Monoclonal antibodies against neuraminidase and uses thereof

Abstract

The application discloses an anti-neuraminidase human monoclonal antibody obtained through multiple rounds of in-vitro screening and recombination modification and application thereof, and the application is used for preparing products for preventing, detecting or treating influenza viruses. The monoclonal antibody provided by the application has significant neuraminidase inhibiting activity and broad-spectrum influenza virus antigen binding capacity, and has excellent prevention and treatment effects on various seasonal influenza viruses, thereby providing a new technical option for resisting influenza infection and having important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to monoclonal antibodies against neuraminidase and their applications. Background Technology

[0002] The influenza virus has two glycoproteins on its surface—hemagglutinin (HA) and neuraminidase (NA)—which work synergistically to promote the viral infection process.

[0003] Hemagglutinin (HA) binds to sialic acid (SA) receptors on the cell surface and plays a crucial role in viral adsorption and membrane fusion, making it a primary target for influenza virus infection prevention. Neuraminidase (NA), also known as sialic acid glycoprotein, is a surface glycoprotein of the influenza virus that cleaves sialic acid, promoting the release of viral particles from infected cells. Simultaneously, neuraminidase can assist the virus in penetrating the mucus layer by cleaving decoy receptors in mucin.

[0004] Neuraminidase is also an important target for antiviral therapy against influenza, and both neuraminidase inhibitors and antibodies have been found to have protective effects. Neuraminidase inhibitors are widely used in antiviral therapy, and antibodies targeting neuraminidase can inhibit neuraminidase activity by inhibiting the active site or binding to epitopes surrounding the active site. Currently reported neuraminidase antibodies 8H2, 6C6, and 3D4 primarily target the N1 subtype, while antibodies CR14011, CR14012, and CR14017 primarily target influenza B virus neuraminidase. However, although several monoclonal antibodies against human neuraminidase have been discovered in recent years, the understanding of their targeted neuraminidase epitopes is still incomplete. Conserved epitopes of the neuraminidase protein cannot be fully elucidated, especially those with broad-spectrum neuraminidase inhibitory activity. Furthermore, the number of existing specific broad-spectrum antibodies against neuraminidase is limited, and they cannot simultaneously target both influenza A and B viruses. The neutralization mechanism of neuraminidase-targeting antibodies is not yet fully understood. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a monoclonal antibody against neuraminidase and its applications.

[0006] The first objective of this invention is to provide a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof.

[0007] A second object of the present invention is to provide the use of the monoclonal antibody or its antigen-binding fragment in the preparation of products for the prevention and / or treatment of influenza viruses.

[0008] A third objective of this invention is to provide a biomaterial.

[0009] A fourth object of the present invention is to provide the use of the said biological material in the preparation of monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

[0010] A fifth object of the present invention is to provide a method for preparing monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

[0011] A sixth objective of this invention is to provide a medicine for the prevention and / or treatment of influenza viruses.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] This invention provides F6 and FF34 antibodies with neuraminidase inhibitory activity, which can strongly bind to the neuraminidase of influenza A and B viruses (A / Brevig Mission / 1 / 1918, A / California / 04 / 2009, A / India / Pun151245 / 2015, A / Switzerland / 9715293 / 2013, A / Red knot / Delaware Bay / 310 / 2016, B / Colorado / 06 / 2017) and exhibit excellent anti-infective and therapeutic efficacy.

[0014] A monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising fragment (1) or fragment (2);

[0015] The fragment (1) comprises a heavy chain variable region (1) and a light chain variable region (1); the heavy chain variable region (1) comprises CDR-F6-H1 to CDR-F6-H3, wherein the amino acid sequence of CDR-F6-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-F6-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-F6-H3 is shown in SEQ ID NO.4; the light chain variable region (1) comprises CDR-F6-L1 to CDR-F6-L3, wherein the amino acid sequence of CDR-F6-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-F6-L2 is GAS, and the amino acid sequence of CDR-F6-L3 is shown in SEQ ID NO.11;

[0016] The fragment (2) comprises a heavy chain variable region (2) and a light chain variable region (2); the heavy chain variable region (2) comprises CDR-FF34-H1 to CDR-FF34-H3, wherein the amino acid sequence of CDR-FF34-H1 is shown in SEQ ID NO.17, the amino acid sequence of CDR-FF34-H2 is shown in SEQ ID NO.18, and the amino acid sequence of CDR-FF34-H3 is shown in SEQ ID NO.19; the light chain variable region (2) comprises light chain variable regions of CDR-FF34-L1 to CDR-FF34-L3, wherein the amino acid sequence of CDR-FF34-L1 is shown in SEQ ID NO.25, the amino acid sequence of CDR-FF34-L2 is AAS, and the amino acid sequence of CDR-FF34-L3 is shown in SEQ ID NO.26.

