CN110642944B

CN110642944B - Antibody for neutralizing human infected H7N9 influenza A virus and application thereof

Info

Publication number: CN110642944B
Application number: CN201910976103.4A
Authority: CN
Inventors: 谢良志; 孙春昀; 张�杰; 宋德勇; 饶木顶; 朱萍霞; 李成红
Original assignee: Sinocelltech Ltd
Current assignee: Sinocelltech Ltd
Priority date: 2014-03-07
Filing date: 2014-03-07
Publication date: 2021-06-11
Anticipated expiration: 2034-03-07
Also published as: WO2015131836A1; CN110642944A; CN104892753B; CN104892753A

Abstract

The present invention relates to humanized antibodies that bind to and neutralize human hemagglutinin protein of influenza a virus infected with H7N9 and antibody fragments that bind to its antigen, including the nucleotide and amino acid sequences of the variable regions of the heavy and light chains of the antibody. The invention also relates to a method for efficiently expressing the complete antibody in a CHO cell system and the like. The invention also relates to the use of said antibodies, associated hemagglutinin protein antigen-binding fragments and epitopes in the diagnosis, treatment and prevention of influenza a virus infection in humans with H7N 9.

Description

Antibody for neutralizing human infected H7N9 influenza A virus and application thereof

The application is a divisional application of an invention patent application with the application number of 201410081962.4, the application date of 2014 is 3, 7 and the invention name is 'an antibody for neutralizing human infection with H7N9 influenza A virus and the application thereof'.

Technical Field

The invention relates to a novel antibody series of hemagglutinin HA protein of A-type H7N9 influenza and humanized antibodies thereof, which can neutralize 100TCID under a smaller dose₅₀The H7N9 virus infects MDCK cells to half, and protects mice under 10LD50 dose to achieve 90% death protection rate, so the antibody can be used for treating and preventing diseases caused by the H7N9 virus. The invention also relates to the binding epitope of the H7N9 hemagglutinin protein bound by the antibody and the amino acid bound by the antibody. The invention also relates to a method for producing the antibody in CHO cells by adopting gene recombination technology。

Background

Influenza viruses are defined by the type of Hemagglutinin (HA) as two types a and B, type a can be subdivided into 16 subtypes, with group I existing with H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes and group II consisting of H3, H4, H7, H10, H14 and H15 subtypes. H1, H2 and H3 in types B and a cause a wider range of influenza diseases in humans, and H5, H7 and H9 subtypes cause sporadic severe infections and may cause new pandemics in humans. From 2 months to 6 months in 2013, people with outbreaks of high pathogenicity and high mortality in China infected with influenza A H7N9 virus, and 132 people in total, of which 43 died [ Rongbao G et al N Engl J Med.2013,368: 1888-. The virus is reassorted by H7 and N9 subtypes of virus that are shed in humans, and no corresponding immunoreactive memory is established in humans, so that severe respiratory distress syndrome can be triggered by the viral infection, leading to death of the patient [ Kageyama T et al, Euro SURvell.2013.18:20453 ]. The human seasonal influenza vaccine consists of A-H1N1, A-H3N2 and type-B3 subtypes, and the immune response established by a human body to the vaccine has no protective effect on H7N9 virus. Therefore, after the H7N9 patient is infected, exogenous antiviral drugs are urgently needed to inhibit the amplification of the virus in vivo. Although there is currently no transmission of H7N9 infection between humans, there have been several experiments that have shown that non-contact airborne transmission can occur between influenza-sensitive animals such as ferrets, pigs [ Herfst S, et al, science.2012; 336: 1534-41; zhu et al, science.2013.341(6142):183-186 ]. Autumn and winter are high-rise seasons of influenza, and effective drugs are needed to control and treat the emergence of new H7N9 influenza cases that may occur.

Currently, the H7N9 virus inhibiting drug is duffy (Oseltamivir) or an analogue thereof (Zanamivir), the small molecule drug is an enzyme activity inhibitor of the NA protein of the influenza virus, and the small molecule drug has the effect of avoiding or reducing the amplification of the virus so as to achieve the treatment effect of reducing the virus titer. The medicine has certain clinical effect, but the virus clearance rate is relatively slow, and duffy resistant strains have been reported, so that more ideal medicines are needed to quickly reduce the virus replication in vivo [ Yunwen Hu et al, Lancet.2013.381:2273-79 ].

It has been reported that influenza critically ill patients are rescued by serum from convalescent patients, and therefore neutralizing antibodies are also an antiviral drug under intense development for prevention and treatment. The hemagglutinin protein (HA), which is the trimeric protein HA0 consisting of HA1 and HA2 subunits and comprising more than 500 amino acids, is the primary target of broad-spectrum and subtype-specific antibodies. The HA1 head part is the binding part of the virus and the sialic acid receptor of the target cell, the virus can be adhered to the cell, the conformational change of the stem HA2 region can be triggered by the environment with low pH value, the virus can be fused with the cell membrane, when the virus is endocytosed in the cell, the genome of the virus is released to the cytoplasm, and the new virus can be replicated in the cell. Antibodies directed against HA can effectively block the process of viral infection of cells, thereby interrupting the viral replication chain. The regions of HA1 that bind sialic acid in the influenza receptor vary widely among subtypes, and the same subtype HAs evolved new variants in the HA1 region, thus, this site HAs no broad spectrum but is the most effective neutralization site [ Kida H et al, vaccine.1985,3,219-222 ]. The existing broad-spectrum antibody mainly aims at a relatively conserved HA2 region, and after the antibody is combined with an HA2 epitope, the allosteric of an HA protein can be prevented by steric hindrance to block the infection process of the virus. The antibody of M2 protein has also been reported to have protective effect in vivo, M2 is the ion channel protein on the surface of virus, and has only 24 amino acids on the surface of virus or cell, and when the virus infects cells, the cell surface antigen expresses a large amount of M2 protein. The anti-M2 antibody binds to virus-replicating cells and kills the cells by ADCC and CDC, thereby exerting a virus-inhibitory effect. Many different types of anti-hemagglutinin and anti-M2 antibodies are currently in development, a small number of antibodies have entered clinical phase I and II studies, and no antibody drug is currently on the market.

Despite decades of research, rapid identification of the most effective emergency drugs for emergencies, such as H7N9 virus outbreak, remains a very crucial and challenging task, and thus it is necessary to identify the most effective drugs through various channels (chemical, traditional and biological).

Disclosure of Invention

The invention mainly relates to an antibody and an antigen binding fragment thereof for binding human H7N9 influenza A virus hemagglutinin protein, wherein the antibody is obtained by screening H7N9(A/Anhui/1/2013) hemagglutinin protein from an antibody library constructed from spleen and bone marrow tissues of rabbits immunized by H7N 7A/Netherlands/219/2003 and H7N9(A/Anhui/1/2013) hemagglutinin protein HA, and the binding epitope of the antibody is a region on the surface of HA1 bound with sialic acid. Accordingly, the invention encompasses antibodies and antigen binding fragments thereof that neutralize human infection with H7N9 and related H7 influenza a virus infection.

