Antibody specifically binding to influenza A virus hemagglutinin

Abstract

The invention relates to an antibody which is used for being specifically combined with influenza A virus hemagglutinin and comprises a first immunoglobulin or / and a second immunoglobulin, wherein the first immunoglobulin has a first variable structural region with an amino acid sequence shown as SEQ ID NO: 1, a second variable structural region with an amino acid sequence shown as SEQ ID NO: 2, and a third variable structural region with an amino acid sequence shown as SEQ ID NO: 3; wherein the second immunoglobulin has a fourth variable structural region with an amino acid sequence shown as SEQ ID NO: 4, a fifth variable structural region with an amino acid sequence shown as SEQ ID NO: 5 and a sixth variable structural region with an amino acid sequence shown as SEQ ID NO: 6. Compared with a traditional IgG antibody, the single-domain antibody is adopted, the molecular weight is relatively small, better lung permeability is achieved, hidden epitopes on some antigens can be recognized, and therefore more accurate targeted therapy is achieved. The bispecific antibody disclosed by the invention can be combined with the influenza A virus HA in a broad-spectrum manner and can be used for neutralizing the influenza A virus HA in a broad-spectrum manner.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to an antibody that specifically binds to hemagglutinin of influenza A virus. Background Art

[0002] Influenza virus is one of the main pathogens causing human respiratory infections. According to its type, it can be divided into influenza A virus (IAV), influenza B virus (IBV), and influenza C virus (ICV), and most seasonal influenza is caused by influenza A and B viruses. Influenza A virus is particularly prone to causing large-scale or even global epidemics due to its high variability and ability to cross species. Influenza virus is a single-stranded, negative-sense, segmented RNA virus that contains 8 RNA segments and encodes 8 proteins. Among these, two membrane proteins, hemagglutinin (HA) and neuraminidase (NA), are the main targets of neutralizing antibodies. HA is responsible for the virus invasion process, while NA is involved in the release of the virus from host cells. In particular, the HA protein, as a key factor for virus invasion, has become an important target for developing influenza neutralizing antibodies.

[0003] Compared with small molecule drugs, antibody drugs have shown higher safety and stronger clearance effects in the treatment of influenza. However, traditional monoclonal antibody drugs have limitations such as insufficient broad-spectrum, large molecular weight, and the need for intravenous injection. Given that influenza virus mainly affects the respiratory and lung regions, the use of nebulized inhalation preparations can provide a more effective treatment route, which not only increases the local drug concentration but also improves the convenience and acceptance of drug use by patients. Therefore, it is particularly important to develop new, highly efficient and broad-spectrum neutralizing antibodies against influenza virus, especially influenza A virus, that can be administered by inhalation. Summary of the Invention

[0004] The purpose of the present invention is to provide an antibody that specifically binds to hemagglutinin of influenza A virus for the deficiencies in the prior art.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide an antibody for specifically binding to hemagglutinin of influenza A virus, including:

[0007] A first immunoglobulin single variable domain, and / or a second immunoglobulin single variable domain;

[0008] Among them, the first immunoglobulin single variable domain has a first variable region with an amino acid sequence as shown in SEQ ID NO: 1, a second variable region with an amino acid sequence as shown in SEQ ID NO: 2, and a third variable region with an amino acid sequence as shown in SEQ ID NO: 3;

[0009] Among them, the second immunoglobulin single variable domain has a fourth variable region with an amino acid sequence as shown in SEQ ID NO: 4, a fifth variable region with an amino acid sequence as shown in SEQ ID NO: 5, and a sixth variable region with an amino acid sequence as shown in SEQ ID NO: 6.

[0010] SEQ ID NO: 1: GFSFPFAA.

[0011] SEQ ID NO: 2: ISATGDRI.

[0012] SEQ ID NO: 3: NFPRGLEFLDDF.

[0013] SEQ ID NO: 4: GSIFHING.

[0014] SEQ ID NO: 5: IFSGGST.

[0015] SEQ ID NO: 6: AARKWTGNVWAF.

[0016] Preferably, the amino acid sequence of the first immunoglobulin single variable domain is as shown in SEQ ID NO: 7-9.

[0017] SEQ ID NO: 7: QVKLEESGGGLVQPGGSLRLSCVAS GFSFPFAA MNWHR QVPGKERELVAS ISATG DRI NYADSVKGRFTISRDNLKNTLYLQMSALKPEDSA VYYC NFPRGLEFLDDF WGQGTQVTVSS.

[0018] SEQ ID NO: 8: EVQLVESGGGLVQPGGSLRLSCAAS GFSFPFAA MSWVR QAPGKGLEWVSA ISATG DRI YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AIYYC NFPRGLEFLDDF WGQGTLVTVSS.