[0017] Preferably, the heavy chain variable region (1) further comprises FR-F6-H1 to FR-F6-H4, wherein the amino acid sequence of FR-F6-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-F6-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-F6-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-F6-H4 is shown in SEQ ID NO.8; the light chain variable region (1) further comprises FR-F6-L1 to FR-F6-L4, wherein the amino acid sequence of FR-F6-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-F6-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-F6-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-F6-L4 is shown in SEQ ID NO.15.

[0018] More preferably, the amino acid sequence of the heavy chain variable region (1) is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region (1) is as shown in SEQ ID NO.9.

[0019] Preferably, the heavy chain variable region (2) further comprises FR-FF34-H1 to FR-FF34-H4, wherein the amino acid sequence of FR-FF34-H1 is shown in SEQ ID NO.20, the amino acid sequence of FR-FF34-H2 is shown in SEQ ID NO.21, the amino acid sequence of FR-FF34-H3 is shown in SEQ ID NO.22, and the amino acid sequence of FR-FF34-H4 is shown in SEQ ID NO.23; the light chain variable region (2) further comprises FR-FF34-L1 to FR-FF34-L4, wherein the amino acid sequence of FR-FF34-L1 is shown in SEQ ID NO.27, the amino acid sequence of FR-FF34-L2 is shown in SEQ ID NO.28, the amino acid sequence of FR-FF34-L3 is shown in SEQ ID NO.29, and the amino acid sequence of FR-FF34-L4 is shown in SEQ ID NO.30.

[0020] More preferably, the amino acid sequence of the heavy chain variable region (2) is at least 95% identical to that of SEQ ID NO.16; and the amino acid sequence of the light chain variable region (2) is at least 95% identical to that of SEQ ID NO.24.

[0021] More preferably, the amino acid sequence of the heavy chain variable region (2) is shown in SEQ ID NO.16; and the amino acid sequence of the light chain variable region (2) is shown in SEQ ID NO.24.

[0022] Preferably, the monoclonal antibody or its antigen-binding fragment further comprises an Fc segment. The present invention does not have a particular limitation on the species origin of the Fc segment, including but not limited to human Fc segments, mouse Fc segments, rabbit Fc segments, or biologically acceptable modifications or mutations thereof.

[0023] More preferably, the Fc segment is a human Fc segment or a biologically acceptable modification or mutation thereof.

[0024] Preferably, the fragment (1) further comprises a heavy chain constant region (1) and a light chain constant region (1), the amino acid sequence of the heavy chain constant region (1) being shown in SEQ ID NO.31 and the amino acid sequence of the light chain constant region (1) being shown in SEQ ID NO.32.

[0025] Preferably, the fragment (2) further comprises a heavy chain constant region (12) and a light chain constant region (2), the amino acid sequence of the heavy chain constant region (2) being shown in SEQ ID NO.31 and the amino acid sequence of the light chain constant region (2) being shown in SEQ ID NO.32.

[0026] The use of any of the monoclonal antibodies or their antigen-binding fragments in the preparation of products for the prevention and / or treatment of influenza viruses should also be within the scope of protection of this invention.

[0027] A biological material, which is any one of the following (1) to (3):

[0028] (1) A nucleic acid molecule encoding any of the monoclonal antibodies or their antigen-binding fragments;

[0029] (2) A carrier containing the nucleic acid molecules described in (1);

[0030] (3) Host cells containing the vector in (2).

[0031] This invention does not have any special limitations on the source and type of host cells. Prokaryotic and eukaryotic cells that can conventionally carry the vector, such as Escherichia coli, HEK293 cells, and CHO cells, can all achieve the purpose of this invention.

[0032] Preferably, the host cell in (3) is a mammalian cell.

[0033] More preferably, the host cell described in (3) is a 293T cell.

[0034] The application of the biomaterials in the preparation of monoclonal antibodies against neuraminidase or their antigen-binding fragments should also be within the scope of protection of this invention.

[0035] The application of the biomaterials in the preparation of products for the prevention and / or treatment of influenza viruses should also be within the scope of protection of this invention.

[0036] A method for preparing a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, comprising culturing the host cells in (3) of the biological material under conditions that allow expression of the monoclonal antibody or the antigen-binding fragment thereof, and recovering the monoclonal antibody or the antigen-binding fragment thereof from the cultured host cell culture.

[0037] This invention does not have any special limitations on the source and type of host cells. Prokaryotic and eukaryotic cells that can conventionally carry the vector, such as Escherichia coli, HEK293 cells, and CHO cells, can all achieve the purpose of this invention.

[0038] Preferably, the host cell is a mammalian cell.

[0039] More preferably, the host cell described herein is a 293T cell.

[0040] An medicament for the prevention and / or treatment of influenza viruses, comprising any of the said monoclonal antibodies or their antigen-binding fragments, and a pharmaceutically acceptable carrier and / or excipient.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The monoclonal antibody provided by this invention has significant neuraminidase inhibitory activity and broad-spectrum anti-influenza virus antigen binding ability. It has excellent preventive and therapeutic effects against a variety of seasonal influenza viruses, providing a new technical option for combating influenza infection and has important application value. Attached Figure Description

[0043] Figure 1 The results of SDS-PAGE assays for F6 and FF34 antibodies are shown.

