Influenza A virus antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an influenza A virus antibody as well as a preparation method and application thereof. The antibody or the antigen binding fragment thereof can specifically recognize and bind to influenza A virus HA protein and / or HA1 protein, and has good affinity with the influenza A virus HA protein and / or HA1 protein; meanwhile, the antibody has a remarkable neutralizing effect on influenza A viruses, particularly, the neutralizing capacity on H3 subtype epidemic strains A / Darwinin / 6 / 2021 and A / Massachuetts / 18 / 2022 in recent years is remarkably enhanced, and the IC50 value is remarkably lower than that of reported similar antibodies; the compound is high in binding activity, high in binding speed and remarkable in neutralizing capacity, particularly has the advantages when rapidly neutralizing viruses, can effectively cope with rapid changes of influenza virus strains, and has potential clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to influenza A virus antibodies and preparation methods and applications thereof. Background Art

[0002] Influenza A virus, especially the H3 subtype, is an RNA virus that is prone to mutation and has extremely high genetic variability. It is the main pathogen that causes seasonal influenza. The virus binds to host cell receptors through the hemagglutinin (HA) protein on its surface, then invades the cell and initiates the infection process. The HA protein is composed of two subunits, HA1 and HA2. The HA1 subunit carries the main antigenic site and is the key area for the action of neutralizing antibodies.

[0003] The HA protein of the H3N2 subtype frequently undergoes genetic variation, and these cumulative mutations may lead to antigenic drift, allowing the virus to evade the host's immune system and trigger new infections and outbreaks. In recent years, newly emerged influenza strains such as A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 have shown high prevalence and pathogenicity during the flu season. Existing antibodies have poor neutralization effects on these new strains, which highlights the urgent need to develop new broad-spectrum neutralizing antibodies. The development of broad-spectrum neutralizing antibodies that can recognize and neutralize 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 biological materials 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, 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: In a first aspect of the present invention, there is provided an influenza A virus antibody or an antigen-binding fragment thereof, wherein the influenza A virus antibody or the antigen-binding fragment thereof comprises: a1) HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or, LCDR1, LCDR2 and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or 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).

[0014] In some embodiments, the CDRs are defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering systems.

[0015] In some embodiments, 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 shown in SEQ ID NO: 5, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 7; and / or a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 8, a LCDR2 having an amino acid sequence of AAS, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; or 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); Wherein, the CDR is defined according to the IMGT numbering system.

[0016] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: 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 / or 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; or 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); Wherein, the CDRs are defined according to the Kabat numbering system.

[0017] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: 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 / or 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; or d2) 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, 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); Wherein, the CDRs are defined according to the AbM numbering system.

[0018] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: 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 / or 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; or 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); Wherein, the CDRs are defined according to the Chothia numbering system.

[0019] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: 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 / or 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; or 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); Wherein, the CDR is defined according to the Contact numbering system.

[0020] Those skilled in the art will appreciate that the above amino acid substitutions are conservative substitutions.

[0021] In some embodiments, 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.

[0022] 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, minks, 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.

[0023] 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.

[0024] 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, minks, 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.

[0025] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: A heavy chain variable region (VH) comprising the amino acid sequence shown in 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 shown in 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.

[0026] 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.

[0027] 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 includes at least a portion of the heavy chain constant region of an immunoglobulin derived from humans, or a mutant thereof.

[0028] In some embodiments, the light chain constant region may include at least a portion of an immunoglobulin light chain constant region 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 includes a light chain constant region derived from a human immunoglobulin, or a mutant thereof.

[0029] 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.

[0030] In some embodiments, the light chain constant region may include light chain constant regions derived from κ-type and λ-type immunoglobulins.

[0031] 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.

[0032] 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.

[0033] In some embodiments, the influenza A virus antibody or antigen-binding fragment thereof comprises: A heavy chain comprising the amino acid sequence shown in 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 shown in 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.

[0034] 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.

[0035] The third aspect of the present invention provides a multispecific antibody or an 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 of the first aspect of the present invention.

