A class of fully human antibodies that inhibit the neuraminidase activity of H3N2 influenza virus and their applications

Abstract

This invention relates to a class of fully human antibodies that inhibit the neuraminidase activity of H3N2 influenza virus and their applications, belonging to the field of biomedical technology. Addressing the shortcomings of existing technologies, such as the susceptibility of H3N2 subtypes of influenza A virus to antigenic mutations, the tendency for traditional small-molecule drugs to develop resistance, and the narrow protective spectrum of existing antibody drugs, this invention provides a class of fully human antibodies that recognize the same epitope group and inhibit the neuraminidase activity of H3N2 influenza virus. The CDR regions of these fully human antibodies exhibit high overall similarity, establishing a conserved antibody-binding backbone. Simultaneously, their heavy chain variable region CDR3 exhibits specific sequence variations, forming a structural feature of "conserved backbone + variant HCDR3". The fully human antibodies provided by this invention have low immunogenicity risk and broad application prospects in the prevention, treatment, and related diagnostic reagents and test kits for H3N2 influenza.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a class of fully human antibodies that inhibit the activity of neuraminidase of H3N2 influenza virus and their applications. Background Technology

[0002] Influenza A virus is a major pathogen causing seasonal influenza and pandemics worldwide, posing a serious threat to human public health. Based on antigenic differences in its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza A virus can be divided into several subtypes. Epidemiological data show that in recent years, the H3N2 subtype influenza strain has often led to higher hospitalization and mortality rates, particularly among the elderly and immunocompromised individuals, where the harm is especially severe.

[0003] Neuraminidase, an enzyme on the surface of the influenza virus, is a tetrameric type II transmembrane glycoprotein with sialic acid cleavage. During the viral infection cycle, its primary function is to cleave sialic acid at the ends of host cell surface glycoproteins and glycolipids, thereby promoting the release of newly synthesized viral particles from the infected cell surface and preventing viral particle aggregation. Furthermore, NA can also facilitate viral entry into host cells by penetrating the sialic acid barrier in respiratory mucus. Given its crucial role in viral replication and transmission, NA has been an important target for the development of anti-influenza drugs.

[0004] Currently, the main methods for preventing and controlling influenza include vaccination and small-molecule antiviral drugs. While vaccination is the most effective means of preventing influenza, the influenza virus (especially H3N2) is highly susceptible to antigenic drift, often leading to a mismatch between vaccine strains and circulating strains, resulting in less than ideal vaccine efficacy (effectiveness below 40% in some seasons). Furthermore, traditional chicken embryo vaccine production processes may cause adaptive mutations in H3N2 strains, further affecting vaccine efficacy. Neuraminidase inhibitors (NAIs), such as oseltamivir and zanamivir, are first-line drugs for the clinical treatment of influenza; these drugs inhibit viral release by competitively binding to the active site of NAIs. However, with the widespread use of NAIs, several studies have reported mutations in H3N2 virus strains that exhibit resistance to oseltamivir or reduced sensitivity (such as E119V and R292K mutations).

[0005] In recent years, research on monoclonal antibodies as an antiviral therapy has attracted much attention. However, the vast majority of anti-influenza monoclonal antibodies currently under development primarily target the HA protein, aiming to block viral adsorption and entry into cells. Although HA antibodies have highly efficient neutralizing activity, HA is a major recognition target of the host immune system and is subjected to enormous immune pressure, making it highly susceptible to antigenic mutations to evade antibody neutralization. As a result, antibodies against HA typically have a narrow spectrum of coverage.

[0006] In contrast, antibody research targeting NA has lagged behind. Early studies suggested that NA's immunogenicity was weaker than HA's, and that its antibody neutralization ability was not strong. However, recent research indicates that NA has unique advantages as a target: NA's antigenic drift rate is slower than HA's, and the structure of its catalytic active site is relatively conserved across different strains. Therefore, developing monoclonal antibodies against NA, especially those targeting the H3N2 subtype of NA, has gradually become a new research hotspot. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies, such as the ease with which the H3N2 subtype of influenza A virus undergoes antigenic mutation, the tendency for traditional small molecule drugs to develop resistance, and the narrow protective spectrum of existing antibody drugs. It provides a class of fully human antibodies that inhibit the neuraminidase activity of H3N2 influenza virus and their applications.

