Anti-h7 subtype influenza virus antibodies and uses thereof

Abstract

The application belongs to the technical field of antibodies, and discloses an anti-H7 subtype influenza virus antibody and application thereof. The application provides an anti-H7 subtype influenza virus antibody or an antigen binding fragment thereof, which has strong binding activity and binding capacity with H7 subtype influenza virus HA protein, has strong neutralization activity on H7 subtype influenza virus, and can be used for detecting H7 subtype influenza virus and / or HA protein of H7 subtype influenza virus, and preventing and / or treating diseases caused by H7 subtype influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to antibodies against H7 subtype influenza virus and their applications. Background Technology

[0002] Influenza viruses belong to the Orthomyxoviridae family and are a class of enveloped, single-stranded, negative-sense, segmented RNA viruses. Influenza viruses are serotyped based on the antigenicity of their surface glycoproteins—hemagglutinin (HA) and neuraminidae (NA)—e.g., H5N1, H7N9, H10N8. Waterfowl and wild birds are natural hosts for influenza viruses. Before the emergence of H7N9 avian influenza virus, H7 subtype influenza viruses existed only in poultry and wild birds, with occasional human infections. The transmission of avian influenza viruses from animals to humans is constrained by factors such as differences in sialic acid receptors, limited viral RNA genome replication in the cell nucleus, and the innate immune response in humans. Most avian influenza infected individuals have a history of contact with poultry, but the efficiency of human-to-human transmission of avian influenza viruses is relatively low.

[0003] Since the first report of H7N9 avian influenza infection in humans in March 2013, there have been five peaks in human infections. Measures such as banning live poultry markets, mandatory H7N9 vaccination for farmed poultry, and culling H7N9-infected poultry have reduced H7N9 virus contamination in live poultry markets and farms, effectively preventing human cases of H7N9 infection. However, the battle between the H7N9 virus and humans has not ceased. Low-pathogenic H7N9 viruses, which emerged in 2013, are less pathogenic to chickens, showing no clinical symptoms after challenge; while highly pathogenic strains with multiple basic amino acids in the HA gene are 100% lethal to poultry. Since the mandatory immunization of poultry with inactivated H7N9 vaccines in 2017, transmission of the H7N9 virus to humans has been effectively blocked, but new variants emerged in 2019. This indicates that the H7N9 virus is still in a period of active mutation, and effective measures are urgently needed to eliminate the impact of H7N9 avian influenza virus mutations.

[0004] Therapeutic neutralizing antibodies are an effective means of combating H7N9 virus infection. Since Milstein et al. reported the use of mouse hybridoma technology to screen monoclonal antibodies in 1975, monoclonal antibodies have attracted attention as a treatment option for various infections, including influenza. Monoclonal antibodies inhibit influenza virus replication through several mechanisms, including neutralizing the virus by binding to the HA (hypoallergenic glycoside) to interfere with receptor binding and fusion, and stimulating an endogenous immune response through the function of the antibody Fc fragment. HA is a trimeric protein with an immunogenically dominant globular head that binds to sialic acid receptors on host cells. Amino acid mutations accumulate in the HA head, allowing the virus to evade the neutralizing effects of previous infections or immune-induced antibodies, and are one of the main factors contributing to antigenic variation. The HA stem is highly conserved, and conserved epitopes in the stem can induce a broad spectrum of neutralizing antibodies, making it an important candidate antigen for universal influenza vaccines. Summary of the Invention

[0005] The first aspect of the present invention is to provide an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof.

[0006] A second aspect of the present invention aims to provide a nucleic acid molecule encoding an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof, as described in the first aspect of the present invention.

[0007] A third aspect of the present invention aims to provide expression cassettes, recombinant vectors, or transgenic cell lines comprising nucleic acid molecules of the second aspect of the present invention.

[0008] A fourth aspect of the present invention is to provide a coupling agent.

[0009] The fifth aspect of this invention aims to provide the application of the anti-H7 subtype influenza virus antibody or its antigen-binding fragment of the first aspect of this invention, the nucleic acid molecule of the second aspect of this invention, the expression cassette of the third aspect of this invention, the recombinant vector or transgenic cell line and / or the conjugate of the fourth aspect of this invention.

[0010] The sixth aspect of this invention aims to provide a product.

[0011] The seventh aspect of this invention is to provide a medicine.

[0012] The object of the eighth aspect of the present invention is to provide a method for preparing an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof, as described in the first aspect of the present invention.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof, said antibody against H7 subtype influenza virus or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region;

[0015] The heavy chain variable region includes CDR1, CDR2 and CDR3;

[0016] The amino acid sequence of CDR1 in the heavy chain variable region is as follows:

[0017] a) GFTDSYT (SEQ ID NO.5); or

[0018] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 5;

[0019] The amino acid sequence of CDR2 in the heavy chain variable region is as follows:

[0020] a) IVPVVGVP (SEQ ID NO. 6); or

[0021] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 6;

[0022] The amino acid sequence of CDR3 in the heavy chain variable region is as follows:

[0023] a)ARDLGDGYGSNSGYFDF(SEQ ID NO.7); or

[0024] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 7;

[0025] The variable region of the light chain includes CDR1, CDR2 and CDR3;

[0026] The amino acid sequence of CDR1 in the light chain variable region is as follows:

[0027] a) QGIGSW (SEQ ID NO. 8); or

[0028] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 8;

[0029] The amino acid sequence of CDR2 in the light chain variable region is as follows:

[0030] a) RAS; or

[0031] b) Sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity with RAS;

[0032] The amino acid sequence of CDR3 in the light chain variable region is as follows:

[0033] a) QQHDDSPLT (SEQ ID NO.9); or

[0034] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.9.