[0036] The 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 of the present invention, wherein the biomaterial comprises any one of n1) to n9): 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 invention; n2) an expression cassette comprising the nucleic acid molecule described in n1); n3) a vector comprising the nucleic acid molecule described in n1); n4) a vector comprising the expression cassette described in n2); n5) a cell comprising the nucleic acid molecule described in n1); n6) a cell comprising the expression cassette described in n2); n7) a cell comprising the vector described in n3); n8) a cell comprising the vector described in n4); 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; The cell described in any one of n5) to n9) does not contain any propagation material.

[0037] Those skilled in the art will appreciate that nucleotides in a nucleic acid molecule may be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In some embodiments, any of the vectors 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.

[0042] In some embodiments, the carrier can be selected from nanoparticles, liposomes, exosomes, microbubbles or gene guns.

[0043] In some embodiments, any of the cells in n5)-n9) can be host cells conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule 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, the eukaryotic cell can include, for example, CHO cells, HEK293 cells, BHK cells, NS0 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.

[0044] In some embodiments, any of the cells in n5)-n9) may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptor of the present invention (i.e., modified immune cells).

[0045] 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 in the fourth aspect of the present invention.

[0046] The sixth aspect of the present invention provides a conjugate, which includes the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and a conjugated portion.

[0047] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label or a therapeutic agent.

[0048] In some embodiments, the detectable marker can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art, and examples thereof 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 avidin (e.g., streptavidin) modified with the above-mentioned markers. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent 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 as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through a linker of different lengths to reduce potential steric hindrance.

[0049] In some embodiments, the detectable marker may include, but is not limited to, an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), a colored substance, biotin, and the like.

[0050] 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.

[0051] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, it can be a chemical group such as polyethylene glycol (PEG), methyl, ethyl or sugar group.

[0052] The seventh aspect of the present invention provides a pharmaceutical composition, which includes: 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.

[0053] In some embodiments, the pharmaceutical composition may further include additional pharmaceutically active agents.

[0054] 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.

[0055] 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.

[0056] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ophthalmically, topically, or the like.

[0057] 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.

[0058] The eighth aspect of the present invention provides a diagnostic or therapeutic kit, which includes: 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.

[0059] In some embodiments, the kit may further include instructions and / or an administration device.

[0060] In some embodiments, the kit can be used to diagnose influenza A virus infection or a disease caused by the virus.

[0061] In some embodiments, the kit can be used to prevent and / or treat influenza A virus infection or diseases caused by it.

[0062] The 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 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.

[0063] In some embodiments, the sample is selected from at least one of body fluids, tissues, cells, and excrement of the subject to be tested.

[0064] In some embodiments, the body fluid includes at least one of blood and lymph.

[0065] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood.

[0066] In some embodiments, the excreta include at least one of urine, feces, and tears.

[0067] 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).

[0068] In some embodiments, the subject to be tested includes a human.

[0069] 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).

[0070] In the present invention, the disease caused by influenza A virus infection includes influenza A.

[0071] The beneficial effects of the present invention are: The present invention provides an influenza A virus antibody or an antigen-binding fragment thereof, which can specifically recognize and bind to the influenza A virus HA protein and / or HA1 protein and has good affinity thereto; at the same time, it has a significant neutralizing effect on the influenza A virus, in particular, the neutralizing ability of 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 that have been reported; it has high binding activity, fast binding speed, and significant neutralization ability, especially has advantages in rapid virus neutralization, and can 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.

[0072] Furthermore, the fully humanized influenza A virus antibody or its antigen-binding fragment has a lower immunogenicity and risk of adverse reactions, and can be produced in large quantities through mammalian cell lines, ensuring the stability and clarity of the components. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The OD values ​​of the binding of Flu-B0203 antibody to hemagglutinin HA trimer are shown.

[0074] Figure 2 The affinity kinetic curve of Flu-B0203 antibody and hemagglutinin HA1 monomer is shown.

