Monoclonal antibody for resisting H7N9 influenza virus and application thereof

Abstract

The invention discloses an anti-H7N9 influenza virus monoclonal antibody and an application thereof. The anti-H7N9 influenza virus monoclonal antibody disclosed by the invention has relatively high affinity with various H7N9 viruses, the affinity with H7N9-AH13HA is at a pM level, and the affinity with H7N9-SF003HA and H7N9-SD12HA is at an nM level; the neutralizing activity (IC50) on H7N9 / AH13 and H7N1 / SF003 can reach 0.01 mu g / mL, and the neutralizing activity on H7N9 / AH13 and H7N1 / SF003 can reach 0.005 mu g / mL; the hemagglutination titer to H7N9 / AH13 is 0.97 mu g / mL, the hemagglutination titer to H7N1 / SF003 is 1.95 mu g / mL, and both the H7N9 / AH13 and the SF003 have very high neutralization titers; the kit can be used for detecting the H7N9 influenza virus and / or the HA protein of the H7N9 influenza virus, and preventing and / or treating diseases caused by the H7N9 influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, and more specifically, it relates to a monoclonal antibody against H7N9 influenza virus and its application. Background Technology

[0002] The H7N9 influenza virus, an influenza A virus belonging to the Orthomyxoviridae family, poses a serious threat to human health. Influenza A viruses are highly diverse and adaptable, capable of causing different types of influenza diseases, including seasonal influenza and avian influenza. The H7N9 virus has attracted global attention since 2013 due to its ability to infect humans and cause severe illness and death, resulting in a serious public health problem. As of the end of 2017, a total of 1,568 laboratory-confirmed cases of human H7N9 virus infection had been reported globally, with 616 deaths, a mortality rate of 39.3%. The main mutations in the H7N9 virus are point mutations in the HA protein, which enhance the virus's binding ability to human receptors and promote cross-host transmission.

[0003] Currently, prevention and treatment measures for H7N9 avian influenza virus infection are limited. There is no commercially available human H7N9 influenza virus vaccine; the main treatment methods are supportive medical interventions and small molecule drugs such as neuraminidase inhibitors (NAIs). However, the problem of H7N9 influenza virus developing resistance to neuraminidase inhibitors is becoming increasingly serious, posing significant challenges to clinical treatment. Therefore, finding more effective treatment methods has become particularly important.

[0004] Monoclonal antibodies (mAbs) are expected to be an ideal treatment option for H7N9 virus infection due to their specificity and good safety profile. Monoclonal antibodies block influenza virus replication by binding to the hemagglutinin (HA) protein, thereby inhibiting viral infection of host cells, suppressing viral membrane fusion, and exerting cell-mediated cytotoxic effects through the function of the antibody's Fc fragment. Currently, several human monoclonal antibodies have been discovered, primarily targeting the HA and NA proteins of the H7N9 virus, especially the HA protein. These antibodies are mainly divided into two categories: those induced after vaccination and those induced after natural infection with the H7N9 influenza virus.

[0005] Although some antibodies have been discovered, the continuous evolution and mutation of the H7N9 virus may cause mutations at key antigenic sites, reducing the efficacy of existing antibodies. Therefore, researchers need to continuously screen for new H7N9 influenza virus antibodies to discover more antigenic recognition epitopes and enhance antibody diversity and broad-spectrum efficacy. Statistical analysis of overlapping epitopes in antibody libraries can explore conserved epitopes in viral evolution, providing new strategies and methods for the prevention and treatment of H7N9 influenza. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an antigen-binding fragment (Fab), which is composed of a variable region (VH) of the heavy chain and a variable region (VL) of the light chain, and directly participates in antigen recognition and binding.

[0007] The second objective of this invention is to provide a monoclonal antibody against H7N9 influenza virus. The monoclonal antibody consists of four polypeptide chains, including two identical heavy chains (H chains) and two identical light chains (L chains), which are connected by disulfide bonds and non-covalent bonds to form a symmetrical "Y"-shaped structure.