[0008] One of the objectives of this invention is to provide a class of fully human antibodies that inhibit the activity of H3N2 influenza virus neuraminidase. The fully human antibody binds to the same epitope group on the H3N2 influenza virus neuraminidase protein, thereby blocking the activity of H3N2 influenza virus neuraminidase and inhibiting the virus. The fully human antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH and the light chain variable region VL comprise: (1) The following C24-H-CDR1~3 and C24-L-CDR1~3, wherein C24-H-CDR1~3 and C24-L-CDR1~3 are defined according to the IMGT numbering system; The C24-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 1~3, respectively have one or more amino acid substitutions, deletions or additions, or have amino acid sequences with at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 1~3; The C24-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 5, respectively, have one or more amino acid sequences with substitutions, deletions or additions of amino acids, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 5; or, (2) The following C71-H-CDR1~3 and C71-L-CDR1~3, wherein C71-H-CDR1~3 and C71-L-CDR1~3 are defined according to the IMGT numbering system; The C71-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 6~8, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 6~8. The C71-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 9, respectively, have one or more amino acid sequences with substitutions, deletions or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 9; or, (3) The following C3009-H-CDR1~3 and C3009-L-CDR1~3, wherein C3009-H-CDR1~3 and C3009-L-CDR1~3 are defined according to the IMGT numbering system; The C3009-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 10~12, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 10~12; The C3009-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 13, respectively, have one or more amino acid sequences with substitutions, deletions or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 13; or, (4) The following C184-H-CDR1~3 and C184-L-CDR1~3, wherein C184-H-CDR1~3 and C184-L-CDR1~3 are defined according to the IMGT numbering system; The C184-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 14~16, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 14~16. The C184-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 17, aspartic acid D-alanine A-alanine A, and SEQ ID No. 9, respectively, have one or more amino acid sequences with substitutions, deletions, or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 17, aspartic acid D-alanine A-alanine A, and SEQ ID No. 9.

[0009] In a preferred embodiment of the present invention, the heavy chain variable region VH and the light chain variable region VL retain the neuraminidase binding activity and enzyme activity inhibitory activity of H3N2 influenza virus, wherein the heavy chain variable region VH and the light chain variable region VL are: (1) The following heavy chain variable region C24-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.18, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.18; The light chain variable region C24-L described below has an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO.19, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.19. or, (2) The following heavy chain variable region C71-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.20, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.20; The light chain variable region C71-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.21, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.21. or, (3) The following heavy chain variable region C3009-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.22, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.22; The light chain variable region C3009-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.23, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.23. or, (4) The following heavy chain variable region C184-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.24, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.24; The light chain variable region C184-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.25.

[0010] In a preferred embodiment of the present invention, the fully human antibody is selected from any one of immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin E, or immunoglobulin D.

[0011] In a preferred embodiment of the present invention, the fully human antibody includes one or more of the following: Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, biantibody, single-domain antibody, chimeric antibody, bispecific antibody, or multispecific antibody.

[0012] A second objective of this invention is to provide a nucleic acid, wherein the nucleic acid is an amino acid sequence encoding the aforementioned fully human antibody.

[0013] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid.

[0014] A fourth objective of this invention is to provide a host cell containing the aforementioned nucleic acid or expression vector.

[0015] A fifth objective of this invention is to provide a pharmaceutical composition comprising: the aforementioned fully human antibody, nucleic acid, expression vector or host cell, and pharmaceutically acceptable carrier and / or excipient; the pharmaceutical composition further comprising other pharmaceutically active agents selected from M2 protein ion channel inhibitors and neuraminidase inhibitors; the M2 protein ion channel inhibitors include, but are not limited to, amantadine or rimantadine, and the neuraminidase inhibitors include, but are not limited to, oseltamivir.

[0016] The sixth objective of this invention is to provide a kit comprising the aforementioned fully human antibody; the kit further comprises a second antibody that specifically recognizes the antibody, the second antibody bearing a detectable label; the detectable label is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin; the enzyme is horseradish peroxidase or alkaline phosphatase; the chemiluminescent reagent is an acridinium ester compound, luminol and its derivatives, or ruthenium derivatives; the fluorescent dye is fluorescein or fluorescent protein.