[0075] Figure 3 The IC values ​​of Flu-B0203 antibody and pseudovirus neutralization are shown. 50 value. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0077] 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.

[0078] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein 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.

[0079] HA protein is the most important 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 main object of host acquired immune system recognition. The HA protein trimer is structurally divided into a spherical head and a long fibrous stem. The head is composed of 3 HA1 molecules and the stem is composed of 3 HA2 molecules. After the influenza virus infects the host, it will induce a strong immune response, leading to the production of neutralizing antibodies. Neutralizing antibodies prevent the virus from binding to host cells and attaching to host cells by specifically binding to the receptor binding site of the HA protein.

[0080] The term "about" as used herein 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.

[0081] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted.

[0082] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs are available from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare 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.

[0083] 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 a 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).

[0084] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0085] It should be noted that, if the specific conditions are not specified in the examples, the experimental conditions are carried out according to conventional conditions, manufacturer recommendations or publicly reported experimental conditions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. If the manufacturer of the reagents used is specified, similar products from other manufacturers are substitutable.

[0086] The quantitative tests in the following examples were performed three times unless otherwise specified, and the results were averaged. Example 1. Flow cytometric sorting of B cells and screening and purification of influenza A virus neutralizing antibodies

[0087] 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: 1. PBMC isolation and memory B cell sorting 10 mL of venous blood was collected from volunteers who were infected with influenza virus in the second half of 2023 and produced protective antibodies and placed in anticoagulant tubes containing ethylenediaminetetraacetic acid (EDTA). PBMCs were separated by Ficoll gradient centrifugation. Recombinant influenza virus HA 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 sorting antigen, and single antigen-specific memory B cells were sorted from PBMCs into 96-well PCR (Polymerase Chain Reaction) plates using an Astrios EQ (BeckMan Coulter) flow cytometer, so that each well contained one B cell. The 96-well plate containing B cells was placed in a refrigerator at -80°C for future use.

[0088] 2. Single-cell PCR amplification of fully human monoclonal antibodies 1) Reverse transcription PCR: Add all IgG1 subtype-specific primers for heavy chain, κ light chain, and λ light chain and Maxima H Minus reverse transcriptase (Thermo) to a 96-well PCR plate containing a single B cell, reverse transcribe at 50°C for 30 min, and inactivate the reverse transcriptase at 85°C for 5 min. Store the obtained cDNA product at -80°C.

[0089] 2) Nested PCR: First round reaction: 2µL cDNA product was used as template, and TransStart(®) FastPfu DNA Polymerase (Transgene, AP221), dNTPs and nested PCR primers were added. Reaction conditions: pre-denaturation at 98℃ for 5min, followed by 40 PCR cycles, each cycle was: 98℃×30s, 55℃ (heavy chain VH, kappa light chain Vκ) / 50℃ (lambda light chain Vλ)×1min, 72℃×1min, and finally extension at 72℃ for 5min.

[0090] Second round of reaction: Using the PCR product of the first round of reaction as a template, add TransStart(®) FastPfu DNA Polymerase (Transgene, AP221) and nested PCR primers. Reaction conditions: pre-denaturation at 98°C for 5 min, then 35 PCR cycles, each cycle is: 98°C × 30s, 58°C (heavy chain VH) / 60°C (κ light chain Vκ) / 64°C (λ light chain Vλ) × 1min, 72°C × 1min, and finally extend at 72°C for 5min to obtain nested PCR amplification products.

[0091] 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.

[0092] 3) Agarose gel electrophoresis 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 frame was constructed for the sequence.

[0093] 3. Acquisition and identification of antibody sequences IgBLAST software was used to analyze and process the DNA sequence with the human genome reference sequence database GRCh38 as a reference. The VDJ gene fragments of the heavy chain and light chain in the antibody sequence were identified by this software, and the number of somatic hypermutations (SHM) was counted. Calculation formula: SHM mutation rate = mutation count / V gene alignment nucleic acid length. The CDR region was identified with the help of ANARCI software, and the CDR region and variable region sequences of the antibody (named Flu-B0203) are shown in Table 1, and the antibody characteristic information is shown in Table 2.

