WO2013044840A1 - 高致病性禽流感的中和分子及其制备方法 - Google Patents

高致病性禽流感的中和分子及其制备方法 Download PDF

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WO2013044840A1
WO2013044840A1 PCT/CN2012/082277 CN2012082277W WO2013044840A1 WO 2013044840 A1 WO2013044840 A1 WO 2013044840A1 CN 2012082277 W CN2012082277 W CN 2012082277W WO 2013044840 A1 WO2013044840 A1 WO 2013044840A1
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binding molecule
seq
amino acid
antibody
acid sequence
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PCT/CN2012/082277
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English (en)
French (fr)
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周保罗
胡红星
周伯平
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中国科学院上海巴斯德研究所
深圳市第三人民医院
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Publication of WO2013044840A1 publication Critical patent/WO2013044840A1/zh

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is in the field of biotechnology and immunology; more specifically, the present invention relates to neutralizing molecules of highly pathogenic avian influenza and methods for their preparation. Background technique
  • the highly pathogenic avian influenza H5N1 virus has infected about 500 million birds since 1997, and more and more people are infected in Asia, Europe and Africa. Although human infections have been transmitted by birds so far, the H5N1 virus has evolved and evolved to evolve new strains with human transmission capacity. The widespread spread of this new virus combined with the lack of pre-existing immunity to the H5N1 virus will cause significant morbidity and death to humans - the main manifestations of the highly pathogenic avian influenza H5N1 virus are severe pneumonia, lymph Cell reduction, hyper-lymphokineemia, and high viral load in the respiratory tract 2 _ ⁇ .
  • the virus can usually be cultured from the patient's cerebrospinal fluid, feces, sputum and serum samples.
  • H5N1 virus can develop resistance to seven pairs tricyclodecanol amine ion channel blocker drugs.
  • neuridine inhibitors such as oseltamivir have a certain effect on seasonal influenza, the effect on H5N1 virus is still controversial. Animal experiments show efficacy only neuraminidase inhibitor drugs prior to infection or after infection administration to play an instant effect 2, and H5N1 virus neuraminidase inhibitor oseltamivir other drugs may also be resistant 8. Therefore, it is urgent to find a way to effectively treat avian influenza and control the spread of avian influenza in humans.
  • Monoclonal and polyclonal antibodies antibody therapy has been effectively used to treat a variety of diseases hepatitis A, hepatitis B, rabies, varicella and cytomegalovirus infections 9. Infants can also gain immunity against influenza virus through acquired antibody immunization1 ( ⁇ 3 . Monoclonal antibodies isolated from survivors of the 1918 Spanish influenza pandemic can effectively reduce influenza mortality by 50% 14 . Entering the plasma of a patient with H5N1 infection can effectively reduce the viral load of patients infected with H5N1 virus and can fully recover. 15 Influenza mice, ferrets, horses and human-derived influenza antibodies can be effectively injected into mice. Prevention and treatment of influenza 1 ⁇ — 25. Recently, Koudstaal et al.
  • the hemagglutinin gene ( ⁇ ) is the most variable gene in the avian influenza virus genome. From the sequence of ⁇ , from
  • Branch 2 can be further divided into 5 sub-branches.
  • Sub-branch 2.3 can be further divided into four Asian sub-branches 28 of 2.3.1, 2.3.2, 2.3.3 and 2.3.4.
  • the highly pathogenic avian influenza H5N1 virus that has been infected with humans so far is divided into 0, 1, 2 and 7 branches.
  • the highly pathogenic avian influenza H5N1 virus that is more prevalent in China belongs to 2.3.4 Asian sub-branches 27 ' 28 .
  • the highly pathogenic avian influenza H5N1 virus that infects poultry and birds in Southeast Asia and East Asia also belongs to the 2.3.4 Asian sub-sector 29 . Studies have shown that at least 5 in humans There are four different antigens 3Q .
  • a binding molecule which recognizes avian influenza virus hemagglutinin HA1 and binds to an epitope on the N-terminal region of hemagglutinin, the epitope comprising the following:
  • the epitope further comprises the following sites:
  • the N-terminal region is the amino acid region 51 to 260 of the hemagglutinin amino acid sequence.
  • the binding molecule (eg, 65C6 or an analog thereof) comprises the heavy chain CDR1 set forth in SEQ ID NO: 7, the heavy chain CDR2 set forth in SEQ ID NO: 8, SEQ ID NO: 9
  • the heavy chain CDR3 is shown.
  • the binding molecule (eg, 65C6 or an analog thereof) comprises the light chain CDR1 of SEQ ID NO: 10, the light chain CDR2 of SEQ ID NO: 1 1 , SEQ ID NO: 12 Light chain CDR3 as shown.
  • the binding molecule (eg, 65C6 or an analog thereof) comprises the heavy chain CDR1 set forth in SEQ ID NO: 7, the heavy chain CDR2 set forth in SEQ ID NO: 8, SEQ ID NO: 9
  • the heavy chain CDR3 is shown; and the light chain CDR1 of SEQ ID NO: 10, the light chain CDR2 of SEQ ID NO: 1 1 , and the light chain CDR3 of SEQ ID NO: 12.
  • the binding molecule (e.g., 65C6 or an analog thereof) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1.
  • the binding molecule (e.g., 65C6 or an analog thereof) comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2.
  • the binding molecule (e.g., 65C6 or an analog thereof) comprises:
  • the binding molecule comprises the heavy chain CDR1 set forth in SEQ ID NO: 13, the heavy chain CDR2 set forth in SEQ ID NO: 14, the heavy chain CDR3 set forth in SEQ ID NO: 15; Or
  • the light chain CDR1 set forth in SEQ ID NO: 16 the light chain CDR2 set forth in SEQ ID NO: 17, and the light chain CDR3 set forth in SEQ ID NO: 18 are included.
  • the binding molecule (e.g., 100F4 or an analog thereof) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3.
  • the binding molecule (e.g., 100F4 or an analog thereof) comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4.
  • the binding molecule comprises:
  • a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4.
  • the binding molecule comprises the heavy chain CDR1 set forth in SEQ ID NO: 19, the heavy chain CDR2 set forth in SEQ ID NO: 20, and the heavy chain CDR3 set forth in SEQ ID NO: 21; / or
  • the binding molecule (e.g., 3C1 1 or an analog thereof) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5.
  • the binding molecule (e.g., 3C1 1 or an analog thereof) comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 6.
  • the binding molecule comprises: a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain variable region, the light chain variable The region has the amino acid sequence shown in SEQ ID NO: 6.
  • the binding molecule is a human monoclonal antibody, Fab, F(ab'), F(ab') 2 , Fv, dAb, Fd, a complementarity determining region (CDR) fragment, a single-chain antibody (scFv), a bivalent single chain antibody, a single chain phage antibody, a bispecific diabody, a triple chain antibody, a four chain antibody;
  • the binding molecule is a human monoclonal antibody; more preferably, Human monoclonal antibodies whose heavy chain constant regions select one of the heavy regions of one of the heavy chain types in the lower panel: IgG1, IgG2a, IgG2b and IgG3, and one of its light chain constant regions select one of the lower regions of the light chain type: ⁇ chain And the ⁇ chain; more preferably, the human monoclonal antibody has a heavy chain constant region and a light chain constant region having the amino acid sequences shown by Genebank No. ACK87036 and ACK
  • the CDR1, CDR2 and CDR3 regions are arranged in series from amino acid to carboxyl terminus.
  • the CDR1 is preceded by a CDR1 and a CDR2, a CDR2 and a CDR3 region, and after the CDR3, a framework region; preferably, the framework region has an amino acid length of 6-40 Preferably; preferably from 8 to 35; more preferably from 10 to 32.
  • a polynucleotide is provided which encodes a binding molecule as described above.
  • an expression vector comprising:
  • a host cell comprising the expression vector; or a polynucleotide in which the polynucleotide is integrated into the genome is provided.
  • the host cell is a Drosophila S2 cell.
  • a method of producing a binding molecule comprising: culturing a host cell as described above to express the binding molecule.
  • binding molecule for the preparation of a composition (e.g., a drug) for preventing, ameliorating or treating an avian influenza virus infection.
  • a composition e.g., a drug
  • the avian influenza virus is a virus of the H5 subtype.
  • the avian influenza virus is an H5N1 virus.
  • the avian influenza virus is a H5 subtype virus other than the 7.2 branch of H5N1; more preferably an H5N1 virus other than the 7.2 branch of H5N1.
  • a pharmaceutical composition comprising an effective amount of a binding molecule as described above, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an effective amount of another anti-influenza drug selected from the group consisting of: an alkylamine drug or an influenza virus neuraminidase inhibitor.
  • the alkylamine drug comprises amantadine or rimantadine; or the influenza virus neuraminidase inhibitor comprises: oseltamivir or zanamivir.
  • a method of preventing, ameliorating or treating an avian influenza virus infection comprising administering to a patient an effective amount of a binding molecule of any of the foregoing.
  • a method for identifying an avian influenza virus comprising: contacting a binding molecule of any of the foregoing with a sample to be detected, and observing binding of the binding molecule to a sample to be detected In case, if the binding molecule binds to the sample to be detected, the avian influenza virus is present in the sample.
  • an immunogen (vaccine) against avian influenza virus comprising an epitope which binds to any of the binding molecules described above.
  • the epitope comprises the following sites:
  • the epitope further comprises the following sites:
  • the immunogen does not include hemagglutinin of the full-length avian influenza H5N1 virus.
  • FIG. la Schematic diagram of the construction of an antibody expression vector.
  • MT-P represents the MT promoter
  • Bip represents the signal peptide coding region
  • VL- ⁇ represents the light chain lambda variable region
  • VL- ⁇ represents the light chain ⁇ variable region
  • VH represents the heavy chain variable region
  • CL-in 1 represents a light chain human 1 constant region
  • CL-K 1 represents a light chain ⁇ 1 constant region
  • CH- ⁇ 1 represents a heavy chain ⁇ 1 constant region
  • poly-A is a sequence containing an adenine nucleotide chain.
  • FIG. 1 Trypan blue staining results of purified antibodies against 65C6, 100F4, 3C11 and TG15.
  • HC represents a band of a heavy chain
  • LC represents a band of a light chain.
  • Figure 5a-b shows the change in body weight and survival rate of mice within 14 days after HPAI H5N1 A/Shenzhen/406H/06 virus inoculation.