[0044] Figure 2 The antigen binding of neuraminidase monoclonal antibodies was detected by ELISA; A is F6 antibody; B is FF34 antibody.

[0045] Figure 3 The enzyme activity inhibitory activity of the neuraminidase monoclonal antibody was detected in the MUNANA assay. The target antigens A to H were N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / California / 4 / 2009(H1N1), N1-AVietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), NAB-B / Colorado / 06 / 2017(Victoria), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), N4-A / Red knot / Delaware Bay / 310 / 2016(H10N4) and N9-A / Shanghai / 02 / 2013(H7N9). The negative control was DPBST containing 1 v / v% BSA.

[0046] Figure 4The inhibitory activity of the neuraminidase monoclonal antibody was detected by ELISA. The target antigens A to H were N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / California / 4 / 2009(H1N1), N1-AVietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), NAB-B / Colorado / 06 / 2017(Victoria), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), N4-A / Red knot / Delaware Bay / 310 / 2016(H10N4) and N9-A / Shanghai / 02 / 2013(H7N9). The negative control was DPBST containing 1 v / v% BSA.

[0047] Figure 5 To detect the protective efficacy of neuraminidase monoclonal antibodies in animal experiments; A shows mouse body weight monitoring under prophylactic protection against A / California / 4 / 2009(H1N1) antibody; B shows mouse survival curves under prophylactic protection against A / California / 4 / 2009(H1N1) antibody; C shows mouse body weight monitoring under therapeutic protection against A / California / 4 / 2009(H1N1) antibody; D shows the protective efficacy against A / California / 4 / 2009(H1N1) antibody. E represents the survival curve of mice under antibody therapeutic protection; E represents the weight monitoring of mice under antibody prophylactic protection against A / Guizhou / 54 / 1989(H3N2); F represents the survival curve of mice under antibody prophylactic protection against A / Guizhou / 54 / 1989(H3N2); G represents the weight monitoring of mice under antibody therapeutic protection against A / Guizhou / 54 / 1989(H3N2); H represents the survival curve of mice under antibody therapeutic protection against A / Guizhou / 54 / 1989(H3N2). Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Preparation of neuraminidase monoclonal antibody

[0050] 1. Isolation and purification of PBMCs

[0051] Ten volunteers infected with seasonal influenza in 2019–2020 were recruited, and blood samples were collected. Personal dry cell (PBMCs) were isolated using density gradient centrifugation. The obtained PBMCs were sorted by flow cytometry. PB-CD19+ (Biolegend, 302232) and APC-CD27+ flow cytometry antibodies (Biolegend, 302810) were added at a ratio of 1:100, and PE-CD38+ flow cytometry antibody (Biolegend, 303516) was added at a ratio of 1:200. The cells were incubated at 4°C in the dark for 30 min, then 2 mL of PBS (containing 0.2 v / v % BSA) was added, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 2 mL of PBS (containing 0.2 v / v % BSA). Cells positive for all three antibodies (i.e., CD19) were then centrifuged. + CD27 + CD38 high Individual plasmablasts B cells were sorted into 96-well plates.

[0052] 2. Nested PCR

[0053] Place the 96-well plate on ice and perform single-cell RT-PCR. Prepare the RT-PCR premix according to the instructions of the Qiagen OneStep RT-PCR Kit. Add 15 μL of the premix to each well, carefully cap the plate, and perform the reaction using the procedure recommended in the OneStep protocol.

[0054] Nested PCR was performed after the reaction was completed. The reaction system is shown in Table 1. The first round of nested PCR program is shown in Table 2, and the second round of nested PCR program is shown in Table 3. The primers used in both rounds of nested PCR were from Table 1 of the existing technology “Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen (DOI:10.1038 / nprot.2009.3)”. The first round used “RT-PCR” primers, and the second round used “Nested PCR” primers.

[0055] Table 1 Nested PCR Reaction System

[0056]

[0057] Table 2. First-round nested PCR program

[0058]

[0059] Table 3. Second round nested PCR program

[0060]

[0061] After the second round of nested PCR, variable region fragments of the heavy and light chains of two neuraminidase monoclonal antibodies (F6 antibody and FF34 antibody) were obtained.

[0062] According to the IMGT method for defining domains, the specific domain divisions of the F6 antibody and the FF34 antibody are as follows:

[0063] The amino acid sequence of the heavy chain variable region of the F6 antibody is: EVQLVESGGGLVQPGGSLRLSCAAS GFSFTTYE MNWVRQAPGKGLEWVSH ISSRGLVI YYADSVKGRFTMSRDTAKNSLYLQMDSLTVADTAVYYC ARHYFDRDWGYSGM DL W GQGTTVTVSS (SEQ ID NO.1); wherein, the underlined parts are, in order, the complementary determination regions CDR-F6-H1 (SEQ ID NO.2), CDR-F6-H2 (SEQ ID NO.3) and CDR-F6-H3 (SEQ ID NO.4), and the remaining parts are, in order, the skeleton regions FR-F6-H1 (SEQ ID NO.5), FR-F6-H2 (SEQ ID NO.6), FR-F6-H3 (SEQ ID NO.7) and FR-F6-H4 (SEQ ID NO.8).

