Influenza A virus antibodies and their preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to influenza A virus antibodies, preparation methods, and applications thereof. The antibodies or antigen-binding fragments thereof can specifically recognize and bind to the HA protein and / or HA1 protein of influenza A virus, and have good affinity therewith; at the same time, they have a significant neutralizing effect on influenza A virus, especially the neutralizing ability of the recent H3 subtype epidemic strains A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 is significantly enhanced, IC 50 The value is significantly lower than that of similar antibodies that have been reported; it has high binding activity, fast binding speed, and significant neutralization ability, especially in rapidly neutralizing viruses. It can also effectively respond to the rapid changes of influenza virus strains and has potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to influenza A virus antibodies and a preparation method and application thereof. Background Art

[0002] Influenza A virus, particularly the H3 subtype, is a highly mutable RNA virus with extremely high genetic variability. It is the primary pathogen causing seasonal influenza. The virus invades cells and initiates infection by binding to host cell receptors via the hemagglutinin (HA) protein on its surface. The HA protein is composed of two subunits, HA1 and HA2. The HA1 subunit carries the primary antigenic site, a key region for neutralizing antibodies.

[0003] The HA protein of the H3N2 subtype undergoes frequent genetic variation. These cumulative mutations can lead to antigenic drift, allowing the virus to evade the host immune system, triggering new infections and outbreaks. In recent years, newly emerged influenza strains such as A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 have demonstrated high prevalence and pathogenicity during flu seasons. Existing antibodies have poor neutralization efficacy against these new strains, highlighting the urgent need to develop novel broadly neutralizing antibodies. The development of broadly neutralizing antibodies capable of recognizing and neutralizing multiple influenza strains is of great significance for the treatment and prevention of influenza. Summary of the Invention

[0004] The first aspect of the present invention aims to provide influenza A virus antibodies or antigen-binding fragments thereof.

[0005] The second aspect of the present invention aims to provide a chimeric antigen receptor.

[0006] The third aspect of the present invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0007] The fourth aspect of the present invention aims to provide a biomaterial related to the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect.

[0008] The fifth aspect of the present invention aims to provide a method for preparing the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect.

[0009] The sixth aspect of the present invention aims to provide a conjugate.

[0010] The seventh aspect of the present invention aims to provide a pharmaceutical composition.

[0011] The eighth aspect of the present invention aims to provide a diagnostic or therapeutic kit.

[0012] The purpose of the ninth aspect of the present invention is to provide an application of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, an influenza A virus antibody or an antigen-binding fragment thereof is provided, wherein the influenza A virus antibody or the antigen-binding fragment thereof comprises:

[0015] a1) HCDR1, HCDR2 and HCDR3 comprised in a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or, LCDR1, LCDR2 and LCDR3 comprised in a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or

[0016] a2) HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or, LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1).

[0017] In some embodiments, the CDRs are defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering systems.

[0018] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0019] b1) a VH comprising the following three CDRs: a HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, a HCDR2 having the amino acid sequence shown in SEQ ID NO: 6, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 7; and / or a VL comprising the following three CDRs: a LCDR1 having the amino acid sequence shown in SEQ ID NO: 8, a LCDR2 having the amino acid sequence of AAS, and a LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0020] b2) a VH comprising the following three CDRs: HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in b1); and / or a VL comprising the following three CDRs: LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in b1);

[0021] Wherein, the CDR is defined according to the IMGT numbering system.

[0022] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0023] c1) a VH comprising the following three CDRs: a HCDR1 having the amino acid sequence of SEQ ID NO: 10, a HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a HCDR3 having the amino acid sequence of SEQ ID NO: 12; and / or a VL comprising the following three CDRs: a LCDR1 having the amino acid sequence of SEQ ID NO: 13, a LCDR2 having the amino acid sequence of SEQ ID NO: 14, and a LCDR3 having the amino acid sequence of SEQ ID NO: 9; or

[0024] c2) a VH comprising the following three CDRs: HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in c1); and / or a VL comprising the following three CDRs: LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in c1);

[0025] Wherein, the CDRs are defined according to the Kabat numbering system.

