Anti-H3N2 influenza virus antibody and application thereof

Abstract

The invention provides an anti-H3N2 influenza virus antibody and application thereof. The amino acid sequence of a heavy chain variable region of the antibody is as shown in SEQ ID No.1, and the amino acid sequence of a light chain variable region of the antibody is as shown in SEQ ID No.2. The antibody provided by the invention has very strong binding activity, affinity and neutralizing ability to different H3N2-HA, and has a certain cross effect with an H10 subtype. The kit can be used for detecting the H3N2 / H10 subtype influenza virus and / or the HA protein of the H3N2 / H10 subtype influenza virus, and preventing and / or treating diseases caused by the H3N2 / H10 subtype influenza virus.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, immunology and antibody technology, and more specifically, an anti-H3N2 influenza virus antibody and its application. Background Technology

[0002] The H3N2 influenza virus belongs to the influenza A virus family and is characterized by two major glycoproteins on its surface: hemagglutinin (HA) and neuraminidase (NA). HA is responsible for the binding of the virus to host cells, while NA is involved in the release of the virus from host cells. Since 1968, the H3N2 influenza virus has been circulating globally, causing seasonal influenza outbreaks. Seasonal influenza virus infection poses a serious threat to human health, with an estimated 3-5 million severe influenza cases and 250,000-500,000 deaths worldwide each year. The prevalence of H3N2 influenza virus, especially among children, the elderly, and patients with chronic diseases, presents a continuing challenge to global public health.

[0003] Therapeutic antibodies have long been reported for the treatment of viral infectious diseases such as influenza. In vitro antibody production technology is an important branch of modern biotechnology, and it is of great significance for research in virology, vaccine development, and the preparation of therapeutic antibodies. The following are some of the main in vitro antibody production technologies:

[0004] Hybridoma technology was the earliest method used to produce monoclonal antibodies. In 1975, George Keller... First proposed and implemented by Dr. [Name] and Dr. Sidney Bolon (César Milstein), this technique involves fusing B cells capable of producing specific antibodies with myeloma cells to form hybridoma cells. These hybridoma cells possess both the antibody-producing ability of B cells and the unlimited proliferation characteristic of myeloma cells. Through screening and cloning, hybridoma cell lines capable of stably producing a single specific antibody can be obtained.

[0005] Phage display technology is a method that utilizes the surface of bacteriophages to display specific proteins or peptides. In antibody production, this technology involves inserting antibody gene fragments into the phage genome, causing the phage surface to display variable regions (Fab fragments) of the antibody. By screening for phages that bind to specific antigens, genes that produce specific antibodies can be rapidly identified and amplified. A drawback is the high requirement for antibody affinity and specificity, which may necessitate further validation and optimization.

[0006] Cell culture and protein expression systems, including insect cell (such as Sf9 and Sf21 cells) and mammalian cell (such as CHO and 293 cells), are used for antibody production. Mammalian cell expression systems can produce bioactive proteins that more closely resemble natural proteins. By transfecting plasmids encoding antibody genes into these cells, recombinant antibodies can be produced in large quantities in vitro, making them suitable for large-scale production. In addition, there are yeast- and plant-based expression systems that can also be used for antibody production. However, their drawbacks include expensive culture media and complex growth conditions.

[0007] Transcription activator-like effector nucleases (TALENs) and CRISPR / Cas9 technology. TALENs were first proposed in 2011 and developed by Hockemeyer et al., while CRISPR / Cas9 technology was first widely used in gene editing in 2012, proposed by Jennifer Doudna and Emmanuelle Charpentier. These gene editing technologies can be used to precisely modify the genome of cells, including B cells. These technologies can specifically recognize and cut specific DNA sequences, knock out or knock in specific genes to study key genes in antibody production, or directly generate specific antibody genes in B cells. However, these technologies also have significant drawbacks: high cost, delivery efficiency issues, potential immune responses, off-target effects, and the risk of gene integration. Currently, many studies have developed antibodies against the H3N2 influenza virus, but these antibodies have poor affinity and lack the potential to become broad-spectrum antibodies. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an antibody against H3N2 influenza virus.

[0009] A second objective of the present invention is to provide a DNA molecule encoding the aforementioned antibody against the H3N2 influenza virus.

[0010] A third object of the present invention is to provide an expression cassette, recombinant vector or host cell containing the above-mentioned DNA molecules.

[0011] A fourth objective of this invention is to provide a host cell containing the aforementioned nucleic acid molecule or expression vector.

