WO2015131836A1 - 一种h7型甲型流感病毒的中和抗体及其用途 - Google Patents

一种h7型甲型流感病毒的中和抗体及其用途 Download PDF

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WO2015131836A1
WO2015131836A1 PCT/CN2015/073698 CN2015073698W WO2015131836A1 WO 2015131836 A1 WO2015131836 A1 WO 2015131836A1 CN 2015073698 W CN2015073698 W CN 2015073698W WO 2015131836 A1 WO2015131836 A1 WO 2015131836A1
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seq
antibody
variable region
chain variable
amino acid
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PCT/CN2015/073698
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English (en)
French (fr)
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谢良志
孙春昀
张�杰
宋德勇
饶木顶
朱萍霞
李成红
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神州细胞工程有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates to the field of biological products, and in particular to a neutralizing antibody of the H7 influenza A virus and use thereof, and to a method for producing the antibody in CHO cells by genetic recombination technology.
  • the influenza virus is defined by the type of hemagglutinin (HA) as A and B.
  • the type A can be subdivided into 16 subtypes, of which group I has H1, H2, H5, H6, H8, H9, H11, H12. , H13 and H16 subtypes exist, and group II consists of H3, H4, H7, H10, H14 and H15 subtypes.
  • H1, H2 and H3 in type B and type A cause a wider range of influenza diseases in humans, and H5, H7 and H9 subtypes cause sporadic serious infections in humans and may cause new pandemics.
  • influenza A H7N9
  • the virus is reconstituted by viruses of H7 and N9 subtypes distributed in humans, and no corresponding immune response memory is established in the human body. Therefore, the virus infection can cause severe respiratory distress syndrome, leading to death of patients [Kageyama T et al. People, Euro SUrvell. 2013.18:20453].
  • the human seasonal influenza vaccine consists of three subtypes, A-H1N1, A-H3N2 and B.
  • the H7N9 virus inhibitory drug is Oseltamivir or its analog (Zanamivir), which is an inhibitor of the enzymatic activity of the influenza virus NA protein, and their effect is to avoid or reduce the expansion of the virus, thereby achieving Reduce the therapeutic effect of viral titers.
  • the drug has a certain clinical effect, but the rate of virus clearance is still relatively slow, and there have been reports of Tamiflu strains, so there is a need for more effective drugs to rapidly reduce the replication of the virus in the body [Yunwen Hu Et al., Lancet. 2013.381: 2273-79].
  • Hemagglutinin protein is the primary target of broad-spectrum and subtype-specific antibodies consisting of the HA1 and HA2 subunits comprising a trimeric protein HA0 comprising more than 500 amino acids. Its HA1 head is the site where the virus binds to the sialic acid receptor of the target cell, allowing the virus to adhere to the cell, and the pH is low. The conformational change of the HA2 region of the stem can cause the virus to fuse with the cell membrane.
  • the virus When the virus is endocytosed inside the cell, the genome of the virus is released into the cytoplasm, and the new virus can be replicated in the cell. Antibodies against HA can effectively block the process by which the virus infects cells, thereby disrupting the viral replication chain.
  • the region of HA1 that binds to the influenza receptor sialic acid is quite different, and the same subtype also evolves new variants in the HA1 region. Therefore, this site does not have broad-spectrum properties. However, it is the most effective neutralization site [Kida H et al., Vaccine. 1985, 3, 219-222].
  • the current broad-spectrum antibodies are mainly directed to the relatively conserved HA2 region.
  • the antibodies After binding to the HA2 epitope, the antibodies can block the viral infection process by steric hindrance to prevent the HA protein from being altered. Antibodies against the M2 protein have also been reported to have an in vivo protective effect. M2 is an ion channel protein on the surface of the virus. It has only 24 amino acids on the surface of the virus or cells. When the virus infects the cells, the cell surface antigen expresses a large amount of M2 protein. The anti-M2 antibody binds to the virus-replicating cells and kills the cells by the mechanism of ADCC and CDC, thereby exerting a virus suppressing effect. At present, there are many different types of anti-hemagglutinin and anti-M2 antibodies in the research and development stage, a small number of antibodies have entered clinical phase I and phase II studies, and there are currently no antibody drugs on the market.
  • Another object of the invention is to provide the use of the antibody.
  • a further object of the invention is to provide a method of preparing the antibody.
  • the neutralizing human antibody against H7N9 influenza A virus provided by the present invention is an antibody constructed from rabbit spleen and bone marrow tissues immunized with H7N7A/Netherlands/219/2003 and H7N9 (A/Anhui/1/2013) hemagglutinin protein HA.
  • the library was obtained by screening with hemagglutinin protein of H7N9 (A/Anhui/1/2013), and the binding epitope of the antibody was the region where the surface of HA1 binds to sialic acid.
  • the invention encompasses antibodies or antigen-binding fragments thereof that are neutralized by human infection with influenza A H7N9 and related H7 influenza A viruses.
  • an antibody or antigen-binding fragment thereof which neutralizes human infection with H7N9 influenza virus is provided.
  • the present invention provides an antibody or antigen-binding fragment thereof which neutralizes human infection with H7N9 influenza A virus, and has at least one CDR sequence and SEQ in a complementarity determining region (CDR) sequence ID NO: Any of 2, 8, 13, 15, 18 and 19 has at least 61% sequence homology.
  • CDR complementarity determining region
  • the light chain CDR1 thereof comprises the amino acid sequence of any one of SEQ ID NO: 1-6
  • the light chain CDR2 thereof comprises SEQ ID NO: 7-10.
  • An amino acid sequence, the light chain CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11-14, wherein The antibody is capable of neutralizing the H7N9 influenza A virus.
  • the antibody or antigen-binding fragment thereof of the neutralizing human infected H7N9 influenza A virus provided by the present invention comprises the heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, and comprises SEQ ID NO: 16-18.
  • Exemplary antibodies of the invention include rabbit-derived antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, H7N9-R002 and humanized antibodies H7N9-H002.
  • the homology of the light chain CDR1 ranges from 61% to 100%
  • the homology of the light chain CDR2 ranges from 62%.
  • the light chain CDR3 has a homology ranging from 80% to 100%.
  • the heavy chain CDR1 has a homology ranging from 100%
  • the heavy chain CDR2 has a homology ranging from 87% to 100%
  • the heavy chain CDR3 has a homology ranging from 100%.
  • the antibody neutralizes a human infected with H7N9 influenza A virus. All CDR sequence homology is based on H7N9-R002.
  • At least one CDR of an antibody or antibody fragment of the invention comprises a sequence having at least 95% sequence homology to any one of SEQ ID NOs: 1-19, wherein the antibody neutralizes human infection with H7N9 Influenza A virus.
  • amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the antibodies and antibody fragments thereof of the invention are set forth in SEQ ID NO: 28 and SEQ ID NO: 37
  • amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the antibody of interest are set forth in SEQ ID NO: 56 and SEQ ID NO: 57.
  • the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having about 80%, 85%, 90%, 95%, 97%, 98 of the sequence set forth in SEQ ID NO:28 %, 99% or 100% identical amino acid sequence.
  • the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having about 92%, 95%, 97%, 98%, 99 of the sequence set forth in any one of SEQ ID NO:37 % or 100% identical amino acid sequence.
  • the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having about 80%, 85%, 90%, 95%, 97%, 98 of the sequence set forth in SEQ ID NO:56 %, 99% or 100% identical amino acid sequence.
  • the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having about 92%, 95%, 97%, 98%, 99 of the sequence of any of SEQ ID NO: 57 % or 100% identical amino acid sequence.
  • an antibody of the invention comprises a light chain variable region having the same as any of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27 or 28.
  • Amino acid sequences of about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the sequence are identical.
  • the antibody of the invention and antibody fragments thereof comprise a heavy chain variable region having any of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36 or 37
  • the amino acid sequence of about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the sequence is the same.
  • the invention comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20 and SEQ ID NO: a heavy chain variable region of the 29 amino acid sequence; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 30;
  • the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 31; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 And a heavy chain variable region of the amino acid sequence of SEQ ID NO: 32; or the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and the amino acid sequence of SEQ ID NO: 33;
  • the antibody comprises a light chain variable region comprising the amino acid sequence set forth in S
  • H7N9 influenza A virus which is H7N9-R002 and variants thereof H7N9-R003, H7N9-R006, H7N9- R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021.
  • the invention comprises an antibody or antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO: 38 and a SEQ ID NO: 39 core a heavy chain variable region of a nucleotide sequence; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO: 40 and a heavy chain variable region of the nucleotide sequence of SEQ ID NO: 41;
  • the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO: 42 and a heavy chain variable region of SEQ ID NO: 43; or the antibody comprises a nucleotide comprising SEQ ID NO: 44 a light chain variable region of the sequence and a heavy chain variable region of the nucleotide sequence of SEQ ID NO: 45; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO: 46 and SEQ ID NO: a heavy chain variable region of
  • the present invention comprises a neutralizing human infected H7N9 influenza A virus antibody or antigen-binding fragment thereof, wherein the antibody is derived from H7N7 (A/Netherlands/219/2003) hemagglutinin A rabbit antibody library of proteins whose antibodies are humanized antibodies, which are designed by humanization of rabbit antibodies, and antibodies thereof are produced by eukaryotic expression.
  • the present invention provides a polynucleotide comprising the antibody of the present invention or an antigen-binding fragment thereof Nucleic acid molecule.
  • Humanized monoclonal antibodies, CHOs expressing the antibodies of the invention, 293 transfected cell lines, and nucleotide sequences encoding the antibodies of the invention are all included within the scope of the invention.
  • the antibodies of the invention can be prepared by any method known in the art.
  • the sequence of an antibody is inserted into a corresponding eukaryotic expression vector, and transfected into a cell, such as a technique of obtaining an antibody by transient expression of 293 cells, and a technique of stably expressing an antibody using CHO cells.
  • High density cell culture techniques are commonly employed to achieve high antibody yields.
  • the antibody can be purified by various methods such as centrifugation, filtration, affinity, charge, molecular weight, and hydrophobicity.
  • antibody fragments of the invention are prepared by cloning or expressing portions of the heavy or light chain sequences.
  • Antibody fragments can include scFv, Fab, Fab', F(ab') 2 and Fv fragments.
  • Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabody.
  • Any suitable host cell/vector system can be used to encode the expression of the DNA sequences of the antibody molecules and fragments thereof of the invention.
  • Suitable host systems include E. coli, yeast cells, insect cells, and mammalian host cells.
  • Mammalian host cells include, but are not limited to, CHO, HEK293, PER.C6, NSO, SP20.
  • An antibody of the invention can be prepared by i) expressing a nucleic acid sequence of the invention in a suitable host cell, and ii) isolating the expressed antibody product, iii) purifying the antibody.
  • the present invention provides a vector comprising the antibody nucleic acid molecule of the present invention or a cell expressing the antibody of the present invention or an antigen-binding fragment thereof.
  • the antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, or based on the modified Any other gene containing this antibody gene, obtains an antibody product having the biological activity of neutralizing H7N9 influenza A virus, or a complex obtained by in vitro labeling or cross-linking method, and is clinically used for treatment and H7N9 type A Specific antibody drugs for influenza virus-related diseases.
  • the present invention provides an immunogenic polypeptide comprising a hemagglutinin protein and an antibody binding epitope which bind to the antibody of the present invention or an antigen-binding fragment thereof, and an isofunctional antibody.