[0026] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0027] d1) a VH comprising the following three CDRs: a HCDR1 having the amino acid sequence of SEQ ID NO: 15, a HCDR2 having the amino acid sequence of SEQ ID NO: 16, and a HCDR3 having the amino acid sequence of SEQ ID NO: 12; and / or a VL comprising the following three CDRs: a LCDR1 having the amino acid sequence of SEQ ID NO: 13, a LCDR2 having the amino acid sequence of SEQ ID NO: 14, and a LCDR3 having the amino acid sequence of SEQ ID NO: 9; or

[0028] d2) a VH comprising the following three CDRs: HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in d1); and / or a VL comprising the following three CDRs: LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in d1);

[0029] Wherein, the CDRs are defined according to the AbM numbering system.

[0030] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0031] e1) a VH comprising the following three CDRs: a HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, a HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or a VL comprising the following three CDRs: a LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, a LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and a LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0032] e2) a VH comprising the following three CDRs: HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in e1); and / or a VL comprising the following three CDRs: LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in e1);

[0033] Wherein, the CDRs are defined according to the Chothia numbering system.

[0034] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0035] f1) a VH comprising the following three CDRs: a HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, a HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and / or a VL comprising the following three CDRs: a LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, a LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and a LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; or

[0036] f2) a VH comprising the following three CDRs: HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in f1); and / or a VL comprising the following three CDRs: LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in f1);

[0037] Wherein, the CDR is defined according to the Contact numbering system.

[0038] Those skilled in the art will understand that the above amino acid substitutions are conservative substitutions.

[0039] In some embodiments, the heavy chain variable region of the influenza A virus antibody or antigen-binding fragment thereof further includes a framework region of the heavy chain variable region.

[0040] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of an immunoglobulin derived from mice, primates, cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks or geese, or a mutant thereof; and further includes the framework region of the heavy chain variable region of an immunoglobulin derived from humans, or a mutant thereof.

[0041] In some embodiments, the light chain variable region of the influenza A virus antibody or antigen-binding fragment thereof further comprises a framework region of the light chain variable region.

[0042] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region of an immunoglobulin derived from mice, primates, cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks or geese, or a mutant thereof; and further includes the framework region of the light chain variable region of an immunoglobulin derived from humans, or a mutant thereof.

[0043] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0044] a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0045] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof may further include a heavy chain constant region and / or a light chain constant region.

[0046] In some embodiments, the heavy chain constant region may include at least a portion of the heavy chain constant region of an immunoglobulin derived from mice, primates, cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks or geese, or a mutant thereof; and further include at least a portion of the heavy chain constant region of an immunoglobulin derived from humans, or a mutant thereof.

[0047] In some embodiments, the light chain constant region can include at least a portion of a light chain constant region of an immunoglobulin derived from a mouse, primate, cow, horse, cattle, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; and further includes a light chain constant region of an immunoglobulin derived from a human, or a mutant thereof.

[0048] In some embodiments, the heavy chain constant region can include a heavy chain constant region derived from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM immunoglobulin.

[0049] In some embodiments, the light chain constant region can include light chain constant regions derived from kappa-type and lambda-type immunoglobulins.

[0050] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; further, a fully human antibody.

[0051] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof may include, but is not limited to, a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (scFv), a dsFv, or a Fd fragment.

[0052] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises:

[0053] A heavy chain comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0054] The second aspect of the present invention provides a chimeric antigen receptor, which includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain includes the antibody or antigen binding fragment thereof according to the first aspect of the present invention.