[0019] SEQ ID NO:9: EVQLVESGGGLVQPGGSLRLSCAAS GFSFPFAA MNWVR QAPGKGLEWVSA ISATG DRI NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AIYYC NFPRGLEFLDD FWGQGTLVTVSS。

[0020] Preferably, the amino acid sequence of the second single variable domain of the immunoglobulin is as shown in SEQ ID NO:10.

[0021] SEQ ID NO:10: EVQLVESGGDSVQPGGSLRLSCVAS GSIFHING MGWYRQAPGRQRELVAA IFSGG ST NYADSVKGRFTISGDNAKNTAYLQMNSLKPEDTAVYYC AARKWTGNVWAF WGQGTQVTVSS。

[0022] Furthermore, the antibody is a bispecific antibody, which is composed of the first single variable domain of the immunoglobulin and the second single variable domain of the immunoglobulin connected by a linker, and the C-terminus of the first single variable domain of the immunoglobulin is connected to the N-terminus of the second single variable domain of the immunoglobulin through a linker.

[0023] Even further, the amino acid sequence of the linker is as shown in SEQ ID NO:11.

[0024] SEQ ID NO:11: GGGGSGGGGSGGGGSGGGGS。

[0025] The amino acid sequence of the bispecific antibody is as shown in SEQ ID NO:12.

[0026] SEQ ID NO:12: QVKLEESGGGLVQPGGSLRLSCVAS GFSFPFAA MNWHRQVPGKERELVAS ISATG DRI NYADSVKGRFTISRDNLKNTLYLQMSALKPEDSAVYYC NFPRGLEFLDDF WGQGTQVTVSS GGGGSGGGGSGG GGSGGGGS EVQLVESGGDSVQPGGSLRLSCVAS GSIFHING MGWYRQAPGRQRELVAA IFSGGSTNYADSVKGRFTISGDNAKNTAYLQMNSLKPEDTAVYYC AARKWTGNVWAF WGQGTQVTVSS。

[0027] The second aspect of the present invention is to provide a fusion protein, which is a fusion of the above-mentioned antibody and a heterologous protein.

[0028] Preferably, the heterologous protein is human immunoglobulin Fc, and more preferably the Fc of human IgG1.

[0029] The third aspect of the present invention is to provide a conjugate, which is a conjugate of the above-mentioned antibody, or the above-mentioned fusion protein, and an effector molecule.

[0030] Preferably, the effector molecule is a detectable label, and more preferably one of a fluorescent label, a radioactive label, avidin, biotin, or an enzyme.

[0031] The present invention also provides a nucleotide molecule for encoding the above-mentioned antibody. The present invention also provides a vector or recombinant vector comprising the nucleotide molecule, optionally operably linked to a regulatory sequence. The present invention also provides a transformant comprising the vector or recombinant vector, as well as a method for producing and optionally recovering the antibody or antigen-binding fragment. The transformant described in the present invention can be any prokaryotic cell or eukaryotic cell, including but not limited to bacterial cells (such as Escherichia coli), insect cells (such as using a baculovirus expression system), yeast, or mammalian cells (such as CHO or BHK cell lines). Other suitable transformants are known to those skilled in the art. Preferably, the transformant is an Escherichia coli cell, which has a short culture cycle and low production cost.

[0032] The present invention also provides a pharmaceutical composition, comprising: a prophylactically or therapeutically effective amount of the above-mentioned antibody, or fusion protein, or conjugate, or nucleotide molecule, or vector or recombinant vector, or transformant, and a pharmaceutically or physiologically acceptable carrier, excipient, or stabilizer. The pharmaceutical composition includes but is not limited to freeze-dried formulations, aqueous solution formulations, liposome, or capsule formulations, etc. The concentration of the antibody, bispecific antibody, fusion protein, conjugate, nucleotide molecule, vector or recombinant vector, transformant described in the present invention can vary from about 0.1 wt% to 100 wt%.

[0033] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0034] Compared with traditional IgG antibodies, the present invention uses single-domain antibodies, which have a relatively small molecular weight, better pulmonary permeability, and can recognize hidden epitopes on some antigens, thus achieving more precise targeted therapy.

[0035] The bispecific antibody of the present invention can bind to influenza A virus HA in a broad spectrum and can neutralize influenza A virus HA in a broad spectrum. Brief Description of the Drawings

[0036] Figure 1 Shows the results of detecting the binding of single-domain antibodies to influenza A virus H1N1-HA protein using ELISA.

[0037] Figure 2 Shows the results of detecting the binding of single-domain antibodies to influenza A virus H3N2-HA protein using ELISA.

[0038] Figure 3 To detect the neutralizing activity of single-domain antibodies against live A / California / 07 / 2009 (H1N1) virus.

[0039] Figure 4 To detect the neutralizing activity of single-domain antibodies against live A / Puerto Rico / 8 / 1934 (H1N1) virus.

[0040] Figure 5 To detect the neutralizing activity of single-domain antibodies against live A / WSN / 1933 (H1N1) virus.

