WO2016192652A1 - 抗Flu B的广谱单克隆抗体及其用途 - Google Patents

抗Flu B的广谱单克隆抗体及其用途 Download PDF

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WO2016192652A1
WO2016192652A1 PCT/CN2016/084496 CN2016084496W WO2016192652A1 WO 2016192652 A1 WO2016192652 A1 WO 2016192652A1 CN 2016084496 W CN2016084496 W CN 2016084496W WO 2016192652 A1 WO2016192652 A1 WO 2016192652A1
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virus
influenza
seq
antibody
monoclonal antibody
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French (fr)
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陈毅歆
沈晨光
陈俊煜
王国松
张梦娅
夏宁邵
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厦门大学
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Priority to AU2016270147A priority Critical patent/AU2016270147B2/en
Priority to JP2017562638A priority patent/JP6423550B2/ja
Priority to EP16802575.7A priority patent/EP3305811B1/en
Priority to US15/732,536 priority patent/US10696736B2/en
Publication of WO2016192652A1 publication Critical patent/WO2016192652A1/zh
Priority to HK18106096.0A priority patent/HK1246805A1/zh

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Definitions

  • the invention relates to the field of immunology and molecular virology, in particular to the field of diagnosis, prevention and treatment of influenza B virus.
  • the present invention relates to a broad-spectrum monoclonal antibody against Influenza B virus (Flu B), a cell line producing the antibody, and a composition comprising the antibody (for example, a diagnostic agent and Therapeutic agent).
  • the invention relates to the use of said antibodies.
  • the antibodies of the invention can be used to diagnose, prevent and/or treat infections of influenza B virus and/or diseases caused by such infections (e.g., influenza).
  • Influenza is a respiratory infection caused by influenza virus. Its clinical features are fever, fatigue, body aches and a certain degree of respiratory symptoms. Influenza viruses are a major threat to human health, and their continued rapid antigenic drift has allowed seasonal influenza to spread widely among people. Common human seasonal influenza viruses include seasonal H1N1, seasonal H3N2, and influenza B viruses. According to WHO statistics, seasonal flu causes at least 250,000-500,000 deaths per year (Peter D.C. et al., J Clin Invest. 2008, 118:3273-3275). In addition, the outbreak of influenza is still a major threat to civilization.
  • influenza virus is Orthomyxoviridae, an enveloped single-stranded negative sense RNA virus of the genus Influenza. Its genome consists of 8 segments of RNA encoding more than ten viral proteins. According to the difference in antigenicity and genetic characteristics of viral nuclear protein (NP) and matrix protein (M), influenza virus is divided into three types: A (A), B (B), and C (C) (Horimoto T. et al. , Nat Rev Microbiol, 2005, 3(8): 591-600); Among them, Influenza A Virus (Flu A) has a wide range of hosts, which is highly variable and can cause worldwide pandemics; Influenza B virus (Flu B) can only infect humans and seals. The mutation is slower than influenza A virus, and generally only causes local epidemics; Influenza C virus (Flu C) is the most variable. Slow, weak virulence, usually only infected pregnant women and children with low resistance.
  • NP nuclear protein
  • M matrix protein
  • Influenza A Virus has
  • influenza B virus was first isolated in 1940. Since the 1980s, influenza B virus has been dominated by two sub-ageages with significant differences in antigenicity and genotype, namely the Victoria subfamily and the Yamagata subfamily. In the 1980s, the Victoria sub-system dominated the popularity. In the 1990s, the Yamagata subfamily dominated the epidemic. After 2000, these two sublines were common (Jumat MR. et al., BMC Res Notes, 2014, 7: 863).
  • influenza B virus is also an important cause of flu outbreaks.
  • influenza B virus causes a lot of feeling every year. The person is ill and dying (Lin et al., Virus Res, 2004, 103(1-2): 47-52).
  • the mortality and morbidity caused by influenza B virus is lower than that of influenza A virus H3N2, it is higher than H1N1 subtype (Dreyfus C. et al., Science, 2012, 337 (6100): 1343-8) .
  • Studies have shown that the clinical symptoms caused by influenza B virus are usually the same as the clinical symptoms of influenza A virus (Jumat MR.
  • the first line of defense against influenza is neutralizing antibodies.
  • the main target of vaccine-induced neutralizing antibodies is the hemagglutinin (HA) protein located on the surface of the virus.
  • the HA protein on the surface of the virus is in a trimer structure, and each monomer is composed of two domains, HA1 and HA2.
  • HA1 Located at the head of the trimer, HA1 has a globular structure and contains a receptor binding site, which is a key region for viral infection of host cells.
  • HA1 also contains important antigenic sites that induce the production of protective neutralizing antibodies and is a key target for vaccine design (Wang T.T. et al., Nat Struct Mol Biol. 2009, 16:233-234).
  • HA2 is located at the base of the trimer and has a stalk-like structure containing a fusion peptide that mediates fusion of the viral envelope with the host cell membrane.
  • Some monoclonal antibodies against HA2 are able to neutralize the virus by inhibiting membrane fusion of influenza viruses (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234).
  • Influenza viruses are highly variable, with HA mutations being the most rapid.
  • the traditional influenza vaccine mainly targets the HA protein, and the influenza vaccine is easily disabled due to the viral antigenic drift caused by the HA gene mutation.
  • WHO will choose to use or establish a new influenza virus vaccine strain as a vaccine candidate for the next season's epidemic season based on the monitoring of the variability of influenza strains prevailing in the previous year. New vaccines are vaccinated each year to ensure effective resistance to existing influenza strains. Therefore, the development of "broad-spectrum vaccines" that are not affected by viral mutations has gradually become a hot spot in new vaccine research.
  • influenza virus surface glycoprotein "hemagglutinin (HA)” is the main target for the development of broad-spectrum vaccines and immunotherapeutic drugs for influenza viruses.
  • the so-called “broad-spectrum vaccine” should contain “broad-spectrum neutralizing epitopes” shared by different virus variants, which can directly induce “broad-spectrum neutralizing antibodies” to resist different Infection of viral variants. Therefore, finding a broad-spectrum neutralizing epitope on HA has become an important way to study influenza broad-spectrum vaccines and immunotherapeutic drugs.
  • human mouse monoclonal antibodies specific for influenza B virus have been shown to be effective in treating experimental mice infected with influenza virus in a mouse animal model (Yasugi M. et al., PLoS Pathog, 2013, 9 ( 2)).
  • polyclonal and monoclonal antibodies are effective for preventing infection with viruses such as hepatitis A, hepatitis B, rabies, and respiratory syncytial cells (Sawyer L.A. et al., Antiviral Res. 2000, 47: 57-77).
  • Reports of human convalescent sera were also used during the Spanish flu period of 1918 (Luke T.C. et al., Ann Intern Med. 2006, 145: 599-609). This information suggests that antibodies can be used as an alternative to tools and tools for antiviral therapy.
  • Throsby et al first reported a broad-spectrum neutralizing human monoclonal antibody CR6261 that recognizes HA2, which neutralizes all belonging to Group 1 (including H1, H2, H5, H6, H8 and H9). Type of influenza virus (Throsby M. et al., PLoS One. 2009, 3: e3942). In 2011, Corti D. et al. also obtained a human-type broad-spectrum neutralizing monoclonal antibody against HA2 using similar technology, which was able to neutralize 16 H subtype influenza viruses (Corti D. et al., Science.
  • HA1 of influenza virus is structurally at the very top of the trimer, has a globular structure, contains a large number of neutralizing epitopes and is easily accessible. Therefore, finding a broad-spectrum neutralizing epitope on HA1 would facilitate the development of efficient broad-spectrum influenza vaccines and therapeutic antibodies.
  • Cyrille Dreyfus et al. reported in 2012 the only broad-spectrum neutralizing monoclonal antibody CR8033 that recognizes the HA1 domain of influenza B virus, but this monoclonal antibody only has hemagglutination in the Yamagata subfamily of influenza B virus. Inhibitory activity (Dreyfus C. et al., Science, 2012, 337 (6100): 1343-8).
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Or producing an antigen-binding fragment of an antibody by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs) Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • Fd fragment means an antibody fragment consisting of V H and C H 1 domains
  • Fv fragment means a single arm of V H and V L domains of an antibody, Antibody fragment
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al, Nature 341:544-546 (1989))
  • Fab fragment means by V L , V H , C antibody fragments L and C H 1 domains
  • F (ab ') 2 fragment means antibody fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • the antigen-binding fragment is a single chain antibody (e.g., the scFv), wherein V L and V H domains are paired to form so that it can be produced by a linker to a single polypeptide chain monovalent molecules (see, e.g., Bird Et al, Science 242: 423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 :6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • Antigen binding of antibodies can be obtained from a given antibody (e.g., monoclonal antibodies 12G6, 7G6, 11B10 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods). A fragment (for example, the above antibody fragment), and specifically screens the antigen-binding fragment of the antibody in the same manner as used for the intact antibody.
  • a given antibody e.g., monoclonal antibodies 12G6, 7G6, 11B10 provided herein
  • conventional techniques known to those skilled in the art e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods.
  • a fragment for example, the above antibody fragment
  • specifically screens the antigen-binding fragment of the antibody in the same manner as used for the intact antibody.
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • the terms "monoclonal antibody” and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, A group of identical antibody molecules, in addition to spontaneous mutations that may occur spontaneously.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • monoclonal antibodies can be prepared as follows.
  • the mice or other suitable host animals are first immunized with the immunogen (addition of an adjuvant if necessary).
  • the method of injection of the immunogen or adjuvant is usually subcutaneous injection or intraperitoneal injection.
  • the immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitors, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be Freund's adjuvant or MPL-TDM or the like.
  • lymphocytes can also be obtained by in vitro immunization.
  • the lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • a suitable fusing agent such as PEG
  • the hybridoma cells prepared above may be inoculated into a suitable culture medium, and the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells.
  • hypoxanthine guanine phosphotransferase HGPRT or HPRT
  • hypoxanthine, aminoguanidine, and thymine HAT medium
  • myeloma cells should have a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture fluid.
  • murine myeloma is preferred for myeloma cells, such as MOP-21 or MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of the monoclonal antibody can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107: 220 (1980).
  • the target cell strain can pass the standard described by (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The dilution method was subcloned.
  • a suitable culture solution may be DMEM or RPMI-1640 or the like.
  • hybridoma cells can also be grown in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by the subcloned cells can be cultured from the cell culture medium, Separated from ascites or serum.
  • Monoclonal antibodies can also be obtained by genetic engineering recombinant techniques.
  • a DNA molecule encoding a monoclonal antibody heavy chain and a light chain gene can be isolated from a hybridoma cell by PCR amplification using a nucleic acid primer that specifically binds to the monoclonal antibody heavy chain and light chain genes.
  • the obtained DNA molecule is inserted into an expression vector, and then transfected into a host cell (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and cultured under appropriate conditions.
  • the recombinant antibody expressed can be obtained.
  • chimeric antibody refers to an antibody whose light chain or/and a portion of a heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or Subclass), and another portion of the light or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but in any case, it remains Binding activity to a target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • humanized antibody means that all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by a CDR region of a non-human antibody (donor antibody).
  • An antibody or antibody fragment, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • receptor Some amino acid residues of the framework region (FR) of an antibody may also be replaced by amino acid residues of the corresponding non-human antibody or by amino acid residues of other antibodies to further refine or optimize the performance of the antibody.
  • neutralizing antibody refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence of a target virus (eg, the ability to infect a cell).
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • epitope peptide refers to a peptide segment on an antigen that can be used as an epitope.
  • a single epitope peptide is capable of being specifically recognized/bound by an antibody directed against the epitope.
  • carrier protein refers to a protein that can serve as a carrier for an epitope peptide, ie, it can insert an epitope peptide at a specific position (eg, inside the protein, N-terminus or C-terminus).
  • the epitope peptide can be presented so that the epitope peptide can be recognized by the antibody or the immune system.
  • carrier proteins are well known to those skilled in the art and include, for example, HPV L1 protein (the epitope peptide can be inserted between amino acids 130-131 of the protein or between amino acids 426-427, see Slupitzky Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops [J]. J Gen Virol, 2001, 82: 2799-2804; Varsani, A., etc.
  • Mouse hepatitis virus core protein (substitutable epitope peptides can be substituted for amino acids 79-81 of the protein, see Sabine Gertrud Beterams and Michael Nassal, J. Virol. 1998, 72(6): 4997), CRM197 protein (the epitope peptide can be linked to the N-terminus or C-terminus of the protein or fragment thereof).
  • a linker e.g., a flexible or rigid linker
  • Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete with antigen (eg, influenza virus hemagglutinin protein). High throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Application WO 03/48731.
  • antigen eg, influenza virus hemagglutinin protein
  • antibodies that compete with the monoclonal antibodies of the invention eg, monoclonal antibody 12G6, 7G6, or 11B10 for binding to the same epitope on the influenza virus hemagglutinin protein can be obtained using conventional techniques known to those of skill in the art and Its antigen-binding fragment (ie, antigen-binding portion).
  • the terms "isolated” or “isolated” refer to artificially obtained from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • Term “Isolated” or “isolated” does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain such as, but not limited to, GI698 , ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC); phage such as lambda Phage or M13 phage and animal virus.
  • plasmids include, but are not limited to, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC); phage such as lambda Phage or M13 phage and animal virus.
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • PAC P1 derived artificial chromosomes
  • phage such as lambda Phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between two sequences is the number of matching positions shared by the two sequences divided by the number of bits to compare Set the number ⁇ 100 function. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • the term "immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the mode of immunization.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and 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 antibody e.g., the monoclonal antibody 12G6, 7G6 or 11B10 of the invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10
  • a dissociation equilibrium constant (K D ) of -10 M or less binds to an antigen (eg, influenza virus hemagglutinin protein), for example, as measured using a surface plasmon resonance (SPR) in a BIACORE instrument.
  • an antigen eg, influenza virus hemagglutinin protein
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • hybridomas and “hybridoma cell lines” are used interchangeably and, when referring to the terms “hybridomas” and “hybridoma cell lines”, they also include subclones of hybridomas. And progeny cells. For example, when referring to hybridoma cell line 12G6, 7G6 or 11B10, it also refers to subcloning and progeny cells of hybridoma cell line 12G6, 7G6 or 11B10.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It 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 adjusting agents, surfactants, adjuvants, ionic strength enhancement. Agent.
  • pH adjusting agents 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.
  • adjuvant refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • protein vaccine refers to a polypeptide-based vaccine, which optionally further comprises an adjuvant.
  • the polypeptide in the vaccine may be obtained by genetic engineering techniques or may be obtained by chemical synthesis.
  • nucleic acid vaccine refers to a vaccine based on DNA or RNA (eg, a plasmid, such as an expression plasmid), which optionally further comprises an adjuvant.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, an influenza virus infection or a disease associated with an influenza virus infection
  • an effective amount for treating a disease means a disease sufficient to cure or at least partially arrest a patient who has already had a disease and concurrent The amount of the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • Yamagata subline and “Yamagata subtype influenza B virus” refer to the antigenic and evolutionary relationship of its hemagglutinin protein with the influenza B virus representative strain B/Yamagata/16/ 1988 Influenza virus subfamily in the same evolutionary branch; it includes the following exemplary strains: B/Harbin/7/1994, B/Florida/4/2006, B/Xiamen/891/206, B/Xiamen/ 756/2007, B/Xiamen/1147/2008, B/Xiamen/N697/2012.
  • the terms “Yamagata subline” and “Yamagata subtype influenza B virus” have the same meaning and are used interchangeably.
  • Victoria subline and Victoria subtype influenza B virus refer to the antigenic and evolutionary relationship of its hemagglutinin protein with the influenza B virus representative strain B/Victoria/2/. 1987 Influenza virus subfamily in the same evolutionary branch; including but not limited to the following exemplary strains: B/Hong Kong/330/2001, B/Malaysia/2506/2004, B/Xiamen/3043/2006, B/Xiamen/165/2007, B/Brisbane/60/2008, B/Brisbane/33/2008, B/Xiamen/1346/2008, B/Xiamen/N639/2010, B/Xiamen/N678/2012.
  • the terms "Victoria subline” and "Victoria subtype B influenza virus” have the same meaning and are used interchangeably.
  • hemagglutinin protein and "HA protein” refer to an antigenic glycoprotein encoded by fragment 4 of the influenza virus genome, which is present on the surface of a viral membrane and is synthesized in the endoplasmic reticulum of the cell, The molecular weight is about 76 kD.
  • the HA protein can be hydrolyzed into a HA1 polypeptide (molecular weight 47 kD, also referred to herein as "HA1 domain”) and a HA2 polypeptide (molecular weight 29 kD, also referred to herein as "HA2 domain”), both of which pass disulfide bonds.
  • HA1 polypeptide molecular weight 47 kD
  • HA2 polypeptide molecular weight 29 kD
  • Hemagglutinin proteins are immunogenic and anti-hemagglutinin antibodies can be used to neutralize influenza viruses.
  • Hemagglutinin proteins are well known to those skilled in the art and their amino acid sequences can be found in various public databases, such as NCBI.
  • the HA1 polypeptide/domain is located at the head of the HA protein and has a globular structure. It contains a viral receptor binding site that binds to the sialic acid receptor on the host cell membrane to mediate the virus. Enter the cell.
  • the HA2 polypeptide/domain can assist in the fusion of the viral envelope with the host cell membrane, which plays an important role in the entry of the virus into the host cell.
  • hemagglutination inhibitory activity refers to a functional activity of an antibody or antigen-binding fragment thereof to inhibit coagulation caused by binding of an influenza virus HA protein to a sialic acid receptor on the surface of a red blood cell.
  • An antibody or antigen-binding fragment having hemagglutination inhibitory activity is capable of inhibiting binding of a virus to a cellular receptor.
  • neutralizing activity means that an antibody or antigen-binding fragment thereof binds to an antigenic protein on a virus and thereby reduces or inhibits the virulence of the target virus (eg, the ability to infect cells) Functional activity.
  • An antibody or antigen-binding fragment having neutralizing activity is capable of preventing the release of mature and/or progeny virus of the virus-infected cell and/or progeny virus.
  • the present inventors have unexpectedly discovered through extensive experimental studies that a broad-spectrum neutralizing epitope exists in the HA1 domain of the influenza B virus surface antigen hemagglutinin protein, and antibodies recognizing these epitopes are capable of specifically binding across the HA subline.
  • the hemagglutinin protein of the influenza virus of the Yamagata subfamily and the Victoria subfamily exhibits a broad spectrum of viral binding reactivity and a broad spectrum of ability to neutralize the virus. Therefore, the antibodies of the present invention are particularly suitable for use in the diagnosis, prevention, and treatment of influenza B virus infection or diseases associated with influenza B virus infection (eg, influenza).
  • the invention provides a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementarity determining region (CDR) selected from the group consisting of:
  • VH heavy chain variable region
  • CDR complementarity determining region
  • amino acid sequences are respectively VH CDR1-3 shown in SEQ ID NOs: 13-15; and/or,
  • VL variable region
  • CDR complementarity determining region
  • amino acid sequences are respectively VL CDR1-3 shown in SEQ ID NOs: 4-6;
  • amino acid sequences are VL CDR1-3 as shown in SEQ ID NOS: 16-18, respectively.
