Single-domain antibody for resisting influenza A (H1N1) virus or antigen binding fragment thereof and application of single-domain antibody or antigen binding fragment

Abstract

The invention relates to a single-domain antibody for resisting influenza A (H1N1) virus or an antigen binding fragment thereof and an application of the single-domain antibody or the antigen binding fragment. Specifically, the present invention relates to single domain antibodies or antigen binding fragments thereof against influenza A (H1N1) virus surface hemagglutinin (HA), nucleic acid molecules encoding them, methods of preparing them, and pharmaceutical compositions containing them. The single-domain antibody or the antigen binding fragment thereof can be used for preventing and / or treating influenza A (H1N1) virus infection or diseases related to influenza A (H1N1) virus infection of a subject (such as a human), neutralizing the virulence of the influenza A (H1N1) virus in vitro or in the body of the subject (such as the human), and / or preventing and / or treating the influenza A (H1N1) virus infection or the diseases related to the influenza A (H1N1) virus infection of the subject (such as the human). The present invention relates to a method for reducing the level of influenza A H1N1 virus DNA and / or the serum level of influenza A H1N1 virus in a subject (e.g., a human).

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of molecular virology, immunology and antibodies, in particular to a single-domain antibody or antigen-binding fragment thereof against influenza A H1N1 virus and application thereof. BACKGROUND

[0002] Influenza viruses continue to threaten global public health safety. Seasonal influenza, which is composed of H1N1, H3N2 and B influenza viruses, causes 3-5 million severe cases and 290-650 thousand deaths worldwide each year. About 88,000 people die of respiratory diseases caused by influenza in China each year. The emergence of variant and drug-resistant strains poses a threat to the use of existing vaccines and small molecule drugs. Therefore, developing effective broad-spectrum anti-influenza virus vaccines and drugs is an important demand for our country and even the world. Neutralizing antibodies have been successfully applied in the prevention and treatment of viral infections. The hemagglutinin protein (HA) on the surface of influenza viruses is a key protein that mediates viral infection of host cells and is also a key target for neutralizing antibodies and vaccines. In-depth development of broad-spectrum influenza virus neutralizing antibodies and research on the molecular mechanism of broad-spectrum influenza antibodies in neutralizing different subtypes of influenza viruses will contribute to the development of universal influenza vaccines and broad-spectrum influenza antibody drugs.

[0003] Currently, the prevention of influenza viruses mainly relies on vaccines, and the clinical treatment mainly relies on neuraminidase inhibitors. Every year, the World Health Organization will predict the seasonal influenza subtypes that may prevail the next year, and accordingly produce the corresponding influenza vaccines. Vaccines can stimulate the body to produce specific antibodies against the corresponding virus subtypes, playing a defensive role. After the injection of influenza vaccines into the body, it takes a certain period of time to produce the corresponding antibodies, so vaccines can only play a preventive role. Once an influenza outbreak occurs, they cannot play an effective therapeutic role. Moreover, the differences between influenza virus subtypes are large, and the current seasonal vaccines are not sufficient to cover new epidemic strains, posing a risk of global pandemic. Neuraminidase inhibitors such as peramivir, oseltamivir, and zanamivir are the main drugs used in the clinical treatment of influenza. These drugs inhibit the replication of influenza viruses by inhibiting the neuraminidase (NA) on the surface of influenza viruses, preventing the production and spread of new influenza viruses in the body, thereby inhibiting influenza virus infection. The therapeutic window of such drugs is narrow, and with the passage of time, drug resistance increases, reducing the efficacy. More and more reports show that drug-resistant influenza virus strains have emerged against neuraminidase inhibitors, which makes this class of drugs no longer a panacea. The development of new influenza drugs is imminent.

[0004] Single-domain antibody, also known as nanobody, is a new type of antibody found in camelids and chondrichthyan in recent years, which shows good clinical application value in many disease fields. Nanobody has stable physical and chemical properties, low production cost and strong tissue penetration, which is superior to traditional IgG antibody in many application scenarios. And more and more studies have proved that nanobody has broad-spectrum antiviral ability and can recognize hidden epitopes that traditional antibodies cannot approach.

[0005] Therefore, the development of nanobodies targeting influenza virus will provide new ideas and clinical application value for the prevention and treatment of influenza virus. SUMMARY

[0006] It is found through research that the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to hemagglutinin (HA) of influenza A H1N1 virus developed by the present application can effectively neutralize influenza A H1N1 virus and / or hemagglutinin (HA) of influenza A H1N1 virus, providing new ideas and clinical application value for the prevention and treatment of influenza virus, especially influenza A H1N1 virus. At the same time, the single-domain antibody or antigen-binding fragment thereof of the present application can be used for the development of detection reagents or diagnostic reagents for H1N1 influenza virus or HA antigen thereof.