  • Figure 7a-b shows the change in body weight and survival rate of mice within 14 days after HPAI H5N1 A/Shenzhen/406H/06 virus inoculation.
  • the Fab segment of each antibody forms a fixed 105 degree angle when combined with the HA molecule.
  • hemagglutinin A, 23 single amino acid mutation sites on hemagglutinin (HA).
  • the mutation of these single amino acids enables the 51-260 amino acid fragment of hemagglutinin with the single amino acid mutation displayed on the surface of the brewer's yeast to lose the binding ability to the antibody 65C6.
  • 10 mutant amino acids are buried in the three-dimensional structure of hemagglutinin, and the other 13 mutant amino acids are exposed on the surface.
  • C Seven hemagglutinin proteins of A/Beijing/01/03 strains identified by yeast display and pseudovirus neutralization test, respectively, in the 7.1 subtype of H5N1, 117, 118, 121, 161, 162, 164 The amino acid at position 167 (shown in red).
  • D From the three-dimensional structure of the hemagglutinin protein, the seven amino acids 117, 118, 121, 161, 162, 164 and 167 (shown in red and blue) are entangled with each other.
  • Comparative antibody 65C6 neutralizes the original strain of the 7.1 subclass and the mutation of 5 individual amino acids of the strain and the titer of the combined mutation of 5 amino acids.
  • the inventors have conducted extensive and intensive research to obtain a binding molecule against avian influenza virus containing a unique CDR region, which has a good neutralizing effect on avian influenza virus.
  • the present inventors have also intensively studied the binding site of one of the binding molecules on the avian influenza virus hemagglutinin (HA), and obtained a neutralizing epitope of the binding molecule.
  • HA hemagglutinin
  • the present invention provides a binding molecule that specifically binds to an avian influenza virus.
  • the binding molecule binds to the H5N1 virus of avian influenza.
  • the binding molecules of the invention exhibit good neutralizing activity against avian influenza viruses.
  • the present inventors successfully isolated three human-derived anti-H5 cells from the memory B cells of the convalescent patients infected with the 2.3.4 sub-branched H5N1 virus using highly sensitive HA and NA pseudovirus screening methods and molecular cloning techniques.
  • Monoclonal antibodies to subtype avian influenza viruses 65C6, 100F4 and 3C1 1. All three monoclonal antibodies have good affinity with HA1.
  • 65C6 and 100F4 are capable of neutralizing many species (19 or more) of the H5 subtype of avian influenza virus, which is a broad-spectrum neutralizing antibody; 3C11 is capable of neutralizing 4 or more than 4 H5 subtypes. bird flu virus.
  • a more preferred antibody in the present invention is a 65C6 antibody which has a good neutralizing ability for almost all branches of the H5N1 virus and has a good preventive and therapeutic effect in animals. Electron microscopy and in vitro antibody screening experiments showed that the 65C6 antibody binds to a conserved epitope in the head region of H5 HA, and in vitro mutagenesis experiments showed that the escaped mutant was not screened by the 1st generation antibody screening, showing 65C6 The conserved neutralizing epitope recognized by the antibody is located in the head region of the HA and this epitope is difficult to mutate in all H5N1.
  • the 65C6 antibody alone or in combination with other antibodies or with small molecule inhibitors has great potential for the treatment of infections caused by various branches of H5N1; on the other hand, the common neutralizing epitopes using H5HA As a immunogen, a broad spectrum antiviral antibody against all H5N1 branches may be prepared.
  • the binding molecule of the invention may be an intact immunoglobulin molecule, which may be an antigen binding fragment, including but not limited to Fab, F(ab') F(ab') 2 Fv dAb, Fd, complementarity determining region ( a CDR) fragment, a single chain antibody (scFv), a bivalent single chain antibody, a single chain phage antibody, a bispecific antibody, a triple chain antibody, a four chain antibody, and at least a specific antigen sufficient to confer an avian influenza virus strain Combination of immunoglobulin fragments (multiple) Peptide or fragment thereof.
  • an antigen binding fragment including but not limited to Fab, F(ab') F(ab') 2 Fv dAb, Fd, complementarity determining region ( a CDR) fragment, a single chain antibody (scFv), a bivalent single chain antibody, a single chain phage antibody, a bispecific antibody, a triple chain antibody, a four chain antibody, and at
  • the invention also provides the use of the binding molecule described herein for the manufacture of a medicament for the prevention, alleviation and/or treatment of avian influenza virus infection.
  • This infection can occur in small groups, but it can also be spread worldwide in the form of seasonal epidemics, or spread more globally, with millions of individuals at risk.
  • the present invention provides binding molecules that can neutralize infection of avian influenza virus strains that cause this epidemic as well as a potential global epidemic.
  • the binding molecules of the present invention can be prepared and stored on a large scale because they provide protection against different epidemic strains and are advantageous for preparing for avian influenza outbreaks that may occur in the future.
  • each of the binding molecules may comprise two, three, four, five or all six CDR regions as disclosed herein.
  • a binding molecule of the invention comprises at least two CDRs disclosed herein.
  • the invention also encompasses "functional variants" of the binding molecules described.
  • a variant molecule is considered to be a functional variant of a parental binding molecule if it competes with a parental binding molecule (a binding molecule prior to mutation) for specific binding to an avian influenza virus or a protein fragment thereof.
  • the functional variant is still capable of binding to the HA1 protein of avian influenza virus or a fragment thereof, and has the same or similar binding properties as the binding molecule prior to the mutation (e.g., the same epitope of the identified epitope).
  • Functional variants include, but are not limited to, primary structural sequences that are substantially similar, but which contain, for example, in vitro or in vivo chemical and/or biochemically modified derivatives that are not found in the parent binding molecule.
  • modifications include acetamylation, deuteration, covalent attachment of nucleotide or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxyl groups Chemical, methylation, oxidation, PEGylation, proteolytic processing, phosphorylation, etc.
  • the modification in the amino acid and/or nucleotide sequence of the parent binding molecule does not significantly affect or alter the binding properties of the binding molecule encoded by or containing the amino acid sequence, ie The binding molecule is still able to recognize and bind to its target site.
  • the functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can include both natural and non-natural nucleotides and amino acids.
  • Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar structure or chemical nature.
  • a family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chain amino acids (eg, aspartic acid, glutamic acid), uncharged polar side chain amino acids (eg, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chain amino acids (eg glycine, alanine, valine, leucine) Acid, isoleucine, valine, phenylalanine, methionine), branched side chain amino acids (eg threonine, valine, isoleucine) and aromatic side chain amino acids (eg tyrosine) Acid, phenylalanine, tryptophan).
  • basic side chains eg
  • variants may have non-conservative amino acid substitutions, for example, amino acids are replaced by another amino acid residue having a different structure or chemical nature. Similar small variations can also include amino acid deletions and/or insertions. Guidance for determining which amino acid residues can be substituted, inserted or deleted without eliminating immunological activity can be found using computer programs well known in the art.
  • a functional variant may comprise a truncation of an amino acid sequence at the amino terminus or the carboxy terminus or both.
  • Functional variants of the invention may have the same or different, higher or lower binding affinities than the parent binding molecule, but still bind to avian influenza virus or fragments thereof.
  • a functional variant of the invention may have increased or decreased binding affinity for HA1 or a fragment thereof of an avian influenza virus H5 subtype virus as compared to a parent binding molecule.
  • Functional variants within the scope of the invention have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more preferably the parent binding molecules described herein.
  • Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally sequence amino acid sequences for comparison and to identify similar or identical amino acid residues.
  • Functional variants can be obtained by altering the parental binding molecule or a portion thereof using common molecular biology methods known in the art including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and Heavy chain and / or light chain shuffling method.
  • the term (; human) binding molecule it also encompasses functional variants of the (human) binding molecule.
  • the antigen-binding properties of a binding molecule are generally described by three specific regions located in the heavy and light chain variable regions, referred to as complementarity determining regions (CDRs), which partition the variable regions into The four framework regions (FR), the amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction. These CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the binding molecule.
  • the amino acid sequence of the same type of binding molecule can be compared to determine which amino acids constitute the FR or CDR regions.
  • the substitution, insertion or deletion may occur in a region other than the CDR region, such as the FR region of an antibody heavy or light chain; since the FR region does not participate in direct binding to the antigen, an appropriate change in the region is Yes.
  • the binding molecule is a monoclonal antibody comprising a human constant region (e.g., a human constant region IgH sequence and an IgKappa sequence).
  • a human constant region e.g., a human constant region IgH sequence and an IgKappa sequence.
  • the heavy chain variable region, the light chain variable region and the complementarity determining region (CDR) of the heavy chain variable region and the light chain variable region of the anti-avian influenza virus monoclonal antibody are uniquely different from the existing ones.
  • the present invention comprises: a monoclonal antibody having a corresponding amino acid sequence of the monoclonal antibody, and a monoclonal antibody having the variable region chain of the monoclonal antibody.
  • the present invention also encompasses any antibody having a light chain and a heavy chain comprising said complementarity determining regions (CDRs), and the CDR regions are 90% or more (more preferably 95% or more) with the CDRs of the monoclonal antibodies of the present invention. The homology of any antibody.
  • the CDR region of the anti-avian influenza virus monoclonal antibody of the present invention is novel, and it is aimed at a unique epitope on the HA1 protein of the avian influenza virus, and the technical concept is different from the existing anti-avian influenza virus antibody. .
  • the monoclonal antibodies of the present invention may be of all human origin, and the heavy chain, light chain variable region and constant region thereof are all derived from human antibodies. Therefore, it has a particularly excellent function of recognizing and neutralizing the avian influenza virus, and has the characteristics of low immunogenicity and high safety.
  • the invention encompasses an immunoconjugate comprising at least one binding molecule of the invention and further The step comprises at least one other therapeutic molecule, therapeutic agent or detectable substance.
  • the label suitable for treatment and/or prevention may be a toxin or a functional part thereof, an antibiotic, an enzyme, an enhanced phagocytosis or an immunostimulatory other binding molecule.
  • An immunoconjugate comprising a detectable substance can be used diagnostically, for example, to assess whether a subject has been infected with an avian influenza virus strain or to monitor the occurrence or progression of an avian influenza virus infection as part of a clinical laboratory procedure to, for example, determine a prescribed treatment regimen. efficacy.
