Anti-h7 subtype influenza virus hemagglutinin protein bispecific neutralizing antibody bsab-h7 and application thereof

Abstract

This invention discloses a bispecific neutralizing antibody, BsAb-H7, for combating H7 subtype influenza virus and its applications. The invention uses two monoclonal antibodies targeting different antigenic epitopes of the H7 subtype influenza virus hemagglutinin protein as parent antibodies: antibody 1F6 targeting epitope M173 and antibody 3B10 targeting epitope N168. The invention also provides a method for expressing and purifying the bispecific neutralizing antibody BsAb-H7, which is expressed via secretion in CHO-S cells and purified by affinity chromatography to obtain the target protein. This bispecific neutralizing antibody BsAb-H7 can specifically bind simultaneously to different antigenic epitopes of the H7 subtype influenza virus hemagglutinin protein, inhibiting H7 subtype influenza virus replication through neutralization. This invention provides an effective tool for the prevention and treatment of H7 subtype influenza virus infection and can be widely applied in combination with other drugs and in other research.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering antibody technology, specifically relating to a novel bispecific neutralizing antibody that can simultaneously target the hemagglutinin protein antigenic epitopes M173 and N168 of the H7 subtype influenza virus, namely, the anti-H7 subtype influenza virus hemagglutinin protein bispecific neutralizing antibody BsAb-H7 and its applications. It can be used to prepare drugs for the treatment or prevention of H7 subtype influenza A virus infection. Background Technology

[0002] The H7 subtype of influenza A virus includes types such as H7N2, H7N3, H7N7, and H7N9, which are widespread in poultry worldwide. Among them, the H7N7 and H7N9 subtypes, since being confirmed to be able to infect humans across species in Europe in 2003 and Asia in 2013 respectively, have become a major emerging infectious disease threat of global concern. It is noteworthy that the H7N9 subtype influenza virus is usually low pathogenicity in poultry and difficult to detect, but it can cause severe pneumonia, acute respiratory distress syndrome, and even multiple organ failure in humans, with a clinical mortality rate as high as 40%. As of recent years, more than 1,565 laboratory-confirmed human infections have been reported globally, mainly concentrated in the Yangtze River Delta and Pearl River Delta regions of China, where live poultry trading is active, exhibiting a significant epidemiological characteristic of winter and spring poultry and a high correlation with live poultry markets.

[0003] The H7N9 subtype influenza virus exhibits high genetic variability and cross-species adaptation potential. Its hemagglutinin protein can enhance its affinity for human upper respiratory tract α-2,6-sialic acid receptors through mutations in key epitopes (such as Q226L and G186V), thereby promoting effective viral attachment and invasion into human cells. Simultaneously, the virus continuously circulates and accumulates adaptive mutations in avian hosts, giving it potential for human-to-human transmission and posing a significant pandemic risk. Currently, H7 subtype influenza prevention and control still face severe challenges. Regarding vaccines, traditional chicken embryo-dependent production cycles are long, and frequent viral antigenic drift often leads to mismatches between vaccine strains and circulating strains, making it difficult to provide an effective immune barrier in the early stages of an outbreak. In terms of treatment, while neuraminidase inhibitors (such as oseltamivir) are first-line drugs, drug-resistant strains are constantly emerging, and some patients, even with standard treatment, rapidly progress to severe illness, highlighting the limitations of existing therapies.

[0004] Against this backdrop, the development of novel and highly effective antiviral strategies is urgently needed. Monoclonal antibodies, due to their high specificity and good safety profile, have become an important direction in antiviral biological agents. Bispecific antibodies, by simultaneously recognizing two different antigenic epitopes, can not only enhance neutralizing efficacy but also effectively inhibit viral immune escape. Based on this, this study designed a novel bispecific neutralizing antibody, BsAb-H7, using the CrossMab platform. One end (1F6) specifically binds to the M173 epitope of the hemagglutinin protein of the H7 subtype influenza virus, while the other end (3B10) targets the N168 epitope. This bispecific antibody synergistically blocks the binding of the virus to host cell receptors, significantly enhances neutralizing activity, and reduces the risk of viral escape through a dual recognition mechanism. BsAb-H7 combines high affinity, strong neutralizing capacity, and good safety, providing an innovative biological agent strategy for the prevention and treatment of H7 subtype influenza and laying a technological foundation for addressing potential pandemics of other influenza viruses. Summary of the Invention

