fusion protein of the present invention after codon optimization is as shown in SEQ ID NO.2.

[0011] On the one hand, the present invention also discloses a genetically engineered NK cell, which is obtained by introducing the nucleotide sequence shown in SEQ ID NO.2 into natural killer cells of CD56 + CD3 - through a lentiviral vector and amplifying and culturing for 14 days under the condition of 5% O2 + 5% CO2.

[0012] Preferably, the proportion of CD16 + cells in the genetically engineered NK cells of the present invention is 89.6% ± 4.1%, the proportion of cells expressing the fusion protein is 83.6% ± 3.4%, the IL-15 secretion amount is 420 ± 35 pg / 10 6 cells / 24 h, and the positive rate of CD107a is 41.3% ± 2.7%.

[0013] On the one hand, the present invention also discloses a pharmaceutical composition, which comprises an effective amount of the genetically engineered NK cells and an effective amount of bispecific antibody.

[0014] Preferably, the bispecific antibody of the present invention comprises an anti-EGFR Fab region, an anti-HER2 Fab region, and an Fc region.

[0015] Preferably, the anti-EGFR Fab region of the present invention is an anti-EGFR arm Fab region, and the amino acid sequences of its heavy chain variable region and light chain variable region are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0016] Preferably, the anti-HER2 Fab region of the present invention is an anti-HER2 arm Fab region, and the amino acid sequences of its heavy chain variable region and light chain variable region are as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0017] Preferably, the Fc region of the present invention comprises an Fc domain composed of a Kb chain and a He chain, and the amino acid sequences of the Kb chain and the He chain are as shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0018] In one aspect, the present invention also discloses an application of the genetically engineered NK cells as described above in the preparation of anti-tumor drugs.

[0019] The present invention provides an engineered NK cell based on a fusion protein and a preparation method thereof. The fusion protein integrates a high-affinity CD16a variant, the active core of IL-15 and the functional domain of its receptor IL-15Rα to form a transmembrane fusion protein with optimized structure and complementary functions. This design shows significant advantages at multiple levels, specifically including the following aspects:

[0020] 1. Significantly improved membrane localization and expression efficiency: By introducing the signal peptide and transmembrane region of CD16a into the fusion construct, the accurate localization of the fusion protein on the cell membrane is effectively promoted. Flow cytometry results show that the double-positive expression rate of the fusion protein in HEK293T cells is as high as 82.4% ± 3.8%, which is significantly higher than 0.3% ± 0.1% of the group without CD16a fusion, indicating that this construct can achieve high-efficiency expression and functional manifestation.

[0021] 2. Achieving autocrine IL-15 signal transduction in cells: The introduction of the active core of IL-15 and the functional domain of IL-15Rα into the fusion protein can trigger the self-activation and survival mechanism of NK cells without relying on exogenous cytokines, significantly enhancing the functional plasticity and maintenance ability of the cells themselves, and avoiding the dependence of traditional NK cells on the supplementation of IL-2 or IL-15.

[0022] 3. Enhancing the activation ability of the STAT5 signal axis: Co-culture experiments of the fusion protein in the 293T model show that it can significantly increase the level of p-STAT5 (OD450 = 1.78 ± 0.11), which is 2.7 times that of the control group (OD450 = 0.65 ± 0.07), proving that this fusion structure can effectively activate the IL-15-mediated JAK / STAT signaling pathway, which is the key mechanism for maintaining the long-term survival and functional activity of NK cells.

[0023] 4. Improving NK cell expansion and functional phenotypes: Under the condition of simulating the hypoxic tumor microenvironment, the genetically engineered NK cells expressing this fusion protein show excellent proliferation ability (amplification multiple 45.7 ± 3.2), the CD16 positive ratio is as high as 89.6% ± 4.1%, and the fusion protein is continuously expressed (the proportion of cells expressing the fusion protein is 83.6% ± 3.4%), which is greatly improved compared with the control group, indicating that this system has good stability and adaptability.

[0024] 5. Enhancement of ADCC and natural killer activity: In the ADCC induction experiment of engineered NK cells, the positive rate of CD107a was 41.3% ± 2.7%, which was significantly higher than that of the untransfected control group (11.5% ± 1.2%). The target cell lysis rate reached 71.6% ± 4.3%, far exceeding that of ordinary NK cells (24.8% ± 3.4%). This indicates that this construct not only enhances the Fc-mediated antibody-dependent cytotoxicity but also improves the efficiency of the natural killer pathway.