[0035] Preferably, the amino acid sequence of the heavy chain variable region of the anti-H7 subtype influenza virus antibody or its antigen-binding fragment is as follows:

[0036] a) EVQLVQSGAEVKRPGSSVKVSCKASGFTFDSYTFNWVRQAPGQGLEWMGAIVPVVGVPTYAEKFQGRVTISADTSKTTAFMELTGLTSEDTAVYYCARDLGDGYGSNSGYFDFWGQGALVTVSS (SEQ ID NO.3); or

[0037] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.3;

[0038] The amino acid sequence of the light chain variable region of the anti-H7 subtype influenza virus antibody or its antigen-binding fragment is as follows:

[0039] a)DIVLTQSPSSSLSASVGDRITITCRASQGIGSWLAWYQHKPGEAPKLLIYRASTLATGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQHDDSPLTFGGGTKVEIK(SEQ ID NO.4); or

[0040] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.4.

[0041] Preferably, the H7 subtype influenza virus is the H7N9 subtype influenza virus.

[0042] A second aspect of the invention provides a nucleic acid molecule encoding an antibody against an H7 subtype influenza virus or an antigen-binding fragment thereof, as described in the first aspect of the invention.

[0043] Preferably, the nucleic acid molecule comprises nucleic acid molecule A encoding the heavy chain variable region of the antibody against the H7 subtype influenza virus or its antigen-binding fragment, and nucleic acid molecule B encoding the light chain variable region of the antibody against the H7 subtype influenza virus or its antigen-binding fragment;

[0044] The nucleotide sequence of the nucleic acid molecule A is shown in SEQ ID NO.1;

[0045] The nucleotide sequence of the nucleic acid molecule B is shown in SEQ ID NO.2.

[0046] A third aspect of the invention provides an expression cassette, recombinant vector, or transgenic cell line comprising the nucleic acid molecule of the second aspect of the invention.

[0047] Preferably, the transgenic cell line does not contain any plant or animal species.

[0048] A fourth aspect of the present invention provides a conjugate comprising an antibody against H7 subtype influenza virus or its antigen-binding fragment and a conjugation portion as described in the first aspect of the present invention.

[0049] The coupling component is at least one of the following: a detection marker, a radionuclide, an enzyme, a toxin, and a chemotherapeutic agent.

[0050] Preferably, the detection marker is a fluorescent or luminescent marker.

[0051] Preferably, the radionuclide is a diagnostic isotope.

[0052] Preferably, the diagnostic isotope is at least one selected from Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

[0053] Preferably, the toxin is Pseudomonas aeruginosa exotoxin.

[0054] The fifth aspect of the present invention provides the application of the anti-H7 subtype influenza virus antibody or its antigen-binding fragment of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette of the third aspect of the present invention, the recombinant vector or transgenic cell line, and the conjugate of the fourth aspect of the present invention.

[0055] Application of any one of (1) to (4) in any one of (5) to (6);

[0056] (1) The antibody against H7 subtype influenza virus or its antigen-binding fragment of the first aspect of the present invention;

[0057] (2) The nucleic acid molecule of the second aspect of the present invention;

[0058] (3) The expression cassette, recombinant vector, or transgenic cell line of the third aspect of the present invention;

[0059] (4) The coupling of the fourth aspect of the present invention;

[0060] (5) Prepare products for detecting H7 subtype influenza virus and / or H7 subtype influenza virus HA protein;

[0061] (6) Prepare drugs for the prevention and / or treatment of diseases caused by H7 subtype influenza virus.

[0062] Preferably, the product described in (5) is at least one of reagents, test plates and kits.

[0063] Preferably, the H7 subtype influenza virus is the H7N9 subtype influenza virus.

[0064] In a sixth aspect, the present invention provides a product comprising at least one of the following: an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof from the first aspect of the present invention and a conjugate from the fourth aspect of the present invention.

[0065] Preferably, the product is at least one of a reagent, a test plate, and a kit.

[0066] Preferably, the product is used to detect H7 subtype influenza virus and / or the HA protein of H7 subtype influenza virus.

[0067] Preferably, the H7 subtype influenza virus is the H7N9 subtype influenza virus.

[0068] A seventh aspect of the present invention provides a medicament comprising at least one of (1) to (4);

[0069] (1) The antibody against H7 subtype influenza virus or its antigen-binding fragment of the first aspect of the present invention;

[0070] (2) The nucleic acid molecule of the second aspect of the present invention;

[0071] (3) The expression cassette, recombinant vector, or transgenic cell line of the third aspect of the present invention;

[0072] (4) The coupling of the fourth aspect of the present invention.

[0073] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0074] Preferably, the drug is used for the prevention and / or treatment of diseases caused by H7 subtype influenza viruses.

[0075] Preferably, the H7 subtype influenza virus is the H7N9 subtype influenza virus.

[0076] Preferably, the coupling portion of the conjugate is a toxin and / or a chemotherapeutic agent.