[0017] The seventh objective of this invention is to provide the application of the above-mentioned fully human antibody, nucleic acid, expression vector, host cell or pharmaceutical composition in the preparation of a drug, which is used to prevent or treat diseases caused by influenza A virus H3N2 infection, or as a passive immunizing agent or long-acting prophylactic biological agent against influenza A virus H3N2. The diseases mentioned include influenza pneumonia, acute respiratory infection, mild influenza, and severe influenza; The drug may be used alone or in combination with other pharmaceutically active agents; The other pharmaceutically active agents are antiviral drugs for influenza; The anti-influenza virus drugs are M2 protein ion channel inhibitors and neuraminidase inhibitors. The M2 protein ion channel inhibitors include, but are not limited to, amantadine and rimantadine, and the neuraminidase inhibitors include, but are not limited to, oseltamivir.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention, through affinity purification and mass spectrometry identification, obtains a class of fully human antibodies capable of specifically binding to H3N2 influenza A virus neuraminidase (NA); compared with the prior art, the fully human antibodies provided by the present invention that inhibit the activity of H3N2 influenza A virus neuraminidase have the following beneficial effects: 1. Unique sequence features endow broad-spectrum activity and high efficacy: Sequence alignment analysis shows that the CDR regions of the fully human antibodies provided by this invention that inhibit the activity of H3N2 influenza A virus neuraminidase are highly similar overall, establishing a conserved backbone and stability for antibody binding; at the same time, they exhibit specific sequence variations in the CDR3 region of the heavy chain variable region; this structural feature of "conserved backbone + variant HCDR3" enables these antibodies to not only broadly recognize the neuraminidase of different H3N2 strains, but also achieve high-efficiency virus inhibition through the unique CDR3 structure of the heavy chain variable region.

[0019] 2. High Affinity Binding Capacity and Definite Epitope Mechanism: Enzyme-linked immunosorbent assay (ELISA) results show that the fully human antibody provided in this invention, which inhibits the activity of H3N2 influenza A virus neuraminidase, exhibits significant binding ability against the A / H3N2 / Kansas / 14 / 2017 strain neuraminidase protein, demonstrating extremely high affinity. Mutant binding experiments confirmed that the binding epitope of this type of antibody is critically dependent on the asparagine (N200) residue at position 200 of the NA protein; the N200S mutation completely disrupts the antibody's binding activity, indicating that N200 is the key site mediating the binding of this type of antibody. This definitive epitope characteristic provides a solid structural basis for the antibody's specific mechanism of action.

[0020] 3. Broad-spectrum and highly efficient enzyme activity inhibition: Enzyme-linked lectin assay (ELLA) showed that the fully human antibody provided in this invention, which inhibits the neuraminidase activity of H3N2 influenza A virus, exhibited highly efficient inhibitory effects (IC50) on the neuraminidase protein of both the A / H3N2 / Kansas / 14 / 2017 strain and the recently circulating A / H3N2 / Jilin-Chaoyang / 1230 / 2024 strain. 50 (<0.5 μg / mL), demonstrating excellent broad-spectrum inhibitory activity and potential to combat viral mutations.

[0021] 4. Significant antiviral activity: Plaque assays showed that the fully human antibody provided in this invention, which inhibits the neuraminidase activity of H3N2 influenza A virus, can effectively inhibit plaque formation of strain A / H3N2 / Jilin-Chaoyang / 1230 / 2024, thereby blocking viral amplification and exhibiting excellent in vitro antiviral activity.

[0022] 5. Low immunogenicity risk: As a fully human antibody, the fully human antibody provided by this invention that inhibits the neuraminidase activity of H3N2 influenza A virus has a significantly lower immunogenicity risk in the human body than murine or chimeric antibodies, making it suitable for repeated administration and long-term prophylactic use.

[0023] 6. Broad Application Prospects: The fully human antibody provided by this invention, which inhibits the activity of H3N2 influenza A virus neuraminidase, exhibits high affinity, broad-spectrum enzyme inhibition ability, and highly efficient antiviral activity against H3N2 influenza virus neuraminidase. This type of antibody provides new candidate drugs for the prevention and treatment of H3N2 influenza and has broad application prospects in influenza prevention, treatment, related diagnostic reagents, and test kits. Attached Figure Description