  • the epitope is located at the head of the HA protein, and is a region where the HA protein binds to the sialic acid sugar receptor, specifically including three parts: i) the outermost ⁇ -sheet region of the head of the HA1, the ⁇ -sheet layer The region is 154-162 aa; ii) the sialic acid receptor binds to the most important HA1 loop region, 213-218 aa; iii) a 3-dimensional overhang, a region consisting of Gln53, Gln65, Arg81, Glu82 and Ser84.
  • the H7N9-H002 antibody provided by the present invention binds to the amino acid site of the H7N9 virus hemagglutinin protein and comprises the following amino acids: hemagglutinin trimeric protein A chain: 53Q, 88Y, 121R, 125A, 126T, 127S, 130R, 131R, 132S, 133G, 134S, 135S, 142W, 144L, 148D, 184K, 185L, 213Q, 216G, 217L.
  • the invention further provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention, a nucleotide molecule of the invention, a vector comprising the nucleic acid molecule of the invention, an antibody or antibody fragment expressing the same The cell, or an immunogenic polypeptide of the invention, and a pharmaceutically acceptable diluent or carrier.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody and/or antibody fragment of the invention and/or a nucleic acid encoding such an antibody and/or an epitope recognized by an antibody of the invention.
  • the pharmaceutical compositions may also contain a pharmaceutically acceptable carrier.
  • the carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, and inactive viral particles.
  • the pharmaceutical compositions of the present invention typically have a pH of from 5.5 to 8.5.
  • the pH can be 7.4, and the pH can be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen free and is isotonic to human body fluids.
  • the pharmaceutical composition may comprise an effective amount of one or more antibodies of the invention and/or an epitope polypeptide comprising an antibody that binds to the invention, ie sufficient to treat, ameliorate or prevent a desired disease or condition or sufficient to exhibit detectable The amount of curative effect.
  • An antibody or antigen-binding fragment thereof of the present invention a nucleotide sequence of the present invention, a vector comprising the nucleotide sequence of the present invention, a cell expressing the vector of the present invention, an epitope comprising the antibody or antibody fragment of the present invention, and Use of the immunogenic polypeptide thereof, or the pharmaceutical composition of the present invention (i) for the preparation of a medicament for the treatment and prevention of human infection with H7N9 influenza A virus; (ii) for the preparation of a vaccine against influenza A virus of H7N9 or Iii) Use in the identification of human infected H7N9 influenza A virus is included in the scope of the present invention.
  • the use of the antibody or antigen-binding fragment of the present invention to detect whether a human hemagglutinin antigen of H7N9 influenza A virus has a specific conformation of a specific conformation to monitor the quality of the H7N9 virus or hemagglutinin protein vaccine is also in the present invention.
  • the use of the antibody or antigen-binding fragment of the present invention to detect whether a human hemagglutinin antigen of H7N9 influenza A virus has a specific conformation of a specific conformation to monitor the quality of the H7N9 virus or hemagglutinin protein vaccine is also in the present invention.
  • the antibodies and antibody fragments of the invention, or derivatives and variants thereof, are useful in the treatment, prevention, and diagnosis of human infection with H7N9 influenza A virus infection.
  • the invention provides i) antibodies, antibody fragments or variants thereof and derivatives thereof; ii) methods of production of the antibodies of the invention; iii) epitopes capable of binding to the antibodies of the invention; or iv) ligands, preferably for treatment An antibody capable of binding to an epitope that binds to an antibody of the invention.
  • an antibody, antibody fragment, epitope or composition of the invention is administered to a subject in need of treatment and prevention.
  • subjects include, but are not limited to, patients infected with H7N9 influenza A virus, subjects at risk of H7N9 influenza A virus infection or susceptible to H7N9 influenza A virus infection, such as subjects with poor immune function. Due to the broad spectrum protective effect of the antibodies provided in the present invention against subtypes of the virus, the antibodies of the present invention have similar quality and prophylactic effects against other H7 series of influenza antibodies.
  • the antibodies or antibody fragments of the invention can be used for passive immunization or active immunization.
  • the antibodies and fragments thereof of the present invention are also useful as kits for diagnosing H7N9 influenza virus infection.
  • an epitope capable of binding to an antibody of the invention can be used in a kit to monitor the efficiency of the vaccination program by detecting the presence of a protective anti-H7N9 influenza A virus antibody.
  • the antibodies, antibody fragments or variants and derivatives thereof of the invention can also be used in kits to monitor the production of vaccines having the desired immunogenicity.
  • the invention also provides a method of preparing a medicament comprising the step of mixing a monoclonal antibody with one or more pharmaceutically acceptable carriers. Including obtaining (e.g., by expression and purification) a monoclonal antibody, the step of mixing it with a pharmaceutical carrier can be carried out at different times by different people at different locations.
  • the antibody of the present invention is selected from a rabbit antibody immunized with HA protein, and a pharmaceutically acceptable antibody is obtained by humanization.
  • the eukaryotic expression vector was constructed, transfected into CHO cell line, and the cells were cultured in high density, and the antibodies were isolated and purified. Optimization of antibodies, including codon optimization and antibody affinity maturation optimization.
  • the present invention covers all nucleic acids, vectors, sequences, antibodies and the like used and prepared in these steps.
  • the invention includes a method of reducing a human infection with an H7N9 influenza A virus infection or reducing the risk of human infection with an H7N9 influenza A virus comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody of the invention or antigen binding thereof Antibody fragment.
  • the invention encompasses an epitope that specifically binds to an antibody of the invention, or an antigenic fragment thereof, for use in: (i) preparation of a medicament for treating human infection with H7N9 influenza A virus; (ii) detection of a therapeutic person The structure of a drug infected with H7N9 influenza A virus; (iii) as a vaccine; or (iv) screening for a ligand capable of neutralizing human infection with H7N9 influenza A virus.
  • the present invention is based on the discovery and isolation of an enhanced immune matured animal-derived antibody that specifically neutralizes H7N9 human infectious virus from an individual immunized with H7N7 (hemagglutinin HA protein is highly homologous to H7N9 virus) or H7N9 virus hemagglutinin protein. .
  • the neutralizing activity of these antibodies against the H7N9 virus is expected, and the hemagglutinin protein epitope recognized by the antibody should be part of the H7N9 virus subunit vaccine.
  • the antibodies and antigen-binding fragments thereof provided by the present invention can bind to a variety of partially variant H7N9 influenza viruses.
  • the present invention provides neutralizing antibodies and antigen-binding fragments thereof against HA of different humans infected with H7N9 influenza A virus.
  • an antibody of the invention and antigen-binding fragments thereof specifically bind to a sialic acid receptor binding epitope of an H7N9 influenza A virus.
  • the epitope to which the antibody of the present invention binds may be a linearly continuous or conformationally discontinuous amino acid sequence.
  • the antibodies of the invention and antigen-binding fragments thereof inhibit the hemagglutination activity of H7N9 influenza A virus HA.
  • Epitopes recognized by the antibodies of the invention can have a variety of uses.
  • a purified or synthetic form of the epitope and its mimotope can be used to increase the immune response (ie, preparation of the antibody as a vaccine or for other uses), or to screen for antibodies that immunoreact with the epitope or its immunological epitope. serum.
  • Epitopes of the invention can also be used to screen for ligands that bind to the epitope.
  • ligands include, but are not limited to, antibodies (different species), antibody fragments, peptides, and the like that are similar viral proteins that prevent epitopes and thus prevent infection.
  • the epitopes of the invention can also be used in the development and application of diagnostic tools. Such applications include direct testing and post-marking use.
  • the invention also encompasses an antibody or antibody fragment thereof that competes with an antibody of the invention or with an antibody of the invention, ie, an antibody that binds to the same epitope.
  • an antibody or antibody fragment thereof that competes with an antibody of the invention or with an antibody of the invention, ie, an antibody that binds to the same epitope.
  • an antibody or antibody fragment thereof that competes with an antibody of the invention or with an antibody of the invention, ie, an antibody that binds to the same epitope.
  • the antibodies of the invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., alpha, gamma or mu heavy chain), but are predominantly IgG.
  • the antibody can be an IgGl, IgG2, IgG3 or IgG4 subclass.
  • the antibodies of the invention may have a kappa or lambda light chain.
  • antibody fragment refers to any fragment of an antibody of the invention that retains the antigen-binding activity of an antibody, as well as fragments having a similarity of 60%.
  • Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv.
  • antibody as used herein includes antibodies and antigen-binding fragments thereof.
  • neutralizing antibody refers to the ability of an antibody to neutralize, ie, prevent, inhibit, reduce, hinder, or interfere with viral initiation and/or maintenance of the host against infection.
  • neutralizing antibody or “antibody to neutralize” are used interchangeably herein.
  • these antibodies can be used alone, or as a prophylactic or therapeutic agent in combination with a drug such as a NA inhibitor or vaccine in a suitable formulation, and include any modification and improvement after antibody-based use.
  • the present invention provides an antibody which specifically binds to a receptor binding region in the HA1 globular head of H7N9 and H7 virus HA, and the high affinity binding of the antibody to the H7N9 virus prevents the binding of the virus to the receptor, thereby Prevents viruses from invading cells and inhibits viral replication and spread.
  • the antibody and antigen-binding fragment of the present invention have high affinity and high neutralizing potency, and the antibody concentration of the present invention required for neutralizing 50% of influenza A virus can be, for example, 200 ng/ml or less.
  • the 50% influenza A virus that neutralizes 100 TCID50A/Anhui/1/2013 virus requires an antibody concentration of about 25 ng/ml for the invention and an affinity for the antibody of the order of nM ( 10-9 M). It can be seen that the neutralized human antibody infected with H7N9 influenza A virus can neutralize 100 TCID 50 of H7N9 virus to half of infected MDCK cells at a small dose, and protect mice against 10LD 50 dose, reaching 90%. The rate of death protection, so the antibody can be used for the treatment and prevention of diseases caused by the H7N9 virus. It can also be used to prepare drugs or vaccines against the diseases caused by the H7N9 influenza A virus, which plays an important role in safeguarding public health interests and safeguarding human health. .
  • Figure 1 Hemagglutination inhibitory activity of rabbit-derived anti-H7N9 antibody against H7N9 (A/Anhui/1/2013) influenza virus hemagglutinin HA protein (2 ⁇ g/mL).
  • Figure 1a is a photograph of agglutination of a hemagglutination plate
  • Figure 1b is an agglutination data analysis of the antibody.
  • Figure 2 Humanized H7N9 antibody H7N9-H002 and original rabbit antibody against 4 H7N9 (A/Anhui/1/2013, A/Shanghai/1/2013, A/Hangzhou/1/2013, A/Pigeon/Shanghai/ S1069/2013) Hemagglutination inhibitory activity of influenza virus hemagglutinin HA protein.
  • FIG. 3 Microneutralization (MN) activity of humanized H7N9 antibody H7N9-H002 and naive rabbit antibody against H7N9 (A/Anhui/1/2013) virus.
  • Figure 4 Humanized H7N9 antibody H7N9-H002 variable region sequence
  • Figure 4a Light chain variable region of humanized antibody Nucleotide and amino acid sequences
  • Figure 4b Heavy chain variable region nucleotide and amino acid sequences of humanized antibodies.
  • Figure 5 Animal protective effect of humanized H7N9 antibody H7N9-H002 in the influenza prevention group;
  • Figure 5a Survival rate of the mouse,
  • Figure 5b Proportion of weight loss of the mouse.
  • the antibody doses of the prophylaxis group were 1 mg/kg, 3 mg/kg, 10 mg/kg and 20 mg/kg, respectively, and the virus was H7N9 (A/Anhui/1/2013).