[0077] The eighth aspect of the present invention provides a method for preparing an antibody against H7 subtype influenza virus or an antigen-binding fragment thereof, as described in the first aspect of the present invention, by culturing a transgenic cell line as described in the third aspect of the present invention.

[0078] The beneficial effects of this invention are:

[0079] This invention provides an antibody against H7 subtype influenza virus or its antigen-binding fragment, which exhibits strong binding activity to the HA protein of H7 subtype influenza virus (EC). 50 It exhibits strong neutralizing activity (0.091 ug / mL) against H7 subtype influenza virus, with a binding capacity (binding constant Kon value of 2.71+E051 / Ms, dissociation constant Koff value of <1.00E-07 1 / s, and affinity constant KD value of <1.00E-12M). It can be used to detect H7 subtype influenza virus and / or the HA protein of H7 subtype influenza virus, and to prevent and / or treat diseases caused by H7 subtype influenza virus. Attached Figure Description

[0080] Figure 1 This is an SDS-PAGE result of the monoclonal antibody against H7N9 subtype influenza virus prepared in Example 2.

[0081] Figure 2 This is a graph showing the results of enzyme-linked immunosorbent assay (ELISA) in Example 3, which detected the binding activity of monoclonal antibody 41E10 against H7N9 subtype influenza virus to HA antigen.

[0082] Figure 3 This is a graph showing the results of detecting the binding ability of the monoclonal antibody 41E10 against the H7N9 subtype influenza virus to the HA protein using biomembrane interference technology in Example 3.

[0083] Figure 4 This is a graph showing the results of the neutralizing activity assay for the monoclonal antibody 41E10 against the H7N9 subtype influenza virus in Example 3. Detailed Implementation

[0084] The present invention will be further described in detail below through specific embodiments.

[0085] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0086] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0087] Example 1: Isolation of monoclonal antibodies against H7N9 subtype influenza virus

[0088] Inactivated H7N9 subtype influenza virus vaccine was prepared for immunization of Chinese rhesus macaques (specific methods are referenced in: Pan WQ et al., Patient-derived avian influenza A(H5N6)virus is highly pathogenic in mice but can be effectively treated by anti-influenza polyclonal antibodies, Emerging Microbes & Infections, 2018, 7:107.). Peripheral blood mononuclear cells (PBMCs) were isolated from the macaques, and B cells that specifically bind to the HA protein were sorted by flow cytometry. The heavy and light chain sequences of the specific antibody were amplified by single-cell RT-PCR (specific methods are referenced in: Zhang F et al., Phenotypic characterization of Chinese rhesus macaque plasmablasts for cloning antigen-specific monoclonal antibodies, Frontiers in Immunology, 2019, 10:2426.). The specific methods are as follows:

[0089] 1. Preparation of inactivated vaccine against H7N9 subtype influenza virus

[0090] (1) Nanjing Genscript Company was commissioned to synthesize the HA and NA genes of influenza virus strain A / Anhui / 1 / 2013 (H7N9) (sequence numbers EPI439507 and EPI439509, respectively, www.gisaid.org database).

[0091] (2) The synthesized HA and NA genes, along with six internal genes of the A / Perto Rico / 8 / 1934 (H1N1) virus (i.e., PB2, PB1, PA, NP, M, NS1, and NS2, accession numbers AF389115, AF389116, AF389117, AF389119, AF389121, and AF389122), were cloned into bidirectional expression plasmid pM containing the human RNA polymerase I promoter and the CMV promoter, respectively, to obtain recombinant plasmids. 24 hours before transfection, 293T cells were passaged, and cell counts were performed, with the density controlled at 1 x 102. 6 cells / mL. Take 1 x 10⁻⁶ cells / mL. 6For each cell transfection, 0.5 μg of each of the eight plasmids and 4 μg (1 mg / mL) of PEI were required. 4 μg of plasmid and 4 μg of PEI were dissolved separately in 250 μL of opti-MEM medium (Gibco) and incubated at 25°C for 15 minutes. An equal volume of plasmid and PEI was then mixed and incubated at 25°C for 15 minutes before being added to the prepared cells. After 6 hours, 500 μL of opti-MEM medium was added, and the cells were cultured for 48 hours. 100 μL of the transfection product was then used to amplify the recombinant virus (named PR8-H7N9) in 10-day-old SPF chicken embryos. The genome of the recombinant virus was amplified in full length by RT-PCR primers. The eight pairs of primers used for gene amplification were those reported in the literature (Hoffman et al., Universal primer set for the full-length amplification of allinfluenza A viruses, Archive of Virology, 2001, 146: 2275-2289) (Ba-PB2-1, Ba-PB2-2341R; Bm-PB1-1, Bm-PB1-2341R; Bm-PA-1, Bm-PA-2233R; Bm-HA-1, Bm-NS-890R; Bm-NP-1, Bm-NP-1565R; Ba-NA-1, Ba-NA-1413R; Bm-M-1, Bm-M-1027R; Bm-NS-1, Bm-NS-890R). After the PCR product was confirmed to be identical to the plasmid sequence, the virus was amplified again. The viral genome variation was monitored at each generation of amplified virus. The virus from one generation amplified in SPF chicken embryos was inactivated with 4% paraformaldehyde (Sigma) at 4°C for 24 hours. The HA protein (approximately 30% of the total virus) was quantified using a protein quantification kit (Thermofisher). The inactivated virus was mixed with aluminum adjuvant (Sigma) at a 1:1 volume ratio and used to immunize two Chinese macaques (3-5 years old). Each macaque was inoculated with approximately 1 mL of the mixture containing 15 μg of HA protein and adjuvant, with 0.5 mL in each of the macaque's upper limbs. Booster immunizations were given on days 21 and 56 post-immunization, using the same dosage as the initial immunization.