[0008] A third objective of this invention is to provide a nucleic acid molecule encoding the aforementioned antigen-binding fragment or a monoclonal antibody against the H7N9 influenza virus.

[0009] A fourth objective of this invention is to provide an expression vector containing the aforementioned nucleic acid molecules.

[0010] A fifth object of the present invention is to provide a host cell containing the above-mentioned nucleic acid molecule or expression vector.

[0011] A sixth object of the present invention is to provide the application of the above-mentioned antigen-binding fragment, anti-H7N9 influenza virus monoclonal antibody, nucleic acid molecule, expression vector or host cell.

[0012] The seventh object of the present invention is to provide a reagent kit.

[0013] The eighth object of the present invention is to provide a medicine.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] An antigen-binding fragment (Fab) comprising a heavy chain variable region and a light chain variable region;

[0016] The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 in the heavy chain variable region is GYSISSGY, the amino acid sequence of CDR2 in the heavy chain variable region is YISYLGDT, and the amino acid sequence of CDR3 in the heavy chain variable region is VSPPPDWNDFIFAH.

[0017] The light chain variable region comprises three complementarity-determining regions, CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 in the light chain variable region is QSLLSSNGYTH, the amino acid sequence of CDR2 in the light chain variable region is YGF, and the amino acid sequence of CDR3 in the light chain variable region is MQALQTPLT.

[0018] The antigen-binding fragment of the present invention has high titer, strong specificity, no heterologous serum reaction, no cross-contamination, and clear composition. It avoids both allergic reactions caused by heterologous serum proteins and the problems of limited sources of human immunoglobulins and potential pathogen contamination.

[0019] Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1: QVQLQESGPGLVKPSETLALTCTVSGYSISSGYGWTWIRQPPGKGLEWLGYIS YLGDTDITPSFKGRVAISIDTSKNQFSLKLTSVTAADTAVYFCVSPPPDWNDFI FAHWGQGVPVTVSS;

[0020] The amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 2: DIQMTQSPLSLPVTLGEPASISCRSSQSLLSSNGYTHLSWYLQRPGQSPQLLILY GFDRASGVPDRFSASGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGT KVEIK.

[0021] The present invention also provides a monoclonal antibody against H7N9 influenza virus, wherein the monoclonal antibody contains the above-mentioned antigen-binding fragment.

[0022] Furthermore, the anti-H7N9 influenza virus monoclonal antibody also comprises a heavy chain constant region and a light chain constant region; the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0023] The amino acid sequence of the constant region of the light chain is shown in SEQ ID No. 4: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC.

[0024] The present invention also provides a nucleic acid molecule encoding the above-mentioned antigen-binding fragment or the above-mentioned anti-H7N9 influenza virus monoclonal antibody.

[0025] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID No. 5: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGGCCCTCACCTGCACTGTTTCTGGTTACTCCATCAGTAGTGGTTATGGCTGGACCTGGATCCGCCAGCCCCCGGGGAAGGGGCTGGAGTGGCTTGGGTACATCTCTTATCTTGGGGACACTGACATCACCCCGTCCTTCAAGGGTCGAGTCGCCATTTCAATAGACACGTCCAAGAATCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCCGTATATTTCTGTGTGAGTCCCCCCCCTGACTGGAATGATTTCATATTTGCCCACTGGGGCCAGGGAGTCCCGGTCACCGTCTCCTCAG;

[0026] The nucleotide sequence of the nucleic acid molecule encoding the variable region of the light chain is shown in SEQ ID No. 6: GACATCCAGATGACTCAGTCTCCACTCTCTCTGCCCGTCACCCTTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTTCTGAGCAGTAATGGATATACACATTTGAGTTGGTACCTGCAGAGGCCAGGCCAGTCTCCACAGCTCCTGATCCTTTATGGTTTTGATCGGGCCTCCGGGGTCCCCGACAGGTTCAGTGCCAGTGGGTCAGGCACAGATTTTACACTGAAAATCAGCAGGGTGGAGGCTGAAGACGTAGGGGTCTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC.