[0041] Figure 6 To detect the neutralizing activity of single-domain antibodies against live A / Guizhou / 54 / 1989 (H3N2) virus.

[0042] Figure 7 To detect the neutralizing activity of single-domain antibodies against A / Qinghai / (H5N1) pseudovirus.

[0043] Figure 8 To detect the neutralizing activity of single-domain antibodies against A / Thailand / (H5N1) pseudovirus.

[0044] Figure 9 Shows the construction and verification of a broad-spectrum bispecific single-domain antibody.

[0045] Figure 10 Shows the results of the preventive and protective activity of aerosolized single-domain antibodies against H1N1 influenza virus in mice.

[0046] Figure 11 Shows the results of the preventive and protective activity of aerosolized single-domain antibodies against H3N2 influenza virus in mice.

[0047] Figure 12 Shows the results of the therapeutic and protective activity of aerosolized single-domain antibodies against H3N2 influenza virus in mice.

[0048] Figure 13SDS-PAGE gel images of the humanized HA05 and HA06 sequences for 501 people.

[0049] Figure 14 Shows the affinity of the humanized sequence HA06 to H1-HA detected by BLI.

[0050] Figure 15 Shows the neutralizing activity of the humanized sequence HA06 against the H1N1 strain detected by Microneutralization. Detailed implementation

[0051] To more thoroughly understand the invention, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0052] In the present invention, "antibody" means a protein composed of one or more domains encoded by all or part of a substantially recognized immunoglobulin gene. The recognized immunoglobulin genes, for example in humans, include kappa (κ), lambda (λ), and heavy chain loci, which contain numerous variable region genes, as well as constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α) encoding IgM, IgD, IgG, IgE, and IgA isotypes respectively. Antibodies in the present invention mean to include full-length antibodies, their single chains, and all their parts, domains or fragments, as well as natural antibodies, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic purposes or other purposes further specified below from any organism. The term "antibody" includes antibody fragments, which are well known in the art, such as Fab, Fab’, F(ab’)2, Fv, scFv or the antigen-binding domain of an antibody (such as a VHH domain or a VH / VL domain), or antibody fragments produced by modifying a complete antibody or resynthesizing those antibodies using recombinant DNA technology. The term "antibody" includes monoclonal as well as polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing antibodies, or inhibitory antibodies, or stimulatory antibodies. Antibodies of the present invention can be non-human antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

[0053] Specifically included within the definition of "antibody" are non-glycosylated antibodies. Preferably, the non-glycosylated antibodies are used in the present invention, which means antibodies lacking sugar attachment at position 297 in the Fc region, where the numbering is according to the EU system of Kabat. The non-glycosylated antibodies can be deglycosylated antibodies, which are antibodies from which the Fc sugars have been removed, for example, by chemical or enzymatic methods. Optionally, the non-glycosylated antibodies can be non-glycosylated or unglycosylated antibodies, which are antibodies without Fc sugar expression, for example, by mutating one or more residues encoding glycosylated forms or by expressing in organisms such as bacteria that do not attach sugars to proteins.

[0054] "IgG" as used in the present invention means a polypeptide belonging to the antibody class, which is essentially encoded by the well-recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. "Immunoglobulin (Ig)" in the present invention means a protein composed of one or more polypeptides essentially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have many structural forms, including, but not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains. Known Ig domains in the antibody IgG class are VH, Cγi, Cγ2, Cγ3, VL, and CL.

[0055] "Antigen" as used in the present invention means a compound, composition, or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into the animal, and can be a protein, carbohydrate, lipid, or other pathogen.

[0056] "Single immunoglobulin variable domain" as used in the present invention means an immunoglobulin variable domain capable of specifically binding to an epitope without pairing with other immunoglobulin variable domains. An example of a single immunoglobulin variable domain in the context of the present invention is a "domain antibody", such as the VH domain antibody in the present invention.

[0057] "Variable heavy chain (VH)" as used in the present invention means the region of the immunoglobulin heavy chain with a relatively large variation in the amino acid sequence near the N-terminus, which is the variable domain of the heavy chain in a conventional 4-chain antibody. It is particularly used to distinguish the VHH antibodies of camelids.

[0058] As used herein, "identity" refers to the similarity between nucleotide or amino acid sequences, also known as sequence identity. Sequence identity is typically measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences. When aligned using standard methods, homologs or variants will have a relatively high degree of sequence identity. Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv Appl. Math., 2:482, 1981; Needlema and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; and Altschul et al., Nature Genet., 1994, 6:119-129.

[0059] The NCBI Basic Local Alignment Search Tool (BLAST TM) (Altschul et al., J. Mol. Biol., 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0060] As used herein, "bispecific antibody" refers to an artificial antibody containing two specific antigen-binding sites.

[0061] As used herein, "fully human" means that the antibody is encoded entirely by human antibody genes.

[0062] As used herein, "fusion protein" refers to the expression product of a fusion gene, or two or more proteins fused by biological or chemical means.

