Influenza A virus neutralizing antibody, its preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to an influenza A virus neutralizing antibody, and a preparation method and application thereof. The antibody or its antigen-binding fragment can specifically recognize and bind to the HA protein and / or HA1 protein of influenza A virus, and has good affinity with it; at the same time, it has good neutralizing activity against influenza A virus; it has high binding activity, fast binding speed, and remarkable neutralizing ability, especially has advantages in quickly neutralizing the virus, can effectively cope with 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 HA protein and / or HA1 protein of influenza A virus in a sample.

Description

Technical Field

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

[0002] Influenza viruses are highly variable RNA viruses. Especially the influenza A H3 subtype, due to its high variability, causes influenza pandemics worldwide every year, posing a major threat to public health. The hemagglutinin (HA) protein of influenza virus is the key protein for the virus to enter host cells and is also the main target of neutralizing antibodies. The HA protein consists of two subunits, HA1 and HA2, where the HA1 subunit contains most of the antigenic sites and is the main binding region for neutralizing antibodies.

[0003] In recent years, new influenza strains have emerged continuously. H3N2 strains such as A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 have shown high prevalence and pathogenicity during recent influenza seasons. However, the existing antibodies have poor neutralizing effects on these new strains, which further highlights the urgency and importance of developing novel broad-spectrum neutralizing antibodies. Summary of the Invention

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

[0005] The purpose of the second aspect of the present invention is to provide chimeric antigen receptors.

[0006] The purpose of the third aspect of the present invention is to provide multispecific antibodies or their antigen-binding fragments.

[0007] The purpose of the fourth aspect of the present invention is to provide biological materials related to the antibodies or their antigen-binding fragments of the first aspect of the present invention, the chimeric antigen receptors of the second aspect, or the multispecific antibodies or their antigen-binding fragments of the third aspect.

[0008] The purpose of the fifth aspect of the present invention is to provide preparation methods for the antibodies or their antigen-binding fragments of the first aspect of the present invention, the chimeric antigen receptors of the second aspect, or the multispecific antibodies or their antigen-binding fragments of the third aspect.

[0009] The purpose of the sixth aspect of the present invention is to provide conjugates.

[0010] The purpose of the seventh aspect of the present invention is to provide pharmaceutical compositions.

[0011] The purpose of the eighth aspect of the present invention is to provide diagnostic or therapeutic kits.

[0012] The object of the ninth aspect of the present invention is to provide the use of the antibody or its antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, or the multispecific antibody or its antigen-binding fragment of the third aspect, the biological material of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

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

[0014] The first aspect of the present invention provides an influenza A virus antibody or its antigen-binding fragment, and the influenza A virus antibody or its antigen-binding fragment includes:

[0015] 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

[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 system.

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

[0019] b1) VH including the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, HCDR2 having the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 7; and / or, VL including the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 8, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0020] b2) The VH comprising the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions as compared to HCDR1, HCDR2, and HCDR3 shown in b1); and / or, the VL comprising the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions as compared to LCDR1, LCDR2, and LCDR3 shown in b1);

[0021] Wherein, the CDRs are defined according to the IMGT numbering system.

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

[0023] c1) The VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, the VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0024] c2) The VH comprising the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions as compared to HCDR1, HCDR2, and HCDR3 shown in c1); and / or, the VL comprising the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions as compared to LCDR1, LCDR2, and LCDR3 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 its antigen-binding fragment comprises:

[0027] d1) The VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, the VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0028] d2) The VH comprising the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions or additions as compared with the HCDR1, HCDR2, and HCDR3 shown in d1); and / or, the VL comprising the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions or additions as compared with the LCDR1, LCDR2, and LCDR3 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 its antigen-binding fragment comprises:

[0031] e1) The VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, the VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or

[0032] e2) The VH comprising the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions or additions as compared with the HCDR1, HCDR2, and HCDR3 shown in e1); and / or, the VL comprising the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions or additions as compared with the LCDR1, LCDR2, and LCDR3 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 its antigen-binding fragment comprises:

[0035] f1) a VH comprising the following 3 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, a VL comprising the following 3 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; or

[0036] f2) a VH comprising the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions or additions as compared with the HCDR1, HCDR2, and HCDR3 shown in f1); and / or, a VL comprising the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions or additions as compared with the LCDR1, LCDR2, and LCDR3 shown in f1);

[0037] Wherein, the CDRs are defined according to the Contact numbering system.