  • Detectable moieties/substances include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions.
  • the label used to label the monoclonal antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, such as immunohistochemical staining (tissue) sample, flow cytometry, Laser scanning cytometry detection, fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), bioassay (eg, phagocytometry assay), Western blotting applications, and the like.
  • Suitable labels for detection/analysis/diagnostic techniques and/or methods known in the art are well known to those skilled in the art.
  • the binding molecules of the invention can be conjugated/attached to one or more antigens.
  • these antigens are antigens recognized by the immune system of the subject to whom the molecule-antigen conjugate is bound.
  • the antigens may be identical to each other, but may also be different. Conjugation methods for attaching antigens and binding molecules are well known in the art and include, but are not limited to, the use of crosslinkers.
  • the immunoconjugate can be produced as a fusion protein comprising a binding molecule of the invention and a suitable therapeutic molecule. , therapeutic or detectable substances.
  • the fusion protein can be produced by methods known in the art, for example by constructing a nucleic acid molecule comprising a nucleotide sequence encoding a binding molecule and a core encoding a suitable label, and subsequently expressing the nucleic acid molecule. Glycosidic acid sequence.
  • nucleic acid molecule encoding at least one binding molecule, a functional variant thereof or an immunoconjugate of the invention.
  • nucleic acid molecules can be used as intermediates for cloning.
  • the nucleic acid molecule is isolated or purified.
  • the sequence of the DNA molecule can be obtained by conventional techniques or by hybridoma technology.
  • a variant of a nucleic acid molecule is a nucleic acid sequence which can be directly translated to provide the same amino acid sequence as the sequence translated from the parent nucleic acid molecule by using a standard genetic code.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DNA sequence encoding the binding molecule (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the sequences of the binding molecules of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These ones
  • the vector can be used to transform a suitable host cell to enable it to express a protein.
  • the vector of the present invention is, for example, a plasmid expression vector containing a viral promoter, and an IgH of an anti-avian influenza virus binding molecule heavy chain variable region (VH) and a constant region is inserted in the expression vector, respectively (from The constant region of human IgH) fusion sequence and light chain variable region VL is fused to human Ig kappa (constant region from human Ig kappa) fusion sequence.
  • VH anti-avian influenza virus binding molecule heavy chain variable region
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • bacterial cells such as Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf ; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc.
  • a host cell particularly suitable for use in the present invention is a eukaryotic host cell, such as a fly S2 cell.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the binding molecule of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • binding molecules of the invention may also be produced in transgenic non-human mammals such as rabbits, goats or cows and secreted, for example, into their milk.
  • transgenic non-human mammals such as rabbits, goats or cows and secreted, for example, into their milk.
  • a neutralizing molecule of the present invention recognizes a neutralizing epitope conserved on the spherical end of the distal membrane region of HA, and the antibody is excellent in the H5N1 virus. Therefore, an immunogen based on the epitope of the antibody 65C6 can be designed to induce an immune response which can neutralize various (sub)types of the H5N1 virus.
  • the immunogen preferably comprises the following epitopes: Ser of position 121 of the amino acid sequence of hemagglutinin; and Arg of position 162 of the amino acid sequence of hemagglutinin, the above epitope is Binding to a molecule-bound epitope.
  • the immunogen preferably further comprises the following epitopes: amino acid sequence relative to hemagglutinin 117th Lie at position 118 relative to the amino acid sequence of hemagglutinin; Lys at position 161 relative to the amino acid sequence of hemagglutinin; Tyr at position 164 relative to the amino acid sequence of hemagglutinin; or relative to blood cells Thr of the 167th amino acid sequence of the lectin.
  • Suitable immunogens can be designed based on the epitopes shown above to induce the production of some new broad-spectrum neutralizing binding molecules (such as antibodies).
  • the design of the immunogen can be referred to some techniques known in the art, with the principle of exposing the above-described neutralizing epitope to the surface of its spatial structure.
  • the binding molecules of the invention can be used to prepare compositions for inhibiting avian influenza viruses.
  • composition for inhibiting avian influenza virus or avian influenza virus infection-associated disease comprising: an effective amount of a binding molecule of the present invention; and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • a “pharmaceutically acceptable carrier” should be compatible with the binding molecules of the present invention, i.e., can be blended therewith without substantially reducing the effectiveness of the composition under normal circumstances.
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween®; wetting agents such as sodium lauryl sulfate Colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphat
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease state, mode of administration, and the like.
  • the administration method can be, for example, injection or other treatment.
  • the binding molecules of the invention may be used in unseparated or isolated form. Furthermore, the binding molecules of the invention may be used alone or in a mixture comprising at least one of the binding molecules (or variants or fragments thereof) of the invention. In other words, the binding molecules can be used in combination, for example, as a pharmaceutical composition comprising two or more binding molecules, variants or fragments thereof of the invention.
  • binding molecules having different but complementary activities can be combined in one therapeutic regimen to achieve the desired prophylactic, therapeutic or diagnostic effect, but alternatively, binding molecules with the same activity can be combined in a single therapeutic regimen to achieve the desired prophylaxis. , therapeutic or diagnostic effects.
  • the mixture further comprises at least one additional therapeutic agent.
  • the pharmaceutical composition may comprise two or more binding molecules having neutralizing activity against an avian influenza virus.
  • the binding molecule exhibits synergistic neutralizing activity when applied in combination.
  • the composition comprises at least two binding molecules having neutralizing activity, characterized in that the binding molecule plays a role in neutralizing avian influenza virus Synergy.
  • the term "synergistic" means that when used in combination, the combined effect of the binding molecules is higher than the additive effect when applied alone.
  • the synergistic binding molecules can bind to different structures on the same or different fragments of the avian influenza virus. The way to calculate synergy is through a combination of index calculations. The concept of the combination index (CI) has been described by Chou and Talalay (1984).
  • the composition may also comprise a binding molecule having neutralizing activity and a non-neutralizing avian influenza virus specific binding molecule.
  • the non-neutralizing and neutralizing avian influenza virus-specific binding molecules may also act synergistically in neutralizing the avian influenza virus H5 subtype.
  • binding molecules or pharmaceutical combinations of the invention can be detected in a suitable animal model system prior to use in humans.
  • animal model systems include, but are not limited to, mice, ferrets, and monkeys.
  • binding molecules of the invention may also be administered in combination with other anti-influenza drugs, such as, but not limited to, alkylamines (amantadine and rimantadine); 2) influenza virus neuraminidase Inhibitors (oseltamivir and zanamivir).
  • alkylamines amantadine and rimantadine
  • influenza virus neuraminidase Inhibitors oseltamivir and zanamivir.
  • the present invention also provides a pharmaceutical composition comprising the binding molecule of the present invention and the above-mentioned anti-influenza drug.
  • the dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response).
  • a suitable dosage range may, for example, be from 0.01 to 500 mg/kg body weight, preferably from 0.1 to 50 mg/kg body weight.
  • a bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation.
  • the molecules and compositions of the invention are preferably sterile. Methods for making these molecules and compositions sterile are well known in the art. Other molecules for diagnosis, prevention, and/or treatment can be administered in a dosage regimen similar to the binding molecules of the invention.
  • the patient can be administered prior to, concurrently with, or subsequent to administration of one or more human binding molecules or pharmaceutical compositions of the invention.
  • Accurate dosing regimens for human patients are usually selected during clinical trials. Detection reagents and kits
  • the binding molecules of the invention can be used to prepare reagents or kits for detecting influenza viruses.
  • sample to be tested or “sample to be tested” encompasses a variety of sample types, including biologically derived blood and other body fluid samples, solid tissue samples such as biopsy tissue samples or tissue culture, or derived therefrom. The cells or their offspring.
  • sample types including biologically derived blood and other body fluid samples, solid tissue samples such as biopsy tissue samples or tissue culture, or derived therefrom.
  • the cells or their offspring also encompasses samples that have been treated by any means after they have been obtained, such as treatment with a reagent, solubilization, or enrichment of certain components such as proteins or polynucleotides.
  • the term encompasses a variety of clinical samples from any species, as well as cultured cells, cell supernatants, and cell lysates.
  • kits for the convenient, rapid and accurate detection of avian influenza viruses e.g., H5N1 can be prepared.
  • the present invention provides a test kit for detecting the presence or absence of avian influenza virus in a sample, which contains the binding molecule of the present invention.
  • a detection kit for specifically detecting an avian influenza virus can be conveniently prepared.
  • an antigen to be tested is coated on a solid phase carrier by an indirect ELISA method, and the binding molecule of the present invention is used for detection.
  • the binding molecule is an antibody and can be detected according to the principle of the double-antibody sandwich method.
  • the double-anti-sandwich method conventionally fixes a primary antibody (such as the monoclonal antibody of the present invention) to a carrier, and then reacts the primary antibody with the antigen, and then reacts with the secondary antibody after washing (the secondary antibody carries a detectable signal, Alternatively, it may be combined with a substance carrying a detectable signal, and finally a chemiluminescence or enzyme-linked color reaction detection signal may be performed.
  • the double-antibody sandwich method is particularly useful for the detection of antigens with two or more epitopes.
  • the kit may further comprise other detection reagents or auxiliary reagents in addition to the binding molecules of the present invention, such as reagents conventionally used in ELISA kits, and these reagents.
  • detection reagents or auxiliary reagents in addition to the binding molecules of the present invention, such as reagents conventionally used in ELISA kits, and these reagents.
  • the properties and methods for their formulation are well known to those skilled in the art, such as color developers, labels, secondary antibodies, anti-antibodies, sensitizers and the like. It will be understood by those skilled in the art that various variations of the detection kits are included in the present invention as long as the binding molecules of the present invention are utilized as reagents for recognizing avian influenza virus.
  • kits for indicating the method of using the reagent loaded therein may be included in the kit.
  • various immunologically related methods can be used to detect the HA protein or its content in the sample, thereby knowing whether the donor of the sample to be tested is infected with the avian influenza virus. Methods are all included in the present invention. Preferably, the method is for the purpose of non-disease diagnosis.