[0024] Figure 1 The following diagram shows the screening and identification steps and results of the fully human antibody targeting the H3N2 subtype neuraminidase of influenza A virus in Example 1; A is a schematic diagram of the screening process, B is an SDS-PAGE electrophoresis analysis diagram, and C is an ELISA binding activity diagram of the enriched antibody. Figure 2 The image shows the sequence alignment results of the fully human antibody in Example 2; A is the sequence alignment result of the heavy chain variable region (VH), and B is the sequence alignment result of the light chain variable region (VL). Figure 3 The graph shows the inhibitory effect of the fully human antibody on the neuraminidase activity of different H3N2 strains in Example 3 (enzyme-linked agglutinin assay, ELLA); A is strain A / H3N2 / Kansas / 14 / 2017, and B is strain A / H3N2 / Jilin-Chaoyang / 1230 / 2024. Figure 4 The image shows the detection results (enzyme-linked immunosorbent assay, ELISA) of the binding of the fully human antibody to the A / H3N2 / Kansas / 14 / 2017 strain neuraminidase protein in Example 4. Figure 5 The figure shows the results of the plaque inhibition experiment of the fully human antibody against the A / H3N2 / Jilin-Chaoyang / 1230 / 2024 strain of virus in Example 5. Detailed Implementation

[0025] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0027] Example 1: Screening and identification of fully human antibodies targeting H3N2 subtype neuraminidase In this embodiment, total IgG was purified from plasma of healthy individuals. Specific antibodies were enriched using streptavidin affinity chromatography with biotinylated neuraminidase protein (N2 protein), and the antibody sequences were identified using mass spectrometry. Figure 1 As shown in Part A of the diagram.

[0028] 1. Volunteer blood collection With informed consent obtained and in compliance with relevant ethical requirements, peripheral venous blood was collected from one volunteer (number 3). A sterile vacuum blood collection tube (containing anticoagulant) was used to collect the venous blood. After collection, the blood collection tube was gently inverted to mix the anticoagulant and then temporarily stored at 4°C.

[0029] 2. Plasma separation Centrifuge the whole blood collected in step 1 at 2000 × g for 15 minutes at 4°C. After centrifugation, the blood will separate into three layers: the upper layer is pale yellow plasma, the middle layer is a white membrane layer, and the lower layer is red blood cells. Transfer the upper plasma layer into a clean centrifuge tube, being careful not to aspirate the middle white membrane layer or the lower red blood cells. To further remove residual cells, centrifuge the collected plasma again at 12000 rpm for 10 minutes at 4°C. Take the supernatant and inactivate it at 56°C for 1 hour. Aliquot and store at -80°C.

[0030] 3. Purification of total serum IgG Total IgG was isolated and purified from plasma using the high affinity of Protein A for human IgG Fc fragments. The specific steps were as follows: The plasma aliquots from step 2 were diluted 1:5 with 1×PBS buffer, and particulate matter was removed by filtration through a 0.45 μm filter. 1 mL of Protein A beads (GenScript) was taken and equilibrated with 10 column volumes (CV) of 1×PBS (pH 7.4) until the pH of the eluent stabilized at 7.4. The pretreated plasma sample was then added to the chromatography column, with a flow rate not exceeding 1 mL / min. After the sample had completely passed through the beads, the column was washed with at least 20 column volumes of 1×PBS until the flow-through was clear of Protein A. 280Values ​​were returned to baseline, and elution was performed using 0.1 M glycine-hydrochloric acid elution buffer (pH 2.8). The eluent was collected stepwise, and the pH was immediately adjusted to neutral by adding 1 M Tris-HCl (pH 8.6) neutralization solution to obtain purified serum total IgG.

[0031] 4. Measurement and storage of serum total IgG concentration The OD of the purified serum total IgG in step 3 was determined using a UV spectrophotometer. 280 The antibody concentration was calculated, and the solution was replaced with buffer using an ultrafiltration tube (MWCO 100 kD) to concentrate it to the required concentration (1-5 mg / mL). After aliquoting, the solution was stored at -80°C.

[0032] 5. Resin preparation and equilibration Take 120 μL of streptavidin agarose resin (Thermo Fisher Scientific) and put it into a 1.5 mL centrifuge tube. Add 500 μL of phosphate buffer (PBS, pH 7.4), centrifuge at 2000 × g for 1 minute at 4°C, discard the supernatant, and wash once to obtain equilibrated resin.

[0033] 6. Antigen conjugation Add 200 μg of biotinylated N2 protein to the equilibrated resin obtained in step 5, and add 400 μL of PBS and mix well. Incubate at room temperature for 30 minutes (mixing gently every 5 minutes during this period), and then incubate on ice for 30 minutes (mixing gently every 10 minutes during this period). After incubation, centrifuge at 2000 × g for 2 minutes at 4°C, discard the supernatant, and retain the resin precipitate conjugated with the antigen to obtain the antigen-conjugated resin (labeled W0).

