Preparation method of nk cells and application thereof in tumor immunotherapy

Abstract

The present application relates to a kind of fusion protein and its application in enhancing natural killer (NK) cell immune function.The fusion protein includes CD16a signal peptide, IL-15R alpha functional region, flexible connecting peptide, IL-15 active core sequence and high affinity mutant CD16a transmembrane domain, can be effectively positioned cell membrane and keep the functional activity of IL-15.It is introduced into NK cell by lentiviral vector, and the obtained genetically engineered NK cell stably expresses the fusion protein under low oxygen environment, significantly improves cell proliferation capacity, ADCC function and tumor killing activity.In vitro experiment shows that the engineered NK cell exhibits excellent killing effect in breast cancer PDX model, especially under the action of combined bispecific antibody, and the killing rate is as high as 97.1%.The present application provides a new and effective molecular tool for cellular immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to a preparation method of NK cells and its application in tumor immunotherapy. BACKGROUND

[0002] Natural killer (NK) cells are important effector cells of the innate immune system, which can effectively clear virus-infected cells and tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and non-MHC-dependent direct killing mechanism. Therefore, NK cell-based immunotherapy is considered as an important direction of anti-tumor therapy. However, current NK cell therapy still faces many key bottlenecks in clinical application, which limits its wide application and efficacy.

[0003] Firstly, the down-regulation of CD16 expression seriously affects the ADCC function of NK cells. Although IL-2 stimulation commonly used in in vitro expansion process can promote proliferation, it significantly reduces CD16 (FcyRIIIa) expression. Studies have shown that after 7 days of IL-2 culture, the proportion of CD16 positive in NK cells decreases from >90% to <30%, directly weakening its ability to mediate antibody-dependent killing.

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

[0005] Thirdly, the lack of targeting is also a current treatment difficulty. Traditional NK cell therapy relies on non-specific antibody-mediated ADCC, however, solid tumors generally lack high expression and specific target antigens, and the penetration of antibodies is limited. For example, the objective remission rate of trastuzumab in the treatment of HER2-positive breast cancer is only 12-24%.

[0006] Although the existing improvement strategies have improved the efficacy to some extent, there are still obvious limitations. For example, IL-15 can prolong the survival of NK cells, but it is easy to induce severe cytokine storm; CAR-NK enhances targeting, but the heterogeneity of solid tumor antigens brings the risk of immune escape; high affinity CD16 variants can enhance ADCC, but they do not significantly improve the immunosuppressive microenvironment.

[0007] Therefore, it is urgent to develop a new NK cell treatment strategy that integrates targeted activation, metabolic adaptation and microenvironment regulation, in order to achieve more efficient and persistent immune clearance of solid tumors. SUMMARY

[0008] The application provides a novel fusion protein and its application in preparing genetically engineered NK cells, aiming to improve the membrane positioning ability, endogenous IL-15 signal transmission ability and CD16-mediated ADCC function of NK cells, thereby enhancing the anti-tumor activity thereof.

[0009] Therefore, in one aspect, the application discloses a fusion protein, the amino acid sequence of which is shown as SEQ ID NO. 1.

[0010] Preferably, the codon-optimized nucleotide sequence of the fusion protein of the application is shown as SEQ ID NO. 2.

[0011] In one aspect, the application further discloses a genetically engineered NK cell, which is obtained by introducing the nucleotide sequence shown as SEQ ID NO. 2 into CD56 + CD3 - natural killer cells through a lentiviral vector, and is obtained after 14-day expansion culture under the condition of 5% O2+5% CO2.

[0012] Preferably, the genetically engineered NK cell of the application has a CD16 + cell ratio of 89.6%±4.1%, a fusion protein-expressing cell ratio of 83.6%±3.4%, an IL-15 secretion amount of 420±35 pg / 10 6 cells / 24h, and a CD107a positive rate of 41.3%±2.7%.

[0013] In one aspect, the application further discloses a pharmaceutical composition, which comprises an effective amount of the genetically engineered NK cell and an effective amount of a bispecific antibody.