  • Figure 6 Animal protective effect of humanized H7N9 antibody H7N9-H002 in the influenza treatment group;
  • Figure 6a Survival rate of the mouse,
  • Figure 6b Proportion of weight loss of the mouse.
  • the antibody dose in the treatment group was 20 mg/kg, and the administration time was 3 hours, 1 day and 3 days after the challenge, and the virus was H7N9 (A/Anhui/1/2013).
  • Figure 7 Animal protective effect of humanized H7N9 antibody H7N9-H002 in the influenza treatment group
  • Figure 7a Survival rate of the mouse
  • Figure 7b Proportion of weight loss of the mouse.
  • the antibody dose of the treatment group was 40 mg/kg
  • the administration time was 1 day and 3 days after the challenge
  • the virus was H7N9 (A/Anhui/1/2013).
  • Figure 8 Schematic diagram of H7N9 (A/Anhui/1/2013) influenza virus hemagglutinin HA protein: humanized H7N9 antibody H7N9-H002 binding to the HA protein epitope is represented by blue; HA protein receptor sialic acid The bound epitope is shown in red; the portion of the antibody binding epitope that crosses the sialic acid binding epitope is indicated by yellow.
  • Figure 9 ELISA binding data of humanized H7N9 antibody H7N9-H002 and H7N9 different strain HA protein.
  • Figure 10 ELISA binding data of humanized H7N9 antibody H7N9-H002 and H7 class virus HA.
  • FIG. 11 In vitro neutralizing activity of H7N9-H002 antibody against various viruses.
  • the CDRs of the antibody heavy chain refer to H-CDR1, H-CDR2, H-CDR3, respectively, and the CDRs of the antibody light chain are L-CDR1, L-CDR2 and L-CDR3, respectively.
  • the positions of the CDR amino acids are defined as CDR1-IMGT positions 27-38, CDR2-IMGT positions 56-65, and CDR3-IMGT positions 105-117 according to the IMGT numbering system.
  • Exemplary antibodies of the invention include rabbit-derived antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021 and H7N9-R002.
  • Tables 1 and 2 provide the amino acid sequences of the six CDRs of the heavy and light chains of exemplary antibodies of the invention, respectively.
  • H7N9 Prior to the outbreak of H7N9 virus, a rabbit antibody library of H7N7 (A/Netherlands/219/2003) and H7N9 (A/Anhui/1/2013) hemagglutinin protein was constructed and expressed in 293 mammalian transient expression and insect system expression.
  • the hemagglutinin protein of H7N9 virus (A/Anhui/1/2013) was subjected to panning of the rabbit antibody library, and 30 anti-human infected H7N9 virus hemagglutinin-specific antibodies were obtained by screening.
  • the antibody sequence was constructed into a eukaryotic expression vector and a milligram amount of antibody was produced in a 293 mammalian transient expression system.
  • the CDRs of 11 antibodies with good hemagglutination activity were analyzed. Among them, 9 antibodies were derived from the same germline gene family, and the light chain was derived from IGKV1S56*01.
  • the amino acid sequence variation of CDR1-3 is shown in the table. 1.
  • the heavy chain is derived from the gene family IGHV1S34*01, and the amino acid sequences of the three CDR1-3s are shown in Table 2.
  • the amino acid sequences and homology of the light chain variable region and the heavy chain variable region are shown in Table 3.
  • the binding regions of antibodies that share the same CDR sequence or a small number of mutated CDR sequences are similar, and the results are also consistent with the hemagglutination inhibitory activity of these antibodies.
  • H7N9-R002 rabbit antibody was used for humanized design to obtain H7N9-H002 humanized antibody.
  • the humanized transformation technique was based on the most classic CDR transplantation method and aimed at rabbit source.
  • the humanized antibody gene sequence was obtained by whole-gene synthesis and constructed into a eukaryotic expression vector to produce a milligram to a gram-level protein antibody in a 293 mammalian transient expression system.
  • the specific experiment was as follows: the H7N9-H002 antibody was biotinylated and immobilized on the surface of the streptavidin chip to dilute the recombinant hemagglutinin protein to different Concentration, detection of binding and dissociation rates with antibodies, calculation of affinity constants for obtaining antibody binding antigen proteins.
  • the test results are shown in Table 5.
  • the guinea pig erythrocyte agglutination inhibition assay was used to detect the anticoagulant ability of the humanized H7N9-H002 antibody and the original rabbit antibody H7N9-R002.
  • the specific experiment is as follows: the serially diluted antibody was incubated with different agglutination units of different HA proteins for 1 h, and then an equal volume of 1% guinea pig red blood cells was added, and incubated at room temperature for 30 minutes to obtain a concentration of hemagglutination inhibition (Fig. 1 and Figure 2).
  • the H7N9-H002 humanized antibody possesses a higher hemagglutination inhibitory titer relative to the original rabbit antibody, and the concentration of the neutralizing antibody is in the range of 0.031 to 0.25 ⁇ g/mL for different recombinant influenza hemagglutinin proteins.
  • the virus neutralization ability of humanized H7N9-H002 antibody and the original rabbit antibody H7N9-R002 was detected by virus micro-neutralization assay.
  • the specific experiments were as follows: different concentrations of diluted antibody and 100 ⁇ 50% TICD 50 of H7N9 (A/Anhui/ 1/2013) The virus was incubated at 37 ° C for 1 hour. 100 ⁇ L of the virus and antibody mixture was added to MDCK cells, and after 2 hours, the culture supernatant was aspirated, and the cells were washed with MEM, and then replaced with 100 ⁇ L of MEM medium containing 1 ⁇ g/mL of TPCK-treated trypsin.
  • the humanized antibody has a significantly reduced virus neutralization titer compared to the original rabbit antibody, and the antibody titer that inhibits the H7N9 virus in vitro is as high as 0.025 ⁇ g/mL.
  • Host cells expressing a glycosylated anti-H7N9 monoclonal antibody can be derived from a variety of tissues, but are preferably vertebrate cells. Available cell lines are SV40 transformed monkey kidney CV1 cell line (ATCC CRL 1651), human embryonic kidney cells (293 or suspension cultured 293) [Graham et al., J. Gen Virol.
  • infant hamsters Renal cells BHK-21ATCC CCL 10
  • Chinese hamster ovary cells CHO/dhfr-ATCC CRL 9096
  • monkey kidney cells CV1ATCC CRL90
  • African green monkey kidney cells VEO-76ATCC CRL 1587
  • human uterine cancer cells HELA ATCC CCL 2
  • canine kidney cells MDCK, ATCC CCL 34
  • human lung cells W138, ATCC CCL95
  • human liver cells Hep G2, HB 8065
  • cells which produce an anti-H7N9 monoclonal antibody can be cultured in various media.
  • Commercially available media such as DMEM, MEM, Ham's F12, RPMI-1640 (Gibco) can be used for the culture of host cells.
  • the medium in Ham et al., Meth. Enz. 1979 58:44; Barnes et al., Anal. Biochem. 1980 102: 255; U. S. Pat. Nos. 4, 767, 707; 4, 657, 866; 4, 927, 762 can be used to culture host cells.
  • the culture conditions of the host cells such as temperature, pH, and the like, are also conventional conditions well known to those skilled in the art.
  • Transfection of a humanized anti-H7N9 monoclonal antibody into a mammalian host cell can be carried out using conventional techniques well known to those skilled in the art.
  • the host cell is a CHO/dhfr-cell
  • the following DNA transfection method can be selected by the method of calcium phosphate coprecipitation [Jordan et al., Nucleic Acids Res. 199624:4], liposome packaging method (such as lipofectamine 2000). [Audouy S. et al., Mol Membr Biol. 20018 (2): 129], electroporation and microinjection [Morrison et al., Science 1985 229: 1202].
  • Lipofectamine 2000 (Invitrogen) transfection method, according to the instructions for use, four N2-1, N5-4, N6-3, N8-3 intact antibody sequence expression vector pIRESneo3d-anti-H7N9 each prepared 1.5 ⁇ g plasmid and A mixture of 4.5 ⁇ L of Lipofectamine 2000 was co-transfected into 6 ⁇ 10 5 cells in one well of a 6-well plate, and after overnight transfection, the cells were evenly distributed into a 96-well plate.
  • the intact antibody-transfected CHO cells were screened under DMEM + 5% dFBS + 1.0 mg/mL G418, and the screening medium was changed every 2-3 days, and 20 or more screening cell clones were obtained for 2-3 weeks.
  • the cells were transferred to a 24-well plate at the same density, and cultured for 5 days in 0.5 mL of DMEM + 5% dFBS medium, and the expression level of the humanized anti-H7N9 monoclonal antibody in the culture supernatant was measured by ELISA.
  • the antibody production cells were cultured by high-density cell culture process, and a kilogram antibody production process of 0.8-1.4 g/L yield was obtained through process optimization.
  • H7N9-H002 antibody at a concentration of up to 25 mg/mL was prepared into PBS buffer (Na 2 HPO 4 10 mM, KH 2 PO 4 1.8 mM, NaCl 137 mM, KCl 2.7 mM, pH 7.4).
  • Example 4 In vivo prophylactic and therapeutic effects of humanized antibodies in a challenge model
  • H7N9 challenge model selection of 4-6 week old BALB / c female mice explore LD50 (LD 50) required for viral titer. After groping, it was determined that the viral dose of 10 times the LD 50 was 2.5 ⁇ 10 6 TCID 50 .
  • mice in each group were intravenously injected with 1,3,10 or 20 mg/kg of H7N9-H002 humanized antibody. After 24 hours, nasal infection was 10 times LD 50 of H7N9 (A/Anhui/ 1/2013) Virus. Mortality of the mice was observed within 2 weeks, changes in body weight were recorded, and virus titer and histopathological staining analysis were performed in the 1 mg/kg dose group. The results showed that 10 mice in the vehicle group died at 11 days, and all the dose groups received a 100% mortality protection rate (Fig. 5). The body weight data also had a significant correlation with the dose (Fig. 5). ), only in the 1 mg/kg dose group, the mice showed a brief and significant decrease in body weight.
  • the viral titer of the lung and nasal cavity of the mice decreased by about 100-fold (Table 6), so that the amount of virus in the body can be effectively reduced, the immune system is overreacted to the virus, and the body weight is effectively reduced. The decline and the damage of the virus to the lung tissue.
  • mice In the treatment effect study, a group of 10 mice was infected with H7N9 (A/Anhui/1/2013) virus with 10 times LD 50 in the nasal cavity.
  • the first dose group was infected for 3 hours, and intravenous injection of 20 mg after 1 day and 3 days.
  • the antibody in kg
  • the second dose group was injected with 40 mg/kg of antibody intravenously 1 day and 3 days after infection.
  • Mortality of the mice was observed within 2 weeks, changes in body weight were recorded, 20 mg/kg was detected, and virus titer and histopathological staining analysis were performed in the dose group after 1 day.
  • mice in the vehicle group died at 10 days, and the survival rate of the mice in the drug-administered group was as high as 90% after the infection at the 20 mg/kg dose for 3 hours and the infection at the 40 mg/kg dose for 1 day (Fig. 6 and 7).
  • Mortality and body weight data for the mice correlate with the dose administered and the time of administration, indicating that the antibody has a definitive therapeutic effect.
  • the virus titer of the lungs and nasal cavity administered 1 day after infection in the 20 mg/kg dose group decreased by more than 1000-fold, significantly reducing the live virus level of the lesion tissue (Table 7). Compared with the vehicle group, pathological staining of lung tissue also showed more obvious symptoms of inflammation reduction.
