Monoclonal antibody against neuraminidase and application thereof

Abstract

The invention discloses an anti-neuraminidase human monoclonal antibody obtained through multiple rounds of in-vitro screening and recombination transformation and application of the anti-neuraminidase human monoclonal antibody. The application refers to preparation of products for preventing, detecting or treating influenza viruses. The monoclonal antibody provided by the invention has remarkable neuraminidase inhibitory activity and broad-spectrum influenza virus antigen binding capacity, has excellent prevention and treatment effects on various seasonal influenza viruses, provides a new technical choice for resisting influenza infection, and has important application value.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, in particular to anti-neuraminidase monoclonal antibodies and applications thereof. Background Art

[0002] There are two glycoproteins on the surface of influenza viruses: 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 decisive role in the process of virus adsorption and membrane fusion, and is the main target for preventing influenza virus infection. Neuraminidase (NA), also known as sialidase, is a glycoprotein on the surface of influenza virus that can cleave sialic acid and promote the release of virus particles from infected cells. At the same time, neuraminidase can help the virus pass through the mucus layer by cleaving the decoy receptors in mucus proteins.

[0004] Neuraminidase is also an important target for anti-influenza virus treatment, and both neuraminidase inhibitors and neuraminidase antibodies have been found to have protective effects. Neuraminidase inhibitors are widely used in antiviral treatment, and antibodies targeting neuraminidase can inhibit the activity of neuraminidase by inhibiting the active center or binding to epitopes around the active site. Currently, neuraminidase antibodies 8H2, 6C6, and 3D4 have been reported, mainly targeting the N1 subtype, and antibodies CR14011, CR14012, and CR14017 are mainly targeted at influenza B virus neuraminidase. However, although several monoclonal antibodies to human neuraminidase have been discovered by those skilled in the art in recent years, the understanding of the neuraminidase epitopes they target is still incomplete, and the conserved epitopes of neuraminidase proteins cannot be fully resolved, especially for those epitopes with broad neuraminidase inhibitory activity. Moreover, the number of currently available specific broad-spectrum antibodies against neuraminidase is limited, and they cannot target both influenza A and B viruses at the same time, and the neutralization mechanism of antibodies targeting neuraminidase is not yet fully understood. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides anti-neuraminidase monoclonal antibodies and applications thereof.

[0006] The first object of the present invention is to provide an anti-neuraminidase monoclonal antibody or an antigen-binding fragment thereof.

[0007] The 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 preventing and / or treating influenza virus.

[0008] The third object of the present invention is to provide a biomaterial.

[0009] The fourth object of the present invention is to provide the use of the biomaterial in preparing anti-neuraminidase monoclonal antibodies or antigen-binding fragments thereof.

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

[0011] The sixth object of the present invention is to provide a drug for preventing and / or treating influenza virus.

[0012] In order to achieve the above object, the present invention is implemented by the following scheme:

[0013] The present invention provides F6 antibodies and FF34 antibodies with neuraminidase inhibitory activity, which can strongly bind to neuraminidase across 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-infection and therapeutic effects.

[0014] An anti-neuraminidase monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises 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 SEQ ID NO.16; the amino acid sequence of the light chain variable region (2) is at least 95% identical to SEQ ID NO.24.

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

[0022] Preferably, the monoclonal antibody or antigen-binding fragment thereof further comprises an Fc segment. The present invention has no special limitation on the species origin of the Fc segment, including but not limited to human Fc segment, mouse Fc segment, rabbit Fc segment 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) is shown in SEQ ID NO.31, and the amino acid sequence of the light chain constant region (1) is 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) is shown in SEQ ID NO.31, and the amino acid sequence of the light chain constant region (2) is shown in SEQ ID NO.32.

[0026] The use of any of the monoclonal antibodies or antigen-binding fragments thereof in the preparation of products for preventing and / or treating influenza viruses should also be within the scope of protection of the present invention.

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

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

[0029] (2) a vector comprising the nucleic acid molecule described in (1);

[0030] (3) A host cell comprising the vector described in (2).

[0031] The present invention has no special limitation on the source and type of host cells, including but not limited to Escherichia coli, HEK293 cells, CHO cells and other conventional prokaryotic and eukaryotic cells that can carry the vector can achieve the purpose of the present invention.