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

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

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

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

[0018] In one aspect, the application also discloses the use of the genetically engineered NK cell in the preparation of an anti-tumor drug.

[0019] The application provides an engineered NK cell based on a fusion protein and a preparation method thereof, the fusion protein integrating a high-affinity CD16a variant, an IL-15 active core and a functional domain of its receptor IL-15Rα to form a transmembrane fusion protein with optimized structure and complementary functions. The design exhibits significant advantages in multiple aspects, including the following aspects.

[0020] 1. Membrane localization expression efficiency is significantly improved: by introducing the signal peptide and the transmembrane region of CD16a into the fusion construction, the accurate localization of the fusion protein on the cell membrane is effectively promoted. The flow detection 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 improved compared with the 0.3%±0.1% of the non-fusion CD16a group, indicating that the construction can realize high-efficiency expression and functional externalization.

[0021] 2. Realize self-secretion type IL-15 signal transmission: the introduction of the IL-15 active core and the IL-15Rα functional domain in the fusion protein can trigger the self-activation and survival mechanism of NK cells without relying on exogenous cytokines, significantly enhance the functional plasticity and maintenance ability of the cells themselves, and avoid the dependence of traditional NK cells on IL-2 or IL-15 supplement.

[0022] 3. Enhance the activation ability of STAT5 signal axis: the co-culture experiment of the fusion protein in the 293T model shows that it can significantly improve the p-STAT5 level (OD450=1.78±0.11), which is 2.7 times of the control group (OD450=0.65±0.07), proving that the fusion structure can effectively activate the JAK / STAT signal pathway mediated by IL-15, which is the key mechanism to maintain the long-term survival and functional activity of NK cells.

[0023] 4. Improve the expansion and functional phenotype of NK cells: under the simulation conditions of the hypoxic tumor microenvironment, the genetically engineered NK cells expressing the fusion protein exhibit excellent proliferation capacity (expansion multiple 45.7±3.2), the CD16 positive proportion 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 significantly improved compared with the control group, indicating that the system has good stability and adaptability.

[0024] 5. Improve ADCC and natural killer activity: In ADCC induction experiments, the CD 107a positive rate of the engineered NK cells was 41.3% ± 2.7%, which was significantly higher than that of the untransfected control group (11.5% ± 1.2%), and the target cell lysis rate reached 71.6% ± 4.3%, which was much higher than that of ordinary NK cells (24.8% ± 3.4%), indicating that the construction not only enhanced the Fc-mediated antibody-dependent cellular cytotoxicity, but also improved the efficiency of the natural killer pathway.

[0025] 6. Enhance bispecific antibody-mediated tumor targeting killing effect: The engineered NK cells constructed by the application can be used in cooperation with bispecific antibodies targeting tumor antigens and CD16, which significantly enhances the targeting recognition and lysis ability of tumor cells. The in vitro killing experiment results show that in breast cancer PDX model target cells, the use of engineered NK cells alone can achieve a lysis rate of up to 82.5% ± 4.1%, and after combined with bispecific antibodies, the killing efficiency is further improved to 97.1% ± 2.6%, achieving a synergistic amplification effect. This mechanism fully utilizes the advantage of high expression of CD16 in the fusion protein, forms a highly affine effector ring with the CD16 binding arm of the bispecific antibody, enhances the formation of the immune synapse and the exocytosis killing reaction, and is especially suitable for solid tumors and other indications with strong antigen heterogeneity.

[0026] 7. Good convertibility and safety basis: The fusion protein constructed by the application uses human sequences, avoiding the immune rejection reaction caused by the introduction of xenogeneic proteins. At the same time, stable transduction is achieved using a lentiviral vector, and combined with clinical level culture conditions, it has good GMP process adaptability, facilitating industrialization promotion and clinical development.