[0007] Antibody or antigen-binding fragment thereof

[0008] Therefore, in a first aspect, the present application provides a single-domain antibody or antigen-binding fragment thereof capable of specifically binding to hemagglutinin (HA) of influenza A H1N1 virus, comprising:

[0009] a heavy chain variable region (VHH) comprising a complementarity determining region 1 (CDR1) as shown in SEQ ID NO: 2 or a variant thereof, a complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 3 or a variant thereof, and a complementarity determining region 3 (CDR3) as shown in SEQ ID NO: 4 or a variant thereof,

[0010] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions, e.g. conservative substitutions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0011] wherein the CDRs are defined by the IMGT numbering system.

[0012] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a VHH comprising: a CDR1 as set forth in SEQ ID NO: 2, a CDR2 as set forth in SEQ ID NO: 3, and, a CDR3 as set forth in SEQ ID NO: 4,

[0013] wherein the CDRs are defined by the IMGT numbering system.

[0014] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a complementarity determining region 1 (CDR1) or a variant thereof, a complementarity determining region 2 (CDR2) or a variant thereof, and a complementarity determining region 3 (CDR3) or a variant thereof, contained within a heavy chain variable region (VHH) as set forth in SEQ ID NO: 1 ;

[0015] wherein the variant has one or several (e.g. 1, 2, or 3) amino acid substitutions, deletions, or additions as compared to the sequence from which it is derived; preferably the substitutions are conservative substitutions.

[0016] Preferably, the substitutions are conservative substitutions.

[0017] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a CDR1, a CDR2, and a CDR3 contained within a heavy chain variable region (VHH) as set forth in SEQ ID NO: 1 ;

[0018] wherein the 3 HCDRs contained within the VHH are defined by the Kabat, IMGT, Chothia, or AbM numbering system.

[0019] In some embodiments, the VHH of the single-domain antibody or antigen-binding fragment thereof further comprises a framework region (FR) of an immunoglobulin.

[0020] In some embodiments, the VHH of the single-domain antibody or antigen-binding fragment thereof comprises: a framework region 1 (FR1) as set forth in SEQ ID NO: 5 or a variant thereof, a framework region 2 (FR2) as set forth in SEQ ID NO: 6 or a variant thereof, a framework region 3 (FR3) as set forth in SEQ ID NO: 7 or a variant thereof, and a framework region 4 (FR4) as set forth in SEQ ID NO: 8 or a variant thereof, the variant having one or more (e.g. 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions as compared to the sequence from which it is derived, or having a sequence identity of 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%, at least 99%, or 100%; preferably the substitutions are conservative substitutions.

[0021] In some embodiments, the VHH of the single-domain antibody or antigen-binding fragment thereof comprises a FR1 as set forth in SEQ ID NO: 5, a FR2 as set forth in SEQ ID NO: 6, a FR3 as set forth in SEQ ID NO: 7, and a FR4 as set forth in SEQ ID NO: 8.

[0022] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a VHH, which comprises an amino acid sequence as set forth in SEQ ID NO: 1, or a variant thereof,

[0023] wherein the variant has one or more (e.g., 1, 2, 3, 4, or 5) substitutions, deletions, or additions of amino acids compared to the sequence as set forth in SEQ ID NO: 1, or a sequence with 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%, at least 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 1; preferably, the substitutions are conservative substitutions.

[0024] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a VHH, which comprises an amino acid sequence as set forth in SEQ ID NO: 1.

[0025] In some embodiments, the single-domain antibody or antigen-binding fragment thereof is optionally linked to an Fc domain, a tag peptide, or a combination thereof, through a linker peptide.

[0026] Preparation of antibodies

[0027] The antibodies of the present application can be prepared in various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the heavy chain gene of the antibody of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed.

[0028] In a second aspect, the present application also provides an isolated nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof described above.

[0029] In a third aspect, the present application also provides a vector comprising the isolated nucleic acid molecule of the second aspect; preferably, the vector is a cloning vector or an expression vector.

[0030] In a fourth aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule of the second aspect or the vector of the third aspect.

[0031] Fifthly, the present invention also provides a method for preparing the single-domain antibody or antigen-binding fragment thereof as described in the first aspect, comprising culturing the host cell as described in the fourth aspect under conditions that allow expression of the single-domain antibody or antigen-binding fragment thereof, and recovering the single-domain antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0032] Derived antibodies

[0033] The single-domain antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the single-domain antibody or antigen-binding fragment does not adversely affect its binding to the hemagglutinin protein on the surface of the H1N1 influenza A virus. Therefore, the single-domain antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the single-domain antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., an avidin or a multihistidine tag) capable of mediating the binding of the antibody or antigen-binding fragment to another molecule.