[0094]

[0095]

[0096] 4. Expression plasmid construction and antibody preparation The nucleic acid sequences encoding the heavy and light chains of paired immunoglobulins (Flu-B0203) (amino acid sequences are 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 based on Gibson assembly, and then the two plasmids were co-transfected into HEK293F cells, cultured in a 37°C 5% CO2 incubator for 72 h, and the supernatant was collected by centrifugation at 300 g for 5 min. The monoclonal antibodies secreted in the cell culture medium were purified by Protein A affinity chromatography, and the collected antibodies were replaced with PBS, and the antibody concentration and purity were tested.

[0097] 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.Structure and Function Analysis of an Antibody Recognizing All Influenza ASubtypes. 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. . Example 2. ELISA detection of antibody binding activity

[0098] 1. Experimental process The full-length hemagglutinin HA protein (Beijing Sino Biological Technology Co., Ltd., 40992-V08B, InfluenzaA H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA Protein (His Tag)) was used as the antigen, and the antigen was diluted to 1 μg / mL with coating solution and then coated on a 96-well ELISA plate with a volume of 100 μL per well, overnight at 4°C, and blocked with blocking solution at 37°C for 2 hours. The purified Flu-B0203 antibody and other control antibodies (S5V2-29 and FluA-20) expressed in Example 1 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 h. After washing, 100 μL of goat anti-human IgG (H+L)-HRP (1:2000 dilution) was added and incubated at 37°C for 1 h. After adding the substrate colorimetric solution, the plate was placed at room temperature and away from light for 10 min. The reaction was terminated with 2 M sulfuric acid, and the OD value at 450 / 630 nm was detected.

[0099] 2. Results The results are as follows Figure 1 As shown, the expressed and purified Flu-B0203 antibody showed a highly 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 a stronger binding ability. Example 3. Affinity constant and affinity kinetics analysis of SPR detection antibody Flu-B0203

[0100] 1. Capture method test The antibody affinity test was conducted by capture method, with 1*HBS-EP (Cytiva) as the buffer. The 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 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 40992-V08H1, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA1 Protein (HisTag)) was passed through the chip in increasing concentrations (1.5625 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) to obtain signal curves. Each concentration was taken as 1 cycle. After completing 1 cycle, the chip was regenerated with 4 M MgCl2 to return to the original state without antibody capture, and the regeneration time was 60 s. The obtained signal curve was analyzed using Biacore 8K System software to obtain the affinity activity detection graph of Flu-B0203 antibody and HA1 monomer.

[0101] 2. Results 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 has a higher affinity to the HA1 monomer than the S5V2-29 and FluA-20 control antibodies. In addition, the higher binding constant (Ka value) of the Flu-B0203 antibody shows its rapid binding properties, which means that it can quickly bind to the virus and exert a neutralizing effect.

[0102] Example 4. Evaluation of pseudovirus neutralization activity

[0103] 1. Fake virus packaging Pseudoviruses were prepared based on the human immunodeficiency virus (HIV-1) packaging system. Plasmids encoding the HA and neuraminidase (NA) genes of the H3N2 virus were cloned into the pSV1.0 vector. Table 4 shows the GISAID (Global Influenza and Respiratory Virus Information Exchange Database) registration numbers of the HA and NA genes of the 8 pseudovirus strains used. HEK-293FT cells were co-transfected with the HA plasmid, NA plasmid, and HIV backbone plasmid (pSG3.Δenv-FlucΔnef) using the transfection reagent Lipofectamine 2000 (Invitrogen). The culture supernatant was collected 48 hours after transfection, centrifuged at 4000 rpm for 10 minutes, and concentrated using a 30KD ultrafiltration centrifuge tube.

[0104] For the above-mentioned viral backbone plasmid sequence and experimental conditions, see 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.