[0012] A fifth object of the present invention is to provide the application of the above-mentioned anti-H3N2 influenza virus antibody, DNA molecule, expression cassette, recombinant vector or host cell.

[0013] The sixth object of the present invention is to provide a reagent kit.

[0014] The seventh object of the present invention is to provide a medicine.

[0015] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0016] An antibody against H3N2 influenza virus, comprising a heavy chain variable region and a light chain variable region; the amino acid sequence of CDR1 of the heavy chain variable region is SLTVNNNY, the amino acid sequence of CDR2 of the heavy chain variable region is SFGSGTT, and the amino acid sequence of CDR3 of the heavy chain variable region is AREYVEGPGTGGHFDL.

[0017] The amino acid sequence of CDR1 in the light chain variable region is TLPWQY, the amino acid sequence of CDR2 in the light chain variable region is KDS, and the amino acid sequence of CDR3 in the light chain variable region is LSSPNTGPWV.

[0018] Seasonal influenza vaccination can induce the production of specific and broad-spectrum neutralizing antibodies against the influenza virus surface glycoprotein hemagglutinin. This invention screens valuable neutralizing antibodies for the prevention and treatment of influenza infection. The anti-H3N2 influenza virus antibodies screened by this invention exhibit strong binding activity, affinity, and neutralizing capacity against different H3N2-HA antibodies. Simultaneously, the 31D7 antibody shows some cross-binding neutralizing affinity activity against H10N3 and H10N8, demonstrating potential as a broad-spectrum antibody.

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

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

[0021] Furthermore, the antibody also includes constant regions, namely heavy chain constant regions and light chain constant regions.

[0022] Preferably, the amino acid sequence of the heavy chain constant region is as 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: SSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0024] The present invention also provides a DNA molecule that encodes any of the above-described antibodies against the H3N2 influenza virus.

[0025] Furthermore, the nucleotide sequence encoding the variable region of the heavy chain of the antibody against H3N2 influenza virus is shown in SEQ ID No. 5: CAAGTGCAGCTGGTCGAGAGCGGCGGAGGAGTGGTGCAGCCTGGAGGCAGCCTGCGGCTGTCCTGCGCCCCTAGCAGCCTTACAGTGAACAACAATTACATGGGCTGGGTCAGACAGAGCCCCGGCAAAGGCCTGGAATGGGTGTCCCTGTCTTTTGGCTCTGGCACCACCGTGTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGCACAACAGCAAGAACATCCTGTACCTGCAGATGAGCAGACTGAGAGTGGAAGATACCGCCGTGTATTACTGTGCTAGAGAGTACGTGGAGGGCCCAGGAACAGGCGGCCACTTCGACCTGTGGGGCCGGGGCGTGCTGGTGACCGTTTCTAGC.

[0026] Furthermore, the nucleotide sequence encoding the variable region of the light chain of the antibody against H3N2 influenza virus is shown in SEQ ID No. 6: CTCCCCGTGCTGACACAACCTCCATCTGTGTCCGTGTCCCCTGGACAGACCGCCAGAATCACCTGTAGCGGCGAGACACTGCCTTGGCAGTACGTGTCTTGGTATCAGCAGAGACCCGGCCAGGCCCCTGTGCTGCTGATCTTCAAGGACAGCGAGCGGCCTAGCGGAGTGCCAGAAAGATTCAGCGGCTCTACCAGCGGCACCACCGTCACCCTGACCATCAGCGGCGTGCTGGCTGAAGATGAGGCCGACTACTACTGCCTGAGCAGCCCCAACACCGGCCCTTGGGTTTTTGGCGGAGGTACCAAGCTGACAGTGCTC.

[0027] Furthermore, the nucleotide sequence encoding the constant region of the heavy chain of the anti-H3N2 influenza virus antibody is shown in SEQ ID No.As shown in Figure 7: GCTTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA。.

[0028] Furthermore, the nucleotide sequence encoding the constant region of the light chain of the antibody against H3N2 influenza virus is shown in SEQ ID No. 8: TCGAGTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG.

[0029] The present invention also provides expression cassettes, recombinant vectors or host cells containing any of the DNA molecules described above.

[0030] Preferably, the expression vector comprises pcmv3-IgG-H and pcmv3-IgG-K / L.

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

[0032] This invention also provides a method for preparing the above-mentioned anti-H3N2 influenza virus antibody, specifically including the following steps:

[0033] (1) Collect peripheral blood samples from seasonal influenza vaccine recipients, establish a high-throughput in vitro culture system for memory B cells, clone the antibody heavy chain and light chain variable region genes of hemagglutinin-specific B cells, and obtain the DNA fragment of the antibody variable region by PCR.