  • the invention provides a method of detecting avian influenza virus in vitro (non-diagnostic or therapeutic), comprising the steps of:
  • the binding molecule of the present invention is applied to the (al) solid phase carrier, thereby binding the avian influenza virus in the sample to be tested to the binding molecule to form the "avian influenza virus - the binding molecule of the present invention” a solid phase carrier of the element complex; (a3) a test substance which specifically binds to the binding molecule of the present invention is applied to the solid phase carrier of (a2) to form a binding molecule-testing substance having the "avian influenza virus-inventive agent of the present invention” a solid phase carrier of the ternary complex; the test object carries a label;
  • a concentration standard curve is prepared, and the influenza virus content in the sample to be tested can be obtained by comparing the concentration standard curve.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the manufacturer's recommended conditions. . Percentages and parts are by weight unless otherwise stated.
  • the Shenzhen patient was diagnosed with the highly pathogenic H5N1 avian influenza virus in June 2006 and was cured by entering the plasma of a convalescent patient infected with the highly pathogenic H5N1 avian influenza virus. Blood samples were collected 6 months after the patient's recovery, and peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation. Plasma and peripheral blood mononuclear cell sample stored at 31 -80 ° C. animal
  • mice were 6-8 weeks old.
  • Female BALB/c mice were purchased from Charles River Laboratories (L'Arbresle, France) and were housed in a vacuum, microbial isolation device, and air was filtered through a HEPA filter. 12-hour light and 12-hour dark cycle.
  • the challenge experiment was conducted in the Biosafety Level 3 laboratory of the Pasteur Institute in Cambodia. Mice were anesthetized by intraperitoneal injection of 75 mg/kg sodium pentobarbital prior to inoculation.
  • Culture virus packaging cell line 293FT was purchased from Invitrogen) as a complete DMEM medium [high sugar,
  • Virus 10% fetal bovine serum, 2 mM L-glutamic acid, 1 mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (100 g/ml); Invitrogen Life Technologies] contains 0.5 mg/ml of G418.
  • the culture medium of MDCK cells purchased from American Tissue Culture Company
  • the culture solution of Drosophila S2 cells was complete SFM containing 10% (v/v) FBS, 50 U/ml penicillin, 50 g. /ml streptomycin and 2 mM L-glutamic acid]
  • S2 cell culture temperature was 28 °C.
  • H5N1 viruses A/Shenzhen/406H/06 and A/Cambodia/P0322095/05 were obtained from Shenzhen Donghu Hospital and Pasteur Institute of Cambodia, respectively. Virus propagation in MDCK cells, virus-containing supernatant after the last aliquots stored at -80 ° C 32.
  • Half Computing Group infective dose the virus by serial dilution, and infected MDCK cells, calculate the median tissue infection dose 33 by Reed and Muench formula.
  • mice in each group were instilled with 50 ul of 10 times serial dilution of virus for 14 days, and mice with a weight loss of more than 35% were euthanized. Finally, the half-lethal amount is calculated by the Reed and Muench formula.
  • the H5 virus consists of 10 branches and 5 branches 2 sub-branches, wherein branches 0, 1, 2.1, 2.2, 2.3 and 7 are isolated from humans and the rest are isolated from birds. Construction of codon-optimized H5 virus and HI HA and flag tag Nl NA The method and method for producing influenza HA/NA pseudoviruses are described in the previously published article 34 '35 .
  • VSV-G-embedded pseudovirus A pseudovirus embedded in the VSV-G viral envelope protein.
  • Method of embedding please refer to the method described in Vaccine 27: 6777-6790 (2009).
  • HA is obtained by a conventional synthesis method.
  • a method for screening serum neutralizing antibodies during rehabilitation based on a pseudovirus neutralization assay and a method for screening supernatant neutralizing antibodies of Drosophila S2 transfected cell lines are as described above . Briefly, supernatants were incubated with sputum and sputum (eg A/Shenzhen/406H/06) coated pseudovirus for 1 hour at 37 ° C and then added to MDCK cells. After overnight incubation, the cells were washed once with PBS and supplemented with complete medium. After 48 hours, the luciferase activity was determined according to the procedure in the instructions of the BrightGlo Luciferase kit.
  • sputum and sputum eg A/Shenzhen/406H/06
  • the percent inhibition is calculated as: (relative value of luciferase in pseudovirus in complete culture - relative value of luciferase in pseudovirus containing complete dilution of antibody) / fluorescence of pseudovirus in complete culture
  • the relative value of the enzyme is XI 00%.
  • the virus was incubated with an equal volume serial dilution of human monoclonal antibody 65C6 at room temperature. Then add an equal volume of 0.5% chicken red blood cells and incubate for 30 minutes at room temperature. The red blood cells are small dots at the bottom of the hole, and the edges are smooth and tidy, which is considered to be inhibition of blood coagulation. Construction of a vector containing human immunoglobulin heavy and light chain constant regions and stable expression in Drosophila S2 cells facilitates cloning of human monoclonal antibodies. The B-cell total RNA transformed by Epstein-Barr virus was extracted and reverse transcribed into cDNA.
  • the fragments encoding the constant regions of the antibody ⁇ 1, ⁇ and ⁇ were amplified by PCR and ligated to the ⁇ cloning vector for sequencing.
  • the correct antibodies ⁇ and ⁇ were constant.
  • the fragment of the region was digested with Bglll and Pmel and ligated into the same pMT/Bip empty vector (purchased from Invitrogen) to obtain MT/Bip/ ⁇ constant, and pMT/Bip/ ⁇ constant plasmid.
  • the correct antibody ⁇ constant region fragment was digested with Xhol and Pmel and ligated into the same digested pMT/Bip empty vector to obtain pMT/Bip/ ⁇ constant vector.
  • the primer sequences for amplifying each constant region fragment are as follows:
  • CD22+ positive cells were isolated by magnetic beads coupled to the human anti-human CD22+ antibody by Milteny, the specific steps of which refer to the instructions for the product.
  • the isolated CD22+ cells were plated in a 96-well plate per 30 cells, supplemented with 10% fetal bovine serum in RPMI1640 cell culture medium, CpG 2006, Epstein-Barr virus and irradiated peripheral blood mononuclear cells as trophoblast cells. .
  • the cell supernatant was collected two weeks later, and positive clones were screened by a pseudovirus neutralization experiment. After a subcloning, the cells in the extracted RNA positive subclones, reverse transcribed into the cDNA, was amplified by PCR of the light chain and heavy chain antibody 38.
  • the amplified PCR product was ligated to the T-vector and digested with Sfil, BsiWI (kappa chain), Sffl Xhol ( ⁇ chain) and Sfil, Apal ( ⁇ chain) and ligated to the same digested PMT/Bip. On the carrier.
  • subclones still contain B cells that secrete other unrelated antibodies.
  • the inventors transformed a mixed plasmid containing the heavy chain of the antibody into Escherichia coli, and then plated, randomly picked out a single bacterial clone, and extracted the plasmid.
  • the S1 cells were transiently transfected with the mixed plasmid of the heavy chain of the individual antibody and the light chain, and after 3 days of induction by CdCl 2 , the supernatant was collected by the neutralization of the pseudovirus to select the correct heavy chain plasmid clone, and then correctly
  • the cloning plasmid was sent for sequencing to obtain the sequence of the heavy chain of the antibody.
  • the invention The human identified the gene sequence of the light chain of the antibody.
  • the plasmid containing the heavy and light chains of the antibody was co-transfected with SCo cells with pCoBlast (purchased from Invitrogen; with a blasticidin resistance gene). After 72 hours, 25 ug/ml blasticidin was added for screening and a stable cell line was obtained after two weeks.
  • the stably transfected cells were subjected to limiting dilution, and a high-yield monoclonal stable cell line was selected by measuring the amount of antibody in the supernatant of each subclone cell. Production and purification of human monoclonal antibodies produced by stable Drosophila S2 cells
  • Wave Bioreactor 20/50 EHT with a WAVEPOD Control Unit was used to produce human monoclonal antibodies. Briefly, 150 ml of expressed human monoclonal antibody (1 to 2 million per ml) was steadily transferred to the 1-L cell bag. The starting speed of the wave bioreactor was set to 22 rpm and the maximum angle of 8° was adjusted to 26 rpm at 9 degrees on the third day. The filtered air was passed through the cell culture bag at a rate of 0.15 L/min. The pH of the solution was between 6.0 and 6.3. Six days after the initial culture, perfusion began, and the perfusion rate gradually increased from 0.3 to 1.5 culture volumes (CV) per day to maintain a glucose concentration of not less than 4 g/l. After 10 days, 5 uM of chromium chloride was added to the cell culture solution. After 5 days of induction, the supernatant was collected.
  • CV culture volumes
  • the collected supernatant was centrifuged at 12,000 x g for 10 minutes at 4 ° C and filtered through a 0.45 ⁇ filter.
  • the filtered supernatant was concentrated 5 times in a QuixStand Benchtop system with a 50 KD Hollow Fiber Cartridge (Model UFP-50-C-4MA).
  • the concentrated supernatant was centrifuged at 12 000 x g for 4 minutes at 4 ° C and filtered through a 0.45 ⁇ filter.
  • a 5 ml pre-packed Protein G column was loaded by adding 1 mM of PMSF.
  • the eluted fractions were desalted by a HiTrap desalting column and finally the antibody was dissolved in PBS.
  • Antibody concentration was determined by the BCA method.
  • the enzyme-linked immunosorbent kit for detecting human antibody IgG was purchased from Mabtech AB (Sweden). For specific procedures, refer to the manufacturer's reagent instructions. Briefly, antibodies against human IgG were diluted to lug/ml in PBS pH 7.4 and added to 96-well enzyme-linked immunoplates overnight at 4 °C. The next day, the plate was washed with PBS and blocked with PBST containing 0.1% BSA for 1 hour. A certain proportion of the diluted cell culture supernatant or purified human monoclonal antibody was added to the well, and a human antibody standard from 0.1 to 500 ng/ml was also added to the well for 2 hours at room temperature.
  • VLP virus-like particle
  • HA/NA VLPs are virus-like particles expressing influenza virus HA/NA; HIV-1 VLPs are virus-like particles expressing HIV-1 envelope proteins; their preparation methods are described in Vaccine 27: 6777-6790, published in 2009. The method described in the article.
  • Membrane surface resonance (SPR) analysis was performed on a BIAcore T100 (Biacore AB, Sweden) instrument according to the manufacturer's instructions, and antibodies 3C11, 65C6, 100F4 and an unrelated TG15 antibody (for antibodies that do not recognize avian influenza virus) were used separately.