In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that neutralizes H7-associated influenza A virus, comprising any one of sequences (a) - (H), wherein

(a) A light chain variable region having light chain CDR1 of SEQ ID NO: 1. light chain CDR2 is SEQ ID NO: 7. light chain CDR3 is SEQ ID NO: 11 and the heavy chain variable region of which heavy chain CDR1 is SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 16. heavy chain CDR3 is SEQ ID NO: 19;

(b) a light chain variable region having light chain CDR1 of SEQ ID NO: 2. light chain CDR2 is SEQ ID NO: 8. light chain CDR3 is SEQ ID NO: 12 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(c) a light chain variable region having light chain CDR1 of SEQ ID NO: 3. light chain CDR2 is SEQ ID NO: 7. light chain CDR3 is SEQ ID NO: 11 and the heavy chain variable region of which heavy chain CDR1 is SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(d) a light chain variable region having light chain CDR1 of SEQ ID NO: 2. light chain CDR2 is SEQ ID NO: 8. light chain CDR3 is SEQ ID NO: 13 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(e) a light chain variable region having light chain CDR1 of SEQ ID NO: 2. light chain CDR2 is SEQ ID NO: 8. light chain CDR3 is SEQ ID NO: 12 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(f) a light chain variable region having light chain CDR1 of SEQ ID NO: 4. light chain CDR2 is SEQ ID NO: 9. light chain CDR3 is SEQ ID NO: 14 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(g) a light chain variable region having light chain CDR1 of SEQ ID NO: 5. light chain CDR2 is SEQ ID NO: 10. light chain CDR3 is SEQ ID NO: 11 and the heavy chain variable region of which heavy chain CDR1 is SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

(h) a light chain variable region having light chain CDR1 of SEQ ID NO: 6. light chain CDR2 is SEQ ID NO: 8. light chain CDR3 is SEQ ID NO: 13 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19.

in one embodiment, the antibody comprises any one of sequences (a) - (h), wherein

(a) A light chain variable region having the amino acid sequence of SEQ ID NO: 20; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29;

(b) a light chain variable region having the amino acid sequence of SEQ ID NO: 21; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 30, of a nitrogen-containing gas;

(c) a light chain variable region having the amino acid sequence of SEQ ID NO: 22; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31;

(d) a light chain variable region having the amino acid sequence of SEQ ID NO: 23; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 32, a first step of removing the first layer;

(e) a light chain variable region having the amino acid sequence of SEQ ID NO: 24; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 33;

(f) a light chain variable region having the amino acid sequence of SEQ ID NO: 25; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 34;

(g) a light chain variable region having the amino acid sequence of SEQ ID NO: 26; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 35;

(h) a light chain variable region having the amino acid sequence of SEQ ID NO: 27; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 36.

in one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is an isolated antibody.

In one embodiment, the antigen binding fragment is a Fab, Fab ', F (ab')2, Fv, scFv and the antibody is a scFv-based multi-chain antibody.

In one embodiment, the H7-related influenza a virus is H7N 9.

In another aspect, the invention relates to a nucleic acid molecule of a polynucleotide encoding the aforementioned antibody or antigen-binding fragment thereof.

In another aspect, the invention relates to a vector comprising the nucleic acid molecule as described above.

In another aspect, the invention relates to a cell expressing the aforementioned antibody or antigen-binding fragment thereof.

In one embodiment, the cell comprises a nucleic acid molecule as described above.

In one embodiment, the cell comprises the vector as described above.

In another aspect, the invention relates to the use of the aforementioned antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of influenza a virus infection.

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof as described above or a nucleic acid molecule as described above or a vector as described above or a cell as described above in the preparation of (i) a medicament for the treatment of an H7-related influenza a virus infection; (ii) h7-related influenza a virus vaccine detection agent; or (iii) H7-related influenza A virus infection.

In one embodiment, the H7-related influenza a virus is H7N 9.

In another embodiment of the invention, the invention comprises an antibody or antigen binding fragment thereof that neutralizes human infection with influenza A H7N9 virus, wherein said antibody is derived from a library of rabbit antibodies to the hemagglutinin protein of H7N7(A/Netherlands/219/2003), wherein the antibodies are humanized antibodies, and wherein the antibodies are humanized by design from rabbit antibodies, and wherein the antibodies are produced by expression in eukaryotic cells.

In yet another aspect, the invention includes hemagglutinin proteins and antibody binding epitopes comprising antibodies or antigen binding fragments thereof that bind to the invention, and immunogenic polypeptides from which the antibodies can be obtained that are isofunctional.

The invention further includes a pharmaceutical composition comprising an antibody of the invention and antigen binding fragments thereof, a nucleotide molecule of the invention, a vector comprising a nucleic acid molecule of the invention, a cell expressing an antibody or antibody fragment of the invention, or an immunogenic polypeptide of the invention, and a pharmaceutically acceptable diluent or carrier.

The antibody and antigen binding fragment thereof of the present invention, the nucleotide sequence of the present invention, the vector comprising the nucleotide sequence of the present invention, the cell expressing the vector of the present invention, the epitope comprising the antibody or antibody fragment binding thereto of the present invention and the immunogenic polypeptide thereof, or the pharmaceutical composition of the present invention (i) in the preparation of a medicament for the treatment and prevention of human infection with influenza a virus of H7N 9; (ii) use in vaccines or (iii) in the diagnosis of human infection with influenza a virus H7N9 is included within the scope of the invention. Further, the use of the antibodies or antigen binding fragments of the invention to detect whether the hemagglutinin antigen of human infected H7N9 influenza A virus has a specific epitope in the correct conformation to monitor the quality of the H7N9 virus or hemagglutinin protein vaccine is also within the scope of the invention.

In another aspect, the invention includes a method of reducing infection in a human by influenza a virus infection H7N9, or reducing the risk of infection in a human by influenza a virus infection H7N9, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody, or antigen-binding antibody fragment thereof, of the invention.

In another aspect, the invention includes an epitope that specifically binds to an antibody or antigenic fragment thereof of the invention for use in: (i) preparing a medicament for treating human infection with influenza a virus H7N 9; (ii) detecting the structure of a medicament for treating human infection with H7N9 influenza A virus; (iii) as a vaccine; or (iv) screening for ligands that neutralize human infection with influenza A virus H7N 9.

Drawings

FIG. 1: hemagglutination inhibitory activity of rabbit-derived anti-H7N9 antibody against H7N9(A/Anhui/1/2013) influenza virus hemagglutinin HA protein (2. mu.g/mL). 1a is a photograph of agglutination of the hemagglutination plate, and 1b is an analysis of agglutination data of the antibody.

FIG. 2: hemagglutination inhibitory activity of humanized H7N9 antibody H7N9-H002 and original rabbit antibody against 4H 7N9(A/Anhui/1/2013, A/Shanghai/1/2013, A/Hangzhou/1/2013, A/Pigeon/Shanghai/S1069/2013) influenza virus hemagglutinin HA proteins.