[0063] As used herein, "conjugate" refers to an antibody or protein molecule linked to another small or large molecule by biological or chemical means.

[0064] The "amino acid" used in the present invention means one of the 20 naturally occurring amino acids or any unnatural analog, which may be located at the specifically defined positions. The "protein" in the present invention means at least two covalently linked amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be composed of naturally occurring amino acids and peptide bonds, or composed of synthetic peptide mimetic structures, which are the "analogs". Therefore, the "amino acid" or "peptide residue" used in the present invention means naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in the (R) or (S) configuration. In a preferred embodiment, the amino acid is in the (S) or L-configuration. If unnatural side chains are used, non-amino acid substitutions may be used, for example to prevent or delay in vivo degradation.

[0065] The "polypeptide" used in the present invention means a polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acid is an α-amino acid, the L-optical isomer or D-optical isomer can be used. The terms "polypeptide" or "protein" as used in the present invention are intended to cover any amino acid sequence and include modified sequences, such as glycoproteins. The term "polypeptide" is particularly intended to cover naturally occurring proteins, as well as recombinantly or synthetically produced proteins.

[0066] The "nucleotide" used in the present invention means a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and their synthetic unnatural analogs) through phosphodiester bonds. Therefore, the term includes nucleotide polymers in which the nucleotides and the bonds between them include unnatural synthetic analogs, such as but not limited to phosphorothioates, phosphoroamidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNA), etc. For example, these polynucleotides can be synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, usually not more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence in which "U" replaces "T" (i.e., A, U, G, C).

[0067] The present invention uses conventional symbols to describe nucleotide sequences: the left-hand end 5'-end of a single-stranded nucleotide sequence; the left-hand direction of a double-stranded nucleotide sequence is called the 5'-direction. The direction of adding 5'- to 3'-nucleotides to a nascent RNA transcript is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the coding strand.

[0068] The "plasmid" used in the present invention refers to a plasmid artificially constructed on the basis of a natural plasmid to adapt to laboratory operations. A nucleic acid molecule can be introduced into a host cell to generate a transformed host cell. The vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.

[0069] The "host cell" used in the present invention, also known as the recipient cell, refers to a host cell that receives an exogenous gene in transformation and transduction (infection).

[0070] The "pharmaceutically acceptable carrier" used in the present invention refers to a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for delivering one or more therapeutic compounds or molecules (such as one or more antibodies), as well as additional pharmaceutical agents.

[0071] The "prophylactically or therapeutically effective amount" in the present invention refers to a certain amount of a specific agent sufficient to achieve the desired effect in a subject being treated with the agent. The exact dose will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. The dose range can be 0.01 - 100 mg / kg body weight or more, such as 0.1, 1, 10, or 50 mg / kg body weight, preferably 1 - 10 mg / kg. As is well known in the art, adjustments may be necessary for antibody or Fc fusion degradation, systemic or local drug delivery and the rate of new protease synthesis, as well as age, body weight, general health, gender, diet, time of administration, drug interactions, and the severity of the disease condition, and can be determined by those skilled in the art by conventional experimental methods. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat, or improve an infection or disease. The therapeutically effective amount of an agent for preventing, improving, and / or treating a subject will depend on the subject being treated, the type and severity of the affliction, and the mode of administration of the therapeutic composition.

[0072] As used herein, "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, that serves as a template for the synthesis of other polymers and macromolecules in biological processes with a defined nucleotide sequence, or a defined amino acid sequence and the resulting biological properties. Thus, if the transcription and translation of the mRNA produced by the gene results in a protein in a cell or other biological system, the gene encodes the protein. The coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as the transcription template, gene or cDNA) can be said to encode the protein, or other products of the gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0073] Unless otherwise indicated, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless the context clearly dictates otherwise, the singular terms "a," "an," and "the" include plural referents. It should also be understood that all base sizes or amino acid sizes, and all molecular weight or molecular weight values given for nucleic acids or polypeptides are approximate and are provided for description purposes. Although methods and materials similar or equivalent to those described in this invention may be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprising" means "including." All publications, patent applications, patents, and other references mentioned in this invention are incorporated herein by reference in their entirety. In case of conflict, the present specification (including the term explanations) shall prevail. Additionally, the materials, methods, and examples are illustrative only and not limiting.

[0074] Standard recombinant DNA techniques and molecular cloning techniques used in the examples are well known in the art (Ausubel, F.M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and materials and methods suitable for microbial growth are well known in the art. Major chemical and biological reagents are purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

[0075] The present invention will be described in detail below in conjunction with specific examples.