[0034] Therefore, in a sixth aspect, the present invention also provides a multispecific antibody comprising the single-domain antibody or its antigen-binding fragment described in the first aspect.

[0035] In some implementations, the multispecific antibody specifically binds to the surface hemagglutinin (HA) protein of the H1N1 influenza A virus and additionally specifically binds to one or more other targets.

[0036] In a seventh aspect, the invention also provides a coupling comprising:

[0037] (1) The single-domain antibody or its antigen-binding fragment as described in the first aspect, or the multispecific antibody as described in the sixth aspect; and

[0038] (2) Coupling part,

[0039] The coupling portion is selected from the following: detectable markers, drugs, toxins, cytokines, or combinations thereof;

[0040] Preferably, the detectable marker is selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase), chemiluminescent reagents (e.g., acrid esters, luminol and its derivatives, ruthenium derivatives), fluorescent markers (e.g., fluorescein, fluorescent protein), biotin, and radionuclides.

[0041] The detectable markers described in this invention are any substances that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), 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)), luminescent materials (e.g., chemiluminescent materials such as acridinium esters), and magnetic beads (e.g., Dynabeads). ® ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0042] In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0043] Pharmaceutical Compositions and Therapeutic Uses

[0044] In an eighth aspect, the present invention also provides a pharmaceutical composition comprising the single-domain antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antibody described in the sixth aspect, or the conjugate described in the seventh aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0045] In an eighth aspect, the present invention also provides the use of the single-domain antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antibody described in the sixth aspect, the conjugate described in the seventh aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a medicament, wherein the medicament is used for:

[0046] (i) Prevention and / or treatment of H1N1 influenza virus infection or illness associated with H1N1 influenza virus infection in subjects (e.g., humans),

[0047] (ii) for neutralizing the virulence of influenza A H1N1 virus in vitro or in subjects (e.g., humans), and / or,

[0048] (iii) To reduce the serum levels of influenza A H1N1 virus DNA and / or influenza A H1N1 virus in subjects (e.g., humans).

[0049] In another aspect, the present invention provides a method for: preventing and / or treating a subject (e.g., a human) from infection with or related to influenza A (H1N1) virus, for neutralizing the virulence of influenza A (H1N1) virus in vitro or in a subject (e.g., a human), and / or for reducing the serological level of influenza A (H1N1) virus DNA and / or influenza A (H1N1) virus in a subject (e.g., a human), the method comprising: administering to a subject in need an effective amount of the single-domain antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the multispecific antibody described in the sixth aspect, the conjugate described in the seventh aspect, or the pharmaceutical composition described in the eighth aspect.

[0050] The drugs and drug compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0051] Reagent kit and detection uses

[0052] In a ninth aspect, the present invention also provides a kit containing the single-domain antibody or its antigen-binding fragment described in the first aspect, the multispecific antibody described in the sixth aspect, and the conjugate described in the seventh aspect.

[0053] In some embodiments, the single-domain antibody or antigen-binding fragment of the present invention carries a detectable label. In other embodiments, the kit further includes a second antibody that specifically recognizes the single-domain antibody or antigen-binding fragment of the present invention. Preferably, the second antibody further includes a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (e.g., horseradish peroxidase).

[0054] In a tenth aspect, the present invention also provides a method for detecting the presence or level of influenza A H1N1 virus or its surface hemagglutinin protein (HA) in a sample, comprising:

[0055] (1) Contact the sample with the single-domain antibody or its antigen-binding fragment described in the first aspect, the conjugate described in the seventh aspect, or the kit described in the ninth aspect;

[0056] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of influenza A H1N1 virus or hemagglutinin (HA) on the surface of influenza A H1N1 virus in the sample.

[0057] In some implementations, the method is an immunological detection, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CIA), radioimmunoassay (RIA), or fluorescence immunoassay.

[0058] In some embodiments, the single-domain antibody or antigen-binding fragment of the present invention further includes a detectable label. In other embodiments, the method further includes detecting the antibody or antigen-binding fragment of the present invention using a second antibody carrying a detectable label. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0059] In an eleventh aspect, the present invention provides a method for diagnosing whether a subject is infected with influenza A (H1N1) virus, comprising: detecting the presence of the influenza A (H1N1) virus HA protein in a sample from said subject using a single-domain antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the single-domain antibody or antigen-binding fragment thereof of the present invention further includes a detectable marker. In other embodiments, the method further includes using a second antibody carrying a detectable marker to detect the single-domain antibody or antigen-binding fragment thereof of the present invention.