[0038] Those skilled in the art should 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 its antigen-binding fragment further comprises the framework region of the heavy chain variable region.

[0040] In some embodiments, the framework region of the heavy chain variable region comprises the framework region of the heavy chain variable region of an immunoglobulin derived from a mouse, primate, bovine, horse, bovine, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; further comprising the framework region of the heavy chain variable region of an immunoglobulin derived from a human or a mutant thereof.

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

[0042] In some embodiments, the framework region of the light chain variable region comprises the framework region of the light chain variable region of an immunoglobulin derived from a mouse, primate, bovine, horse, bovine, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; further comprising the framework region of the light chain variable region of an immunoglobulin derived from a human or a mutant thereof.

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

[0044] 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.

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

[0046] In some embodiments, the heavy chain constant region may comprise at least a portion of the heavy chain constant region of an immunoglobulin derived from mouse, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; and further comprises at least a portion of the heavy chain constant region of a human immunoglobulin or a mutant thereof.

[0047] In some embodiments, the light chain constant region may comprise at least a portion of the light chain constant region of an immunoglobulin derived from mouse, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; and further comprises the light chain constant region of a human immunoglobulin or a mutant thereof.

[0048] In some embodiments, the heavy chain constant region may comprise the heavy chain constant region of an immunoglobulin selected from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM.

[0049] In some embodiments, the light chain constant region may comprise the light chain constant regions of κ-type and λ-type immunoglobulins.

[0050] In some embodiments, the influenza A virus antibody or its antigen-binding fragment may 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 its antigen-binding fragment may include, but is not limited to, Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv or Fd fragment.

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

[0053] 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 therewith; 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 therewith.

[0054] In a second aspect of the present invention, there is provided a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding domain comprises the antibody or its antigen-binding fragment of the first aspect of the present invention.

[0055] In a third aspect of the present invention, there is provided a multispecific antibody or its antigen-binding fragment comprising two or more (such as three or four) antigen-binding domains, wherein one antigen-binding domain comprises the antibody or its antigen-binding fragment of the first aspect of the present invention.

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

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

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

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

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

[0061] n5) A cell comprising the nucleic acid molecule of n1);

[0062] n6) A cell comprising the expression cassette of n2);

[0063] n7) A cell comprising the vector of n3);

[0064] n8) A cell comprising the vector of n4);

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

[0066] Any of the cells of n5)-n9) does not contain propagation material.

[0067] Those skilled in the art should understand that nucleotides in the nucleic acid molecule can be replaced according to 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 an antigen-binding fragment thereof according to the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or an antigen-binding fragment thereof according to the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the antibody or an antigen-binding fragment thereof according to 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 an antigen-binding fragment thereof according to the first aspect of the present 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 an antigen-binding fragment thereof according to the first aspect of the present 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 one of the carriers 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, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector can include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, phage vectors, adenoviral vectors, adeno-associated vectors, or herpes simplex vectors.

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

[0073] In some embodiments, any one of the cells in n5)-n9) can be a host cell conventionally used in the art, as long as it can stably express the nucleic acid molecule carried by the expression vector as the above-mentioned antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment 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, 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.

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

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

[0076] The sixth aspect of the present invention provides a conjugate, which includes the antibody or its antigen-binding fragment of the first aspect of the present invention; and a conjugate part.

[0077] In some embodiments, the conjugate part can include, but is not limited to, a detectable marker or a therapeutic agent.