[0092] 2. Determination of serum titer in macaques

[0093] (1) Peripheral blood was collected 14 days after the third immunization. The serum was separated by centrifugation (3000 rpm, 10 minutes) using standard methods. Serum titers were determined using a virus micro-neutralization assay and a hemagglutination inhibition assay. Serum titers of 1:1024 or higher were suitable for PBMC separation and flow cytometry. The HA titer was determined according to the WHO Influenza Virology Laboratory Manual. The virus titer was adjusted to 8 HA units, and then 50 μL of serum diluted 1:100 was diluted 1:2. The diluted macaque serum was added to 50 μL per well of a 96-well hemagglutination plate, followed by an equal volume of 50 μL of 8 HA units of virus. The plate was incubated at room temperature (approximately 25°C) for 30 minutes, then 1% chicken erythrocytes were added and incubated for another 30 minutes. Based on the agglutination of erythrocytes by the virus, the hemagglutination inhibition titer of the serum was determined to be 1:2560.

[0094] (2) Micro-neutralization assay to determine the neutralizing titer of macaque serum and antibodies

[0095] First, the infectivity of the PR8-H7N9 virus strain (Tissue Culture Infectious Dose 50, TCID50) was determined. Canine kidney cells (MDCK) were seeded in 96-well cell culture plates to 70-90% confluence density, and the cell density was adjusted to 1 x 10⁻⁶ cells / well. 6 Cells / well, add 1 mL of culture medium (DMEM medium + 10% fetal bovine serum), and dilute 60 μL of the virus stock solution 10-fold (0.3% BSA in phosphate buffer), with each gradient repeated in four wells. After incubating at 37°C in a 5% CO2 incubator for 48 hours, discard the virus culture medium, add 80% acetone to fix the cells for 10 minutes, wash 3 times with phosphate buffer, block with 5% defatted buffer at room temperature for 2 hours, wash 3 times with buffer, add anti-NP protein antibody (Pan Influenza A Nucleoprotein Antibody, Mouse MAb, Shenzhou Yiqiao Co., Ltd., #40205-MM16, 1:2000 dilution, 100 μL / well), and incubate at room temperature for 1 hour. After washing three times with buffer, add 100 μL of HRP-labeled anti-mouse IgG (diluted 1:5000), incubate at room temperature for 1 hour, wash six times with buffer, add 100 μL of substrate TMB / well, incubate at room temperature in the dark for 10–15 minutes for color development, add stop solution (1M sulfuric acid) 100 μL / well, and measure the OD value of each well using a microplate reader (OD450). A positive OD value is defined as 2.1 times or greater than the average OD value of the cell-negative wells (without virus, but with an equal volume of 0.3% BSA in phosphate buffer). Calculate the viral titer according to the Reed-Muench method to obtain the final TCID. 50 / 100uL is 10-3.4 / 100uL.

[0096] (3) The serum to be tested was first inactivated in a 56°C water bath for 30 minutes. The inactivated serum was then diluted three times with phosphate buffer, with three replicates for each dilution. 50 μL of the diluted serum was mixed with 50 μL of 100 TCID50 solution. 50 10-1 . 4 A 100 μL virus dilution was added to each well of MDCK cell culture. The cells were incubated at 37°C with 5% CO2 for 1 hour. The serum and virus mixture was discarded, and the cells were washed once with PBS. Then, culture medium (DMEM containing 0.3% BSA and 1 μg / mL TPCK-treated trypsin) was added. After 48 hours of culture, the cells were fixed with 80% acetone for 10 minutes, and the presence of the virus was detected using an antibody against the NP protein of influenza A virus. The neutralizing activity of serum or antibody against viral replication was calculated to be 1:5120 (based on a serum titer >1:1280) according to the Reed-Muench method.

[0097] 3. Flow cytometry sorting of HA-specific B cells

[0098] Biotin was used to label HA protein, which was then mixed with flow cytometry antibody (CD3-CD14-CD56-CD19+CD20+CD27+IgG+HA-biotin-SA-APC) to stain PBMCs. HA-specific B cells were then sorted into 96-well PCR plates (1 cell / well). RT-PCR amplification was performed using macaque-specific antibody heavy and light chain variable region amplification primers, and the amplified PCR products were sequenced. The specific method is as follows:

[0099] H7N9 (A / Anhui / 1 / 2013(H7N9)) HA protein was purchased from Sinocare Pharmaceutical Co., Ltd., Cat. No. 40103-V08H. Biotin labeling of the HA protein was performed using the EZ-Link Sulfo-NHS-biotin labeling kit (Thermofisher #21217) according to the kit's instructions. Flow cytometry antibody staining was performed as described in the literature (Zhang F et al., Phenotypic characterization of Chinese rhesus macaque plasmablasts for cloning antigen-specific monoclonal antibodies, Frontiers in Immunology, 2019, 10:2426.). CD3-pacific blue, CD14-PE, CD56-PE, CD19-PE-Cy7, CD20-PE-CF594, CD27-PerCP-5.5, IgG-APC-H7, and Streptavidi-APC were purchased from BD Biosciences. Antibody dosage should be followed according to the instruction manual, diluted 1:100 to 1:200 for staining PBMCs. Single HA-specific B cells should be sorted using a BD FACSAria II instrument, 1 cell / well. HA-specific B cells should be aliquoted into 96-well PCR plates, and 10 μL of cell lysis buffer (including reverse transcription buffer, carrier RNA, and RNase inhibitor from Reverse Transcription Kit #18080051) should be added. The plates should then be centrifuged at 1000 rpm for 1 minute. The cell plates should be flash-frozen on dry ice for 1 minute and then placed on ice to continue the reverse transcription reaction, transing RNA into cDNA.

[0100] Subsequently, the variable regions of the macaque antibody heavy and light chains were amplified using two rounds of PCR amplification with primers listed in the literature (Zhang F et al., Phenotypic characterization of Chinese rhesus macaque plasmablasts for cloning antigen-specific monoclonal antibodies, Frontiers in Immunology, 2019, 10:2426.). The primers for the first round of PCR amplification were located in the macaque antibody heavy chain (VH1a-1, VH1b-1, VH1c-1, VH2-1, VH3-1, VH4-1, VH5-1, VH6-1, VH7-1) and kappa (VK1a-1, VK1b-1, VK2-1, VK3-1, VK4 / 5-1, VK6 / 7-1), and lambda chain (VL-1, VL...). The upstream leader region of the variable region genes (VL3a-1, VL3b-1, VL3c-1, VL4a-1, VL4b-1, VL5 / 11-1, VL6-1, VL8-1, VL9-1) was used for amplification, and the downstream primers were located in the constant regions of the IgG chain (IgG-1), kappa chain (Kappa-1), and lambda chain (Lambda-1), respectively. The template used for amplification was the cDNA obtained in the previous step. The PCR products from the first round were used as templates for a second round of amplification. The primers for the second round were located inside the primers from the first round. The upstream primers were located on the heavy chain (VH1a-2, VH1b-2, VH2-2, VH3-2, VH4a-2, VH4b-2, VH5 / 7-2, VH6-2) and the kappa chain (VK1-2, VK2a-2, VK2b-2, VK3a-2, VK3b-2, VK3c-2, VK3d-2, VK4-2, VK5-2, VK6-2, VK...). The FR1 initiation positions of the 7-2) and lambda chains (VL1a-2, VL1b-2, VL1c / 5b-2, VL2a-2, VL2b-2, VL3-2, VL4a-2, VL4b-2, VL5a-2, VL5b-2, VL6-2, VL7-2, VL8-2, VL9 / 11-2, VL10-2) were used, with downstream primers located in the constant regions of the IgG chain (IgG-2), kappa chain (Kappa-2), and lambda chain (Lambda-2), respectively. After gel electrophoresis, 350–500 bp of the PCR product was excised and the recovered PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for 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: The gene sequence of the heavy chain variable region is: GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATTCACCTTCGACAGCTATACTTTCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGCGATCGTCCCTGTTGTCGGTGTACCCACCTACGCAGAGAAGTTCCAGGGCAGAGTCACGATTTCCGCGGACACGTCCAAAACTACCGCCTTCATGGAATTGACCGGCCTGACCTCTGAGGACACGGCCGTCTACTATTGTGCGAGAGATTTAGGCGACGGCTACGGTAGTAATTCAGGATACTTCGATTTCTGGGGCCAGGGCGCCCTGGTCACCGTCTCCTCA (SEQ ID NO.1), the gene sequence of the light chain variable region is: GACATTGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTAGGAGACAGAATCACCACTTGCAGAGCCAGTCAGGGTATTGGCAGCTGGTTGGCCTGGTATCAACACAAACCAGGGGAGGCCCCTAAACTCCTGATCTATAGGGCG TCCACTCTGGCAACAGGGGTCCCATCGAGGTTCAGTGGAAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATATCGCCACATATTACTGTCAACAACATGATGATTCCCCGCTCACTTTCGGCGGAGGGACCAAAGTGGAAATCAAA(SEQ ID NO.2),The amino acid sequences of the heavy chain variable region and the light chain variable region were then determined: the amino acid sequence of the heavy chain variable region is: EVQLVQSGAEVKRPGSSVKVSCKASGFTFDSYTFNWVRQAPGQGLEWMGAIVPVVGVPTYAEKFQGRVTISADTSKTTAFMELTGLTSEDTAVYYCARDLGDGYGSNSGYFDFWGQGALVTVSS (SEQ ID NO. 3), and the amino acid sequence of the light chain variable region is: DIVLTQSPSSLSASVGDRITITCRASQGIGSWLAWYQHKPGEAPKLLIYRASTLATGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQHDDSPLTFGGGTKVEIK (SEQ ID NO. 4).

[0101] Furthermore, referring to IMGT ( www.imgt.org The amino acid sequences of the antibody's six CDR regions were determined by comparing the macaque germline gene sequences from the website. The results showed that the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region were: GFTDDSYT (SEQ ID NO.5), IVPVVGVP (SEQ ID NO.6), and ARDLGDGYGSNSGYFDF (SEQ ID NO.7), respectively; while the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region were: QGIGSW (SEQ ID NO.8), RAS, and QQHDDSPLT (SEQ ID NO.9), respectively.