[0064] The amino acid sequence of the light chain variable region of the F6 antibody is: EVVLTQSPGTLSLSPGERATLSCRAS QSLGTNY LAWYQHKPGQSPRLLID GAS TRAIGI PDRFSAGSGTDFTLTVSRLEPEDFAVYYC QHYGNPYT FGQGTKLEIK (SEQ ID NO. 9); wherein, the underlined parts are, in order, the complementary determination regions CDR-F6-L1 (SEQ ID NO. 10), CDR-F6-L2 and CDR-F6-L3 (SEQ ID NO. 11), and the remaining parts are, in order, the skeleton regions FR-F6-L1 (SEQ ID NO. 12), FR-F6-L2 (SEQ ID NO. 13), FR-F6-L3 (SEQ ID NO. 14) and FR-F6-L4 (SEQ ID NO. 15).

[0065] The amino acid sequence of the heavy chain variable region of the FF34 antibody is: EVQLVESGGGLVEPGGSLRLSCAAS GLTFSTYEMNWVRQAPGKGLEWISH ISSSGFAI Y YADSVRGRFTISRDIAKQSLDLQMNSLRAEDTAVYYC ARDYYNRDLGYS GMDV WGQ GTTVTVSS (SEQ ID NO.16); wherein, the underlined parts are, in order, the complementary determination regions CDR-FF34-H1 (SEQ ID NO.17), CDR-FF34-H2 (SEQ ID NO.18) and CDR-FF34-H3 (SEQ ID NO.19), and the remaining parts are, in order, the skeleton regions FR-FF34-H1 (SEQ ID NO.20), FR-FF34-H2 (SEQ ID NO.21), FR-FF34-H3 (SEQ ID NO.22) and FR-FF34-H4 (SEQ ID NO.23).

[0066] The amino acid sequence of the light chain variable region of the FF34 antibody is: EIVLTQSPGTLSLSPGERATLSCRAS QYISSMN LAWYQQKPGQAPRLLIS AAS SRATGIP DRFSGSGSGTDFTLTISGLEPEDFAVYYC QKYGSPYT FGQGTKVEIK (SEQ ID NO.24); wherein, the underlined part is the complementary determination region CDR-FF34-L1 (SEQ ID NO.25), CDR-FF34-L2 and CDR-FF34-L3 (SEQ ID NO.26) in sequence, and the remaining part is the skeleton region FR-FF34-L1 (SEQ ID NO.27), FR-FF34-L2 (SEQ ID NO.28), FR-FF34-L3 (SEQ ID NO.29) and FR-FF34-L4 (SEQ ID NO.30) in sequence.

[0067] 3. Expression, purification, and identification of neuraminidase monoclonal antibodies

[0068] (1) Construction of recombinant plasmids

[0069] The coding sequence of the variable region (SEQ ID NO.1) of the heavy chain of the F6 antibody was inserted into the pcDNA3.1 vector expressing the constant region (SEQ ID NO.31) of the heavy chain to obtain the heavy chain expression plasmid of the F6 antibody, denoted as pcDNA3.1-F6-H.

[0070] The coding sequence of the light chain variable region (SEQ ID NO. 9) of the F6 antibody was inserted into the pcDNA3.1 vector expressing the light chain constant region (SEQ ID NO. 32) to obtain the light chain expression plasmid of the F6 antibody, denoted as pcDNA3.1-F6-L.

[0071] The coding sequence of the heavy chain variable region (SEQ ID NO.16) of the FF34 antibody was inserted into the pcDNA3.1 vector expressing the heavy chain constant region (SEQ ID NO.31) to obtain the heavy chain expression plasmid of the FF34 antibody, denoted as pcDNA3.1-FF34-H.

[0072] The coding sequence of the light chain variable region (SEQ ID NO.24) of the FF34 antibody was inserted into the pcDNA3.1 vector expressing the light chain constant region (SEQ ID NO.32) to obtain the light chain expression plasmid of the FF34 antibody, denoted as pcDNA3.1-FF34-L.

[0073] (2) Antibody expression

[0074] Antibody expression was achieved by transiently transfecting 293T cells with plasmids. Before transfection, ensure that the 293T cells grow uniformly in the cell culture dish and that the number of passages does not exceed 30, otherwise the cells will not effectively express antibodies. The transfection steps are as follows: Aspirate 2.5 mL of antibody-free and serum-free DMEM into a 15 mL centrifuge tube. First, add the heavy chain expression plasmids and corresponding light chain expression plasmids of the antibodies constructed in the previous step, then add 100 μL of polyethyleneimine (PEI) to the centrifuge tube, vortex to mix, and incubate at room temperature for 15 min. Slowly add the solution after standing for 15 min to the culture medium of 293T cells and mix well. Incubate overnight at 37°C, then change the medium and incubate for another 5 days.