[0078] In some embodiments, the detectable label can be any substance detectable by means such as fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, 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.) microspheres, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In some embodiments, such labels are applicable to immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances or enzymes. In some embodiments, the detectable label as described above can be linked to the antibody or its antigen-binding fragment of the present invention through linkers of different lengths to reduce potential steric hindrance.

[0079] In some embodiments, the detectable label may include, but is not limited to, enzymes (such as horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances), colored substances, biotin, etc.

[0080] In some embodiments, the therapeutic agent may include, for example, but is not limited to, drugs for preventing and / or treating influenza A virus infection or diseases caused thereby.

[0081] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (such as increasing the serum half-life), and may be, for example, chemical groups such as polyethylene glycol (PEG), methyl, ethyl or glycosyl.

[0082] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising: the antibody or its antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or its antigen-binding fragment 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 an additional pharmaceutically active agent.

[0084] In some embodiments, the additional pharmaceutically active agent can be a drug with biological activity, such as a drug capable of preventing and / or treating influenza A virus infection or diseases caused thereby.

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

[0086] In some embodiments, the pharmaceutical composition can be administered by, for example, parenteral, subcutaneous injection, sublingual, rectal, nasal, intravenous injection, intramuscular injection, oral, ocular, topical, etc.

[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, which includes: the antibody or its antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or its antigen-binding fragment of the third aspect, the biological material 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 a dosing device.

[0090] In some embodiments, the kit can be used for diagnosing influenza A virus infection or diseases caused thereby.

[0091] In some embodiments, the kit can be used for preventing and / or treating influenza A virus infection or diseases caused thereby.

[0092] The ninth aspect of the present invention provides the use of the antibody or its antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or its antigen-binding fragment of the third aspect, the biological material of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in the preparation of a product for any one of c1)-c4):

[0093] c1) Diagnosing influenza A virus infection or diseases caused thereby;

[0094] c2) Preventing and / or treating influenza A virus infection or diseases caused thereby;

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

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

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

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

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

[0100] In some embodiments, the excreta 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 orangutans, 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 to be tested includes humans.

[0103] In the present invention, the influenza A virus includes influenza A virus subtype H3; further includes H3N2 subtype, for example, but not limited to, at least one of A / HongKong / 4801 / 2014 (H3N2), A / Hong Kong / 45 / 2019 (H3N2), A / Darwin / 6 / 2021 (H3N2), A / Massachusetts / 18 / 2022 (H3N2).

[0104] In the present invention, the diseases caused by influenza A virus infection include influenza A.

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

[0106] The present invention provides an influenza A virus antibody or its antigen-binding fragment, which can specifically recognize and bind to influenza A virus HA protein and / or HA1 protein, and has good affinity with it; at the same time, it has good neutralizing activity against influenza A virus. In particular, the neutralizing ability against the prevalent strains A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022 of H3 subtype in recent years is significantly enhanced, IC 50The value is significantly lower than that of reported antibodies of the same kind; it has high binding activity, fast binding speed, and significant neutralizing ability. Especially, it has advantages in quickly neutralizing the virus, can effectively cope with 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 a sample.

[0107] Furthermore, the fully humanized influenza A virus antibody or its antigen-binding fragment has low immunogenicity and a low risk of adverse reactions, and can be prepared in large quantities through mammalian cell lines, ensuring the stability and clarity of the components. Brief Description of the Drawings

[0108] Figure 1 Shows the binding OD value of the Flu-B0254 antibody to the hemagglutinin HA trimer.

[0109] Figure 2 Shows the affinity kinetics curve of the Flu-B0254 antibody to the hemagglutinin HA1 monomer.

[0110] Figure 3 Shows the IC 50 value of the Flu-B0254 antibody against pseudovirus neutralization. Detailed Description of the Embodiments

[0111] To make the objectives, technical solutions, and advantages of the present invention clearer, the following further elaborates on the present invention in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing 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 pertains. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0113] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, referring to "a cell" includes multiple such cells and equivalents known to those skilled in the art, and so on.