[0005] The purpose of this invention is to provide a bispecific neutralizing antibody, BsAb-H7, that can simultaneously and specifically target the hemagglutinin protein antigenic epitopes N168 and M173 of the H7 subtype influenza virus for the prevention and treatment of H7 subtype influenza virus infection, as well as the nucleic acid molecule encoding the bispecific neutralizing antibody BsAb-H7.

[0006] A bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus, comprising variable region sequences of two parental full-length antibodies against hemagglutinin protein antigenic epitope M173 and epitope N168 of H7 subtype influenza virus; the amino acid sequence of the heavy chain of the parental antibody 1F6 against hemagglutinin protein antigenic epitope M173 of H7 subtype influenza virus is shown in SEQ ID NO.1, and the amino acid sequence of the light chain of the parental antibody 1F6 against hemagglutinin protein antigenic epitope M173 of H7 subtype influenza virus is shown in SEQ ID NO.3; the amino acid sequence of the heavy chain of the parental antibody 3B10 against hemagglutinin protein antigenic epitope N168 of H7 subtype influenza virus is shown in SEQ ID NO.5, and the amino acid sequence of the light chain of the parental antibody 3B10 against hemagglutinin protein antigenic epitope N168 of H7 subtype influenza virus is shown in SEQ ID NO.7.

[0007] The nucleic acid molecule encoding the bispecific neutralizing antibody BsAb-H7, wherein the nucleotide sequence of the heavy chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.2, and the nucleotide sequence of the light chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.4; the nucleotide sequence of the heavy chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.6, and the nucleotide sequence of the light chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.8.

[0008] An expression vector containing the aforementioned nucleic acid molecules is provided, capable of transfecting eukaryotic CHO-S cells and expressing the protein. The expression vector is derived from the eukaryotic expression vector pcDNA3.4.

[0009] A host cell containing the above-mentioned expression vector.

[0010] The sequence is as follows: SEQ ID NO.1 Heavy chain: Amino acid sequence (121 AA) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 EVLLQQSGPELVKPGASVKIPCKASGYRFTDYNMDWVKQSHGKSLEWIGDINPTNGGTLYNQKFKGKATLTVDKSSSTSYMELRSLTPEDSAVYYCTRSIYHDYDGYFDYWGQGTTLSVSS SEQ ID NO.2 Heavy chain: DNA sequence (363 bp) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GAGGTCCTGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATACCCTGCAAGGCTTCTGGATACAGATTCACTGACTACAACATGGACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTACCAATGGTGGTACTCTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACATCCTACATGGAACTCCGCAGCCTGACACCTGAGGACTCTGCAGTCTATTACTGTACAAGAAGTATCTACCATGATTACGACGGCTACTTTGACTACTGGGGCCAAGGCACCACTCTCTCAGTCTCCTCA SEQ ID NO.3 Light chain: Amino acid sequence (107 AA) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYRTPLTFGAGTKLELK SEQ ID NO.4 Light chain: DNA sequence (321 bp) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTTATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGGTCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGAACTCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO.5 Heavy chain: Amino acid sequence (117 AA) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 EVQLQQSGAEVVKPGASVKLSCTDFGFNIKDTYIHWVKKRPEQGLEWIGRIDPANDNTKYGPKFQGKGTITADTSSNTAYLQLSSLTFEDTAVYYCARVYGGKFDVWGAGTTVTVSS SEQ ID NO.6 Heavy chain: DNA sequence (351 bp) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGGTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGATTTTGGCTTCAACATTAAAGACACCTATATACACTGGGTGAAGAAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGATAATACTAAATATGGCCCGAAGTTTCAGGGCAAGGGCACTATAACAGCAGACACATCCTCCAACACCGCCTACCTGCAGCTCAGCAGCCTGACATTTGAGGACACTGCCGTCTATTACTGTGCTAGAGTCTACGGCGGGAAATTCGATGTCTGGGGCGCAGGGACCACCGTCACCGTCTCCTCA SEQ ID NO.7 Light chain: Amino acid sequence (107 AA) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 DILMTQSPSSMSVSLGDTVSITCHASQGISNNIGWLQQKPGKSFKGLIYHGTILEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQWPYTFGGGTKLEIK SEQ ID NO.8 Light chain: DNA sequence (321 bp) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGCAATAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCATCT TGGAAGATGGAGTTCCATCCAGGTTCAGTGGCAGTGGATCTGGCGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTGGCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA The second objective of this invention is to provide a method for preparing the bispecific neutralizing antibody BsAb-H7, which is achieved through the following steps and technical solutions: (1) The mouse monoclonal antibody 1F6 targeting the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus and the mouse monoclonal antibody 3B10 targeting the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus were obtained by screening using hybridoma technology.