[0034] (2) The DNA fragment described in step (1) is ligated into the expression vector, transferred 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 B cells in step (1) include CD3. - / CD19 - / CD27 - / CD80 + B cells;

[0037] Preferably, the host cells in step (3) include 293T cells.

[0038] The antibodies prepared by the above-described method of this invention exhibit high purity, no immunogenicity, and clear composition, eliminating potential risks such as pathogen contamination. Furthermore, this antibody preparation method is standardized, controllable, low-cost, simple, efficient, and suitable for standardized production. This invention establishes a complete in vitro culture and screening system for antibodies using memory B cells. Using this system, H3N2 subtype virus hemagglutinin-specific neutralizing antibodies were isolated from peripheral blood memory B cells of seasonal influenza vaccine recipients. This can be used for screening human antibodies against emerging and re-emerging infectious diseases.

[0039] Further research in this invention shows that the screened antibody 31D7 exhibits EC50 activity against the HA protein of various H3N2 strains. 50 The values ​​were all between 0.01 and 0.04 μg / mL, demonstrating its strong binding ability as a monoclonal antibody selected for targeting the H3N2 virus. More noteworthy is the EC50 binding of the 31D7 antibody to H10N3-HA. 50 The value was 2.01 μg / mL, indicating that 31D7 has the potential to cross-bind with other influenza subtypes. 31D7 exhibits a strong affinity for H3N2-HA, and the 31D7 antibody also shows some affinity for H10N3-HA. The 31D7 antibody has neutralizing ability against all four strains of the H3N2 subtype, and also has some neutralizing ability against H10N8. The 31D7 antibody can significantly inhibit the replication of the H3N2 subtype virus and also has cross-neutralizing activity against the H10N8 subtype. In other words, the 31D7 antibody of this invention has strong binding activity, affinity, and neutralizing ability against different H3N2-HA strains. Furthermore, the 31D7 antibody has some cross-binding and neutralizing affinity activity against H10N3 and H10N8, and has the potential to become a broad-spectrum antibody.

[0040] Therefore, the present invention also provides the use of any of the above-described anti-H3N2 influenza virus antibodies, any of the above-described DNA molecules or expression cassettes, recombinant vectors or host cells in the preparation of products for detecting H3N2 / H10 subtype influenza virus and / or the HA protein of H3N2 / H10 subtype influenza virus, or in the preparation of medicaments for the prevention and / or treatment of diseases caused by H3N2 / H10 subtype influenza virus.

[0041] Furthermore, the H10 subtype includes H10N3 or H10N8.

[0042] The present invention also provides a kit containing any of the above-described anti-H3N2 influenza virus antibodies.

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

[0044] The present invention also provides a pharmaceutical composition comprising any of the above-described anti-H3N2 influenza virus antibodies, any of the described DNA molecules or expression cassettes, recombinant vectors, or host cells.

[0045] Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

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

[0047] The anti-H3N2 influenza virus antibody of this invention exhibits strong binding activity, affinity, and neutralizing ability against different H3N2-HA proteins. A 50% binding and neutralizing effect requires only tens of ng, and the affinity is in the nM range. Simultaneously, the anti-H3N2 influenza virus antibody of this invention shows certain cross-binding, neutralizing, and affinity activity against H10N3 and H10N8, demonstrating the potential to become a broad-spectrum antibody. It can be used to detect the HA protein of H3N2 / H10 subtype influenza viruses and / or to prevent and / or treat diseases caused by H3N2 / H10 subtype influenza viruses. Furthermore, the antibody of this invention has high purity, is non-immunogenic, has a clear composition, and does not pose a potential risk of pathogen contamination. Attached Figure Description

[0048] Figure 1 The result of SDS-PAGE of the purified 31D7 antibody in Example 4 is shown in the figure: lane 1 is the marker, lanes 2-3 are 31D7 and MEDI8852.

[0049] Figure 2 The graph shows the results of ELISA detection of the binding ability of 31D7 antibody with different H3N2 and H10N3 hemagglutinin proteins in Example 5.

[0050] Figure 3 This is the ECG used in Example 5 to detect the binding ability of the 31D7 antibody to different H3N2 and H10N3 hemagglutinin proteins using ELISA. 50 Result value.

[0051] Figure 4 The graph shows the results of detecting the affinity of 31D7 antibody with different hemagglutinin proteins HA using biomembrane interference (BLI) technology in Example 5.