[0075] (3) Purification

[0076] Add 150 μL of protein A agarose beads to a new 50 mL centrifuge tube, add 1×PBS to 50 mL, centrifuge at 3000 rpm for 10 min at 4°C, and remove the PBS using a vacuum pump. Collect the cell supernatant from the culture dish into a 50 mL centrifuge tube, centrifuge at 3000 rpm for 10 min at 4°C, then pour the supernatant into the 50 mL centrifuge tube containing the agarose beads, add 1×PBS to fill the tube to 50 mL, and incubate overnight at 4°C with a shaker. The next day, remove from the refrigerator and centrifuge at 3000 rpm for 10 min at 4°C. Remove the supernatant using a vacuum pump; at this point, the protein is bound to the agarose beads and remains at the bottom of the tube. Elute with 1×PBS twice to remove other proteins. After removing the PBS, add 2.5 mL of 0.1 M Glycine-HCl (pH 2.7), incubate at room temperature with a horizontal shaker for 10 min, and then centrifuge; at this point, the protein is in the supernatant. Transfer the supernatant to a new 15 mL centrifuge tube, add 120 μL of 1M Tris-HCl (pH=9) to adjust the pH to 7.0, then transfer the entire liquid to a 30 kDa ultrafiltration tube and centrifuge at 3000 rpm and 4 °C for 10 min. Discard the centrifugation waste liquid. Add PBS to the ultrafiltration tube three times to replace the supernatant, and finally concentrate to a volume of 200 μL to obtain the monoclonal antibody. Transfer it to a 1.5 mL centrifuge tube and store at 4 °C for later use.

[0077] (4) Identification

[0078] Take 8 μL of purified monoclonal antibody, add 2 μL of 5X reducing loading buffer and mix well. Boil the sample in a 100℃ metal bath for 5 min to break disulfide bonds. Perform SDS-PAGE analysis. The results are as follows: Figure 1 As shown, under reducing conditions, the disulfide bonds between the antibody heavy and light chains are broken, resulting in antibody heavy chain bands of approximately 50 kDa and antibody light chain bands of approximately 25 kDa. This indicates that monoclonal antibodies F6 and FF34 were successfully expressed in 293T cells and secreted into the cell supernatant. Thus, two neuraminidase monoclonal antibodies were successfully prepared, denoted as F6 antibody and FF34 antibody.

[0079] Example 2: Antigen binding of neuraminidase monoclonal antibody

[0080] 1. Antigen preparation and purification

[0081] Referring to the "antibody expression" procedure in Example 1, antibodies from A / Califomia / 04 / 2009 (H1N1), A / India / Pun151245 / 2015 (H1N1), A / Brevig Mission / 1 / 1918 (H1N1), A / Switzerland / 9715293 / 2013 (H3N2), A / Kansas / 14 / 2017 (H3N2), A / Japan / 305 / 1957 (H2N2), A / Swine / Missouri / 2124514 / 2006 (H2N3), and A / Red knot / Delaware were expressed. The extracellular domain fragments of the NA from Bay / 310 / 2016(H10N4), B / Jiangsu01 / 2015(Victoria), B / Colorado / 06 / 2017(Victoria), and B / Phuket / 3073 / 2013-like(Yamagata) were recombined into the pcDNA3.1 vector to construct recombinant expression plasmids for each NA. Expression was achieved by transient transfection of 293T cells with the plasmids. Cell supernatant was collected, filtered through a 0.22 μm filter membrane, and then purified using a Ni+ Sepharose High-Performance chromatography column to obtain 11 proteins, which were used as coating antigens and are designated as follows: N1-A / Califomia / 04 / 2009(H1N1), N1-A / India / Pun151245 / 2015(H1N1), N1-A / BrevigMission / 1 / 1918(H1N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N2-A / Kansas / 14 / 2017(H3N2), N2-A / Japan / 305 / 1957(H2N2), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), N4-A / Red knot / Delaware Bay / 310 / 2016(H10N4), NAB-B / Jiangsu01 / 2015(Victoria), NAB-B / Colorado / 06 / 2017(Victoria) and NAB-B / Phuket / 3073 / 2013-like(Yamagata).