[0011] (2) Sequencing of the heavy and light chain variable region genes of mouse monoclonal antibodies 1F6 and 3B10.

[0012] (3) Construction of bispecific neutralizing antibody BsAb-H7 using molecular biology methods: The amino acid sequences of the variable regions of the light and heavy chains of 1F6 and 3B10 were spliced ​​with the amino acid sequences of the constant regions of the light and heavy chains of human IgG1 antibody to construct four heavy and light chain sequences of the bispecific antibody. Simultaneously, clonal recombination was performed to construct the recombinant vector of bispecific neutralizing antibody BsAb-H7. The target plasmid was transformed into E. coli DH5α competent cells, plated, and positive single clones were picked for sequencing comparison. The cells with correct sequencing results were selected for amplification and preservation.

[0013] (4) Four target plasmids were co-transfected into CHO-S cells for expression. The culture supernatant was collected by low-temperature centrifugation. The antibody in the supernatant was separated and purified by Protein A affinity purification method. The expression and assembly of the expression product were identified by polyacrylamide gel electrophoresis.

[0014] (5) This invention yielded a bispecific neutralizing antibody against H7 subtype influenza virus, namely BsAb-H7. The affinity between the antibody and the antigen was analyzed by enzyme-linked immunosorbent assay (ELISA), and the antiviral ability of the antibody at the cellular level was analyzed by in vitro neutralization assay. At the same time, the preventive and therapeutic effects of the antibody were tested in mice. The results showed that BsAb-H7 can effectively bind to and neutralize H7 subtype influenza virus.

[0015] This invention discloses a bispecific neutralizing antibody, BsAb-H7, for combating H7 subtype influenza virus and its applications, belonging to the field of genetic engineering antibody technology. This invention uses antibody 1F6, targeting the H7 subtype influenza virus hemagglutinin protein epitope M173, and antibody 3B10, targeting the H7 subtype influenza virus hemagglutinin protein epitope N168, as parental antibodies. Based on the CrossMab platform, the Fab arm functional domain (CH1-CL) of the bispecific IgG antibody is exchanged using genetic engineering technology, and BsAb-H7 is expressed in a mammalian eukaryotic expression system. This invention also provides a method for expressing and purifying the bispecific neutralizing antibody BsAb-H7, which is expressed via secretory expression in CHO-S cells and purified by affinity chromatography to obtain the target protein. This bispecific neutralizing antibody BsAb-H7 can specifically bind simultaneously to both the H7 subtype influenza virus hemagglutinin protein epitopes M173 and N168, inhibiting the H7 subtype influenza virus through neutralization. This invention provides an effective tool for the prevention and treatment of H7 subtype influenza virus infection, and can be extended to use in combination with other drugs and in other research.

[0016] The present invention comprises two parental monoclonal antibodies, 1F6 and 3B10, for a bispecific neutralizing antibody. One end of 1F6 is characterized by its specific targeting of the H7 subtype influenza virus hemagglutinin protein epitope M173; the other end of 3B10 specifically targets the H7 subtype influenza virus hemagglutinin protein epitope N168. Humanized human IgG1 antibody is used as the backbone to reduce potential immunogenicity from murine antibodies. Simultaneously, a "crossover" mechanism is employed to exchange the light chain CL and heavy chain CH1 of 3B10, thus avoiding heterologous mismatch during bispecific antibody assembly without altering the antibody's structure and maintaining its affinity for the antigen. Protein expression is performed using a mammalian cell expression system to ensure the biological activity of the bispecific antibody.