FIG. 3: micro-neutralizing (MN) activity of humanized H7N9 antibody H7N9-H002 and original rabbit antibody against H7N9(A/Anhui/1/2013) virus.

FIG. 4: humanized H7N9 antibody H7N9-H002 variable region sequence; 4 a: the light chain variable region nucleotide and amino acid sequences of humanized antibodies, 4 b: the heavy chain variable region nucleotide and amino acid sequences of the humanized antibodies.

FIG. 5: the animal protective effect of humanized H7N9 antibody H7N9-H002 in the flu-preventing group; 5 a: survival of mice, 5 b: body weight loss ratio of mice. The antibody dose of the preventive group was 1mg/kg, 3mg/kg, 10mg/kg and 20mg/kg, respectively, and the virus was H7N9 (A/Anhui/1/2013).

FIG. 6: the animal protection effect of humanized H7N9 antibody H7N9-H002 in influenza treatment group; 6 a: survival of mice, 6 b: body weight loss ratio of mice. The antibody dose in the treatment group was 20mg/kg, the administration time was 3 hours, 1 day and 3 days after challenge, and the virus was H7N9 (A/Anhui/1/2013).

FIG. 7: animal protection effect of humanized H7N9 antibody H7N9-H002 in influenza treatment group, 7 a: survival of mice, 7 b: body weight loss ratio of mice. The antibody dose in the treatment group was 40mg/kg, the administration time was 1 day and 3 days after challenge, and the virus was H7N9 (A/Anhui/1/2013).

FIG. 8: structural schematic diagram of HA protein of H7N9(A/Anhui/1/2013) influenza virus hemagglutinin: the epitope to which humanized H7N9 antibody H7N9-H002 binds the HA protein is indicated by blue; the epitope to which sialic acid of the HA protein receptor binds is shown in red; the portion of the antibody binding epitope that intersects the sialic acid binding epitope is indicated in yellow.

FIG. 9: data for ELISA binding of humanized H7N9 antibody H7N9-H002 to HA protein of different strains of H7N 9.

FIG. 10: data for ELISA binding of humanized H7N9 antibody H7N9-H002 to H7-class viral HA.

Detailed Description

The present invention is based on the discovery and isolation of an animal-derived antibody that specifically neutralizes the enhanced immune maturation of the H7N9 human infectious virus from individual animals immunized with the H7N7 (hemagglutinin HA protein is highly homologous to the H7N9 virus) or H7N9 virus hemagglutinin protein. The neutralizing activity of these antibodies against H7N9 virus is expected and the hemagglutinin protein epitope recognized by the antibodies should be part of the H7N9 virus subunit vaccine.

In one aspect, the invention provides antibodies and antigen-binding fragments thereof that neutralize human influenza a virus infection with H7N9, which in one embodiment can bind to a variety of partially variant H7N9 influenza viruses.

In another aspect of the invention, the invention provides neutralizing antibodies against HA of influenza a virus infected with H7N9 from different humans, and antigen binding fragments thereof. In one embodiment, the antibodies and antigen binding fragments thereof of the invention specifically bind to a sialic acid receptor binding epitope of influenza a virus, H7N 9. In another embodiment, the antibodies and antigen binding fragments thereof of the invention inhibit the hemagglutination activity of influenza a virus HA, H7N 9.

Humanized monoclonal antibodies, CHO, 293 transfected cell lines expressing the antibodies of the invention and nucleotide sequences encoding the antibodies of the invention are included within the scope of the invention.

As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody, as well as fragments that are 60% similar. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fab ', F (ab')2, Fv, or scFv. As used herein, the term "antibody" includes antibodies and antigen-binding fragments thereof.

As used herein, "neutralizing antibody" refers to an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a virus to initiate and/or maintain a host against infection. The terms "neutralizing antibody" or "… neutralizing antibody" are used interchangeably herein. These antibodies may be used alone or as a prophylactic or therapeutic agent in combination with a NA inhibitor or a vaccine or the like in a suitable formulation, as described herein, and include any modification or improvement based on the antibodies.

The antibodies and antigen binding fragments of the invention have high affinity and high neutralizing potency. The concentration of the antibody of the present invention required to neutralize 50% of influenza a virus may be, for example, 200ng/ml or less. In one example, a concentration of about 25ng/ml of an antibody of the present invention is required to neutralize 50% of influenza A virus of 100TCID50A/Anhui/1/2013 virus, with antibody affinities of both nM (10) grades^-9M)。

Antibodies of the invention

The invention provides an antibody which HAs specific binding to a receptor binding region in the HA1 globular head of HA of H7N9 and H7 viruses, and the high-affinity binding of the antibody to the H7N9 virus can prevent the binding of the virus to the receptor, thereby preventing the virus from invading cells and inhibiting the replication and spread of the virus.

Exemplary antibodies of the invention include rabbit antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, and H7N 9-R002.

The CDRs of the antibody heavy chain are referred to as H-CDR1, H-CDR2, and H-CDR3, respectively, and similarly, the CDRs of the antibody light chain are referred to as L-CDR1, L-CDR2, and L-CDR3, respectively. The positions of the CDR amino acids are defined according to the IMGT numbering system as CDR1-IMGT positions 27-38, CDR2-IMGT positions 56-65 and CDR3-IMGT positions 105-117.

Tables 1 and 2 provide the amino acid sequences of the 6 CDRs of the heavy and light chains, respectively, of an exemplary antibody of the invention.

Table 1: amino acid sequence of light chain CDR1-3 of H7N9 rabbit neutralizing antibody

a: all CDR sequence homologies were normalized to H7N9-R002

Table 2: amino acid sequence of heavy chain CDR1-3 of H7N9 rabbit neutralizing antibody

a: all CDR sequence homologies were normalized to H7N9-R002

In one embodiment, the antibody or antibody fragment of the invention comprises at least one CDR having a sequence with at least 95% sequence homology to any one of SEQ ID NOs 1-19, wherein said antibody neutralizes human infected H7N9 influenza a virus.

Exemplary antibodies of the invention include rabbit antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, H7N9-R002 and humanized antibodies H7N 9-H002. in one embodiment, the amino acid sequences of the light chain (VL) and heavy chain variable regions (VH) of the antibodies of the invention and antibody fragments are set forth in SEQ ID NO:28 and SEQ ID NO:37, and the amino acid sequences of the light chain variable regions (VL) and heavy chain variable regions (VH) of the humanized antibodies are set forth in SEQ ID NO:56 and SEQ ID NO: 57.

In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO 28. In another embodiment, the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having an amino acid sequence that is about 92%, 95%, 97%, 98%, 99% or 100% identical to any of the sequences set forth in SEQ ID NO 37.

In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 56. In another embodiment, the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having an amino acid sequence that is about 92%, 95%, 97%, 98%, 99% or 100% identical to any of the sequences set forth in SEQ ID NO. 57.

In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to any of

SEQ ID NOs

20, 21, 22, 23, 24, 25, 26, 27 or 28. In another embodiment, the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to any of

SEQ ID NOs

29, 30, 31, 32, 33, 34, 35, 36 or 37.