[0102] Example 2: Construction, expression, and purification of monoclonal antibody expression vector against H7N9 subtype influenza virus

[0103] Using the gene sequence of the heavy chain variable region (SEQ ID NO.1) as a template, and primers HF: CTAGTAGCAACTGCAACCGGTGAGGTCCAGCTGGTACAG (SEQ ID NO.10), HR: ACCGATGGGCCCTTGGTCGACTGAGGAGACGGTGACCAG (SEQ ID NO.11) as primers, PCR product 1 was obtained. Using the gene sequence of the light chain variable region (SEQ ID NO.2) as a template, and primers LF: CTAGTAGCAACTGCAACCGGTGACATTGTGCTGACCCAG (SEQ ID NO.12), LR: TGCAGCCACCGTACGCTCGAGGTTTGATTTCCACTTTGG (SEQ ID NO.11) as primers, PCR product 1 was obtained. Using primer NO.13, PCR product 2 was obtained. PCR product 1 and the corresponding human IgG1 antibody heavy chain expression vector (accession number: FJ475055) were digested with AgeI and SalI. PCR product 2 and the human kappa chain expression vector (accession number: FJ475056) were digested with AgeI and XhoI. The purified products were then transformed into Top10 competent cells using T4 DNA ligase to construct the corresponding heavy chain clones and kappa chain clones, which were sent to Shanghai Sangon Biotech for sequencing. Plasmids were extracted from the correctly sequenced clones. 0.5 μg each of the antibody heavy chain plasmid (clone) and kappa chain plasmid (clone) were dissolved in Opti-MEM medium to a final volume of 100 μL. 10 μL of PEI (1 mg / mL) transfection reagent was dissolved in 90 μL of Opti-MEM medium and incubated at room temperature for 15 minutes. Then, the plasmids and PEI were mixed, incubated at room temperature for 15 minutes, and added to 1x10 μL of Opti-MEM medium. 6 293T cells were cultured at 37°C for 5 days using a shaker at 125 rpm and 8% CO2. The supernatant was then collected by centrifugation at 4000 rpm for 30 minutes at 25°C, filtered through a 0.22 μM filter, and subsequently purified using a Protein A purification kit: first, the cells were equilibrated with 0.1 M Tris buffer at pH 7.0, then loaded with the antibody and eluted with 0.1 M Tris buffer at pH 7.0; followed by elution with 0.1 M Tris buffer at pH 8.0. The eluent was collected and dialyzed against PBS buffer. The purified antibody (named anti-H7N9 subtype influenza virus monoclonal antibody 41E10) was analyzed at 280 nm using a Nanodrop One instrument to determine the absorbance and calculate the antibody concentration. The purified antibody was then analyzed by SDS-PAGE. The SDS-PAGE results are shown below. Figure 1The results showed two bands with molecular weights of approximately 50 kDa and 25 kDa under reducing conditions, corresponding to the variable and constant regions of the heavy chain and light chain, respectively. The purified monoclonal antibody bands were consistent with expectations.

[0104] Example 3: Performance Analysis of Monoclonal Antibody 41E10 Against H7N9 Subtype Influenza Virus

[0105] 1. Detection of the binding activity of monoclonal antibody 41E10 against H7N9 subtype influenza virus to HA antigen

[0106] The binding affinity of the anti-H7N9 subtype influenza virus monoclonal antibody 41E10 to the H7N9 subtype influenza virus hemagglutinin HA protein was detected by enzyme-linked immunosorbent assay (ELISA). Specifically, the H7N9 subtype influenza virus hemagglutinin HA protein (purchased from Shenzhou Yiqiao Pharmaceutical Co., Ltd., Cat. No. 40103-V08H) was coated at 1 μg / mL into a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed 6 times with PBS-T solution (0.2% Tween-20), and 300 μL of blocking buffer (PBS, 5% skim milk powder) was added to each well, and the plate was blocked at room temperature for 1 hour. Wash the plate three times with PBS-T. Serially dilute monoclonal antibody 41E10 threefold with PBS dilution buffer (PBS, 5% FBS, 2% BSA, 1% Tween-20) (initial concentration 30 μg / mL). Add 100 μL of 41E10 dilution buffer (100 μL of PBS dilution was added to the control group) to the ELISA plate and incubate at 37°C for 1 hour. Wash the plate six times with PBS-T. Add 100 μL of horseradish peroxidase-labeled goat anti-human IgG antibody (1:5000 dilution, Abcam) and incubate at room temperature for 1 hour. Wash the plate six times with PBS-T. Add 100 μL of TMB chromogenic substrate (ThermoFisher) and incubate at room temperature in the dark for 30 minutes. Terminate the reaction with 2M H2SO4 and read the absorbance at 405 nm using a microplate reader. The results are as follows: Figure 2 As shown, the 41E10 antibody can bind to the HA protein of the H7N9 subtype influenza virus, with a binding capacity of EC. 50 It is 0.03 ug / mL.

[0107] 2. Detection of the binding affinity between the monoclonal antibody 41E10 against H7N9 subtype influenza virus and HA protein using biomembrane interferometry.