[0105]

[0106] 2. Pseudovirus titration and neutralization activity detection When performing pseudovirus titration experiments, the Promega luciferase assay kit was used for quantitative analysis of pseudoviruses. First, the pseudovirus was diluted 5 times in a 96-well culture plate, and each well contained 100 μL of the diluted virus solution. 100 μL of MDCK cells treated with trypsin were inoculated into each well to make each well contain 3×10 cells. 4 After incubating the culture plate at 37°C for 48 h, the culture supernatant was gently removed and discarded. Then 100 μL of Bright-Glo luciferase substrate (Promega) was added to each well. After incubation at room temperature for 2 min, 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 infection dose (TCID 50 ) to assess the infectivity of the pseudovirus.

[0107] For neutralization assays, serially dilute the antibody to be tested 2-fold in a 96-well plate, with 100 μL of dilution per well. Then add 50 μL of 5000 TCID 50Pseudovirus (RLU value is 10 6 ), mix well and incubate at 37℃ for 1h. Add 100μL MDCK cell suspension to the 96-well plate to make each well contain 3×10 cells. 4 The cells were incubated at 37°C for 48 h, and then the luciferase activity was detected according to the above method. 50 The neutralizing potency of the antibodies was assessed by a four-parameter nonlinear regression model.

[0108] 3. Results 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 (in 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 antibody has limited neutralizing effect on the virus strain, and the change in color depth directly reflects the strength of the antibody's neutralizing activity.

[0109] The results showed that Flu-B0203 antibody not only has a neutralizing effect on early strains such as A / HongKong / 01 / 1968, but also has a strong neutralizing ability against new strains 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 against most strains. 50 value, which means that it can achieve neutralization effect at lower concentrations, thus demonstrating its potential as a broad-spectrum neutralizing antibody.

[0110] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All 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) having the amino acid sequence shown in SEQ ID NO: 1; and / or, LCDR1, LCDR2 and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:

2.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: 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 shown in SEQ ID NO: 5, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 7; and / or a VL comprising the following three CDRs: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 8, a LCDR2 having an amino acid sequence of AAS, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; the CDRs are 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 / or 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 / or 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 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 / or 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) VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and / or, VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence 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, characterized in that: 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 shown in 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 shown in 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. Any one of a1)-a2) proteins: a1) a protein comprising an antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the antigen binding domain comprises the antibody or antigen binding fragment thereof according to any one of claims 1 to 3, and the protein is a chimeric antigen receptor; a2) A protein comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the protein is a multispecific antibody or antigen-binding fragment thereof.

5. A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the protein according to claim 4, wherein the biological material comprises any one of n1) to n9): n1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the protein according to claim 4; n2) an expression cassette comprising the nucleic acid molecule described in n1); n3) a vector comprising the nucleic acid molecule described in n1); n4) a vector comprising the expression cassette described in n2); n5) a cell comprising the nucleic acid molecule described in n1); n6) a cell comprising the expression cassette described in n2); n7) a cell comprising the vector described in n3); n8) a cell comprising the vector described in n4); n9) A cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the protein according to claim 4; The cell described in any of n5)-n9) does not contain any propagation material.

6. A conjugate comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and, coupling part.

7. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the protein according to claim 4, the biomaterial according to claim 5, or the conjugate according to claim 6; and a pharmaceutically acceptable carrier.

8. A diagnostic or therapeutic kit comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the protein according to claim 4, the biomaterial according to claim 5, the conjugate according to claim 6, or the pharmaceutical composition according to claim 7.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the protein according to claim 4, the biomaterial according to claim 5, the conjugate according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a product, wherein the product is used 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.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the protein according to claim 4, the biomaterial according to claim 5, the conjugate according to claim 6, the pharmaceutical composition according to claim 7, the diagnostic or therapeutic kit according to claim 8, or the use according to claim 9, characterized in that: The influenza A virus comprises influenza A virus H3 subtype; or The diseases caused by influenza A virus infection include influenza A.

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