Table 3: sequences of the light and heavy chain variable regions of the H7N9 rabbit neutralizing antibody

a: all sequence homologies of the variable regions were normalized to H7N9-R002

The invention also includes an antibody or antibody fragment thereof that competes with, i.e., binds to the same epitope as, an antibody of the invention or an antibody fragment thereof. Including but not limited to H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, H7N9-R002 and humanized antibodies H7N 9-H002.

The antibodies of the invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., alpha, gamma or mu heavy chain), but are predominantly IgG. In the IgG isotype, the antibody may be of the IgG1, IgG2, IgG3 or IgG4 subclasses. The antibodies of the invention may have a kappa or lambda light chain.

Preparation of antibodies

The antibodies of the invention may be prepared by any method known in the art. For example, the sequence of the antibody is inserted into a corresponding eukaryotic expression vector, and transfected into cells, such as a technology for obtaining the antibody by adopting 293 cell transient expression and a technology for obtaining the antibody by adopting CHO cell stable expression. High density cell culture techniques are commonly used to obtain high yields of antibodies.

Antibody purification techniques, including techniques for making pharmaceutical grade antibodies, are also known in the art. Antibodies can be purified by various chromatographic methods such as centrifugation, filtration, affinity, charge, molecular weight, hydrophobicity, etc.

The manner of preparation of the antibody fragments of the invention is also known in the art and includes enzymatic digestion with pepsin or papain, or the antibody fragments may be obtained by cloning or expressing the sequences of portions of the heavy or light chains. Antibody fragments may include scFv, Fab ', F (ab')₂And Fv fragments. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies.

Any suitable host cell/vector system may be used for the expression of the DNA sequences encoding the antibody molecules of the invention and fragments thereof. Suitable host systems include E.coli, yeast cells, insect cells and mammalian host cells. Mammalian host cells include, but are not limited to, CHO, HEK293, per.c6, NSO, SP 2.

The antibody of the present invention can be prepared by the following steps: i) expressing the nucleic acid sequences of the invention in a suitable host cell, and ii) isolating the expressed antibody product, iii) purifying the antibody.

Antibody epitopes

The antibodies of the invention can be used to assay the epitopes to which they bind. The present inventors have found that neutralizing H7N9 influenza a virus infection antibodies bind to an epitope on the HA protein. In one embodiment, the antibody HAs been analyzed for binding epitopes to sialic acid binding regions in the globular head of HA. The epitope bound by the antibody of the invention may be a linear contiguous or conformationally discontinuous sequence of amino acids.

The epitopes recognized by the antibodies of the invention can have a variety of uses. Epitopes and their mimotopes in purified or synthetic form may be used to enhance the immune response (i.e., as vaccines or for antibody preparation for other uses), or to screen sera for antibodies immunoreactive with the epitope or an immunological epitope thereof.

The epitopes of the invention may also be used to screen for ligands that bind to the epitope. Such ligands include, but are not limited to, antibodies (of different species), antibody fragments, peptides, and other similar viral proteins that can block an epitope and thus prevent infection.

The epitopes of the invention may also be used in the development and application of diagnostic tools. Such applications include direct testing and post-labeling use.

Pharmaceutical composition

The invention provides a pharmaceutical composition comprising an antibody and/or antibody fragment of the invention and/or a nucleic acid encoding such an antibody and/or an epitope recognized by an antibody of the invention. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, and inactive viral particles.

The pharmaceutical compositions of the present invention generally have a pH of 5.5-8.5, in some embodiments, the pH may be 7.4, and the pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen-free and isotonic with respect to human body fluids.

The pharmaceutical composition may comprise an effective amount of one or more antibodies of the invention and/or comprise an epitope polypeptide that binds to an antibody of the invention, i.e., an amount sufficient to treat, ameliorate or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect.

Medical treatment and use

The antibodies and antibody fragments or derivatives and variants thereof of the invention are useful for the treatment, prevention and diagnosis of influenza a virus infection in humans with H7N9 infection.

The present invention provides i) an antibody, antibody fragment or variant thereof and derivatives thereof; ii) the mode of production of the antibody of the invention; iii) an epitope capable of binding to an antibody of the invention; or iv) a ligand, preferably an antibody capable of binding an epitope that binds to an antibody of the invention, useful in therapy.

In one embodiment, an antibody, antibody fragment, epitope, or composition of the invention is administered to a subject in need of treatment and prevention. Such subjects include, but are not limited to, patients infected with influenza a virus H7N9, subjects at risk of infection with influenza a virus H7N9 or susceptible to infection with influenza a virus H7N9, e.g., immunocompromised subjects. Due to the broad spectrum protective effect of the antibodies provided in the present invention against viruses within subtypes, the antibodies of the present invention may have similar quality and prophylactic effects against influenza antibodies of other H7 series.

The antibodies or antibody fragments of the invention can be used for passive immunization or active immunization.

The antibodies and fragments thereof described in the present invention may also be used in kits for diagnosing H7N9 for influenza infection. Furthermore, epitopes capable of binding to the antibodies of the invention may be used in kits to monitor the efficiency of vaccination programs by detecting the presence of protective anti-H7N9 influenza a virus antibodies. The antibodies, antibody fragments or variants and derivatives thereof of the present invention may also be used in kits to monitor the production of vaccines with desired immunogenicity.

The invention also provides a method of preparing a medicament comprising the step of admixing a monoclonal antibody and one or more pharmaceutically acceptable carriers. The steps involving obtaining (e.g., by expression and purification) monoclonal antibodies, mixing them with the pharmaceutical carrier can be performed at different times, with different personnel at different locations.

The antibody of the invention is screened from a rabbit antibody immunized by HA protein, and a medicinal antibody is obtained by humanization modification. Constructing a eukaryotic expression vector, transfecting a CHO cell line, carrying out high-density culture expression on the cell, and producing the antibody by separation and purification. Optimization of the antibody, including codon optimization and antibody affinity maturation optimization. The present invention covers all nucleic acids, vectors, sequences, antibodies, etc. used and prepared in these steps.

Examples

Exemplary embodiments of the invention are provided in the following examples. The following examples are given by way of illustration only and are presented to assist one of ordinary skill in the art in utilizing the present invention. The examples are not intended to limit the scope of the invention in any way.

Example 1 analysis of Rabbit-derived neutralizing antibody Activity against H7N9 Virus and sequence information

Rabbit antibody libraries of hemagglutinin proteins of H7N7(A/Netherlands/219/2003) and H7N9(A/Anhui/1/2013) were constructed before outbreak of H7N9 virus, and were panned with hemagglutinin proteins of H7N9 virus (A/Anhui/1/2013) obtained by transient expression in 293 mammals and expression in insect systems, and screened to obtain 30 strains of hemagglutinin-specific antibodies against human infection H7N9 virus. Antibody sequences are constructed into eukaryotic expression vectors and milligram quantities of antibodies are produced in a 293 mammalian transient expression system. Antibodies were tested for binding to various H7N9 viral hemagglutinin proteins (table 4), and hemagglutination inhibition assays were used to detect antibody activity and to detect the ability of the antibodies to inhibit guinea pig hemoglobin hemagglutination of the H7N9 viral hemagglutinin protein. When 2. mu.g/mL of recombinant H7N9(A/Anhui/1/2013) hemagglutinin protein was added in the agglutination assay, 11 of the rabbit monoclonal antibodies had hemagglutination inhibition activity, and all of the 11 antibodies had hemagglutination inhibition ability with high sensitivity (FIG. 1).