[0108] The binding kinetics of the antibody to the HA protein were detected using a FortiBio OtectRED96 instrument, as follows: The probe was pre-equilibrated by soaking it in pure water for 10 minutes, and the detection was performed at room temperature (25°C). First, the probe was immersed in sterile water for 60 seconds to obtain the baseline. Then, protein capture was performed by immersing the probe in a 10 μg / mL solution of HA protein (purchased from Shenzhou Yiqiao Pharmaceutical Co., Ltd., Cat. No. 40103-V08H) for 200 seconds. After zeroing the probe again, it was immersed in buffer (0.02% Tween-20 PBS) for 120 seconds to remove unbound HA protein. The probe was then immersed in a 66.7 nM, 3-fold serially diluted 41E10 antibody solution (diluted with PBS diluent (PBS, 5% FBS, 2% BSA, 1% Tween-20)) for 300 seconds to obtain the dynamic binding curve of antibody 41E10 and HA protein. For dissociation, the probe was placed in buffer for 300 seconds. Protein binding causes a change in the thickness of the biofilm, resulting in a relative shift in the interference light waves, which is detected by the spectrometer, forming an interference spectrum. The real-time shift of the interference spectrum shows the interaction between the protein and antibody. Based on this result, a dynamic curve for the dissociation of antibody-protein binding was established. In data analysis, the data from the sample wells were subtracted from the data from the buffer control wells, and non-specific background values ​​were also subtracted, using a 1:1 binding model. Overall curve fitting was performed on the binding constant Kon, dissociation constant Koff, and affinity constant KD at different 41E10 antibody dilutions to obtain these values. The results are as follows: Figure 3 As shown: the binding constant Kon value of the 41E10 antibody to the HA protein is 2.71+E05 1 / Ms, the dissociation constant Koff value is <1.00E-071 / s, and the affinity constant KD value is <1.00E-12M.

[0109] 3. Detection of hemagglutination inhibition and neutralizing activity of the 41E10 monoclonal antibody against H7N9 subtype influenza virus.

[0110] The hemagglutination inhibition experiment was conducted as follows: The titer of the virus solution prepared in step 1 of Example 1 was adjusted to 8 HA units. Then, the 41E10 antibody was diluted 2-fold (initial concentration 100 μg / mL, diluted with PBS diluent (PBS, 5% FBS, 2% BSA, 1% Tween-20)) and added to each well of a 96-well hemagglutination plate at a volume of 50 μL. An equal volume of 50 μL of the 8 HA unit virus solution was then added. The plate was incubated at room temperature (approximately 25°C) for 30 minutes. Then, 100 μL of 1% chicken erythrocytes was added, and the plate was incubated for another 30 minutes. The hemagglutination inhibition titer of the serum was determined based on the agglutination of erythrocytes by the virus. The hemagglutination inhibition titer of the 41E10 antibody against H7N9 virus was 0.39 μg / mL.

[0111] The neutralizing activity was measured as follows: 41E10 antibody was serially diluted three times (starting concentration 100 μg / mL, diluted with PBS diluent (PBS, 5% FBS, 2% BSA, 1% Tween-20)). Each dilution was replicated in four wells. 50 μL of the diluted antibody (50 μL of PBS diluent was added to the control group) was mixed with 100 TCID50 (10... -1.4 50 μL of a virus dilution (100 μL / mL) was added to each MDCK cell culture well. The mixture was incubated at 37°C with 5% CO2 for 1 hour. The antibody-virus mixture was discarded, and the cells were washed once with PBS. Then, culture medium (DMEM containing 0.3% BSA and 1 μg / mL TPCK-treated trypsin) was added. After 48 hours of culture, the cells were fixed with 80% acetone for 10 minutes, and the presence of the virus was detected using an antibody against the H7N9 subtype influenza virus NP protein. The neutralizing activity of the antibody against viral replication was calculated using the Reed-Muench method. Figure 4 As shown, the neutralizing activity of the 41E10 antibody against the H7N9 virus was 0.091 ug / mL.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. SEQUENCE LISTING <110> The First Affiliated Hospital of Guangzhou Medical University <120> Anti-H7 subtype influenza virus antibodies and their applications <130> <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 372 <212> DNA <213> Artificial sequence <400> 1 gaggtccagc tggtacagtc tggggctgag gtgaagaggc ctgggtcctc agtgaaggtt 60 tcctgcaagg cttctggatt caccttcgac agctatactt tcaactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagcg atcgtccctg ttgtcggtgt acccacctac 180 gcagagaagt tccagggcag agtcacgatt tccgcggaca cgtccaaaac taccgccttc 240 atggaattga ccggcctgac ctctgaggac acggccgtct actattgtgc gagagattta 300 ggcgacggct acggtagtaa ttcaggatac ttcgatttct ggggccaggg cgccctggtc 360 accgtctcct ca 372 <210> 2 <211> 321 <212> DNA <213> Artificial sequence <400> 2 gacattgtgc tgacccagtc tccatcctcc ctgtctgcgt ctgtaggaga cagaatcacc 60 atcacttgca gagccagtca gggtattggc agctggttgg cctggtatca acacaaacca 120 ggggaggccc ctaaactcct gatctatagg gcgtccactc tggcaacagg ggtcccatcg 180 aggttcagtg gaagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagatatcg ccacatatta ctgtcaacaa catgatgatt ccccgctcac tttcggcgga 300 gggaccaaag tggaaatcaa a 321 <210> 3 <211> 124 <212> PRT <213> Artificial sequence <400> 3 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Thr Phe Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Val Pro Val Val Gly Val Pro Thr Tyr Ala Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Lys Thr Thr Ala Phe 65 70 75 80 Met Glu Leu Thr Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Asp Gly Tyr Gly Ser Asn Ser Gly Tyr Phe Asp 100 105 110 Phe Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> artificial sequence <400> 4 Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Asp Asp Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 8 <212> PRT <213]] Artificial sequence <400> 5 Gly Phe Thr Phe Asp Ser Tyr Thr 1 5 <210> 6 <211> 8 <212> PRT <213]] Artificial sequence <400> 6 Ile Val Pro Val Val Gly Val Pro 1 5 <210> 7 <211> 17 <212> PRT <213]] Artificial sequence <400> 7 Ala Arg Asp Leu Gly Asp Gly Tyr Gly Ser Asn Ser Gly Tyr Phe Asp 1 5 10 15 Phe <210> 8 <211> 6 <212> PRT <213> Artificial sequence <400> 8 Gln Gly Ile Gly Ser Trp 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <400> 9 Gln Gln His Asp Asp Ser Pro Leu Thr 1 5 <210> 10 <211> 39 <212> DNA <213> Artificial sequence <400> 10 ctagtagcaa ctgcaaccgg tgaggtccag ctggtacag 39 <210> 11 <211> 39 <212> DNA <213> Artificial sequence <400> 11 accgatgggc ccttggtcga ctgaggagac ggtgaccag 39 <210> 12 <211> 39 <212> DNA <213> Artificial sequence <400> 12 ctagtagcaa ctgcaaccgg tgacattgtg ctgacccag 39 <210> 13 <211> 39 <212> DNA <213> Artificial sequence <400> 13 tgcagccacc gtacgctcga ggtttgattt ccactttgg 39