[0060] In a twelfth aspect, the use of the single-domain antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of the H1N1 influenza A virus HA protein in a sample, or for diagnosing whether a subject is infected with the H1N1 influenza A virus.

[0061] Terminology Definition

[0062] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0063] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as referring to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody).

[0064] Single-domain antibodies are also called nanobodies, and the two terms are used interchangeably. Typically, a nanobody consists of four framework regions and three complementarity-determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0065] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide containing a fragment of a single-domain antibody that retains the ability to specifically bind to the same antigen bound by the single-domain antibody and / or competes with the single-domain antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety." See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen-binding fragment of the nanobodies of the present invention can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the nanobodies of the present invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody may be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody to contain only a portion of FR1 and / or FR4, or lack one or both of those backbone regions, as long as it substantially retains antigen binding and specificity.

[0066] Antigen-binding fragments of a single-domain antibody can be obtained from a given single-domain antibody (e.g., the nanobody provided by this invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and antigen-binding fragments of the single-domain antibody can be specifically screened in the same manner as those used for intact nanobodies.

[0067] In this article, unless the context clearly indicates otherwise, when referring to the term "single-domain antibody," it includes not only the complete single-domain antibody but also the antigen-binding fragment of the single-domain antibody.

[0068] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc etal., Dev. Comparat. Immunol. 27:55-77, 2003).

[0069] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, IMGT, Chothia, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the IMGT numbering system.

[0070] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0071] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D"" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0072] As used herein, the term "isolated" refers to something obtained artificially from its natural state. If a substance or component is found in nature that has been "isolated," it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, for a naturally occurring, unisolated polynucleotide or polypeptide in a living animal, a high-purity identical polynucleotide or polypeptide isolated from its natural state is called "isolated." The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0073] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0074] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0075] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0076] The twenty common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “peptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala. Additionally, as used herein, the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably.

[0077] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin.

[0078] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., infection with or associated with the H1N1 influenza virus) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received). In this application, the antibody of the present invention has the ability to neutralize the H1N1 influenza virus, thereby enabling its use in preventing / preventing infection of unaffected subjects or their cells with the H1N1 influenza virus. Furthermore, the antibody of the present invention has the ability to clear the H1N1 influenza virus (i.e., it can clear the H1N1 influenza virus DNA and / or the H1N1 influenza virus HA protein in the body, and clear the H1N1 influenza virus and cells infected with the H1N1 influenza virus in the body), and can thus be used to treat H1N1 influenza virus infection or diseases related to H1N1 influenza virus infection in sick subjects.

[0079] As used in this article, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0080] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., infection with or related to the H1N1 influenza virus) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., infection with or related to the H1N1 influenza virus); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0081] Beneficial effects of the invention

[0082] The anti-H1N1 influenza A virus HA single-domain antibody of the present invention can not only effectively bind to the H1N1 influenza A virus HA protein, but also effectively neutralize the H1N1 influenza A virus in serum. Therefore, the single-domain antibody of the present invention provides an effective means for the prevention and / or treatment of H1N1 influenza A virus infection and has significant clinical application value. Attached Figure Description

[0083] Figure 1 SDS-PAGE results of 1024 single-domain antibody against H1N1 influenza virus.

[0084] Figure 2 Results of the binding ability of 1024 single-domain antibodies to the HA protein of different strains of influenza A H1N1 virus, as detected by ELISA.

[0085] Figure 3 Neutralization test results of different concentrations of 1024 single-domain antibody against different strains of influenza A (H1N1) virus.

[0086] Sequence information

[0087] The sequence information involved in this article is shown in Table 1 below.

[0088] Table 1 Detailed Implementation

[0089] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0090] Example 1: Construction of a single-domain antibody phage library