[0034] 7. Washing after coupling The resin containing the conjugated antigen obtained in step 6 was washed twice with 1000 μL PBS, and the supernatant collected from the washing step was labeled as W1 and W2, respectively.

[0035] 8. Antibody incubation and enrichment Mix 12000 μg of serum total IgG obtained in step 4 with the resin obtained in step 7, and dilute with PBS to a total volume of about 8 mL to obtain a mixture. Incubate the mixture at 4°C for 4 hours.

[0036] 9. Washing removes non-specific binding. After the incubation in step 8 is completed, discard the supernatant by centrifugation (the precipitate is labeled W3), and wash twice with 2 mL PBS (the supernatants are labeled W4 and W5 respectively), wash once with 2 mL PBS containing 0.05% NP-40 (the supernatant is labeled W6), and wash three times with 1 mL PBS (the supernatants are labeled W7, W8, and W9 respectively).

[0037] 10. Washing and Neutralization Add 200 μL of 0.1 M glycine-hydrochloric acid (pH 2.8) to the centrifuge tube from step 9, let stand at room temperature for 3 minutes, then centrifuge to collect the supernatant. Immediately add neutralization solution (this fraction is designated E1). Repeat the elution step until no protein is detected in the eluent (referred to sequentially as E2, E3...En). Then, use A... 280 The protein content of each eluted fraction was monitored by absorbance.

[0038] 11. Protein concentration determination The absorbance (A) of each eluted fraction in step 10 was measured at 280 nm using a UV spectrophotometer. 280 ), calculate protein concentration.

[0039] 12. SDS-PAGE electrophoresis analysis The eluted fraction and washing control from step 10 were subjected to SDS-PAGE electrophoresis for analysis. The results are as follows: Figure 1 As shown in section B, N2-specific antibodies in serum were successfully enriched.

[0040] 13. ELISA Validation of Enriched Antibodies The binding activity of the enriched antibody to N2 protein was detected by ELISA. The negative control group consisted of antibody 2A10 and total IgG before enrichment.

[0041] (1) Coating: Dilute the antigen to 1 μg / mL, add 50 μL / well to a 96-well microplate (NEST), and incubate overnight at 4°C; (2) Blocking: Discard the coating solution, add 170 μL of PBST containing 3% BSA, and incubate at room temperature for 1 hour; (3) Add antibody: Wash the coated plate three times with TBST using a plate washer, and perform serial dilution of the antibody in the plate to make the initial concentration 15 μg / mL. Incubate at room temperature for 1.5 hours. (4) Add secondary antibody: Wash the coated plate 6 times with TBST using a plate washer, add HRP-labeled anti-human IgG antibody (1:10000 dilution, Jackson), 50 μL / well, and incubate at room temperature for 1.5 hours; (5) Color development and detection: The coated plate was washed 6 times with TBST using a plate washer, TMB substrate was added and reacted in the dark for 5-7 minutes, 2 M sulfuric acid was added to terminate the reaction, and the 450 nm OD value was measured.

[0042] The results are as follows Figure 1 As shown in section C, the binding activity of the enriched antibody to N2 is significantly stronger than that of the total IgG before enrichment, indicating that the N2-specific antibody was successfully enriched.

[0043] 14. Mass spectrometry (MS) Elution fractions E1, E2, and E3 with high protein concentrations were collected, mixed, and subjected to mass spectrometry analysis to identify the amino acid sequence of the antibody.

[0044] like Figure 1 As shown, in this embodiment, N2-specific antibodies were enriched from the plasma of healthy individuals, and the antibody sequences were identified, successfully obtaining a fully human antibody targeting the neuraminidase activity of the H3N2 influenza virus; the identified fully human antibody is as follows: The amino acid sequence of the heavy chain variable region (C24-H) of the fully human antibody C24 is shown in SEQ ID NO.18, and the amino acid sequence of the light chain variable region (C24-L) is shown in SEQ ID NO.19. The amino acid sequence of the heavy chain variable region (C71-H) of the fully human antibody C71 is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region (C71-L) is shown in SEQ ID NO.21. The fully human antibody C3009 has the following amino acid sequence: the heavy chain variable region (C3009-H) is shown in SEQ ID NO.22, and the light chain variable region (C3009-L) is shown in SEQ ID NO.23. The amino acid sequence of the heavy chain variable region (C184-H) of the fully human antibody C184 is shown in SEQ ID NO.24, and the amino acid sequence of the light chain variable region (C184-L) is shown in SEQ ID NO.25.