[0114] Hemagglutinin HA is a key trimeric protein on the surface of influenza virus, which is composed of two subunits, HA1 and HA2, connected by disulfide bonds. It plays a crucial role in the infection process of influenza virus, including binding to receptors on the surface of host cells, promoting the fusion of virus and host cell membranes, and participating in the packaging of virus particles, and is a key factor determining virus pathogenicity. As the main recognition target of the host immune system, HA protein can stimulate the host to produce a strong immune response, especially the production of neutralizing antibodies. These neutralizing antibodies specifically recognize and bind to HA protein, blocking the binding of virus and host cells, thereby inhibiting virus infection and transmission.

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

[0116] The "percent sequence identity" or "identity percent" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within a 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. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted since nucleotides or amino acids from the target sequence are counted and nucleotides or amino acids from the reference sequence are not counted.

[0117] The percent sequence identity can be calculated by the following process: Determine the number of positions in which the same amino acid residue or nucleic acid base occurs in both of two 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 percent sequence identity. The comparison of sequences and the determination of the percent sequence identity between two sequences can be done using software readily available for online use and download. Suitable software programs are available from a variety of sources for the alignment of protein and nucleotide sequences. One suitable program for determining the percent sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website of the National Center for Biotechnology Information of the U.S. government (blast.ncbi.nlm.nih.gov). Bl2seq uses the BLASTN or BLASTP algorithm for comparison between 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, which are part of the EMBOSS suite of bioinformatics programs and are 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 alter the expected properties of a protein / polypeptide containing 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 of an amino acid residue with another amino acid residue having a similar side chain, such as a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute 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. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0119] Examples and drawings are provided below to assist in understanding the present invention. However, it should be understood that these examples and drawings are only for illustrative purposes and 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.

[0120] It should be noted that for those not specified with specific conditions in the examples, they are carried out under conventional conditions, manufacturer's suggestions or experimental conditions reported in the public. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially. For those reagents indicating the manufacturer, similar products of other manufacturers are substitutable.

[0121] For the quantitative tests in the following examples, three repeated experiments are set up, and the results are averaged.

[0122] Example 1. Sorting of B cells by flow cytometry 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 capable of specifically binding to the full-length hemagglutinin HA and HA1 monomer were screened. Further, candidate antibody Flu-B0254 was screened as follows:

[0124] 1. PBMC isolation and memory B cell sorting

[0125] 10 mL of venous blood was collected from volunteers infected with influenza virus and producing protective antibodies in the second half of 2023 and placed in an anticoagulant tube containing Ethylene Diamine Tetraacetic Acid (EDTA). PBMCs were isolated by Ficoll gradient centrifugation. Using recombinant influenza virus HA protein (Beijing Sino Biological Inc., 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 PBMCs into a 96-well PCR (Polymerase Chain Reaction) plate using an Astrios EQ (BeckMan Coulter) flow cytometer, with each well containing 1 B cell. The 96-well plate containing B cells was placed in a -80°C refrigerator for storage for later use.

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

[0127] 1) Reverse transcription PCR: All IgG1 subtype-specific primers for the heavy chain, κ light chain, and λ light chain, as well as Maxima H Minus reverse transcriptase (Thermo), were added to the 96-well PCR plate containing a single B cell. Reverse transcription was carried out at 50°C for 30 min, and the reverse transcriptase was inactivated at 85°C for 5 min. The obtained cDNA product was stored at -80°C.

[0128] 2) Nested PCR:

[0129] First-round reaction: Using 2 μL of cDNA product as a template, add TransStart(®) FastPfu DNA Polymerase (Transgene, AP221), dNTPs, and nested PCR primers. Reaction conditions: Pre-denaturation at 98°C for 5 min, followed by 40 PCR cycles, each cycle consisting of: 98°C for 30 s, 55°C (heavy chain VH, κ light chain Vκ) / 50°C (λ light chain Vλ) for 1 min, 72°C for 1 min, and finally extension at 72°C for 5 min.