[0055] The third aspect of the present invention provides a multispecific antibody or antigen-binding fragment thereof, which comprises two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0056] A fourth aspect of the present invention provides a biomaterial related to the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect, wherein the biomaterial comprises any one of n1) to n9):

[0057] n1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the present invention;

[0058] n2) an expression cassette comprising the nucleic acid molecule described in n1);

[0059] n3) a vector comprising the nucleic acid molecule described in n1);

[0060] n4) a vector comprising the expression cassette described in n2);

[0061] n5) a cell comprising the nucleic acid molecule described in n1);

[0062] n6) a cell comprising the expression cassette described in n2);

[0063] n7) a cell comprising the vector described in n3);

[0064] n8) a cell comprising the vector described in n4);

[0065] n9) a cell comprising the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the present invention;

[0066] The cell of any one of n5) to n9) does not contain propagation material.

[0067] It will be understood by those skilled in the art that nucleotides in a nucleic acid molecule can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0068] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0069] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the invention comprises: SEQ ID NO: 3, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0070] In some embodiments, the nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the invention comprises: SEQ ID NO: 4, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0071] In some embodiments, any of the vectors described in n3)-n4) can be an expression vector. In some embodiments, the expression vector can include a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but is not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector can include, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0072] In some embodiments, the carrier can be selected from nanoparticles, liposomes, exosomes, microbubbles or gene guns.

[0073] In some embodiments, any of the cells described in n5)-n9) can be host cells conventionally used in the art, as long as the expression vector can stably express the nucleic acid molecule carried therein as the above-mentioned antibody or antigen-binding fragment thereof, chimeric antigen receptor, or multispecific antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell can include, for example, Escherichia coli, and the eukaryotic cell can include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0074] In some embodiments, the cell described in any of n5)-n9) may be an immune cell. In some embodiments, the immune cell may include, but is not limited to, T cells, NK cells, DCs, and macrophages. In these embodiments, the immune cell may express the chimeric antigen receptor described above (i.e., be a modified immune cell).

[0075] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect, which is obtained by culturing the cells of the fourth aspect of the present invention.

[0076] The sixth aspect of the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and a coupling moiety.

[0077] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label or a therapeutic agent.

[0078] In some embodiments, the detectable label can be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical, chemical, or other means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding to modified avidins (e.g., streptavidin) of the above labels. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label described above can be linked to the antibody or antigen-binding fragment thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0079] In some embodiments, the detectable label may include, but is not limited to, an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), a colored substance, biotin, and the like.

[0080] In some embodiments, the therapeutic agent may include, for example, but not limited to, a drug for preventing and / or treating influenza A virus infection or a disease caused by the same.

[0081] In some embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), methyl, ethyl, or sugar group.

[0082] The seventh aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, or the conjugate of the sixth aspect; and a pharmaceutically acceptable carrier.

[0083] In some embodiments, the pharmaceutical composition may further include additional pharmaceutically active agents.

[0084] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating influenza A virus infection or a disease caused by the virus.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0086] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ophthalmically, topically, or the like.

[0087] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0088] The eighth aspect of the present invention provides a diagnostic or therapeutic kit comprising: the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0089] In some embodiments, the kit may further include instructions and / or an administration device.

[0090] In some embodiments, the kit can be used to diagnose influenza A virus infection or diseases caused by the virus.

[0091] In some embodiments, the kit can be used to prevent and / or treat influenza A virus infection or diseases caused by it.

[0092] A ninth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in the preparation of a product, wherein the product is used for any of c1) to c4):

[0093] c1) Diagnosis of influenza A virus infection or diseases caused by it;

[0094] c2) prevention and / or treatment of influenza A virus infection or diseases caused by it;

[0095] c3) detecting the presence or level of influenza A virus hemagglutinin HA protein in the sample;

[0096] c4) detecting the presence or level of influenza A virus hemagglutinin HA1 protein in the sample.

[0097] In some embodiments, the sample is selected from at least one of body fluids, tissues, cells, and excretions of the subject to be tested.