Table 4: hemagglutination inhibition antibody obtained by screening 11 strains and subtype broad-spectrum HA protein binding capacity thereof

The CDRs of the 11 antibodies with good hemagglutination activity were analyzed, 9 of them were from the same germline gene family, the light chain was from IGKV1S56 × 01, the amino acid sequence variation of CDR1-3 is shown in Table 1, the heavy chain was from gene family IGHV1S34 × 01, and the amino acid sequence of 3 CDR1-3 is shown in Table 2. The amino acid sequences and homologies of the light chain variable region and the heavy chain variable region are shown in table 3. The binding regions of antibodies sharing the same CDR sequences or less mutated CDR sequences were similar, and the results were also consistent with the hemagglutination inhibition activity of these antibodies.

Example 2 humanized antibodies have high viral neutralizing Activity

According to the expression level and the neutralizing activity of the antibody, a rabbit antibody H7N9-R002 is selected for humanized design to obtain a humanized antibody H7N9-H002, and the humanized modification technology is designed for the rabbit antibody according to the most classical CDR grafting method. The humanized antibody gene sequence is obtained by means of whole gene synthesis and is constructed into eukaryotic expression vector to produce milligram level to gram level protein antibody in 293 mammal transient expression system. The humanized antibody with rabbit antibody affinity is screened through affinity detection with H7N9 hemagglutinin protein to obtain humanized antibody H7N9-H002 with the highest affinity, and the humanized antibody has the variable region of the antibody light chain with amino acid sequence of SEQ ID No. 56, nucleotide sequence of SEQ ID No. 58, variable region of the antibody heavy chain with amino acid sequence of SEQ ID No. 57 and nucleotide sequence of SEQ ID No. 59. The affinity of the humanized antibody and various recombinant H7N9 hemagglutinin proteins is detected, and the specific experiment is as follows: biotinylation of the H7N9-H002 antibody is combined and fixed on the surface of a streptavidin chip, the recombinant hemagglutinin protein is diluted to different concentrations, the combination and dissociation rate of the recombinant hemagglutinin protein and the antibody is detected, and the affinity constant of the antibody combined with the antigen protein is calculated. The results are shown in Table 5.

Table 5: affinity detection of H7N9-H002 humanized antibody and different recombinant H7N9 hemagglutinin proteins

Virus	kon(1/Ms)^(a)	kdis(1/s)^(b)	K_D(M)
				A/Anhui/1/2013	2.32E+04	1.72E-04	7.42E-09
A/Shanghai/1/2013	3.00E+04	1.63E-04	5.42E-09
				A/Hangzhou/1/2013	3.74E+04	1.37E-04	3.68E-09
A/Pigeon/Shanghai/S1069/2013	3.96E+04	1.13E-04	2.85E-09
				A/Hangzhou/3/2013	4.55E+04	2.81E-04	6.16E-09

a: constant of binding rate

b: off rate constant

The anti-agglutination ability of the humanized H7N9-H002 antibody and the original rabbit antibody H7N9-R002 was tested by a guinea pig erythrocyte agglutination inhibition assay. The specific experiment is as follows: the serially diluted antibodies were incubated with 4 agglutination units of different HA proteins for 1h, and an equal volume of 1% guinea pig red blood cells was added and incubated at room temperature for 30 minutes to obtain the concentration of hemagglutination inhibition (FIGS. 1 and 2). Compared with the original rabbit antibody, the H7N9-H002 humanized antibody has higher hemagglutination inhibition titer, and the concentration of the neutralizing antibody is in the range of 0.031-0.25 mu g/mL aiming at different recombinant influenza hemagglutinin proteins.

The virus micro-neutralization experiment is adopted to detect the virus neutralization capacity of the humanized H7N9-H002 antibody and the original rabbit antibody H7N9-R002, and the specific experiment is as follows: antibodies diluted at different concentrations were conjugated to 100X 50% TICD₅₀The H7N9(A/Anhui/1/2013) virus of (1) was incubated at 37 ℃ for 1 hour. mu.L of the virus and antibody mixture was added to MDCK cells and cultured, and after 2 hours, the culture supernatant was aspirated, and after washing the cells with MEM, 100. mu.L of MEM medium containing 1. mu.g/mL of TPCK-treated pancreatin was replaced. After 3 days of incubation at 37 ℃, CPE (viral plaques) were counted. The lowest antibody concentration at which 50% of MDCK cells are free of infection is the neutralizing concentration of the antibody. The data are shown in FIG. 3, and the humanized antibody has obviously reduced virus neutralization titer compared with the original rabbit antibody, and the titer of the antibody for inhibiting H7N9 virus in vitro is as high as 0.025 mu g/mL.

Example 3 recombinant production of humanized anti-H7N9 antibody

Host cells expressing glycosylated anti-H7N9 monoclonal antibodies can be derived from a variety of tissues, but are preferably vertebrate cells. Cell lines which may be used are SV 40-transformed monkey kidney CV1 cell line (ATCC CRL 1651), human embryonic kidney cells (293 or 293 in suspension culture [ Graham et al, J.Gen Virol.197736: 59 ], baby hamster kidney cells (BHK-21 ATCC CCL 10), Chinese hamster ovary cells (CHO/dhfr-ATCC CRL 9096), monkey kidney cells (CV1 ATCC CRL90), African green monkey kidney cells (VERO-76 ATCC CRL 1587), human uterine cancer cells (HELA ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), human lung cells (W138, ATCC CCL95), human liver cells (Hep G2, HB 8065) and the like.

In the present invention, cells such as CHO cells producing the anti-H7N9 monoclonal antibody can be cultured in various media. Commercial culture media such as DMEM, MEM, Ham's F12, RPMI-1640(Gibco) can be used for the culture of host cells. Furthermore, Ham et al, meth.Enz.197958: 44; barnes et al, anal. biochem.1980102: 255; U.S. Pat. nos.4,767, 707; 4,657,866, respectively; 4,927,762 can be used to culture the host cells. The culture conditions of the host cells, such as temperature, pH, etc., are also conventional conditions well known to those skilled in the art.