[0052] Figure 5 The figure shows the results of the neutralizing activity of the 31D7 antibody against different strains of H3N2 influenza virus and H10N8 influenza virus in Example 5.

[0053] Figure 6The IC50 of the 31D7 antibody in Example 5 against different strains of H3N2 influenza virus and H10N8 influenza virus was measured. 50 Result value. 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] Seasonal influenza vaccination can induce the production of specific and broad-spectrum neutralizing antibodies against the influenza virus surface glycoprotein hemagglutinin. This invention establishes a complete in vitro culture and screening system for memory B cells. Using this system, H3N2 subtype virus hemagglutinin-specific neutralizing antibodies can be isolated from peripheral blood memory B cells of seasonal influenza vaccine recipients. Valuable neutralizing antibodies can be screened for use in the prevention and treatment of influenza infection.

[0057] Example 1: Isolation, screening, and identification of antibodies against influenza virus H3N2 subtype

[0058] For volunteers who received the seasonal influenza vaccine, with the written consent of their institution's ethics committee and the patient themselves, peripheral blood was drawn for antibody isolation.

[0059] 1. Collection of peripheral blood samples from seasonal influenza vaccine recipients for PBMC cell isolation:

[0060] Prepare lymphocyte separation medium, DMPBS containing 2% FBS, and 1640 medium containing 2% FBS. Add 5 mL of lymphocyte separation medium to a 15 mL centrifuge tube. Shake the blood sample well and slowly add it to the lymphocyte separation medium. Centrifuge at 1000g for 30 minutes. After separation, collect the PBMCs from the middle layer, wash once with 10 mL of 1640 medium, and centrifuge at 400g for 6 minutes. Wash again with 10 mL of PBS containing FBS, centrifuge at 400g for 6 minutes, discard the supernatant, and count the cells. Cryopreserve the cells in 90% FBS + 10% DMSO and store in liquid nitrogen.

[0061] 2. Irradiated 293T-mCD40L cell line:

[0062] Resuscitate one tube of 293T-mCD40L cells and expand the culture to 20 10cm cell culture dishes. Once confluence reaches 90%, digest the cells with trypsin and collect them into 50mL centrifuge tubes, centrifuging at 350g for 5 minutes. Resuspend the cells in DMEM containing 10% FBS. Expose the cells to X-ray irradiation at 5000 rads. After counting, freeze 1,500,000 cells per tube in liquid nitrogen.

[0063] 3. Prepare the culture medium for B cells:

[0064] Prepare a B cell culture medium system (one 384-well plate) with the following specific quantities: 15 mL Complete IMDM medium, 750 ng IL-2 (50 ng / mL), 750 ng IL-21 (50 ng / mL), and one tube of irradiated mCD40L cell line (5000 cells / well).

[0065] 4. Magnetic bead sorting of CD27+ memory B cells:

[0066] PBMC resuscitation: Rapidly dissolve one tube of PBMCs at 37°C, add to 5 mL of IMDM medium, centrifuge at 350g for 5 minutes, and discard the supernatant. Magnetic bead labeling of non-memory B cells: This includes cell counting, centrifugation, resuspending, adding B Cell Biotin-Antibody Cocktail, mixing and incubating for 10 minutes, then adding Anti-Biotin MicroBeads and incubating again, followed by washing and resuspending. Next, magnetic bead removal of non-B cells: This includes LS column preparation, column flushing, cell loading, and negative selection for collecting memory B cells. Finally, magnetic bead labeling of memory B cells and collection of positive memory B cells: This includes centrifugation, resuspending, adding CD27 MicroBeads, and finally collecting the cells using an LS column for cell counting.

[0067] 5. Culture and ELISA detection:

[0068] CD27+ memory B cells, obtained through magnetic bead sorting, were added in groups of four to each well of a 384-well plate and cultured in a 37°C, 5% CO2 cell culture incubator. On day 12, IgG expression was detected by ELISA, including steps such as coating, blocking, incubation with primary antibody, addition of enzyme-labeled secondary antibody, addition of substrate chromogenic solution, termination of the reaction, and measurement of OD value. On day 13, H3N2 (A / Perth / 16 / 2009) antigen was detected by ELISA, following the same procedure as IgG expression detection. The result was interpreted as follows: under the detection instrument at 450nm (410nm for ABST chromogenic assay), with the blank control well zeroed, the OD value was measured; a result greater than 2.1 times the specified negative control OD value was considered positive.