  • the monoclonal antibody comprises a heavy chain variable region (VH) selected from the group consisting of:
  • VH as shown in SEQ ID NO: 19;
  • the monoclonal antibody comprises a light chain variable region (VL) selected from the group consisting of:
  • VL as shown in SEQ ID NO: 20;
  • the monoclonal antibody comprises:
  • Amino acid sequences such as VH CDR1-3 shown in SEQ ID NO: 1-3, and VL CDR1-3 having an amino acid sequence as shown in SEQ ID NO: 4-6, respectively;
  • amino acid sequences are VH CDR1-3 shown in SEQ ID NOS: 7-9, respectively, and the VL CDR1-3 having the amino acid sequence shown in SEQ ID NO: 10-12, respectively;
  • amino acid sequences are VH CDR1-3 shown in SEQ ID NOS: 13-15, respectively, and the VL CDR1-3 shown in SEQ ID NOs: 16-18, respectively.
  • the monoclonal antibody comprises:
  • VH as shown in SEQ ID NO: 19 and VL as shown in SEQ ID NO: 20;
  • the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody (eg, scFv), mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (eg, human mouse chimeric antibody) or bispecific or multispecific antibody.
  • the monoclonal antibody comprises a non-CDR region and the non-CDR region is from a species other than a murine, such as from a human antibody.
  • the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line 12G6, 7G6 or 11B10, said hybridoma cell line 12G6, 7G6 Both 11B10 and 11B10 are deposited with the China Type Culture Collection (CCTCC) and have the accession numbers CCTCC NO: C201527, CCTCC NO: C201435 and CCTCC NO: C201432, respectively.
  • CTCC China Type Culture Collection
  • the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the HA1 domain of a hemagglutinin protein of at least 2 sublines of influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the HA1 domain of the hemagglutinin protein of the Yamagata subline and the Victoria subline of influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof has hemagglutination inhibitory activity against the Yamagata subline influenza B virus and the Victoria subtype influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is neutralizing and is capable of neutralizing the Yamagata subline influenza B virus and the Victoria subtype influenza B virus.
  • the monoclonal antibody or antigen-binding fragment thereof has one or more activities selected from the group consisting of: (a) an influenza B virus that inhibits at least two sublines (eg, Yamagata subfamily) And the Victoria subtype of influenza B virus enter the host cell; (b) inhibit the release of at least 2 subline influenza B viruses (such as the Yamagata subline and the Victoria subtype of influenza B virus) from the host cell; c) inhibiting membrane fusion of at least 2 subline influenza B viruses (eg, Yamagata subline and Victoria subtype influenza B virus) with host cells; (d) triggering influenza B virus against at least 2 sublines (for example, the ADCC effect of the Yamagata subfamily and the Victoria subtype of influenza B virus); and (e) the triggering of influenza B viruses against at least two sublines (eg, the Yamagata subfamily and the Victoria subfamily influenza B virus)
  • the role of CDC is selected from the group consisting of: (a) an influenza B virus that inhibits at least two sublines (eg, Yama
  • the monoclonal antibody or antigen-binding fragment thereof has at least one, at least two, at least three, at least four, or five of the above activities. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof has all five of said activities. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof neutralizes influenza B virus by the five activities described, and thereby prevents and treats infection with influenza B virus.
  • the invention provides a monoclonal antibody or antigen-binding fragment thereof, It is capable of blocking/blocking at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% of the binding of the influenza B virus or its hemagglutinin protein to a monoclonal antibody selected from the group consisting of Preferably at least 95% or preferably at least 99%:
  • the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the HA1 domain of a hemagglutinin protein of at least 2 sublines of influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the HA1 domain of the hemagglutinin protein of the Yamagata subline and the Victoria subline of influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof has hemagglutination inhibitory activity against the Yamagata subline influenza B virus and the Victoria subtype influenza B virus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is neutralizing and is capable of neutralizing the Yamagata subline influenza B virus and the Victoria subtype influenza B virus.
  • the monoclonal antibody or antigen-binding fragment thereof has one or more activities selected from the group consisting of: (a) an influenza B virus that inhibits at least two sublines (eg, Yamagata subfamily) And the Victoria subtype of influenza B virus enter the host cell; (b) inhibit the release of at least 2 subline influenza B viruses (such as the Yamagata subline and the Victoria subtype of influenza B virus) from the host cell; c) inhibiting membrane fusion of at least 2 subline influenza B viruses (eg, Yamagata subline and Victoria subtype influenza B virus) with host cells; (d) triggering influenza B virus against at least 2 sublines ADCC (eg influenza virus of Yamagata subfamily and Victoria subfamily) And (e) triggering the CDC effect against at least 2 sublines of influenza B viruses, such as the Yamagata subline and the Victoria subtype of influenza B virus.
  • an influenza B virus that inhibits at least two sublines (eg, Yamagata subfamily) And the Victoria subtype of influenza B virus enter the host cell
  • the monoclonal antibody or antigen-binding fragment thereof has at least one, at least two, at least three, at least four, or five of the above activities. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof has all five of said activities. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof neutralizes influenza B virus by the five activities described, and thereby prevents and treats infection with influenza B virus.
  • the epitope recognized by such a monoclonal antibody is identical to the epitope recognized by the monoclonal antibody 12G6, 7G6 or 11B10, or spatially overlapping, such that the monoclonal antibody can reduce the monoclonal antibody 12G6, 7G6 or 11B10 and hemagglutinin protein.
  • the combination of HA1 domains is at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, or preferably at least 99%.
  • a method can be determined by a conventional method such as the method described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) to reduce the binding of a certain monoclonal antibody to a known monoclonal antibody (for example, type B). The ability of the influenza virus hemagglutinin protein).
  • An exemplary method comprises: pre-coating an antigen on a microplate, and then co-adding the serially diluted unlabeled test antibody and a specific concentration of the labeled known mAb to the pre-coated micro Incubation was carried out in the well plates, and then the number of known antibodies bound to the plates under different dilutions of the antibody to be tested was determined after washing.
  • the antigen is pre-coated onto a 96-well microtiter plate and the ability of the test monoclonal antibody to block the labeled known monoclonal antibody is determined by radiolabeling or enzymatic labeling.
  • Anti-idiotypic antibodies can be produced using monoclonal antibodies of the invention by methods known in the art (Schulman JL. et al, Monographs in allergy, 1986, 22: 143-9). Anti-idiotypic antibodies are unique types of antibodies that specifically recognize/bind the antibodies used to prepare them (ie, as the idiotype of the variable regions of the antibodies used to prepare them) Epitopes), which are capable of mimetic/reconstructing epitopes recognized by the antibodies used to make them. The monoclonal antibodies of the present invention can also be used to prepare such anti-idiotypic antibodies, and the anti-idiotype antibodies thus obtained are also included in the scope of the present invention. Thus, in one aspect, the invention also provides an anti-idiotypic antibody that specifically targets a unique form of a monoclonal antibody of the invention.
  • the invention also provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • nucleic acid molecules can be isolated from hybridoma cells, or can be obtained by genetic engineering recombinant techniques or chemical synthesis methods.
  • the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises:
  • amino acid sequence is the VH CDR1-3 of SEQ ID NO: 1-3;
  • amino acid sequence is VH CDR1-3 of SEQ ID NOs: 7-9;
  • amino acid sequence is VH CDR1-3 of SEQ ID NOS: 13-15.
  • the antibody heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29.
  • the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody light chain variable region, wherein the antibody light chain variable region comprises:
  • amino acid sequence is VL CDR1-3 of SEQ ID NO: 4-6;
  • amino acid sequence is VL CDR1-3 of SEQ ID Nos: 10-12; or
  • amino acid sequence is VL CDR1-3 of SEQ ID NOS: 16-18.
  • the antibody light chain variable region has the amino acid sequence set forth as SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24;
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30.
  • the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region as defined above, and an energy as defined above A nucleic acid sequence encoding a variable region of an antibody light chain.
  • the invention provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention as defined above.
  • the invention provides a vector comprising an isolated nucleic acid molecule as defined above.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • the vectors of the invention are, for example, plasmids, cosmids, phage, and the like.
  • host cells comprising the isolated nucleic acid molecule or vector of the invention are also provided.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • a method of making a monoclonal antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell of the invention under suitable conditions, and recovering the monoclonal of the invention from the cell culture An antibody or antigen-binding fragment thereof.
  • the invention provides a hybridoma cell line selected from the group consisting of:
  • Hybridoma cell line 11B10 deposited at the China Center for Type Culture Collection (CCTCC), and having the accession number CCTCC NO: C201432.
  • amino acid sequence of the heavy chain variable region of monoclonal antibody 12G6 is set forth in SEQ ID NO: 19 (the exemplary nucleotide sequence of which is set forth in SEQ ID NO: 25), and the light chain can be The amino acid sequence of the variable region is set forth in SEQ ID NO: 20 (the exemplary nucleotide sequence of which is shown in SEQ ID NO: 26).
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 12G6 are SEQ ID NO: 1-3, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are respectively SEQ ID NO: 4-6.
  • the amino acid sequence of the heavy chain variable region of monoclonal antibody 7G6 is set forth in SEQ ID NO: 21 (the exemplary nucleotide sequence thereof is set forth in SEQ ID NO: 27), and the light chain can be The amino acid sequence of the variable region is set forth in SEQ ID NO: 22 (the exemplary nucleotide sequence of which is set forth in SEQ ID NO: 28).
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 7G6 are SEQ ID NOS: 7-9, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are SEQ ID NOS: 10-12, respectively.
  • the amino acid sequence of the heavy chain variable region of monoclonal antibody 11B10 is set forth in SEQ ID NO: 23 (the exemplary nucleotide sequence of which is set forth in SEQ ID NO: 29), and the light chain can be The amino acid sequence of the variable region is set forth in SEQ ID NO: 24 (the exemplary nucleotide sequence of which is set forth in SEQ ID NO: 30).
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 11B10 are SEQ ID NOS: 13-15, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are SEQ ID NOS: 16-18, respectively.
  • the invention provides a composition comprising a monoclonal antibody or antigen binding fragment thereof, an anti-idiotypic antibody, an isolated nucleic acid molecule, a vector or a host cell as described above.
  • the invention provides a kit comprising a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the kit further comprises a second antibody that specifically recognizes a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the second antibody further comprises a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials and enzymes (e.g., horseradish peroxidase), and the like.
  • the invention provides for the detection of influenza B virus or its hemagglutinin protein A method of presenting or leveling in a sample, which comprises using a monoclonal antibody of the present invention or an antigen-binding fragment thereof.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label.
  • the method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • the influenza B virus is selected from the group consisting of the Yamagata subline and the Victoria subtype influenza B virus.
  • the present invention provides a method of diagnosing whether a subject is infected with an influenza B virus, comprising: detecting an influenza B virus or a hemagglutinin protein thereof using the monoclonal antibody of the present invention or an antigen-binding fragment thereof The presence of a sample from the subject.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label.
  • the influenza B virus is selected from the group consisting of Yamagata subfamily and Victoria subtype influenza B virus.
  • influenza B virus is selected from the group consisting of Yamagata subfamily and Victoria subtype influenza B virus.
  • the sample includes, but is not limited to, excretion from a subject (eg, a mammal, preferably a human), oral and nasal secretions, and the like.
  • the monoclonal antibody is an antibody comprising: a VH CDR 1-3 having an amino acid sequence as set forth in SEQ ID NO: 1-3, respectively, and/or an amino acid sequence such as SEQ ID NO: VL CDR1-3 shown by 4-6; preferably, it comprises: VH as shown in SEQ ID NO: 19 and/or VL as shown in SEQ ID NO: 20; more preferably, it is a single Anti-12G6.
  • the monoclonal antibody is an antibody that is packaged
  • the amino acid sequence is VH CDR1-3 as shown in SEQ ID NOs: 7-9, respectively, and/or the VL CDR1-3 of the amino acid sequence shown in SEQ ID NOs: 10-12, respectively; preferably, it includes: VH shown in SEQ ID NO: 21 and/or VL as shown in SEQ ID NO: 22; more preferably, it is mAb 7G6.
  • the monoclonal antibody is an antibody comprising: a VH CDR1-3 having an amino acid sequence as set forth in SEQ ID NOs: 13-15, respectively, and/or an amino acid sequence such as SEQ ID NO: 16-18 to VL CDR1-3; preferably, it comprises: VH as shown in SEQ ID NO: 23 and/or VL as shown in SEQ ID NO: 24; more preferably, it is a single Resistance to 11B10.
  • enzyme immunoassay enzyme immunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • radioimmunoassay chemiluminescence immunoassay
  • fluorescent immunoassay e.g., fluorescent immunoassay, immunochromatography, competition, and the like.
  • the detection method can include the following steps: (i) allowing the antibody or antigen-binding fragment thereof to bind to the target virus or antigen (ie, influenza B virus or its hemagglutinin protein) Combining the monoclonal antibody or antigen-binding fragment thereof of the present invention with a sample to be tested under conditions of forming an antibody or antigen-binding fragment thereof - influenza B virus or its hemagglutinin protein complex; (ii) detecting the complex The presence of the substance to determine if the sample contains influenza B virus or its hemagglutinin protein.
  • the target virus or antigen ie, influenza B virus or its hemagglutinin protein
  • the detection method can include the steps of: (i) adsorbing the first antibody onto the solid support; (ii) adding to the support described above, possibly containing the influenza B virus Or a suspected test sample of its hemagglutinin protein; (iii) adding a second antibody with a label to the above support; (iv) detecting the presence of the marker to determine influenza B virus or its hemagglutinin Whether the protein is present in the sample.
  • the above detection methods can be used to detect a target antigen or antibody.
  • the competition method is used to compare antigens in a sample with a known amount of labeled antigens.
  • the immunological assay based on competition method generally comprises adding a sample containing an unknown amount of the target antigen and a pre-quantized labeled target antigen to the prior art to coat the monoclonal antibody of the present invention with a known physical or chemical method. On the phase support; then, after a period of incubation, the support is rinsed and the amount or level of label bound to the support is detected.
  • a sandwich-based immunological assay can include adding a sample containing an unknown amount of a target antigen to a solid support that has been pre-coated with the monoclonal antibody of the present invention, either physically or chemically; The labelled monoclonal antibody of the invention is added for reaction; after a period of incubation, the support is rinsed and the amount or level of label bound to the support is detected.
  • the label may be a radioisotope, an enzyme, a substrate for an enzyme, a luminescent substance such as isoluminol and acridinium ester, a fluorescent substance such as fluorescein and rhodamine, a colored substance such as latex particles and colloidal gold, and the like.
  • enzymes for labeling include, but are not limited to, peroxidases (such as horseradish peroxidase HRP), alkaline phosphatase, beta-galactosidase, acetylcholinesterase, and glucose oxidase.
  • Suitable enzyme substrates include, for example, 2,2'-azino-bis(3-ethylbenzothiapyrrolidin-6sulfonic acid), luminol-hydrogen peroxide, o-phenylenediamine-hydrogen peroxide (for peroxidase), p-nitrophenyl phosphate, 4-methylphosphatidyl ketone, 3-(2'-helixadamantane)-4-methoxy-4-(3"-phosphoryl) Phenyl-1,2-diethoxyalkane (for alkaline phosphatase), p-nitrophenyl- ⁇ -D-galactose and methylumbelliferyl- ⁇ -D-galactose (for beta galactoside Fluorescent substances for labeling include, but are not limited to, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, phycoerythr
  • labels include, but are not limited to, quantum dot labels, chromophore labels, affinity ligand labels, electromagnetic spin labels, heavy atom labels, epitope labels (such as FLAG or HA epitopes), and the ability to form complexes. Binding pair (eg, streptavidin/biotin, anti- Biotin/biotin or antigen/antibody complexes (eg rabbit IgG and anti-rabbit IgG)).
  • Binding pair eg, streptavidin/biotin, anti- Biotin/biotin or antigen/antibody complexes (eg rabbit IgG and anti-rabbit IgG)).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody of the invention, or an antigen binding fragment thereof, or an anti-idiotypic antibody, and a pharmaceutically acceptable carrier and/or excipient.
  • the monoclonal antibody is selected from the group consisting of:
  • a monoclonal antibody comprising: a VH CDR1-3 having an amino acid sequence as shown in SEQ ID NO: 1-3, respectively, and/or a VL CDR1-3 having an amino acid sequence as shown in SEQ ID NO: 4-6, respectively.
  • it comprises: VH as shown in SEQ ID NO: 19 and/or VL as shown in SEQ ID NO: 20; more preferably, it is a monoclonal antibody produced by hybridoma cell line 12G6,
  • the hybridoma cell line 12G6 is deposited in the China Center for Type Culture Collection (CCTCC) and has the accession number CCTCC NO: C201527;
  • a monoclonal antibody comprising: a VH CDR1-3 having an amino acid sequence as shown in SEQ ID NOs: 7-9, respectively, and/or a VL CDR1-3 having an amino acid sequence as shown in SEQ ID NOs: 10-12, respectively.
  • it comprises: VH as shown in SEQ ID NO: 21 and/or VL as shown in SEQ ID NO: 22; more preferably, it is a monoclonal antibody produced by hybridoma cell line 7G6,
  • the hybridoma cell line 7G6 is deposited with the China Type Culture Collection (CCTCC) and has the accession number CCTCC NO: C201435; or
  • a monoclonal antibody comprising: a VH CDR1-3 having an amino acid sequence as shown in SEQ ID NO: 13-15, respectively, and/or a VL CDR1-3 having an amino acid sequence as shown in SEQ ID NOs: 16-18, respectively.
  • it comprises: VH as shown in SEQ ID NO: 23 and/or VL as shown in SEQ ID NO: 24; more preferably, it is a monoclonal antibody produced by hybridoma cell line 11B10,
  • the hybridoma cell line 11B10 is deposited with the China Center for Type Culture Collection (CCTCC) and has the accession number CCTCC NO: C201432.
  • the pharmaceutical composition further comprises other pharmaceutically active agents (eg, anti-influenza virus drugs such as M2 protein ion channel inhibitors (eg, amantadine and rimantadine) and neuraminidase) Inhibitor (eg, oseltamivir)).
  • pharmaceutically active agents eg, anti-influenza virus drugs such as M2 protein ion channel inhibitors (eg, amantadine and rimantadine) and neuraminidase) Inhibitor (eg, oseltamivir)).
  • M2 protein ion channel inhibitors eg, amantadine and rimantadine
  • neuraminidase eg, oseltamivir
  • the invention provides a method for neutralizing the virulence of an influenza B virus in a sample comprising contacting a sample comprising an influenza B virus with a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • a sample comprising an influenza B virus with a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • Such methods can be used for therapeutic purposes, or for non-therapeutic purposes (eg, the sample is a cell sample, not a patient or a sample from a patient).
  • the influenza B virus is selected from the group consisting of Yamagata subfamily and Victoria subtype influenza B virus.
  • a monoclonal antibody or antigen-binding fragment thereof of the invention for the preparation of a medicament for neutralizing the virulence of influenza B virus in a sample.
  • the invention provides a monoclonal antibody or antigen-binding fragment thereof as described above for use in neutralizing the virulence of influenza B virus in a sample.
  • a monoclonal antibody of the invention or an antigen-binding fragment thereof, or an anti-idiotypic antibody, for use in the manufacture of a pharmaceutical composition for preventing or treating a subject's influenza B virus Infection or disease associated with influenza B virus infection (eg, flu).
  • the invention provides a monoclonal antibody, or antigen-binding fragment thereof, or an anti-idiotypic antibody, as described above, for use in preventing or treating influenza B virus infection or infection with influenza B virus in a subject Related diseases (such as influenza).