[0130] Second-round reaction: Using the PCR product of the first-round 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, followed by 35 PCR cycles, each cycle consisting of: 98°C for 30 s, 58°C (heavy chain VH) / 60°C (κ light chain Vκ) / 64°C (λ light chain Vλ) for 1 min, 72°C for 1 min, and finally extension at 72°C for 5 min to obtain the nested PCR amplification product.

[0131] For the sequences of the IgG1 subtype-specific primers targeting the heavy chain, κ light chain, and λ light chain, and 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] Take 5 μL of the nested PCR amplification product for detection by 1.5% agarose gel electrophoresis. Select paired positive clones for sequencing to obtain the antibody variable region sequence, and construct a linear expression frame for this sequence.

[0134] 3. Obtaining and identification of antibody sequences

[0135] Using the IgBLAST software, with the human genome reference sequence database GRCh38 as a reference, the DNA sequence was analyzed and processed. Through this software, the VDJ gene segments of the heavy and light chains in the antibody sequence were identified, and the number of somatic hypermutations (SHM) was counted. Calculation formula: SHM mutation rate = mutation count / nucleic acid length of V gene alignment. The ANARCI software was used to identify the CDR region. The CDR region and variable region sequences of the Flu-B0254 antibody are shown in Table 1, and the antibody characteristic information is shown in Table 2.

[0136]

[0137]

[0138] 4. Expression plasmid construction and antibody preparation

[0139] The coding nucleic acid sequences of the paired immunoglobulin (Flu-B0254) heavy and light chains (amino acid sequences are shown in Table 1) were submitted to GenScript Biotech Corporation for gene synthesis. Based on Gibson assembly, the heavy and light chain sequences were cloned into the pcDNA3.4 expression vector respectively. Subsequently, the two plasmids were co-transfected into HEK293F cells and cultured in a 37°C 5% CO2 incubator for 72 h. The supernatant was collected by centrifugation at 300 g for 5 min. The monoclonal antibody secreted in the cell culture medium was purified by Protein A affinity chromatography. The collected antibody was buffer-exchanged with PBS, and the antibody concentration and purity were detected.

[0140] For the sequences of the control antibodies S5V2-29, FluA-20, CR8020, FI6v3, MEDI8852, CR8043 and the antibody preparation conditions in the present invention, see Watanabe A, 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; Ekiert DC, Friesen RH, Bhabha G, et al. A highly conserved neutralizingepitope on group 2 influenza A viruses. Science. 2011;333(6044):843-850; CortiD, Voss J, Gamblin SJ, et al. A neutralizing antibody selected from plasmacells that binds to group 1 and group 2 influenza A hemagglutinins. Science.2011;333(6044):850-856; Kallewaard NL, Corti D, Collins PJ, et al. Structureand Function Analysis of an Antibody Recognizing All Influenza A Subtypes.Cell. 2016;166(3):596-608; Friesen RH, Lee PS, Stoop EJ, et al. A commonsolution to group 2 influenza virus neutralization. Proc Natl Acad Sci U S A.2014;111(1):445-450。.

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

[0142] 1. Experimental procedure

[0143] Using the full-length hemagglutinin HA protein (Beijing Sino Biological Inc., 40992-V08B, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA Protein (His Tag)) as the antigen, the antigen was diluted to 1 μg / mL with the coating buffer and then coated on a 96-well ELISA plate, with a volume of 100 μL per well and coated overnight at 4°C. The plate was blocked at 37°C for 2 h with the blocking buffer. The purified Flu-B0254 antibody expressed in Example 1 and other control antibodies (S5V2-29 and FluA-20) were diluted to 1 μg / mL, and 100 μL of each was added to the blocked ELISA plate and incubated at 37°C for 1 h. After washing the plate, 100 μL of goat anti-human IgG(H+L)-HRP (diluted 1:2000) was added and incubated at 37°C for 1 h. After adding the substrate chromogenic solution, it was placed in the dark at room temperature for 10 min, and the reaction was terminated with 2 M sulfuric acid. The OD values at wavelengths of 450 / 630 nm were measured.