Transfection of mammalian host cells with the humanized anti-H7N9 monoclonal antibody can be carried out using conventional techniques well known to those skilled in the art. When the host cell is a CHO/dhfr cell, calcium phosphate coprecipitation method [ Jordan et al, Nucleic Acids Res.199624: 4 ], liposome packaging method (e.g., lipofectamine 2000) [ Audouy S.et al, Mol Membr biol.200118 (2):129 ], electroporation method and microinjection method [ Morrison et al, Science 1985229:1202 ] can be used as DNA transfection methods. We used the Lipofectamine 2000(Invitrogen) transfection method, according to the instructions, 4 expression vectors pIRESneo3d-anti-H7N9 of the complete antibody sequence N2-1, N5-4, N6-3, N8-3 to prepare a mixture of 1.5. mu.g plasmid and 4.5. mu.L Lipofectamine 2000, co-transfect 6X 105 cells in one well of a 6-well plate, and after overnight transfection, distribute the cells evenly into 1 96-well plate.

Whole antibody transfected CHO cells were screened in DMEM + 5% dFBS +1.0mg/mL G418, the screening medium was changed every 2-3 days, and 20 selected cell clones were obtained each for 2-3 weeks. The cells were transferred to a 24-well plate at the same density, cultured in 0.5mL DMEM + 5% dFBS medium for 5 days, and the expression amount of the humanized anti-H7N9 monoclonal antibody in the culture supernatant was measured by ELISA.

In order to make the humanized anti-H7N9 monoclonal antibody cell screened by G418 more efficiently express, high expression water is selectedThe flat cells were expressed by MTX amplification. Specifically, recombinant CHO cells were cultured in DMEM + 5% dFBS medium, gradually increasing MTX concentration was added during the culture, the expression level of the antibody was continuously monitored during the culture, the increase of the MTX concentration was stopped when the MTX concentration increased to a level that is not tolerated by the cell growth, and the expression amount of the humanized anti-H7N9 monoclonal antibody in the culture medium was measured by ELISA after five days of culture in a T25 flask. And (3) culturing antibody production cells by adopting a high-density cell culture process, and obtaining a kilogram-level antibody production process with the yield of 0.8-1.4 g/L through process optimization. H7N9-H002 antibody was prepared in PBS buffer (Na) at a concentration of up to 25mg/mL₂HPO₄ 10mM,KH₂PO₄ 1.8mM,NaCl 137mM,KCl 2.7mM,pH7.4)。

Example 4 in vivo prophylactic and therapeutic Effect of humanized antibodies in challenge model

The H7N9 model for counteracting toxic substance is selected from BALB/c mother mouse of 4-6 weeks old, and half Lethal Dose (LD) is searched₅₀) The required virus titer. Through groping, determine 10 times LD₅₀The virus dose of (A) is 2.5X 10⁶TCID₅₀。

In the prevention effect study, 10 mice are selected in each group, 1, 3, 10 or 20mg/kg of H7N9-H002 humanized antibody is injected intravenously, and after 24 hours, the nasal cavity is infected with 10-fold LD₅₀H7N9 (A/Anhui/1/2013). Mice were observed for mortality over 2 weeks, body weight changes were recorded, virus titers were tested in the 1mg/kg dose group and histopathological staining was analyzed. The results showed that 10 mice in the vehicle group all died at day 11, whereas all the dosing groups achieved 100% mortality protection (fig. 5), and the body weight data also had a clear correlation with the dose (fig. 5), except that a short, significant drop in body weight of the mice occurred in the 1mg/kg dose group. In the dose group of 1mg/kg, the viral titer in the lung and nasal cavity of the mice was reduced by about 100 times (Table 6), so that the viral load in vivo could be effectively reduced, the excessive response of the immune system to the virus could be alleviated, and the weight loss and the damage of the virus to the lung tissue could be effectively reduced.

Table 6: comparison of viral titers in the antibody-administered group and vehicle group in lung and nasal tissues (1mg/kg dose-prevented group)

In the study of therapeutic effect, 10 mice were combined and nasal cavity was infected with 10-fold LD₅₀The H7N9(A/Anhui/1/2013) virus of (1), the first dose group was infected 3 hours, 20mg/kg of antibody was intravenously injected after 1 day and 3 days, and the second dose group was infected 1 day and 3 days, and 40mg/kg of antibody was intravenously injected. Mice were observed for mortality over 2 weeks, body weight changes were recorded, 20mg/kg was detected, and virus titers and histopathological staining were analyzed in the dose groups after 1 day. The results showed that 10 mice in the vehicle group all died at 10 days, and the survival rate of the mice in the group administered 3 hours after the infection with the 20mg/kg dose and the group administered 1 day after the infection with the 40mg/kg dose was as high as 90% (fig. 6 and 7). Mortality and body weight data (figures 6 and 7) of the mice correlated with the dose and time of administration, indicating that the antibody had a clear therapeutic effect. The viral titer in the lungs and nasal cavity administered 1 day after infection in the 20mg/kg dose group decreased by over 1000-fold, significantly reducing the live virus level in focal tissues (table 7). Pathological staining of lung tissue also showed more pronounced symptoms of reduced inflammation relative to vehicle group.

Table 7: comparison of Virus Titers in antibody-administered group and vehicle group in Lung and nasal tissues (20mg/kg dose treatment group)

a: below the limit of virus detection

Example 5 epitope analysis of H7N9-H002 binding to HA protein

The humanized antibody H7N9-H002 HAs HA hemagglutination inhibiting activity, and the binding region of the antibody is in HA1 subunit of HA protein and HAs great overlap with the binding region of HA receptor sialic acid. By adopting Discovery Studio 3.5 protein structure simulation software, the spatial structure of the H7N9-H002 antibody is simulated, and more accurate antibody conformation is obtained. The binding site of H7N9-H002 on the crystal structure of H7N7(A/Netherlands/219/2003) [ PDB entry:4DJ6] was analyzed using Discovery Studio 3.5 protein interaction site docking software. Since the amino acid homology of the hemagglutinin proteins of H7N 7A/Netherlands/219/2003 and H7N 9A/Anhui/1/2013 is as high as 94.6%, and H7N9-H002 can well bind to the hemagglutinin protein of H7N 7A/Netherlands/219/2003, the region of the hemagglutinin protein where the antibody binds to H7N 7A/Netherlands/219/2003 is considered to be the region of the hemagglutinin protein where the antibody binds to H7N 9A/Anhui/1/2013. This region contains mainly 3 parts: i) the outermost beta-sheet (154-162aa) region of the HA1 head; ii) sialic acid receptor binds to the most important HA1 loop region (213-218 aa); iii) a 3-dimensional overhang region consisting of Gln53, Gln65, Arg81, Glu82 and Ser 84. The results of the specific bound amino acid analysis are shown in FIG. 8 and Table 8.