[0027] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region is shown in SEQ ID No.As shown in Figure 7: GCTTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA;.

[0028] The nucleotide sequence of the nucleic acid molecule encoding the light chain constant region is shown in SEQ ID No. 8: CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG.

[0029] The present invention also provides an expression vector containing the above-mentioned nucleic acid molecules.

[0030] The present invention also provides a host cell containing the above-mentioned nucleic acid molecules or expression vectors.

[0031] Preferably, the host cells include 293F cells.

[0032] The present invention also provides a method for preparing the aforementioned anti-H7N9 influenza virus monoclonal antibody, the method comprising the following steps:

[0033] (1) Collect B cells from immunized individuals and obtain DNA fragments of antibody variable regions by PCR;

[0034] (2) The DNA fragment described in step (1) is ligated into an expression vector, transformed into competent cells, and then single clones are selected for screening after culturing.

[0035] (3) The selected expression vector is transferred into the host cell, cultured and the supernatant is collected, and the antibody is separated and purified.

[0036] Preferably, the immunized individuals in step (1) include Chinese macaques. Chinese macaques share high genetic homology with humans. This invention uses Chinese macaques immunized with the H7N9 influenza virus vaccine to stimulate B cells to produce neutralizing antibodies, and the affinity of the antibodies is improved through multiple immunizations.

[0037] Preferably, the B cells in step (1) include CD3. - / CD20 + / CD27 + IgG + / H7-HA-HIS + Memory B cells.

[0038] In this invention, CD3 antibodies are first used to distinguish T cells and B cells, followed by screening for B cells with the CD20 phenotype using CD20 antibodies, and then using CD27 antibodies... + IgG + Antibody screening was used to select memory B cells with high expression, and finally H7-HA-HIS was selected. + Targeting memory B cell populations specifically expressing H7 antibodies, and obtaining CD3 - / CD20 + / CD27 + IgG + / H7-HA-HIS + Memory B cells greatly improve antibody separation efficiency.

[0039] In this invention, the primers for PCR in step (1) were designed according to Nature Protocol, 2009, 4(3), 372-384 and Journal of Immunological Methods, 2008, 329(1-2), 112-124.

[0040] Preferably, the host cell in step (3) includes 293F cells.

[0041] The present invention also provides the use of the above-mentioned antigen-binding fragment, anti-H7N9 influenza virus monoclonal antibody, nucleic acid molecule, expression vector or host cell in the preparation of products for detecting H7N9 influenza virus and / or H7N9 influenza virus HA protein, or in the preparation of medicaments for the prevention and / or treatment of diseases caused by H7N9 influenza virus.

[0042] The present invention also provides a kit containing the above-mentioned antigen-binding fragment or anti-H7N9 influenza virus monoclonal antibody.

[0043] Preferably, the kit also includes a washing solution.

[0044] The present invention also provides a drug comprising the above-mentioned antigen-binding fragment, an anti-H7N9 influenza virus monoclonal antibody, a nucleic acid molecule, an expression vector, or a host cell.

[0045] Preferably, the drug further includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients, or diluents.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The monoclonal antibody against H7N9 influenza virus of the present invention exhibits high affinity for various H7N9 viruses, with affinity for H7N9-AH13 HA at the pM level (Kd < 1 pM), and affinity for H7N9-SF003 HA and H7N9-SD12 HA at the nM level (Kd 0.54 nM and 0.60 nM, respectively); it also demonstrates neutralizing activity (IC50) against H7N9 / AH13 and H7N1 / SF003. 50 The concentrations can reach 0.01 μg / mL and 0.005 μg / mL; the hemagglutination titer for H7N9 / AH13 is 0.97 μg / mL, and the hemagglutination titer for H7N1 / SF003 is 1.95 μg / mL, both showing high neutralizing titers; it can be used to detect H7N9 influenza virus and / or the HA protein of H7N9 influenza virus, and to prevent and / or treat diseases caused by H7N9 influenza virus. Attached Figure Description

[0048] Figure 1 This is an electrophoresis diagram of the recombinant vector, where DL2000 represents the molecular weight of DNA.