[0069] Example 2: Obtaining the antibody heavy and light chain variable region gene using RT-PCR amplification

[0070] 1. Cells were lysed from H3N2-positive wells, and RNA was extracted:

[0071] Prepare lysis buffer, stop solution, and RT system. Lyse the positive cells obtained in Example 1 and perform reverse transcription, including cell washing, adding lysis buffer and stop solution, and finally adding the RT system. Set the RT reaction conditions as follows: 37°C for 15 min; 50°C for 5 min; 98°C for 5 min; and store at 4°C.

[0072] 2. Nested PCR amplification of antibody genes:

[0073] Two rounds of PCR were used, each in a 25 μL system. The first round of PCR consisted of 3.5 μL of reverse transcription product cDNA, external primers designed and preserved in the lab, KOD FX enzyme, buffe, and dNTPs. The reaction conditions were as follows: 95°C for 3 minutes, gradient annealing between 98°C and 67°C (towards 58°C) for 10 seconds, 45 seconds, and 2 minutes at each annealing temperature, with 3 cycles at each temperature, 30 cycles at 55°C, and a final extension at 68°C for 7 minutes. The second round of PCR was set up similarly to the first round, with internal primers designed and preserved in the lab. The reaction conditions were: 95°C for 3 minutes, 98°C, 62°C, and 68°C for 10 seconds, 45 seconds, and 1 minute for a total of 50 cycles, followed by a final extension at 68°C for 7 minutes. After the reaction, a 2% agarose gel was prepared, electrophoresed, and the DNA concentration was determined and sequenced. The heavy chain variable region gene sequence is CAAGTGCAGCTGGTCGAGAGCGGCGGAGGAGTGGTGCAGCCTGGAGGCAGCCTGCGGCTGTCCTGCGCCCCTAGCAGCCTTACAGTGAACAACAATTACATGGGCTGGGTCAGACAGAGCCCCGGCAAAGGCCTGGAATGGGTGTCCCTGTCTTTTGGCTCTGGCACCACCGTGTACGCC GACAGCGTGAAGGGCAGATTCACCATCAGCCGGCACAACAGCAAGAACATCCTGTACCTGCAGATGAGCAGACTGAGAGTGGAAGATACCGCCGTGTATTACTGTGCTAGAGAGTACGTGGAGGGCCCAGGAACAGGCGGCCACTTCGACCTGTGGGGCCGGGGCGTGCTGGTGACCGTTTCTAGC(SEQ ID No.5).

[0074] The light chain variable region gene sequence is CTCCCCGTGCTGACACAACCTCCATCTGTGTCCGTGTCCCTGGACAGACCGCCAGAATCACCTGTAGCGGCGAGACACTGCCTTGGCAGTACGTGTCTTGGTATCAGCAGAGACCCGGCCAGGCCCCTGTGCTGCTGATCTTCAAGGACAGC GAGCGGCCTAGCGGAGTGCCAGAAAGATTCAGCGGCTCTACCAGCGGCACCACCGTCACCCTGACCATCAGCGGCGTGCTGGCTGAAGATGAGGCCGACTACTACTGCCTGAGCAGCCCCAACACCGGCCCTTGGGTTTTTGGCGGAGGTACCAAGCTG(SEQ ID No.6)

[0075] Furthermore, the amino acid sequences of the heavy chain variable region and the light chain variable region were determined:

[0076] The amino acid sequence of the heavy chain variable region is: QVQLVESGGGVVQPGGSLRLSCAPSSLTVNNNYMGWVRQSPGKGLEWVSLS FGSGTTVYADSVKGRFTISRHNSKNILYLQMSRLRVEDTAVYYCAREYVEGPGTGGHFDLWGRGVLVTVSS (SEQ ID No. 1);

[0077] The amino acid sequence of the light chain variable region is: LPVLTQPPSVSVSPGQTARITCSGETLPWQYVSWYQQRPGQAPVLLIFKDSER PSGVPERFSGSTSGTTVTLTISGVLAEDEADYYCLSSPNTGPWVFGGGTKLTV L (SEQ ID No. 2).

[0078] The CDR1, CDR2, and CDR3 of the variable regions of the heavy and light chains are shown in Table 1.

[0079] Table 1. CDR1, CDR2, and CDR3 (IMGT scheme) of the variable region of the heavy and light chains.

[0080]

[0081]

[0082] The light chain constant region gene sequence is TCGAGTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCA AACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG(SEQ ID No.8).

[0083] The amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3.

[0084] The amino acid sequence of the constant region of the light chain is shown in SEQ ID No. 4.

[0085] Example 3: Construction of an expression vector for antibody heavy and light chains

[0086] 1. Antigen vector cloning: Reaction system configuration: Total volume: 5μL: Nested PCR product: pCMV3-IgG / K / L vector.