  • the present invention provides a method for preventing or treating a disease associated with influenza B virus infection or influenza B virus infection (eg, influenza) in a subject, comprising administering to a subject in need thereof A prophylactically or therapeutically effective amount of a monoclonal antibody of the invention or an antigen binding fragment thereof or an anti-idiotypic antibody, or a pharmaceutical composition of the invention, is administered.
  • a disease associated with influenza B virus infection or influenza B virus infection eg, influenza
  • the subject is a mammal, such as a human.
  • the monoclonal antibodies of the invention, or antigen-binding fragments thereof, or anti-idiotypic antibodies, or pharmaceutical compositions of the invention can be administered to a subject by any suitable route of administration.
  • routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, Intrathecal, or nasal route.
  • the monoclonal antibody is an antibody comprising: a VH CDR 1-3 having an amino acid sequence as set forth in SEQ ID NO: 1-3, respectively, and/or an amino acid sequence such as SEQ ID NO: VL CDR1-3 shown by 4-6; preferably, it comprises: VH as shown in SEQ ID NO: 19 and/or VL as shown in SEQ ID NO: 20; more preferably, it is a single Anti-12G6.
  • the monoclonal antibody is an antibody comprising: a VH CDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 7-9, respectively, and/or an amino acid sequence such as SEQ ID NO: VL CDR1-3 shown by 10-12; preferably, it comprises: VH as shown in SEQ ID NO: 21 and/or VL as shown in SEQ ID NO: 22; more preferably, it is a single Resistance to 7G6.
  • the monoclonal antibody is an antibody comprising: a VH CDR1-3 having an amino acid sequence as set forth in SEQ ID NOs: 13-15, respectively, and/or an amino acid sequence such as SEQ ID NO: 16-18 to VL CDR1-3; preferably, it comprises: VH as shown in SEQ ID NO: 23 and/or VL as shown in SEQ ID NO: 24; more preferably, it is a single Resistance to 11B10.
  • compositions provided by the present invention may be used alone or in combination, or may be combined with other pharmaceutically active agents (for example, anti-influenza virus drugs such as M2 protein ion channel inhibitors (for example, amantadine and rimantadine) and A neuraminidase inhibitor (eg, oseltamivir) is used in combination.
  • anti-influenza virus drugs such as M2 protein ion channel inhibitors (for example, amantadine and rimantadine) and A neuraminidase inhibitor (eg, oseltamivir) is used in combination.
  • the monoclonal antibodies of the invention and antigen-binding fragments thereof have significant advantages compared to the prior art.
  • the monoclonal antibodies and antigen-binding fragments thereof of the present invention are capable of specifically binding to the hemagglutinin protein of influenza B virus of at least two sublines (eg, Yamagata subline and Victoria subline), exhibiting a broad spectrum of viruses
  • the ability to bind to a reactive and broad-spectrum neutralizing virus has a particularly significant advantage for preventing or treating a subject's influenza B virus infection or a disease associated with influenza B virus infection, such as influenza.
  • Figure 1 shows the three-dimensional structure of key amino acid positions in the epitopes recognized by monoclonal antibodies 12G6 (Figure 1A), 7G6 ( Figure 1B), 11B10 ( Figure 1C).
  • Figure 1A the receptor binding site for HA is labeled cyan and the amino acid site recognized by monoclonal antibody 12G6 is marked red.
  • the results showed that the key epitope amino acids recognized by monoclonal antibody 12G6 were located at positions 156, 176 and 183 of HA.
  • Figure IB the receptor binding site for HA is labeled cyan and the amino acid site recognized by monoclonal antibody 7G6 is marked red.
  • Figure 2 shows the use of mAb 7G6 and 11B10 against influenza virus B/Floria/04/2006 (Yamagata) (Fig. 2A), B/Malaysia/2506/2004 (Victoria) (Fig. 2B), A/Brisbane/20/2007
  • H3N2 Fig. 2C
  • H1N1 Fig. 2D
  • the abscissa is the dilution factor of the monoclonal antibody
  • the ordinate is the ELISA test result (OD 450 ).
  • Figure 3 shows the protective effect of monoclonal antibody 12G6 on B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA) influenza B virus-infected mice.
  • FIGS 3A and 3B show the negative control group (PBS-NC), the B/Florida/04/2006 virus infection control group (Flu B Viral cont), and the treatment group (12G6-10 mg/kg) mice, respectively. Survival and weight changes.
  • Figures 3C and 3D show the survival rate and body weight of the negative control group (PBS-NC), B/Brisbane/60/2008 virus-infected control group (Flu B Viral cont), and treatment group (12G6-10 mg/kg), respectively. Variety.
  • mice in the negative control group showed no significant body weight fluctuation during the whole experiment, and the two virus-infected control mice showed significant weight loss.
  • the B/Florida/04/2006 virus control group mice all died 8 days after infection, and the B/Brisbane/60/2008 virus control group mice all died 10 days after the virus infection.
  • the 10 mg/kg dose of 12G6 antibody injection intervention restored the body weight of the infected mice and allowed the infected mice to survive for 14 days with a therapeutic effect of 100%.
  • Figure 4 shows the protective effect of mAb 7G6 on B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA) influenza B virus-infected mice.
  • Figure 4A and Figure 4B show the survival rate and body weight of the negative control group (PBS-NC), B/Florida/04/2006 virus infection control group (Flu B Viral cont) and treatment group (7G6-10 mg/kg) mice, respectively.
  • Figure 4C and Figure 4D show the survival rate and body weight of the negative control group (PBS-NC), B/Brisbane/60/2008 virus infection control group (Flu B Viral cont) and treatment group (7G6-10 mg/kg) mice, respectively. Variety.
  • mice in the negative control group showed no significant body weight fluctuation during the whole experiment, and the two virus-infected control mice showed significant weight loss.
  • the B/Florida/04/2006 virus control group mice all died 8 days after infection, and the B/Brisbane/60/2008 virus control group mice all died 5 days after the virus infection.
  • the 10 mg/kg dose of 7G6 antibody injection intervention restored the body weight of the infected mice and allowed the infected mice to survive for 14 days with a therapeutic effect of 100%.
  • Figure 5 shows the protective effect of mAb 11B10 on B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA) influenza B virus-infected mice.
  • 5A and 5B show the survival rate and body weight of a negative control group (PBS-NC), a B/Florida/04/2006 virus-infected control group (Flu B Viral cont), and a treatment group (11B10-10 mg/kg), respectively.
  • Figures 5C and 5D show the survival rate and body weight of the negative control group (PBS-NC), B/Brisbane/60/2008 virus-infected control group (Flu B Viral cont), and treatment group (11B10-10 mg/kg), respectively. Variety.
  • mice in the negative control group showed no significant body weight fluctuation during the whole experiment, and the two virus-infected control mice showed significant weight loss.
  • the B/Florida/04/2006 virus control group mice all died 8 days after infection, and the B/Brisbane/60/2008 virus control group mice all died 8 days after the virus infection.
  • the 10 mg/kg dose of 11B10 antibody injection intervention restored the body weight of the infected mice and allowed the infected mice to survive for 14 days with a therapeutic effect of 100%.
  • Figure 6 shows the results of immunofluorescence analysis of MDCK cells infected with influenza B virus B/Floria/04/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria), wherein the influenza B virus is infected Polyclonal antiserum against monoclonal antibody 12G6, anti-B/Florida/4/2006 virus (B/FL. antiserum), polyclonal antiserum against B/Maylaysia/2506/2004 virus (B/Mal) . Antiserum) or PBS (no antibody control) was incubated.
  • Figure 7 shows the results of staining MDCK cells infected with influenza B virus B/Florida/4/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria) with Giemsa dye, wherein the MDCK cells are After virus infection, incubation was carried out with antibody 12G6 at 0 ⁇ g/ml, 5 ⁇ g/ml, 20 ⁇ g/ml or 100 ⁇ g/ml, respectively.
  • Figure 8 shows the results of immunoblot analysis of NP proteins in culture supernatants and cell lysates for detecting MDCK cells using influenza B virus B/Florida/4/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria) was infected and, after infection, medium (no antibody control) or the indicated concentration of monoclonal antibody 12G6 (2 ⁇ g/ml, 0.2 ⁇ g/ml or 0.02 ⁇ g/ml; diluted in Incubation was carried out in the medium or at a specified concentration of a negative control antibody (20 ⁇ g/ml or 2 ⁇ g/ml; diluted in the medium).
  • Figure 9 shows ADCC effects (Figure 9A) and CDC effects (Figure 9A) triggered by monoclonal antibody 12G6 and negative control antibodies against influenza viruses Massachusetts/02/2012-like (Yamagata) and B/Brisbane/60/2008 (Victoria) ( Figure 9B) Analytical results; wherein, Ctr: negative control antibody (anti-HIV mAb 5G6).
  • the present invention relates to the following biomaterials that have been deposited at the China Center for Type Culture Collection (CCTCC, Wuhan, China, Wuhan University):
  • Hybridoma cell line 12G6 the accession number is CCTCC NO: C201527, and the preservation time is April 10, 2015;
  • Hybridoma cell line 7G6 the accession number is CCTCC NO: C201435, and the preservation time is March 26, 2014;
  • Hybridoma cell line 11B10 the accession number is CCTCC NO: C201432, and the preservation time is March 26, 2014.
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Guide to Editing Molecular Biology, Third Edition, John Wiley & Sons, Inc., 1995; restriction endonucleases are used according to the conditions recommended by the product manufacturer. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially. The invention is described by way of example, and is not intended to limit the scope of the invention.
  • Example 1 Preparation of monoclonal antibodies against influenza B virus HA protein
  • B/Xiamen/891/2006 (Yamagata), B/Xiamen/1346/2008 (Victoria), B/Xiamen/N697/2012 (Yamagata), B/Xiamen/3043/2006 (Victoria) are all separated by the laboratory. Influenza B strain.
  • Hybridoma cells secreting monoclonal antibodies are obtained using standard in vivo immunization methods and PEG fusion methods. For details, see Ed Harlow et al., "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988. The brief process is as follows:
  • the titer of the above inactivated virus was adjusted to 128 HA, and then immunization was carried out by sequential immunization. Briefly, the virus B/Xiamen/891/2006 (Yamagata) was first mixed and emulsified in equal volume with Freund's complete adjuvant (CFA), and then the mice were immunized for the first time; the immunization protocol was multiple injections of muscles through the limbs. Each mouse was injected with 400 ul of emulsified virus solution.
  • CFA Freund's complete adjuvant
  • Virus B/Xiamen/1346/2008 (Victoria), B/Xiamen/N697/2012 (Yamagata), B/Xiamen/3043/2006 (Victoria) mixed with Freund's incomplete adjuvant (IFA) and emulsified, then separately Mice were boosted on the 14th, 28th, and 42th day after the first immunization. Finally, the mice were boosted with spleen on the 56th day after the first immunization. The immunogen was an equal volume mixture of the above viruses, and each mouse was injected with a volume of 100 ul. Three days after the end of immunization, the mouse spleens were taken for fusion experiments.
  • the mouse spleen was taken, ground to obtain a spleen cell suspension, which was then mixed with mouse myeloma cells SP2/0 in the logarithmic growth phase, and subjected to cell fusion under the action of PEG1500.
  • the fused cells were resuspended in 400 ml of fusion medium and dispensed into 20 96-well cell culture plates for culture.
  • the fusion medium was RPMI 1640 complete screening medium containing HAT and 20% FBS.
  • the fused cells were cultured on a 96-well cell culture plate for 10 days, and then the cell supernatant was aspirated for hemagglutination inhibition test (HI) and ELISA.
  • the viruses used for the assay were B/Xiamen/891/2006 (Yamagata) and B/Xiamen/1346/2008 (Victoria).
  • HI detection antibodies secreted by positive wells should be able to inhibit the agglutination of influenza B virus and red blood cells; for ELISA, antibodies secreted by positive wells should be able to specifically bind to influenza B virus coated on polystyrene plates. reaction.
  • the cloned positive clones were cloned three times to obtain a hybridoma cell strain capable of stably secreting antibodies. Finally, 41 hybridoma cell lines resistant to influenza B virus hemagglutinin including 12G6, 7G6, and 11B10 were obtained.
  • the ascites containing the monoclonal antibody was subjected to precipitation treatment with a 50% ammonium sulfate solution.
  • the obtained precipitate was then dissolved in PBS and purified in an AKTA system using a Protein A column to obtain a purified monoclonal antibody.
  • the purity of the purified monoclonal antibody was identified by SDS-PAGE.
  • Example 2 Identification of a broad-spectrum monoclonal antibody recognizing influenza A virus hemagglutinin across the HA subline
  • the HI detection method is carried out in accordance with the WHO operation guide. Based on the reactivity of the monoclonal antibody with each influenza virus strain, three broad-spectrum monoclonal antibodies that recognize the Yamagata and Victoria sublines of the influenza B virus (ie, the influenza A virus hemagglutinin across the HA subline) were identified. 12G6, 7G6 and 11B10 (Table 2).
  • Monoclonal antibodies 12G6, 7G6 and 11B10 specifically reacted with the Yamagata subtype B influenza virus and the Victoria subtype B influenza virus, showing broad-spectrum reactivity across the HA subline.
  • monoclonal antibody 7G6 can react with influenza B virus, Yamagata subtype B influenza virus and Victoria subtype B influenza virus, which have not been sub-systematic in the early years.
  • influenza B viruses used for detection except for the B/Harbin/7/1994 (Yamagata subfamily) and B/Great Lakes/1739/1954 strains, the monoclonal antibody 7G6 can The specific response of the influenza B virus shows an extremely broad response spectrum.
  • Monoclonal antibodies 12G6 and 11B10 specifically react with some of the early unincorporated influenza B viruses, some of the Yamagata subtypes of influenza B virus, and some of the Victoria subtypes of influenza B virus. Among them, the response spectrum of the monoclonal antibody 11B10 to the Yamagata subfamily and the Victoria subfamily virus was wider than that of the monoclonal antibody 12G6; and the monoclonal antibody 12G6 was more responsive to the early influenza B virus than the 11B10.
  • the monoclonal antibodies 12G6, 7G6 and 11B10 are specific, broad-spectrum monoclonal antibodies capable of recognizing influenza B virus across the HA subline.
  • Table 2 Hemagglutination inhibition test activity of monoclonal antibodies 12G6, 7G6 and 11B10 (HI titer)
  • HI titer indicates that the monoclonal antibody can completely inhibit the maximum dilution factor of viral HA activity; wherein, ⁇ 100, it means no reaction.
  • Neutralization titer is an important indicator for evaluating whether a monoclonal antibody has the potential to prevent and treat disease.
  • the neutralization activity of monoclonal antibodies 12G6, 7G6 and 11B10 against the influenza B virus representative strains of each subline was examined by microwell cell neutralization assay (refer to Hulse-Post et al., PNAS. 2005). 102:10682-7). The experimental results are shown in Table 3. Three monoclonal antibodies (12G6, 7G6, and 11B10) have broad-spectrum cross-neutralizing activity against influenza B virus of the early sub-system, influenza B virus of the Yamagata sub-line, and influenza B virus of the Victoria sub-line.
  • the neutralizing activity of monoclonal antibody 7G6 is basically the same as that of HI activity; except for B/Harbin/7/1994 (Yamagata subfamily) and B/Great Lakes/1739/1954 did not show neutralizing activity, and monoclonal antibody 7G6 has neutralizing activity against all influenza B viruses to be tested, showing a broad spectrum of neutralizing activity and polarity. Strong reactivity.
  • the neutralizing activity response spectrum of monoclonal antibody 11B10 is slightly broader than the HI activity response spectrum. In particular, monoclonal antibody 11B10 was able to neutralize almost all influenza B viruses from 1962 to 2012 (except B/Harbin/7/1994).
  • the neutralizing activity response spectrum of monoclonal antibody 12G6 is quite different from the HI activity response spectrum, which can neutralize all influenza B viruses between 1940 and 2012, showing extremely strong and extremely broad-spectrum neutralizing activity.
  • monoclonal antibodies 12G6, 7G6 and 11B10 were used to induce selection of influenza B virus strains with escape mutations.
  • the selected escape virus was subjected to plaque purification, amplification culture, gene retrieval, sequencing and escape mutation site structure localization, thereby determining the region where the epitope recognized by the monoclonal antibodies 12G6, 7G6 and 11B10 is located and the key table identified. Amino acid.
  • Escaped mother virus B virus selected from influenza B virus B/Singapore/3/1964 and B/Xiamen/1346/2008 (Victoria subline) as escape virus screening.
  • RT-PCR was performed on the obtained escape virus strain to obtain a structural gene of the virus. Subsequently, the structural gene is sequenced and aligned with the sequence of the structural gene of the escaped mater virus to identify escape mutations and mutation sites.
  • escape mutation results showed that the escape mutation sites were located in the gene encoding the HA1 domain of the influenza A virus hemagglutinin protein.
  • the escape mutation results of monoclonal antibody 12G6 showed that the escape mutation site involved 156, 176 and 183 of the HA protein (SEQ ID NO: 39) of B/Xiamen/1346/2008 (Victoria subline). Amino acid residues (counted from the signal peptide, the same below). Based on B/Xiamen/1346/2008 (Victoria subline) virus escape screening, 15 escape virus strains were obtained, of which 4 strains were mutated at the 156th amino acid residue of HA (G156W), and 4 strains were in HA.
  • the escape mutation screening results of mAb 7G6 showed that the escape mutation site involved amino acid residues 156, 165, and 180 of the HA protein of B/Singapore/3/1964 (SEQ ID NO: 40).
  • Six escape virus strains were obtained based on B/Singapore/3/1964 virus escape screening. Among them, 6 strains were mutated at the 156th amino acid residue of HA protein (G156W), and 4 strains were 165 amino acid residues in HA protein. Mutation occurred at the base (K165E), and 6 strains were mutated at the amino acid residue at position 180 of the HA protein (N180T). The above three amino acid positions were identified as key epitope amino acids recognized by monoclonal antibody 7G6, and their localization on the three-dimensional structure of HA is shown in Fig. 1B.
  • the escape mutation screening results for mAb 11B10 showed that the escape mutation site involved the amino acid residue at position 180 of the HA protein (SEQ ID NO: 39) of B/Xiamen/1346/2008 (Victoria subline).
  • Four escape virus strains were obtained based on B/Xiamen/1346/2008 (Victoria subline) virus escape screening, all of which were mutated at the 180th amino acid residue of the HA protein (N180K). This indicates that the amino acid at position 180 of the HA protein is a key epitope amino acid recognized by the monoclonal antibody 11B10, and its localization on the three-dimensional structure of the HA is shown in Fig. 1C.
  • 1 ul of reverse transcription primer was added to each tube, and one of the reverse transcription primers added was MVJkR (5'-CCGTTTGKATYTCCAGCT TGGTSCC-3') (SEQ ID NO: 31) for amplification of the light chain variable region gene;
  • One tube of the added reverse transcription primer was MVDJhR (5'-CGGTGACCGWGGTBCCTTGRCCCCA-3') (SEQ ID NO: 32) for amplification of the heavy chain variable region gene.
  • Add 1 ul dNTP (Shanghai Shenggong) to each tube and place in a 72 ° C water bath for 10 min, then immediately put it in an ice bath for 5 min.
  • MVJkR SEQ ID NO: 31
  • MVDJhR SEQ ID NO: 32
  • the template is the two cDNAs synthesized in the previous step.
  • the PCR conditions were: 94 ° C for 5 min, 35 cycles (94 ° C for 40 s, 53 ° C for 1 min, 72 ° C for 50 s), 72 ° C for 15 min.