[0017] Another object of the present invention is to provide the bispecific neutralizing antibody BsAb-H7 that can effectively bind to and neutralize H7 subtype influenza virus and the method of using it.

[0018] Application of bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus in drugs for the prevention and / or treatment of H7 subtype influenza virus.

[0019] The advantage of this invention is that it provides a bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus, and the antiviral effect of the antibody has been verified in cells and animals, which will provide a new reference solution for the prevention and treatment of H7 subtype influenza virus. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the construction of the gene expression vector for the bispecific neutralizing antibody BsAb-H7.

[0021] Figure 2 This image shows the expression, purification, and identification of the bispecific neutralizing antibody BsAb-H7.

[0022] Figure 3 This study describes the in vitro neutralizing effect of the bispecific neutralizing antibody BsAb-H7. Note: For the in vitro neutralizing effect against H7 subtype influenza virus (A / Zhejiang / DTID-ZJU01 / 2013(H7N9)), each dilution contains 3 replicates. The unit for different antibody dilutions is micrograms per milliliter.

[0023] Figure 4 This study describes the in vivo prophylactic effect of the bispecific neutralizing antibody BsAb-H7. Note: A: Mouse body weight change curve; B: Mouse survival curve.

[0024] Figure 5 This study describes the in vivo therapeutic effect of the bispecific neutralizing antibody BsAb-H7. Note: A: Mouse body weight change curve; B: Mouse survival curve. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Example 1. Construction of bispecific neutralizing antibody BsAb-H7 (1) Mouse monoclonal antibody 1F6 targeting the H7 subtype influenza virus hemagglutinin protein antigenic epitope M173 and mouse monoclonal antibody 3B10 targeting the H7 subtype influenza virus hemagglutinin protein antigenic epitope N168 were obtained by screening using hybridoma technology: ① Six-week-old BALB / c mice were selected and immunized with purified H7 subtype influenza virus hemagglutinin protein; ② The mice prepared in ① were sacrificed and spleen lymphocytes were obtained. The mouse spleen lymphocytes were fused with mouse myeloma cells by polyethylene glycol fusion method. The fused cells were appropriately diluted and seeded into feeder cell culture plates for culture; ③ The above culture was cultured in hypoxanthine-phosphoribosyltransferase selective medium. When the cell colonies grew to a suitable size, the cell culture supernatant was aspirated for antibody identification and positive clones were screened; ④ Hybridoma cells were cloned using the limiting dilution method, and the culture supernatant was used for enzyme-linked immunosorbent assay to identify positive clones. The limiting dilution clones were repeated several times until the positive well rate of hybridoma cells reached 100%. The cloned hybridoma cells were expanded for antibody identification and physicochemical property analysis. ⑤ Positive hybridoma cells were inoculated into BALB / c mice via intraperitoneal injection of 0.5 ml of paraffin oil per mouse, followed by inoculation with 5 × 10⁶ cells per mouse. 6 Ten days later, ascites fluid was collected, centrifuged, and antibody titers were determined. Monoclonal antibodies were then purified. ⑧ Monoclonal antibodies in the ascites fluid were purified using the Protein G affinity purification method.

[0027] (2) Sequencing of the heavy and light chain variable region genes of murine monoclonal antibodies 1F6 and 3B10: ① Collect 5×10 6 ① Target monoclonal hybridoma cells were lysed with 1 mL of Trizol and RNA was extracted; ② Using the extracted RNA as a template, cDNA was synthesized using a reverse transcription kit; ③ Using the cDNA obtained by reverse transcription as a template, PCR amplification was performed using specific primers, the amplified fragment was inserted into an expression vector, and sequencing was performed. The amino acid sequence of the heavy chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the bispecific neutralizing antibody is shown in SEQ ID NO.1; the amino acid sequence of the light chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.3; the amino acid sequence of the heavy chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.5; and the amino acid sequence of the light chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.7.