  • the humanized antibody H7N9-H002 has HA-induced hemagglutination inhibitory activity, and the binding region of the antibody is on the HA1 subunit of the HA protein, and has a large overlap with the binding region of the receptor sialic acid of HA.
  • Discovery Studio 3.5 protein structure simulation software we simulated the spatial structure of the H7N9-H002 antibody and obtained a more accurate antibody conformation.
  • the binding site of H7N9-H002 on the crystal structure of H7N7 (A/Netherlands/219/2003) [PDB entry: 4DJ6] was analyzed using the Discovery Studio 3.5 protein interaction site docking software.
  • H7N7A/Netherlands/219/2003 and H7N9A/Anhui/1/2013 have a hemagglutinin protein amino acid homology of up to 94.6%, and H7N9-H002 can bind well to H7N7A/Netherlands/219/2003 hemagglutinin protein. Therefore, it is considered that the region in which the antibody binds to the hemagglutinin protein of H7N7A/Netherlands/219/2003 is also a region that binds to the H7N9A/Anhui/1/2013 hemagglutinin protein.
  • This region consists mainly of three parts: i) the outermost ⁇ -sheet (154-162aa) region of the HA1 head; ii) the sialic acid receptor binding to the most important HA1 annular region (213-218aa); iii) a 3-dimensional The prominent, composed of Gln53, Gln65, Arg81, Glu82 and Ser84.
  • the specific combined amino acid analysis results are shown in Figures 8 and 8.
  • H7N9-H002 binds to the amino acid position of the H7N9 virus hemagglutinin protein
  • H7N9 virus strains with amino acid point mutations using the insect baculovirus expression system (A/Anhui/1/2013, A/Shanghai/1/2013, A/Hangzhou/1/2013, A/Pigeon/Shanghai/ S1069/2013, A/Hangzhou/3/2013, A/Shanghai/4664T/2013, A/Zhejiang/1/2013) and 7 other strains of H7 [A/turkey/Italy/4602/99 (H7N1) , A/chicken/SK/HR-00011/2007 (H7N3), A/turkey/Italy/214845/2002 (H7N3), A/equine/Kentucky/1a/1975 (H7N7), A/mallard/Netherlands/33/ 2006 (H7N8), A/chicken/Netherlands/1/03 (H7N7), A/Netherlands/219/2003 (H7N7)] A total of 14 kinds of HA, HA protein
  • H7N9-H002 was added and allowed to stand at room temperature for 1 h. After 1 h, the plate was washed, and the detection antibody goat anti-human IgG Fc/HRP was added. After 1 h at room temperature, the plate was washed, TMB color was added, and the OD450 was determined after termination. The color depth of the color reaction and the concentration of HA protein in the well were determined. Just proportional. The results of ELISA binding are shown in Figures 9 and 10, and the data show that the antibodies bind well to the HA proteins of these viruses. Based on the mechanism of virus action and antigen-antibody binding ability, H7N9-H002 will have a good virus neutralization effect on H7N9 virus variants and other types of H7 viruses.
  • Example 7 H7N9-H002 can neutralize newly erupted H7N9 virus in vitro
  • the H7N9-H002 antibody has good virus neutralization activity for all viruses, and the neutralizing activity against H7N9A/Xinjiang/75802/2014 is low, and the neutralizing ability of other viruses can be 3 to 45 times higher. This result shows that the H7N9-H002 antibody has a broad spectrum of ability to neutralize the H7N9 virus.
  • the present invention provides an antibody or antigen-binding fragment of neutralizing influenza A virus having high affinity and high neutralizing efficiency, and the antibody concentration of the present invention required for neutralizing 50% of influenza A virus may be 200 ng/ml or less, for example,
  • the 50% influenza A virus of 100 TCID50A/Anhui/1/2013 virus requires the antibody of the present invention to have a concentration of about 25 ng/ml, and the affinity of the antibody is nM (10 -9 M). It can be seen that the neutralized human antibody infected with H7N9 influenza A virus can neutralize 100 TCID 50 of H7N9 virus to half of infected MDCK cells at a small dose, and protect mice against 10LD 50 dose, reaching 90%.
  • the rate of death protection and thus the antibody of the present invention can be used for the treatment and prevention of diseases caused by the H7N9 virus, and can also be used for preparing a drug or vaccine against the disease caused by the H7N9 influenza A virus, which is important for safeguarding public health interests and maintaining human health. effect.

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Abstract

本发明提供了结合并中和H7型甲型流感病毒的中和抗体，所述抗体的轻、重链可变区的核苷酸序列与SEQ ID NO:38-55、58或59中任一个的核苷酸序列至少75%相同，可中和人感染H7N9甲型流感病毒血凝素蛋白；还提供了完整抗体在CHO等细胞中高效表达的方法，以及所述抗体、相关血凝素蛋白抗原结合片段和表位在人感染H7N9甲型流感病毒感染的诊断、治疗和预防中的用途。

Description

一种H7型甲型流感病毒的中和抗体及其用途 技术领域
本发明涉及生物制品领域，具体地，涉及一种H7型甲型流感病毒的中和抗体及其用途，还涉及采用基因重组技术在CHO细胞中生产该抗体的方法。
背景技术
流感病毒通过血凝素(HA)的类型定义为A和B两种类型，A型可细分为16种亚型，其中组I有H1，H2，H5，H6，H8，H9，H11，H12，H13和H16亚型存在，组II由H3，H4，H7，H10，H14和H15亚型组成。B型和A型中的H1，H2和H3在人类中引发较广泛的流感疾病，H5，H7和H9亚型在人类中引起散发性严重感染并可能引起新的大流行病。2013年的2月到6月，在中国爆发了高致病性和高死亡率的人感染甲型H7N9流感病毒，共132人感染，其中43人死亡【Rongbao G等人.N Engl J Med.2013,368:1888-1897】。该病毒由在人类中散发的H7和N9亚型的病毒重配而成，人体中没有建立相应的免疫反应记忆，因此该病毒感染可引发剧烈的呼吸道窘迫综合征，导致病人死亡【Kageyama T等人，Euro SUrvell.2013.18:20453】。人季节性流感疫苗由A-H1N1，A-H3N2和B型3种亚型组成，人体对该类疫苗建立的免疫应答对H7N9病毒没有保护作用。因此H7N9病人感染后，急需外源的抗病毒药物来抑制病毒在体内的扩增。尽管，目前还没有人之间H7N9感染的传播，但已有多个实验表明，在流感敏感性动物如雪貂、猪之间可以发生非接触性的空气传播【Herfst S,等人，Science.2012；336:1534-41；H.Zhu等人，Science.2013.341(6142):183-186】。秋冬季为流感的高发季节，需要有效的药物来控制和治疗可能发生的新的H7N9流感病例的出现。
当前，对H7N9病毒抑制药物为达菲(Oseltamivir)或其类似物(Zanamivir)，该小分子药物为流感病毒NA蛋白的酶活性抑制剂，它们的效果是避免或减少病毒的扩增，从而达到降低病毒滴度的治疗效果。该药物在临床上有一定的效果，但其病毒清除的速率还是比较慢，并且已有出现达菲耐受株的报道，因此需要有更理想的药物来快速降低病毒在体内的复制【Yunwen Hu等人，Lancet.2013.381:2273-79】。