[0091] Alpacas were immunized with the hemagglutinin (HA) antigen of the influenza A H1N1 (A / Victoria / 4897 / 2022) virus (Fortunebio, IAVICH1-HA2022H1), and peripheral blood mononuclear cells (PBMCs) were collected afterward. PBMCs were lysed with Trizol, centrifuged at 12000 rpm to remove the precipitate, and the supernatant was collected. 200 μl of chloroform was added, vortexed for 15 s, incubated at room temperature for 15 min, and then centrifuged at 12000 rpm for 15 min. The clear layer was carefully aspirated, and an equal volume of isopropanol was added, and the mixture was incubated at room temperature for 10 min. The precipitate was centrifuged at 12000 rpm for 10 min, and washed twice with 75% ethanol. The ethanol was removed, and the RNA precipitate was dissolved in DEPC water. cDNA was synthesized using a Bio-Rad reverse transcription kit (bio-rad, Cat: 1708891). Using the cDNA as a template, the variable region of a single-domain antibody was amplified using specific primers. The VHH gene was amplified using primers from the literature (Nat Protoc. 2014 March ;9(3): 674–693) in two rounds of PCR amplification. The first round primers were located upstream in the leader region and downstream in the CH2 constant region (CALL001: 5'-GTCCTGGCTGCTCTTCTACAAGG-3', CALL002: 5'-GGTACGTGCTGTTGAACTGTTCC-3'). Smaller fragments from the first round PCR product were recovered and used as templates for the second round of amplification. The second round primers were located upstream in the variable region FR1 and downstream in the variable region FR4 (VHH-For: 5'-TACCGTGGCCCAGGCGGCCCVAWGGRTTGAHCAAAMACCGAVA-3'Sfi I (restriction enzyme site, used for plasmid ligation), VHH-Back: 5'-GGTGGCCGGCCTGGCCTTYMACAGTCAGAAWRGTGCCKCY-3'Sfi I). The amplified DNA fragment was purified and constructed into the pcantab5e phage vector (laboratory-preserved). It was then electroporated into *E. coli* TG1 competent cells (LGC, Cat: 60502), and immediately added to 1 ml of recovery medium. The cells were incubated at 37°C for 1 hour. 10 μL of the electroporated bacterial culture was then serially diluted 10-fold with culture medium to a final volume of 1:10. -780 μL of each gradient was plated and incubated overnight. Library volume was calculated as: clone number × dilution factor × 12.5. All remaining electroporated bacterial cultures were plated in large petri dishes and incubated overnight at 37°C. Colonies were scraped from the plates with a plating stick, resuspended in culture medium, and a final concentration of 20% glycerol was added. The culture was then stored at -80°C for later use. This yielded a phage library. Forty-eight clones were selected for culture PCR to determine the library's positive conversion rate, and sequencing was used to verify antibody diversity. The library showed a 100% positive conversion rate and 87.2% diversity.

[0092] Take 100 μL of the phage library prepared above and inoculate it into 50 mL of 2YT medium containing ampicillin and 1% glucose. Incubate at 37°C until the logarithmic growth phase. Add helper phage M13K07 at a 20-fold concentration, mix well, and incubate at 37°C for 20 minutes, then shake for 30 minutes. Centrifuge, discard the medium, add 50 mL of 2YT ampicillin-kanamycin medium, and incubate overnight at 30°C with shaking. Centrifuge the next day, collect the supernatant, add 1 / 4 volume of 20% PEG / 2.5 M NaCl, precipitate the recombinant phage at 4°C for 2 hours, centrifuge to collect the phage pellet, dissolve in 5 mL of PBS, repeat the precipitation once, dissolve the phage in PBS, add glycerol to a final concentration of 15%, aliquot and store at -80°C for later use to obtain the phage display library. Simultaneously, take 10 μL and perform a 10-fold serial dilution, infect the TG1 logarithmic growth phase, incubate overnight, and calculate the phage library titer using the same method as for the bacterial library.

[0093] Example 2: Screening of single-domain antibody phage libraries

[0094] Three types of hemagglutinin (HA) antigens of influenza A H1N1 virus (antigen sequences derived from influenza virus vaccine strains published by the WHO in the Northern Hemisphere, sequence numbers: (1) A / Victoria / 2570 / 2019 (type / isolation region / strain number / isolation year), WEY08940.1; (2) A / Victoria / 4897 / 2022, WEY08903.1; (3) A / Wisconsin / 67 / 2022, WBO08838.1) were diluted with carbonate buffer (CBS) and coated onto 96-well microplates at 1 μg / well, and coated at 4°C for 12 hours. The antigens were discarded, and blocking buffer (5% skim milk dissolved in PBST) was added at 200 μL / well. The plates were blocked at 37°C for 2 hours and washed 4 times with 0.05% PBST. Take 100 times the volume of recombinant phage and dilute it in 100 μL of blocking buffer. Add the phage to the wells coated with HA antigen and incubate at room temperature for 2 hours. Discard the phage and wash 10 times with 0.1% PBST for 2 minutes each time. Elute with 100 μL of triethylamine for 10 minutes. Take out the eluent and immediately add 1 M Tris-HCl (pH 7.4) to neutralize the eluent. Add the neutralized eluent to 3 mL of logarithmic growth phase TG1, let stand for 30 minutes, shake and incubate for 30 minutes. Take 10 μL for 10-fold serial dilution and calculate the library volume. Centrifuge the rest and resuspend the bacteria in 300 μL of medium. Spread the bacteria on 2YT solid medium plates and incubate overnight at 37°C. The next day, calculate the library volume of the serially diluted plates. Scrape the bacteria off the bacterial bank plates with a spreader and resuspend them in 3 mL of 2YT medium. Add glycerol to a final concentration of 20% and freeze at -80°C for later use. The first round of bacterial bank is obtained.