[0049] Figure 2 The results of electrophoresis and staining of antibody 41B5 are shown. In the figure, Marker represents the molecular weight of the protein, MEDI8852 is the positive control antibody, and Loading buffr is the negative control.

[0050] Figure 3 The binding activity of antibody 41B5 to H7N9-HA protein was measured.

[0051] Figure 4 The results show the affinity assay results between antibody 41B5-IgG and H7N9-HA.

[0052] Figure 5 The results show the affinity test results between 41B5-Fab and H7N9-HA.

[0053] Figure 6 The in vitro trace neutralizing activity of antibody 41B5 against H7N9-PR8 virus was measured. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0055] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0056] The H7N9 virus continues to mutate in poultry without infecting human subjects. To address this challenge, this invention proposes immunizing Chinese rhesus monkeys with highly homologous antibody genes to human antibodies, and screening for H7N9 HA-specific neutralizing antibodies from Chinese rhesus monkeys vaccinated with H7N9 influenza. This invention not only deepens our understanding of viral immune responses but may also contribute to finding more effective vaccines and treatments, and developing effective protective strategies against H7N9 influenza virus infection.

[0057] Example 1: Mononuclear cell isolation and memory B cell sorting

[0058] (1) Blood samples were collected from the upper limb veins of Chinese macaques that had been vaccinated against H7N9 for two weeks and collected in anticoagulant tubes containing heparin. Mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation, and the PBMC suspension was aspirated, washed three times with PBS, and the supernatant was discarded.

[0059] (2) After filtering the cell suspension through a 40 μm filter membrane and counting the cells, the target cell population was sorted from the PBMCs using a BD FACSria flow cytometer. The morphologically intact single cells were sorted into 96-well PCR plates, and 20 μL of single-cell lysis buffer (containing 0.2 μL of RNasin inhibitor (Promega, Madison, WI), 5 μL of 5× first chain buffer, 1.25 μL of 0.1M DTT and 0.0625 μL of IGEPAL (Sigma-Aldrich, St. Louis, MO)) was added to each well to ensure that each well contained one memory B cell, followed by brief centrifugation.

[0060] Example 2: Isolation of antibody variable region gene from single B cells using single-cell PCR

[0061] (1) Single-cell RT-PCR: Reverse transcription (RT-PCR) was immediately performed on the PCR plate containing sorted cells. For the RT reaction, 1 μL of random hexamer (150 ng / μL, Promega), 2 μL of dNTPs (10 mM each) and 0.5 μL of SuperScript III (Invitrogen) were added to each well, and then incubated at 42 °C for 1 h. PCR amplification was performed under the following conditions: 42 °C for 10 min; 25 °C for 10 min, 50 °C for 90 min, 98 °C for 5 min, and 4 °C constant. The obtained cDNA product was stored at -80 °C.