[0025] 6. Enhancement of the tumor-targeted killing effect mediated by bispecific antibodies: The engineered NK cells constructed in the present invention can be used in combination with bispecific antibodies targeting tumor antigens and CD16, significantly enhancing their ability to target and lyse tumor cells. The results of in vitro killing experiments showed that in the target cells of the breast cancer PDX model, the use of engineered NK cells alone achieved a lysis rate of up to 82.5% ± 4.1%, while the killing efficiency further increased to 97.1% ± 2.6% after combination with the bispecific antibody, achieving a synergistic amplification effect. This mechanism makes full use of the advantage of high expression of CD16 in the fusion protein to form a highly affinity effector loop with the CD16-binding arm of the bispecific antibody, enhancing the formation of immune synapses and exocytic killing reactions, especially suitable for indications with strong antigen heterogeneity such as solid tumors.

[0026] 7. Having a good basis for transformability and safety: The fusion protein constructed in the present invention uses human-derived sequences, avoiding immune rejection reactions caused by the introduction of xenogeneic proteins. At the same time, lentiviral vectors are used to achieve stable transduction, combined with clinical-grade culture conditions, with good adaptability to GMP processes, facilitating industrial promotion and clinical development.

[0027] In summary, the fusion protein provided by the present invention and its application in genetically engineered NK cells not only achieve an innovative combination of the structural functions of NK cell receptors but also significantly enhance their anti-tumor immune effects, especially the targeted killing ability in combination with bispecific antibody therapy, with broad clinical application prospects, providing a new solution for the development of a new generation of NK cell immunotherapy products with "self-carried growth factors + antibody response". Brief Description of the Drawings

[0028] Figure 1 Schematic diagram of the structure of the fusion polypeptide.

[0029] Figure 2 Flow cytometry detection results of the membrane localization of the fusion protein.

[0030] Figure 3 Flow cytometry detection results of NK cells.

[0031] Figure 4 Western blot was used to detect the expression of the fusion protein, where CD16-IL15 is the fusion protein.

[0032] Figure 5 Flow cytometry detection results of the fusion protein.

[0033] Figure 6 SPR response curve and heat map. Specific implementation mode

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] 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.

[0036] Example 1: Design and verification of the fusion polypeptide

[0037] I. Overview of the fusion protein design

[0038] 1. CD16a signal peptide (guiding secretion and membrane localization): MWQLLLPTALLLLVSAGMRT;

[0039] 2. IL-15Rα chain functional domain (key region for binding IL-15): GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR;

[0040] 3. Flexible linker peptide (ensuring domain independence): GGGSGGGSGGGS;

[0041] 4. IL-15 active core (survival and proliferation promoting functional domain): VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI;

[0042] 5. CD16a transmembrane region (V158 high-affinity variant, phenylalanine (F) at position 158 of the original sequence → valine (V), enhancing antibody binding): TGLYFSVKTNIRSSTRDWKDHKFKW;

[0043] 6. The complete amino acid sequence of the fusion protein is shown in SEQ ID NO.1, and its structural diagram is as Figure 1 shown.

[0044] II. Experimental steps

[0045] 1. Gene construction and plasmid transfection

[0046] 1.1 Sequence synthesis and cloning: Synthesize the full-length cDNA (as shown in SEQ ID NO.2) after codon optimization of the amino acid sequence shown in SEQ ID NO.1; clone it into the pcDNA3.1(+) expression vector, and the restriction enzyme sites are HindIII / XbaI.

[0047] 1.2 Cell transfection: Culture HEK293T cells to a certain cell density and seed them in 6-well plates. When the cells grow to 70%-80% confluence, perform transfection. Add 1 mL of transfection reagent to each well, which contains 2.5 μg of Plasmid DNA, 5 μL of P3000 Reagent, 7.5 μL of Lipofectamine 3000, and the balance is Opti-MEM. After transfection, continue to culture in an incubator containing 5% CO2 at 37 °C for 48 hours.

[0048] 2. Detection of membrane localization expression by flow cytometry

[0049] 2.1 Antibody staining: Anti-IL-15Rα labeled with PE (1:50), anti-CD16 labeled with APC (1:50).

[0050] 2.2 Staining procedure: Collect the cells 48 h after transfection, wash them with PBS, fix them with 4% PFA for 10 min, add the antibody mixture and stain for 30 min (protected from light at 4 °C), and detect with a FACS analyzer.