[0144] 2. Results

[0145] The results are as Figure 1 shown. The purified Flu-B0254 antibody can bind to the hemagglutinin HA trimer protein, and the binding levels are similar to those of the reported S5V2-29 and FluA-20 antibodies in the literature, with OD values all reaching above 4, indicating strong binding activity.

[0146] Example 3. SPR detection of the affinity constant and kinetic analysis of antibody Flu-B0254

[0147] 1. Capture assay

[0148] The antibody affinity test was performed using the capture method. The buffer was 1*HBS-EP (Cytiva). The CM5 chip was first conjugated with anti-human Fc (Cytiva). Then the capture antibody was diluted to a concentration of 5 μg / mL, and the binding time was 60 s. The analyte hemagglutinin HA1 monomer protein (Beijing Sino Biological Inc., 40992-V08H1, Influenza A H3N2 (A / Massachusetts / 18 / 2022; A / Thailand / 8 / 2022) Hemagglutinin / HA1 Protein (HisTag)) was flowed over the chip successively at gradually increasing concentrations (1.5625 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) to obtain signal curves respectively. Each concentration was taken as one cycle. After one cycle was completed, the chip was regenerated with 4M MgCl2 to return to the original state without the captured antibody, and the regeneration time was 60 s. The obtained signal curves were analyzed using Biacore 8K System software to obtain the affinity activity detection graph of the Flu-B0254 antibody and the HA1 monomer.

[0149] 2. Results

[0150] As shown in Table 3 and Figure 2 the equilibrium dissociation constant KD value of the Flu-B0254 antibody for the hemagglutinin HA1 monomer reached the order of magnitude of 10 -10 M, indicating that this antibody has a higher affinity than the S5V2-29 and FluA-20 neutralizing antibodies reported in the literature. At the same time, the relatively high Ka value indicates that Flu-B0254 has a fast binding rate and can effectively bind and neutralize the virus.

[0151]

[0152] Example 4. Evaluation of Pseudovirus Neutralization Activity

[0153] 1. Pseudovirus Packaging

[0154] Pseudoviruses were prepared based on the Human Immunodeficiency Virus Type 1 (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 Initiative on Sharing All Influenza Data) accession numbers of the HA and NA genes of the 4 pseudovirus strains used. The HA plasmid, NA plasmid, and HIV backbone plasmid (pSG3.Δenv-FlucΔnef) were co-transfected into HEK-293FT cells using the transfection reagent Lipofectamine 2000 (Invitrogen). The culture supernatant was collected 48 h after transfection, centrifuged at 4000 rpm for 10 min, and concentrated using a 30KD ultrafiltration centrifugal tube.

[0155] The sequence of the viral backbone plasmid and the experimental conditions are described in 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] For the pseudovirus titration experiment, the Promega luciferase assay kit was used to quantitatively analyze the pseudovirus. First, the pseudovirus was serially diluted 5-fold in a 96-well culture plate, with each well containing 100 μL of the diluted virus solution. 100 μL of trypsinized MDCK cells were seeded into each well, resulting in 3×10 4 cells per well. After incubating the 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 incubating at room temperature for 2 min, 150 μL of the lysate was transferred to a new 96-well plate, and the luciferase activity was measured using a Glomax 96 microplate photometer. The 50% tissue culture infective dose (TCID 50 ) was calculated by the Reed-Muench method to evaluate the infectivity of the pseudovirus.

[0159] For the neutralization assay, the antibody to be tested was serially diluted 2-fold in a 96-well culture plate, with each well containing 100 μL of the dilution. Then, 50 μL of 5000 TCID50 pseudovirus (RLU value is 10 6 ), after mixing, place it at 37°C for incubation for 1 h. Add 100 μL of MDCK cell suspension to a 96-well plate, so that the number of cells per well is 3×10 4 cells, incubate at 37°C for 48 h, and then perform luciferase activity detection according to the above method. The IC50 is determined by a four-parameter non-linear regression model to evaluate the neutralizing potency of the antibody.