[0062] (2) Nested PCR amplification of antibody genes: The variable regions of the IgG heavy and light chains were independently amplified by nested PCR. The forward primers for the first round of PCR amplification were located in the frame regions of the variable regions of the antibody heavy, kappa, and lambda chains, respectively, while the reverse primers were located in the corresponding constant regions. The template used for amplification was cDNA obtained through reverse transcription. PCR amplification was performed under the following conditions: 94℃ for 5 min; 50 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min, followed by 72℃ for 10 min and then cooling to 4℃. Using the PCR products from the first round as templates, a second round of amplification was performed. The forward primers for the second round of PCR amplification were located at the FR1 initiation positions of the heavy, kappa, and lambda chains, respectively, while the reverse primers were located in the corresponding constant regions. PCR amplification was performed under the following conditions: 94℃ for 5 min; 50 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min, followed by 72℃ for 10 min and then cooling to 4℃. PCR products were analyzed by electrophoresis on a 2% agarose gel. Bands between 350 and 500 bp were excised, and the recovered PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing. Based on the sequencing results, the gene sequences of the heavy chain variable region and light chain variable region of the antibody paired with the amplification well were determined.

[0063] The gene sequence of the heavy chain variable region is: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGGCCCTCACCTGCACTGTTTCTGGTTACTCCATCAGTAGTGGTTATGGCTGGACCTGGATCCGCCAGCCCCCGGGGAAGGGGCTGGAGTGGCTTGGGTACATCTCTTATCTTGGGGACACTGACA TCACCCCGTCCTTCAAGGGTCGAGTCGCCATTTCAATAGACACGTCCAAGAATCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCCGTATAATTTCTGTGTGAGTCCCCCCCTGACTGGAATGATTTCATATTTGCCCACTGGGGCCAGGGAGTCCCGGTCACCGTCTCCTCAG(SEQ ID No.5);

[0064] The gene sequence of the light chain variable region is: GACATCCAGATGACTCAGTCTCCACTCTCTCTGCCCGTCACCCTTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTTTGAGCAGTAATGGATATACACATTTGAGTTGGTACCTGCAGAGGCCAGGCCAGTCTCCACAGCTCCTGATCCTTTA TGGTTTTGATCGGGCCTCCGGGGTCCCCGACAGGTTCAGTGCCAGTGGGTCAGGCACAGATTTTACACTGAAAATCAGCAGGGTGGAGGCTGAAGACGTAGGGGTCTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC(SEQ IDNo.6);

[0065] Furthermore, the amino acid sequences of the heavy chain variable region and the light chain variable region were determined:

[0066] The amino acid sequence of the heavy chain variable region is: QVQLQESGPGLVKPSETLALTCTVSGYSISSGYGWTWIRQPPGKGLEWLGYIS YLGDTDITPSFKGRVAISIDTSKNQFSLKLTSVTAADTAVYFCVSPPPDWNDFIFAHWGQGVPVTVSS (SEQ ID No. 1);

[0067] The amino acid sequence of the light chain variable region is: DIQMTQSPLSLPVTLGEPASISCRSSQSLLSSNGYTHLSWYLQRPGQSPQLLILY GFDRASGVPDRFSASGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGT KVEIK (SEQ ID No. 2).

[0068] Furthermore, the amino acid sequences of the six CDR regions of the antibody were determined by comparing the sequences with those of human and rhesus monkey antibodies using IMGT / V-QUEST (http: / / www.imgt.org / ). The results showed that the amino acid sequence of CDR1 in the heavy chain variable region was GYSISSGY, the amino acid sequence of CDR2 in the heavy chain variable region was YISYLGDT, and the amino acid sequence of CDR3 in the heavy chain variable region was VSPPPDWNDFIFAH.

[0069] The amino acid sequence of CDR1 in the light chain variable region is QSLLSSNGYTH, the amino acid sequence of CDR2 in the light chain variable region is YGF, and the amino acid sequence of CDR3 in the light chain variable region is MQALQTPLT.

[0070] The gene sequence of the heavy chain constant region is shown in SEQ ID No. 7;

[0071] The gene sequence of the light chain constant region is shown in SEQ ID No. 8.

[0072] The amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3;

[0073] The amino acid sequence of the constant region of the light chain is shown in SEQ ID No. 4.