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

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

[0034] The use of the biomaterial in the preparation of anti-neuraminidase monoclonal antibodies or antigen-binding fragments thereof should also be within the protection scope of the present invention.

[0035] The use of the biological material in the preparation of products for preventing and / or treating influenza viruses should also be within the protection scope of the present invention.

[0036] A method for preparing an anti-neuraminidase monoclonal antibody or an antigen-binding fragment thereof, comprising culturing the host cell described in (3) in the biological material under conditions allowing 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] The present invention has no special limitation on the source and type of host cells, including but not limited to Escherichia coli, HEK293 cells, CHO cells and other conventional prokaryotic and eukaryotic cells that can carry the vector can achieve the purpose of the present invention.

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

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

[0040] A drug for preventing and / or treating influenza virus, comprising any one of the monoclonal antibodies or antigen-binding fragments thereof, 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 the present invention has significant neuraminidase inhibitory activity and broad-spectrum anti-influenza virus antigen binding ability, has excellent prevention and treatment effects on a variety of seasonal influenza viruses, provides a new technical option for resisting influenza infection, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure shows the SDS-PAGE detection results of F6 antibody and FF34 antibody.

[0044] Figure 2 This is an ELISA experiment to detect the antigen binding of neuraminidase monoclonal antibodies; A is F6 antibody; B is FF34 antibody.

[0045] Figure 3 The MUNANA experiment was used to detect the enzyme inhibition ability of neuraminidase monoclonal antibodies; 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 1v / v% BSA.

[0046] Figure 4The ELISA experiment was used to detect the enzyme activity inhibition ability of neuraminidase monoclonal antibodies; 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 1v / v% BSA.

[0047] Figure 5 Animal experiments to detect the protective ability of neuraminidase monoclonal antibodies; A is the weight monitoring of mice under the preventive protection of antibodies against A / California / 4 / 2009(H1N1); B is the survival curve of mice under the preventive protection of antibodies against A / California / 4 / 2009(H1N1); C is the weight monitoring of mice under the therapeutic protection of antibodies against A / California / 4 / 2009(H1N1); D is the weight monitoring of mice under the therapeutic protection of antibodies against A / California / 4 / 2009(H1N1) A is the survival curve of mice under the therapeutic protection of antibodies against A / Guizhou / 54 / 1989(H3N2); E is the weight monitoring of mice under the preventive protection of antibodies against A / Guizhou / 54 / 1989(H3N2); F is the survival curve of mice under the preventive protection of antibodies against A / Guizhou / 54 / 1989(H3N2); G is the weight monitoring of mice under the therapeutic protection of antibodies against A / Guizhou / 54 / 1989(H3N2); H is the survival curve of mice under the therapeutic protection of antibodies against A / Guizhou / 54 / 1989(H3N2). DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1 Preparation of Neuraminidase Monoclonal Antibody

[0050] 1. Isolation and purification of PBMC

[0051] Ten volunteers infected with seasonal influenza in 2019-2020 were recruited, blood samples were collected, and PBMCs were isolated by density gradient centrifugation. The obtained PBMCs were sorted by flow cytometry, and 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 antibodies (Biolegend, 303516) were added at a ratio of 1:200. The cells were incubated at 4°C in the dark for 30 minutes, and 2 mL of PBS (containing 0.2 v / v% BSA) was added. Centrifuge at 1000 rpm for 5 minutes. The supernatant was discarded and 2 mL of PBS containing 0.2 v / v% BSA was added to resuspend the cells. All three antibodies were positive (i.e., CD19 + 、CD27 + 、CD38 high ) were sorted into 96-well plates.

[0052] 2. Nested PCR

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

[0054] After completion, nested PCR was performed, 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 the two rounds of nested PCR were from "Table 1" in the prior art "Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen (DOI: 10.1038 / nprot.2009.3)", where "RT-PCR" primers were used in the first round and "Nested PCR" primers were used in the second round.

[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, the variable region fragments of the heavy chain and light chain of two neuraminidase monoclonal antibodies (F6 antibody and FF34 antibody) were obtained.