[0051] 2.3 Results ( Figure 2 ): The proportion of double-positive (CD16+IL15Rα+) cells in the experimental group was 82.4% ± 3.8%, and that in the control group (wild-type CD16a, no amino acid mutation, and no IL-15 fusion part) was 0.3% ± 0.1%.

[0052] 3. NK cell activation experiment

[0053] 3.1 Cell co-culture: Mix the 293T transfected cells with the isolated and identified NK cells (as described in Example 2) at a ratio of 1:1 and co-culture them in a medium containing 10% FBS RPMI-1640 for 24 hours.

[0054] 3.2 Measurement index: After cell lysis, detect the expression of p-STAT5 by ELISA.

[0055] 3.3 The results showed that the OD450nm value of the experimental group was 1.78 ± 0.11; the OD450nm value of the control group (wild-type CD16a, no amino acid mutation, and no IL-15 fusion part) was 0.65 ± 0.07.

[0056] The above results indicate that the fusion protein designed in the present invention was successfully expressed in 293T cells, showing good membrane localization ability and IL-15 functional activity. It can effectively activate the STAT5 signaling pathway of NK cells, significantly superior to the unfused CD16 control group, and has the application potential as an enhanced effector receptor in NK cell immunotherapy.

[0057] Example 2: Construction and functional verification of genetically engineered NK cells expressing the fusion protein

[0058] I. Experimental materials and reagents

[0059] 1. Donor source: Peripheral blood of healthy volunteers (meeting ethical requirements).

[0060] 2. Sorting reagent: CD56 MicroBeads (Miltenyi Biotec).

[0061] 3. Lentiviral vector: pLVX-EF1α (Clontech).

[0062] 4. Transfected cells: Lenti-X 293T (Takara).

[0063] 5. Transfection reagent: PEIpro (Polyplus).

[0064] 6. Cell culture medium: X-VIVO 15 (Lonza) + 10% human AB serum.

[0065] 7. Cytokines: rhIL-2 (PeproTech,) and rhIL-21 (PeproTech).

[0066] 8. Flow antibodies: APC-CD16, PE-IL-15Rα, FITC-CD56, PerCP-CD3 (all purchased from BioLegend).

[0067] II. Experimental procedures

[0068] 1. Isolation and identification of peripheral blood NK cells

[0069] 1.1 Isolation: PBMCs were isolated by Ficoll gradient centrifugation;

[0070] 1.2 Magnetic bead sorting: CD56 + CD3 - NK cells were purified using CD56 MicroBeads;

[0071] 1.3 Flow cytometry verification: The proportion of CD56 + CD3 - reached 95.2% ± 1.1%, asFigure 3 as shown

[0072] 2. Lentiviral vector construction and packaging

[0073] 2.1 Inserted sequence: Construct an expression cassette containing SEQ ID NO.2;

[0074] 2.2 Transfect Lenti-X 293T cells: Using the three-plasmid method (pLVX-CD16-IL15 + psPAX2 + pMD2.G), and transfect with PEIpro;

[0075] 2.3 Virus collection: Collect the virus supernatant at 48 h and 72 h, and concentrate it after filtration through a 0.45 μm filter;

[0076] 2.4 Titer detection: The virus titer was determined by qPCR to be 1×10 9 TU / mL.

[0077] 3. NK cell infection and expansion culture

[0078] 3.1 Infection conditions: MOI = 20, containing 8 μg / mL polybrene, and infect by centrifugation (1200 g, 90 min, 32 °C);

[0079] 3.2 Culture system: X-VIVO 15 + 10% AB serum + IL-2 (100 U / mL) + IL-21 (10 ng / mL);

[0080] 3.3 Hypoxic culture: Expand in a 5% O2 + 5% CO2 incubator for 14 days, and change the medium by half every 3 days.

[0081] III. Detection methods and data analysis

[0082] 1. Verification of fusion protein expression

[0083] 1.1 Western blot: Double verification with anti-CD16 and anti-IL-15 antibodies, and specific bands were observed for the fusion protein (as Figure 4 shown);

[0084] 1.2 Flow cytometry: Double staining with CD16-APC and IL-15Rα-PE, the double-positive rate of the experimental group was 83.6% ± 3.4%( Figure 5 ), and that of the control group was 0%.

[0085] 2. Detection of NK cell phenotype and function, and the results are shown in Table 1.