  • the amino coupling kit was immobilized on a CM5 chip, and the HA protein of the serially diluted (from 2090 nM to 84 nM) soluble recombinant A/Anhui/2017Ol strain was at 25 °C. Flowed through the chip surface for 180 s at a constant speed of 50 ⁇ /minute.
  • the data was processed by BIAcore T100 evaluation software (version 3.2). Detection of 65C6 antibody and HA complex by negative electron microscopy
  • Soluble hemagglutinin was digested from the H5N1 virus (A/Shenzhen/406H/06) according to the previously described method of purifying the enzymatic reaction with Bromelian 39 .
  • the digested soluble hemagglutinin forms an immune complex with the 65C6 antibody as previously described 39 .
  • soluble hemagglutinin first diluted to 50 ⁇ ⁇ / ⁇ 1 with PBS (pH 7.2) and applied to carbon film.
  • Antibody 65C6 was gradually added to the carbon film coated with soluble hemagglutinin until all of the soluble hemagglutinin complexed with the antibody. It was then transferred to another thin carbon sheet by blotting and dried in air.
  • the amount of antibody 65C6 is selected to be the minimum amount that can form a complex. The preventive and therapeutic effects of antibody 65C6 on highly pathogenic avian influenza in mice
  • mice 8 groups of female BALB/c mice (6 per group, 6 to 8 weeks, mean weight 20 g) were injected intraperitoneally with 50 ul of PBS containing 15 mg/kg, 5 mg/kg and 1 mg/kg. 65C6 or 15mg/kg of the control antibody TG15. After 4 hours, 24 mice were intranasally instilled with 50 ul of PBS containing 5 MLD 50 of A/Shenzhen/406H/06. Another 24 mice were nasally instilled with 50 ul of PBS containing 5 MLD 5 . HPAI H5N1 A/Cambodia/P0322095/05. In the following 14 days, mice were weighed daily, and survival was recorded. Mice with a body weight loss of more than 35% were euthanized. On the fourth day, one mouse per group was used to take tissue for histopathological section analysis.
  • mice 4 groups of female BALB/c mice (6 per group, 6 to 8 weeks, mean weight 20 g) were injected intranasally with 50 ul of PBS containing 5 MLD 5 .
  • A/Shenzhen/406H/06 The other 4 groups of female BALB/c mice were instilled with 50 ul of PBS containing 5 MLD 50 of HPAI H5N1 A/Cambodia/P0322095/05.
  • 1 ml of PBS was administered intraperitoneally with 40 mg/kg of 65C6 or 40 mg/kg of the control antibody TG15.
  • mice were weighed daily to record survival, mouse body Mice with a weight drop of more than 35% were euthanized. On day 4, each group of mice took his lung tissue for histopathological section analysis. Pathological analysis
  • the removed lung tissue is subjected to a predetermined treatment and sectioned.
  • the sections were fixed for HE staining to provide a basis for pathological analysis.
  • the inventors made a 5-fold serial dilution of A/Shenzhen/406H/06 virus stock solution with 2 ug/ml of 65C6 and 7.8 ug/ml.
  • the 100F4 was incubated at 37 ° C for one hour and then added to MDCK cells.
  • Cell cytopathic (CPE) was observed over the next 72 to 96 hours.
  • the virus supernatant in the wells in which the CPE appeared at the highest dilution of the virus was collected and the next round of passage was repeated as in the previous method.
  • 65C6 did not produce significant escape mutations, while 100F4 clearly produced escape mutations, and two 100F4 escape mutants were obtained by plaque assay.
  • the primers for amplifying the gene fragment of the heavy chain variable region, the kappa chain variable region, and the ⁇ chain variable region are as follows (in which the black cleavage indicates the cleavage site):
  • Amplification ⁇ chain break region (SEQ ID NO: )
  • a series of antibody heavy and light chain pair co-transfection experiments were then performed in Drosophila S2 cells to identify pairs of heavy and light chains that produced potent neutralizing antibodies.
  • Six supernatants were found to have 99% neutralization activity from the supernatant of approximately 16,000 Epstein-Barr virus-transfected B cells.
  • Three strains of monoclonal antibodies capable of secreting 65C6, 100F4 and 3C1 1 were identified from Drosophila S2 cell lines transfected with hundreds of heavy light chain pairs.
  • a Drosophila S2 cell line expressing a TG15 human monoclonal antibody against HIV-lgp41 was also prepared for use in a negative control.
  • the preparation method is the same as that of the Drosophila S2 cell line expressing 65C6.
  • the heavy chain variable regions of the three antibodies 65C6, 100F4 and 3C11 are 5-a*03 5-a*03 and 4-61*03, respectively.
  • the light chain variable regions of the three antibodies 65C6, 100F4 and 3C11 are VK3D, respectively. -15*01, VK2D-28*01 and ⁇ 1-40*01, the VH and VL chain protein sequences of the antibodies are shown in Table 2.
  • the amino acid sequence of the heavy chain (VH) of 65C6 is SEQ ID NO: 1; and the amino acid sequence of the light chain (VL) is SEQ ID NO: 2.
  • the amino acid sequence of the heavy chain (VH) of 100F4 is SEQ ID NO: 3; the amino acid sequence of the light chain (VL) is SEQ ID NO: 4.
  • the amino acid sequence of the 3C11 heavy chain (VH) is SEQ ID NO:
  • the amino acid sequence of the light chain (VL) is SEQ ID NO: 6.
  • the sequence numbers of their CDR regions are shown in Table 2.
  • SEQ ID NO: 7 SEQ ID NO: 8 FR3 CDR3 FR4 ALLTTVTTFEY
  • FIG. 9 shows the results of trypan blue staining of purified antibodies of 65C6, 100F4, 3C11 and TG15.
  • the stained bands of the heavy chain (; 50 kDa) and light chain (24-26 kDa) of the antibody are clearly visible and of high purity.
  • Example 2 Human monoclonal antibody 65C6, 100F4 and 3C11 antigen specificity and affinity experiments
  • Western blotting was used to detect antigen-specific experiments of human monoclonal antibodies.
  • HIV-1, HA and NA virus-like particles were transfected with SDS/PAGE and then with PVDF membrane, and then with antibodies 65C6, 100F4, 3C11 and TG15.
  • the reaction was carried out and the specificity of the antibody was analyzed according to the blot.
  • the negative control antibody TG15 as shown in Figure lb specifically binds to the envelope protein on the HIV-1 virus but does not bind to HA and NA on the influenza virus.
  • the immune serum (Immune sera) of the mouse used in the positive control was specific to HA on the influenza virus.
  • HAj and HA 2 bind but not bind to envelope proteins on HIV-1 viruses.
  • Antibodies 65C6, 100F4 and 3C11 are specific for HA. It binds to HAi but does not bind to the envelope proteins of HA 2 and HIV-1. This suggests that the epitopes recognized by antibodies 65C6, 100F4 and 3C11 are in the HAi region of influenza hemagglutinin proteins.
  • the method of surface plasmon resonance is used to determine the affinity of antigen antibodies.
  • the results are shown in Figure lc, showing the binding and free curves of different concentrations of hemagglutinin to antibodies 100F4, 65C6 and 3C11.
  • the affinity (KD) of 100F4, 65C6 and 3C11 antibodies to hemagglutinin was estimated to be 2.42 ⁇ 1 ( ⁇ 9 , 4.14 X 1 ( ⁇ 8 and 7.02 X 1 ( ⁇ 8 , see Table 3.
  • this The inventors concluded that the 100F4, 65C6 and 3C11 antibodies have good affinity for hemagglutinin.
  • Figure 3 and Table 4 are the results of the neutralization activity test of the antibodies 100F4, 65C6, 3C11 and TG15 against 19 all H5N1 and 1 H1N1 subclass pseudoviruses and VSV-G embedded pseudoviruses, negative control antibodies TG15 has no neutralizing activity against 19 H5N1 and 1 H1N1 subclass pseudoviruses and VSV-G embedded pseudoviruses.
  • Antibody 3C11 has good neutralizing activity against four H5N1 pseudoviruses (A/Hong Kong/156/97, A/Turkey/65-595/2006, A/Xingjiang/1/2006 and A/Beijing/01/2003) (IC95 values were 0.516, 4.04, 5.612 and 3.465 g/ml, respectively).
  • antibody 100F4 neutralized all 19 H5N1 subclass pseudoviruses well.
  • the neutralizing rate of the antibody 100F4 to the six H5N1 pseudoviruses is up to Up to 95%; at a concentration less than lg/ml, the neutralizing rate of antibody 100F4 against 13 H5N1 pseudoviruses can reach 95%, and the concentration required for the remaining 6 H5N1 pseudoviruses to reach 95% It is between 1.022 and 8.122 g/ml.
  • the binding rate of antibody 65C6 to hemagglutinin was lower than that of antibody 100F4 (as shown in Table 4), the neutralizing activity was higher than that of antibody 100F4.
  • the neutralizing rate of antibody 65C6 to 16 H5N1 pseudoviruses can reach 95%; at a concentration less than lg/ml, antibody 65C6 neutralizes 17 of the H5N1 pseudoviruses The rate was 95%, and the concentration required to achieve 95% neutralization of the remaining two H5N1 pseudoviruses was only 1.085 g/ml and 1.528 g/ml (Table 4). The inventors thus concluded that antibody 65C6 can efficiently neutralize all 19 H5N1 subclass pseudoviruses.
  • the inventors also performed a hemagglutination inhibition test (Table 5), and the results showed that the antibody 65C6 completely inhibited all at a concentration of 0.3 g/ml and 2.7 g/ml. Hemagglutination activity of six H5N1 viruses; however, this antibody did not inhibit the hemagglutination activity of H1N1, H2N2 and H3N2 viruses. From this it can be concluded that the neutralizing epitope recognized by antibody 65C6 is shared by HA of all H5 subtypes, but not in HA of H1, H2 and H3 subtypes.
  • the inventors used antibodies to screen for escaped mutants. After one or two generations of 100F4 antibody screening, escape strains can be detected; as the number of passages increases, the activity of the anti-100F4 antibody mutant is more and more strong; after 11 generations, the antibody concentration is 1600 ⁇ ⁇ / ⁇ 1 In the case of the mutant strain can also escape. Subsequently, two mutant strains were cloned by plaque method and their complete sputum sequences were tested and compared. It was found that the mutant strains could escape when the antibody concentration was 1600 ⁇ ⁇ / ⁇ 1. One of the mutant strains has eight single amino acid mutations in the ⁇ sequence, six of which are in the HA1 region.