有报道用康复病人的血清救活了流感危重病人，因此中和抗体也是正在热点研发的可用于预防和治疗的抗病毒药物。血凝素蛋白(HA)是广谱和亚型特异抗体的首要目标，该蛋白是由HA1和HA2亚基组成包含500多个氨基酸的三聚体蛋白HA0。其HA1头部是病毒与靶细胞的唾液酸受体结合的部位，可使病毒与细胞黏附，低pH值的环境 可引发茎部HA2区域的构象变化可使病毒与细胞膜融合，当病毒内吞到细胞内部后，病毒的基因组被释放到细胞质中，新的病毒可以在细胞内复制。针对HA的抗体可以有效的阻止病毒感染细胞的过程，从而中断病毒复制链。在不同的亚型中，与流感受体唾液酸结合的HA1的区域差异较大，并且同一亚型在HA1区域也会不断进化出新的变体，因此，该位点虽不具有广谱性，但为最有效的中和位点【Kida H等人，Vaccine.1985,3,219-222】。目前的广谱抗体主要针对相对保守的HA2区域，该类抗体结合HA2表位后，可通过空间位阻来阻止HA蛋白的变构来阻断病毒的感染过程。M2蛋白的抗体也有报道显示具有体内的保护效果，M2为病毒表面的离子通道蛋白，在病毒或细胞表面只有24个氨基酸，当病毒感染细胞后，该细胞表面抗原表达大量的M2蛋白。抗M2的抗体可以结合病毒复制细胞，通过ADCC和CDC的机理来杀死该细胞，起到病毒抑制效果。目前已有多种不同类型的抗血凝素和抗M2的抗体在研发阶段，少量的抗体已进入临床I和II期研究，目前还没有上市的抗体药物。
尽管经过了数十年的研究，针对突发事件，比如H7N9病毒爆发事件，快速的找出最有效的应急药物仍是非常紧要和挑战的任务，因此需要通过多种渠道(化药、中药和生物药)找出最有效的药物。
发明内容
本发明的目的在于提供一种结合人感染甲型H7N9流感病毒血凝素蛋白的抗体或其抗原结合片段。
本发明的另一目的在于提供该抗体的应用。
本发明的再一目的在于提供制备该抗体的方法。
本发明提供的中和人感染H7N9甲型流感病毒的抗体是从H7N7A/Netherlands/219/2003和H7N9(A/Anhui/1/2013)血凝素蛋白HA免疫的兔脾和骨髓组织构建的抗体库中用H7N9(A/Anhui/1/2013)的血凝素蛋白筛选获得，该抗体的结合表位为HA1表面与唾液酸结合的区域。因此，本发明包含了中和人感染甲型H7N9和相关H7甲型流感病毒感染的抗体或其抗原结合片段。
在本发明的一个具体实例中，提供了一种中和人感染H7N9流感病毒的抗体或其抗原结合片段。在本发明的另一个实施例中，本发明还提供了一种中和人感染H7N9甲型流感病毒的抗体或其抗原结合片段，其互补决定区(CDR)序列中至少有一个CDR序列与SEQ ID NO:2,8,13,15,18和19中的任一个有至少61％的序列同源性。
进一步地，本发明提供的抗体或其抗原结合片段中，其轻链CDR1包括具有SEQ ID NO:1-6任一所示的氨基酸序列，其轻链CDR2包括具有SEQ ID NO:7-10任一所示的氨基酸序列，其轻链CDR3包括具有SEQ ID NO:11-14任一所示的氨基酸序列，其中所述 抗体能够中和H7N9甲型流感病毒。
进一步地，本发明所提供的中和人感染H7N9甲型流感病毒的抗体或其抗原结合片段中，包括含有SEQ ID NO:15所示氨基酸序列的重链CDR1，含有SEQ ID NO:16-18任一所述氨基酸序列的重链CDR2和含有SEQ ID NO:19所示氨基酸的重链CDR3。
本发明的示例性抗体包括兔源抗体H7N9-R003，H7N9-R006，H7N9-R019，H7N9-R031，H7N9-RA401，H7N9-RA403，H7N9-RA595，H7N9-R021、H7N9-R002和人源化抗体H7N9-H002。在本发明的一个具体实施例中，本发明所包含的抗体及其抗原结合片段中，轻链CDR1的同源性范围为61％-100％，轻链CDR2的同源性范围为62％-100％，轻链CDR3的同源性范围为80％-100％。在本发明的另一个实施例中，重链CDR1的同源性范围为100％，重链CDR2的同源性范围为87％-100％，重链CDR3的同源性范围为100％。其中所述抗体中和人感染H7N9甲型流感病毒。所有的CDR序列同源性都以H7N9-R002为标准。
在一个实施例中，本发明的抗体或抗体片段至少一个CDR含有与SEQ ID NO:1-19中的任一个具有至少95％的序列同源性的序列，其中所述抗体中和人感染H7N9甲型流感病毒。
在一个实施例中，本发明的抗体及其抗体片段的轻链可变区(VL)和重链可变区(VH)的氨基酸序列见SEQ ID NO:28和SEQ ID NO:37，其人源化抗体的轻链可变区(VL)和重链可变区(VH)的氨基酸序列见SEQ ID NO:56和SEQ ID NO:57。
在一个实施例中，本发明的抗体及其抗体片段包含轻链可变区，其具有与SEQ ID NO:28中所述序列约80％、85％、90％、95％、97％、98％、99％或100％相同的氨基酸序列。在另一实施例中，本发明的抗体及其抗体片段包含重链可变区，其具有与SEQ ID NO:37中任一所述序列约92％、95％、97％、98％、99％或100％相同的氨基酸序列。
在一个实施例中，本发明的抗体及其抗体片段包含轻链可变区，其具有与SEQ ID NO:56中所述序列约80％、85％、90％、95％、97％、98％、99％或100％相同的氨基酸序列。在另一个实施例中，本发明的抗体及其抗体片段包含重链可变区，其具有与SEQ ID NO:57中任一所述序列约92％、95％、97％、98％、99％或100％相同的氨基酸序列。
在一个实施例中，本发明的抗体及其抗体片段包含轻链可变区，其具有与SEQ ID NO:20，21，22，23，24，25，26，27或28中任一所述序列约70％、75％、80％、85％、90％、95％、97％、98％、99％或100％相同的氨基酸序列。在另一实施例中，本发明的抗体及其抗体片段包含重链可变区，其具有与SEQ ID NO:29，30，31，32，33，34，35，36或37中任一所述序列约70％、75％、80％、85％、90％、95％、97％、98％、99％或100％相同的氨基酸序列。
在本发明的一个具体实施例中，本发明包含一种抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含含SEQ ID NO:20所示氨基酸序列的轻链可变区和SEQ ID NO:29氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:21氨基酸序列的轻链可变区和SEQ ID NO:30氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:22氨基酸序列的轻链可变区和SEQ ID NO:31氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:23氨基酸序列的轻链可变区和SEQ ID NO:32氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:24氨基酸序列的轻链可变区和SEQ ID NO:33氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:25氨基酸序列的轻链可变区和SEQ ID NO:34氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:26氨基酸序列的轻链可变区和SEQ ID NO:35氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:27氨基酸序列的轻链可变区和SEQ ID NO:36氨基酸序列的重链可变区；或所述抗体包含含SEQ ID NO:28氨基酸序列的轻链可变区和SEQ ID NO:37氨基酸序列的重链可变区；并且其中所述抗体中和人感染H7N9甲型流感病毒，是H7N9-R002和其变异体H7N9-R003,H7N9-R006,H7N9-R019,H7N9-R031,H7N9-RA401,H7N9-RA403,H7N9-RA595,H7N9-R021。
在本发明的一个具体实施例中，本发明包含一种抗体或其抗原结合片段，其中所述抗体包含含SEQ ID NO:38核苷酸序列的轻链可变区和SEQ ID NO:39核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:40核苷酸序列的轻链可变区和SEQ ID NO:41核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:42核苷酸序列的轻链可变区和SEQ ID NO:43核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:44核苷酸序列的轻链可变区和SEQ ID NO:45核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:46核苷酸序列的轻链可变区和SEQ ID NO:47核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:48核苷酸序列的轻链可变区和SEQ ID NO:49核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:50核苷酸序列的轻链可变区和SEQ ID NO:51核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:52核苷酸序列的轻链可变区和SEQ ID NO:53核苷酸序列的重链可变区；或所述抗体包含含SEQ ID NO:54核苷酸序列的轻链可变区和SEQ ID NO:55核苷酸序列的重链可变区；并且其中所述抗体中和人感染H7N9甲型流感病毒，是H7N9-R002和其变异体H7N9-R003,H7N9-R006,H7N9-R019,H7N9-R031,H7N9-RA401,H7N9-RA403,H7N9-RA595,H7N9-R021。
在本发明的另一个实施方案中，本发明包含一种中和人感染H7N9甲型流感病毒抗体或其抗原结合片段，其中所述抗体来源于H7N7(A/Netherlands/219/2003)血凝素蛋白的兔抗体文库，其抗体是人源化抗体，由兔抗体经人源化设计而来，其抗体采用真核细胞表达生产。
进一步地，本发明提供了一种含有本发明的抗体或其抗原结合片段的多核苷酸的 核酸分子。人源化单克隆抗体，表达本发明抗体的CHO，293转染细胞系以及编码本发明的抗体的核苷酸序列都包含在本发明的范围内。
本发明的抗体可以用本领域已知的任何方法来准备。例如将抗体的序列***到相应的真核表达载体中，转染进入细胞，如采用293细胞瞬时表达获得抗体的技术，采用CHO细胞稳定表达获得抗体的技术。通常采用高密度细胞培养技术来获得抗体高产量。
抗体的纯化技术，包括制备药物级抗体的技术也是本领域已知的。可以利用离心、过滤，亲和、电荷、分子量、疏水性等多种色谱法来纯化抗体。
本发明的抗体片段的制备方式也是本领域已知的，包括用胃蛋白酶或木瓜蛋白酶的酶消化，或者抗体的片段可以通过克隆或表达部分的重链或轻链的序列来获得。抗体片段可以包括scFv、Fab、Fab’、F(ab’)2和Fv片段。示例性分子包括但不限于双特异性Fab2、三特异性Fab3、双特异性scFv和双抗体。
任何合适的宿主细胞/载体***可以用于编码本发明的抗体分子及其片段的DNA序列的表达。合适的宿主***包括大肠杆菌、酵母细胞、昆虫细胞和哺乳动物宿主细胞。哺乳动物宿主细胞包括但不限于CHO，HEK293，PER.C6、NSO、SP20。
本发明的抗体可以通过以下步骤制备：i)在合适的宿主细胞中表达本发明的核酸序列，和ii)分离表达的抗体产物，iii)纯化抗体。
本发明提供了一种含有本发明的抗体核酸分子的载体或表达本发明抗体或其抗原结合片段的细胞。利用本发明提供的中和人感染H7N9甲型流感病毒抗体的核酸分子，可以在原核细胞、酵母细胞、真核细胞及任何重组***中表达和生产此抗体，或以此为基础的改建后的含有此抗体基因的任何其它基因，获得具有中和H7N9甲型流感病毒生物学活性的抗体产物，或利用体外标记或交联的方法获得的复合物，制成临床上用于治疗与H7N9甲型流感病毒相关的疾病的特异性抗体药物。
更进一步地，本发明提供了含有结合本发明的抗体或其抗原结合片段的血凝素蛋白及抗体结合表位，及可以获得同功能抗体的免疫原性多肽。