[0076] Example 1 Production of Influenza Virus Hemagglutinin HA Antigen Protein

[0077] The HA base sequences from H1N1 (A / California / 07 / 2009) and H3N2 (A / New York / 55 / 2004) were respectively fused with the sequences of AVI-Tag, Folden, and 6×His and constructed into the pTT expression vector, and expressed using 293F cells (the protein was harvested 96 hours after transfection). The target protein was obtained from the cell supernatant through Ni affinity chromatography, and the obtained protein was verified for the binding activity of the antigen to the known antibody by ELISA. The results showed that the protein purity of H1N1-HA and H3N2-HA reached over 95% after purification and had biological activity, which could meet the experimental requirements for the next step.

[0078] Example 2 Screening for high-affinity single-domain antibodies against the hemagglutinin HA antigen of influenza A virus

[0079] The alpacas were immunized 5 times at two-week intervals, immunized with H1N1-HA and H3N2-HA antigens respectively. After immunization, blood was collected to isolate peripheral blood mononuclear cells (PBMC) and the binding activity to different antigens was detected. As the number of immunizations increased, antibodies against H1N1-HA and H3N2-HA gradually accumulated in the alpacas. After the immunization was completed, blood was taken from the jugular vein and PBMC were separated from the peripheral blood by density gradient centrifugation using lymphocyte separation medium (Ficoll). Subsequently, magnetic bead sorting was used to separate B cells in the PBMC sample by the specific affinity of anti-CD19+ antibody-conjugated magnetic beads to the CD19 + molecule on the surface of B cells. For the isolated B cells, total RNA was first extracted by the Trizol method and reverse transcribed to synthesize the first strand of cDNA. Subsequently, a series of degenerate primers for single-chain antibody amplification were designed, and cDNA was used as a template to obtain the VH+CH2 fragment. Using this fragment as a template, primers were further designed to amplify the nanobody (VHH) fragment.

[0080] The VHH nucleic acid fragment was then cloned into the phage display vector pComb3x (Addgene, product number: 63891), and the ligation product was electrotransformed into the electrocompetent TG1 of Escherichia coli to construct a phage display library of single-domain antibodies with a library capacity of approximately 10 9 Then, antibodies were screened against biotin-labeled H1N1-HA and H3N2-HA proteins. The specific steps were as follows: The biotin-labeled HA protein was immobilized on streptavidin-coated magnetic beads, and 10 12 phages displaying antibodies were incubated with 4 μg H1-HA, 2 μg H3-HA, and 1 μg H1-HA antigens for two hours at room temperature in the 1st, 2nd, and 3rd rounds respectively. The phages used for each round of screening were all 1012 Pick the phages obtained from the third-round screening, infect TG1 cells, and randomly select clones for monoclonal phage ELISA, followed by further sequencing to identify single-domain antibodies that bind to the HA protein.

[0081] The specific steps of monoclonal ELISA are as follows: Pick the monoclonal clones obtained from the third-round screening into a 96-well shaking culture plate, shake at 37°C and 220 rpm for 2 - 3 hours, then add IPTG at a ratio of 1:1000 and induce expression overnight at 30°C. Coat the H1N1-HA and H3N2-HA proteins in a 96-well half-well plate (Corning) at a quantity of 100 μg per well, incubate overnight at 4°C, wash three times with 0.1% PBST, block with 3% milk for 1 hour, then wash three times with 0.1% PBST, and then add the cultured bacterial solution from the previous step as the primary antibody, incubate at 37°C for 1.5 hours, then wash three times with 0.1% PBST, and then add the secondary antibody anti-Flag-HRP (Sigma-Aldrich) diluted 1:5000, incubate at 37°C for 45 minutes, wash five times with 0.1% PBST, add ABTS (Invitrogen) for color development, and read the OD405 value on an enzyme-linked immunosorbent assay (ELISA) reader. Consider monoclonal clones with an OD value more than 5 times that of the negative control as positive. Send the bacterial solution of the positive monoclonal clones to a sequencing company for sequencing.

[0082] Example 3 Detection of the binding ability of single-domain antibodies to H1N1-HA and H3N2-HA proteins

[0083] Expression and purification of positive single-domain antibodies: Select monoclonal clones with enriched sequences according to the sequencing results. The preparation of their soluble expression products is basically carried out according to the literature (Cell Host Microbe. 2017. 22(4):471 - 483.e5.). Specifically: Transfer the plasmid into HB2151 competent cells, pick a single colony from the overnight-grown ampicillin plate, inoculate it into SB bacterial culture medium, and express for 12 - 14 hours under IPTG induction conditions at 30°C. Harvest the bacteria, lyse them, and purify the single-domain antibodies from them using Ni-TNA (Yeasen Biotech).

[0084] Activity detection: Coat 100 ng of HA protein in a 96-well half-well plate (Corning) overnight at 4°C. After washing three times with 0.1% PBST, block with 3% milk for 1 hour. Then wash three times with 0.1% PBST, add single-domain antibodies starting from 2 μmol and diluted in a 3-fold concentration gradient, and incubate for 1.5 hours. After washing three times with 0.1% PBST, incubate with anti-FLAG-HRP antibody (Sigma-Aldrich) for 45 minutes. Then wash five times with 0.1% PBST and add ABTS (Invitrogen) for color development. The results showed that single-domain antibody 501 (SEQ ID NO: 7, the same below) had a strong ability to bind to H1N1-HA protein ( Figure 1 ), and single-domain antibody 602 (SEQ ID NO: 10, the same below) had a strong ability to bind to H3N2-HA protein ( Figure 2 ).