  • the fragment of interest was recovered and cloned into pMD 18-T vector and sent to Shanghai Boya for sequencing. By blasting the sequencing results, the gene sequence encoding the variable region of the antibody is determined, and the corresponding amino acid sequence is determined.
  • variable region gene of the antibody was cloned from the hybridoma cell lines 12G6, 7G6, 11B10, and referred to the Kabat method (Kabat EA, Wu TT, Perry HM, Gottesman KS, Coeller K. Sequences of proteins of immunological interest , US Department of Health and Human Services, PHS, NIH, Bethesda, 1991) Determines the amino acid sequence of the CDR region (complementary determinant region) of the mAb. The results are shown in Tables 8a-8b.
  • Table 8b Amino acid and nucleotide sequences of the variable regions of the monoclonal antibodies 12G6, 7G6, 11B10
  • Example 6 Application of monoclonal antibodies 7G6 and 11B10 for detecting influenza B virus
  • Preparation of influenza virus Take a certain amount of the following influenza virus strains B/Floria/04/2006 (Yamagata), B/Malaysia/2506/2004 (Victoria), A/Brisbane/20/2007 (H3N2), A /NewCalidonia/20/1999(H1N1) at 4°C Inactivated with 0.03% formalin solution.
  • the inactivated virus was subjected to sucrose density gradient centrifugation on an ultracentrifuge, and centrifuged at 25,200 rpm for 3 hours at 4 °C.
  • the virus pellet was dissolved in 1 x PBS overnight at 4 °C.
  • the ultracentrifuged virus was tested by HA titer assay to determine the titer of the virus solution.
  • Monoclonal antibody monoclonal antibodies 7G6 and 11B10 prepared as above; the concentration of the monoclonal antibody was 1 mg/ml.
  • the titer of the ultracentrifuged virus was adjusted to 128 HA and pre-coated on a 96-well polystyrene plate at 200 ul per well. Subsequently, the 96-well plate was blocked with a blocking solution.
  • the mAb to be tested was diluted to 0.1 mg/ml as an initial concentration, and 15 double-fold dilutions were performed.
  • the diluted monoclonal antibody was added to the above-mentioned ELISA plate in a volume of 100 ul per well, and incubated at 37 ° C for 30 minutes.
  • the plate was washed 5 times with ELISA wash (PBST), followed by 100 ul of diluted HRP-labeled secondary antibody and incubated at 37 ° C for 30 minutes. After washing the plate with PBST for 5 times, the developer was added and the color was developed for 20 min. Subsequently, the absorbance of A450 was read on a microplate reader.
  • PBST ELISA wash
  • mAb 7G6 and 11B10 have influenza B viruses (B/Floria/04/2006 (Yamagata) and B/Malaysia/2506/2004 (Victoria)) for both Yamada and Victoria HA sublines. Strong binding reactivity, but no specific reactivity to representative strains of influenza A virus (such as A/Brisbane/20/2007 (H3N2) and A/NewCalidonia/20/1999 (H1N1)).
  • Example 7 Application of monoclonal antibodies 12G6, 7G6, 11B10 for the treatment of influenza virus infection
  • the monoclonal antibodies of the invention are highly potent against influenza B strains of at least two HA sublines (eg, Yamagata and Victoria) of different isolation and isolation times. Neutralizing activity.
  • the monoclonal antibody of the present invention is characterized by neutralization of influenza B virus against at least two HA sublines (eg, Yamagata and Victoria). Wide spectrum, medium and high titer.
  • the present inventors validated the monoclonal antibody of the present invention against Yamagata in a biosafety laboratory based on a virus-infected animal model of the Yamagata subfamily of the influenza B virus and the Victoria subfamily.
  • the specific plan is as follows:
  • mice Balb/C mice, SPF, 6-8 weeks old, female, weighing approximately 20 g.
  • BR60-MA Mouse adaptation strain of Victoria subtype B influenza virus: B/Brisbane/60/2008, referred to as BR60-MA.
  • mice were sent to the biosafety laboratory one day in advance, grouped into 5 cages, labeled G1, G2, ..., and the body weight of each mouse was recorded. The detailed scheme is shown in Table 9.
  • Viral infection pre-diluted the Yamagata subfamily virus B/Florida/04/2006 to 10 5 TCID 50 /ul, Victoria sub-virus B/Brisbane/60/2008 pre-diluted to 10 6 TCID 50 /ul, mouse virus inoculation The amount is 50ul / only. Prior to inoculation, mice were anesthetized with isoflurane and then infected with the virus via nasal infusion.
  • a dose of antibody was administered to mice in the antibody-treated group 24 h after virus infection (dpi.1) at a dose of 10 mg/kg and an injection volume of 100 ul/mouse.
  • mice were infected with a one-week dose of Yamagata subtype influenza B virus FL04-MA and Victoria subtype B influenza virus BR60-MA, respectively.
  • One day after infection mice in the treatment group were injected with antibodies via the tail vein.
  • the therapeutic effect of the monoclonal antibody was judged by measuring the body weight of each group of mice and calculating the survival rate.
  • the experimental results are shown in Figures 3-5.
  • Monoclonal antibody 12G6 can inhibit the entry of influenza B virus into host cells.
  • Virus B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria);
  • HA-specific antibodies monoclonal antibody 12G6, polyclonal antiserum against B/Florida/4/2006 virus, and polyclonal antiserum against B/Maylaysia/2506/2004 virus;
  • GAM-FITC FITC-labeled goat anti-mouse antibody, green fluorescence
  • MDCK cells MDCK cells
  • the cells were subjected to DAPI staining (blue fluorescence) using a commercially available kit; and, using rabbit polyclonal antiserum against the influenza B virus NP protein (used as primary antibody) and GAM-FITC (used as two) Anti-, green fluorescence) immunofluorescence analysis of cells.
  • B/Brisbane/60/2008 When incubated with polyclonal antiserum against B/Maylaysia/2506/2004 virus, B/Brisbane/60/2008 (Victoria) could not enter MDCK cells (cells showed blue fluorescence), while B/Florida/4/ In 2006 (Yamagata), it can enter MDCK cells (the cells show green fluorescence).
  • B/Florida/4/ In 2006 Yamagata
  • the monoclonal antibody 12G6 was used for incubation, neither of the two subline influenza viruses tested could enter the MDCK cells (the cells exhibited blue fluorescence).
  • Monoclonal antibody 12G6 can inhibit the fusion of influenza B virus with cell membrane.
  • Virus B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria);
  • HA specific antibody monoclonal antibody 12G6
  • MDCK cells MDCK cells
  • the indicated concentration of antibody 12G6 (0 ⁇ g/ml, 5 ⁇ g/ml, 20 ⁇ g/ml or 100 ⁇ g/ml) was added to the cells and incubated at 37 ° C for 30 minutes. Then, the antibody solution was removed, and the cells were incubated in 10 mM MES and 10 mM HEPES (pH 5.5) at 37 ° C for 2 minutes (the HA protein was able to undergo allosteric conditions under acidic conditions and promote fusion of the viral envelope with the cell membrane). After washing three times with the medium (after washing, the virus/cell culture environment is restored from acidic to neutral), the virus-infected cells are followed at 37 ° C. Continue to culture for 3 hours.
  • the cells were fixed, stained with Giemsa dye, and observed for cell fusion. If the antibody is capable of inhibiting fusion of the viral envelope with the cell membrane, the syncytia resulting from membrane fusion will not be observed after staining. Conversely, if the antibody does not inhibit fusion of the viral envelope with the cell membrane, then after staining, syncytia that undergo membrane fusion will be observed.
  • Monoclonal antibody 12G6 is capable of inhibiting the release of influenza B virus from host cells.
  • Virus B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria)
  • HA specific antibody mAb 12G6, and negative control antibody (anti-HIV mAb 5G6);
  • MDCK cells MDCK cells
  • MDCK cells were seeded in 96-well plates at a density of 40,000 per well. After 4 hours, an excessive amount of influenza B virus was added to the cells to carry out cell infection. After 3 hours of infection, the virus solution was aspirated and the cells were washed 3 times with PBS to remove the uninfected virus. Add medium (no antibody control) to the cell culture plate, or the specified concentration of monoclonal antibody 12G6 (2 ⁇ g/ml, 0.2 ⁇ g/ml or 0.02 ⁇ g/ml; diluted in the medium), or the specified concentration Negative control antibody (20 ⁇ g/ml or 2 ⁇ g/ml; diluted in medium) and incubation continued for 16-18 hours at 37 °C.
  • medium no antibody control
  • the cell supernatant and the cell lysate were separately collected, and subjected to immunoblot analysis using rabbit polyclonal antiserum against the influenza B virus NP protein (used as a primary antibody) and GAM-HRP (used as a secondary antibody).
  • Monoclonal 12G6 has ADCC and CDC activity.
  • ADCC and CDC activities of mAb 12G6 were tested according to the method described by Srivastava V. et al., J Virol, 2013 May, 87(10): 5831-40 et al.
  • Virus B/Massachusetts/02/2012-like (Yamagata subfamily), and B/Brisbane/60/2008 (Victoria subfamily);
  • HA specific antibody 12G6, and negative control antibody (anti-HIV mAb 5G6);
  • MDCK cells MDCK cells, and murine NK cells
  • PKH-67 SIGMA-ALDRICH, catalog number: PKH67GL; used as a conventional cell membrane dye
  • 7-AAD eBioscience, catalog number: 00-6993-50; used as a nucleic acid dye for identifying dead cells
  • NK cells i.e., effector cells
  • mouse NK cell isolation kit NK Cell Isolation Kit II mouse, manufacturer: MACS, catalog number: 130-096-892
  • MDCK cells i.e., target cells
  • MOI multiplicity of infection
  • 100 ⁇ l of the cells were seeded in a 96-well plate at a cell concentration of 1 ⁇ 10 5 cells/mL.
  • the membrane of MDCK cells was stained with PKH-67 dye.
  • the antibody to be tested was diluted to the specified concentration (20 ⁇ g/ml, 2 ⁇ g/ml, or 0.5 ⁇ g/ml), and added to the cells of the culture plate in a volume of 50 ⁇ l/well, and then incubated at 37 ° C. minute. Subsequently, for the ADCC assay, a volume of 100 ⁇ l of effector cells was added to the cells of the culture plate according to a ratio of effector cells to target cells of 50:1; for CDC assay, 100 ⁇ l of 100-fold diluted guinea pig serum was added to the cells of the culture plate. As a complement.
  • the plate was incubated at 37 ° C for 2 hours, then the dye 7-AAD was added in a volume of 1 ⁇ l/well and incubated for 5 minutes. After the incubation, the cells were analyzed by flow cytometry and the percentage of dead target cells was calculated.
  • the above experiment was repeated in the absence of antibody, used as a background control, which represents the spontaneous lysis rate of infected target cells incubated with effector cells.
  • the above experiment was repeated with 1% Triton X-100 (instead of the antibody to be tested) as a positive control, which represents the maximum lysis rate of infected target cells incubated with effector cells.
  • the fluorescent staining status of various types of cells is as follows: live effector cells, no fluorescence; dead effector cells, 7-AAD staining (far red light); live target cells, PKH-67 staining (green light); dead Target cells, PKH-67 and 7-AAD double stained (far red and green).