本发明发现抗原表位位于HA蛋白的头部，是HA蛋白与唾液酸糖受体结合的区域，具体包括3个部分：i)HA1头部最外侧的β片层区域，所述β片层区域为154～162aa；ii)唾液酸受体结合最重要的HA1环形区域，213-218aa；iii)一个3维的突出、由Gln53,Gln65,Arg81,Glu82和Ser84组成的区域。具体地，本发明提供的H7N9-H002抗体结合H7N9病毒血凝素蛋白的氨基酸位点包含以下氨基酸：血凝素三聚体蛋白A链：53Q，88Y，121R，125A，126T，127S，130R,131R,132S,133G,134S,135S,142W,144L,148D,184K,185L,213Q，216G，217L。
本发明进一步提供一种药物组合物，其包含本发明的抗体或其抗原结合片段、本发明的核苷酸分子、含有本发明的核酸分子的载体、表达本发明的抗体或抗体片段的 细胞、或本发明的免疫原性多肽以及药学可接受的稀释剂或载体。
本发明提供了一种药物组合物，其包含本发明的抗体和/或抗体片段和/或编码这样的抗体的核酸和/或由本发明的抗体所识别的表位。药物组合物还可以含有药学可接受的载体。载体本身不应诱导对接受所述组合物的个体有害的抗体的产生，且不应当有毒。合适的载体可以是大的、缓慢代谢的大分子，例如蛋白质、多肽、脂质体、多糖、聚乳酸、聚乙醇酸、聚氨基酸、氨基酸共聚物和非活性病毒颗粒。
本发明的药物组合物一般具有5.5-8.5的pH值，在一些实施方案中，pH可以为7.4，可以通过使用缓冲液来维持pH值。组合物可以是无菌和/或不含热原的，是对于人体液呈等渗的。
药物组合物可包含有效量的本发明的一种或多种抗体和/或包含结合本发明的抗体的表位多肽，即足以治疗、改善或预防期望的疾病或疾病状况或者足以表现出可检测的疗效的量。
本发明的抗体或其抗原结合片段、本发明的核苷酸序列、含本发明的核苷酸序列的载体、表达本发明的载体的细胞、包含结合本发明的抗体或抗体片段的表位及其免疫原性多肽、或本发明的药物组合物(i)在制备人感染H7N9甲型流感病毒的治疗和预防药物中的用途；(ii)在制备H7N9甲型流感病毒疫苗中的用途或(iii)在人感染H7N9甲型流感病毒的鉴别中使用都包括在本发明的范围内。进一步，本发明的抗体或抗原结合片段来检测人感染H7N9甲型流感病毒的血凝素抗原是否具有正确构象的特异表位来监控H7N9病毒或血凝素蛋白疫苗的质量的用途也在本发明的范围之内。
医学治疗和用途
本发明的抗体和抗体片段或其衍生物及变体可用于人感染H7N9甲型流感病毒感染的治疗、预防和诊断。本发明提供了i)抗体、抗体片段或其变体及其衍生物；ii)本发明中抗体的生产方式；iii)能够结合本发明抗体的表位；或iv)配体，优选可用于治疗的能够结合表位的抗体，所述表位结合本发明的抗体。
在一个实施例中，向需要治疗和预防的受试者给予本发明的抗体、抗体片段、表位或组合物。这样的受试者包括但不限于感染H7N9甲型流感病毒的病人，面临H7N9甲型流感病毒感染风险或对H7N9甲型流感病毒易感染的受试者，例如免疫功能低下的受试者。由于本发明中提供的抗体对亚型内病毒的广谱保护效应，本发明抗体可对其他H7系列的流感抗体具有类似的质量和预防效果。
本发明的抗体或抗体片段可用于被动免疫或主动免疫。
本发明中所述抗体及其片段也可用于诊断H7N9流感病毒感染的试剂盒。此外，能够结合本发明的抗体的表位可以用在试剂盒中，以通过检测保护性抗H7N9甲型流感病毒抗体的存在来监测疫苗接种程序的效率。本发明所述的抗体、抗体片段或其变体及衍生物也可以用在试剂盒中，以监测具有期望的免疫原性的疫苗的生产。
本发明还提供一种制备药物的方法，该方法包括将单克隆抗体与一种或多种药学可接受的载体混合的步骤。包括获得(如通过表达和纯化)单克隆抗体，将其与药物载体混合的步骤可以在不同的时间，由不同的人员在不同的地点进行。
本发明的抗体是从HA蛋白免疫的兔抗体中筛选，通过人源化改造获得可药用的抗体。构建真核表达载体，转染CHO细胞系，细胞高密培养表达、分离纯化生产抗体。抗体的优化，包括密码子的优化和抗体亲和力成熟优化。本发明覆盖这些步骤中使用和制备的所有核酸、载体、序列、抗体等。
本发明包括一种减少人感染H7N9甲型流感病毒感染或降低人感染H7N9甲型流感病毒感染风险的方法，其包括向有需求的个体使用治疗或预防有效量的本发明的抗体或其抗原结合抗体片段。
在另一方面，本发明包括一种特异的结合本发明的抗体或其抗原片段的表位，其用于：(i)制备治疗人感染H7N9甲型流感病毒的药物；(ii)检测治疗人感染H7N9甲型流感病毒药物的结构；(iii)作为疫苗；或(iv)筛选能中和人感染H7N9甲型流感病毒的配体。
本发明基于从H7N7(血凝素HA蛋白与H7N9病毒高度同源)或H7N9病毒血凝素蛋白免疫动物个体中发现和分离特异性中和H7N9人感染病毒的增强性免疫成熟的动物源性抗体。这些抗体对H7N9病毒的中和活性是预期的，并且该抗体识别的血凝素蛋白表位应该是H7N9病毒亚单位疫苗的一部分。本发明提供的抗体及其抗原结合片段可以结合多种部分变异的H7N9流感病毒。
本发明提供了针对不同人感染H7N9甲型流感病毒的HA的中和抗体及其抗原结合片段。在一个实施例中，本发明的抗体及其抗原结合片段特异性的结合在H7N9甲型流感病毒的唾液酸受体结合表位。本发明的抗体所结合的表位可以是线性连续的或构象不连续的氨基酸序列。在另一个具体实施例中，本发明的抗体及其抗原结合片段可以抑制H7N9甲型流感病毒HA的血凝活性。
本发明的抗体所识别的表位可以具有多种用途。纯化或合成形式的表位及其模拟表位可以用于提高免疫应答(即作为疫苗或用于其它用途的抗体制备)，或用于筛选与表位或其免疫表位发生免疫反应的抗体的血清。
本发明的表位还可以用于筛选结合所述表位的配体。这样的配体包括但不限于抗体(不同种属)、抗体片段、肽等其他可以阻止表位因而预防感染的类似病毒蛋白。本发明的表位还可以用于诊断工具的研发和应用。这样的应用包括直接测试和标记后的使用。
本发明还包括一种抗体或其抗体片段，其与本发明的抗体或与本发明的抗体竞争即结合相同的表位的抗体。包括但不限于H7N9-R003，H7N9-R006，H7N9-R019，H7N9-R031，H7N9-RA401，H7N9-RA403，H7N9-RA595，H7N9-R021、H7N9-R002 及其人源化抗体H7N9-H002的单克隆抗体。
本发明的抗体可以是任何同种型(如IgA，IgG，IgM，即α、γ或μ重链)，但主要为IgG。在IgG同种型中，抗体可以是IgG1，IgG2，IgG3或IgG4亚类。本发明的抗体可以具有κ或λ轻链。
如本文所用，术语“抗原结合片段”或“抗体片段”指保持抗体的抗原结合活性的本发明的抗体的任何片段以及相似度在60％的片段。示例性的抗体片段包括但不限于单链抗体、Fab，Fab’，F(ab’)2,Fv或scFv。如本文所用，术语“抗体”包括抗体及其抗原结合片段。
如本文所用，“中和抗体”指的是抗体可以中和，即预防、抑制、减少、阻碍或干扰病毒引发和/或保持宿主抵抗感染的能力。术语“中和抗体”或“中和…的抗体”在本文中互换使用。如本文所述，这些抗体可以单独使用，或作为预防剂或治疗剂在合适的制剂中与NA抑制剂或疫苗等药物联合使用，并包括以抗体为基础的任何修饰和改良后使用。
本发明提供了一种对H7N9和H7类病毒HA的HA1球状头部中受体结合区域有特异性结合的抗体，该抗体高亲和力的与H7N9病毒结合可阻止病毒与受体的结合，因此可阻止病毒侵入细胞而抑制病毒复制和扩散。本发明的抗体和抗原结合片段具有高亲和力和高中和效力，中和50％甲型流感病毒所需的本发明的抗体浓度可以例如为200ng/ml或更低。在一个实施例中，中和100TCID50A/Anhui/1/2013病毒的50％甲型流感病毒所需要本发明的抗体浓度约为25ng/ml，抗体的亲和力均为nM级(10-9M)。可见，本发明提供的中和人感染H7N9甲型流感病毒的抗体可在较小的剂量下中和100TCID50的H7N9病毒到半数感染MDCK细胞，并且针对10LD50剂量下保护小鼠，达到90％的死亡保护率，因而该抗体可用于针对H7N9病毒所引发疾病的治疗和预防也可用于制备针对H7N9甲型流感病毒所引发疾病的药物或疫苗，对保障公共卫生利益，维护人类健康具有重要作用。
附图说明
图1：兔源抗H7N9抗体对H7N9(A/Anhui/1/2013)流感病毒血凝素HA蛋白(2μg/mL)的血凝抑制活性。图1a为血凝板的凝集照片，图1b为抗体的凝集数据分析。
图2：人源化H7N9抗体H7N9-H002和原始兔抗体对4种H7N9(A/Anhui/1/2013,A/Shanghai/1/2013,A/Hangzhou/1/2013,A/Pigeon/Shanghai/S1069/2013)流感病毒血凝素HA蛋白的血凝抑制活性。
图3：人源化H7N9抗体H7N9-H002和原始兔抗体对H7N9(A/Anhui/1/2013)病毒的微中和(MN)活性。
图4：人源化H7N9抗体H7N9-H002可变区序列；图4a：人源化抗体的轻链可变区 核苷酸和氨基酸序列，图4b：人源化抗体的重链可变区核苷酸和氨基酸序列。
图5：人源化H7N9抗体H7N9-H002在流感预防组的动物保护效果；图5a：小鼠的存活率，图5b：小鼠的体重下降比例。预防组的抗体剂量分别为1mg/kg，3mg/kg，10mg/kg和20mg/kg，病毒为H7N9(A/Anhui/1/2013)。
图6：人源化H7N9抗体H7N9-H002在流感治疗组的动物保护效果；图6a：小鼠的存活率，图6b：小鼠的体重下降比例。治疗组的抗体剂量为20mg/kg，给药时间为攻毒后3小时，1天和3天，病毒为H7N9(A/Anhui/1/2013)。
图7：人源化H7N9抗体H7N9-H002在流感治疗组的动物保护效果，图7a：小鼠的存活率，图7b：小鼠的体重下降比例。治疗组的抗体剂量为40mg/kg，给药时间为攻毒后1天和3天，病毒为H7N9(A/Anhui/1/2013)。
图8：H7N9(A/Anhui/1/2013)流感病毒血凝素HA蛋白的结构示意图：人源化H7N9抗体H7N9-H002结合该HA蛋白的表位由蓝色表示；HA蛋白受体唾液酸结合的表位由红色显示；抗体结合表位与唾液酸结合表位交叉的部分由黄色表示。
图9：人源化H7N9抗体H7N9-H002与H7N9不同毒株HA蛋白的ELISA结合数据。
图10：人源化H7N9抗体H7N9-H002与H7类的病毒HA的ELISA结合数据。
图11：H7N9-H002抗体对多种病毒的体外中和活性。
具体实施方式
在以下的实施例中提供了本发明的示例性的实施方案。以下的实施例仅通过示例的方式给出，并用于帮助普通技术人员使用本发明。所述实施例并不能以任何方式来限制本发明的范围。
实施例1兔源抗H7N9病毒中和抗体活性和序列信息分析
抗体重链的CDR分别指H-CDR1，H-CDR2，H-CDR3，同样，抗体轻链的CDR分别是L-CDR1，L-CDR2和L-CDR3。CDR氨基酸的位置根据IMGT编号***定义为CDR1-IMGT位置27-38，CDR2-IMGT位置56-65以及CDR3-IMGT位置105-117。本发明示例性抗体包括兔源抗体H7N9-R003，H7N9-R006，H7N9-R019，H7N9-R031，H7N9-RA401，H7N9-RA403，H7N9-RA595，H7N9-R021和H7N9-R002。表1和2分别提供了本发明示例性抗体的重链和轻链的6个CDR的氨基酸序列。
表1 H7N9兔中和抗体的轻链CDR1-3的氨基酸序列
Figure PCTCN2015073698-appb-000001
Figure PCTCN2015073698-appb-000002
a：所有的CDR序列同源性都以H7N9-R002为标准
表2 H7N9兔中和抗体的重链CDR1-3的氨基酸序列
Figure PCTCN2015073698-appb-000003
Figure PCTCN2015073698-appb-000004
a：所有的CDR序列同源性都以H7N9-R002为标准
表3 H7N9兔中和抗体的轻链和重链可变区的序列
Figure PCTCN2015073698-appb-000005
Figure PCTCN2015073698-appb-000006
Figure PCTCN2015073698-appb-000007
a：所有的可变区序列同源性都以H7N9-R002为标准
在H7N9病毒爆发前，已构建了H7N7(A/Netherlands/219/2003)和H7N9(A/Anhui/1/2013)血凝素蛋白的兔抗体文库，用293哺乳动物瞬时表达和昆虫***表达获得的H7N9病毒(A/Anhui/1/2013)的血凝素蛋白对该兔抗体文库进行淘洗，筛选获得30株抗人感染H7N9病毒血凝素蛋白特异性抗体。将抗体序列构建到真核表达载体中，在293哺乳动物瞬时表达***中生产获得毫克级量的抗体。测试抗体对多种H7N9病毒血凝素蛋白的结合能力(表4)，同时采用血凝抑制试验来检测抗体的活性，检测抗体抑制H7N9病毒血凝素蛋白的豚鼠血红细胞凝集的能力。当凝集试验中加入2μg/mL的重组H7N9(A/Anhui/1/2013)血凝素蛋白时，其中有11株有血凝抑制活性的兔单克隆抗体，并且这11株抗体都拥有高灵敏度的血凝抑制能力(图1)。