[0062] According to the IMGT method, the domains of F6 antibody and FF34 antibody are specifically divided 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 the complementary determining 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 the framework 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 (SEQID 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 (SEQID NO.9); wherein the underlined parts are the complementary determining regions CDR-F6-L1 (SEQ ID NO.10), CDR-F6-L2 and CDR-F6-L3 (SEQ ID NO.11), and the remaining parts are the framework 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); the underlined parts are the complementary determining regions CDR-FF34-H1 (SEQID NO.17), CDR-FF34-H2 (SEQ ID NO.18) and CDR-FF34-H3 (SEQ ID NO.19), and the remaining parts are the framework regions FR-FF34-H1 (SEQ ID NO.20), FR-FF34-H2 (SEQ ID NO.21), FR-FF34-H3 (SEQ IDNO.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 parts are the complementary determining regions CDR-FF34-L1 (SEQ ID NO.25), CDR-FF34-L2 and CDR-FF34-L3 (SEQ ID NO.26), and the remaining parts are the framework regions 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).

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

[0068] (1) Construction of recombinant plasmid

[0069] The coding sequence of the heavy chain variable region (SEQ ID NO.1) of the F6 antibody was inserted into the pcDNA3.1 vector expressing the heavy chain constant region (SEQ ID NO.31), thereby obtaining the heavy chain expression plasmid of the F6 antibody, which was recorded 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), thereby obtaining the light chain expression plasmid of the F6 antibody, which was recorded 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), thereby obtaining the heavy chain expression plasmid of the FF34 antibody, which was recorded 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 (SEQID NO.32), thereby obtaining the light chain expression plasmid of the FF34 antibody, which was recorded as pcDNA3.1-FF34-L.

[0073] (2) Antibody expression

[0074] Antibody expression is achieved by transient transfection of 293T cells with plasmids. Before transfection, ensure that 293T cells grow evenly in the cell culture dish, and the number of passages should not exceed 30 times, otherwise the cells cannot effectively express antibodies. The transfection steps are as follows: aspirate 2.5mL of antibody-free and serum-free DMEM into a 15mL centrifuge tube, first add the heavy chain expression plasmid and the corresponding light chain expression plasmid of each antibody constructed in the previous step, and then add 100μL of polyethyleneimine (Polyethylenimine, PEI) to the centrifuge tube, vortex to mix, and incubate at room temperature for 15min. Slowly add the solution after standing for 15min to the culture medium of 293T cells and mix. After culturing overnight in a 37℃ incubator, change the medium and culture in the incubator for 5d.

[0075] (3) Purification

[0076] Add 150μL agarose beads to a new 50mL centrifuge tube, add 1×PBS to 50mL, centrifuge at 3000rpm and 4℃ for 10min, and remove PBS with a vacuum pump. Collect the cell supernatant in the culture dish into a 50mL centrifuge tube, centrifuge at 3000rpm and 4℃ for 10min, then pour the supernatant into the 50mL centrifuge tube containing agarose beads, add 1×PBS to fill the centrifuge tube to 50mL, and shake overnight at 4℃. Take it out of the refrigerator on the second day, centrifuge at 3000rpm and 4℃ for 10min. Use a vacuum pump to suck out the supernatant. At this time, the protein binds to the agarose beads and remains at the bottom of the tube. Add 1×PBS twice to elute the impurities. After removing the PBS, add 2.5mL 0.1MGlycine-HCL (pH2.7), shake on a horizontal shaker at room temperature for 10min, and then centrifuge. At this time, the protein is in the supernatant. The supernatant was transferred to a new 15 mL centrifuge tube, and 120 μL 1M Tris-HCL (pH = 9) was added to adjust the pH to 7.0, and then all the liquid was transferred to a 30 kDa ultrafiltration tube, and centrifuged at 3000 rpm, 4 ° C, for 10 min, and the waste liquid was discarded. PBS was added to the ultrafiltration tube for replacement three times, and finally concentrated to a volume of 200 μL to obtain the monoclonal antibody, which was transferred to a 1.5 mL centrifuge tube and stored at 4 ° C for future 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 the disulfide bonds and perform SDS-PAGE detection. The results are as follows: Figure 1 As shown, under reducing conditions, due to the destruction of the disulfide bonds between the antibody light and heavy chains, antibody heavy chain bands of about 50 kDa and antibody light chain bands of about 25 kDa appeared. This indicates that monoclonal antibodies F6 and FF34 were successfully expressed in 293T cells and secreted into the cell supernatant. So far, two neuraminidase monoclonal antibodies were successfully prepared, denoted as F6 antibody and FF34 antibody.