[0082] 2. ELISA experiment

[0083] Add 2 μg / mL of each coating antigen, 50 μL per well, to each well of a 96-well ELISA plate. Cap the plate and incubate overnight at 4°C in a humidified chamber. Remove the plate after overnight coating, wash 6 times with PBST, and blot dry. Prepare ELISA blocking buffer, add 150 μL to each well for blocking, and incubate at 37°C in a humidified chamber for 1 hour. After blocking, wash 6 times with PBST and blot dry. Add 90 μL of 10 μg / mL of the target antibody (F6 or FF34 antibody) to row A of the dilution plate, and 60 μL of PBS to the remaining rows. Then, sequentially transfer 30 μL from A to G to the next row, dilute, and transfer 50 μL to each well of the ELISA plate. Incubate at 37°C in a humidified chamber for 1 hour. After incubation, wash 6 times with PBST and discard any remaining liquid in the ELISA plate. Add 75 μL of goat anti-human IgG-HRP secondary antibody diluted 1:2000 to each well and incubate in a humidified chamber at 37°C for 1 h. Wash 6 times with PBST, discard any remaining liquid in the ELISA plate, and add 100 μL of chromogenic solution to each well from low to high concentration. Incubate in the dark and record the reaction time. Read the OD values ​​in the ELISA reader at 5 min, 10 min, and 15 min of reaction.

[0084] like Figure 2 As shown in Figure A, antibody F6 can bind to multiple antigens, including N1-A / California / 04 / 2009(H1N1), N1-A / India / Pun1512245 / 2015, N1-A / BrevigMission / 1 / 1918(H1N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N2 / Kansas / 14 / 2017(H3N2), N2-A / Japan / 305 / 1957(H2N2), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), N4-A / Red knot / Delaware Bay / 310 / 2016(H10N4), and NAB-B / Jiangsu / 01 / 2015(Victoria). Figure 2As shown in Figure B, antibody FF34 can bind to multiple antigens, including N1-A / California / 04 / 2009 (H1N1), N1-A / India / Pun1512245 / 2015, N1-A / BrevigMission / 1 / 1918 (H1N1), N2-A / Switzerland / 9715293 / 2013 (H3N2), N2-A / Japan / 305 / 1957 (H2N2), N3-A / Swine / Missouri / 2124514 / 2006 (H2N3), NAB-B / Colorado / 06 / 2017 (Victoria), and NAB-B / Phuket / 3073 / 2013-like (Yamagata). These results indicate that both F6 and FF34 antibodies possess broad-spectrum influenza virus antigen binding capabilities.

[0085] Example 3: Enzyme activity inhibition ability of neuraminidase monoclonal antibody

[0086] 1. Antigen preparation and purification

[0087] Following the "antibody expression" procedure in Example 1, extracellular domain fragments of NA from A / Califomia / 04 / 2009(H1N1), A / Brevig Mission / 1 / 1918(H1N1), A / Vietnam / 1203 / 2004(H5N1), A / Switzerland / 9715293 / 2013(H3N2), A / Swine / Missouri / 2124514 / 2006(H2N3), A / Red knot / Delaware Bay / 310 / 2016(H10N4), B / Colorado / 06 / 2017(Victoria), and A / Shanghai / 02 / 2013(H7N9) were recombined into the pcDNA3.1 vector to construct recombinant expression plasmids for each NA. Expression was achieved by transiently transfecting 293T cells with the plasmids. Cell supernatant was collected, filtered through a 0.22 μm filter membrane, and then purified using a Ni+ Sepharose High-Performance chromatography column to obtain 11 proteins, which were designated as target antigens, and are listed as follows: N1-A / Califomia / 04 / 2009(H1N1), N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / Vietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), N4-A / Red knot / Delaware Bay / 310 / 2016(H10N4), NAB-B / Colorado / 06 / 2017(Victoria) and N9-A / Shanghai / 02 / 2013(H7N9).

[0088] 2. The MUNANA assay was used to identify the effect of antibodies against the active site of nuclease (NA).

[0089] The working concentration of each target antigen was determined as follows: 90 μL of each target antigen (30 μg / mL) was added to row A, and 60 μL of DPBST (containing 1 v / v% BSA) was added to the remaining rows. 30 μL of each target antigen was transferred sequentially from row A to row G. After dilution, 50 μL of each antigen was transferred to a 96-well plate. 50 μL of 33.3 mM MUNANA substrate was added, and the plate was incubated at 37°C in the dark for 1 h. After incubation, 100 μL of stop solution (11 mL anhydrous ethanol + 2.225 mL 0.5 M NaOH) was added, and the fluorescence signal was read to calculate the working concentration of each target antigen.

[0090] The enzyme activity inhibition capacity of F6 antibody and FF34 antibody was determined as follows: 120 μL of the test antibody (F6 antibody or FF34 antibody) at 32 μg / mL was added to row A, and 60 μL of PBS was added to the remaining rows. Then, 60 μL of each antibody was transferred sequentially from A to G to the next row, followed by the addition of an equal volume of antigen. The mixture was incubated at 37°C for 45 min. After incubation, 100 μL of the solution was transferred to each well of a 96-well plate, and 50 μL of 33.3 mM MUNANA substrate was added to each well. The plate was incubated for 1 h, and 100 μL of stop solution was added to each well. The fluorescence signal was read, and the enzyme activity inhibition capacity of each test antibody was calculated.