表4 11株筛选获得的血凝抑制抗体及其亚型广谱HA蛋白结合能力
Figure PCTCN2015073698-appb-000008
对这11株有很好的血凝活性的抗体的CDR进行分析，其中有9株抗体来源于同一个胚系基因族，轻链来源于IGKV1S56*01，其CDR1-3的氨基酸序列变异见表1，重链来源于基因族IGHV1S34*01，其3个CDR1-3的氨基酸序列见表2。轻链可变区和重链可变区的氨基酸序列和同源性见表3。抗体共用相同的CDR序列或少量突变的CDR序列抗体的结合区域比较类似，该结果也与这些抗体都具有血凝抑制活性相吻合。
实施例2人源化抗体的生物学功能验证
根据抗体的表达水平和中和活性，选用了H7N9-R002这株兔抗体进行人源化设计获得H7N9-H002人源化抗体，人源化改造技术是按照最经典的CDR移植方法并针对兔源抗体进行的设计。通过全基因合成的方式获得人源化抗体基因序列，并构建到真核表达载体中，在293哺乳动物瞬时表达***中生产出毫克级到克级蛋白量的抗体。通过与H7N9血凝素蛋白的亲和力检测筛选保留兔抗体亲和力的人源化抗体，获得亲 和力最高的人源化抗体H7N9-H002，其抗体轻链的可变区的氨基酸序列为SEQ ID NO:56，核苷酸序列为SEQ ID NO:58，抗体重链的可变区的氨基酸序列为SEQ ID NO:57，核苷酸序列为SEQ ID NO:59。检测该人源化抗体与多种重组H7N9血凝素蛋白的亲和力，具体实验如下：将H7N9-H002抗体生物素化后结合固定在链亲和素芯片的表面，稀释重组血凝素蛋白到不同浓度，检测与抗体的结合和解离速率，计算获得抗体结合抗原蛋白的亲和力常数。检测结果见表5。
表5 H7N9-H002人源化抗体与不同重组H7N9血凝素蛋白的亲和力检测
病毒 kon(1/Ms)(a) kdis(1/s)(b) KD(M)
A/Anhui/1/2013 2.32E+04 1.72E-04 7.42E-09
A/Shanghai/1/2013 3.00E+04 1.63E-04 5.42E-09
A/Hangzhou/1/2013 3.74E+04 1.37E-04 3.68E-09
A/Pigeon/Shanghai/S1069/2013 3.96E+04 1.13E-04 2.85E-09
A/Hangzhou/3/2013 4.55E+04 2.81E-04 6.16E-09
a：结合速率常数；b：解离速率常数
采用豚鼠红细胞凝集抑制试验来检测人源化H7N9-H002抗体与原兔抗体H7N9-R002的抗凝集能力。具体实验如下：将系列稀释的抗体与4个凝集单位的不同HA蛋白进行1h的温育，再加入等体积的1％豚鼠血红细胞，室温下温育30分钟，获得血凝抑制的浓度(图1和图2)。相对于原兔抗体，H7N9-H002人源化抗体拥有更高的血凝抑制效价，针对不同的重组流感血凝素蛋白，中和抗体浓度在0.031～0.25μg/mL的范围。
采用病毒微中和实验检测人源化H7N9-H002抗体与原兔抗体H7N9-R002的病毒中和能力，具体实验如下：不同浓度稀释的抗体与100×50％TICD50的H7N9(A/Anhui/1/2013)病毒在37℃温育1小时。100μL的病毒和抗体混合物加入到MDCK细胞中培养，2小时后，吸去培养上清液，并用MEM清洗细胞后，替换为100μL的含1μg/mL TPCK处理的胰酶的MEM培养基。37℃培养3天后，计数CPE(病毒斑)。50％的MDCK细胞无感染的最低抗体浓度为抗体的中和浓度。该数据见图3，人源化抗体比原兔抗体拥有明显降低的病毒中和滴度，其体外抑制H7N9病毒的抗体滴度高达0.025μg/mL。
实施例3人源化抗H7N9抗体的重组生产
表达糖基化的抗H7N9单克隆抗体的宿主细胞可来源于多种组织，但优选为脊椎动物细胞。可用的细胞系有SV40转化的猴肾CV1细胞系(ATCC CRL 1651)，人胚胎肾细胞(293或悬浮培养的293)【Graham et al.,J.Gen Virol.197736:59】，婴幼仓鼠肾细胞(BHK-21ATCC CCL 10)，中国仓鼠卵巢细胞(CHO/dhfr-ATCC CRL 9096)，猴肾细胞(CV1ATCC CRL90)，非洲绿猴肾细胞(VERO-76ATCC CRL 1587)，人子宫癌细胞(HELA ATCC CCL 2)，犬肾细胞(MDCK,ATCC CCL 34)，人肺细胞(W138， ATCC CCL95)，人肝细胞(Hep G2,HB 8065)等。
本发明中，产生抗H7N9单克隆抗体的细胞，如CHO细胞可在多种培养基中培养。商业化的培养基如DMEM，MEM，Ham’s F12，RPMI-1640(Gibco)都可用于宿主细胞的培养。此外，Ham et al.,Meth.Enz.197958:44；Barnes et al.,Anal.Biochem.1980102:255；U.S.Pat.Nos.4,767,707；4,657,866；4,927,762中的培养基均可用于培养宿主细胞。宿主细胞的培养条件，如温度，pH值等也是本领域技术人员熟知的常规条件。
人源化抗H7N9单克隆抗体转染哺乳动物宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主细胞为CHO/dhfr-细胞时，可选用如下的DNA转染方法有磷酸钙共沉淀法【Jordan et al.,Nucleic Acids Res.199624:4】，脂质体包装法(如lipofectamine 2000)【Audouy S.et al.,Mol Membr Biol.200118(2):129】，电穿孔法和显微注射法【Morrison et al.,Science 1985229:1202】。我们采用Lipofectamine 2000(Invitrogen)转染法，按其使用说明，4种N2-1、N5-4、N6-3、N8-3完整抗体序列的表达载体pIRESneo3d-anti-H7N9各制备1.5μg质粒和4.5μL Lipofectamine 2000的混合物，共转染6孔板的一个孔中6×105个细胞，过夜转染后，将细胞平均分配到1块96孔板中。
完整抗体转染的CHO细胞在DMEM+5％dFBS+1.0mg/mL G418下进行筛选，每2-3天更换筛选培养基，培养2-3周各获得20多个筛选细胞克隆。将细胞按相同的密度传至24孔板中，在0.5mL DMEM+5％dFBS培养基中培养5天，ELISA检测培养基上清液中人源化抗H7N9单克隆抗体的表达量。
为了使G418筛选的人源化抗H7N9单克隆抗体细胞更高效的表达，选较高表达水平的细胞进行MTX扩增表达。具体操作为，重组CHO细胞在DMEM+5％dFBS培养基中培养，并在培养过程中加入逐步增加的MTX浓度，培养过程中持续监测抗体的表达水平，当MTX浓度增加到细胞生长无法耐受时停止增加MTX浓度，在T25培养瓶中培养五天后ELISA检测培养基清液中人源化抗H7N9单克隆抗体的表达量。采用高密细胞培养工艺培养抗体生产细胞，经过工艺优化获得0.8～1.4g/L产量的公斤级抗体生产工艺。将浓度高达25mg/mL的H7N9-H002抗体制备到PBS缓冲液中(Na2HPO410mM,KH2PO41.8mM,NaCl 137mM,KCl 2.7mM,pH7.4)。
实施例4人源化抗体在攻毒模型中的体内预防和治疗效果
H7N9的攻毒模型选用4-6周龄的BALB/c母鼠，摸索半数致死剂量(LD50)所需要的病毒滴度。经过摸索，确定10倍的LD50的病毒剂量为2.5×106TCID50。
预防效果研究中，每组选用10只小鼠，静脉注射1，3，10或20mg/kg的H7N9-H002人源化抗体，24小时后，鼻腔感染10倍LD50的H7N9(A/Anhui/1/2013)病毒。观察2周内小鼠的死亡率，记录体重变化，检测1mg/kg剂量组的病毒滴度和组织病理 学染色分析。结果显示，溶媒组的10只小鼠在11天时全部死亡，而所有的给药剂量组都获得了100％的死亡保护率(图5)，体重数据也与剂量具有明显的相关性(图5)，只是在1mg/kg的剂量组中出现小鼠的体重短暂明显下降。在1mg/kg的剂量组中，小鼠肺和鼻腔的病毒滴度下降约100倍(表6)，因此可以有效的降低体内病毒量，减轻免疫***对病毒的过度反应，有效的减少了体重的下降和病毒对肺组织的损伤。
表6 肺和鼻腔中抗体给药组和溶媒组的病毒滴度比较(1mg/kg剂量预防组)
Figure PCTCN2015073698-appb-000009
治疗效果研究中，10只小鼠一组，鼻腔感染10倍LD50的H7N9(A/Anhui/1/2013)病毒，第一个剂量组为感染3小时，1天和3天后静脉注射20mg/kg的抗体，第二个剂量组为感染1天和3天后静脉注射40mg/kg的抗体。观察2周内小鼠的死亡率，记录体重变化，检测20mg/kg，1天后给药剂量组的病毒滴度和组织病理学染色分析。结果显示，溶媒组的10只小鼠在10天时全部死亡，而20mg/kg剂量感染3小时后给药组和40mg/kg剂量感染1天后给药组的小鼠存活率都高达90％(图6和7)。小鼠的死亡率和体重数据(图6和7)与给药剂量和给药时间成相关性，显示该抗体具有明确的治疗效果。20mg/kg剂量组感染1天后给药的肺和鼻腔的病毒滴度下降1000倍以上，明显的降低了病灶组织的活病毒水平(表7)。相对于溶媒组，肺组织病理染色也显示有较明显的炎症减轻症状。
表7 肺和鼻腔中抗体给药组和溶媒组的病毒滴度比较(20mg/kg剂量治疗组)
Figure PCTCN2015073698-appb-000010
a：低于病毒检测极限
实施例5H7N9-H002结合HA蛋白的表位分析
人源化抗体H7N9-H002具有HA致血凝的抑制活性，该抗体的结合区域在HA蛋白的HA1亚基上，并且与HA的受体唾液酸的结合区域有很大的重叠。采用Discovery Studio 3.5蛋白结构模拟软件，我们对H7N9-H002抗体的空间结构进行模拟，获得了较为准确的抗体构象。采用Discovery Studio 3.5蛋白相互作用位点对接软件，分析了H7N9-H002在H7N7(A/Netherlands/219/2003)[PDB entry:4DJ6]晶体结构上的结合位点。由于H7N7A/Netherlands/219/2003和H7N9A/Anhui/1/2013的血凝素蛋白氨基酸同源性高达94.6％，并且H7N9-H002可以很好的结合H7N7A/Netherlands/219/2003的血凝素蛋白，因此认为抗体结合H7N7A/Netherlands/219/2003的血凝素蛋白的区域同样为结合H7N9A/Anhui/1/2013血凝素蛋白的区域。该区域主要包含3个部分：i)HA1头部最外侧的β片层(154-162aa)区域；ii)唾液酸受体结合最重要的HA1环形区域(213-218aa)；iii)一个3维的突出、由Gln53,Gln65,Arg81,Glu82和Ser84组成的区域。具体的结合氨基酸分析结果见图8和表8。
表8：H7N9-H002结合H7N9病毒血凝素蛋白的氨基酸位点
Figure PCTCN2015073698-appb-000011
实施例6H7N9-H002与H7N9的不同毒株及与其他类型的H7病毒的结合情况
采用昆虫杆状病毒表达***表达7株有氨基酸点突变的H7N9病毒毒株(A/Anhui/1/2013，A/Shanghai/1/2013，A/Hangzhou/1/2013，A/Pigeon/Shanghai/S1069/2013，A/Hangzhou/3/2013，A/Shanghai/4664T/2013，A/Zhejiang/1/2013)和7株其他类型的H7毒株[A/turkey/Italy/4602/99(H7N1)，A/chicken/SK/HR-00011/2007(H7N3)，A/turkey/Italy/214845/2002(H7N3)，A/equine/Kentucky/1a/1975(H7N7)，A/mallard/Netherlands/33/2006(H7N8)，A/chicken/Netherlands/1/03(H7N7)，A/Netherlands/219/2003(H7N7)]共计14种HA，将HA蛋白按照0.0125μg/mL和0.025μg/mL 2种浓度包被在96孔ELISA板上(0.0125μg/ml～0.4μg/ml)，4℃包被过夜。次日洗板，室温封闭1h后，加入2μg/mL的H7N9-H002，室温作用1h。1h后洗板，加入检测抗体山羊抗人IgG Fc/HRP，室温下作用1h后，洗板，加入TMB显色，终止后测定OD450.显色反应的颜色深浅与孔内包被的HA蛋白浓度成正比。ELISA结合的结果见图9和图10，数据显示抗体都能很好的与这些病毒的HA蛋白结合。基于病毒作用的机理、抗原抗体结合能力，H7N9-H002对H7N9病毒的变异体及其他类型的H7病毒都将有很好的病毒中和效果。