[0079] Example 2 Antigen Binding of Neuraminidase Monoclonal Antibodies

[0080] 1. Antigen preparation and purification

[0081] Referring to the "Antibody Expression" procedure in Example 1, antibodies from A / California / 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), A / Red knot / Delaware The NA extracellular domain fragments of 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 respectively to construct the recombinant expression plasmids of each NA, and the expression was achieved by transiently transfecting 293T cells with the plasmids. The cell supernatant was collected, filtered through a 0.22 μm filter membrane, and purified by Ni+Sepharose High-Performance chromatography column to obtain 11 proteins, which were used as coating antigens and were named 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), and 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 2μg / mL of each coated antigen to a 96-well ELISA plate at 50μL per well, cover the lid, and place in a humidified box at 4℃ for overnight coating. Take out the overnight coated ELISA plate, wash the plate 6 times with PBST, and pat dry. Prepare ELISA blocking solution, add 150μL to each well for blocking, and incubate in a humidified box at 37℃ for 1h. After blocking, wash 6 times with PBST and pat dry. Add 90μL of 10μg / mL of the test antibody (F6 antibody or FF34 antibody) to row A of the dilution plate, add 60μL PBS to the remaining rows, and then transfer 30μL from A to G to the next row in sequence. After dilution, transfer 50μL per well to the ELISA plate, incubate in a humidified box at 37℃ for 1h. After incubation, wash 6 times with PBST and discard the residual liquid in the ELISA plate. Add 75 μL of goat anti-human IgG-HRP secondary antibody diluted at a ratio of 1:2000 to each well and incubate in a wet box at 37°C for 1 hour. Wash with PBST 6 times, discard the residual liquid in the ELISA plate, add 100 μL of colorimetric solution to each well from low concentration to high concentration, protect from light and record the time, and read the OD value in the ELISA reader at 5min, 10min and 15min of reaction.

[0084] like Figure 2 As shown in A, F6 antibody can bind to multiple antigens including N1-A / California / 04 / 2009(H1N1), N1-A / India / Pun1512245 / 2015, N1-A / Brevig Mission / 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 B, FF34 antibody can bind to multiple antigens including N1-A / California / 04 / 2009(H1N1), N1-A / India / Pun1512245 / 2015, N1-A / Brevig Mission / 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). The above results show that both F6 antibody and FF34 antibody have broad-spectrum influenza virus antigen binding ability.

[0085] Example 3 Neuraminidase monoclonal antibody enzyme activity inhibition ability

[0086] 1. Antigen preparation and purification

[0087] Referring to the "Antibody Expression" operation method in Example 1, the NA extracellular domain fragments 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) and B / Colorado / 06 / 2017 (Victoria), A / Shanghai / 02 / 2013 (H7N9) were recombined into the pcDNA3.1 vector, respectively, to construct the recombinant expression plasmids of each NA, and the expression was achieved by transient transfection of 293T cells with the plasmids. The cell supernatant was collected, filtered through a 0.22 μm filter membrane, and purified by Ni+Sepharose High-Performance chromatography column to obtain 11 proteins, which were used as target antigens and were named 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. MUNANA assay identifies the effect of antibodies on NA enzyme activity sites

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

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

[0091] The experimental results are as follows Figure 3 As shown in A to H, F6 antibody is active 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 has an enzyme activity inhibitory effect; the FF34 antibody has an enzyme activity inhibitory effect on 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] Determine the working concentration of each target antigen as follows: add 25μg / mL fetuin to a 96-well ELISA plate at 50μL per well, incubate at 4°C for 24h, then wash the plate 3 times with PBST and pat dry. Add 90μL of each target antigen with a starting concentration of 30μg / mL to row A, add 60μL DPBST (containing 1v / v% BSA) to the remaining rows, and transfer 30μL from A to G to the next row in sequence. After dilution, transfer 50μL per well to the ELISA plate coated with fetuin and incubate at 37°C for 20h. After incubation, wash the plate 6 times with PBST and pat dry. Dilute HRP-labeled peanut agglutinin at a ratio of 1:400, add 100μL to each well, and incubate in the dark for 2h. Wash the plate 9 times with PBST, discard the residual liquid in the ELISA plate, add 100 μL of colorimetric solution to each well, react in the dark and record the time, read the OD value in the ELISA reader at 5 min, 10 min and 15 min of reaction, and calculate the working concentration of each target antigen.