[0028] (3) Design and synthesis of the four strands of the bispecific neutralizing antibody BsAb-H7: ① The bispecific neutralizing antibody BsAb-H7 heavy and light chain genes were constructed by ligating the heavy chain variable region of 1F6 and the heavy chain constant region of human IgG1 (CH1+CH2+CH3), the light chain variable region of 1F6 and the light chain constant region of human IgG1 (CL), the heavy chain variable region of 3B10, the light chain constant region of human IgG1 (CL) and the heavy chain constant region (CH2+CH3), and the light chain variable region of 1F6 and the heavy chain constant region of human IgG1 (CH1) using overlap PCR technology; ② The target genes were cloned into the eukaryotic expression vector pcDNA respectively. 3.4; ③ Transform the plasmids into E. coli DH5α competent cells, plate the transformed bacterial solution on LB plates, and incubate overnight upside down at 37°C; ④ Randomly select 10 single clones and add them to 4 ml of liquid LB medium. After amplification culture at 37°C for 8 hours with shaking, aspirate 2 μL of bacterial solution for bacterial PCR and sequencing to identify and screen recombinant plasmid DNA; ⑤ Expand and preserve the bacterial solution with correct sequencing; ⑥ Extract plasmids using a plasmid extraction kit to obtain four sets of recombinant plasmids pcDNA 3.4-1F6-H / L-Chain and pcDNA 3.4-3B10-H / L-Chain.

[0029] The results are attached. Figure 1 A schematic diagram of the construction of the gene expression vector for the bispecific neutralizing antibody BsAb-H7.

[0030] Example 2. Expression, purification and identification of bispecific neutralizing antibody BsAb-H7 The recombinant plasmid expressing the four strands of the bispecific neutralizing antibody BsAb-H7 was co-transfected into CHO-S cells.

[0031] (1) Linearization of transfected plasmids: The four groups of recombinant plasmids were linearized, digested overnight at 37°C, and the plasmids were recovered. The degree of linearization of plasmid DNA was detected by 1% agarose gel electrophoresis. The plasmid DNA concentration was detected by UV spectrophotometer and then stored at -20°C for later use.

[0032] (2) Cell passage culture: CHO-S cells were cultured in FreeStyle™ CHO expression medium at 37°C and 5% CO2 until they reached the logarithmic growth phase.

[0033] (3) Four recombinant plasmids, pcDNA 3.4-1F6-H / L-Chain and PcDNA 3.4-3B10-H / L-Chain, were co-transfected into CHO-S cells at a ratio of 1:1:1:1 using electroporation. The medium was changed the next day, and the cells were passaged to gradually expand the cell culture scale. The cell culture medium was collected, and the samples were filtered through a 0.22-micron filter membrane and purified by Protein A affinity chromatography to obtain a large quantity of the target protein. The molecular weight and assembly were identified by 8% non-reduced and 12% reduced polyacrylamide gel electrophoresis, respectively, to preliminarily verify the target protein.

[0034] The results are attached. Figure 2 The bispecific neutralizing antibody BsAb-H7 was successfully expressed and correctly assembled.