  • the ADCC and CDC activities were calculated as follows:
  • ADCC activity or CDC activity (percentage of dead target cells in the test group - percentage of dead target cells in the background control) / (percentage of dead target cells in the positive control - percentage of dead target cells in the background control) * 100%
  • the antibody 12G6 has multiple functional activities: it can inhibit the entry of the virus into the host cell, inhibit the fusion of the virus with the cell membrane, inhibit the release of the virus from the host cell, and trigger antibody-dependent cell-mediated cells against the virus. Toxicity (ADCC) and complement dependent cytotoxicity (CDC) effects.
  • the antibody 12G6 can neutralize the influenza B virus by these five activities, and thereby prevent and treat the infection of the influenza B virus.

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Abstract

提供了一种抗乙型流感病毒（Flu B）的广谱单克隆抗体，产生所述抗体的细胞株，以及包含所述抗体的组合物，还提供了所述抗体用于诊断、预防和/或治疗Flu B的感染和/或由所述感染引起的疾病的用途。

Description

抗Flu B的广谱单克隆抗体及其用途 技术领域
本发明涉及免疫学领域和分子病毒学领域，特别是乙型流感病毒的诊断、预防和治疗领域。具体而言，本发明涉及抗乙型流感病毒(Influenza B Virus,简称Flu B)的广谱单克隆抗体，产生所述抗体的细胞株，以及包含所述抗体的组合物(例如，诊断剂和治疗剂)。此外，本发明还涉及所述抗体的用途。本发明的抗体可用于诊断、预防和/或治疗乙型流感病毒的感染和/或由所述感染引起的疾病(例如流感)。
背景技术
流行性感冒(Influenza)简称流感，是由流感病毒引起的呼吸道传染病，其临床特点是引起发热、乏力、全身酸痛及伴随一定程度上的呼吸道症状。流感病毒是人类健康的一大威胁，并且其持续快速的抗原性漂移使得季节性流感在人群中广泛传播。常见的人类季节性流感病毒包括季节性H1N1、季节性H3N2和乙型流感病毒。据WHO统计，季节性流感每年造成至少250,000-500,000人死亡(Peter D.C.等人,J Clin Invest.2008,118:3273-3275)。此外，流感大暴发仍是人类面临的一个重大威胁。自从流感病毒被发现以来，人类历史上共出现了五次世界范围内的流感大流行，共计造成了数千万人的死亡，其中1918年的西班牙流感大暴发造成全球约2000-5000万人死亡。20世纪暴发的其它流感大暴发(H1N1)还包括，1957年的亚洲流感(H2N2)大暴发及1968年的香港流感(H3N2)大暴发，二者均造成了严重的公共卫生威胁及人类社会大恐慌(Xu R.等人，Science.2010,328:357-360)。进入21世纪，流感大暴发仍没有停下脚步。2009年在墨西哥暴发并迅速蔓延全球的新甲流(H1N1)大流行又一次给人类社会敲响警钟。据WHO统计，截至2010年8月6日，全球200多个国家和地区报导的确诊死亡病例共计18449例(WHO Pandemic(h1n1)2009- update 112.6Aug,2010)。当流感病毒大流行结束后，流感病毒往往会演变成季节性流感继续流行，并且在流行过程中通过抗原性漂移，持续危害人类健康。此外，人类还面临着高致病性禽流感的威胁。高致病性H5N1禽流感病毒已成为2003年“非典”疫情之后，人类面临的最大传染病威胁之一。2003年至今，全球共报告600例人感染禽流感H5N1病例，其中死亡353例，死亡率接近60％。(WHO：http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html)。如此高的死亡率不禁使人们担心一旦病毒在人群中传播，将给人类社会带在致命打击。总之，由流感病毒引起的流感是人类面临的一个重大传染病。
流感病毒为正粘病毒科(Orthomyxoviridae)，流感病毒属的有包膜的单链负义RNA病毒。其基因组由8条分节段的RNA组成，编码十种以上病毒蛋白。根据病毒核蛋白(NP)和基质蛋白(M)的抗原性及其基因特性的差异，将流感病毒分为甲(A)、乙(B)、丙(C)三型(Horimoto T.等人,Nat Rev Microbiol,2005,3(8):591-600)；其中，甲型流感病毒(Influenza A Virus,简称Flu A)的宿主广泛，变异较快，能引起世界范围的大流行；乙型流感病毒(Influenza B Virus,简称Flu B)只能感染人和海豹，变异相对甲型流感病毒较慢，一般只引起局部地区的流行；丙型流感病毒(Influenza C Virus,简称Flu C)变异最慢，致病力弱，通常只能感染抵抗力较低的孕妇和小孩。
乙型流感病毒首次分离于1940年。自从20世纪80年代开始，乙型流感病毒由抗原性和基因型都有着显著区别的两种亚系(l ineages)主导流行，即，Victoria亚系和Yamagata亚系。在20世纪80年代，Victoria亚系主导流行。在20世纪90年代，Yamagata亚系主导流行。在2000年之后，这两种亚系共同流行(Jumat MR.等人，BMC Res Notes，2014，7:863)。
虽然流感疫情主要是由甲型流感病毒引发，但是乙型流感病毒也是引起流感疫情暴发的重要原因。每三个流感流行季节，就有一个是由乙型流感病毒病毒主导；并且，乙型流感病毒每年都造成大量的感 染者致病和死亡(Lin等人，Virus Res，2004，103(1-2):47-52)。虽然乙型流感病毒所导致的致死率和致病率比甲型流感病毒H3N2亚型低，但是比H1N1亚型高(Dreyfus C.等人，Science，2012，337(6100):1343-8)。已有研究表明：乙型流感病毒所引起的临床症状通常与甲型流感病毒的临床症状无异(Jumat MR.等人，BMC Res Notes，2014，7:863)，其所导致的重症率也与甲型流感病毒无显著区别(Su S.等人，Clin Infect Dis，2014，59(2):252-5)。总之，最近几年越来越多的研究表明，对乙型流感病毒的预防及治疗的相关研究具有重要的临床意义。
防止流感的第一道防线是中和性抗体。疫苗诱导的中和抗体的主要靶点是位于病毒表面的血凝素(HA)蛋白。病毒表面的HA蛋白呈三聚体结构，每个单体由HA1和HA2两个结构域组成。HA1位于三聚体的头部，呈球状结构，含有受体结合位点，是病毒感染宿主细胞的关键区域。HA1还含有重要的抗原性位点，可诱导机体产生保护性中和抗体，是疫苗设计的关键靶点(Wang T.T.等人,Nat Struct Mol Biol.2009,16:233-234)。HA2位于三聚体的基部，呈柄状结构，含有融合肽，可介导病毒包膜与宿主胞膜的融合。一些针对HA2的单抗能够通过抑制流感病毒的膜融合，从而起到中和病毒的效果(Wang T.T.等人,Nat Struct Mol Biol.2009,16:233-234)。
流感病毒具有高度变异性，其中尤其以HA变异最迅速。目前传统流感疫苗主要针对HA蛋白，容易因HA基因变异引发的病毒抗原性漂移而使流感疫苗失效。为保持流感疫苗持续有效，WHO每年都要根据上一年度流行的流感病毒株的变异监测情况，选择沿用或是确立新的流感病毒疫苗株用作下一年度流行季节的流感疫苗候选株，通过每年接种新疫苗来确保对现有流行的流感病毒株保持有效抵抗。因此，研制不受病毒变异影响的“广谱疫苗”逐渐成为新型疫苗研究的热点。流感病毒表面糖蛋白“血凝素(HA)”是研制流感病毒广谱疫苗和免疫治疗药物的最主要靶点。所谓的“广谱疫苗”应含有不同病毒变异株共有的“广谱中和表位”，可直接诱导“广谱中和抗体”以抵御不同 病毒变异株的感染。因此，寻找HA上的广谱中和表位成了流感广谱疫苗及免疫治疗药物研究的重要途径。
此外，在小鼠动物模型中，对乙型流感病毒特异的人源HA单抗已被证明能有效地治疗被流感病毒感染的实验小鼠(Yasugi M.等人，PLoS Pathog，2013，9(2))。在临床上，多克隆及单克隆抗体可有效地用于预防甲肝、乙肝、狂犬、呼吸道合胞等病毒的感染(Sawyer L.A.等人,Antiviral Res.2000,47:57-77)。在1918年西班牙流感期间亦有用人恢复期血清进行治疗的报道(Luke T.C.等人,Ann Intern Med.2006,145:599-609)。这些信息都提示，抗体可作为抗病毒治疗的替代方法和工具。
目前已有多篇文献报导了针对甲型流感病毒HA的广谱单抗及广谱表位。早在2009年，Throsby等人首次报导了一株识别HA2的广谱中和人源性单抗CR6261，其可中和所有属于Group 1(包含H1,H2,H5,H6,H8及H9等亚型)的流感病毒(Throsby M.等人,PLoS One.2009,3:e3942)。2011年，Corti D.等人应用类似技术也获得了一株针对HA2的人源性广谱中和单抗，其能够中和16种H亚型流感病毒(Corti D.等人,Science.2011,333:850-856)。Yoshida等人在2008年报导了一株针对HA1的广谱中和单抗S139/1，其能中和H1，H2，H3和H13亚型的部分流感病毒株(Yoshida R.等人,PLoS Pathog.2009,5:e1000350)。
而相对于甲型流感病毒，与乙型流感病毒相关的广谱单抗及广谱表位却鲜有文献报导。直到2012年，Dreyfus C等人才发现了三株能中和两个亚系的乙型流感病毒的HA广谱单抗，CR8033、CR8071和CR9114(Dreyfus C.等人，Science，2012，337(6100):1343-8)。2013年，Yasugi M等人发现了另外一株跨乙型流感病毒亚系的广谱中和单抗5A7(Yasugi M.等人，PLoS Pathog，2013，9(2))。
上述研究所获得的单抗的识别表位大多被定位于HA2上融合肽附近的一个保守区域(其主要功能是介导流感病毒膜融合)，而不是识别具有免疫优势性的HA1结构域。这使得这些抗体及所识别的保守表位 在预防和治疗中的应用前景增加了不确定性。有研究表明，识别HA2的中和单抗对天然病毒的中和活性比假病毒的降低了100-1000倍，原因可能与天然病毒的HA2表位不易暴露、较难接近有关(Sui J.等人,Nat Struct Mol Biol.2009,16:265-273；Cort i D.等人,J Cl in Invest.2010,120:1663-1673)。
相比于HA2，流感病毒的HA1在结构上处于三聚体的最顶部，呈球状结构，含大量的中和表位且易于接近。因此，寻找HA1上的广谱中和表位将有利于研制出高效的广谱流感疫苗及治疗性抗体。Cyrille Dreyfus等人在2012年报导了目前为止的唯一一株识别乙型流感病毒HA1结构域的广谱中和单抗CR8033，但该单抗只对乙型流感病毒的Yamagata亚系具有血凝抑制活性(Dreyfus C.等人，Science，2012，337(6100):1343-8)。因此，开发识别乙型流感病毒HA1上更广谱的中和表位的广谱中和单抗，并进行表位的精确定位研究，对乙型流感广谱治疗性抗体及广谱疫苗的研制具有重要指导意义。本领域需要开发更多的、更广谱的识别乙型流感病毒HA1的广谱单克隆抗体。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3) 组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544-546(1989))；术语“Fab片段”意指由VL、 VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如,scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423-426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Hol l iger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体12G6、7G6、11B10)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除 可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
例如，可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)免疫注射小鼠或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融合以获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。上述制备的杂交瘤细胞可以接种到合适的培养液中生长，培养液中优选含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如，对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞，在培养液中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。其中，骨髓瘤细胞首选鼠源骨髓瘤，如MOP-21或MC-11小鼠肿瘤衍生株(THE Salk Institute Cell Distribution Center,San Diego,Calif.USA)，和SP-2/0或X63-Ag8-653细胞株(American Type Culture Collection,Rockville,Md.USA)。另外也有研究报道，利用人骨髓瘤和人鼠异源骨髓瘤细胞株制备人单抗(Kozbor,J.Immunol.,133:3001(1984)；Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987)。生长杂交瘤细胞的培养液用于检 测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107:220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子***表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体 抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，“中和抗体”是指，能清除或显著降低目标病毒的毒力(例如，感染细胞的能力)的抗体或抗体片段。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“表位肽”是指，抗原上能够用作表位的肽段。在一些情况下，单独的表位肽即能够被针对所述表位的抗体特异性识别/结合。在另一些情况下，可能需要将表位肽与载体蛋白融合，以便表位肽能够被特异性抗体识别。如本文中所使用的，术语“载体蛋白”是指这样的蛋白，其可以充当表位肽的载体，即，其可以在特定位置处(例如蛋白内部，N端或C端)***表位肽，以便该表位肽能够呈现出来，从而该表位肽能够被抗体或免疫***识别。此类载体蛋白是本领域技术人员熟知的，包括例如，HPV L1蛋白(可以将表位肽***在所述蛋白的第130-131位氨基酸之间或在第426-427位氨基酸之间，参见Slupetzky,K.等Chimeric papillomavirus-like  particles expressing a foreign epitope on capsid surface loops[J].J Gen Virol,2001,82:2799-2804；Varsani,A.等Chimeric human papillomavirus type 16(HPV-16)L1particles presenting the common neutralizing epitope for the L2minor capsid protein of HPV-6and HPV-16[J].J Virol,2003,77:8386-8393)，HBV核心抗原(可以用表位肽替换所述蛋白的第79-81位氨基酸，参见Koletzki,D.,et al.HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein[J].Biol Chem,1999,380:325-333)，土拨鼠肝炎病毒核心蛋白(可以用表位肽替换所述蛋白的第79-81位氨基酸，参见Sabine
Figure PCTCN2016084496-appb-000001
Gertrud Beterams and Michael Nassal，J.Virol.1998,72(6):4997)，CRM197蛋白(可以将表位肽连接至该蛋白或其片段的N末端或C末端)。任选地，可以在表位肽与载体蛋白之间使用连接体(例如柔性或刚性连接体)，以促进二者各自的折叠。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原(例如，流感病毒血凝素蛋白)的结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO 03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的单克隆抗体(例如，单克隆抗体12G6、7G6或11B10)竞争结合流感病毒血凝素蛋白上的相同表位的抗体及其抗原结合片段(即，抗原结合部分)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分 离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“大肠杆菌表达***”是指由大肠杆菌(菌株)与载体组成的表达***，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698,ER2566,BL21(DE3),B834(DE3),BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草芽孢杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位 置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代 相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中使用的，术语“免疫原性(immunogenicity)”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫***能形成抗体或致敏T淋巴细胞的特异性免疫应答。免疫原性是抗原最重要的性质，一种抗原能否成功地诱导宿主产生免疫应答取决于三方面的因素：抗原的性质、宿主的反应性和免疫方式。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体12G6、7G6或11B10)以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，流感病毒血凝素蛋白)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株12G6、7G6或11B10时，其还指杂交瘤细胞株12G6、7G6或11B10的亚克隆和后代细胞。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“蛋白疫苗”是指，基于多肽的疫苗，其任选地还包含佐剂。疫苗中的多肽可以是通过基因工程技术获得的，也可以是通过化学合成方法获得的。如本文中所使用的，术语“核酸疫苗”是指，基于DNA或RNA(例如质粒，如表达质粒)的疫苗，其任选地还包含佐剂。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如流感病毒感染或与流感病毒感染相关的疾病)有效量是指，足以预防，阻止，或延迟疾病(例如流感病毒感染或与流感病毒感染相关的疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发 症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“Yamagata亚系”和“Yamagata亚系乙型流感病毒”是指，其血凝素蛋白的抗原性和进化关系与乙型流感病毒代表株B/Yamagata/16/1988处于同一个进化分支的乙型流感病毒亚系；其包括下列示例性毒株：B/Harbin/7/1994，B/Florida/4/2006，B/Xiamen/891/206，B/Xiamen/756/2007，B/Xiamen/1147/2008，B/Xiamen/N697/2012。术语“Yamagata亚系”和“Yamagata亚系乙型流感病毒”具有相同的含义，可互换使用。
如本文中所使用的，术语“Victoria亚系”和“Victoria亚系乙型流感病毒”是指，其血凝素蛋白的抗原性和进化关系与乙型流感病毒代表株B/Victoria/2/1987处于同一个进化分支的乙型流感病毒亚系；其包括但不限于下列示例性毒株：B/Hong Kong/330/2001，B/Malaysia/2506/2004，B/Xiamen/3043/2006，B/Xiamen/165/2007，B/Brisbane/60/2008，B/Brisbane/33/2008，B/Xiamen/1346/2008，B/Xiamen/N639/2010，B/Xiamen/N678/2012。术语“Victoria亚系”和“Victoria亚系乙型流感病毒”具有相同的含义，可互换使用。
如本文中所使用的，术语“血凝素蛋白”和“HA蛋白”是指由流感病毒基因组的片段4编码的抗原性糖蛋白，其存在于病毒膜表面，并且在细胞内质网合成，分子量为约76kD。HA蛋白可水解成为HA1多肽(分子量47kD，在本文中也称为“HA1结构域”)和HA2多肽(分子量为29kD，在本文中也称为“HA2结构域”)，二者通过二硫键相连形成具有典型的Ⅰ型膜蛋白结构的HA分子。血凝素蛋白具有免疫原性，并且抗血凝素抗体可以用于中和流感病毒。血凝素蛋白是本领域技术人员熟知的，其氨基酸序列可见于各种公共数据库，例如NCBI。HA1多肽/结构域位于HA蛋白头部，呈球状结构，其含有病毒的受体结合位点，能够与宿主细胞膜上的唾液酸受体相结合，从而介导病毒 进入细胞。HA2多肽/结构域则可以协助病毒包膜与宿主细胞膜相互融合，其在病毒进入宿主细胞的过程中具有重要作用。
如本文中所使用的，术语“血凝抑制活性”是指抗体或其抗原结合片段抑制由流感病毒HA蛋白与红细胞表面的唾液酸受体相结合而引起的凝血现象的功能活性。具有血凝抑制活性的抗体或抗原结合片段能够抑制病毒与细胞受体的结合。
如本文中所使用的，术语“中和活性”是指，抗体或其抗原结合片段与病毒上的抗原蛋白相结合，并由此降低或抑制目标病毒的毒力(例如，感染细胞的能力)的功能活性。具有中和活性的抗体或抗原结合片段能够阻止病毒感染细胞和/或子代病毒的成熟和/或子代病毒的释放。
本发明人经过大量的实验研究出人意料地发现：乙型流感病毒表面抗原血凝素蛋白HA1结构域中存在着广谱中和表位，并且识别这些表位的抗体能够跨HA亚系特异性结合Yamagata亚系和Victoria亚系的乙型流感病毒的血凝素蛋白，显示出广谱的病毒结合反应性及广谱的中和病毒的能力。因此，本发明的抗体特别适合用于诊断、预防和治疗乙型流感病毒感染或与乙型流感病毒感染相关的疾病(例如流感)。
在一个方面，本发明提供了一种单克隆抗体或其抗原结合片段，其包含，选自下列的重链可变区(VH)互补决定区(CDR)：
(1)氨基酸序列分别如SEQ ID NO:1-3所示的VHCDR1-3；
(2)氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3；和
(3)氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3；和/或，
选自下列的轻链可变区(VL)互补决定区(CDR)：
(4)氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；