[0098] In some embodiments, the body fluid includes at least one of blood and lymph.

[0099] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood.

[0100] In some embodiments, the excretion includes at least one of urine, feces, and tears.

[0101] In some embodiments, the subject to be tested includes mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0102] In some embodiments, the subject includes a human.

[0103] In the present invention, the influenza A virus includes influenza A virus H3 subtype; further includes H3N2 subtype, for example, but not limited to at least one of A / HongKong / 01 / 1968 (H3N2), A / Wisconsin / 67 / 2005 (H3N2), A / Perth / 16 / 2009 (H3N2), A / Victoria / 361 / 2011 (H3N2), A / HongKong / 4801 / 2014 (H3N2), A / Hong Kong / 45 / 2019 (H3N2), A / Darwin / 6 / 2021 (H3N2), and A / Massachusetts / 18 / 2022 (H3N2).

[0104] In the present invention, the disease caused by influenza A virus infection includes influenza A.

[0105] The beneficial effects of the present invention are:

[0106] The present invention provides influenza A virus antibodies or antigen-binding fragments thereof, which can specifically recognize and bind to influenza A virus HA protein and / or HA1 protein and have good affinity thereto; at the same time, they have a significant neutralizing effect on influenza A virus, in particular, the neutralizing ability against the recent H3 subtype epidemic strains A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 is significantly enhanced, IC 50The value is significantly lower than that of similar antibodies reported previously; it has high binding activity, fast binding speed, and significant neutralizing ability, especially in rapidly neutralizing viruses. It can also effectively respond to the rapid changes of influenza virus strains and has potential clinical application value; it can be used to prepare products for diagnosing, preventing and / or treating influenza A virus infection or diseases caused by it, or detecting the presence or level of influenza A virus HA protein and / or HA1 protein in samples.

[0107] Furthermore, fully humanized influenza A virus antibodies or antigen-binding fragments thereof have lower immunogenicity and adverse reaction risks, and can be produced in large quantities through mammalian cell lines, ensuring the stability and clarity of the components. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 The OD values of the binding of the Flu-B0203 antibody to the hemagglutinin HA trimer are shown.

[0109] Figure 2 The affinity kinetic curve of Flu-B0203 antibody and hemagglutinin HA1 monomer is shown.

[0110] Figure 3 The IC values of Flu-B0203 antibody neutralizing pseudovirus are shown. 50 value. DETAILED DESCRIPTION

[0111] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0112] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0113] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0114] The HA protein is the primary component on the surface of influenza virus particles. In addition to playing an important role in receptor binding, fusion, packaging, and pathogenicity, it is also the primary target of recognition by the host's acquired immune system. The HA protein trimer is structurally divided into a globular head and a long, fibrous stem. The head is composed of three HA1 molecules, and the stem is composed of three HA2 molecules. After the influenza virus infects the host, it induces a strong immune response, leading to the production of neutralizing antibodies. Neutralizing antibodies specifically bind to the receptor binding site of the HA protein, preventing the virus from binding to host cells and attaching to them.

[0115] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0116] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical, matched positions shared by the sequences over the comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target and reference sequences. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0117] Percent sequence identity can be calculated by the following process: determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and downloading. Suitable software programs are available from various sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses the BLASTN or BLASTP algorithms to compare two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0118] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0119] The following examples and accompanying drawings are provided to assist in understanding the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0120] It should be noted that, where specific conditions are not specified in the examples, the experiments were conducted according to conventional conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments used without manufacturer information are commercially available. For reagents with manufacturer information, similar products from other manufacturers are considered substitutes.

[0121] The quantitative tests in the following examples were performed three times unless otherwise specified, and the results were averaged.