[0035] Example 3. Antiviral effect of bispecific neutralizing antibody BsAb-H7 (1) Micro-neutralization experiment: ① The half-maximal dose of H7 subtype influenza virus (A / Zhejiang / DTID-ZJU01 / 2013(H7N9)) was titrated; ② MDCK cells were seeded in 96-well culture plates, 2×10⁶ cells per well. 4① Incubate cells at 37°C for 24 hours in a 5% CO2 saturated incubator; ② Dilute the virus with virus culture medium containing 0.2% trypsin to a 100-fold half-cell infection dose of 50 μL; ③ Quantitatively dilute 50 μg / mL of bispecific neutralizing antibody BsAb-H7 with virus culture medium to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) in 96-well plates, 50 μL per well; ④ Add 50 μL of 100% CO2 saturated incubator to the wells containing the antibody. Add 50 μL of virus solution at 1 / 2 half-tissue cell infection dose, mix well, and perform 3 replicates for each dilution; the last column serves as a control, with 3 wells as negative cell controls (100 μL of virus culture medium added to each well) and 3 wells as positive cell controls (100 μL of virus solution at 100 times the half-tissue cell infection dose added to each well), and incubate for 2 hours in a 37°C incubator saturated with 5% carbon dioxide; ⑥ Remove the prepared 96 1. Wash cells once with phosphate-buffered saline (PBS) in a 96-well MDCK cell culture plate. 2. Transfer the prepared liquid from the 96-well plate in step ⑤ into the cell culture plate and incubate for 2 hours in a 37°C incubator saturated with 5% CO2. 3. Remove the 96-well cell culture plate and wash cells twice with PBS. Add 200 μL of virus culture medium to each well and incubate for 72 hours in a 37°C incubator saturated with 5% CO2. 4. After 72 hours of culture, take 50 μL of culture supernatant from each well of the 96-well cell culture plate and transfer it to a hemagglutination plate. Add 50 μL of 1% chicken red blood cells to each well of the hemagglutination plate. 5. Observe the results after 30 minutes. The results show that BsAb-H7 can neutralize H7 subtype influenza virus at concentrations greater than 0.39 μg / mL, exhibiting a good in vitro neutralization effect.

[0036] The results are attached. Figure 3 Detection of the in vitro neutralization effect of bispecific neutralizing antibody BsAb-H7.

[0037] (2) Mouse prevention experiment: ① Titration of the median lethal dose (LD50) of H7 subtype influenza virus (A / chicken / Jiangxi / C25 / 2014(H7N7)) in mice; ② Grouping of mice: 7-week-old female BALB / C mice, 5 mice per group, for a total of five groups, numbered as Group 1 to Group 5; ③ Weighing and recording the weight of each mouse; ④ Mice in Group 1 and Group 3 were injected intraperitoneally with 1 mg / kg body weight of bispecific neutralizing antibody BsAb-H7, and mice in Group 2 and Group 4 were injected intraperitoneally with 10 mg / kg body weight of bispecific neutralizing antibody. The study involved administering the bispecific neutralizing antibody BsAb-H7. Group 5 received an injection of 10 mg / kg body weight of mouse IgG1-related antibody. Groups 1, 2, and 5 were then intranasally inoculated with 50 μL of H7 subtype influenza virus, diluted to a 5-fold median lethal dose. Groups 1, 2, and 5 received the same antibody (BsAb-H7 or mouse IgG1-related antibody) 12 hours after injection. Groups 3 and 4 received the same antibody (BsAb-H7) 48 hours after injection. Body weight was observed and recorded daily. The results showed that the bispecific neutralizing antibody BsAb-H7 effectively prevented H7 subtype influenza virus infection in mice, achieving 100% protection at a concentration of 1 mg / kg body weight when administered 12 hours post-infection.

[0038] The results are attached. Figure 4 The bispecific neutralizing antibody BsAb-H7 has a preventive effect in vivo.

[0039] (3) Mouse treatment experiment: ① Mouse grouping: 7-week-old female BALB / C mice, 5 mice per group, a total of seven groups, numbered as Group 1 to Group 7 respectively; ② Weigh and record the weight of each mouse; ③ Dilute the H7 subtype influenza virus to 10 times the median lethal dose of 50 μL, and inoculate all mice in Group 1 to Group 7 with the H7 subtype influenza virus intranasally, 50 μL per mouse; ③ 12 hours after infection, mice in Group 1, Group 2 and Group 3 were injected intraperitoneally with 1, 3 and 10 mg / kg body weight of bispecific neutralizing antibody BsAb-H7, respectively, and mice in Group 7 were injected intraperitoneally with 10 mg / kg body weight of mouse IgG1 type unrelated antibody; ④ 48 hours after infection, mice in Group 4, Group 5 and Group 6 were injected intraperitoneally with 1, 3 and 10 mg / kg body weight of bispecific neutralizing antibody BsAb-H7, respectively; ⑤ Observe and record the weight every day. The results showed that the bispecific neutralizing antibody BsAb-H7 could effectively treat H7 subtype influenza virus infection in mice, and the therapeutic effect was closely related to the treatment time. At a concentration of 3 mg / kg body weight, it could still achieve 100% protective efficiency 48 hours after infection.