(5)氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；和
(6)氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3。
在某些优选的实施方案中，所述的单克隆抗体包括选自下列的重链可变区(VH)：
(1)如SEQ ID NO:19所示的VH；
(2)如SEQ ID NO:21所示的VH；和
(3)如SEQ ID NO:23所示的VH。
在某些优选的实施方案中，所述的单克隆抗体包括选自下列的轻链可变区(VL)：
(1)如SEQ ID NO:20所示的VL；
(2)如SEQ ID NO:22所示的VL；和
(3)如SEQ ID NO:24所示的VL。
在某些优选的实施方案中，所述的单克隆抗体包含：
(1)氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；
(2)氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；或者
(3)氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3。
在某些优选的实施方案中，所述的单克隆抗体包括：
(1)如SEQ ID NO:19所示的VH和如SEQ ID NO:20所示的VL；
(2)如SEQ ID NO:21所示的VH和如SEQ ID NO:22所示的VL；或
(3)如SEQ ID NO:23所示的VH和如SEQ ID NO:24所示的VL。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、小鼠抗体、兔抗体、人源化抗体、全人抗体、嵌合抗体(例如，人鼠嵌合抗体)或双特异或多特异抗体。
在某些优选的实施方案中，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在某些优选的实施方案中，所述单克隆抗体是由杂交瘤细胞株12G6、7G6或11B10产生的单克隆抗体，所述杂交瘤细胞株12G6、7G6 和11B10均保藏于中国典型培养物保藏中心(CCTCC)，且分别具有保藏号CCTCC NO:C201527、CCTCC NO:C201435和CCTCC NO:C201432。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够特异性结合至少2个亚系的乙型流感病毒的血凝素蛋白的HA1结构域。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够特异性结合Yamagata亚系和Victoria亚系的乙型流感病毒的血凝素蛋白的HA1结构域。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段对Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒具有血凝抑制活性。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段是中和性的，能够中和Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有选自下列的一种或多种活性：(a)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)进入宿主细胞；(b)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)从宿主细胞中释放；(c)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)与宿主细胞的膜融合；(d)触发针对至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)的ADCC作用；和(e)触发针对至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)的CDC作用。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有上述活性中的至少1种、至少2种、至少3种、至少4种、或5种。某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有所述的全部5种活性。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段通过所述的5种活性来中和乙型流感病毒，并由此预防和治疗乙型流感病毒的感染。
在另一个方面，本发明提供了一种单克隆抗体或其抗原结合片段， 其能够封闭/阻断乙型流感病毒或其血凝素蛋白与选自下列的单克隆抗体的结合的至少50％，优选至少60％，优选至少70％，优选至少80％，优选至少90％，优选至少95％或优选至少99％：
(1)由杂交瘤细胞株12G6产生的单克隆抗体，所述杂交瘤细胞株12G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201527；
(2)由杂交瘤细胞株7G6产生的单克隆抗体，所述杂交瘤细胞株7G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201435；和
(3)由杂交瘤细胞株11B10产生的单克隆抗体，所述杂交瘤细胞株11B10保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201432。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够特异性结合至少2个亚系的乙型流感病毒的血凝素蛋白的HA1结构域。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够特异性结合Yamagata亚系和Victoria亚系的乙型流感病毒的血凝素蛋白的HA1结构域。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段对Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒具有血凝抑制活性。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段是中和性的，能够中和Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有选自下列的一种或多种活性：(a)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)进入宿主细胞；(b)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)从宿主细胞中释放；(c)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)与宿主细胞的膜融合；(d)触发针对至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)的ADCC 作用；和(e)触发针对至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)的CDC作用。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有上述活性中的至少1种、至少2种、至少3种、至少4种、或5种。某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有所述的全部5种活性。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段通过所述的5种活性来中和乙型流感病毒，并由此预防和治疗乙型流感病毒的感染。
此类单抗所识别的表位与单抗12G6、7G6或11B10识别的表位相同，或者在空间上存在重叠，从而此类单抗能够降低单抗12G6、7G6或11B10与血凝素蛋白的HA1结构域的结合至少50％，优选至少60％，优选至少70％，优选至少80％，优选至少90％，优选至少95％，或优选至少99％。
可以采用常规方法如Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,Ed Harlow and David Lane(1988)中描述的方法，测定某一待测单抗降低某一已知单抗结合抗原(例如，乙型流感病毒的血凝素蛋白)的能力。一个示例性的方法包括：先把抗原预包被在微孔板上，然后把系列稀释的未标记的待测抗体以及特定浓度的经标记的已知单抗共同加入上述预包被后的微孔板中进行孵育，然后在洗涤后测定在不同稀释度的待测抗体下，已知抗体结合到板上的数量。待测抗体竞争已知抗体结合抗原的能力越强，已知抗体结合抗原的能力就越弱，结合到板上的已知抗体就越少。通常，将抗原预包被在96孔微孔板上，并利用放射标记法或酶标记法测定待测单抗阻断经标记的已知单抗的能力。
可通过本领域已知的方法，利用本发明的单克隆抗体来生产抗-独特型抗体(Schulman JL.等人，Monographs in allergy，1986，22:143-9)。抗-独特型抗体是特异性地识别/结合用于制备它们的抗体的独特型的抗体(即，以用于制备它们的抗体的可变区的独特型作为 抗原表位)，其能够模拟/重建用于制备它们的抗体所识别的抗原表位。本发明的单克隆抗体也可用于制备此类抗-独特型抗体，并且，由此所获得的抗-独特型抗体也包括在本发明的范围内。因此，在一个方面，本发明还提供了一种抗独特型抗体，其特异性地针对本发明的单克隆抗体的独特型。
本发明还提供了分离的核酸分子，其编码本发明的单克隆抗体或其抗原结合片段。此类核酸分子可以从杂交瘤细胞中分离得到，也可以利用基因工程重组技术或化学合成方法获得。
因此，在另一个方面，本发明提供了分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中所述抗体重链可变区包含：
(1)氨基酸序列为SEQ ID NO:1-3的VH CDR1-3；
(2)氨基酸序列为SEQ ID NO:7-9的VH CDR1-3；或
(3)氨基酸序列为SEQ ID NO:13-15的VH CDR1-3。
在某些优选的实施方案中，所述抗体重链可变区具有如SEQ ID NO:19，SEQ ID NO:21或SEQ ID NO:23所示的氨基酸序列。
在某些优选的实施方案中，所述核酸分子具有如SEQ ID NO:25，SEQ ID NO:27或SEQ ID NO:29所示的核苷酸序列。
在另一个方面，本发明提供了分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中所述抗体轻链可变区包含：
(1)氨基酸序列为SEQ ID NO:4-6的VL CDR1-3；
(2)氨基酸序列为SEQ ID NO:10-12的VL CDR1-3；或
(3)氨基酸序列为SEQ ID NO:16-18的VL CDR1-3。
在某些优选的实施方案中，所述抗体轻链可变区具有如SEQ ID NO:20，SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列；
在某些优选的实施方案中，所述核酸分子具有如SEQ ID NO:26，SEQ ID NO:28或SEQ ID NO:30所示的核苷酸序列。
在另一个方面，本发明提供了分离的核酸分子，其包含如上文所定义的能够编码抗体重链可变区的核酸序列，以及如上文所定义的能 够编码抗体轻链可变区的核酸序列。
在另一个方面，本发明提供了分离的核酸分子，其编码如上文所定义的本发明的单克隆抗体或其抗原结合片段。
在另一个方面，本发明提供了一种载体，其包含如上文所定义的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。
在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，还提供了包含本发明的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
在另一个方面，还提供了制备本发明的单克隆抗体或其抗原结合片段的方法，其包括，在合适的条件下培养本发明的宿主细胞，和从细胞培养物中回收本发明的单克隆抗体或其抗原结合片段。
在另一方面，本发明提供了选自下列的杂交瘤细胞株：
1)杂交瘤细胞株12G6，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201527；
2)杂交瘤细胞株7G6，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201435；和
3)杂交瘤细胞株11B10，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201432。
如本申请所证实的，单克隆抗体12G6的重链可变区的氨基酸序列如SEQ ID NO:19所示(其示例性核苷酸序列如SEQ ID NO:25所示)，且轻链可变区的氨基酸序列如SEQ ID NO:20所示(其示例性核苷酸序列如SEQ ID NO:26所示)。
单克隆抗体12G6重链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO:1-3；轻链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ  ID NO:4-6。
如本申请所证实的，单克隆抗体7G6的重链可变区的氨基酸序列如SEQ ID NO:21所示(其示例性核苷酸序列如SEQ ID NO:27所示)，且轻链可变区的氨基酸序列如SEQ ID NO:22所示(其示例性核苷酸序列如SEQ ID NO:28所示)。
单克隆抗体7G6重链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO:7-9；轻链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO:10-12。
如本申请所证实的，单克隆抗体11B10的重链可变区的氨基酸序列如SEQ ID NO:23所示(其示例性核苷酸序列如SEQ ID NO:29所示)，且轻链可变区的氨基酸序列如SEQ ID NO:24所示(其示例性核苷酸序列如SEQ ID NO:30所示)。
单克隆抗体11B10重链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO:13-15；轻链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO:16-18。
在另一个方面，本发明提供了一种组合物，其包含如上文所描述的单克隆抗体或其抗原结合片段、抗独特型抗体、分离的核酸分子、载体或宿主细胞。
在另一个方面，本发明提供了一种试剂盒，其包括本发明的单克隆抗体或其抗原结合片段。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在某些优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的单克隆抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。
在另一个方面，本发明提供了检测乙型流感病毒或其血凝素蛋白 在样品中的存在或其水平的方法，其包括，使用本发明的单克隆抗体或其抗原结合片段。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆抗体或其抗原结合片段。所述方法可以用于诊断目的(例如，所述样品是来自患者的样品)，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。在某些优选的实施方案中，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
在另一个方面，本发明提供了诊断受试者是否感染了乙型流感病毒的方法，其包括：使用本发明的单克隆抗体或其抗原结合片段检测乙型流感病毒或其血凝素蛋白在来自所述受试者的样品中的存在。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆抗体或其抗原结合片段。优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测乙型流感病毒或其血凝素蛋白在样品中的存在或其水平，或用于诊断受试者是否感染了乙型流感病毒。优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
在某些优选的实施方案中，所述样品包括但不限于来自受试者(例如哺乳动物，优选人)的***物、口腔和鼻腔分泌物等。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:19所示的VH和/或如SEQ ID NO:20所示的VL；更优选地，其是单抗12G6。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包 括：氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:21所示的VH和/或如SEQ ID NO:22所示的VL；更优选地，其是单抗7G6。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:23所示的VH和/或如SEQ ID NO:24所示的VL；更优选地，其是单抗11B10。
使用抗体或其抗原结合片段来检测目标病毒或抗原(例如，乙型流感病毒或其血凝素蛋白)在样品中的存在或其水平的一般方法是本领域技术人员所熟知的。在某些优选的实施方案中，所述检测方法可以使用酶联免疫吸附(ELISA)、酶免疫检测、化学发光免疫检测、放射免疫检测、荧光免疫检测、免疫色谱法、竞争法及类似检测方法。
在某些优选的实施方案中，所述检测方法可包括以下几个步骤：(i)在允许抗体或其抗原结合片段与目标病毒或抗原(即，乙型流感病毒或其血凝素蛋白)结合形成抗体或其抗原结合片段-乙型流感病毒或其血凝素蛋白复合物的条件下，将本发明的单克隆抗体或其抗原结合片段与待测样品接触；(ii)检测所述复合物的存在，以确定样品中是否含有乙型流感病毒或其血凝素蛋白。
在某些优选的实施方案中，所述检测方法可包括以下几个步骤：(i)将第一抗体吸附到固相支持物上；(ii)在上述支持物中加入可能含有乙型流感病毒或其血凝素蛋白的可疑待测样品；(iii)在上述支持物中加入带有标记物的第二抗体；(iv)检测该标记物的存在从而判定乙型流感病毒或其血凝素蛋白是否存在于所述样品中。
此外，通过利用竞争法或夹心法的原理，上述检测方法可以用于检测目标抗原或抗体。
竞争法用于比较样品中抗原和一种已知量的标记抗原竞争结合本 发明所述单克隆抗体的数量关系。基于竞争法的免疫学检测通常包括，将含有未知数量的目标抗原的样品和预先定量的经标记的目标抗原加入到事先用已知的物理或化学方法把本发明所述单抗包被到固相支持物上；然后，在孵育一段时间后，冲洗所述支持物，并检测结合到所述支持物上的标记物的量或水平。
在夹心法中，样品中的目标抗原被夹在包被单抗和标记单抗之间，然后通过检测标记单抗上的标记物的量或水平，可检测并定量抗原的存在。例如，基于夹心法的免疫学检测可包括，将含有一种未知数量目标抗原的样品加入到用物理或化学方法预先包被了本发明所述单克隆抗体的固相支持物上进行反应；然后，加入经标记的本发明所述单克隆抗体进行反应；在孵育一段时间后，冲洗所述支持物，并检测结合到所述支持物上的标记物的量或水平。
标记物可以是放射性同位素、酶、酶的底物、发光物质如异鲁米诺和吖啶酯、荧光物质如荧光素和罗丹明、有色物质如乳胶颗粒和胶体金等。例如，标记用的酶包括但不限于，过氧化物酶(如辣根过氧化物酶HRP)、碱性磷酸酶、β半乳糖苷酶、乙酰胆碱酯酶和葡萄糖氧化酶。合适的酶底物包括例如，2,2'-连氮基-双(3-乙基苯并噻吡咯啉-6磺酸)、鲁米诺-过氧化氢、邻苯二胺-过氧化氢(针对过氧化物酶)、对硝基苯磷酸盐、4-甲基磷酸伞型酮、3-(2'-螺旋金刚烷)-4-甲氧基-4-(3"-磷酰基)苯基-1,2-二乙氧基烷(针对碱性磷酸酶)、对硝基苯-β-D-半乳糖和甲基伞形酮-β-D-半乳糖(针对β半乳糖苷酶)。标记用的荧光物质包括但不限于，荧光素、异硫氰酸荧光素、罗丹明、四甲基罗丹明、伊红、绿色荧光蛋白、藻红蛋白、香豆素、甲基香豆素、芘、孔雀绿、二苯乙烯、荧光黄、Cascade蓝、二氯三嗪基荧光素、丹磺酰氯、藻红蛋白、荧光镧系络合物、Cy3、Cy5等等。放射性同位素包括但不限于，14C、123I、124I、131I、35S或3H。
其它的标记物包括但不限于，量子点标记、生色团标记、亲和配体标记、电磁自旋标记、重原子标记、表位标记(如FLAG或HA表位)、以及能够形成配合物的结合对(例如，链霉抗生物素蛋白/生物素、抗 生物素蛋白/生物素或抗原/抗体配合物(如兔IgG和抗-兔IgG))。
将标记物结合到抗原或抗体上的方法是本领域已知的，包括但不限于，顺丁烯二酰亚胺法(J.Biochem.(1976),79,233)、生物素活化法(J.Am.Chem.Soc.(1978),100,3585)、疏水结合法、酯活化法或异氰酸酯法("Enzyme免疫测定techniques",published in 1987 by Igaku Shoin)。
在另一个方面，本发明提供了一种药物组合物，其包含本发明的单克隆抗体或其抗原结合片段，或者抗独特型抗体，以及药学上可接受的载体和/或赋形剂。在某些优选的实施方案中，所述单克隆抗体选自下列：
(1)单克隆抗体，其包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:19所示的VH和/或如SEQ ID NO:20所示的VL；更优选地，其是由杂交瘤细胞株12G6产生的单克隆抗体，所述杂交瘤细胞株12G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201527；
(2)单克隆抗体，其包括：氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:21所示的VH和/或如SEQ ID NO:22所示的VL；更优选地，其是由杂交瘤细胞株7G6产生的单克隆抗体，所述杂交瘤细胞株7G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201435；或者
(3)单克隆抗体，其包括：氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:23所示的VH和/或如SEQ ID NO:24所示的VL；更优选地，其是由杂交瘤细胞株11B10产生的单克隆抗体，所述杂交瘤细胞株11B10保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201432。
在某些优选的实施方案中，所述药物组合物还包含其他药学活性剂(例如抗流感病毒药物，例如M2蛋白离子通道抑制剂(例如，金刚烷胺和金刚乙胺)和神经氨酸酶抑制剂(例如，奥司他韦))。
在另一个方面，本发明提供了用于中和样品中乙型流感病毒的毒力的方法，其包括，将包含乙型流感病毒的样品与本发明的单克隆抗体或其抗原结合片段接触。此类方法可以用于治疗目的，或非治疗目的(例如所述样品是细胞样品，而不是患者或来自患者的样品)。优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段用于制备药物的用途，所述药物用于中和样品中乙型流感病毒的毒力。在另一个方面，本发明提供了如上文所描述的单克隆抗体或其抗原结合片段，其用于中和样品中乙型流感病毒的毒力。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段或者抗独特型抗体在制备药物组合物中的用途，所述药物组合物用于预防或治疗受试者的乙型流感病毒感染或与乙型流感病毒感染相关的疾病(例如流感)。在另一个方面，本发明提供了如上文所描述的单克隆抗体或其抗原结合片段或者抗独特型抗体，其用于预防或治疗受试者的乙型流感病毒感染或与乙型流感病毒感染相关的疾病(例如流感)。
在另一个方面，本发明提供了用于预防或治疗受试者的乙型流感病毒感染或乙型流感病毒感染相关的疾病(例如流感)的方法，其包括，给有此需要的受试者施用预防或治疗有效量的本发明的单克隆抗体或其抗原结合片段或者抗独特型抗体，或者本发明的药物组合物。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
可通过任何适当的施用途径来将本发明的单克隆抗体或其抗原结合片段或者抗独特型抗体或者本发明的药物组合物施用给受试者。此类施用途径包括但不限于，口服、口腔、舌下、局部、肠胃外、直肠、 叶鞘内、或鼻腔途径。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:19所示的VH和/或如SEQ ID NO:20所示的VL；更优选地，其是单抗12G6。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:21所示的VH和/或如SEQ ID NO:22所示的VL；更优选地，其是单抗7G6。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:23所示的VH和/或如SEQ ID NO:24所示的VL；更优选地，其是单抗11B10。