[0086] Table 1 Summary of NK cell phenotype and function detection

[0087]

[0088] *Indicates significant difference compared with the untransfected group, p < 0.01 (n = 3, t-test)

[0089] IV. Conclusion

[0090] This genetically engineered NK cell can stably express the fusion protein, maintain high expression under hypoxic conditions, significantly enhance the proliferation ability and ADCC function, and provide a strong cellular basis for tumor immunotherapy.

[0091] Example 3: In vitro killing experiment

[0092] 1. Preparation of target cells: The breast cancer PDX tissue of patient GC-024 was cultured in vitro in RPMI 1640 + 10% FBS. The target cells were labeled with CFSE (5 μM, Invitrogen), incubated at room temperature for 10 min, and washed twice with PBS after terminating the staining.

[0093] 2. Preparation of effector cells (NK cells): Ordinary NK cells and genetically engineered NK cells were respectively derived from the PBMC of healthy volunteers and prepared by magnetic bead sorting and lentiviral transduction (see Example 2 for details).

[0094] 3. Double antibody treatment group: Pre-add bispecific antibody (see Example 5 for details) and incubate at 37 °C for 30 minutes.

[0095] 4. Co-culture settings:

[0096] (1) Effector-to-target ratio setting: 1:1, 5:1, 10:1.

[0097] (2) Culture system: Add 1 × 10 4 target cells per well to a 96-well U-bottom plate, add the corresponding proportion of effector cells, and the final volume is 200 μL.

[0098] (3) Culture time: Co-culture for 24 hours at 37 °C in a 5% CO2 incubator.

[0099] 5. LDH release detection: Use CytoTox Non-Radioactive Cytotoxicity Assay (Promega) to detect the LDH in the supernatant. Read the OD490nm value, and use the blank well and the target cell autolysis well as references to calculate the killing rate.

[0100] 6. Apoptosis detection (flow cytometry): Collect the cells, wash them with PBS, and then use the Annexin V-FITC / PI kit (BD Biosciences). Incubate at room temperature in the dark for 15 min. Collect 10,000 cells using a flow cytometer and analyze the apoptosis ratio (Annexin V+ / PI-) with FlowJo.

[0101] 7. Experimental results (Tables 2 and 3), genetically engineered NK cells showed significantly enhanced tumor killing activity. The combination of bispecific antibodies further enhanced the targeting effect, with a killing rate as high as 97.1%, showing potential for synergistic anti-tumor effects.

[0102] Table 2 NK cell killing rate at different effector-to-target ratios (LDH method)

[0103]

[0104] *Note: * Compared with the normal NK group, p < 0.01, with statistical significance.

[0105] Table 3 Proportion of Annexin V+ / PI- apoptotic cells in different groups at an effector-to-target ratio of 5:1

[0106]

[0107] Example 4: In vivo efficacy evaluation in PDX mouse model

[0108] 1. Establishment of animal model:

[0109] (1) Model animals: NOD / SCID mice, 6 - 8 weeks old, female, body weight about 20 ± 2 g.

[0110] (2) Inoculation method: Inoculate GC-027 gastric cancer PDX tissue blocks (about 3 mm 3 ), implant subcutaneously under the right axilla.

[0111] (3) Follow-up: Start group treatment after the tumor diameter reaches about 5.5 - 6 mm (about 100 mm 3 ).

[0112] 2. Grouping and administration (n = 8 for each group): As shown in Table 4.

[0113] Table 4 Grouping and administration

[0114]

[0115] 3. Efficacy evaluation:

[0116] (1) Tumor measurement: Measure the tumor volume every 3 days (formula: V = 0.5 × length × width2), for 28 days continuously.

[0117] (2) Survival analysis: Observe each group for 90 days continuously, record the death time, and plot the survival curve using Kaplan-Meier method.

[0118] 4. Experimental results

[0119] (1) Tumor measurement results showed (Table 5) that genetically engineered NK cells exhibited significantly enhanced tumor killing activity. The combination of bispecific antibodies further enhanced the targeting effect, and the two had a synergistic anti-tumor effect.

[0120] Table 5 Summary of experimental results

[0121]

[0122] *Note: Compared with the PBS group, p < 0.01, significant tumor suppression effect.

[0123] (2) Kaplan-Meier survival curve: The median survival time of the control group was 28 days, that of the engineered NK monotherapy group was 57 days, and that of the combination treatment group was > 90 days.