[0027] In summary, the fusion protein provided by the application and its application in genetically engineered NK cells not only realize the innovative combination of NK cell receptor structure and function, but also significantly enhance its anti-tumor immune effect, especially the targeting killing ability in combination with bispecific antibody therapy, which has broad clinical application prospects, and provides a new solution for the development of a new generation of "self-growth factor + antibody response" NK cell immunotherapy products. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Structure diagram of the fusion polypeptide.

[0029] Figure 2 Fusion protein membrane positioning flow detection results.

[0030] Figure 3 NK cell flow detection results.

[0031] Figure 4 Western blot detection of fusion protein expression, wherein CD16-IL15 is a fusion protein.

[0032] Figure 5 Fusion protein flow cytometry results.

[0033] Figure 6 SPR response curve and heat map. DETAILED DESCRIPTION

[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 art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] 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 application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

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

[0037] I. Overview of fusion protein design

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

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

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

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

[0042] 5. CD16a transmembrane region (V158 high-affinity variant, original sequence phenylalanine (F) at position 158 -> valine (V), enhances 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 shown in Figure 1

[0044] II. Experimental procedures

[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) of the amino acid sequence shown in SEQ ID NO. 1 after codon optimization; clone it into the pcDNA3.1(+) expression vector, and the restriction enzyme cutting site is HindIII / XbaI.

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

[0048] 2. Flow cytometry detection of membrane localization expression

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

[0050] 2.2 Staining procedure: Collect cells 48 hours after transfection, wash with PBS, fix with 4% PFA for 10 minutes, add antibody mixture for staining for 30 minutes (4°C, dark), 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, without amino acid mutation, and without IL-15 fusion part) was 0.3%±0.1%.

[0052] 3. NK cell activation experiment

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

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

[0055] 3.3 The results show that the OD450nm value of the experimental group is 1.78±0.11, and that of the control group (wild-type CD16a, without amino acid mutation, and without IL-15 fusion part) is 0.65±0.07.

[0056] The above results show that the fusion protein designed in the application is successfully expressed in 293T cells, and has good membrane positioning ability and IL-15 functional activity. It can effectively activate the STAT5 signal pathway of NK cells, which is significantly better than the CD16 control group without fusion, and has application potential as an enhanced effector receptor in NK cell immunotherapy.

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

[0058] I. Experimental materials and reagents

[0059] 1. Donor source: peripheral blood of healthy volunteers (in accordance with ethical requirements).

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

[0061] 3. Lentivirus 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 cytometry antibodies: APC-CD16, PE-IL-15Rα, FITC-CD56, PerCP-CD3 (all purchased from BioLegend).

[0067] II. Experimental steps

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

[0069] 1.1 Isolation: Ficoll gradient centrifugation method to separate PBMCs;

[0070] 1.2 Magnetic bead sorting: CD56 MicroBeads were used to purify CD56 + CD3 - NK cells.

[0071] 1.3 Flow verification: CD56 + CD3 - ratio reached 95.2% ± 1.1%, asFigure 3 as shown.

[0072] 2. Lentivirus vector construction and packaging

[0073] 2.1 Insert sequence: construction of expression cassette containing SEQ ID NO. 2;

[0074] 2.2 Transfection of Lenti-X 293T cells: three-plasmid method (pLVX-CD16-IL15 + psPAX2 + pMD2.G), using PEIpro transfection;

[0075] 2.3 Virus collection: virus supernatant was collected at 48h and 72h, and concentrated after 0.45 pm filtration;

[0076] 2.4 Titer detection: qPCR method was used to determine the virus titer to be 1 x 10 9 TU / mL.

[0077] 3. NK cell infection and expansion culture

[0078] 3.1 Infection conditions: MOI = 20, containing 8 pg / mL polybrene, centrifugal infection (1200g, 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: expanded for 14 days in a 5% O2+5% CO2 incubator, with half-volume medium replacement every 3 days.

[0081] III. Detection method and data analysis

[0082] 1. Fusion protein expression verification

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

[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% (as shown), and that of the control group was 0%. Figure 5

[0085] 2. NK cell phenotype and function detection, 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 non-transfected group, p < 0.01 (n = 3, t test)

[0089] IV. CONCLUSION

[0090] The genetically engineered NK cells can stably express the fusion protein, maintain high expression in a hypoxic environment, significantly improve the proliferation capacity and ADCC function, and provide a strong cell basis for tumor immunotherapy.