Table 8: H7N9-H002 binds to amino acid site of hemagglutinin protein of H7N9 virus

Example 6 binding of different strains of H7N9-H002 and H7N9 to other types of H7 viruses

7H 7N9 virus strains with amino acid point mutations (A/Anhui/1/2013, A/Shanghai/1/2013, A/Hangzhou/1/2013, A/Pigeon/Shanghai/S1069/2013, A/Hangzhou/3/2013, A/Shanghai/4664T/2013, A/Zhejiang/1/2013) and 7 other types of H7 strains [ A/turn/Italy/4602/99 (H7N1), A/turn/SK HR/00011/2007 (H7N3), A/turn/Italy/214845/2002 (H7N3), A/equene/Kentucky/1 a/1975(H7N7), A/mallallard/Netherian/33/2006 (H587N 8), A/Netherian/587N 587/2017 (H587/2017N), A/Netherlands/219/2003(H7N7) ] totaling 14 HA species, HA protein was coated on 96-well ELISA plates (0.0125. mu.g/mL-0.4. mu.g/mL) at 2 concentrations of 0.0125. mu.g/mL and 0.025. mu.g/mL and coated overnight at 4 ℃. The next day, the plate was washed, blocked at room temperature for 1 hour, and then 2. mu.g/mL H7N9-H002 was added and allowed to react at room temperature for 1 hour. Washing the plate after 1h, adding a detection antibody goat anti-human IgG Fc/HRP, washing the plate after acting for 1h at room temperature, adding TMB for color development, and determining OD450 after the color development reaction is stopped, wherein the color depth of the color development reaction is in direct proportion to the concentration of the HA protein coated in the hole. The results of ELISA binding are shown in fig. 9 and 10, and the data show that both antibodies bind well to the HA proteins of these viruses. Based on the mechanism of the virus action and the antigen-antibody binding capacity, the H7N9-H002 has good virus neutralization effect on the H7N9 virus variant and other types of H7 viruses.

Sequence listing

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<120> an antibody for neutralizing human infection with influenza A virus H7N9 and use thereof

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Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

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Leu Ile Tyr Phe Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe

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Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

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Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe

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Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

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Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Asp Tyr Ser Gly

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Gly Ile Tyr Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Leu

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Asn Asn Leu Ala Trp Phe Gln Gln Lys Leu Gly Gln Pro Pro Thr Leu

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Leu Ile Trp Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

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Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

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Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Asp Tyr Ser Gly

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Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Glu Ser Val Tyr Lys Asn

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Asn Asn Leu Ala Trp Phe Gln Gln Lys Leu Gly Gln Pro Pro Thr Leu

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Leu Ile Trp Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

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Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

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Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

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Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

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Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

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Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Gly

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Arg Ser Thr Ile Thr Arg Asn Thr Asp Leu Asn Thr Val Thr Leu Lys

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Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

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Gln Ser Val Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

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Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

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Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

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Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser

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Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

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Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

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Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

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<211> 111

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 31

Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

20 25 30

Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

35 40 45

Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

85 90 95

Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 32

<211> 111

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 32

Gln Ser Val Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

20 25 30

Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

35 40 45

Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

85 90 95

Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 33

<211> 111

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 33

Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

20 25 30

Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

35 40 45

Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

85 90 95

Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 34

<211> 112

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 34

Gln Glu Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn

20 25 30

Glu Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys

50 55 60

Ser Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu

65 70 75 80

Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Ser Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 35

<211> 112

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 35

Gln Gln Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn

20 25 30

Glu Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys

50 55 60

Ser Arg Ser Thr Ile Thr Arg Asn Thr Asp Leu Asn Thr Val Thr Leu

65 70 75 80

Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Ser Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 36

<211> 111

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 36

Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Tyr Ser Asn Glu

20 25 30

Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

35 40 45

Ala Ile Ala Gly Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Ser

85 90 95

Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 37

<211> 112

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 37

Gln Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Arg Val Ser Gly Leu Ser Leu Phe Ser Asn

20 25 30

Glu Ile Asn Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Gly Gly Gly Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys

50 55 60

Ser Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu

65 70 75 80

Arg Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Ala Tyr Phe Cys Ala

85 90 95

Ser Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 38

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 38

gaccctatgc tgacccagac tgcatcgccc gtgtctgcag ctgtgggagg cacagtcacc 60

atcaattgcc aggccagtca gagtgtttat aataaaaata acttagcctg gtatcagcag 120

aaactagggc agcctcccaa actcctgatc tatgctgcat ccactctgac atctggggtc 180

ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgg tatttatgct 300

ttcggcggag gaaccaaggt ggagatcaaa 330

<210> 39

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 39

cagtcgttgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggagtggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaag gtcgatccac catcaccaga aacaccgacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt cactgtctct tca 333

<210> 40

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 40

gagctcgtga tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60

atcagttgcc agtccagtga gagtgtttat aagaacaaca acttagcctg gtttcagcag 120

aaactagggc agcctcccaa gctcctgatc tatgatgcat ccaaactggc atctggggtc 180

ccatcgcggt tcagtggcag tgggtctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtag taattatgct 300

ttcggcggag gaaccaagct ggagatcaaa 330

<210> 41

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 41

cagtcggtgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggaatggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaaa gccgatccac catcaccaga aacaccaacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt cactgtctct tca 333

<210> 42

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 42

gagcctatgc tgacccagac tgcatcgtcc gtgtctgcag ctgtgggagg ctcagtcacc 60

atcagttgcc agtccagtca gagtgttgat agtaacaacc aattatcctg gtatcagcag 120

aaaccagggc agcctcccaa actcctgatc tatgctgcat ccactctgac atctggggtc 180

ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgg tatttatgct 300

ttcggcggag ggaccgaggt ggtggtcaaa 330

<210> 43

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 43

cagtcgttgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggaatggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaaa gccgttccac catcaccaga aacaccaacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt caccgtctct tca 333

<210> 44

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 44

gaccctatgc tgacccagac tgcagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60

atcagttgcc agtccagtga gagtgtttat aagaacaaca acttagcctg gtttcagcag 120

aaactaggcc agcctcccac gctcctgatc tgggatgcat ccaaactggc atctggggtc 180

ccatcgcggt tcagtggcag tgggtctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgt taattatgct 300

ttcggcggag ggaccgaggt ggtcgtcaaa 330

<210> 45

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 45

cagtcggtgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggaatggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaaa gccgatccac catcaccaga aacaccaacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt cactgtctct tca 333

<210> 46

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 46

gagctcgtga tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60

atcagttgcc agtccagtga gagtgtttat aagaacaaca acttagcctg gtttcagcag 120

aaactagggc agcctcccaa gctcctgatc tatgatgcat ccaaactggc atctggggtc 180

ccatcgcggt tcagtggcag tgggtctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtag taattatgct 300

ttcggcggag ggaccgaggt ggtcgtcaaa 330

<210> 47

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 47

cagtcgttgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggaatggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaaa gccgttccac catcaccaga aacaccaacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt cactgtctct tca 333