[0074] Example 3: Construction of an expression vector for recombinant antibodies

[0075] The PCR products of the antibody heavy chain variable region gene and light chain variable region gene, which were positive by gel electrophoresis and showed good pairing between the heavy and light chains, were ligated into heavy chain vectors and light chain vectors, respectively, using the TA cloning method to construct expression vectors for the anti-H7N9 antibody. These expression vectors were then transformed into DH5α competent bacteria and cultured overnight at 37°C on ampicillin-containing plates. Single colonies were picked and cultured in LB broth (containing ampicillin) at 37°C for 3-5 hours using shake. PCR was then performed using specific primers under the following conditions: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 100 s, 30 cycles; 72°C for 5 min, 4°C constant. The PCR products were identified by 2% agarose gel electrophoresis, and bacterial cultures containing the target band (approximately 500-750 bp) were sent for sequencing. The results are as follows: Figure 1 As shown, transformants containing antibody heavy and light chain genes were identified in the positive transformants, with a DNA length of approximately 600 bp, ranging from 500 to 750 bp.

[0076] Example 4: Expression and purification of antibody 41B5

[0077] Positive plasmids were transformed into DH5α strains for large-scale amplification, and recombinant plasmids were rapidly extracted. Subsequently, paired IgH and IgL gene plasmids were transiently co-transfected into the Expi293F expression system at a 1:1 (IgH:IgL) ratio using EZCell transfection reagent (Life-iLab Biotech, AC04L092). 18 to 22 hours after transfection, 5% feed was added, and the cells were cultured in a shaker at 37°C and 5% CO2 for 4 to 5 days. Cell supernatant was then collected. The collected cell supernatant was centrifuged to remove cell debris, and the antibody was then purified using a Protein A purification cartridge (L00210, Nanjing Genscript Biotech Co., Ltd.). The purification process involved equilibration with 0.1M Tris buffer at pH 7.0, followed by loading, elution with Tris buffer at the same pH, and finally elution with 0.1M Tris buffer at pH 8.0. The collected eluent was further dialyzed in PBS buffer to purify the obtained antibody 41B5. The absorbance at 280 nm was measured using a Nanodrop One spectrophotometer (ThermoScientific), and the antibody concentration was calculated accordingly. The purified antibody was analyzed by SDS-PAGE, and the results are shown below. Figure 2 As shown, under reduction conditions, two bands of approximately 50 kDa and 25 kDa were observed, corresponding to the variable and constant regions of the heavy chain and the variable and constant regions of the light chain of the antibody, respectively. The purified antibody bands were consistent with the expected results.

[0078] Test Case

[0079] 1. Enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of antibody 41B5 to H7N9 virus HA.

[0080] The HA proteins of three H7N9 viruses (H7N9 / AH13, H7N9 / SF003, and H7N9 / SD12) were diluted in PBS buffer. For coating ELISA plates, 0.1 μg / well of PBS-diluted HA protein was added at a rate of 100 μL / well. Coating was incubated overnight (or at least 12 hours) at 4°C. The HA protein was discarded, and blocking buffer (5% skim milk dissolved in PBS) was added at 200 μL / well. The plates were blocked at room temperature for 2 hours, washed, and 0.1% HCl was added to each well. Wash the plate 3 times with PBST (300 μL / well), discard the liquid, and blot dry. Dilute the 41B5 and MEDI8852 antibodies separately in PBS, starting from 10 μg / mL, and perform 3-fold serial dilutions for a total of 11 dilutions. Add 100 μL / well to each well of the ELISA plate and incubate at 37°C for 2 hours. Wash the plate 3 times with PBST, add HRP-labeled anti-human IgG secondary antibody, and incubate at 37°C for 1 hour. Wash the plate 6 times with PBST, discard the liquid, add 100 μL / well of TMB chromogenic substrate, and incubate at 37°C in the dark for 15 minutes. Stop the incubation by adding 50 μL of 2M H2SO4. Measure the absorbance at 450 nm. Each treatment is repeated twice. Calculate the EC50 of HA protein binding to the antibody. 50 Value, result as Figure 3 As shown in Table 1, antibody 41B5 exhibits good binding activity to the HA of different H7N9 viruses.