[0091] Example 3: In vitro killing experiment

[0092] 1. Target cell preparation: 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 stopping the staining.

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

[0094] 3. Bi-specific antibody treatment group: pre-added bi-specific antibody (see Example 5 for details), incubated at 37°C for 30 min.

[0095] 4. Co-culture setup:

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

[0097] (2) Culture system: 1 x 10 4 cells / well of target cells were added to the 96-well U-bottom plate, and the corresponding proportion of effector cells was added, with a final volume of 200 μL.

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

[0099] 5. LDH release detection: CytoTox Non-Radioactive Cytotoxicity Assay (Promega) was used to detect the supernatant LDH. The OD490nm value was read, and the blank hole and target cell self-lysis hole were used as reference to calculate the killing rate.

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

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

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

[0103]

[0104] *Note: p < 0.01, statistically significant compared with the normal NK group.

[0105] Table 3 Annexin V+ / PI- apoptotic cell ratio at an effector target ratio of 5:1

[0106]

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

[0108] 1. Establishment of animal model:

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

[0110] (2) Inoculation method: inoculate GC-027 gastric cancer PDX tissue block (about 3mm 3 ) and implant subcutaneously in the right axillary.

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

[0112] 2. Grouping and administration (n = 8 per 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 x length x width2), for 28 days.

[0117] (2) Survival analysis: each group is observed for 90 days, the death time is recorded, and the survival curve is plotted by Kaplan-Meier.

[0118] 4. Experimental results

[0119] (1) Tumor measurement results show (Table 5) that genetically engineered NK cells exhibit significantly enhanced tumor killing activity. The combination of double antibodies further enhances the targeting effect, and both have a synergistic anti-tumor effect.

[0120] Table 5 Summary of experimental results

[0121]

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

[0123] (2) Kaplan-Meier survival curve: the median survival of the control group is 28 days, the median survival of the engineered NK single-drug group is 57 days, and the median survival of the combination therapy group is >90 days.

[0124] 5. Experimental summary

[0125] (1) Immune mechanism analysis: the fusion protein synchronizes IL-15 autocrine signal and 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 increases by 2.3 times), and enhances oxidative phosphorylation capacity (Seahorse detects OCR increases by 1.8 times).

[0126] (3) Targeting and persistence: double antibodies bind through CD16 high affinity (SPR detects KD=1.2x10 -9 M), guiding NK cells to enrich in tumors. 28 days after transplantation, engineered NK cells can still be detected in peripheral blood (accounting for 12.3% vs. ordinary NK cells 0.3%).

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

[0128] The above results show that the genetically engineered NK cells constructed by the fusion protein and combined with tumor-specific double antibodies achieve efficient, targeted, persistent, and safe solid tumor clearance, and have significant application prospects in clinical transformation.

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

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

[0131] 1. Constructing schematic and structural innovation: The present invention provides a bispecific antibody structure, which ensures the correct pairing of heterologous heavy chains, significantly improving the assembly efficiency and yield of the bispecific antibody. 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 Anti-EGFR arm Fab region (sequence optimized by humanization and affinity enhancement mutation)

[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 Anti-HER2 arm Fab region (sequence optimized by humanization and affinity enhancement mutation)

[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. Preparation method optimization

[0143] 1. Expression vector construction: A double expression vector using CMV promoter is used to express heavy chain and light chain simultaneously. The coding region is optimized for CHO cell codon bias, with GC content adjusted to ~55% to improve translation efficiency. Mammalian optimized signal peptides (such as IgK or IgH signal peptides) are introduced to enhance secretion efficiency.