[0045] Example 2: Sequence alignment analysis of fully human antibodies Germline gene alignment analysis was performed on the amino acid sequences of the heavy and light chain variable regions of the four fully human antibodies (C24, C71, C3009 and C184) obtained in Example 1 using the NCBI IgBLAST and IMGT / V-QUEST databases, and multiple alignment analysis was performed using bioinformatics software (ClustalW).

[0046] The results are as follows Figure 2As shown, sequence analysis reveals that the overall sequences of the complementarity-determining regions (CDRs) of these fully human antibodies are highly similar; simultaneously, each antibody strain possesses its own unique amino acid variation characteristics in the heavy chain CDR3 (HCDR3) region. These sequence characteristics suggest that the fully human antibodies provided by this invention achieve efficient recognition of NA proteins through specific variations in HCDR3 while maintaining overall conformational stability.

[0047] Example 3: Enzyme-linked lectin assay (ELLA) to assess the inhibitory effect of antibody on NA activity 1. Antibody expression preparation: The variable region genes (VH and VL) of the four fully human antibodies (C24, C71, C3009 and C184) identified in Example 1 were cloned into a fully human IgG1 expression vector, and HEK293F cells were transfected with PEI (Yisheng Bio) for transient expression. After culturing for 5 days, the supernatant was collected, purified using Protein A beads, and the concentration was determined. 2. Coating: Dilute fetoglobulin (Pusitang) with PBS to 50 μg / mL, add 150 μL / well to a 96-well plate (Thermo Fisher Scientific 442404), and incubate at 4°C for 16 hours; 3. Blocking: Discard the coating solution, add 200 μL of PBS blocking solution containing 1% BSA to each well, and incubate at room temperature for 1 hour; 4. Incubation of NA protein and antibody: Pre-titrate the optimal working concentrations of recombinant NA protein (rNA) from strain A / H3N2 / Kansas / 14 / 2017 and strain A / H3N2 / Jilin-Chaoyang / 1230 / 2024 (select the concentration corresponding to 90% absorbance saturation). Perform 3-fold serial dilutions of the antibody in 96-well U-shaped plates (NEST). Mix the diluted antibody with an equal volume of NA protein (NA protein concentration 2 times the pre-titrated concentration) and incubate at 37°C for 1 hour. 5. Sample loading: Wash the coated plate 6 times with TBST using a plate washer. Transfer the NA-antibody mixture from step 4 to the coated plate, 100 μL / well, and incubate at 37°C for 16 hours; 6. Add HRP-conjugated peanut lectin secondary antibody: Wash the coated plate 6 times with TBST using a plate washer. After washing, add 100 μL of HRP-conjugated peanut lectin secondary antibody (2 μg / mL, Sigma-Aldrich) to each well and incubate at room temperature in the dark for 1 hour and 45 minutes. 7. Detection: The coated plate was washed 6 times with TBST using a plate washer. After washing, TMB substrate (Thermo Fisher Scientific) was added for color development, and the reaction was terminated with 2 M sulfuric acid. The absorbance at 450 nm was measured.

[0048] This embodiment evaluated the inhibitory effect of this type of fully human antibody on NA activity, and the results are as follows: Figure 3 As shown, in the case of recombinant NA protein (rNA) of strain A / H3N2 / Kansas / 14 / 2017, the IC50 values ​​of fully human antibodies C24, C71, C184, and C3009 were [data missing]. 50 The concentrations were 0.046 μg / mL, 0.033 μg / mL, 0.074 μg / mL, and 0.039 μg / mL, respectively; in the target rNA of strain A / H3N2 / Jilin-Chaoyang / 1230 / 2024, the IC50 concentration was... 50 The concentrations were 0.41 μg / mL, 0.33 μg / mL, 0.28 μg / mL, and 0.28 μg / mL, respectively. The results indicate that the fully human antibody provided by this invention, which inhibits the neuraminidase activity of H3N2 influenza virus, has a broad-spectrum and potent inhibitory ability on NA enzyme activity.