[0085] Example 4 Neutralization Activity Detection of Single-Domain Antibodies against Different Strains of Influenza A Virus

[0086] First, use MDCK cells to amplify live viruses of A / California / 07 / 2009 (H1N1); A / Puerto Rico / 8 / 1934 (H1N1); A / WSN / 1933 (H1N1); A / Guizhou / 54 / 1989 (H3N2). The amplification method is as follows: Add 2 mL of virus liquid to the adherent MDCK cells and adsorb for 1 hour. Then supplement with 13 mL of virus amplification medium and culture at 37°C and 5% CO2 for 48 hours. The harvested cell supernatant is the amplified influenza virus. Inoculate MDCK cells into a 96-well cell culture plate. After the cells adhere, add the gradient-diluted virus stock solution to the MDCK cells and then culture at 37°C and 5% CO2 for 18 hours. After the culture is completed, quantify the virus infection amount by detecting the amount of virus NP protein by ELISA. Finally, calculate the virus titer according to the Reed and Muench method to calculate the TCID50 / 100 μL of the virus (the specific method refers to the WHO influenza manual). The amplification method of H5N1 pseudovirus is as follows: Co-transfect 293T cells with a deleted HIV-1 backbone plasmid carrying the luciferase reporter gene, HA plasmid, and NA plasmid. Harvest the supernatant after 48 hours. Add the harvested pseudovirus diluted in a gradient to MDCK cells. After culturing for 12 hours, change to fresh medium and continue to culture for 48 hours. Then detect the relative fluorescence value in the cell lysate using the Luciferase Assay Kit from Promega.

[0087] 1) Neutralization experiment of live virus:

[0088] Mix 50 μL of the antibody diluted in a 3-fold gradient with 50 μL of the influenza virus diluted according to 100×TCID50 / 50 μL, and incubate at 37 °C for 1 hour. Then add it to the MDCK cells seeded in a 96-well cell culture plate and culture at 37 °C under 5% CO2 for 18 hours. After the culture is completed, detect the neutralizing activity of the antibody by ELISA to detect the amount of viral NP protein.

[0089] 2) The pseudovirus neutralization experiment is as follows:

[0090] Mix 50 μL of the antibody diluted in a 3-fold gradient with 50 μL of the virus solution with a fluorescence value of approximately 200,000, and add it to the MDCK cells in a 96-well cell culture plate. Culture at 37 °C under 5% CO2 for 12 hours, then replace with fresh medium and continue to culture for 48 h. Use the Luciferase Assay Kit from Promega to detect the relative fluorescence value in the cell lysate.

[0091] The results showed ( Figures 3 - 8 ), that the single-domain antibody 501 could widely neutralize live H1N1 virus and H5N1 pseudovirus, while the single-domain antibody 602 ( Figure 6 ) that could bind to H3N2-HA could neutralize the live virus of A / Guizhou / 54 / 1989 (H3N2).

[0092] Example 5 Construction and verification of bispecific single-domain antibodies

[0093] Clone the gene sequences of the single-domain antibody 501 and the single-domain antibody 602 into the prokaryotic expression vector pComb3x (Addgene, product number: 63891) by homologous recombination according to the method shown in Figure 9 Figure A. Transform the homologous recombination product into competent Escherichia coli Top10, and obtain the bispecific antibody expression vector with the correct sequence by picking single colonies for sequencing. Transfer the plasmid of the correctly constructed bispecific single-domain antibody into the prokaryotic expression competent HB2151, pick a single colony from the overnight grown ampicillin plate, inoculate it into SB bacterial culture medium, and express for 12 - 14 hours under the induction condition of IPTG at 30 °C. Harvest the bacteria and purify the bispecific single-domain antibody from it using Ni-NTA (purchased from GE Healthcare).

[0094] The binding activities of the bispecific single-domain antibody HA23 (SEQ ID NO: 12) with H1N1-HA and H3N2-HA proteins were determined using BLI technology (Emerg Microbes Infect. 2017. 6(10): e89.). The HA antigens expressed and obtained in Example 1 were specifically biotinylated (APExBIO, product number: K4402), and the biotinylated HA antigens were immobilized on SA sensors (Pall Fortebio), and the serially diluted HA23 protein solutions were used as the analysis samples. The steps were as follows: The HA protein was diluted to 15 μg / ml and then immobilized on the SA biosensor, and it was combined with the serially diluted HA23 protein solutions (concentration range of 500 - 18.5 nM or 200 - 7.4 nM) in the running buffer (PBST), and no antibody was added to the blank control wells. The program was set as: Association, 300 s; Dissociation, 300 s, and the temperature was set at 37 °C. The ForteBio Data analysis 10.1 software was used to analyze the data. Using the corresponding blank control wells as the control to subtract the background, the curves of each concentration were fitted according to the Mass Transport binding mode to obtain the kinetic analysis results, and the equilibrium constant (K D ) value was shown in the report. The results showed that the K D with H1N1-HA was 0.46 nM, and the K D with H3N2-HA was 3.78 nM( Figure 9 B).