  • Another mutant strain has 10 single amino acid mutations, 8 of which are in the HA1 region.
  • the mutation sequence common to both mutants has 6 amino acids in the HA1 region at positions 68, 120, 127, 195, 209 and 313, respectively.
  • escape strain 1 contains 8 single amino acid mutations, 6 of which are in the HA1 region; escape strain 2 has 10 amino acid mutations, 8 of which are in the HA1 region.
  • escape strain 2 has 10 amino acid mutations, 8 of which are in the HA1 region.
  • HA1 there are 6 amino acid mutations shared by these two escaped strains, which are at positions 68, 120, 127, 195, 209 and 313, respectively, suggesting that the above six mutations are related to the recognition of 100F4 (see Figure 4). ).
  • mice were intraperitoneally injected with 15 mg/kg, 5 mg/kg and 1 mg/kg of 65C6 antibody (concentration 200 mg/ml, purity greater than 95%) and 15 The mg/kg control antibody TG15 was injected into the mice, and 5 MLD 5 were added after 4 hours.
  • HPAI H5N1 A/Cambodia/P0322095/05 was injected into the lower respiratory tract of the small ⁇ f via nasal drops.
  • Five MLD 5 o highly pathogenic avian influenza H5N1 A/Shenzhen/406H/06 and HPAI H5N1 A/Cambodia/P0322095/05 were selected to demonstrate that the dose had a mortality rate of 100% in the control group.
  • Figures 5a and b show changes in body weight and survival of mice within 14 days after HPAI H5N1 A/Shenzhen/406H/06 virus inoculation
  • Figures 5c and d show small within 14 days of HPAI H5N1 A/Cambodia/P0322095/05 virus inoculation.
  • Rat weight change and survival rate Injection of control antibody TG15 mice infected with H5N1 A/Shenzhen/406H/06 showed significant disease symptoms and weight loss from 3 days, and all 5 mice died on 8-1 1 day. In contrast, mice injected with 1 mg/kg of 65C6 antibody showed significant disease symptoms and weight loss 4-6 days, and 2 mice died and 3 survived on days 11 and 13.
  • mice injected with 5 mg/kg of 65C6 antibody developed disease symptoms at 5-7 days, but weight loss was not significant. One mouse died at 11 days and the remaining 4 survived. However, mice injected with 15 mg/kg of 65C6 antibody showed no disease symptoms and weight loss, and all survived.
  • mice infected with H5N1 A/Cambodia/P0322095/05 showed significant disease symptoms and weight loss from 3 days, with all 5 mice dying on days 8-11.
  • mice injected with 1 mg/kg of 65C6 antibody showed significant disease symptoms and weight loss 4-6 days, and 1 mouse died and 4 survived at 10 days.
  • mice injected with 5 mg/kg and 15 mg/kg 65C6 antibody showed no disease symptoms and weight loss, and all survived.
  • TG15 antibody-treated mice showed significant pathological changes in lung inflammation 4 days after infection including alveolar wall thickening, inflammatory cell infiltration, and vasodilatation and hyperemia (see figure 6d and 6h).
  • mice injected with 1 mg/kg of 65C6 antibody had a small amount of inflammatory response, and alveolar wall thickening, inflammatory cell infiltration, and vasodilatation and hyperemia were not significant (6c).
  • injection of 5 mg/kg and 15 mg/kg of 65C6 antibody did not show any inflammatory response in the H5N1 A/Shenzhen/406H/06 group.
  • injecting 1 mg/kg, 5 mg/kg and B 15 mg/kg 65C6 antibody did not show any inflammatory reaction in the HPAI H5N1 A/Cambodia/P0322095/05 infected group.
  • Example 6 in vivo therapeutic effect of antibody 65C6
  • mice will have 5 MLD 5 via nasal drops.
  • Highly pathogenic avian influenza H5N1 A/Shenzhen/406H/06 Hessian HPAI H5N1 A/Cambodia/P0322095/05 was injected into the upper respiratory tract of mice, 24, 48 and 32 hours later, 40 mg/kg of 65C6 antibody, 40 mg/ Kg and control antibody TG15 were injected into the peritoneal cavity of mice.
  • Figures 7a and b show changes in body weight and survival of mice within 14 days after HPAI H5N1 A/Shenzhen/406H/06 virus inoculation
  • Figures 7c and d show small within 14 days of HPAI H5N1 A/Cambodia/P0322095/05 virus inoculation.
  • Rat weight change and survival rate The mice injected with the control antibody TG15 after HPAI H5N1 A/Shenzhen/406H/06 and HPAI H5N1 A/Cambodia/P0322095/05 infection showed obvious disease symptoms, weight loss, and all mice died in 8-10 days. .
  • mice injected with 65C6 antibody 24, 48 and 72 hours after infection with HPAI H5N1 A/Cambodia/P0322095/05 showed no significant disease symptoms and weight loss, and all mice survived.
  • 65C6 antibody was injected except for one mouse, and all mice survived without any disease symptoms and weight loss.
  • mice treated with TG15 antibody 24 hours after infection showed obvious pathology of pulmonary inflammation after 4 days of infection. Changes include alveolar wall thickening, inflammatory cell infiltration, and vasodilatation hyperemia (see Figures 8b and d). In contrast, the group of mice treated with the 65C6 antibody 24 hours after infection did not show any significant inflammatory response (see Figures 8a and c).
  • the antibody of the present invention can be used for the treatment of a broad-spectrum viral infection of H5N1, and the present invention is merely illustrative of the therapeutic effect of the antibody of the present invention on the viral infection of each H5N1 branch (dade). Those skilled in the art understand that the antibody of the present invention can also be applied to the same type of H5N1.
  • viruses in the branch including but not limited to 0 branch A/Chicken/Hong Kong/317.5/2001 A/Chicken/Hong Kong/728/97 A/chicken/Hubei/wf/2002, etc., 1 branch A/ Chicken/Kohn Kaen/NIAH330/2004 A/chicken/Phichit/NIAH6-4-0001/2006, etc., 2.1 branch A/Chicken/West Java/GARUT-MAY/2006, A/Duck/Bufeleng/BPP V 1/2005 , A/Duck/Pali/BB VW 1358/2005, etc., 2.2 branches of A/duck/Romania/TL/nov/2007, A/duck/Switzerland V389/2006, A/eagle owl/Sweden/V1218/2006, etc.
  • FIG 9 shows the complex of negatively stained HA and antibody 65C6 observed under electron microscopy and a schematic diagram thereof.
  • Each antibody molecule binds to two HAs.
  • the Fab segment of each antibody binds to the end of the HA and forms a fixed 1 10 degree angle at HA.
  • Figure 9d shows that five HA molecules end to each other to form a polymer, and the antibody molecule binds to the other end of the two HAs in the polymer.
  • yeast display based on region-level and fine-epitope levels was applied to the identification of neutralizing epitopes of antibody 65c6, and yeast display methods at the fine epitope level have been reported previously.
  • yeast display methods at the fine epitope level have been reported previously.
  • the inventors By sequencing analysis of PE-staining negative yeast clones, the inventors identified mutations of 23 individual amino acids that disrupted the binding of antibody 65C6 to HA. From the three-dimensional structure of HA, 13 of them are located on the surface of the HA protein at positions 1, 16, 117, 1 18, 121, 147, 152, 160, 161, 162, 163, 164, 167 and 187. On the other hand, the other 10 amino acid mutations are buried in the HA protein, meaning that these amino acids buried in the protein are not directly in contact with the antibody 65C6. See Figure 10A.
  • the pseudovirus formed by the HA mutation at positions 117, 1 18, 121, 161, 162, 164 and 167 is more tolerant to the original strain of antibody 65C6 (at the inhibition rate of IC95, HA 117 and 162)
  • the mutated pseudovirus increased the tolerance of the antibody by a factor of 2
  • the pseudovirus of the 121st and 161th mutations of the HA increased the tolerance of the antibody by more than 8 fold, see Figure 10B.
  • the positions of the corresponding HA mutant amino acids of all tolerant pseudoviruses are adjacent in the three-dimensional structure of the HA protein, as shown in Figure 1 ( ⁇ and 1).
  • subtype 7.1 HA The corresponding five amino acids in subtype 7.1 HA were replaced with the corresponding amino acids in A/Chicken/Vietnam/NCVD-016/08 7.2 subtype HA, and all five amino acids replacing the subtype 7.2 were constructed. Mutations and use these mutations to package the pseudovirus.
  • Figures 10E and F show the neutralizing activity of antibody 65C6 on these pseudoviral mutations.
  • a pseudovirus with a single amino acid mutation at position 159, 163 or 165 on HA appears to be more susceptible to neutralization by antibody 65C6 compared to the A/Beijing/01/2003 7.1 subtype of the original strain.
  • pseudoviruses with mutations at position 162 or 121 on HA were more tolerant to neutralization of 65C6 (antibody concentrations increased by 1.26 and 3.37, respectively, at IC80 inhibition). It is noteworthy that the tolerance of the antibody 65C6 when the five amino acids were mutated together was greatly enhanced compared to the single point mutation at positions 121 and 162 (Fig. 10F).
  • amino acids at positions 121 and 162 on HA are in the epitope recognized by antibody 65C6, and these two amino acids are better able to be antibody 65C6 in the framework of subtype 7.2. Identified.
  • the present invention has two important findings. One is that the 65C6 antibody has a high neutralizing effect on all 10 branches and 5 subclasses of the highly pathogenic avian influenza H5N1 virus, and the 11th generation in vitro 65C6 antibody screening has not been found. Escaped mutant strain. These results indicate that the neutralizing epitope recognized by the 65C6 antibody includes all H5N1 strains, and that the neutralizing epitope recognized by the 65C6 antibody is difficult to mutate, possibly because the mutation of the epitope will affect the survival of the virus strain itself.
  • the 65C6 antibody is isolated from memory B cells of a convalescent patient infected with the H5N1 virus, this neutralization reaction of the 65C6 antibody is based on a human neutralizing antibody that naturally infects and obtains immunity, and thus The vaccine produced by the neutralizing epitope will not only have neutralizing activity against the H5N1 strain circulating in humans but also have a good neutralizing activity against the H5N1 strain which is currently likely to spread to humans in the future of avian transmission.