[0049] Example 4: Enzyme-linked immunosorbent assay (ELISA) to detect the binding characteristics of antibody to NA protein 1. Coating: The recombinant NA protein (rNA) of strain A / H3N2 / Kansas / 14 / 2017 and the rNA mutant carrying the N200S mutation were diluted to 1 μg / mL and added to 50 μL / well of a 96-well microplate (NEST). The plates were incubated overnight at 4°C. 2. Blocking: Discard the coating solution, add 170 μL of PBST containing 3% BSA, and incubate at room temperature for 1 hour; 3. Add antibody: Wash the coated plate three times with TBST using a plate washer, and serially dilute the four fully human antibodies (C24, C71, C3009 and C184) prepared in Example 4 into the plate to make an initial concentration of 15 μg / mL, and incubate at room temperature for 1.5 hours. 4. Add secondary antibody: Wash the coated plate 6 times with TBST using a plate washer, add 50 μL of HRP-labeled anti-human IgG antibody (1:10000 dilution, Jackson) to each well, and incubate at room temperature for 1.5 hours; 5. Color development and detection: Wash the coated plate 6 times with TBST using a plate washer, add TMB substrate and react for 5-7 minutes in the dark, then add 2 M sulfuric acid to terminate the reaction and measure the 450 nm OD value.

[0050] This embodiment tested the binding characteristics of this type of fully human antibody to the NA protein, and the results are as follows: Figure 4As shown, the fully human antibodies C24, C71, C184, and C3009 exhibited significant dose-dependent binding activity against the rNA of strain A / H3N2 / Kansas / 14 / 2017, indicating that the fully human antibodies provided by this invention that inhibit the neuraminidase activity of H3N2 influenza virus possess strong NA binding ability.

[0051] Furthermore, none of the four antibodies mentioned above showed any binding activity against the A / H3N2 / Kansas / 14 / 2017 rNA mutant strain carrying the N200S mutation. This result not only indicates that the asparagine (N200) residue at position 200 of the NA protein is located within the binding epitope of these antibodies, but also suggests that C24, C71, C184, and C3009 recognize overlapping antigenic epitopes and belong to the same epitope bin.

[0052] Example 5: Plaque assay to detect antiviral activity 1. Cell plating: Place 5 × 10⁶ cells into a plate. 5 One MDCK cell was seeded in a 6-well plate (NEST) and incubated at 37°C in a 5% CO2 incubator for 24 hours; 2. Virus-antibody incubation: Take 70 pfu of A / H3N2 / Jilin-Chaoyang / 1230 / 2024 virus and mix it with the four fully human antibodies (C24, C71, C3009 and C184) prepared in Example 4 (final concentration 15 μg / mL) in Opti-MEM (containing 1% penicillin-streptomycin) and incubate at room temperature for 1 hour; 3. Cell infection: Wash cells with PBS, add 250 μl of virus-antibody mixture per well, and incubate at 37°C for 1 hour, gently shaking every 15-20 minutes during the incubation period; 4. Adding the cover layer: Add 2 mL of cover layer (containing 1×MEM, 0.67% low melting point agarose, DEAE, TPCK-trypsin and 15 μg / ml antibody) to each well and incubate at 37°C for 72 hours; 5. Fixation and staining: Cells were fixed with 4% paraformaldehyde (White Shark Biotechnology), and after removing the covering layer, they were stained with crystal violet (Beyond the Sky), washed with water, and then photographed as plaques.

[0053] This embodiment evaluated the inhibitory ability of this type of fully human antibody against influenza A virus H3N2, and the results are as follows: Figure 5As shown, compared with the control group, antibodies C24, C71, C184, and C3009 significantly inhibited plaque formation of the A / H3N2 / Jilin-Chaoyang / 1230 / 2024 strain of virus, indicating that the fully human antibodies provided by this invention that inhibit the neuraminidase activity of H3N2 influenza virus have a strong inhibitory ability against H3N2 influenza A virus.