[0040] The results are attached. Figure 5 The in vivo therapeutic effect of bispecific neutralizing antibody BsAb-H7.

[0041] It should be understood that the present invention has been described in conjunction with the preferred embodiments. However, after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus, characterized in that, The bispecific neutralizing antibody comprises the variable region sequences of two parental full-length antibodies against hemagglutinin protein epitope M173 and epitope N168 of the H7 subtype influenza virus. The amino acid sequence of the heavy chain of the parental antibody 1F6 against hemagglutinin protein epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.1, and the amino acid sequence of the light chain of the parental antibody 1F6 against hemagglutinin protein epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.

3. The amino acid sequence of the heavy chain of the parental antibody 3B10 against hemagglutinin protein epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.5, and the amino acid sequence of the light chain of the parental antibody 3B10 against hemagglutinin protein epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.

7.

2. The method for preparing the bispecific neutralizing antibody BsAb-H7 according to claim 1, comprising the following steps: 1) Mouse monoclonal antibody 1F6 targeting hemagglutinin protein epitope M173 of H7 subtype influenza virus and mouse monoclonal antibody 3B10 targeting hemagglutinin protein epitope N168 of H7 subtype influenza virus were obtained by screening using hybridoma technology. 2) Sequencing of the heavy and light chain variable region genes of murine monoclonal antibodies 1F6 and 3B10; 3) Construction of bispecific neutralizing antibody BsAb-H7 using molecular biology methods: The amino acid sequences of the variable regions of the light and heavy chains of 1F6 and 3B10 were spliced ​​with the amino acid sequences of the constant regions of the light and heavy chains of human IgG1 antibody to construct four heavy and light chain sequences of the bispecific antibody. Simultaneously, clonal recombination was performed to construct the recombinant vector of bispecific neutralizing antibody BsAb-H7. The target plasmid was transformed into E. coli DH5α competent cells, plated, and positive single clones were selected for sequencing comparison. The cells with correct sequencing results were selected for amplification and preservation. 4) Four target plasmids were co-transfected into CHO-S cells for expression. The culture supernatant was collected by low-temperature centrifugation. The antibody in the supernatant was separated and purified by Protein A affinity purification method. The expression and assembly of the expression product were identified by polyacrylamide gel electrophoresis. 5) Obtain bispecific neutralizing antibody against H7 influenza virus, namely BsAb-H7, and analyze the affinity between the antibody and the antigen by enzyme-linked immunosorbent assay, analyze the antiviral ability of the antibody at the cellular level by in vitro neutralization experiment, and test the preventive and therapeutic effects of the antibody in mice.

3. A nucleic acid molecule encoding the bispecific neutralizing antibody BsAb-H7 of claim 1, characterized in that, The nucleotide sequence of the heavy chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.2, and the nucleotide sequence of the light chain of the parental antibody 1F6 against the hemagglutinin protein antigenic epitope M173 of the H7 subtype influenza virus is shown in SEQ ID NO.4; the nucleotide sequence of the heavy chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.6, and the nucleotide sequence of the light chain of the parental antibody 3B10 against the hemagglutinin protein antigenic epitope N168 of the H7 subtype influenza virus is shown in SEQ ID NO.

8.

4. An expression carrier, characterized in that, Containing the nucleic acid molecule of claim 3, it can be transfected into eukaryotic CHO-S cells and express proteins.

5. The expression vector according to claim 4, characterized in that, The expression vector described is derived from the eukaryotic expression vector pcDNA3.

4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 4 or 5.

7. The use of the bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus as described in claim 1 in the preparation of drugs for the prevention of H7 subtype influenza virus.

8. The application according to claim 7, characterized in that: The drug prevents H7 subtype influenza virus infection through virus neutralization.

9. The use of the bispecific neutralizing antibody BsAb-H7 against H7 subtype influenza virus as described in claim 1 in the preparation of a drug for treating H7 subtype influenza virus.

10. The application according to claim 9, characterized in that: The drug treats H7 subtype influenza virus infection through virus neutralization.

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