[0091] Experimental results are as follows Figure 3 As shown in A to H, the F6 antibody is effective against N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / California / 2009(H1N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N1-A / Vietnam / 1203 / 2004(H5N1), N3-A / Swine / Missouri / 2124514 / 2006(H3N2) and N4-A / Red knot / Delaware. The Bay / 310 / 2106(H10N4) antigen exhibits enzyme activity inhibition; the FF34 antibody exhibits enzyme activity inhibition against N1-A / BrevigMission / 1 / 1918(H1N1), N1-A / California / 2009(H1N1), N2-A / Switzerland / 9715293 / 2013(H3N2) and N3-A / Swine / Missouri / 2124514 / 2006(H3N2).

[0092] 3. Ella assay to identify antibody binding.

[0093] The working concentration of each target antigen was determined as follows: 25 μg / mL fetoglobulin was added to 50 μL per well of a 96-well ELISA plate and incubated at 4°C for 24 h. The plate was then washed three times with PBST and blotted dry. 90 μL of each target antigen (initial concentration 30 μg / mL) was added to row A, and 60 μL of DPBST (containing 1 v / v% BSA) was added to the remaining rows. 30 μL of each diluted antigen was transferred sequentially from row A to row G and then transferred to 50 μL per well of the ELISA plate coated with fetoglobulin. The plate was incubated at 37°C for 20 h. After incubation, the plate was washed six times with PBST and blotted dry. HRP-labeled peanut lectin was diluted 1:400 and added to 100 μL per well, and incubated in the dark for 2 h. Wash the plate 9 times with PBST, discard the residual liquid in the plate, add 100 μL of chromogenic solution to each well, react in the dark and record the time. Read the OD value in the microplate reader at 5 min, 10 min and 15 min respectively, and calculate the working concentration of each target antigen.

[0094] The enzyme activity inhibition ability of F6 and FF34 antibodies was determined as follows: 120 μL of the test antibody (F6 or FF34) at 32 μg / mL was added to row A, and 60 μL of DPBST (containing 1 v / v% BSA) was added to the remaining rows. Then, 60 μL of each of the rows from A to G was transferred to the next row, followed by the addition of an equal volume of antigen. The mixture was incubated at 37°C for 2 h. After incubation, 100 μL of the antigen was transferred to each well of an ELISA plate coated with fetal globulin. The plate was incubated at 37°C for 20 h, washed 6 times with PBST, and dried. HRP-labeled peanut lectin was diluted 1:400, and 100 μL was added to each well. The plate was incubated in the dark for 2 h. Wash the plate 9 times with PBST, discard the residual liquid in the plate, add 100 μL of colorimetric solution to each well, react in the dark and record the time. Read the OD value in the microplate reader at 5 min, 10 min and 15 min respectively, and calculate the enzyme activity inhibition ability of each antibody to be tested.

[0095] Experimental results are as follows Figure 4As shown in A to H, antibody F6 inhibits the enzyme activity of N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / California / 2009(H1N1), N1-A / Vietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N3-A / Swine / Missouri / 2124514 / 2006(H3N2) and N4-A / Red knot / Delaware Bay / 310 / 2106(H10N4); antibody FF34 inhibits the enzyme activity of N1-A / Brevig Mission / 1 / 1918(H1N1), N1-A / California / 2009(H1N1), N1-A / Vietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N3-A / Swine / Missouri / 2124514 / 2006(H3N2) and N4-A / Red knot / Delaware Bay / 310 / 2106(H10N4); The following enzymes exhibit inhibitory activity: Mission / 1 / 1918(H1N1), N1-A / California / 2009(H1N1), N1-A / Vietnam / 1203 / 2004(H5N1), N2-A / Switzerland / 9715293 / 2013(H3N2), N3-A / Swine / Missouri / 2124514 / 2006(H2N3), and NAB-B / Colorado / 06 / 2017(Victoria).

[0096] The above results indicate that both F6 and FF34 antibodies can inhibit the activity of neuraminidase in various types of influenza viruses.

[0097] Example 4: Protective ability of neuraminidase monoclonal antibody

[0098] 1. Antibody prevention experiment

[0099] BALB / c mice aged 6–8 weeks were used as experimental animals. One day before challenge, F6 antibody, FF34 antibody, or PBS were injected intraperitoneally into each mouse at a dose of 10 mg / kg (antibody / body weight). Twenty-four hours later, the mice were challenged with 5×LD50 influenza virus (A / California / 04 / 2009(H1N1) or A / Guizhou / 54 / 1989-H3N2). The body weight of the mice was monitored for 14 consecutive days after challenge. Mice whose body weight was less than 75% of their initial body weight were euthanized, and the survival rate of the mice was calculated.

[0100] like Figure 5 As shown in A-B and E-F, mice infected with A / California / 04 / 2009(H1N1) or A / Guizhou / 54 / 1989-H3N2 showed good protective effects from both FF34 and F6 antibodies, with no significant decrease in body weight and all mice surviving.