[0095] 100 μL of the first-round bacterial culture was inoculated into 50 mL of culture medium and cultured at 37°C with shaking until the logarithmic development phase. The first-round phage display library was then prepared according to the phage display library preparation method described in Example 1. The second and third rounds of screening were performed according to the above steps, with the number of washes increased by 5 and the amount of antigen reduced by 3 times in each round. A single clone was picked and placed in a 96-well plate and cultured overnight at 37°C with shaking. The next day, 5 μL of bacterial culture was transferred to a new 96-well plate (300 μL of culture medium / well) and cultured at 37°C with shaking until the logarithmic development phase. 20 times the amount of helper phage was added, and the plate was incubated at 37°C for 30 minutes, followed by shaking for another 30 minutes. After centrifugation, the culture medium was removed, and 300 μL of 2YT ampicillin medium was added. The plate was then cultured overnight at 30°C with shaking.

[0096] Validation of positive clones by phage ELISA: 100 μL of supernatant was added to an ELISA plate coated with HA antigen (50 ng / well) and incubated at room temperature for 2 hours. A recombinant phage library was used as a positive control, and helper phage as a negative control. The plate was washed 4 times with PBST. HRP-labeled anti-M13 antibody (purchased from Sinopharm) was added at 100 μL / well, and the plate was incubated at 37°C for 1 hour. The plate was washed 6 times with PBST. TMB chromogenic substrate (purchased from Millipore) was added at 100 μL / well, and the plate was incubated at 37°C in the dark for 15 minutes. The incubation was stopped with 50 μL of 1MH2SO4, and the absorbance was measured at 450 nm. Positive clones were picked and sequenced. Antibodies with different sequences were selected for the next step of expression vector construction and purification. The amino acid sequences of the complementarity-determining regions (CDRs) and heavy chain variable regions (VHH) of the obtained 1024 single-domain antibody are shown in Table 1.

[0097] Example 3: Single-domain antibody expression and purification

[0098] Prokaryotic expression: Different antibody sequences obtained in Example 2 were amplified by PCR to produce different antibody sequences with homologous arms at both ends. After recovering the PCR products, the pET28a vector was constructed using homologous recombination. The vector was transformed into E. coli TOP 10 competent cells and cultured overnight at 37°C. Single clones were picked for colony PCR, and the antibody sequences were sequenced to verify them. The correctly sequenced clones were inoculated into 10 mL LB kanamycin medium and cultured overnight with shaking. The plasmid was extracted and transformed into E. coli expression strain Arctic express (Weidi Bio, EC2021). Single clones were picked and cultured in LB medium to the logarithmic growth phase. 0.1 mM IPTG was added, and expression was induced at 16°C for 20 hours. The bacterial pellet was collected, high-pressure lysed, centrifuged, and the supernatant was collected.

[0099] Purification of single-domain antibodies: The collected supernatant was filtered through a 0.22 μm filter membrane and then purified using Ni column affinity: 1 ml of Ni purification medium (Tiandi Renhe, SA1010) was equilibrated with Tris-NaCl (pH 8.0) buffer. After loading the sample, impurities and the target protein were washed with Tris-NaCl (pH 8.0) buffer containing different concentrations (20 mM, 40 mM, 80 mM, 100 mM, 300 mM, 500 mM) imidazole. The fraction with higher purity of the target protein was dialyzed into PBS and concentrated. The purified antibody was then subjected to SDS-PAGE polyacrylamide gel electrophoresis.

[0100] SDS-PAGE electrophoresis image of 1024 single-domain antibody as shown below Figure 1 As shown, a single-domain antibody 1024 against H1N1 influenza virus was obtained with a purity >95%.

[0101] Example 4: Detection of Single-Domain Antibody Binding Ability

[0102] The HA antigen proteins of the three different strains of H1N1 influenza virus described in Example 2 were diluted in CBS buffer and coated onto ELISA plates, 100 ng / 100 μL / well, and incubated at 4°C for 12 hours. The antigen was discarded, and blocking buffer (5% skim milk dissolved in PBST) was added, 200 μL / well, and the plates were blocked at 37°C for 2 hours. The plates were washed with 0.05% PBST, 200 μL / well, 4 times, 2 minutes each time. The liquid was then discarded and the plates were patted dry. The 1024 antibody was diluted in PBST, starting from 100 μg / mL, and a semi-logarithmic dilution was performed, resulting in 12 dilutions. 100 μL / well was added to the ELISA plates and incubated at 37°C for 2 hours. The plates were washed with PBST 4 times, 2 minutes each time. HRP-labeled anti-his secondary antibody was added, and the plates were incubated at 37°C for 1 hour. The plates were washed with PBST 6 times, 2 minutes each time. The liquid was then discarded and the plates were patted dry. TMB chromogenic substrate was added, 100 μL / well, and 100 μL / well. Incubate at 37°C in the dark for 15 minutes with μL / well, add 50 μL of 1M H2SO4 to stop the reaction, and measure the absorbance at 450 nm. Calculate the EC50 value of antigen-antibody binding.