[0122] Example 1. Flow cytometric sorting of B cells and screening and purification of influenza A virus neutralizing antibodies

[0123] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of volunteers infected with influenza virus and producing protective antibodies, and monoclonal antibodies that can specifically bind to the full length of hemagglutinin HA were screened as follows:

[0124] 1. PBMC Isolation and Memory B Cell Sorting

[0125] Ten milliliters of venous blood was collected from volunteers infected with influenza virus in the second half of 2023 and who developed protective antibodies. The blood was placed in anticoagulant tubes containing ethylenediaminetetraacetic acid (EDTA). PBMCs were isolated by Ficoll gradient centrifugation. Single antigen-specific memory B cells were isolated from PBMCs using an Astrios EQ (BeckMan Coulter) flow cytometer using recombinant influenza virus HA protein (Beijing Sino Biological Science and Technology Co., Ltd., 40992-V08B, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA Protein (His Tag)) as the sorting antigen. Single antigen-specific memory B cells were sorted from the PBMCs using a flow cytometer. The cells were sorted into 96-well PCR (polymerase chain reaction) plates, with each well containing one B cell. The 96-well plates containing B cells were stored at −80°C until further use.

[0126] 2. Single-cell PCR amplification of fully human monoclonal antibodies

[0127] 1) Reverse Transcription PCR: IgG1 subtype-specific primers targeting heavy, kappa, and lambda light chains, along with Maxima H Minus reverse transcriptase (Thermo), were added to a 96-well PCR plate containing a single B cell. Reverse transcription was performed at 50°C for 30 min, followed by inactivation of the reverse transcriptase at 85°C for 5 min. The resulting cDNA product was stored at -80°C.

[0128] 2) Nested PCR:

[0129] First-round reaction: Using 2 µL of cDNA product as template, add TransStart® Fast Pfu DNA Polymerase (Transgene, AP221), dNTPs, and nested PCR primers. Reaction conditions: Initial denaturation at 98°C for 5 minutes, followed by 40 cycles of PCR: 98°C for 30 seconds, 55°C (heavy chain VH, kappa light chain Vκ) / 50°C (lambda light chain Vλ) for 1 minute, 72°C for 1 minute, and a final extension at 72°C for 5 minutes.

[0130] Second-round reaction: Using the PCR product from the first-round reaction as a template, TransStart® Fast Pfu DNA Polymerase (Transgene, AP221) and nested PCR primers were added. Reaction conditions included initial denaturation at 98°C for 5 minutes, followed by 35 cycles of 98°C for 30 seconds, 58°C (heavy chain VH) / 60°C (kappa light chain Vκ) / 64°C (lambda light chain Vλ) for 1 minute, and 72°C for 1 minute. Finally, a final extension at 72°C for 5 minutes was performed to generate the nested PCR amplification product.

[0131] For the sequences of IgG1 subtype-specific primers for heavy chain, kappa light chain, and lambda light chain, as well as the sequences of the nested PCR primers, see Liao HX, Levesque MC, Nagel A, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods. 2009;158(1-2):171-179.

[0132] 3) Agarose gel electrophoresis

[0133] 5 μL of nested PCR amplification product was detected by 1.5% agarose gel electrophoresis, and paired positive clones were sequenced to obtain the antibody variable region sequence, and a linear expression cassette was constructed for the sequence.

[0134] 3. Acquisition and identification of antibody sequences

[0135] DNA sequences were analyzed using IgBLAST software, using the Human Genome Reference Sequence Database (GRCh38) as a reference. This software was used to identify the VDJ gene segments of the heavy and light chains within the antibody sequences and to count the number of somatic hypermutations (SHM). The SHM mutation rate was calculated as follows: mutation count / V gene alignment length. CDR regions were identified using ANARCI software. The CDR and variable region sequences of the antibody (designated Flu-B0203) are shown in Table 1. Antibody characteristics are shown in Table 2.