[0124] 5. Experimental summary

[0125] (1) Immunomechanism analysis: The fusion protein synchronizes the IL-15 autocrine signal with CD16 activation through the "signal anchoring" strategy, promotes STAT5 phosphorylation, and activates downstream proliferation and survival signals. Hypoxic culture upregulates mitochondrial complex I (NDUFB6 expression increased by 2.3-fold), enhancing the oxidative phosphorylation capacity (OCR detected by Seahorse increased by 1.8-fold).

[0126] (3) Targeting and persistence: The bispecific antibody binds with high affinity to CD16 (SPR detected KD = 1.2×10 -9 M), guiding NK cells to accumulate in tumors. Engineered NK cells could still be detected in peripheral blood 28 days after transplantation (12.3% vs 0.3% for normal NK cells).

[0127] (4) Safety evaluation: Serum IL-6 and TNF-α levels were both lower than the inflammation risk threshold (IL-6 < 10 pg / mL, TNF-α < 20 pg / mL), and no signs of CRS were observed.

[0128] The above results indicate that the present invention realizes efficient, targeted, persistent, and safe solid tumor clearance by constructing genetically engineered NK cells with a fusion protein and combining with tumor-specific bispecific antibodies, and has significant application prospects in clinical translation.

[0129] Example 5: Preparation and testing of bispecific antibodies

[0130] I. Bispecific antibody design and amino acid sequence

[0131] 1. Construction Schematic and Structural Innovation: The present invention provides a bispecific antibody structure that ensures the correct pairing of heterologous heavy chains, significantly improving the assembly efficiency and yield of bispecific antibodies. The schematic is as follows: [Anti-EGFR Fab (heavy chain + light chain)]—[Anti-HER2 Fab (heavy chain + light chain)]—Fc region (heterodimer).

[0132] 2. Specific Amino Acid Sequences

[0133] 2.1 Fab Region of the Anti-EGFR Arm (The sequence is optimized by humanization and affinity-enhancing mutations)

[0134] (1) Heavy Chain Variable Region (VH): As shown in SEQ ID NO.3.

[0135] (2) Light Chain Variable Region (VL): As shown in SEQ ID NO.4.

[0136] 2.2 Fab Region of the Anti-HER2 Arm (The sequence is optimized by humanization and affinity-enhancing mutations)

[0137] (1) Heavy Chain Variable Region (VH): As shown in SEQ ID NO.5.

[0138] (2) Light Chain Variable Region (VL): As shown in SEQ ID NO.6.

[0139] 2.3 Fc Domain Sequence (Heterodimer)

[0140] (1) Kb Chain (containing K409R mutation): As shown in SEQ ID NO.7.

[0141] (2) He Chain (containing K392D mutation): As shown in SEQ ID NO.8.

[0142] II. Optimization of Preparation Method

[0143] 1. Construction of Expression Vector: A dual-expression vector using the CMV promoter is used to express the heavy chain and light chain simultaneously. The coding region is optimized according to the codon preference of CHO cells, and the GC content is adjusted to ~55% to improve translation efficiency. A mammalian-optimized signal peptide (such as IgK or IgH signal peptide) is introduced to enhance the secretion efficiency.

[0144] 2. Establishment of Stable Cell Lines: In this study, the PiggyBac transposon system was first used to construct stable expression cell lines in CHO-K1 cells. Positive clones were screened in a selection medium containing Zeocin (200 μg / mL), and with the help of the ClonePix2 automatic picking platform, high-expression monoclonal cell lines were obtained through efficient screening based on the size of the fluorescence layer and the expression titer.

[0145] 3. Development of perfusion culture system: In the subsequent production process, a high-density cell culture system based on perfusion culture was developed. CD OptiCHO TM medium was used, supplemented with 6 mM L-glutamine and 0.1% Pluronic F-68, and the initial seeding density was 2×10 6 cells / mL. During the 14-day perfusion process, stable product expression was achieved, and the final average yield reached 8.5 g / L, which was about 3.7 times higher than that of traditional batch culture, significantly improving the production efficiency.

[0146] 3. Purification process: The bispecific antibody constructed in this study was purified through a three-step chromatography purification process to obtain a high-purity product. First, the bispecific antibody was efficiently captured by Protein A affinity chromatography, and the recovery rate exceeded 95%; then anion exchange chromatography (AEX) was used to effectively remove host cell DNA and HCP impurities, and the DNA residue was less than 1 ng / mg; finally, hydrophobic interaction chromatography (HIC) was used to further remove high-molecular aggregates, making the aggregation rate less than 1%. The purity of the final product exceeded 99%, and the endotoxin level was less than 0.1 EU / mg, meeting the clinical-grade formulation standard.