[0095] The neutralization activities of HA23 against different influenza A virus strains were detected by the above virus neutralization experiment. The results showed that HA23 had neutralization activities against live virus of A / California / 07 / 2009 (H1N1); live virus of A / Puerto Rico / 8 / 1934 (H1N1); live virus of A / WSN / 1933 (H1N1); live virus of A / Guizhou / 54 / 1989 (H3N2); pseudovirus of A / Qinghai / (H5N1); pseudovirus of A / Thailand / (H5N1)( Figure 9 C).

[0096] Example 6 Protective Activity of Nebulized Inhalation of Single-Domain Antibody against Influenza A Virus

[0097] First, female Babl / c mice at 6 - 8 weeks old were infected with different titers of A / Puerto Rico / 8 / 1934 (H1N1) and A / Guizhou / 54 / 1989 (H3N2) viruses to verify the median lethal dose (LD50) of the viruses in mice.

[0098] The preventive and protective effects of single - domain antibodies against A / Puerto Rico / 8 / 1934 (H1N1) virus infection in mice were verified by the administration method of aerosol inhalation. One day before the mice were infected with 10 LD50 of A / Puerto Rico / 8 / 1934 (H1N1) virus by nasal drip, the antibodies (501, 602, HA23, 501 + 602) at 25 mg / kg and PBS ( Figure 10 A) were aerosolized. After virus infection, the body weight and survival rate of the mice were continuously detected for 14 days. Mice aerosolized with PBS or irrelevant antibody 602 (an antibody without neutralizing activity against H1N1) all died within 7 - 8 days after virus infection (body weight decreased by more than 25%), while all the mice in the group aerosolized with the broad - spectrum single - domain antibody HA23 targeting HA survived completely and there was no significant decrease in body weight ( Figure 10 C - D). Half of the mice were sacrificed on the 5th day after virus infection, and the lung tissues of the mice were taken to detect the copy number of virus gene M2 by qPCR. The copy number of the virus in the lung tissues of the mice in the aerosolized administration group decreased significantly by 10,000 - fold compared with the PBS and irrelevant antibody groups ( Figure 10 B).

[0099] Next, we verified the protective effect of aerosolized broad - spectrum single - domain antibodies against A / Guizhou / 54 / 1989 (H3N2) virus infection in mice. As in the previous animal experiment design, the antibodies (501, 602, HA23, 501 + 602) at 25 mg / kg and PBS ( Figure 11 A) were aerosolized one day before the mice were infected with the virus by nasal drip. On the 5th day after virus infection, half of the mice in each group were sacrificed, and the lung tissues of the mice were taken to detect the copy number of the M2 gene of the virus genome in the lung tissues by qPCR. The results showed that the copy number of the virus genome in the lungs of the mice in the group aerosolized with the broad - spectrum anti - HA single - domain antibody was significantly lower than that in the PBS group and the irrelevant antibody group ( Figure 11 B). The monitoring of the body weight and survival rate of the mice showed that there was no significant decrease in the body weight of the mice in the group aerosolized with the broad - spectrum anti - HA single - domain antibody, and all of them survived ( Figure 11 C - D).

[0100] Example 7 Therapeutic and Protective Activity of Aerosolized Single - Domain Antibodies against Influenza A Virus

[0101] The therapeutic effect of the selected single-domain antibodies against A / Guizhou / 54 / 1989 (H3N2) influenza virus infection was further verified by the administration method of aerosol inhalation. The experimental protocol was designed as Figure 12 shown in A. Five hours after infecting mice with a lethal dose of the virus, the first antibody drug (25 mg / kg, 501, 602, HA23, 501 + 602) was aerosolized and inhaled. Then, the antibody drug was aerosolized and inhaled again on the 1st and 2nd days, respectively. The body weight and survival rate of the mice were continuously monitored for 14 days. Half of the mice were sacrificed on the 5th day, and the lung tissues of the mice were taken. The viral gene copy number in the lung tissues of the mice was detected by qPCR. The monitoring data of body weight and survival rate showed that the body weight of the mice in the single-domain antibody aerosol inhalation group did not show a significant decrease, and all the mice survived completely. However, the body weight of the mice in the PBS or control antibody aerosol inhalation group decreased to less than 25% on the 5th day, and all the mice died completely ( Figure 12 C-D). And the qPCR results showed that the viral gene copy numbers in the PBS group and the control group were significantly higher than those in the antibody groups with therapeutic effects ( Figure 12 B).