[0144] 2. Establishment of stable cell lines: In this study, PiggyBac transposon system is first used to construct stable expression cell lines in CHO-K1 cells. Positive clones are selected in selection medium containing Zeocin (200 μg / mL), and high-efficiency screening is performed according to the size of the fluorescent circle layer and the expression titer with the help of ClonePix2 automatic picking platform, to obtain high-expression monoclonal cell lines.

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

[0146] 3. Purification process: The bispecific antibody constructed in this study was purified to high purity through a three-step chromatography purification process. First, efficient capture of the bispecific antibody was achieved through Protein A affinity chromatography, with a recovery rate of over 95%; then host cell DNA and HCP impurities were effectively removed through anion exchange chromatography (AEX), with DNA residues less than 1 ng / mg; finally, high molecular aggregates were further removed through hydrophobic interaction chromatography (HIC), with an aggregation rate of less than 1%. The final product purity was over 99%, and the endotoxin level was less than 0.1 EU / mg, meeting the standards of clinical-grade preparations.

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

[0148] 1. Purpose of the experiment: The SPR technique was used to evaluate the binding affinity (KD) of the bispecific antibody of the present application to the target EGFR and HER2, and to compare it with existing commercial bispecific antibodies (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 are Fc-tag purified proteins with a concentration of 1 mg / mL, and the bispecific antibody of the present application, ZW25, and Sym013 (all using the same concentration series dilution).

[0150] 3. Experimental steps

[0151] 3.1 Target immobilization: CM5 chips were installed and the system was pre- equilibrated. The activation procedure was initiated by injecting a mixture of NHS (0.05 M) / EDC (0.2 M) for 60 seconds. Recombinant EGFR or HER2 (50 μg / mL in 10 mM sodium acetate buffer, pH 5.0) was injected for covalent coupling to the chip surface, target coupling level: ~10,000 RU. Unreacted sites were blocked with 1 M ethanolamine (pH 8.5). Channel 1 (blank channel) was used as a reference control (no coupled protein).

[0152] 3.2 Antibody analysis: Antibody dilution series were prepared: concentration range 0.125 nM to 8 nM (2-fold dilutions, 8 concentrations in total). Antibody samples were injected at a flow rate of 30 μL / min, association time 180 seconds, dissociation time 600 seconds. After each round of analysis, the chip surface was regenerated using 10 mM glycine-HCl (pH 2.5). Real-time binding curves (Sensorgrams) were collected at each concentration and reference channel subtraction was performed.

[0153] 3.3 Data analysis: Fitting analysis was performed using BIAevaluation software, using a 1 : 1 Langmuir binding model fit. The ka (association rate constant), kd (dissociation rate constant) were obtained and the KD = kd / ka was calculated.

[0154] 4. Experimental results and analysis, specific results are shown in Figure 6 and Table 6.

[0155] 4.1 High affinity performance: The bispecific antibody of the present application has a binding KD of 1.8 x 10 -9 M for EGFR and 6.1 x 10 - 10 M for HER2, which are significantly better than the commercial control antibodies (Sym013, ZW25).

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

[0157] 4.3 The obvious synergistic feature of dual targeting: Both arms have high affinity, which lays the foundation for subsequent cytotoxic effects and in vivo efficacy.

[0158] Table 6 Affinity data table

[0159]

[0160] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

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

1.

2. The fusion protein of claim 1, wherein, The codon-optimized nucleotide sequence of the fusion protein is shown as 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 are obtained after 14 days of expansion culture under the condition of 5% O2+ 5% CO2.

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

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises an effective amount of the genetically engineered NK cell of claim 3 and an effective amount of the bispecific antibody; The bispecific antibody comprises an anti-EGFR Fab region, an anti-HER2 Fab region and an Fc region; The anti-EGFR Fab region is an anti-EGFR arm Fab region, and the amino acid sequences of the heavy chain variable region and the light chain variable region thereof are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively; The anti-HER2 Fab region is an anti-HER2 arm Fab region, and the amino acid sequences of the heavy chain variable region and the light chain variable region thereof are shown as SEQ ID NO. 5 and SEQ ID NO. 6, respectively; The Fc region 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 shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

Images