  • the 65C6 antibody has a good preventive and therapeutic effect on highly pathogenic avian influenza.
  • Intraperitoneal injection of 5 mg/kg of 65C6 antibody protects mice from lethal doses of highly pathogenic avian influenza H5N1 virus, even after intraperitoneal injection of the antibody 72 hours after infection with highly pathogenic avian influenza H5N1 virus in mice The mice can be made to survive without weight loss. Therefore, the 65C6 antibody has great potential for treating human or zoonotic H5N1 virus infection.
  • Plasma has a better effect 2 ' 21 ' 24 ' 4Q ' 41.
  • human monoclonal antibodies have two advantages, one can be mass-produced, in fact found in the present invention more than lg / L / d Single gram
  • the antibody can be produced by Drosophila S2 cells using a wave bioreactor and perfusion culture method, and the other is that the antibody does not react to the external antigen present in human plasma, and the application of human antibodies replaces other sources. Immunological rejection has also been greatly reduced since antibodies have been used to treat the disease.
  • the epitope recognized by the 65C6 antibody is different from the epitopes recognized by the recently discovered group-specific antibodies C179 42 , CR6261 24 ' 42 and F10 23 , and the epitope recognized by the latter three antibodies is located in HA 2 .
  • the neck area is 23 ' 24 ' 42 .
  • the present inventors successfully screened three effective human monoclonal antibodies 15 from memory B cells of H5N1-recovered patients using the H5N1 pseudovirus strain neutralization assay technique and molecular cloning technology.
  • the neutralizing epitope recognized by the 65C6 antibody is located in the head region of HA1 and the antibody has good neutralizing ability to all branches of the H5N1 virus and has a good preventive and therapeutic effect in mice, and the present invention Human in vitro experiments have shown that this epitope is difficult to mutate 43 '44 . Therefore, on the one hand, the 65C6 antibody alone or in combination with other small molecule inhibitors has great potential for the treatment of infections caused by various branches of H5N1.

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Abstract

提供了一种高致病性禽流感病毒的结合分子及其制备方法，该结合分子对禽流感病毒具有良好的中和作用。还公开了该结合分子在禽流感病毒血球凝集素上的结合位点。

Description

高致病性禽流感的中和分子及其制备方法
技术领域
本发明属于生物技术和免疫学领域； 更具体地， 本发明涉及高致病性禽流感的中和 分子及其制备方法。 背景技术
自 1997年以来高致病性禽流感 H5N1病毒已感染了约 5亿只禽类， 同时在亚洲、 欧 洲和非洲已有越来越多的人被感染。 虽然， 目前为止人类的感染都是由禽类传播的， 但 H5N1 病毒经过重组和进化可能演变出来具有人传人能力的新的病毒株。 这种新病毒的 广泛蔓延加之人们对 H5N1病毒缺少预成的免疫力将会对人类造成重大的发病率和死亡 碎- 感染了高致病性禽流感 H5N1 病毒的主要表现是严重的肺炎、 淋巴细胞减少、 高淋 巴因子血症及呼吸道的高病毒载量 2_ό。 病毒通常可以从患者的脑脊液、 粪便、 痰液和血 清样本中培养出来。 目前对此病的治疗主要是依靠抗病毒药物， 但一些 H5N1病毒株可 以对三环癸胺类离子通道阻滞剂药物产生耐药性 7。 虽然奧塞米韦等神经氨酸抑制剂对 季节性流感有一定的疗效但对于 H5N1病毒的效果还存在争议。 动物实验表明神经氨酸 抑制剂药物的疗效只有在感染前或感染后瞬间给药才能发挥疗效 2， 且 H5N1 病毒对奧 塞米韦等神经氨酸抑制剂类药物也可产生耐药性 8。 因此寻找能有效的治疗禽流感和控 制禽流感在人类中传播的方法是急需的。
应用单克隆抗体和多克隆抗体的抗体疗法已有效的应用于甲肝、 乙肝、 狂犬病、 水 痘及巨细胞病毒感染等多种疾病的治疗 9。 婴儿通过后天的抗体免疫也可获得针对流感 病毒的免疫力 1(Μ3。从 1918年西班牙流感大流行的幸存者体内所分离得到的单克隆抗体 可以有效的将流感的死亡率降低 50%14。 输入感染过 H5N1 的康复病人的血浆可以有效 的降低 H5N1病毒感染病人的病毒载量并可以完全康复 15。 将免疫小鼠、 雪貂、 马和人 获得的流感抗体打入小鼠体内可以有效的预防和治疗流感 1ό— 25。最近， Koudstaal等人发 现小鼠单次注射 15毫克 /公斤的人单克隆抗体 CR6261 比感染后连续 5天注射 10毫克 / 公斤 /天的奧塞米韦更能有效预防和治疗致命的 H5N1和 H1N1感染 2ό。 因此， 应用被动 免疫获得的抗体治疗高致病性禽流感 H5N1病毒人类感染性疾病将是一种有效可行的方 法。
血凝素基因 (ΗΑ)是禽流感病毒基因组中变异最大的基因。 从 ΗΑ 的序列方面看， 自
2000来有 10个 Η5ΗΑ的分支在不同的物种中出现 11。 其中分支 2又可分为 5亚分支。 亚分支 2.3又可分为 2.3.1、 2.3.2、 2.3.3和 2.3.4四个亚亚分支 28。 目前为止感染人类的 高致病性禽流感 H5N1病毒分为 0、 1、 2和 7分支， 在中国比较流行的感染人的高致病 性禽流感 H5N1病毒属于 2.3.4亚亚分支 27' 28。 此外， 在东南亚和东亚感染家禽和鸟类 的高致病性禽流感 H5N1病毒也属于 2.3.4亚亚分支 29。 研究显示， 在人类 Η5ΗΑ至少 有四种不同的抗原 3Q。
此外， 流感病毒通过其遗传漂移和重组来逃逸免疫监视的特性使其一直以来是公共 卫生健康的重大威胁， 导致临床上常用的两类抗病毒药物对其效果都不是很理想， 且有 耐药株出现， 因此寻找新的有效的治疗方法是急需的。
综上， 鉴于禽流感的高变异性， 寻找对尽可能多的禽流感变异病毒株均具有良好的 中和能力的中和分子是非常必要的。 发明内容
本发明的目的在于提供高致病性禽流感的中和分子及其制备方法。
在本发明的第一方面， 提供一种结合分子， 其识别禽流感病毒的血球凝集素 HA1， 并结合于血球凝集素 N端区域上的表位上， 该表位包含以下位点：
血球凝集素氨基酸序列第 121位的 Ser; 和
血球凝集素氨基酸序列第 162位的 Arg。
在另一优选例中， 所述的表位还包含以下位点：
血球凝集素氨基酸序列第 1 17位的 lie;
血球凝集素氨基酸序列第 1 18位的 Pro ;
血球凝集素氨基酸序列第 161位的 Lys;
血球凝集素氨基酸序列第 164位的 Tyr; 或
血球凝集素氨基酸序列第 167位的 Thr。
在另一优选例中， 所述的 N端区域是血球凝集素氨基酸序列第 51〜260位氨基酸区 域。
在另一优选例中，所述结合分子 (例如 65C6或其类似物)包含 SEQ ID NO: 7所示的重 链 CDR1， SEQ ID NO: 8所示的重链 CDR2， SEQ ID NO: 9所示的重链 CDR3。
在另一优选例中， 所述结合分子 (例如 65C6或其类似物)包含 SEQ ID NO: 10所示的 轻链 CDR1， SEQ ID NO: 1 1所示的轻链 CDR2， SEQ ID NO: 12所示的轻链 CDR3。
在另一优选例中，所述结合分子 (例如 65C6或其类似物)包含 SEQ ID NO: 7所示的重 链 CDR1， SEQ ID NO: 8所示的重链 CDR2， SEQ ID NO: 9所示的重链 CDR3 ; 以及 SEQ ID NO: 10所示的轻链 CDR1， SEQ ID NO: 1 1所示的轻链 CDR2， SEQ ID NO: 12所示 的轻链 CDR3。
在另一优选例中， 所述的结合分子 (例如 65C6或其类似物)包含重链可变区， 该重链 可变区具有 SEQ ID NO: 1所示的氨基酸序列。
在另一优选例中， 所述的结合分子 (例如 65C6或其类似物)包含轻链可变区， 该轻链 可变区具有 SEQ ID NO: 2所示的氨基酸序列。
在另一优选例中， 所述的结合分子 (例如 65C6或其类似物)包含：
重链可变区， 该重链可变区具有 SEQ ID NO: 1所示的氨基酸序列； 以及 轻链可变区， 该轻链可变区具有 SEQ ID NO: 2所示的氨基酸序列。
在另一优选例中，所述结合分子包含 SEQ ID NO: 13所示的重链 CDR1， SEQ ID NO: 14所示的重链 CDR2， SEQ ID NO: 15所示的重链 CDR3 ; 和 /或