[0136]

[0137]

[0138] 4. Expression plasmid construction and antibody preparation

[0139] The nucleic acid sequences encoding the paired immunoglobulin (Flu-B0203) heavy and light chains (amino acid sequences shown in Table 1) were submitted to GenScript Biotech Co., Ltd. for gene synthesis. The heavy and light chain sequences were cloned into the pcDNA3.4 expression vector using Gibson assembly. The two plasmids were then co-transfected into HEK293F cells. The cells were cultured at 37°C in a 5% CO2 incubator for 72 hours. The supernatant was collected by centrifugation at 300 g for 5 minutes, and the monoclonal antibodies secreted from the cell culture medium were purified using Protein A affinity chromatography. The collected antibodies were then replaced with PBS, and the antibody concentration and purity were determined.

[0140] Recombinant S5V2-29, FluA-20, 1C04, MEDI8852, CR9114, CR8043, and CR8043. McCarthy KR, Kuraoka M, et al. Antibodies to aConserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism. Cell. 2019;177(5):1124-1135.e16;Bangaru S, Lang S,Schotsaert M, et al. A Site of Vulnerability on the Influenza VirusHemagglutinin Head Domain Trimer Interface. Cell. 2019;177(5):1136-1152.e18;Zost SJ, Lee J, Gumina ME, et al. Identification of Antibodies Targeting theH3N2 Hemagglutinin Receptor Binding Site Following Vaccination of Humans.Cell Rep. 2019;29(13):4460-4470.e8;Kallewaard NL, Corti D, Collins PJ, et al. Cell. 2016;166(3):596-608;Dreyfus C, Laursen NS, Kwaks T, et al.Highly conserved protective epitopes on influenza B viruses. Science. 2012;337(6100):1343-1348;Friesen RH, Lee PS, Stoop EJ, et al. A common solution togroup 2 influenza virus neutralization. Proc Natl Acad Sci US A.2014;111(1):445-450. .

[0141] Example 2. ELISA detection of antibody binding activity

[0142] 1. Experimental process

[0143] Hemagglutinin (HA) full-length protein (Beijing Sino Biological Technology Co., Ltd., 40992-V08B, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA Protein (His Tag)) was used as the antigen. The antigen was diluted to 1 μg / mL in coating buffer and coated on a 96-well ELISA plate with a volume of 100 μL per well. The plates were incubated overnight at 4°C and blocked with blocking buffer at 37°C for 2 h. The purified Flu-B0203 antibody expressed in Example 1 and other control antibodies (S5V2-29 and FluA-20) were diluted to 1 μg / mL, and 100 μL per well was added to the blocked ELISA plate and incubated at 37°C for 1 hour. After washing, 100 μL of goat anti-human IgG (H+L)-HRP (1:2000 dilution) was added and incubated at 37°C for 1 hour. After adding the substrate colorimetric solution, the plate was incubated at room temperature in the dark for 10 minutes. The reaction was stopped with 2 M sulfuric acid, and the OD value at 450 / 630 nm was measured.

[0144] 2. Results

[0145] The results are as follows Figure 1 As shown, the expressed and purified Flu-B0203 antibody showed efficient binding ability to the influenza virus hemagglutinin HA trimer protein, and its binding activity OD value reached 4.9025, which was higher than that of the control antibodies S5V2-29 and FluA-20, indicating that it had stronger binding ability.

[0146] Example 3. Affinity constant and affinity kinetics analysis of antibody Flu-B0203 detected by SPR

[0147] 1. Capture method test

[0148] Antibody affinity testing was performed using a capture method using 1*HBS-EP (Cytiva) buffer. A CM5 chip was first coupled with anti-human Fc (Cytiva). The capture antibody was then diluted to a concentration of 5 μg / mL, and the binding time was 60 s. The analyte, hemagglutinin HA1 monomer (Beijing Sino Biological Science and Technology Co., Ltd., 40992-V08H1, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA1 Protein (HisTag)), was sequentially passed over the chip at increasing concentrations (1.5625 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM), and signal curves were obtained. Each concentration was considered a cycle. After each cycle, the chip was regenerated with 4 M MgCl2 for 60 s to return to its original, antibody-free state. The obtained signal curve was analyzed using Biacore 8K System software to obtain an affinity activity detection graph of Flu-B0203 antibody and HA1 monomer.