[0103] The results are as follows Figure 2 As shown in Table 2, single-domain antibody 1024 exhibits good binding activity to the HA protein of different strains.

[0104] Table 2: Results of the binding ability test between single-domain antibody 1024 and the HA protein of different strains of H1N1 influenza virus

[0105] Example 5: Detection of single-domain antibody neutralizing capacity

[0106] Preparation of H1N1 recombinant influenza virus: H1N1 recombinant influenza virus was prepared using the PM-PR88 plasmid (laboratory-preserved) recombinant virus rescue system. In short, firstly, the HA and influenza neuraminidase (NA) genes in the PM-PR88 plasmid were replaced with the HA and NA genes from Victoria / 2570 / 2019 or Wisconsin / 67 / 2022 (synthesized by GenScript) to obtain the recombinant plasmid. Then, 293T cells (ATCCC, RL-3216) and MDCK cells were mixed at a 6:1 ratio and seeded in 6-well plates, and cultured for 18-22 hours. The recombinant plasmid was mixed with Lipo3000 transfection reagent, incubated for 15 minutes, and then added to the cells washed with PBS. After incubation at 37°C for 4-6 hours, the mixture was discarded, 1 ml of culture medium was added, and TPCK trypsin (Sigma, 4370285) was added to a final concentration of 1 µg / ml. The cells were incubated at 37℃ and 5% CO2 for 24 hours, then frozen and thawed at -80℃. The supernatant was collected by centrifugation to obtain the recombinant virus.

[0107] MDCK-SIAT1 cells (ATCC, CRL-3743) with a monolayer density of 80%-90% were seeded into 96-well plates at a density of 20,000 cells / well and cultured in a CO2 incubator for 18-20 hours. The 1024 single-domain antibody was diluted in DMEM medium containing 2% BSA and 2 µg / ml TPCK trypsin, starting at 100 µg / ml and 3-fold diluted in 9 gradients, with each antibody prepared in triplicate, containing 50 µl of antibody per well. 50 µl of virus solution containing 100 TCID50 was added to each well of antibody, and the cells were incubated at 37°C for 1 hour. 100 µl of the virus-antibody mixture was then added to the cells seeded one day prior. A virus control and a cell control were also prepared. The cells were cultured at 37°C and 5% CO2 for 24 hours. The culture supernatant was discarded, and 100 µl / well of 4% PFA was added to fix the cells at room temperature for 15 minutes. Discard the fixative, wash the plate twice with PBST, add 100 µl / well of 0.2% Triton-X100, permeate at room temperature for 15 min, and wash three times with PBST. Add 200 µl / well of 5% skim milk and block at 37°C for 2 h, then wash three times with PBST. Add 100 µl of diluted anti-influenza A NP antibody (Sino Biotech, 40208-R010) to each well and incubate at 37°C for 1 h. Discard the primary antibody and wash the plate five times with PBST. Add 100 µl of diluted HRP-labeled goat anti-rabbit secondary antibody (Beyotime, A0208) to each well and incubate at 37°C for 1 h, then wash six times with PBST. Add 100 µl of TMB substrate (Millipore, ES001) to each well and incubate at room temperature in the dark for 10-15 minutes for color development. Add 50 µl of 1M sulfuric acid to each well to stop the reaction. Measure the OD450 value using a microplate reader. The Reed-Muench method was used to calculate the half-maximal inhibitory concentration (IC50) of the antibody.

[0108] The results are as follows Figure 3 As shown in Table 3, single-domain antibody 1024 exhibits good neutralizing activity against different strains of H1N1 influenza virus A / Victoria / 2570 / 2019 and A / Wisconsin / 67 / 2022.

[0109] Table 3: Results of neutralizing ability of single-domain antibody 1024 against different strains of H1N1 influenza virus

Claims

1. A single-domain antibody or its antigen-binding fragment capable of specifically binding to the hemagglutinin (HA) protein on the surface of influenza A (H1N1) virus, comprising: The heavy chain variable region (VHH) comprises: complementarity-determining region 1 (CDR1) as shown in SEQ ID NO: 2 or a variant thereof, complementarity-determining region 2 (CDR2) as shown in SEQ ID NO: 3 or a variant thereof, and complementarity-determining region 3 (CDR3) as shown in SEQ ID NO: 4 or a variant thereof. in, The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions); preferably, the substitutions are conservative substitutions. The CDRs are defined by the IMGT numbering system; Preferably, the single-domain antibody or its antigen-binding fragment comprises a VHH, wherein the VHH comprises: CDR1 as shown in SEQ ID NO: 2, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4, wherein the CDRs are defined by the IMGT numbering system.