[0094] The enzyme activity inhibition ability of F6 antibody and FF34 antibody was determined as follows: 120 μL of 32 μg / mL of the antibody to be tested (F6 antibody or FF34 antibody) was added to row A, and 60 μL of DPBST (containing 1 v / v% BSA) was added to the remaining rows, and then 60 μL was transferred from A to G to the next row, and then an equal volume of antigen was added to mix, and incubated at 37°C for 2 hours. After the incubation, 100 μL per well was transferred to the ELISA plate coated with fetuin, incubated at 37°C for 20 hours, and the plate was washed 6 times with PBST and patted dry. HRP-labeled peanut agglutinin was diluted at a ratio of 1:400, 100 μL was added to each well, and incubated for 2 hours in the dark. The plate was washed 9 times with PBST, residual liquid in the ELISA plate was discarded, 100 μL of colorimetric solution was added to each well, reaction was carried out in the dark and the time was recorded, and OD values ​​were read in an ELISA reader at 5 min, 10 min and 15 min of reaction, respectively, and the enzyme activity inhibition ability of each antibody to be tested was calculated.

[0095] The experimental results are as follows Figure 4As shown in A to H in Figure 1, F6 antibody has an inhibitory effect on 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); FF34 antibody has an inhibitory effect on 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); 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) had enzyme activity inhibition.

[0096] The above results indicate that both F6 antibody and FF34 antibody can inhibit the enzymatic activity of neuraminidase of many different types of influenza viruses.

[0097] Example 4 Protective ability of neuraminidase monoclonal antibodies

[0098] 1. Antibody prevention experiment

[0099] BALB / c mice aged 6 to 8 weeks were used as experimental animals. F6 antibody, FF34 antibody or PBS was intraperitoneally injected one day before the challenge. The dosage for each mouse was 10 mg / kg (antibody / body weight). 5×LD50 influenza virus (A / California / 04 / 2009 (H1N1) or A / Guizhou / 54 / 1989-H3N2) was challenged 24 hours later. The weight of the mice was monitored for 14 consecutive days after the challenge. The mice with a body weight less than 75% of the initial body weight were euthanized, and the survival rate (Probability of Survival) of the mice was calculated.

[0100] like Figure 5 As shown in A to B and E to F in the figure, when mice were infected with A / California / 04 / 2009 (H1N1) or A / Guizhou / 54 / 1989-H3N2, both FF34 and F6 antibodies showed good preventive and protective effects, with no significant weight loss and all mice survived.

[0101] 2. Antibody therapy experiments

[0102] BALB / c mice aged 6 to 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). F6 antibody, FF34 antibody or PBS was intraperitoneally injected 24 hours after the challenge. The dosage for each mouse was 10 mg / kg (antibody / body weight). The body weight of the mice was monitored for 14 consecutive days after treatment, and the mice whose body weight was less than 75% of the initial body weight were euthanized.

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

[0104] The above results indicate that F6 antibody and FF34 antibody have excellent preventive and therapeutic effects against seasonal influenza viruses A / California / 04 / 2009 (H1N1) and A / Guizhou / 54 / 1989 (H3N2), and the body weight of mice treated with antibodies remained stable 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 rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An anti-neuraminidase monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or antigen-binding fragment thereof 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 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.

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

24.

6. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a product for preventing, detecting or treating influenza virus.

7. 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 antigen-binding fragment thereof according to any one of claims 1 to 5; (2) a vector comprising the nucleic acid molecule described in (1); (3) A host cell comprising the vector described in (2).

8. Use of the biomaterial according to claim 7 in the preparation of anti-neuraminidase monoclonal antibodies or antigen-binding fragments thereof.

9. A method for preparing an anti-neuraminidase monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The host cell of claim 7 is cultured under conditions that allow expression of the monoclonal antibody or antigen-binding fragment thereof, and the monoclonal antibody or antigen-binding fragment thereof is recovered from the culture of the cultured host cell.

10. A drug for preventing and / or treating influenza virus, characterized in that: The invention comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier and / or excipient.

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