实施例7H7N9-H002可以体外中和新爆发的H7N9病毒
2014年400多例人感染H7N9患者中有330株病毒获得分离和序列鉴定。对其序列分析后，其结果显示抗体的HA结合表位的氨基酸在这些病毒中相对保守。选取了其中的9个H7N9病毒和1种H7N7病毒来采用病毒微中和实验检测人源化H7N9-H002抗体，具体实验如下：不同浓度稀释的抗体与100×50％TICD50的H7N9(A/Xinjiang/73030/2014,A/Fujian/2/2014,A/Xinjiang/75802/2014,A/Environment/Shandong/YT001/2014,A/Hunan/26937/2014,A/Guangdong/0019/2014,A/Pigeon/Shang hai/S1069/2013,A/Shanghai/1/2013)或H7N7(A/Netherlands/219/2003)病毒在37℃温育1小时。100μL的病毒和抗体混合物加入到MDCK细胞中培养，2小时后，吸去培养上清液，并用MEM清洗细胞后，替换为100μL的含1μg/mL TPCK处理的胰酶的MEM培养基。37℃培养3天后，计数CPE(病毒斑)。50％的MDCK细胞无感染的最低抗体浓度为抗体的中和浓度。该数据见图11。H7N9-H002抗体对所有病毒都具有很好的病毒中和活性，而其中针对H7N9A/Xinjiang/75802/2014的中和活性较低，其他病毒的中和能力可高3～45倍。该结果显示，H7N9-H002抗体具有广谱的中和H7N9病毒的能力。
工业实用性
本发明提供中和甲型流感病毒的抗体或抗原结合片段具有高亲和力和高中和效力，中和50％甲型流感病毒所需的本发明的抗体浓度可以为200ng/ml或更低，例如中和100TCID50A/Anhui/1/2013病毒的50％甲型流感病毒所需要本发明的抗体浓度 约为25ng/ml，抗体的亲和力均为nM级(10-9M)。可见，本发明提供的中和人感染H7N9甲型流感病毒的抗体可在较小的剂量下中和100TCID50的H7N9病毒到半数感染MDCK细胞，并且针对10LD50剂量下保护小鼠，达到90％的死亡保护率，因而本发明抗体可用于针对H7N9病毒所引发疾病的治疗和预防也可用于制备针对H7N9甲型流感病毒所引发疾病的药物或疫苗，对保障公共卫生利益，维护人类健康具有重要作用。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Figure PCTCN2015073698-appb-000012
Figure PCTCN2015073698-appb-000013
Figure PCTCN2015073698-appb-000014
Figure PCTCN2015073698-appb-000015
Figure PCTCN2015073698-appb-000016
Figure PCTCN2015073698-appb-000017
Figure PCTCN2015073698-appb-000018
Figure PCTCN2015073698-appb-000019
Figure PCTCN2015073698-appb-000020
Figure PCTCN2015073698-appb-000021
Figure PCTCN2015073698-appb-000022
Figure PCTCN2015073698-appb-000023
Figure PCTCN2015073698-appb-000024
Figure PCTCN2015073698-appb-000025
Figure PCTCN2015073698-appb-000026
Figure PCTCN2015073698-appb-000027
Figure PCTCN2015073698-appb-000028

Claims (27)

  1. 一种H7型甲型流感病毒的中和抗体或与其抗原结合片段，其轻链可变区包含SEQ ID NO:56所示的氨基酸序列或与SEQ ID NO:56所示的氨基酸序列至少有80％同源性的氨基酸序列；其重链可变区包含SEQ ID NO:57所示的氨基酸或与SEQ ID NO:57所示的氨基酸至少有80％同源性的氨基酸序列。
  2. 如权利要求1所述的抗体，其特征在于，所述的H7型甲型流感病毒为H7N9、H7N1、H7N3、H7N7或H7N8甲型流感病毒。
  3. 如权利要求2所述的中和抗体或与其抗原结合片段，其中中和100TCID50H7N9病毒达到半数感染所需要的抗体浓度为0.025μg/mL或更低。
  4. 如权利要求2所述的中和抗体或与其抗原结合片段，其中在H7N9病毒10倍致死剂量的攻击下，可有效的预防和治疗小鼠的死亡，达到预防和治疗兼备的药物效力。
  5. 一种H7型甲型流感病毒的中和抗体或其抗原结合片段，其至少一个CDR序列含有与SEQ ID NO:1-19中的任何一个具有至少95％的序列同源性的序列。
  6. 如权利要求5所述的中和抗体或其抗原结合片段，其包含选自以下任一序列的轻链CDR1：SEQ ID NO:1-6；选自以下序列的轻链CDR2：SEQ ID NO:7-10；选自以下序列的轻链CDR3：SEQ ID NO:11-14。
  7. 如权利要求5所述的中和抗体或其抗原结合片段，其包含选自以下任一序列的重链CDR1：SEQ ID NO:15；选自以下序列的重链CDR2：SEQ ID NO:16-18；选自以下序列的重链CDR3：SEQ ID NO:19。
  8. 如权利要求5～7任一所述的中和抗体或其抗原结合片段，其包含含有以下氨基酸序列的轻链：CDRL1为SEQ ID NO:1、CDRL2为SEQ ID NO:7、CDRL3为SEQ ID NO:11；或者CDRL1为SEQ ID NO:2、CDRL2为SEQ ID NO:8、CDRL3为SEQ ID NO:12；或者CDRL1为SEQ ID NO:3、CDRL2为SEQ ID NO:7、CDRL3为SEQ ID NO:11；或者CDRL1为SEQ ID NO:2、CDRL2为SEQ ID NO:8、CDRL3为SEQ ID NO:13；或者CDRL1为SEQ ID NO:4、CDRL2为SEQ ID NO:9、CDRL3为SEQ ID NO:14；或者CDRL1为SEQ ID NO:5、CDRL2为SEQ ID NO:10、CDRL3为SEQ ID NO:11；或者CDRL1为SEQ ID NO:6、CDRL2为SEQ ID NO:8、CDRL3为SEQ ID NO:13；或者CDRL1为SEQ ID NO:2、CDRL2为SEQ ID NO:8、CDRL3为SEQ ID NO:13。
  9. 如权利要求5～7任一所述的中和抗体或其抗原结合片段，其包含含有以下氨基酸序列的重链：CDRH1为SEQ ID NO:15、CDRH2为SEQ ID NO:16、CDRH3为SEQ ID NO:19；或者CDRH1为SEQ ID NO:15、CDRH2为SEQ ID NO:17、CDRH3为SEQ ID NO:19；或者CDRH1为SEQ ID NO:15、CDRH2为SEQ ID NO:18、CDRH3 为SEQ ID NO:19。
  10. 如权利要求5～7任一所述的中和抗体或其抗原结合片段，其中所述抗体包含有SEQ ID NO:20的氨基酸序列的轻链可变区和含有SEQ ID NO:29的氨基酸序列的重链可变区；或者含有SEQ ID NO:21的氨基酸序列的轻链可变区和含有SEQ ID NO:30的氨基酸序列的重链可变区；或者含有SEQ ID NO:22的氨基酸序列的轻链可变区和含有SEQ ID NO:31的氨基酸序列的重链可变区；或者含有SEQ ID NO:23的氨基酸序列的轻链可变区和含有SEQ ID NO:32的氨基酸序列的重链可变区；或者含有SEQ ID NO:24的氨基酸序列的轻链可变区和含有SEQ ID NO:33的氨基酸序列的重链可变区；或者含有SEQ ID NO:25的氨基酸序列的轻链可变区和含有SEQ ID NO:34的氨基酸序列的重链可变区；或者含有SEQ ID NO:26的氨基酸序列的轻链可变区和含有SEQ ID NO:35的氨基酸序列的重链可变区；或者含有SEQ ID NO:27的氨基酸序列的轻链可变区和含有SEQ ID NO:36的氨基酸序列的重链可变区；或者含有SEQ ID NO:28的氨基酸序列的轻链可变区和含有SEQ ID NO:37的氨基酸序列的重链可变区。
  11. 如权利要求1～7任一项所述的抗体或其抗原结合片段，所述抗体是单克隆抗体、纯化的抗体、分离的抗体、单链抗体、Fab，Fab’、F(ab’)2、Fv、scFv或基于scFv的多链抗体。
  12. 权利要求1～11任一所述的抗体或其抗原结合片段在制备治疗H7型甲型流感病毒感染疾病的药物中的应用。
  13. 权利要求1～11任一所述的抗体或其抗原结合片段在制备治疗H7N9甲型流感病毒感染疾病的药物中的应用。
  14. 与权利要求1～11任一所述的抗体或其抗原结合片段相结合的抗原表位。
  15. 如权利要求14所述的抗原表位，其特征在于，位于HA蛋白的头部，是HA蛋白与唾液酸糖受体结合的区域，具体包括3个部分：i)HA1头部最外侧的β片层区域，所述β片层区域为154～162aa；ii)唾液酸受体结合最重要的HA1环形区域，213-218aa；iii)一个3维的突出、由Gln53,Gln65,Arg81,Glu82和Ser84组成的区域。
  16. 如权利要求15所述的抗原表位，其特征在于，包含以下氨基酸：血凝素三聚体蛋白A链：53Q，88Y，121R，125A，126T，127S，130R,131R,132S,133G,134S,135S,142W,144L,148D,184K,185L,213Q，216G，217L。
  17. 一种包含编码如权利要求1～11任一项所述的抗体或其抗原结合片段的多核苷酸的核酸分子。
  18. 如权利要求17所述的核酸分子，其轻链可变区和重链可变区的核苷酸序列与SEQ ID NO:38～55，58或59中任一个的核苷酸序列至少75％相同。
  19. 如权利要求17或18所述的核酸分子，其特征在于，其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:58、59所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:38、39所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:40、41所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:42、43所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:44、45所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:46、47所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:48、49所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:50、51所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:52、53所示；或其轻链可变区和重链可变区的核苷酸序列分别如SEQ ID NO:54、55所示。
  20. 一种载体，其包含权利要求17～19任一所述的核酸分子。
  21. 一种细胞，其表达如权利要求1～11中任一项所述的中和抗体或其抗原结合片段，或者包含权利要求17～19任一所述的核酸分子，或者包含权利要求20所述的载体。
  22. 一种抗体与其他蛋白或/和多肽的融合蛋白，其特征在于，包含权利要求1～11任一项所述的中和抗体或其抗原结合片段。
  23. 一种药物，其特征在于，含有权利要求1～11任一项所述的中和抗体或其抗原结合片段。
  24. 权利要求1～11任一项所述的中和抗体或其抗原结合片段在疫苗检测中的用途。
  25. 权利要求1～11任一项所述的中和抗体或其抗原结合片段在诊断人感染H7型甲型流感病毒中的用途。
  26. 权利要求1～11任一项所述的中和抗体或其抗原结合片段在诊断人感染H7N9甲型流感病毒中的用途。
  27. 一种减少或降低人感染H7N9或H7型甲型流感病毒感染风险的方法，包括向需要的对象给予治疗有效量的如权利要求1所述的中和抗体或其抗原结合片段。
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