[0087] 2. For transformation, add 5 μL of the above mixture to trans 10 competent cells and incubate on ice for 30 minutes. Incubate at 25°C for 10 minutes, heat shock at 42°C for 90 seconds, and incubate on ice for 3 minutes. Add 300 μL of Amp-free LB medium to a clean bench and incubate at 37°C and 220 rpm for 1 hour. Spread the mixture onto solid medium containing Amp+.

[0088] 3. Bacterial culture PCR cloning verification: Observe the petri dishes, pick 10 single colonies from each dish, add them to liquid medium containing Amp, and incubate at 37℃ and 220 rpm for 3 hours. After performing bacterial culture PCR, prepare a 2% agarose gel and run it to verify the presence of a 500bp target band. Send the bacterial samples containing the target band for sequencing. Perform plasmid extraction on the correctly cloned bacterial cultures.

[0089] Example 4: Expression and purification of 31D7 antibody

[0090] 1. Transfect paired heavy and light chain plasmids to express antibodies.

[0091] 293T cell culture: Cells were pre-seeded in 6-well plates, with 1 × 10^6 cells per well, and cultured overnight at 37°C in a 5% CO2 incubator. Transfection procedure: The next day, transfection was performed after cells reached 90% confluence. Transfection conditions were as follows: Heavy chain plasmid: 2 μg, light chain plasmid: 2 μg, EndoFectin™ Max: 8 μL, Opti-MEM medium: 500 μL. EndoFectin™ Max was added to the plasmids, incubated for 30 minutes, and then added to the prepared 293T cells. The medium was replaced with 293Freestyle medium 6-8 hours after transfection. Cell culture supernatant (containing antibody) was collected after 72 hours.

[0092] 2. Antibody purification

[0093] Prepare Protein A packing material; use 10 mL of Protein A packing material for 1 L of antibody supernatant. Prewash the packing material with 10 volumes of ddH2O and binding buffer. Collect the antibody supernatant and centrifuge at 5000 rpm and 4°C for 10 minutes. Transfer the supernatant to 2 M Tris reagent (pH 8, 2.5 M NaCl) and adjust to a final concentration of 50 mM Tris (pH 8, 150 mM NaCl). Add Protein A packing material to the antibody supernatant and gently shake on a shaker at room temperature for 1 hour. Collect the Protein A packing material into a column. Aspirate the packing material from the column with 20 volumes of binding buffer. Elute the antibody with 3 volumes of elution buffer. Repeat the previous step. Antibody concentration and storage: Concentrate the antibody supernatant using a concentration column to a storage concentration of 1 mg / mL and store at -80°C. Elute the column with 20 volumes of elution buffer, 20 volumes of ddH2O, 3 volumes of 6M guanidine hydrochloride, 20 volumes of ddH2O, and 5 volumes of 20% ethanol, and store at 4°C.

[0094] 3. Verification by SDS-PAGE electrophoresis analysis of Cochlear staining.

[0095] Prepare the separating gel and pour it to approximately 3 cm from the top of the short glass plate (5 mL per plate). Cover with isopropanol to prevent the inhibition of gel polymerization by atmospheric oxygen. After 30 minutes, pour off the covering layer and absorb any remaining liquid with absorbent paper. Prepare the stacking gel and insert the comb. Wait for polymerization to complete for 30 minutes. Place the gel in the electrophoresis tank and add Tris-glycine electrode buffer. Remove the comb and prepare for sample loading (15 μL sample + 5 μL loading buffer per well). Depending on the needs, samples can be treated in two ways: one is by adding mercaptoethanol to break disulfide bonds, and the other is without mercaptoethanol to detect intact antibodies. Samples need to be boiled in water for 10 minutes to process them. Set the initial voltage to 80V (8V / cm). After approximately 40 minutes, when the sample enters the separating gel, change the voltage to 120V (15V / cm). Electrophoresis continues until the bromophenol blue stain reaches 1 cm from the bottom of the separating gel, then turn off the power. When cutting the gel, ensure the sample wells are reversed and mark them, for example, by cutting off a corner. Stain the gel for 1 hour. Place the gel in destaining solution and gently shake for 4-8 hours, changing the solution 3-4 times during this period. Alternatively, destaining can be achieved by heating the water in a microwave and changing the water 3 times. After destaining, observe the gel under light to analyze the electrophoresis results.