[0160] 3. Results

[0161] As Figure 3 shown, the heatmap shows the pseudovirus neutralizing activities of different antibodies against multiple influenza virus strains, with the IC 50 value as the index (unit: μg / mL), and the lower the value, the stronger the neutralizing activity. The blue area in the figure represents a low IC 50 value, indicating that the antibody has a high neutralizing effect on the corresponding strain; the red area represents a high IC 50 value, indicating 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 neutralizing activity.

[0162] The results show that Flu-B0254 has a relatively wide neutralizing effect on the H3 subtype strains of influenza A virus prevalent in recent years. Compared with the potent neutralizing antibodies S5V2-29, FluA-20, CR8020, FI6v3, MEDI8852, CR8043 reported in the literature, Flu-B0254 has a significantly enhanced neutralizing ability in neutralizing recently prevalent strains such as A / Darwin / 6 / 2021 and A / Massachusetts / 18 / 2022, and its potency has achieved an order-of-magnitude improvement. The above findings highlight the potential application value of Flu-B0254 antibody in the prevention and treatment of influenza A virus infection or the diseases caused by it, especially in the face of the challenge of the rapid change of influenza A virus strains.

[0163] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

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

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody against influenza A virus or the antigen-binding fragment thereof comprises: b1) VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, HCDR2 having the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 7; and / or VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 8, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; the CDRs are defined according to the IMGT numbering system; or c1) VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; the CDRs are defined according to the Kabat numbering system; or d1) VH comprising the following 3 CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or VL comprising the following 3 CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; the CDRs are defined according to the AbM numbering system; or e1) The VH comprising the following 3 CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 17, HCDR2 with the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 12; and / or, the VL comprising the following 3 CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 13, LCDR2 with the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 9; the CDRs are defined according to the Chothia numbering system; or f1) The VH comprising the following 3 CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 19, HCDR2 with the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 21; and / or, the VL comprising the following 3 CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 22, LCDR2 with the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 with 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, wherein the heavy chain variable region of the influenza A virus antibody or its antigen-binding fragment further comprises the framework region of the heavy chain variable region; or the light chain variable region of the influenza A virus antibody or its antigen-binding fragment further comprises the framework region of the light chain variable region; or the influenza A virus antibody or its antigen-binding fragment comprises a heavy chain variable region 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 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; or the influenza A virus antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; or the influenza A virus antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; or the influenza A virus antibody or its antigen-binding fragment comprises a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv, a dsFv or an Fd fragment; or the influenza A virus antibody or its antigen-binding fragment 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. A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1-3, said biological material comprising any one of n1)-n9): n1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-3; n2) An expression cassette comprising the nucleic acid molecule of n1); n3) A vector comprising the nucleic acid molecule of n1); n4) A vector comprising the expression cassette of n2); n5) A cell comprising the nucleic acid molecule of n1); n6) A cell comprising the expression cassette of n2); n7) A cell comprising the vector of n3); n8) A cell comprising the vector of n4); n9) A cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3; Any of the cells of n5)-n9) does not contain propagation material.

5. Pharmaceutical composition, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-3, or the biological material according to claim 4; And a pharmaceutically acceptable carrier.

6. Diagnostic or therapeutic kit, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-3, the biological material according to claim 4, or the pharmaceutical composition according to claim 5.

7. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the biological material according to claim 4, or the pharmaceutical composition according to claim 5 in the preparation of a product for any one of c1)-c4): c1) Diagnosing influenza A virus infection or a disease caused thereby; c2) Preventing and / or treating influenza A virus infection or a disease caused thereby; c3) Detecting the presence or level of influenza A virus hemagglutinin HA protein in a sample; c4) Detecting the presence or level of influenza A virus hemagglutinin HA1 protein in a sample; The influenza A virus is the influenza A virus H3N2 subtype.

8. The use according to claim 7, wherein The disease caused by the influenza A virus infection is influenza A.

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