[0149] 2. Results

[0150] As shown in Table 3 and Figure 2 As shown in Figure 2, the equilibrium dissociation constant KD value of Flu-B0203 antibody for hemagglutinin HA1 monomer is 10 -10 The antibody's affinity for HA1 monomers is on the order of M, indicating that it has a higher affinity than the control antibodies S5V2-29 and FluA-20. Furthermore, the high binding constant (Ka value) of the Flu-B0203 antibody demonstrates its rapid binding properties, meaning it can quickly bind to the virus and exert a neutralizing effect.

[0151]

[0152] Example 4. Evaluation of pseudovirus neutralization activity

[0153] 1. Fake virus packaging

[0154] Pseudoviruses were prepared using the human immunodeficiency virus (HIV-1) packaging system. Plasmids encoding the H3N2 virus HA and neuraminidase (NA) genes were cloned into the pSV1.0 vector. Table 4 lists the GISAID (Global Influenza and Respiratory Virus Information Exchange) accession numbers for the HA and NA genes of the eight pseudovirus strains used. HEK-293FT cells were co-transfected with the HA and NA plasmids and the HIV backbone plasmid (pSG3.Δenv-FlucΔnef) using the transfection reagent Lipofectamine 2000 (Invitrogen). Culture supernatants were collected 48 hours after transfection, centrifuged at 4000 rpm for 10 minutes, and concentrated using 30 kD ultrafiltration centrifuge tubes.

[0155] For the above-mentioned viral backbone plasmid sequence and experimental conditions, please refer to Tian Y, Zhao H, Liu Q, et al. Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus. Sci Rep. 2018;8(1):8484.

[0156]

[0157] 2. Pseudovirus titration and neutralization activity detection

[0158] When performing pseudovirus titration experiments, the pseudovirus was quantitatively analyzed using a Promega luciferase assay kit. First, the pseudovirus was diluted 5-fold in a 96-well culture plate, with each well containing 100 μL of the diluted virus solution. 100 μL of trypsin-treated MDCK cells was inoculated into each well to a concentration of 3 × 10 cells per well. 4 After incubating the culture plate at 37°C for 48 hours, the culture supernatant was gently removed and discarded. 100 μL of Bright-Glo luciferase substrate (Promega) was then added to each well. After incubation at room temperature for 2 minutes, 150 μL of the lysate was transferred to a new 96-well plate and luciferase activity was measured using a Glomax 96 microplate luminometer. The 50% tissue culture infectious dose (TCID) was calculated using the Reed-Muench method. 50 ) to assess the infectivity of pseudoviruses.

[0159] For neutralization assays, serially dilute the test antibody 2-fold in a 96-well plate, with 100 μL of dilution per well. Then add 50 μL of 5000 TCID50 Pseudovirus (RLU value is 10 6 ), mix well and incubate at 37℃ for 1h. Add 100μL MDCK cell suspension to 96-well plate to make 3×10 cells per well. 4 The cells were incubated at 37°C for 48 h, and then the luciferase activity was detected according to the above method. 50 Neutralizing potency of antibodies was assessed by a four-parameter nonlinear regression model.

[0160] 3. Results

[0161] like Figure 3 As shown in the figure, the heat map shows the pseudovirus neutralization activity of different antibodies against various influenza virus strains, with IC 50 The value is used as an indicator (unit is μg / mL), and the lower the value, the stronger the neutralization activity. The blue area in the figure indicates low IC 50 The value indicates that the antibody has a high neutralizing effect on the corresponding strain; the red area indicates a high IC 50 The value indicates that the neutralizing effect of the antibody on the virus strain is limited, and the change in color depth directly reflects the strength of the antibody's neutralizing activity.