本发明所提供的药物和药物组合物可以单独使用或联合使用，也可以与其他药学活性剂(例如抗流感病毒药物，例如M2蛋白离子通道抑制剂(例如，金刚烷胺和金刚乙胺)和神经氨酸酶抑制剂(例如，奥司他韦))联合使用。
发明的有益效果
与现有技术相比，本发明的单克隆抗体及其抗原结合片段具有显著的有利方面。特别地，本发明的单克隆抗体及其抗原结合片段能够特异性结合至少两个亚系(例如Yamagata亚系和Victoria亚系)的乙型流感病毒的血凝素蛋白，显示出广谱的病毒结合反应性及广谱的中和病毒的能力，从而对于预防或治疗受试者的乙型流感病毒感染或与乙型流感病毒感染相关的疾病(例如流感)具有特别显著的优势。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了单克隆抗体12G6(图1A)、7G6(图1B)、11B10(图1C)所识别的表位中关键氨基酸位点的三维结构。在图1A中，HA的受体结合位点标记为青色，单抗12G6所识别的氨基酸位点标记为红色。结果显示，单抗12G6识别的关键表位氨基酸位于HA的第156位、第176位和第183位。在图1B中，HA的受体结合位点标记为青色，单抗7G6所识别的氨基酸位点标记为红色。结果显示，单抗7G6识别的关键表位氨基酸位于HA的第156位、第165位和第180位。在图1C中，HA的受体结合位点标记为青色，单抗11B10所识别的氨基酸位点标记为红色。结果显示，单抗11B10识别的关键表位氨基酸位于HA的第180位。
图2显示了使用单抗7G6和11B10对流感病毒B/Floria/04/2006(Yamagata)(图2A)、B/Malaysia/2506/2004(Victoria)(图2B)、A/Brisbane/20/2007(H3N2)(图2C)和A/NewCalidonia/20/1999(H1N1)(图2D)进行ELISA检测的结果。其中，横坐标为单抗稀释倍数，纵坐标为ELISA检测结果(OD450)。结果显示，单抗7G6和11B10对两个亚系的乙型流感病毒B/Floria/04/2006(Yamagata)和B/Malaysia/2506/2004(Victoria)均有强的结合反应性，而对甲型流感病毒A/Brisbane/20/2007(H3N2)和A/NewCalidonia/20/1999(H1N1)无特异反应性。
图3显示了单抗12G6对B/Florida/04/2006(FL04-MA)和B/Brisbane/60/2008(BR60-MA)乙型流感病毒感染的小鼠的保护效果。
图3A和图3B分别显示阴性对照组(PBS-NC)、B/Florida/04/2006病毒感染对照组(Flu B Viral cont)和治疗组(12G6-10mg/kg)小鼠的 存活率和体重变化。图3C和图3D分别显示阴性对照组(PBS-NC)、B/Brisbane/60/2008病毒感染对照组(Flu B Viral cont)和治疗组(12G6-10mg/kg)小鼠的存活率和体重变化。
结果显示，阴性对照组小鼠在整个实验过程中未见明显体重波动，两个病毒感染对照组小鼠均出现明显体重下降。B/Florida/04/2006病毒对照组小鼠在感染后8天全部死亡，B/Brisbane/60/2008病毒对照组小鼠在病毒感染后10天全部死亡。对于这两种乙型流感病毒，10mg/kg剂量的12G6抗体注射干预都能够使感染小鼠的体重得到恢复，并能够使感染小鼠正常存活14天，治疗效果达100％。
图4显示了单抗7G6对B/Florida/04/2006(FL04-MA)和B/Brisbane/60/2008(BR60-MA)乙型流感病毒感染的小鼠的保护效果。
图4A和图4B分别显示阴性对照组(PBS-NC)、B/Florida/04/2006病毒感染对照组(Flu B Viral cont)和治疗组(7G6-10mg/kg)小鼠的存活率和体重变化。图4C和图4D分别显示阴性对照组(PBS-NC)、B/Brisbane/60/2008病毒感染对照组(Flu B Viral cont)和治疗组(7G6-10mg/kg)小鼠的存活率和体重变化。
结果显示，阴性对照组小鼠在整个实验过程中未见明显体重波动，两个病毒感染对照组小鼠均出现明显体重下降。B/Florida/04/2006病毒对照组小鼠在感染后8天全部死亡，B/Brisbane/60/2008病毒对照组小鼠在病毒感染后5天全部死亡。对于这两种乙型流感病毒，10mg/kg剂量的7G6抗体注射干预都能够使感染小鼠的体重得到恢复，并能够使感染小鼠正常存活14天，治疗效果达100％。
图5显示了单抗11B10对B/Florida/04/2006(FL04-MA)和B/Brisbane/60/2008(BR60-MA)乙型流感病毒感染的小鼠的保护效果。
图5A和图5B分别显示阴性对照组(PBS-NC)、B/Florida/04/2006病毒感染对照组(Flu B Viral cont)和治疗组(11B10-10mg/kg)小鼠的存活率和体重变化。图5C和图5D分别显示阴性对照组(PBS-NC)、B/Brisbane/60/2008病毒感染对照组(Flu B Viral cont)和治疗组(11B10-10mg/kg)小鼠的存活率和体重变化。
结果显示，阴性对照组小鼠在整个实验过程中未见明显体重波动，两个病毒感染对照组小鼠均出现明显体重下降。B/Florida/04/2006病毒对照组小鼠在感染后8天全部死亡，B/Brisbane/60/2008病毒对照组小鼠在病毒感染后8天全部死亡。对于这两种乙型流感病毒，10mg/kg剂量的11B10抗体注射干预都能够使感染小鼠的体重得到恢复，并能够使感染小鼠正常存活14天，治疗效果达100％。
图6显示了用乙型流感病毒B/Floria/04/2006(Yamagata)或B/Brisbane/60/2008(Victoria)感染的MDCK细胞的免疫荧光分析结果，其中，所述乙型流感病毒在感染MDCK细胞前分别用单抗12G6、抗B/Florida/4/2006病毒的多克隆抗血清(B/FL.抗血清)、抗B/Maylaysia/2506/2004病毒的多克隆抗血清(B/Mal.抗血清)或PBS(无抗体对照)进行了孵育。
图7显示了用吉姆萨染料对乙型流感病毒B/Florida/4/2006(Yamagata)或B/Brisbane/60/2008(Victoria)感染的MDCK细胞进行染色的结果，其中，所述MDCK细胞在病毒感染后分别用0μg/ml，5μg/ml，20μg/ml或100μg/ml的抗体12G6进行了孵育。
图8显示了用于检测MDCK细胞的培养上清液和细胞裂解液中的NP蛋白的免疫印迹分析结果，其中，所述MDCK细胞用乙型流感病毒B/Florida/4/2006(Yamagata)或B/Brisbane/60/2008(Victoria)进行感染，并且在感染后，用培养基(无抗体对照)或指定浓度的单抗12G6(2μg/ml，0.2μg/ml或0.02μg/ml；稀释于培养基中)或指定浓度的阴性对照抗体(20μg/ml或2μg/ml；稀释于培养基中)进行了孵育。
图9显示了单抗12G6和阴性对照抗体触发的针对流感病毒Massachusetts/02/2012-like(Yamagata)和B/Brisbane/60/2008(Victoria)的ADCC作用(图9A)和CDC作用(图9B)的分析结果；其中，Ctr：阴性对照抗体(anti-HIV mAb 5G6)。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1
Figure PCTCN2016084496-appb-000002
Figure PCTCN2016084496-appb-000003
Figure PCTCN2016084496-appb-000004
Figure PCTCN2016084496-appb-000005
关于生物材料保藏的说明
本发明涉及下列已在中国典型培养物保藏中心(CCTCC，中国，武汉，武汉大学)进行保藏的生物材料：
杂交瘤细胞株12G6，保藏号为CCTCC NO:C201527，保藏时间为2015年4月10日；
杂交瘤细胞株7G6，保藏号为CCTCC NO:C201435，保藏时间为2014年3月26日；和
杂交瘤细胞株11B10，保藏号为CCTCC NO:C201432，保藏时间为2014年3月26日。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗乙型流感病毒HA蛋白的单克隆抗体的制备
1.病毒抗原的制备
分别以四个乙型流感病毒株B/Xiamen/891/2006(Yamagata)、B/Xiamen/1346/2008(Victoria)、B/Xiamen/N697/2012(Yamagata)、B/Xiamen/3043/2006(Victoria)接种MDCK细胞，37℃孵育2天后，收集上清，得到扩增后的病毒。收集活病毒，在4℃下以0.03％福马林溶液灭活。通过HA滴度测定法来确定灭活病毒的滴度。HA滴度测定法的具体步骤参照WHO操作指南。B/Xiamen/891/2006(Yamagata)、B/Xiamen/1346/2008(Victoria)、B/Xiamen/N697/2012(Yamagata)、B/Xiamen/3043/2006(Victoria)均为本实验室自行分离的乙型流感病毒株。
2.实验小鼠
6周龄SPF级雌性Balb/C小鼠由厦门大学实验动物中心提供。
3.杂交瘤的制备
使用标准的体内免疫方式和PEG融合方法获得分泌单抗的杂交瘤细胞，详细方法参见Ed Harlow et al.,“Antibodies A Laboratory Manual”,Cold Spring Harbor Laboratory 1988。简要过程如下：
3.1小鼠免疫：
将上述灭活病毒的滴度调整到128HA，然后采用序贯免疫方式进行小鼠免疫。简言之，首先将病毒B/Xiamen/891/2006(Yamagata)与弗氏完全佐剂(CFA)等体积混合并乳化，然后对小鼠进行首次免疫；免疫方案为经四肢肌肉多点注射，每只小鼠共注射400ul经乳化的病毒液。将病毒B/Xiamen/1346/2008(Victoria)、B/Xiamen/N697/2012(Yamagata)、B/Xiamen/3043/2006(Victoria)与弗氏不完全佐剂(IFA)混合并乳化，然后分别于首次免疫后第14d、28d、42d对小鼠进行加强免疫。最后，于首次免疫后第56d对小鼠进行脾脏免疫加强，免疫原为上述病毒的等体积混合液，每只小鼠注射体积100ul。免疫结束后3天，后取小鼠脾脏进行融合实验。
3.2细胞融合：
取小鼠脾脏，研磨得到脾细胞悬液，然后将其与处于对数生长期的小鼠骨髓瘤细胞SP2/0混合，并在PEG1500作用下进行细胞融合。将融合的细胞重悬于400ml融合培养基，并分装到20块96孔细胞培养板中进行培养。融合培养基为含HAT和20％FBS的RPMI1640完全筛选培养基。
3.3杂交瘤的筛选：
将融合后的细胞在96孔细胞培养板上培养10天，然后吸取细胞上清进行血凝抑制试验(HI)和ELISA检测。检测用的病毒为B/Xiamen/891/2006(Yamagata)和B/Xiamen/1346/2008(Victoria)。对于HI检测，阳性孔分泌的抗体应当能够抑制乙型流感病毒与血红细胞发生凝集；对于ELISA检测，阳性孔分泌的抗体应当能够与包被于聚苯乙烯板上的乙型流感病毒发生特异性反应。对筛选到的阳性克隆进行3次克隆化，得到能稳定分泌抗体的杂交瘤细胞株。最终，获得包括12G6、7G6、11B10在内的41株抗乙型流感病毒血凝素的杂交瘤细胞株。
3.4杂交瘤的培养：
将41株稳定的杂交瘤单抗细胞株在二氧化碳培养箱中进行扩增培养，从96孔板转移至24孔板，再转移至50ml细胞培养瓶中继续培养。然后，收集细胞培养瓶内的细胞，将其注射到小鼠腹腔内。7－10天后，从小鼠腹腔中吸取含有单抗的腹水。
4.单抗的纯化
将含有单抗的腹水用50％的硫酸铵溶液进行沉淀处理。然后将获得的沉淀溶解于PBS中，并使用Protein A柱在AKTA***中进行纯化，以获得经纯化的单抗。通过SDS-PAGE来鉴定经纯化的单抗的纯度。
实施例2：跨HA亚系识别乙型流感病毒血凝素的广谱单抗的鉴定
使用分离自不同时间、不同地区、代表不同变异类型的乙型流感病毒代表株以及用作对照的甲型流感病毒代表株，利用血凝抑制试验 方法(HI)来鉴定上述41株单抗对不同亚系的乙型流感病毒变异株的交叉反应性。HI检测法参照WHO操作指南来进行。根据单抗与各流感病毒株的反应性，鉴定出三个能同时识别Yamagata和Victoria亚系的乙型流感病毒(即，跨HA亚系识别乙型流感病毒血凝素)的广谱单抗12G6、7G6和11B10(表2)。
单抗12G6、7G6和11B10均能与Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒发生特异性反应，显示出跨HA亚系的广谱反应性。其中，单抗7G6能与早年未分亚系的乙型流感病毒、Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒发生反应。对于所有用于检测的乙型流感病毒，除了与B/Harbin/7/1994(Yamagata亚系)和B/Great Lakes/1739/1954这两株病毒株不反应外，单抗7G6能与其余所有的乙型流感病毒发生特异性反应，显示出了极广的反应谱。
单抗12G6和11B10能与部分早年未分亚系的乙型流感病毒、部分Yamagata亚系乙型流感病毒和部分Victoria亚系乙型流感病毒发生特异性反应。其中，单抗11B10对Yamagata亚系和Victoria亚系病毒的反应谱比单抗12G6更广；而单抗12G6对早年未分亚系的乙型流感病毒的反应性比11B10更广。
以上结果表明，单抗12G6、7G6和11B10虽然在与某些病毒株的反应性上略有不同，但均是能够跨HA亚系识别乙型流感病毒的特异性、广谱性单抗。
表2：单抗12G6、7G6和11B10的血凝抑制试验活性(HI滴度)
Figure PCTCN2016084496-appb-000006
Figure PCTCN2016084496-appb-000007
注：HI滴度表示，单抗能够完全抑制病毒HA活性的最大稀释倍数；其中，≤100，表示不反应。
实施例3.单抗12G6、7G6和11B10的中和活性的分析
中和活性(Neutralization titer)是评价单抗是否具有预防和治疗疾病潜能的重要指标。在本实施例中，利用微孔细胞中和实验法检测单抗12G6、7G6和11B10对各亚系的乙型流感病毒代表株的中和活性(该方法参照Hulse-Post等人,PNAS.2005,102:10682-7进行)。实验结果如表3所示。三株单抗(12G6、7G6和11B10)对早年未分亚系的乙型流感病毒、Yamagata亚系的乙型流感病毒和Victoria亚系的乙型流感病毒均具有广谱交叉中和活性。其中，单抗7G6的中和活性与HI活性基本一致；除了对B/Harbin/7/1994(Yamagata亚系)和 B/Great Lakes/1739/1954两株病毒未显示出中和活性外，单抗7G6对所有待测的乙型流感病毒都具有中和活性，显示出了较广的中和活性反应谱和极强的反应性。单抗11B10的中和活性反应谱略广于HI活性反应谱。特别地，单抗11B10能中和1962年-2012年的几乎所有乙型流感病毒(B/Harbin/7/1994除外)。单抗12G6的中和活性反应谱与HI活性反应谱有较大区别，其能中和1940-2012年间的所有乙型流感病毒，显示出了极强的并且极为广谱的中和活性。
表3：单抗12G6、7G6和11B10的中和试验活性
Figure PCTCN2016084496-appb-000008
Figure PCTCN2016084496-appb-000009
注：≤100，表示不反应。
实施例4.单抗12G6、7G6和11B10所识别的关键表位氨基酸的鉴定
在本实施例中，用单抗12G6、7G6和11B10来诱导筛选具有逃逸突变的乙型流感病毒株。对筛选到的逃逸病毒进行空斑纯化、扩增培养、基因调取、测序及逃逸突变位点结构定位，从而确定出单抗12G6、7G6和11B10识别的表位所在的区域以及识别的关键表位氨基酸。
1.材料与方法：
(1)逃逸母病毒：选择乙型流感病毒B/Singapore/3/1964和B/Xiamen/1346/2008(Victoria亚系)作为逃逸突变筛选的母病毒。
(2)单抗：纯化后的12G6、7G6和11B10
(3)逃逸筛选方法：取105TCID50的母病毒与单个单抗混匀，单抗终浓度为1mg/ml，总体积为1ml。病毒-单抗复合物在室温下孵育4小时后，用于感染预先铺于96孔板的MDCK细胞。在用病毒-单抗复合物孵育细胞2小时后，吸去复合物，加入含有50ug/ml单抗的病毒维持液，并在37℃继续培养48小时。随后，对培养上清进行血凝实验测定。测定结果呈阳性的孔中的病毒即为逃逸病毒。经3轮空斑克隆化后，获得稳定的逃逸病毒株。
(4)逃逸病毒株中的逃逸突变位点的定位：对获得的逃逸病毒株进行RT-PCR，得到病毒的结构基因。随后，对所述结构基因进行测序，并与逃逸母病毒的结构基因的序列进行比对，从而鉴定出逃逸突变及突变位点。
(5)关键表位氨基酸在HA三维结构上的定位：从PDB数据库下载A/Florida/4/2006(Yamagata)病毒HA蛋白的三维结构文件(PDB号：4FQJ)。利用三维作图软件Pymol在HA三维结构上对HA的受体结合位点及12G6、7G6、11B10所识别的关键表位氨基酸位点进行定位。
2.结果与分析
逃逸突变结果显示，逃逸突变位点均位于编码乙型流感病毒血凝素蛋白HA1结构域的基因中。
特别地，单抗12G6的逃逸突变结果(表4)显示，逃逸突变位点涉及B/Xiamen/1346/2008(Victoria亚系)的HA蛋白(SEQ ID NO:39)的第156、176及183位氨基酸残基(从信号肽开始计数，下同)。基于B/Xiamen/1346/2008(Victoria亚系)的病毒逃逸筛选获得15株逃逸病毒株，其中，4株病毒在HA的第156位氨基酸残基处发生突变(G156W)，4株病毒在HA的第176位氨基酸残基处发生突变(P176Q)，7株病毒在HA的第183位氨基酸残基处发生突变(T183K)。上述三个氨基酸位点被鉴定为单抗12G6识别的关键表位氨基酸，其在HA三维结构上的定位如图1A所示。
表4：单抗12G6诱导的逃逸病毒株的HA中的氨基酸突变位点
Figure PCTCN2016084496-appb-000010
单抗7G6的逃逸突变筛选结果(表5)显示，逃逸突变位点涉及B/Singapore/3/1964的HA蛋白(SEQ ID NO:40)的第156、165、180位氨基酸残基。基于B/Singapore/3/1964的病毒逃逸筛选获得16株逃逸病毒株，其中，6株在HA蛋白的第156位氨基酸残基处发生突变(G156W)，4株在HA蛋白的165位氨基酸残基处发生突变(K165E),6株在HA蛋白的180位氨基酸残基处发生突变(N180T)。上述三个氨基酸位点被鉴定为单抗7G6识别的关键表位氨基酸，其在HA三维结构上的定位如图1B所示。
表5：单抗7G6诱导的逃逸病毒株的HA中的氨基酸突变位点
Figure PCTCN2016084496-appb-000011
单抗11B10的逃逸突变筛选结果(表6)显示，逃逸突变位点涉及B/Xiamen/1346/2008(Victoria亚系)的HA蛋白(SEQ ID NO:39)的第180位氨基酸残基。基于B/Xiamen/1346/2008(Victoria亚系)的病毒逃逸筛选获得4株逃逸病毒株，其均在HA蛋白的第180位氨基酸残基处发生突变(N180K)。这说明，HA蛋白的第180位氨基酸是单抗11B10识别的关键表位氨基酸，其在HA三维结构上的定位如图1C所示。
表6：单抗11B10诱导的逃逸病毒株的HA中的氨基酸突变位点
Figure PCTCN2016084496-appb-000012
上述实验结果显示，单抗12G6、7G6、11B10识别的表位比较接近，它们所识别的表位均位于流感病毒血凝素蛋白的HA1结构域中，在空间位置上均较接近于受体结合位点(RBS)。
实施例5.单抗12G6、7G6和11B10的轻链基因和重链基因的分离与序列分析
半贴壁培养约107个杂交瘤细胞。吹起贴壁细胞使其悬浮，并转移到新的4ml离心管中。以1500rpm离心3min，收集细胞沉淀。随后，将细胞沉淀重悬于100ul无菌PBS(pH＝7.45)中，并转移到一新的1.5ml离心管中。加入800ul Trizol(Roche,Germany)，轻轻颠倒混匀，静置10min。加入200ul氯仿，剧烈振荡15s，静置10min。随后在4℃以12000rpm离心15min。将上层液体转移至一新的1.5ml离心管中，加入 等体积的异丙醇，混匀，静置10min。随后在4℃以12000rpm离心10min，弃上清，加入600ul 75％乙醇进行洗涤。随后在4℃以12000rpm离心5min，弃上清。沉淀于60℃、真空下干燥5min。将透明的沉淀溶于70ul DEPC H2O中，分装成两管。每管加入1ul反转录引物，其中一管加入的反转录引物为MVJkR(5'-CCGTTTGKATYTCCAGCT TGGTSCC-3')(SEQ ID NO:31)，用于扩增轻链可变区基因；另一管加入的反转录引物为MVDJhR(5'-CGGTGACCGWGGTBCCTTGRCCCCA-3')(SEQ ID NO:32)，用于扩增重链可变区基因。每管再加入1ul dNTP(上海生工)，置于72℃水浴10min，随后立即放到冰浴中放置5min。加入10ul 5x反转录缓冲液，1ul AMV(10u/ul,Pormega)，1ul Rnas in(40u/ul,Promega)，混匀后于42℃将RNA反转录成cDNA。
采用聚合酶链式反应法，使用引物组(如表7所示)以及另外设计合成的两条下游引物MVJkR(SEQ ID NO:31)和MVDJhR(SEQ ID NO:32)(上海博亚公司合成)来分离抗体基因的可变区。MVJkR为用于扩增轻链可变区基因的下游引物，MVDJhR为用于扩增重链可变区基因的下游引物。模板为前一步骤合成的两种cDNA。PCR条件为：94℃ 5min，35个循环的(94℃ 40s，53℃ 1min，72℃ 50s)，72℃ 15min。回收目的片段并克隆至pMD 18-T载体中，送至上海博亚公司测序。通过对测序结果进行blast比对，确定出编码抗体可变区的基因序列，并确定出相应的氨基酸序列。
依上述方法，从杂交瘤细胞株12G6、7G6、11B10中克隆出抗体的可变区基因，并参照Kabat方法(Kabat EA,Wu TT,Perry HM,Gottesman KS,Coeller K.Sequences of proteins of immunological interest,U.S Department of Health and Human Services,PHS,NIH,Bethesda,1991)确定出单抗的CDR区(complementary determinant region，互补决定区)的氨基酸序列。结果如表8a-8b所示。
表7：扩增单抗12G6、7G6、11B10的可变区基因的上游引物序列
Figure PCTCN2016084496-appb-000013
表8a：单抗12G6、7G6、11B10的CDR的氨基酸序列
Figure PCTCN2016084496-appb-000014
Figure PCTCN2016084496-appb-000015
表8b：单抗12G6、7G6、11B10的可变区的氨基酸和核苷酸序列
Figure PCTCN2016084496-appb-000016
Figure PCTCN2016084496-appb-000017
实施例6.单抗7G6和11B10用于检测乙型流感病毒的应用
1.材料与方法
(1)流感病毒的制备：取一定量的以下流感病毒株B/Floria/04/2006(Yamagata)、B/Malaysia/2506/2004(Victoria)、A/Brisbane/20/2007(H3N2)、A/NewCalidonia/20/1999(H1N1)，在4℃ 下用0.03％***液灭活。将灭活病毒在超速离心机上进行蔗糖密度梯度离心，在4℃下以25200rpm离心3小时。病毒沉淀物用1×PBS在4℃下溶解过夜。经超速离心的病毒用HA滴度测定法进行检测，以确定病毒液的滴度。
(2)单抗：如上制备的单抗7G6和11B10；单抗浓度均为1mg/ml。
(3)ELISA实验：
将经超速离心的病毒的滴度调整到128HA，以200ul每孔预包被于96孔聚苯乙烯酶标板上。随后，用封闭液对96孔板进行封闭。将待测单抗稀释至0.1mg/ml作为初始浓度，再进行15次两倍梯度稀释。将稀释后的单抗按照每孔100ul的体积加入上述酶标板，并在37℃孵育30分钟。用ELISA洗涤液(PBST)洗涤酶标板5次，随后加入100ul经稀释的HRP标记的二抗，并在37℃孵育30分钟。用PBST洗涤酶标板5次后，加入显色剂，显色20min。随后，在酶标仪上读取A450的吸收值。
2.结果与分析
ELISA结果示于图2中。如图2所示，单抗7G6和11B10对Yamagata和Victoria两个HA亚系的乙型流感病毒(B/Floria/04/2006(Yamagata)和B/Malaysia/2506/2004(Victoria))均有强的结合反应性，而对甲型流感病毒代表株(例如A/Brisbane/20/2007(H3N2)和A/NewCalidonia/20/1999(H1N1))无特异反应性。
上述结果表明，广谱单抗7G6及11B10可用于至少两个HA亚系的乙型流感病毒的特异性检测。
实施例7.单抗12G6、7G6、11B10用于治疗流感病毒感染的应用