[0101] 2. Antibody therapy experiment

[0102] BALB / c mice aged 6–8 weeks were used as experimental animals. Each mouse was challenged with 5×LD50 influenza virus (A / California / 04 / 2009(H1N1) or A / Guizhou / 54 / 1989-H3N2). 24 hours after challenge, mice were injected intraperitoneally with F6 antibody, FF34 antibody, or PBS. The dosage was 10 mg / kg (antibody / body weight) per mouse. The weight of the mice was monitored for 14 consecutive days after treatment. Mice whose weight was less than 75% of their initial weight were euthanized.

[0103] like Figure 5 As shown in C-D and G-H, mice infected with A / California / 04 / 2009(H1N1) or A / Guizhou / 54 / 1989-H3N2 showed good therapeutic protection with both FF34 and F6 antibodies, with no significant decrease in body weight and all mice surviving.

[0104] The above results indicate that F6 and FF34 antibodies have excellent preventive and therapeutic effects against seasonal influenza viruses A / California / 04 / 2009 (H1N1) and A / Guizhou / 54 / 1989 (H3N2), and the mice treated with both antibodies maintained stable body weight within 14 days.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody against neuraminidase or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises fragment (1) or fragment (2); The fragment (1) comprises a heavy chain variable region (1) and a light chain variable region (1); the heavy chain variable region (1) comprises CDR-F6-H1 to CDR-F6-H3, wherein the amino acid sequence of CDR-F6-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-F6-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-F6-H3 is shown in SEQ ID NO.4; the light chain variable region (1) comprises CDR-F6-L1 to CDR-F6-L3, wherein the amino acid sequence of CDR-F6-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-F6-L2 is GAS, and the amino acid sequence of CDR-F6-L3 is shown in SEQ ID NO.11; The fragment (2) comprises a heavy chain variable region (2) and a light chain variable region (2); the heavy chain variable region (2) comprises CDR-FF34-H1 to CDR-FF34-H3, wherein the amino acid sequence of CDR-FF34-H1 is shown in SEQ ID NO.17, the amino acid sequence of CDR-FF34-H2 is shown in SEQ ID NO.18, and the amino acid sequence of CDR-FF34-H3 is shown in SEQ ID NO.19; the light chain variable region (2) comprises CDR-FF34-L1 to CDR-FF34-L3, wherein the amino acid sequence of CDR-FF34-L1 is shown in SEQ ID NO.25, the amino acid sequence of CDR-FF34-L2 is AAS, and the amino acid sequence of CDR-FF34-L3 is shown in SEQ ID NO.

26.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region (1) further comprises FR-F6-H1 to FR-F6-H4, wherein the amino acid sequence of FR-F6-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-F6-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-F6-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-F6-H4 is shown in SEQ ID NO.8; the light chain variable region (1) further comprises FR-F6-L1 to FR-F6-L4, wherein the amino acid sequence of FR-F6-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-F6-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-F6-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-F6-L4 is shown in SEQ ID NO.

15.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region (1) is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region (1) is shown in SEQ ID NO.

9.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region (2) further comprises FR-FF34-H1 to FR-FF34-H4, wherein the amino acid sequence of FR-FF34-H1 is shown in SEQ ID NO.20, the amino acid sequence of FR-FF34-H2 is shown in SEQ ID NO.21, the amino acid sequence of FR-FF34-H3 is shown in SEQ ID NO.22, and the amino acid sequence of FR-FF34-H4 is shown in SEQ ID NO.23; the light chain variable region (2) further comprises FR-FF34-L1 to FR-FF34-L4, wherein the amino acid sequence of FR-FF34-L1 is shown in SEQ ID NO.27, the amino acid sequence of FR-FF34-L2 is shown in SEQ ID NO.28, the amino acid sequence of FR-FF34-L3 is shown in SEQ ID NO.29, and the amino acid sequence of FR-FF34-L4 is shown in SEQ ID NO.

30.

5. The monoclonal antibody or its antigen-binding fragment according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region (2) is at least 95% identical to that of SEQ ID NO.16; the amino acid sequence of the light chain variable region (2) is at least 95% identical to that of SEQ ID NO.

24.

6. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of products for detecting influenza virus.

7. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of products for the prevention or treatment of influenza virus infection.

8. A biomaterial, characterized in that, It is any one of the following (1) to (3): (1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5; (2) A carrier containing the nucleic acid molecule described in (1); (3) Host cells containing the vector in (2).

9. The use of the biomaterial of claim 8 in the preparation of monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

10. A method for preparing a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, characterized in that, Under conditions that allow expression of the monoclonal antibody or its antigen-binding fragment, the host cell of claim 8 is cultured, and the monoclonal antibody or its antigen-binding fragment is recovered from the cultured host cell culture.

11. A drug for the prevention and / or treatment of influenza virus infection, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

12. A drug for the prevention and / or treatment of influenza virus infection, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5, and a pharmaceutically acceptable excipient.

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