2. A single-domain antibody or antigen-binding fragment thereof capable of specifically binding to the hemagglutinin (HA) protein on the surface of influenza A H1N1 virus, comprising: complementarity-determining region 1 (CDR1) or a variant thereof, complementarity-determining region 2 (CDR2) or a variant thereof, and complementarity-determining region 3 (CDR3) or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO: 1; in, The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions); preferably, the substitutions are conservative substitutions. Preferably, the single-domain antibody or its antigen-binding fragment contains CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VHH) as shown in SEQ ID NO: 1; The three HCDRs contained in the VHH are defined by the Kabat, IMGT, Chothia or AbM numbering system.

3. The single-domain antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The VHH of the single-domain antibody or its antigen-binding fragment also includes the framework region (FR) of the immunoglobulin. Preferably, the VHH of the single-domain antibody or its antigen-binding fragment comprises: frame region 1 (FR1) or a variant thereof as shown in SEQ ID NO: 5, frame region 2 (FR2) or a variant thereof as shown in SEQ ID NO: 6, frame region 3 (FR3) or a variant thereof as shown in SEQ ID NO: 7, and frame region 4 (FR4) or a variant thereof as shown in SEQ ID NO: 8, wherein the variant has one or more (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has a sequence identity of 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%, at least 99%, or 100%; preferably, the substitutions are conserved substitutions; Preferably, the VHH of the single-domain antibody or its antigen-binding fragment comprises: FR1 as shown in SEQ ID NO: 5, FR2 as shown in SEQ ID NO: 6, FR3 as shown in SEQ ID NO: 7, and FR4 as shown in SEQ ID NO:

8.

4. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-3, comprising VHH, said VHH comprising the amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof. in, The variant has one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 1, or has 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%, at least 99%, or 100% sequence identity compared to the sequence shown in SEQ ID NO: 1; preferably, the substitutions are conservative substitutions; Preferably, the single-domain antibody or its antigen-binding fragment comprises VHH, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO:

1.

5. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The single-domain antibody or its antigen-binding fragment may optionally be linked to an Fc domain, a tag peptide, or a combination thereof via a linker peptide.

6. An isolated nucleic acid molecule encoding a single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1-5.

7. A vector comprising the isolated nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method for preparing a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising culturing the host cell of claim 8 under conditions allowing expression of the single-domain antibody or antigen-binding fragment thereof, and recovering the single-domain antibody or antigen-binding fragment thereof from the cultured host cell culture.

10. A multispecific antibody comprising the single-domain antibody or antigen-binding fragment thereof as described in any one of claims 1-5; Preferably, the multispecific antibody specifically binds to the surface hemagglutinin (HA) protein of the H1N1 influenza virus and additionally specifically binds to one or more other targets.

11. Coupled elements, which include: (1) The single-domain antibody or its antigen-binding fragment according to any one of claims 1-5, or the multispecific antibody according to claim 10; and (2) Coupling part, in, The conjugate portion is selected from the following: detectable markers, drugs, toxins, cytokines, or combinations thereof; Preferably, the detectable marker is selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters, luminol and its derivatives, ruthenium derivatives), fluorescent markers (e.g., fluorescein, fluorescent protein), biotin, and radionuclides.

12. A pharmaceutical composition comprising a single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1-5, a multispecific antibody as described in claim 10, or a conjugate as described in claim 11, and a pharmaceutically acceptable carrier and / or excipient.

13. Use of the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, the multispecific antibody according to claim 10, the conjugate according to claim 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament. in, The drug is used to prevent and / or treat H1N1 influenza virus infection or related illness in a subject (e.g., a human), to neutralize the virulence of H1N1 influenza virus in vitro or in a subject (e.g., a human), and / or to reduce H1N1 influenza virus DNA and / or serum levels of H1N1 influenza virus in a subject (e.g., a human).

14. A kit comprising a single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1-5, a multispecific antibody as described in claim 10, or a conjugate as described in claim 11.

15. A method for detecting the presence or level of hemagglutinin (HA) protein of influenza A (H1N1) virus in a sample, comprising: (1) Contact the sample with the single-domain antibody or its antigen-binding fragment as described in any one of claims 1-5, the conjugate as described in claim 11, or the kit as described in claim 14; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of influenza A H1N1 virus or hemagglutinin (HA) on the surface of influenza A H1N1 virus in the sample.

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