[0102] Example 8

[0103] The humanized HA05 (SEQ ID NO:8) and HA06 (SEQ ID NO:9) of 501 were constructed on the pComb3X vector, and the plasmid was transferred into HB2151 competent cells. Single colonies were picked from the ampicillin plates grown overnight and inoculated into SB bacterial culture medium. They were expressed for 12 - 14 hours under the induction condition of IPTG at 30°C. After harvesting the bacteria and lysing them, the single-domain antibodies were purified from them using Ni-TNA (Yeasen Biotech). 6 μg of the single-domain antibody protein was mixed with 5X SDS-PAGE loading buffer and loaded onto a 12-well precast protein gel (Smart-Lifesciences). Electrophoresis was carried out at 160 V for 30 minutes. The gel was added with Coomassie brilliant blue staining solution (Beyotime Biotech) and stained at room temperature for 10 minutes, and then decolorized in double-distilled water until clear bands were shown. As Figure 13The results showed that the molecular weight of the humanized single-domain antibody was about 15 kDa, with a single band and good purity. The binding activity of the humanized antibody HA06 to the H1N1-HA protein was determined using BLI technology (Cell Host Microbe. 2020; 27(6): 891-898.e5). The biotin-labeled H1N1-HA antigen was immobilized on an SA sensor (Pall Fortebio), and the serially diluted HA06 protein solution was used as the analysis sample. The steps were as follows: The HA protein was diluted to 15 μg / ml and then immobilized on the SAbiosensor, and it was allowed to bind to the serially diluted HA06 protein solution (concentration range: 1000 nM - 37 nM) in running buffer (PBST), with no antibody added to the blank control wells. The program was set as follows: Association, 300 s; Dissociation, 300 s, and the temperature was set at 37°C. ForteBio Data analysis 10.1 software was used to analyze the data. The corresponding blank control wells were used as controls to subtract the background, and the curves of each concentration were fitted according to the Mass Transport binding mode to obtain the kinetic analysis results. The equilibrium dissociation constant (KD) values of the binding were reported, and the results showed that the KD for H1N1-HA was 0.87 nM( Figure 14 ). The microneutralization virus neutralization assay was used to detect the neutralization activity of HA06 against influenza A virus strains. The results showed that the neutralization activity of the 501 humanized sequence HA06 against the live virus of A / California / 07 / 2009 (H1N1) was comparable to that of 501( Figure 15 ).

[0104] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the principle of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the present invention can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in the present invention, but will conform to the widest scope consistent with the principles and novel features disclosed in the present invention.

Claims

1. An antibody for specifically binding to hemagglutinin of influenza A virus, characterized in that, Comprising: A first immunoglobulin single variable domain, and / or a second immunoglobulin single variable domain; Wherein, the first immunoglobulin single variable domain has a first variable region with the amino acid sequence shown in SEQ ID NO: 1, a second variable region with the amino acid sequence shown in SEQ ID NO: 2, and a third variable region with the amino acid sequence shown in SEQ ID NO: 3; Wherein, the second immunoglobulin single variable domain has a fourth variable region with the amino acid sequence shown in SEQ ID NO: 4, a fifth variable region with the amino acid sequence shown in SEQ ID NO: 5, and a sixth variable region with the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein The amino acid sequence of the first immunoglobulin single variable domain is as shown in SEQ ID NO: 7-9.

3. The antibody according to claim 1, wherein The amino acid sequence of the second immunoglobulin single variable domain is as shown in SEQ ID NO:

10.

4. The antibody according to claim 1, wherein The C-terminus of the first immunoglobulin single variable domain is connected to the N-terminus of the second immunoglobulin single variable domain through a linker.

5. A fusion protein, characterized in that, The antibody according to any one of claims 1-4 is fused with a heterologous protein.

6. The fusion protein according to claim 5, wherein The heterologous protein is human immunoglobulin Fc.

7. A conjugate, characterized in that, The antibody according to any one of claims 1-4, or the fusion protein according to any one of claims 5-6, is conjugated with an effector molecule.

8. The conjugate according to claim 7, wherein, The effector molecule is a detectable label.

9. A nucleotide molecule, characterized in that, Encoding the antibody according to any one of claims 1-4.

10. A vector or recombinant vector, characterized in that, Comprising: The nucleotide molecule according to claim 9.

11. A transformant, characterized in that, Comprising: The vector or recombinant vector according to claim 10, or the nucleotide molecule according to claim 9 is integrated into the gene of the transformant.

12. A pharmaceutical composition, comprising: A prophylactically or therapeutically effective amount of the antibody according to any one of claims 1-4, or the fusion protein according to any one of claims 5-6, or the conjugate according to any one of claims 7-8, or the nucleotide molecule according to claim 9, or the vector or recombinant vector according to claim 10, or the transformant according to claim 10, and a pharmaceutically acceptable carrier.

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