[0147] III. Target binding ability detection experiment (Surface Plasmon Resonance, SPR)

[0148] 1. Experimental purpose: The SPR technique was used to evaluate the binding affinity (KD) of the bispecific antibody of the present invention to the targets EGFR and HER2, and to compare it with the existing commercial bispecific antibody controls (Sym013, ZW25).

[0149] 2. Materials and instruments: SPR analyzer (Biacore T200), chip type (CM5 sensor chip), coupling reagent (NHS / EDC activation system, ethanolamine), HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4), recombinant human EGFR and HER2 were both Fc-tag purified proteins with a concentration of 1 mg / mL, and the bispecific antibody of the present invention, ZW25, Sym013 (all were serially diluted with the same concentration).

[0150] 3. Experimental steps

[0151] 3.1 Target immobilization: Install the CM5 chip and pre-equilibrate the system. Start the activation procedure: Inject a mixture of NHS (0.05 M) / EDC (0.2 M) for 60 seconds. Inject recombinant EGFR or HER2 (50 μg / mL, 10 mM sodium acetate buffer, pH 5.0) and covalently couple it to the chip surface. The target coupling level is approximately 10,000 RU. Block the unreacted sites with 1 M ethanolamine (pH 8.5). Use channel 1 (empty channel) as a reference control (without coupled protein).

[0152] 3.2 Antibody analysis: Prepare a series of antibody dilutions: The concentration gradient is 0.125 nM to 8 nM (two-fold dilution, a total of 8 concentrations). Inject the antibody samples at a flow rate of 30 μL / min, with a binding time of 180 seconds and a dissociation time of 600 seconds. Regenerate the chip surface with 10 mM Glycine-HCl (pH 2.5) after each round of analysis. Collect the real-time binding curves (Sensorgram) at each concentration and perform reference channel subtraction.

[0153] 3.3 Data analysis: Use BIAevaluation software for fitting analysis and fit with a 1:1 Langmuir binding model. Obtain ka (association rate constant), kd (dissociation rate constant), and calculate KD = kd / ka.

[0154] 4. Experimental results and analysis. The specific results are as Figure 6 shown in

[0155] 4.1 High-affinity performance: The KD of the bispecific antibody of the present invention for EGFR binding is 1.8×10 -9 M, and for HER2 is 6.1×10 - 10 M, both significantly superior to the commercial control antibodies (Sym013, ZW25).

[0156] 4.2 Slow dissociation rate (low kd): It shows that the bispecific antibody forms a stable complex with the target, with a longer binding duration, which helps to enhance the cell killing efficiency.

[0157] 4.3 Obvious dual-target synergy characteristics: Both arms have high affinity, laying the foundation for subsequent cytotoxic effects and in vivo efficacy.

[0158] Table 6 Affinity data table

[0159]

[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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. The fusion protein according to claim 1, characterized in that, The nucleotide sequence of the fusion protein after codon optimization is shown in SEQ ID NO.

2.

3. A genetically engineered NK cell, characterized in that, The genetically engineered NK cells are obtained by introducing the nucleotide sequence shown in SEQ ID NO.2 into CD56 + CD3 - natural killer cells through a lentiviral vector and amplifying and culturing them for 14 days under the condition of 5% O2 + 5% CO2.

4. The genetically engineered NK cell according to claim 3, wherein The CD16 of the genetically engineered NK cells + The proportion of cells was 89.6% ± 4.1%, the proportion of cells expressing the fusion protein was 83.6% ± 3.4%, the IL-15 secretion amount was 420 ± 35 pg / 10 6 cells / 24 h, and the positive rate of CD107a was 41.3% ± 2.7%.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the genetically engineered NK cells as claimed in claim 3 and an effective amount of the bispecific antibody.

6. The composition according to claim 5, characterized in that, The bispecific antibody comprises an anti-EGFR Fab region, an anti-HER2 Fab region and an Fc region.

7. The composition according to claim 6, wherein The anti-EGFR Fab region is the anti-EGFR arm Fab region, and the amino acid sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

8. The composition according to claim 6, wherein The anti-HER2 Fab region is the anti-HER2 arm Fab region, and the amino acid sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

9. The composition according to claim 6, characterized in that, The Fc region comprises an Fc domain composed of a Kb chain and an He chain, wherein the amino acid sequences of the Kb chain and the He chain are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

10. Use of a genetically engineered NK cell as claimed in claim 3 in the preparation of an anti-tumor drug.

Images