Claims

1. An antibody against H7 subtype influenza virus or an antigen-binding fragment thereof, wherein the antibody against H7 subtype influenza virus or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 in the heavy chain variable region is: GFTDDSYT (SEQ ID NO.5). The amino acid sequence of CDR2 in the heavy chain variable region is: IVPVVGVP (SEQ ID NO.6). The amino acid sequence of CDR3 in the heavy chain variable region is: ARDLGDGYGSNSGYFDF (SEQ ID NO.7). The variable region of the light chain includes CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 in the light chain variable region is: QGIGSW (SEQ ID NO.8). The amino acid sequence of CDR2 in the light chain variable region is: RAS; The amino acid sequence of CDR3 in the light chain variable region is: QQHDDSPLT (SEQ ID NO.9).

2. The anti-H7 subtype influenza virus antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody against H7 subtype influenza virus or its antigen-binding fragment is: EVQLVQSGAEVKRPGSSVKVSCKASGFTFDSYTFNWVRQAPGQGLEWMGAIVPVVGVPTYAEKFQGRVTISADTSKTTAFMELTGLTSEDTAVYYCARDLGDGYGSNSGYFDFWGQGALVTVSS (SEQ ID NO.3); The amino acid sequence of the light chain variable region of the antibody against H7 subtype influenza virus or its antigen-binding fragment is: DIVLTQSPSSLSASVGDRITITCRASQGIGSWLAWYQHKPGEAPKLLIYRASTLATGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQHDDSPLTFGGGTKVEIK (SEQ ID NO.4).

3. A nucleic acid molecule encoding the antibody against H7 subtype influenza virus as described in claim 1 or 2, or its antigen-binding fragment.

4. An expression cassette, recombinant vector, or transgenic cell line containing the nucleic acid molecule of claim 3.

5. A conjugate comprising: the anti-H7 subtype influenza virus antibody as described in claim 1 or 2, or its antigen-binding fragment and conjugation portion, The coupling part is a detection marker.

6. Any one of (1) to (3) can be used in any one of (4) to (5); (1) The anti-H7 subtype influenza virus antibody or its antigen-binding fragment as described in claim 1 or 2; (2) The nucleic acid molecule according to claim 3; (3) The expression cassette, recombinant vector, or transgenic cell line as described in claim 4; (4) Prepare products for detecting H7 subtype influenza virus and / or H7 subtype influenza virus HA protein; The product is at least one of reagents, test plates, and kits; (5) Prepare drugs for the prevention and / or treatment of diseases caused by H7 subtype influenza virus.

7. The use of the conjugate of claim 5 in the preparation of products for detecting H7 subtype influenza virus and / or the HA protein of H7 subtype influenza virus; The product is at least one of reagents, test plates, and kits.

8. A product comprising: at least one of the following: an antibody against H7 subtype influenza virus as described in claim 1 or 2 or an antigen-binding fragment thereof, and a conjugate as described in claim 5; The product is at least one of reagents, test plates, and kits.

9. A drug comprising at least one of (1) to (3); (1) The anti-H7 subtype influenza virus antibody or its antigen-binding fragment as described in claim 1 or 2; (2) The nucleic acid molecule according to claim 3; (3) The expression cassette, recombinant vector or transgenic cell line as described in claim 4.

10. The medicament according to claim 9, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

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