<210> 48

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 48

gaccctatgc tgacccagac tgcatcgtcc gtgtctgcaa ctgtgggagg cacagtcacc 60

atcagttgcc agtccagtca gagtgttgat aataacaaag atttagcctg gtttcagcag 120

aaaccagggc actctcctaa gctcctgatc tattttgcgt ccagactggc atctggggtc 180

ccgtcgcggt tcaaaggcag tggatctggg acacacttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgc taattatgct 300

ttcggcggag gaaccaagct ggagatcaaa 330

<210> 49

<211> 336

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 49

caggagcagc tgatggagtc cgggggaggc ctggtcaagc ctggaggatc cctgacactc 60

acctgcacag tctctggatt gtccctctat agcaatgaaa taaattgggt ccgccaggct 120

ccagggaacg ggctggaatg gatcggagcc attgctggta gtggtagcgc cgactacgcg 180

agctgggcga aaagccgttc caccatcacc agaaacacca acctgaacac ggtgactctg 240

aaaatgacca gtctgacagc cgcggacacg gccacctatt tctgtgcgtc attctgggac 300

ttgtggggcc caggcaccct ggtcactgtc tcttca 336

<210> 50

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 50

gaccctatgc tgacccagac tgcatccccc gtgtctgcgg ctgttggagg cacagtcacc 60

atcaattgcc agtccagtca gagtgtctat aataacaacc gcttatcctg gtttcagcag 120

aaaccagggc agcctcccaa gctcctgatc tattctgcat ccactctggc atctggggtc 180

tcatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgg tatttatgct 300

ttcggcggag ggaccgagct ggagatccta 330

<210> 51

<211> 336

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 51

cagcagcagc tgatggagtc cgggggaggc ctggtcaagc ctggaggatc cctgacactc 60

acctgcacag tctctggatt gtccctctat agcaatgaaa taaattgggt ccgccaggct 120

ccagggaacg ggctggaatg gatcggagcc attgctggta gtggtagcgc cgactacgcg 180

agctgggcga aaagtcgatc caccatcacc agaaacaccg acctgaacac ggtgactctg 240

aaaatgacca gtctgacagc cgcggacacg gccacctatt tctgtgcgtc attctgggac 300

ttgtggggcc caggcaccct ggtcaccgtc tcttca 336

<210> 52

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 52

gagctcgtgc tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcagc 60

atcagttgcc agtccagtca gagtgtttat aagaacaaca acttagcctg gtttcagcag 120

aaactaggcc agcctcccac gctcctgatc tgggatgcat ccaaactggc atctggggtc 180

ccatcgcggt tcagtggcag tgggtctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgt taattatgct 300

ttcggcggag gaaccaagct ggagatcaaa 330

<210> 53

<211> 333

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 53

cagtcgctgg aggagtccgg gggaggcctg gtcaagcctg gaggatccct gacactcacc 60

tgcacagtct ctggattgtc cctctatagc aatgaaataa attgggtccg ccaggctcca 120

gggaacgggc tggaatggat cggagccatt gctggtagtg gtagcgccga ctacgcgagc 180

tgggcgaaaa gccgttccac catcaccaga aacaccaacc tgaacacggt gactctgaaa 240

atgaccagtc tgacagccgc ggacacggcc acctatttct gtgcgtcatt ctgggacttg 300

tggggcccag gcaccctggt cactgtctct tca 333

<210> 54

<211> 330

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 54

gagctcgatc tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60

atcagttgcc agtccagtga gagtgtttat aagaacaaca acttagcctg gtttcagcag 120

aaactaggcc agcctcccac gctcctgatc tgggatgcat ccaaactggc atctggggtc 180

ccatcgcggt tcagtggcag tgggtctggg acacagttca ctctcaccat cagcgacctg 240

gagtgtgacg atgctgccac ttactactgt gcaggcgatt atagtggtgt taattatgct 300

ttcggcggag ggaccgaggt ggtcgtcaaa 330

<210> 55

<211> 336

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 55

cagcagcagc tggtggagtc cgggggaggc ctggtcaagc ctggaggatc cctgacactc 60

acctgcagag tctctggatt atccctcttt agcaatgaaa taaactgggt ccgccaggct 120

ccagggaacg ggctggaatg gatcggagcc attggtggtg gtggtagcgc cgactacgcg 180

agctgggcga agagccgatc caccatcacc agaaacacca acctgaacac ggtgactctg 240

agaatgacca gtctgacagc cgcggacacg gccgcctatt tctgtgcgtc attctgggac 300

ttgtggggcc caggcaccct ggtcaccgtc tcttca 336

<210> 56

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Glu Ser Val Tyr Lys Asn

20 25 30

Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu

35 40 45

Leu Ile Trp Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Ala Gly Asp Tyr Ser Gly

85 90 95

Val Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 57

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Leu Phe Ser Asn

20 25 30

Glu Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Gly Gly Gly Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Ser Phe Trp Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 58

<211> 330

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

gacatccaga tgacccagag cccatcctcc ctgtctgcct ctgtgggaga cagggtgacc 60

atcacttgtc agtcctctga gtctgtctac aagaacaaca acctggcttg gtatcaacag 120

aagcctggca agcctccaaa actgctgatt tgggatgcca gcaaactggc atctggagtg 180

ccaagcaggt tctctggctc tggctctggc acagacttca ccctgaccat ctcctccctc 240

caacctgagg atgtggctac ctactactgt gctggagact actctggagt gaactatgcc 300

tttggaggag gcacagaggt ggtggtgaag 330

<210> 59

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

gaggtccaac ttgtggagtc tggaggagga ctggtgaagc ctggaggctc cctgagactg 60

tcctgtgctg cctctggact gtccctgttc agcaatgaga taaactgggt gagacaggct 120

cctggcaagg gattggagtg gattggagcc attggaggag gaggctctgc tgactatgcc 180

tcctgggcta agagcaggtt caccatcagc agggacaatg ccaagaactc cctctacctc 240

caaatgaact ccctgagggc tgaggacaca gcagtctact tctgtgcctc cttctgggac 300

ctgtggggac ctggcaccct ggtgacagtg tcctct 336

Claims

1. An antibody or antigen-binding fragment thereof that neutralizes H7-associated influenza A virus, comprising any one of sequences (a) - (H), wherein

(h) a light chain variable region having light chain CDR1 of SEQ ID NO: 6. light chain CDR2 is SEQ ID NO: 8. light chain CDR3 is SEQ ID NO: 13 and a heavy chain variable region having the heavy chain CDR1 of SEQ ID NO: 15. heavy chain CDR2 is SEQ ID NO: 17. heavy chain CDR3 is SEQ ID NO: 19;

wherein the antigen binding fragment is selected from Fab, Fab ', F (ab')2, Fv or scFv.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises any one of (a) to (h), wherein (a) the light chain variable region has the amino acid sequence of SEQ ID NO: 20; a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29;

3. the antibody or antigen binding fragment thereof of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody or antigen binding fragment thereof of claim 1 or 2, wherein the antibody is an isolated antibody.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a scFv-based multi-chain antibody.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the H7-associated influenza a virus is H7N 9.

7. A nucleic acid molecule encoding a polynucleotide of the antibody or antigen-binding fragment thereof of any one of the preceding claims.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1-6.

10. The cell of claim 9, comprising the nucleic acid molecule of claim 7.

11. The cell of claim 9, comprising the vector of claim 8.

12. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5 in the manufacture of a medicament for the treatment of an influenza a virus infection.

13. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 7, or a vector according to claim 8, a cell according to any one of claims 9 to 11, in the preparation of (i) a medicament for the treatment of an H7-associated influenza a virus infection; (ii) h7-related influenza a virus vaccine detection agent; or (iii) H7-related influenza A virus infection.

14. The use of claim 13, wherein the H7-related influenza a virus is H7N 9.