[0096] SDS-PAGE results are as follows Figure 1 The results showed that under reducing conditions of boiling, the disulfide bonds connecting the antibody heavy and light chains were broken, resulting in the separation of the antibody heavy and light chains and the appearance of two bands. These bands corresponded to the variable and constant regions of the 31D7 heavy chain, with a molecular weight of approximately 50 kDa; and the variable and constant regions of the light chain, with a molecular weight of approximately 25 kDa. The 31D7 antibody was successfully expressed with high purity, and the bands of the purified antibody were consistent with expectations.

[0097] Example 5 Activity analysis of 31D7 antibody

[0098] 1. The binding affinity of 31D7 to hemagglutinin HA

[0099] HA proteins of different influenza virus strains (H3N2 / Perth / 2009, H3N2 / Victoria / 2011, H3N2 / Texas / 2012, H10N3 / Jiangsu / 2021) were diluted in PBS buffer and coated with 100 ng / well, 100 μL / well, and incubated overnight at 4°C. The HA protein dilution was discarded, and blocking buffer (5% skim milk dissolved in PBST) was added, 200 μL / well, and the plates were blocked at 37°C for 2 hours. The plates were washed 4 times with 0.05% PBST, 200 μL / well, for 3 minutes each time. The plates were then shaken dry. Primary antibody incubation: Dilute 31D7 and MEDI8852 antibodies separately in PBST, starting at 10 μg / mL, and perform 3-fold serial dilutions for a total of 9 dilutions. Add 100 μL / well to each well of the plate and incubate at 37°C for 2 hours. Wash the plate 4 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 incubation with 50 μL of 1M H2SO4 and measure the absorbance at 450 nm. Perform duplicates for each concentration in 2 wells and calculate the EC50 of HA protein binding to the antibody. 50 The result is as follows Figure 2 and Figure 3 As shown.

[0100] The binding affinity of the 31D7 antibody to the hemagglutinin protein HA on the surface of different H3N2 viral strains was detected using an ELISA assay. The control antibody, MEDI8852, is a broad-spectrum influenza virus monoclonal antibody that targets a highly conserved region of the hemagglutinin stem. Figure 2-3 As shown, the antibody 31D7 obtained by screening in this invention exhibits good binding activity to HA proteins and also cross-binding activity with H10N3-HA. 31D7 shows EC50 activity against the HA proteins of various H3N2 strains. 50 The values ​​were all between 0.01 and 0.04 μg / mL, demonstrating its strong binding ability as a monoclonal antibody selected for targeting the H3N2 virus. More noteworthy is the EC50 binding of the 31D7 antibody to H10N3-HA. 50 The value was 2.01 μg / mL, indicating that 31D7 has the potential to cross-bind with other subtypes of influenza virus.

[0101] 2. Affinity of different HA antigens to 31D7 antibody

[0102] Biofilm layer interference (BLI) assay was used to determine the affinity activities of 31D7 antibody with different antigens. ForteBio Octet Red96e (Sartorius) was used (referencing the method in the article He et al. 2022 Nature Microbiology). The 31D7 antibody was diluted at an initial concentration of 200 nM in PBS buffer (containing 0.02% Tween-20 and 0.2% BSA). When detecting H3N2-HA antigen, the concentration gradient of 31D7 antibody was from 100 nM to 1.56 nM. When detecting H10N3-HA antigen, the concentration gradient of antibody was from 200 nM to 3.125 nM. Both were 2-fold dilutions, for a total of 7 concentration gradients.

[0103] Figure 4 The figures show the binding and dissociation curves of 31D7 antibody and different HA proteins, respectively. Table 2 shows the antigen-antibody affinity, where Ka is the binding constant, Kdis is the dissociation rate, and KD is the dissociation constant of the antibody and HA protein at equilibrium. A smaller KD indicates less dissociation and stronger affinity. The affinity of 31D7 antibody for different H3N2-HA proteins is 1.55 nM and 1.39 nM, respectively, indicating that 31D7 exhibits a strong affinity for H3N2-HA. Notably, 31D7 antibody also shows some affinity for H10N3-HA, with a dissociation constant of 368 nM.

[0104] Table 2 shows the specific dissociation constant, binding constant, and dissociation rate values.

[0105]

[0106] 3. Virus micro-neutralization assay to detect the neutralizing activity of 31D7 antibody.