利用抗体被动免疫来治疗传染病是一种潜在有效的抗病毒治疗途径。之前的实施例已通过体外微孔中和试验证实：本发明的单抗对不同分离地和分离时间的至少两种HA亚系(例如Yamagata和Victoria)的乙型流感病毒株均有很强的中和活性。本发明单抗的特点是，对至少两种HA亚系(例如Yamagata和Victoria)的乙型流感病毒的中和反 应谱广、中和滴度高。为进一步验证本发明单抗的体内抗流感病毒效果，本发明人基于乙型流感病毒的Yamagata亚系和Victoria亚系的病毒感染动物模型，在生物安全实验室内验证了本发明单抗对Yamagata亚系和Victoria亚系乙型流感病毒感染的Balb/C小鼠的体内抗病毒治疗效果。具体方案如下：
(1)材料与方法
动物：Balb/C小鼠，SPF，6-8周龄，雌性，体重约20g。
单抗：12G6、7G6、11B10
乙型流感病毒的小鼠适应株：
Yamagata亚系乙型流感病毒的小鼠适应株：B/Florida/04/2006，简称FL04-MA；
Victoria亚系乙型流感病毒的小鼠适应株：B/Brisbane/60/2008，简称BR60-MA。
麻醉剂:Isoflorane(异弗烷)
动物分组：提前一天将小鼠送入生物安全实验室，按5只一笼分组，标记成G1、G2、……，并记录下每只小鼠的体重，详细方案如表9所示。
病毒感染：把Yamagata亚系病毒B/Florida/04/2006预先稀释成105TCID50/ul，Victoria亚系病毒B/Brisbane/60/2008预先稀释成106TCID50/ul，小鼠病毒接种量为50ul/只。接种前，先用异弗烷麻醉小鼠，然后病毒经鼻腔接种感染小鼠。
单抗的干预：病毒感染后24h(dpi.1)对抗体治疗组小鼠给予一定剂量的抗体，剂量为10mg/kg，注射体积为100ul/小鼠。
观察记录：在病毒感染后1-14天，每天记录小鼠体重变化情况、存活情况和相应的行为表征情况。
表9：试验方案
Figure PCTCN2016084496-appb-000018
Figure PCTCN2016084496-appb-000019
(2)结果与分析
分别用一周致死剂量的Yamagata亚系乙型流感病毒FL04-MA及Victoria亚系乙型流感病毒BR60-MA感染小鼠。在感染后1天，经尾静脉对治疗组的小鼠注射抗体。通过测量各组小鼠的体重并计算生存率来判断单抗的治疗效果。实验结果示于图3-5中。
图3-5的实验结果显示，阴性对照组小鼠在整个实验过程中未见明显的体重波动，两种病毒的感染对照组小鼠均出现明显的体重下降。FL04-MA病毒对照组小鼠在感染后8天全部死亡；BR60-MA病毒对照组小鼠在病毒感染后5-10天全部死亡。对于这两种乙型流感病毒， 10mg/kg剂量的12G6、7G6、11B10抗体注射干预都能够使感染小鼠的体重得到恢复(图3B、3D、4B、4D、5B、5D)。另外，观察病毒感染后1-14天的小鼠存活情况。结果显示，10mg/kg剂量的12G6、7G6、11B10单抗均能使病毒感染的小鼠正常存活14天，治疗效果达100％(图3A、3C、4A、4C、5A、5C)。上述结果表明，本发明的广谱单抗能够有效预防和治疗两种HA亚系的乙型流感病毒的感染以及由其导致的疾病。
实施例8.单抗12G6的功能
1.单抗12G6能够抑制乙型流感病毒进入宿主细胞。
(1)材料与方法
病毒：B/Florida/4/2006(Yamagata)，和B/Brisbane/60/2008(Victoria)；
HA特异性抗体：单抗12G6，抗B/Florida/4/2006病毒的多克隆抗血清，和抗B/Maylaysia/2506/2004病毒的多克隆抗血清；
特异性针对乙型流感病毒NP蛋白的兔多克隆抗血清；
GAM-FITC(FITC标记的山羊抗小鼠抗体，绿色荧光)；
细胞：MDCK细胞；
将单抗12G6(10μg/ml)，抗B/Florida/4/2006病毒的多克隆抗血清，抗B/Maylaysia/2506/2004病毒的多克隆抗血清，和PBS(无抗体对照)分别与两个亚系的乙型流感病毒在37℃孵育一小时。随后，将孵育后的混合物分别添加至在96孔板内培养的单层MDCK细胞，并继续培养16-18小时。培养结束后，用培养基洗涤细胞三次。然后，使用商品化的试剂盒，对细胞进行DAPI染色(蓝色荧光)；并且，使用针对乙型流感病毒NP蛋白的兔多克隆抗血清(用作一抗)和GAM-FITC(用作二抗，绿色荧光)对细胞进行免疫荧光分析。
(2)结果与分析
实验结果如图6所示。结果显示，当使用PBS来进行孵育时，所测试的两个亚系的乙型流感病毒都能进入MDCK细胞(细胞呈现绿色荧光)。当使用抗B/Florida/4/2006病毒的多克隆抗血清来进行孵育时， B/Florida/4/2006(Yamagata)不能进入MDCK细胞(细胞呈现蓝色荧光)，而B/Brisbane/60/2008(Victoria)则能进入MDCK细胞(细胞呈现绿色荧光)。当使用抗B/Maylaysia/2506/2004病毒的多克隆抗血清来进行孵育时，B/Brisbane/60/2008(Victoria)不能进入MDCK细胞(细胞呈现蓝色荧光)，而B/Florida/4/2006(Yamagata)则能进入MDCK细胞(细胞呈现绿色荧光)。当使用单抗12G6来进行孵育时，所测试的两个亚系的乙型流感病毒都不能进入MDCK细胞(细胞呈现蓝色荧光)。这些结果表明，单抗12G6能够抑制两个亚系的乙型流感病毒进入宿主细胞。
2.单抗12G6能够抑制乙型流感病毒与细胞膜融合。
(1)材料与方法
病毒：B/Florida/4/2006(Yamagata)，和B/Brisbane/60/2008(Victoria)；
HA特异性抗体：单抗12G6
实验试剂：吉姆萨染料，10mM MES(Sigma，目录号：M3671-250G)and 10mM HEPES(Sigma，目录号：H3375，pH 5.5)，和TPCK胰蛋白酶(Sigma，目录号：T1426)；
细胞：MDCK细胞；
以MOI＝0.3的感染复数，用B/Florida/4/2006(Yamagata)和B/Brisbane/60/2008(Victoria)分别感染MDCK细胞。24小时后，细胞用培养基洗涤三次，以去除残余的病毒。随后，向细胞中加入2.5mg/ml的TPCK胰蛋白酶。在37℃孵育15分钟后，细胞用培养基洗涤三次，以去除残余的胰蛋白酶。随后，向细胞中加入指定浓度的抗体12G6(0μg/ml，5μg/ml，20μg/ml或100μg/ml)，并在37℃孵育30分钟。然后，去除抗体溶液，并将细胞在10mM MES and 10mM HEPES(pH 5.5)中、37℃下孵育2分钟(HA蛋白能够在酸性条件下发生变构，并促使病毒包膜与细胞膜融合)。在用培养基洗涤三次后(洗涤后，病毒/细胞的培养环境从酸性恢复至中性)，将感染病毒的细胞在37℃继 续培养3小时。随后，固定细胞，用吉姆萨染料进行染色，并观察细胞是否出现膜融合现象。如果抗体能够抑制病毒包膜与细胞膜融合，那么染色后，将无法观察到膜融合导致的合胞体。反之，如果抗体不能抑制病毒包膜与细胞膜融合，那么染色后，将能够观察到发生了膜融合的合胞体。
(2)结果与分析
实验结果如图7所示。结果显示，当不使用抗体12G6来孵育细胞(即，0μg/ml抗体)时，所测试的两个亚系的乙型流感病毒都能够导致MDCK细胞发生膜融合(即，出现了大量的合胞体)。当使用5μg/ml或20μg/ml的抗体12G6进行孵育时，MDCK细胞的膜融合现象得到了显著的抑制(即，合胞体的数量显著下降)，并且20μg/ml抗体的抑制作用强于5μg/ml的抗体。并且，当使用100μg/ml的抗体12G6进行孵育时，MDCK细胞的膜融合现象被完全抑制(即，无合胞体出现)。这些结果表明，单抗12G6能够抑制两个亚系的乙型流感病毒与细胞膜融合；并且，单抗12G6的该抑制活性是剂量依赖性的。
3.单抗12G6能够抑制乙型流感病毒从宿主细胞中释放。
(1)材料与方法
病毒：B/Florida/4/2006(Yamagata)，和B/Brisbane/60/2008(Victoria)
HA特异性抗体：单抗12G6，和阴性对照抗体(anti-HIV mAb 5G6)；
特异性针对乙型流感病毒NP蛋白的兔多克隆抗血清；
GAM-HRP；
细胞：MDCK细胞；
以40000个/孔的密度将MDCK细胞接种于96孔板中。4小时后，向细胞中加入过量的乙型流感病毒，进行细胞感染。感染3小时后，吸掉病毒液，并用PBS洗涤细胞3次，除去未感染的病毒。向细胞培养板中分别添加培养基(无抗体对照)，或指定浓度的单抗12G6(2μg/ml，0.2μg/ml或0.02μg/ml；稀释于培养基中)，或指定浓度 的阴性对照抗体(20μg/ml或2μg/ml；稀释于培养基中)，并在37℃继续孵育16-18小时。随后，分别收集细胞上清和细胞裂解液，并使用针对乙型流感病毒NP蛋白的兔多克隆抗血清(用作一抗)和GAM-HRP(用作二抗)进行免疫印迹分析。
(2)结果与分析
实验结果如图8所示。结果显示，当不使用抗体或者使用阴性对照抗体来孵育被乙型流感病毒感染的MDCK细胞时，在培养上清液和细胞裂解液中均能够检测到显著量的NP蛋白。这表明，两个亚系的乙型流感病毒能够在MDCK细胞中增殖，并且已释放到了细胞外；并且，阴性对照抗体不能抑制乙型流感病毒从宿主细胞中释放。相比之下，当使用单抗12G6来孵育被乙型流感病毒感染的MDCK细胞时，在细胞裂解液中能够检测到NP蛋白，但是培养上清液中的NP蛋白的量随着单抗12G6浓度的上升而显著下降。当单抗12G6的浓度达到2μg/ml时，在培养上清液中完全无法检测到NP蛋白，这表明乙型流感病毒的释放被完全抑制。这些结果表明，单抗12G6能够抑制两个亚系的乙型流感病毒从宿主细胞释放，并且，单抗12G6的该抑制活性是剂量依赖性的。
4.单抗12G6具有ADCC和CDC活性。
按照Srivastava V.等人，J Virol，2013May，87(10):5831-40等人描述的方法来测试单抗12G6的ADCC和CDC活性。
(1)材料与方法
病毒：B/Massachusetts/02/2012-like(Yamagata亚系)，和B/Brisbane/60/2008(Victoria亚系)；
HA特异性抗体：12G6，和阴性对照抗体(anti-HIV mAb 5G6)；
细胞：MDCK细胞，和鼠NK细胞；
细胞染料试剂：PKH-67(SIGMA-ALDRICH，目录号：PKH67GL；用作常规细胞膜染料)，和7-AAD(eBioscience，目录号：00-6993-50；用作核酸染料，用于鉴定死细胞)；
利用鼠NK细胞分离试剂盒(NK Cell Isolation Kit II mouse， 生产商：MACS，目录号：130-096-892)分离小鼠脾脏中的NK细胞(即，效应细胞)，备用。以MOI＝10的感染复数，用乙型流感病毒感染MDCK细胞(即，靶细胞)。3小时后，以1×105细胞/mL的细胞浓度，将100μl细胞接种于96孔板中。培养1小时后，用PKH-67染料对MDCK细胞的膜进行染色。染色后，将待测抗体分别稀释至指定的浓度(20μg/ml，2μg/ml，或0.5μg/ml)，并以50μl/孔的体积添加至培养板的细胞中，然后在37℃孵育15分钟。随后，对于ADCC测定，按照50∶1的效应细胞与靶细胞的比率，向培养板的细胞中加入100μl体积的效应细胞；对于CDC测定，向培养板的细胞中加入100μl稀释100倍的豚鼠血清作为补体。将培养板在37℃孵育2小时，随后按1μl/孔的体积加入染料7-AAD，并孵育5分钟。孵育结束后，利用流式细胞仪对细胞进行分析，计算死亡靶细胞的百分比。另外，还在不使用抗体的条件下重复上述实验，用作本底对照，其代表着与效应细胞孵育的经感染的靶细胞的自发裂解率。此外，还用1％Tri ton X-100(替代待测抗体)重复上述实验，用作阳性对照，其代表着与效应细胞孵育的经感染的靶细胞的最大裂解率。
各种类型的细胞的荧光染色状态如下：活的效应细胞，无荧光；死的效应细胞，7-AAD染色(远红光)；活的靶细胞，PKH-67染色(绿光)；死的靶细胞，PKH-67和7-AAD双染色(远红光和绿光)。如下计算ADCC和CDC活性：
ADCC活性或CDC活性＝(测试组的死亡靶细胞的百分比-本底对照的死亡靶细胞的百分比)/(阳性对照的死亡靶细胞的百分比-本底对照的死亡靶细胞的百分比)*100％
(2)结果与分析
实验结果如图9所示。结果显示，当使用阴性对照抗体进行孵育时，被病毒感染的MDCK细胞在NK细胞或豚鼠血清存在下未发生明显裂解。这表明，阴性对照抗体不能触发针对所测试的两个亚系的乙型流感病毒的ADCC和CDC作用。相比之下，当使用抗体12G6进行孵育时，被病毒感染的MDCK细胞在NK细胞或豚鼠血清的作用下发生了显 著的裂解；并且，随着所使用的抗体浓度的上升，MDCK细胞的裂解现象得以增强。这些结果表明，单抗12G6能够触发针对所测试的两个亚系的乙型流感病毒的ADCC和CDC作用；并且，单抗12G6的该活性是剂量依赖性的。
上述结果表明，抗体12G6具有多种功能活性：其能够抑制病毒进入宿主细胞，能够抑制病毒与细胞膜的融合，能够抑制病毒从宿主细胞中释放，且能够触发针对病毒的抗体依赖细胞介导的细胞毒性(ADCC)和补体依赖的细胞毒(CDC)作用。抗体12G6可通过这5种活性来中和乙型流感病毒，并由此预防和治疗乙型流感病毒的感染。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (17)

  1. 一种单克隆抗体或其抗原结合片段，其包含，选自下列的重链可变区(VH)互补决定区(CDR)：
    (1)氨基酸序列分别如SEQ ID NO:1-3所示的VHCDR1-3；
    (2)氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3；和
    (3)氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3；
    和/或，
    选自下列的轻链可变区(VL)互补决定区(CDR)：
    (1)氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；
    (2)氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；和
    (3)氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3；
    优选地，所述的单克隆抗体包括，选自下列的重链可变区(VH)：
    (1)如SEQ ID NO:19所示的VH；
    (2)如SEQ ID NO:21所示的VH；和
    (3)如SEQ ID NO:23所示的VH；
    和/或
    选自下列的轻链可变区(VL)：
    (1)如SEQ ID NO:20所示的VL；
    (2)如SEQ ID NO:22所示的VL；和
    (3)如SEQ ID NO:24所示的VL；
    优选地，所述的单克隆抗体包含：
    (1)氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；
    (2)氨基酸序列分别如SEQ ID NO:7-9所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:10-12所示的VL CDR1-3；或者
    (3)氨基酸序列分别如SEQ ID NO:13-15所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:16-18所示的VL CDR1-3；
    优选地，所述的单克隆抗体包括：
    (1)如SEQ ID NO:19所示的VH和如SEQ ID NO:20所示的VL；
    (2)如SEQ ID NO:21所示的VH和如SEQ ID NO:22所示的VL；或
    (3)如SEQ ID NO:23所示的VH和如SEQ ID NO:24所示的VL。
    优选地，所述单克隆抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、小鼠抗体、兔抗体、人源化抗体、全人抗体、嵌合抗体(例如，人鼠嵌合抗体)或双特异或多特异抗体；
    优选地，所述单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体；
    优选地，所述单克隆抗体是由杂交瘤细胞株12G6、7G6或11B10产生的单克隆抗体，所述杂交瘤细胞株12G6、7G6和11B10均保藏于中国典型培养物保藏中心(CCTCC)，且分别具有保藏号CCTCC NO:C201527、CCTCC NO:C201435和CCTCC NO:C201432；
    优选地，所述单克隆抗体或其抗原结合片段能够特异性结合至少2个亚系的乙型流感病毒的血凝素蛋白的HA1结构域；
    优选地，所述单克隆抗体或其抗原结合片段能够特异性结合Yamagata亚系和Victoria亚系的乙型流感病毒的血凝素蛋白的HA1结构域；
    优选地，所述单克隆抗体或其抗原结合片段对Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒具有血凝抑制活性；
    优选地，所述单克隆抗体或其抗原结合片段是中和性的，能够中和Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒；
    优选地，所述单克隆抗体或其抗原结合片段具有选自下列的一种或多种活性：(a)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)进入宿主细胞；(b)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)从宿主细胞中释放；(c)抑制至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)与宿主细胞的膜融合；(d)触发针对至少2个亚系的乙型流感病毒(例如 Yamagata亚系和Victoria亚系的乙型流感病毒)的ADCC作用；和(e)触发针对至少2个亚系的乙型流感病毒(例如Yamagata亚系和Victoria亚系的乙型流感病毒)的CDC作用。
  2. 一种单克隆抗体或其抗原结合片段，其能够阻断乙型流感病毒或其血凝素蛋白与选自下列的单克隆抗体的结合的至少50％，优选至少60％，优选至少70％，优选至少80％，优选至少90％，优选至少95％或优选至少99％：
    (1)由杂交瘤细胞株12G6产生的单克隆抗体，所述杂交瘤细胞株12G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201527；
    (2)由杂交瘤细胞株7G6产生的单克隆抗体，所述杂交瘤细胞株7G6保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201435；和
    (3)由杂交瘤细胞株11B10产生的单克隆抗体，所述杂交瘤细胞株11B10保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201432；
    优选地，所述单克隆抗体或其抗原结合片段能够特异性结合至少2个亚系的乙型流感病毒的血凝素蛋白的HA1结构域；
    优选地，所述单克隆抗体或其抗原结合片段能够特异性结合Yamagata亚系和Victoria亚系的乙型流感病毒的血凝素蛋白的HA1结构域；
    优选地，所述单克隆抗体或其抗原结合片段对Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒具有血凝抑制活性；
    优选地，所述单克隆抗体或其抗原结合片段是中和性的，能够中和Yamagata亚系乙型流感病毒和Victoria亚系乙型流感病毒。
  3. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中所述抗体重链可变区包含：
    (1)氨基酸序列为SEQ ID NO:1-3的VH CDR1-3；
    (2)氨基酸序列为SEQ ID NO:7-9的VH CDR1-3；或
    (3)氨基酸序列为SEQ ID NO:13-15的VH CDR1-3；
    例如，所述抗体重链可变区具有如SEQ ID NO:19，SEQ ID NO:21或SEQ ID NO:23所示的氨基酸序列；
    例如，所述核酸分子具有如SEQ ID NO:25，SEQ ID NO:27或SEQ ID NO:29所示的核苷酸序列。
  4. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中所述抗体轻链可变区包含：
    (1)氨基酸序列为SEQ ID NO:4-6的VL CDR1-3；
    (2)氨基酸序列为SEQ ID NO:10-12的VL CDR1-3；或
    (3)氨基酸序列为SEQ ID NO:16-18的VL CDR1-3；
    例如，所述抗体轻链可变区具有如SEQ ID NO:20，SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列；
    例如，所述核酸分子具有如SEQ ID NO:26，SEQ ID NO:28或SEQ ID NO:30所示的核苷酸序列。
  5. 分离的核酸分子，其编码权利要求1或2的单克隆抗体或其抗原结合片段。
  6. 一种载体，其包含权利要求3-5任一项的分离的核酸分子。
  7. 一种宿主细胞，其包含权利要求3-5任一项的分离的核酸分子或权利要求6的载体。
  8. 制备权利要求1或2的单克隆抗体或其抗原结合片段的方法，其包括，在合适的条件下培养权利要求7的宿主细胞，和从细胞培养物中回收所述单克隆抗体或其抗原结合片段。
  9. 杂交瘤细胞株，其选自：
    1)杂交瘤细胞株12G6，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201527；
    2)杂交瘤细胞株7G6，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201435；和
    3)杂交瘤细胞株11B10，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO:C201432。
  10. 一种抗独特型抗体，其特异性地针对根据权利要求1或2的单克隆抗体的独特型。
  11. 一种组合物，其包含权利要求1或2的单克隆抗体或其抗原结合片段，权利要求3-5任一项的分离的核酸分子，权利要求6的载体，权利要求7的宿主细胞，或权利要求10的抗独特型抗体。
  12. 试剂盒，其包括权利要求1或2的单克隆抗体或其抗原结合片段；
    例如，所述单克隆抗体或其抗原结合片段还包括可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质和酶；
    例如，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质和酶。
  13. 用于检测乙型流感病毒或其血凝素蛋白在样品中的存在或其水平的方法，其包括使用权利要求1或2的单克隆抗体或其抗原结合片段；
    例如，所述单克隆抗体或其抗原结合片段还包括可检测的标记，例如放射性同位素，荧光物质，化学发光物质，有色物质和酶；
    例如，所述方法还包括，使用携带可检测的标记(例如放射性同位 素，荧光物质，发光物质，有色物质和酶)的第二抗体来检测所述单克隆抗体或其抗原结合片段；
    优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
  14. 权利要求1或2的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测乙型流感病毒或其血凝素蛋白在样品中的存在或其水平，或用于诊断受试者是否感染了乙型流感病毒；
    优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒；
    优选地，所述样品为来自受试者(例如哺乳动物，优选人)的***物、口腔或鼻腔分泌物。
  15. 一种药物组合物，其包含权利要求1或2的单克隆抗体或其抗原结合片段或权利要求10的抗独特型抗体，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含其他药学活性剂，例如抗流感病毒药物，例如M2蛋白离子通道抑制剂(例如，金刚烷胺和金刚乙胺)和神经氨酸酶抑制剂(例如，奥司他韦)。
  16. 用于中和样品中乙型流感病毒的毒力的方法，其包括，将包含乙型流感病毒的样品与权利要求1或2的单克隆抗体或其抗原结合片段接触；
    优选地，所述乙型流感病毒选自Yamagata亚系和Victoria亚系乙型流感病毒。
  17. 权利要求1或2的单克隆抗体或其抗原结合片段或权利要求10的抗独特型抗体用于制备药物的用途，所述药物用于预防或治疗受试者的乙型流感病毒感染或与乙型流感病毒感染相关的疾病(例如流 感)；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述药物单独使用，或与其他药学活性剂(例如抗流感病毒药物，例如M2蛋白离子通道抑制剂(例如，金刚烷胺和金刚乙胺)和神经氨酸酶抑制剂(例如，奥司他韦))联合使用。
PCT/CN2016/084496 2015-06-03 2016-06-02 抗Flu B的广谱单克隆抗体及其用途 WO2016192652A1 (zh)

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