[0162] The results showed that the Flu-B0203 antibody not only had a neutralizing effect on early strains such as A / HongKong / 01 / 1968, but also showed strong neutralizing ability against new strains that have been prevalent in recent years, such as A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022. Compared with other reported antibodies (S5V2-29, FluA-20, 1C04, MEDI8852, CR9114, CR8043), Flu-B0203 showed lower IC values against most strains. 50 value, which means that it can achieve neutralization effect at lower concentrations, thus demonstrating its potential as a broadly neutralizing antibody.

[0163] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An influenza A virus antibody or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) of the amino acid sequence set forth in SEQ ID NO: 1; and LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) of the amino acid sequence set forth in SEQ ID NO: 2; The CDRs are defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The influenza A virus antibody or antigen-binding fragment thereof comprises: b1) a VH comprising the following three CDRs: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 6, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 7; and a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 8, a LCDR2 having an amino acid sequence of AAS, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 9; the CDRs being defined according to the IMGT numbering system; or c1) a VH comprising the following three CDRs: a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 12; and a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; the CDRs are defined according to the Kabat numbering system; or d1) a VH comprising the following three CDRs: a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 15, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 16, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 12; and a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; the CDRs being defined according to the AbM numbering system; or e1) a VH comprising the following three CDRs: a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 12; and a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; the CDRs are defined according to the Chothia numbering system; or f1) a VH comprising the following three CDRs: a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 19, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 20, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 21; and a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 22, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 23, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 24; the CDRs are defined according to the Contact numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The heavy chain variable region of the influenza A virus antibody or antigen-binding fragment thereof further comprises a framework region of the heavy chain variable region; or The light chain variable region of the influenza A virus antibody or antigen-binding fragment thereof further comprises a framework region of the light chain variable region; or The influenza A virus antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or The influenza A virus antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; or The influenza A virus antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; or The influenza A virus antibody or antigen-binding fragment thereof includes a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv, a dsFv or a Fd fragment; or The influenza A virus antibody or antigen-binding fragment thereof comprises: A heavy chain comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An expression cassette comprising the nucleic acid molecule of claim 4.

6. A vector comprising the nucleic acid molecule of claim 4.

7. A vector comprising the expression cassette of claim 5.

8. A cell comprising the nucleic acid molecule of claim 4, said cell not comprising propagation material.

9. A cell comprising the expression cassette of claim 5, said cell comprising no propagation material.

10. A cell comprising the vector of claim 6, said cell not comprising propagation material.

11. A cell comprising the vector of claim 7, said cell not comprising propagation material.

12. A cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, said cell not comprising propagation material.

13. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression cassette of claim 5, the vector of any one of claims 6 to 7, or the cell of any one of claims 8 to 12; and a pharmaceutically acceptable carrier.

14. A diagnostic or therapeutic kit comprising: The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression cassette of claim 5, the vector of any one of claims 6 to 7, the cell of any one of claims 8 to 12, or the pharmaceutical composition of claim 13.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression cassette according to claim 5, the vector according to any one of claims 6 to 7, the cell according to any one of claims 8 to 12, the pharmaceutical composition according to claim 13, or the diagnostic or therapeutic kit according to claim 14, wherein: The influenza A virus is the influenza A virus H3N2 subtype.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression cassette according to claim 5, the vector according to any one of claims 6 to 7, the cell according to any one of claims 8 to 12, or the pharmaceutical composition according to claim 13 in the preparation of a product for any one of c1) to c4): c1) Diagnosis of influenza A virus infection or diseases caused by it; c2) prevention and / or treatment of influenza A virus infection or diseases caused by it; c3) detecting the presence or level of influenza A virus hemagglutinin HA protein in the sample; c4) detecting the presence or level of influenza A virus hemagglutinin HA1 protein in the sample; The influenza A virus is the influenza A virus H3N2 subtype.

17. The use according to claim 16, characterized in that The disease caused by influenza A virus infection is influenza A.

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