[0107] The in vitro neutralizing capacity of 31D7 antibody was determined using various recombinant viruses. 31D7 antibody was initially diluted 100 μg / mL in three-fold serial dilutions, with two replicates per concentration (50 μL / well). Viral dilution: The virus was diluted to 100 TCID using viral dilution buffer. 50 Add 50 μL of antibody to a 96-well plate at a concentration of 50 μL / well. Then, add 50 μL / well of antibody to 100 TCID50. 50 Mix 50 μL virus / 50 μL / well and incubate at 37°C in a 5% CO2 incubator for 1 h. Transfer the prepared MDCK cells at a rate of 3 × 10⁻⁶ cells / well. 4Cells / well, 100 μL / well, were added to a 96-well plate after incubation with the virus-antibody mixture and incubated at 37°C in a 5% CO2 incubator for 24 h. Finally, ELISA was used for detection. The primary antibody was Influenza ANP, Rabbit Pab (1:2000 dilution), and the secondary antibody was HRP-conjugated goat anti-rabbit IgG (1:2000 dilution). OD was measured using a microplate reader. 450 A well with a value greater than 2.1 times that of the negative control was designated as a positive well. The IC50 of the antibody was calculated based on the values ​​measured by the ELISA reader. 50 value.

[0108] like Figure 5 and Figure 6 As shown, the 31D7 antibody was found to have neutralizing ability against all four strains of the H3N2 subtype, with an IC50 value of [missing information]. 50 The values ​​were 1.119 μg / mL, 0.047 μg / mL, 0.004 μg / mL, and 0.605 μg / mL, respectively. Importantly, the 31D7 antibody also showed some neutralizing ability against H10N8 / Jiangxi / 2013, with an IC50 value of 1.119 μg / mL, 0.047 μg / mL, 0.004 μg / mL, and 0.605 μg / mL. 50 The value was 4.171 μg / mL. The 31D7 antibody significantly inhibited the replication of the H3N2 subtype virus and also had a cross-neutralizing effect on the H10N8 subtype.

[0109] In summary, the antibodies of this invention exhibit strong binding activity, affinity, and neutralizing ability for different H3N2-HA molecules. Furthermore, it is noteworthy that the antibodies demonstrate cross-binding, neutralizing, and affinity activity against H10N3 and H10N8, suggesting their potential as broad-spectrum antibodies. Moreover, the antibodies of this invention possess high purity, are non-immunogenic, have clearly defined components, and do not pose a potential risk of pathogen contamination. The method for preparing monoclonal antibodies in this invention is standardized, controllable, low-cost, simple, efficient, and suitable for standardized production.

[0110] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented. The above embodiments are preferred implementations of the present invention. Those skilled in the art should understand that any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. An antibody against H3N2 influenza virus, characterized in that, It includes a heavy chain variable region and a light chain variable region; the amino acid sequence of CDR1 of the heavy chain variable region is SLTVNNNY, the amino acid sequence of CDR2 of the heavy chain variable region is SFGSGTT, and the amino acid sequence of CDR3 of the heavy chain variable region is AREYVEGPGTGGHFDL. The amino acid sequence of CDR1 in the light chain variable region is TLPWQY, the amino acid sequence of CDR2 in the light chain variable region is KDS, and the amino acid sequence of CDR3 in the light chain variable region is LSSPNTGPWV.

2. The anti-H3N2 influenza virus antibody 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. The anti-H3N2 influenza virus antibody according to claim 2, 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.

4. A DNA molecule, characterized in that, The antibody against H3N2 influenza virus as described in any one of claims 1 to 3 is encoded.

5. The DNA molecule according to claim 4, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain of the antibody against H3N2 influenza virus is shown in SEQ ID No.

5.

6. The DNA molecule according to claim 4, characterized in that, The nucleotide sequence encoding the variable region of the light chain of the antibody against H3N2 influenza virus is shown in SEQ ID No.

6.

7. An expression cassette, recombinant vector, or host cell containing any one of the DNA molecules described in claims 4 to 6.

8. The use of the anti-H3N2 influenza virus antibody according to any one of claims 1 to 3, the DNA molecule according to any one of claims 4 to 6, or the expression cassette, recombinant vector, or host cell according to claim 6 in the preparation of products for detecting H3N2 / H10 subtype influenza virus and / or the HA protein of H3N2 / H10 subtype influenza virus, or in the preparation of medicaments for the prevention and / or treatment of diseases caused by H3N2 / H10 subtype influenza virus.

9. A reagent kit, characterized in that, The kit contains the anti-H3N2 influenza virus antibody as described in any one of claims 1 to 3.

10. A pharmaceutical composition, characterized in that the pharmaceutical composition contains an anti-H3N2 influenza virus antibody according to any one of claims 1 to 3, a DNA molecule according to any one of claims 4 to 6, or an expression cassette according to claim 6, a recombinant vector, or a host cell.

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