[0081] Table 1. Binding activity of antibody 41B5 against H7N9-HA

[0082]

[0083] 2. Detection of the affinity between antibody 41B5 and H7N9 virus HA protein using biomembrane interferometry (BLI).

[0084] The affinity of antibody 41B5 for the hemagglutinin (HA) proteins of three H7N9 viruses (H7N9 / AH13, H7N9 / SF003, and H7N9 / SD12) was detected using biomembrane interference (BLI) technique. Antibody 41B5 was diluted 10 μg / mL in PBS buffer (containing 0.02% Tween-20 and 0.2% BSA). The concentrations of H7N9-HA proteins (H7N9 / AH13, H7N9 / SF003, and H7N9 / SD12) were 200 nM, 66.66 nM, 22.22 nM, 7.41 nM, 2.47 nM, and 0.82 nM, respectively. The results are as follows: Figure 4 As shown in Table 2, the antibody has a high affinity for the HA trimer protein of the three H7N9 viruses.

[0085] Table 2 shows the antigen-antibody affinity, where Ka is the binding constant, Kd is the dissociation constant, and KD is the dissociation constant of the antibody with H7N9-HA protein in equilibrium. A smaller KD indicates less dissociation and stronger affinity. It can be seen that 41B5 exhibits high affinity for AH13-HA (Kd < 1 pM), while its affinity for SF003-HA and SD12-HA is slightly lower (Kd 0.54 nM and 0.60 nM, respectively). The 41B5 IgG dimer exhibits similar binding phase characteristics with all three trimeric-HA proteins and shows extremely slow dissociation.

[0086] Table 2 Affinity of antibody 41B5-IgG to H7N9-HA antigen

[0087]

[0088] We then evaluated the binding of trimeric-HA to the monomeric mAb fragment (Fab). The results are as follows: Figure 5 As shown in Table 3, compared with 41B5-IgG, 41B5-Fab exhibited decreased affinity for AH13-HA, SF003-HA, and SD12-HA. Comparison of the full-length dimer IgG mAb with the monomeric Fab fragment revealed a significant difference in the dissociation of the HA antibody complex. This indicates that the multivalent binding between the dimer IgG and HA produces an affinity effect, masking the accelerated antibody dissociation. Therefore, the binding to the HA trimer is primarily determined by the antibody binding characteristics.

[0089] Table 3. Affinity of antibody 41B5-Fab to H7N9-HA antigen

[0090]

[0091] 3. In vitro neutralizing ability of antibody 41B5 against H7N9-PR8 virus

[0092] 50 μL of antibody 41B5 (starting concentration 100 μg / mL) was serially diluted 2-fold and added to a 96-well cell culture plate with an equal volume of virus (H7N9 / AH13, H7N1 / SF003) (100 TCID50). 50 Mix. After incubating at 37°C for 1 hour, add 100 μL of Madin-Darby canine kidney (MDCK) cells (3 × 10⁻⁶) to each well. 5Cells / mL. Plates were incubated at 37°C and 5% CO2 atmosphere for 18 hours. Cell monolayers were washed with PBS and fixed in cold 80% acetone for 10 min. Virus presence was detected by ELISA using an anti-influenza A virus nucleoprotein (NP) monoclonal antibody (Abcam). Neutralizing antibody titer was defined as the reciprocal of the highest mAb dilution in MDCK cell culture capable of neutralizing the virus.

[0093] The results are as follows Figure 6 As shown in Table 4, antibody 41B5 can significantly inhibit the replication of AH13 and SF003 viruses.