[0054] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A class of fully human antibodies that inhibit the activity of neuraminidase of H3N2 influenza virus, characterized in that, The fully human antibody binds to the same epitope group on the H3N2 influenza virus neuraminidase protein, blocking the activity of H3N2 influenza virus neuraminidase and inhibiting the virus. The fully human antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH and the light chain variable region VL comprise: (1) The following C24-H-CDR1~3 and C24-L-CDR1~3, wherein C24-H-CDR1~3 and C24-L-CDR1~3 are defined according to the IMGT numbering system; The C24-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 1~3, respectively have one or more amino acid substitutions, deletions or additions, or have amino acid sequences with at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 1~3; The C24-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 5, respectively, have one or more amino acid sequences with substitutions, deletions or additions of amino acids, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 5; or, (2) The following C71-H-CDR1~3 and C71-L-CDR1~3, wherein C71-H-CDR1~3 and C71-L-CDR1~3 are defined according to the IMGT numbering system; The C71-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 6~8, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 6~8. The C71-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 9, respectively, have one or more amino acid sequences with substitutions, deletions or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 9; or, (3) The following C3009-H-CDR1~3 and C3009-L-CDR1~3, wherein C3009-H-CDR1~3 and C3009-L-CDR1~3 are defined according to the IMGT numbering system; The C3009-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 10~12, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 10~12; The C3009-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 13, respectively, have one or more amino acid sequences with substitutions, deletions or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4, aspartic acid D-alanine A-alanine A and SEQ ID No. 13; or, (4) The following C184-H-CDR1~3 and C184-L-CDR1~3, wherein C184-H-CDR1~3 and C184-L-CDR1~3 are defined according to the IMGT numbering system; The C184-H-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 14~16, respectively have one or more amino acid substitutions, deletions or additions, or amino acid sequences that have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 14~16. The C184-L-CDR1~3, compared with the amino acid sequences shown in SEQ ID No. 17, aspartic acid D-alanine A-alanine A, and SEQ ID No. 9, respectively, have one or more amino acid sequences with substitutions, deletions, or additions, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 17, aspartic acid D-alanine A-alanine A, and SEQ ID No.

9.

2. The fully human antibody according to claim 1, characterized in that, The heavy chain variable region VH and the light chain variable region VL retain the neuraminidase binding activity and enzyme activity inhibitory activity of H3N2 influenza virus. The heavy chain variable region VH and the light chain variable region VL are as follows: (1) The following heavy chain variable region C24-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.18, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.18; The light chain variable region C24-L described below has an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO.19, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.

19. or, (2) The following heavy chain variable region C71-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.20, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.20; The light chain variable region C71-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.21, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.

21. or, (3) The following heavy chain variable region C3009-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.22, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.22; The light chain variable region C3009-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.23, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.

23. or, (4) The following heavy chain variable region C184-H has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.24, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.24; The light chain variable region C184-L described below has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO.

25.

3. The fully human antibody according to claim 1, characterized in that, The fully human antibody is selected from any one of immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin E, or immunoglobulin D.

4. The fully human antibody according to claim 1, characterized in that, The fully human antibody includes one or more of the following forms: Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, biantibody, single-domain antibody, chimeric antibody, bispecific antibody, or multispecific antibody.

5. A nucleic acid, characterized in that, The nucleic acid is an amino acid sequence encoding the fully human antibody according to any one of claims 1 to 4.

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

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

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: the fully human antibody of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6 or the host cell of claim 7, and a pharmaceutically acceptable carrier and / or excipient; the pharmaceutical composition further comprises other pharmaceutically active agents selected from M2 protein ion channel inhibitors and neuraminidase inhibitors; the M2 protein ion channel inhibitors include, but are not limited to, amantadine or rimantadine, and the neuraminidase inhibitors include, but are not limited to, oseltamivir.

9. A reagent kit, characterized in that, The kit includes the fully human antibody as described in any one of claims 1 to 4; the kit further includes a second antibody that specifically recognizes the antibody, the second antibody having a detectable label; the detectable label is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides or biotin; the enzyme is horseradish peroxidase or alkaline phosphatase; the chemiluminescent reagent is an acridinium ester compound, luminol and its derivatives or ruthenium derivatives; the fluorescent dye is fluorescein or fluorescent protein.

10. The use of the fully human antibody according to any one of claims 1 to 4, the nucleic acid according to claim 5, the expression vector according to claim 6, the host cell according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament, characterized in that, The drug is used to prevent or treat diseases caused by infection with influenza A virus H3N2, or as a passive immunizing agent or long-acting prophylactic biological agent against influenza A virus H3N2. The diseases mentioned include influenza pneumonia, acute respiratory infection, mild influenza, and severe influenza; The drug may be used alone or in combination with other pharmaceutically active agents; The other pharmaceutically active agents are antiviral drugs for influenza; The anti-influenza virus drugs are M2 protein ion channel inhibitors and neuraminidase inhibitors. The M2 protein ion channel inhibitors include, but are not limited to, amantadine and rimantadine, and the neuraminidase inhibitors include, but are not limited to, oseltamivir.

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