[0094] Table 4 Neutralizing activity of antibody 41B5 against H7N9 / AH13 and H7N1 / SF003

[0095]

[0096]

[0097] 4. Hemagglutination inhibition (HI) assay to detect antibody neutralization titer

[0098] Neutralizing antibodies against influenza mainly include two types: those targeting the head of the HA protein and those targeting the neck of the HA protein. Head antibodies generally recognize amino acids near the sialic acid receptor or the sialic acid binding site of the HA protein. Hemagglutination assays can easily and quickly distinguish between head and neck antibodies. Influenza viruses have the property of agglutinating red blood cells, and antibody 41B5 recognizes the sialic acid binding site (RBS), blocking the virus's ability to agglutinate red blood cells.

[0099] The purified antibody 41B5 was serially diluted 2-fold (starting from 250 μg / mL) in 25 μL of PBS in V-well plates. An equal volume of antigen virus (H7N9 / AH13, H7N9 / SF003) (4 HA units) was added, and the plates were incubated at room temperature for 30 min. Subsequently, 50 μL of 1% chicken red blood cell (RBC) suspension was added to each well. Agglutination patterns were observed after 30 min. Chicken red blood cells normally settle, but after viral infection, hemagglutination occurs, preventing sedimentation. The hemagglutination inhibition titer of the antibody is the highest dilution at which the antibody can inhibit the agglutination of the virus with red blood cells.

[0100] As shown in Table 5, the hemagglutination titer of 41B5 against AH13 was 0.97 μg / mL, and the hemagglutination titer against SF003 was 1.95 μg / mL. This shows that the hemagglutination titer of antibody 41B5 is quite significant, with a very high neutralizing titer.

[0101] Table 5. HI titers of antibody 41B5 against H7N9 / AH13 and H7N9 / SF003

[0102]

[0103] In summary, the antibody of this invention exhibits an affinity for H7N9-AH13 HA at the pM level and for H7N9-SF003 HA and H7N9-SD12 HA at the nM level. It is highly pure, non-immunogenic, and has a clear composition, eliminating potential risks such as pathogen contamination. The method for preparing monoclonal antibodies of this invention is standardized, controllable, low-cost, simple, efficient, and suitable for standardized production.

[0104] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An antigen-binding fragment, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 in the heavy chain variable region is GYSISSGY, the amino acid sequence of CDR2 in the heavy chain variable region is YISYLGDT, and the amino acid sequence of CDR3 in the heavy chain variable region is VSPPPDWNDFIFAH. The light chain variable region comprises three complementarity-determining regions, CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 in the light chain variable region is QSLLSSNGYTH, the amino acid sequence of CDR2 in the light chain variable region is YGF, and the amino acid sequence of CDR3 in the light chain variable region is MQALQTPLT.

2. The antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

3. A monoclonal antibody against H7N9 influenza virus, characterized in that, The monoclonal antibody contains the antigen-binding fragment as described in claim 1 or 2.

4. The anti-H7N9 influenza virus monoclonal antibody according to claim 3, characterized in that, It also includes a heavy chain constant region and a light chain constant region; the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3, and the amino acid sequence of the light chain constant region is shown in SEQ ID No.

4.

5. A nucleic acid molecule encoding the antigen-binding fragment of claim 1 or 2, or the anti-H7N9 influenza virus monoclonal antibody of claim 3 or 4.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The use of the antigen-binding fragment of claim 1 or 2, the anti-H7N9 influenza virus monoclonal antibody of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7 in the preparation of products for detecting H7N9 influenza virus and / or the HA protein of H7N9 influenza virus, or in the preparation of medicaments for the prevention and / or treatment of diseases caused by H7N9 influenza virus.

9. A reagent kit, characterized in that, The kit contains the antigen-binding fragment of claim 1 or 2 and the anti-H7N9 influenza virus monoclonal antibody of claim 3 or 4.

10. A drug, characterized in that the drug comprises the antigen-binding fragment of claim 1 or 2, the anti-H7N9 influenza virus monoclonal antibody of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7.

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