包含 SEQ ID NO: 16所示的轻链 CDR1， SEQ ID NO: 17所示的轻链 CDR2， SEQ ID NO: 18所示的轻链 CDR3。
在另一优选例中， 所述的结合分子 (例如 100F4或其类似物)包含重链可变区， 该重链 可变区具有 SEQ ID NO: 3所示的氨基酸序列。
在另一优选例中， 所述的结合分子 (例如 100F4或其类似物)包含轻链可变区， 该轻链 可变区具有 SEQ ID NO: 4所示的氨基酸序列。
在另一优选例中， 所述的结合分子包含：
重链可变区， 该重链可变区具有 SEQ ID NO: 3所示的氨基酸序列； 以及
轻链可变区， 该轻链可变区具有 SEQ ID NO: 4所示的氨基酸序列。
在另一优选例中，所述结合分子包含 SEQ ID NO: 19所示的重链 CDR1， SEQ ID NO: 20所示的重链 CDR2， SEQ ID NO: 21所示的重链 CDR3 ; 禾口 /或
包含 SEQ ID NO: 22所示的轻链 CDR1， SEQ ID NO: 23所示的轻链 CDR2， SEQ ID
NO: 24所示的轻链 CDR3。
在另一优选例中， 所述的结合分子 (例如 3C1 1或其类似物)包含重链可变区， 该重链 可变区具有 SEQ ID NO: 5所示的氨基酸序列。
在另一优选例中， 所述的结合分子 (例如 3C1 1或其类似物)包含轻链可变区， 该轻链 可变区具有 SEQ ID NO: 6所示的氨基酸序列。
在另一优选例中，所述的结合分子包含：重链可变区，该重链可变区具有 SEQ ID NO: 5所示的氨基酸序列； 以及轻链可变区， 该轻链可变区具有 SEQ ID NO: 6所示的氨基酸 序列。
在另一优选例中， 所述的结合分子是人单克隆抗体、 Fab、 F(ab' )、 F(ab' )2、 Fv、 dAb、 Fd、 互补决定区 (CDR)片段、 单链抗体 (scFv)、 二价单链抗体、 单链噬菌体抗体、 双特异双链抗体、 三链抗体、 四链抗体； 优选的， 所述的结合分子是人单克隆抗体； 更 优选的， 所述的人单克隆抗体其重链恒定区选择下组中重链类型之一的恒定区： IgGl、 IgG2a、 IgG2b和 IgG3， 和其轻链恒定区选择下组轻链类型的恒定区之一： κ链和 λ链； 更优选的， 所述的人单克隆抗体其重链恒定区和轻链恒定区分别具有 Genebank 号 ACK87036和 ACK87038所示的氨基酸序列。
在另一优选例中， 在所述结合分子的重链或轻链中， 所述的 CDR1、 CDR2和 CDR3 区从氨基酸至羧基端依次串联排列。
在另一优选例中，所述的 CDR1之前， CDR1与 CDR2之间， CDR2与 CDR3区之间， CDR3之后，还包括框架区；较佳地，所述的框架区的氨基酸长度为 6-40个；较佳地 8-35 个； 更佳地 10-32个。 在本发明的另一方面， 提供一种多核苷酸， 它编码前面任一所述的结合分子。
在本发明的另一方面， 提供一种表达载体， 所述表达载体中含有：
编码前面任一所述的结合分子的重链的多核苷酸； 和 /或
编码前面任一所述的结合分子的轻链的多核苷酸。
在本发明的另一方面， 提供一种宿主细胞， 所述宿主细胞中含有所述的表达载体； 或其基因组中整合有所述的多核苷酸。
在另一优选例中， 所述的宿主细胞是果蝇 S2细胞。
在本发明的另一方面， 提供一种制备前面任一所述的结合分子的方法， 所述方法包 括： 培养前面所述的宿主细胞， 从而表达所述的结合分子。
在本发明的另一方面， 提供所述的结合分子的用途， 用于制备预防、 缓解或治疗禽 流感病毒感染的组合物 (；如药物)。
在另一优选例中， 所述的禽流感病毒是 H5亚型的病毒。
在另一优选例中， 所述的禽流感病毒是 H5N1病毒。
在另一优选例中， 所述的禽流感病毒是除 H5N1的 7.2分支外的 H5亚型的病毒； 较 佳地为除 H5N1的 7.2分支外的 H5N1病毒。
在本发明的另一方面， 提供一种药物组合物， 它含有有效量的前面所述的结合分子， 以及药学上可接受的载体。
在另一优选例中， 所述的药物组合物还含有有效量的其它抗流感药物， 选自： 烷胺 类药物或流感病毒神经氨酸酶抑制剂。
在另一优选例中， 所述的烷胺类药物包括金刚烷胺或金刚乙胺； 或所述的流感病毒 神经氨酸酶抑制剂包括： 奧司他韦或扎那米韦。
在本发明的另一方面， 提供前面任一所述的结合分子的用途， 用于制备鉴定禽流感 病毒的试剂或试剂盒。
在本发明的另一方面， 提供一种预防、 缓解或治疗禽流感病毒感染的方法， 所述的 方法包括给予患者有效量的前面任一所述的结合分子。
在本发明的另一方面， 提供一种鉴定禽流感病毒的方法， 所述方法包括： 将前面任 一所述的结合分子与待检测样品接触， 观察所述的结合分子与待检测样品的结合情况， 若所述的结合分子与待检测样品发生结合， 则该样品中存在禽流感病毒。
在本发明的另一方面， 提供一种抗禽流感病毒的免疫原 (疫苗)， 其包含有一段能与前 面任一所述的结合分子结合的抗原表位。
在另一优选例中， 所述的抗原表位包含以下位点：
相对于血球凝集素的氨基酸序列第 121位的 Ser; 和
相对于血球凝集素的氨基酸序列第 162位的 Arg。
在另一优选例中， 所述的抗原表位还包含以下位点：
相对于血球凝集素的氨基酸序列第 117位的 lie; 相对于血球凝集素的氨基酸序列第 118位的 Pro;
相对于血球凝集素的氨基酸序列第 161位的 Lys;
相对于血球凝集素的氨基酸序列第 164位的 Tyr; 或
相对于血球凝集素的氨基酸序列第 167位的 Thr。
在另一优选例中， 所述的免疫原不包括全长的禽流感 H5N1病毒的血球凝集素。 本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 la、 抗体表达载体的构建示意图。 其中， MT-P表示 MT启动子， Bip表示信号肽 编码区； VL- λ表示轻链 λ可变区； VL- κ表示轻链 κ可变区； VH表示重链可变区； CL- 入 1表示轻链人 1恒定区； CL- K 1表示轻链 κ 1恒定区； CH- γ 1表示重链 γ 1恒定区； poly-A为含有表达腺嘌吟核苷酸链的序列。
图 lb、 SDS/PAGE电泳鉴定抗体的识别区域。
图 lc、 不同浓度的血凝素与抗体 100F4、 65C6和 3C11的结合和游离曲线。
图 2、 65C6、 100F4、 3C11和 TG15纯化抗体的台盼蓝染色结果。 其中， HC表示重 链的条带， LC表示轻链的条带。
图 3、 抗体 100F4、 65C6、 3C1 1和 TG15对 19个所有 H5N1禾 Π 1个 H1N1亚类的假 病毒以及 VSV-G包埋假病毒的中和活性测试的结果。 以抗体 TG15作为阴性对照。
图 4、 来自于野生型 A/Shenzhen/406H/06与其两株 100F4逃逸株变体中 H5 HA蛋白 序列的对比结果。
图 5a-b、 HPAI H5N1 A/Shenzhen/406H/06病毒接种后的 14天内小鼠的体重改变和存 活率。
图 5c-d、HPAI H5N1 A/Cambodia/P0322095/05病毒接种后的 14天内小鼠的体重改变 和存活率。
图 6、感染 H5N1 A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/P0322095/ 054天后 的肺部组织进行病理切片。 其中，
a、给予 15mg/kg 65C6抗体的经 H5N1 A/Shenzhen/406H/06感染的小鼠肺部组织病理 切片；
b、 给予 5mg/kg 65C6抗体的经 H5N1 A/Shenzhen/406H/06感染的小鼠肺部组织病理 切片；
c、 给予 lmg/kg 65C6抗体的经 H5N1 A/Shenzhen/406H/06感染的小鼠肺部组织病理 切片；
d、 给予 15mg/kg TG15抗体的经 H5N1 A/Cambodia/P0322095/05感染的小鼠肺部组 织病理切片；
e、给予 15mg/kg 65C6抗体的经 H5N1 A/Cambodia/P0322095/05感染的小鼠肺部组织 病理切片；
f、 给予 5mg/kg 65C6抗体的经 H5N1 A/Cambodia/P0322095/05感染的小鼠肺部组织 病理切片；
g、 给予 lmg/kg 65C6抗体的经 H5N1 A/Cambodia/P0322095/05感染的小鼠肺部组织 病理切片；
h、 给予 15mg/kg TG15抗体的经 H5N1 A/Cambodia/P0322095/05感染的小鼠肺部组 织病理切片。
图 7a-b、 HPAI H5N1 A/Shenzhen/406H/06病毒接种后的 14天内小鼠的体重改变和存 活率。
图 7c-d、HPAI H5N1 A/Cambodia/P0322095/05病毒接种后的 14天内小鼠的体重改变 和存活率。
图 8a和 c、 感染 H5N1 A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/ P0322095/05 感染后 24小时经 65C6抗体处理的小鼠组在感染 4天后未出现任何明显的炎症反应。
图 8b和 d、感染 H5N1 A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/ P0322095/05 感染后 24小时经 TG15抗体处理的小鼠在感染 4天后出现明显的肺部炎症病理改变包 括肺泡壁增厚、 炎症细胞浸润和血管扩张充血。
图 9、 电镜下观察到的经负染的 HA和抗体 65C6的复合物及其示意图。
a、 一个抗体与两个 HA分子形成的复合体；
b、 一个抗体与两个 HA分子形成的复合体；
c、 一个抗体与两个 HA分子形成的复合体；
d、 一个抗体与五个 HA分子形成的复合体， 五个 HA分子 C端连接形成 Rossett结 构， 由此推测抗体 65C6是结合在 HA的 N端；
e、 两个抗体与两个 HA分子形成的复合体；
每个抗体的 Fab段与 HA分子结合时都形成固定的 105度的角度。
图 10、 涉及到中和表位的氨基酸。
A、 血球凝集素 (HA)上 23个单氨基酸的突变位点。 这些单个氨基酸的突变能够使酿 酒酵母表面展示的带有该单个氨基酸突变的血球凝集素的 51-260 个氨基酸的片段失去 和抗体 65C6的结合能力。而这其中有 10个突变的氨基酸从血球凝集素的三维结构上看 是埋在里面的， 而另外 13个突变的氨基酸是暴露在表面的。
B、 对 13个单个氨基酸发生突变的 HA形成的假病毒， 要达到 95%的中和效果所需 要的抗体 65C6 的浓度以及相对于中和原始株抗体浓度增加的倍数。 红色所示为对单克 隆抗体 65C6的中和更加耐受的单个氨基酸的突变。
C、通过酵母展示和假病毒中和试验所鉴定出来的 7个分别位于 H5N1的 7.1亚型的 A/Beijing/01/03株的血球凝集素蛋白上 117， 118， 121 , 161， 162， 164禾 Π 167(红色所 示)位的氨基酸。 D、 从血球凝集素蛋白的三维结构上来看那 7个氨基酸 117， 118 , 121， 161 , 162， 164和 167(红色和蓝色所示)相互挨在一起。
E、 比较抗体 65C6中和 7.1亚类的原始株和该株的 5个单个氨基酸的突变以及 5个 氨基酸的联合突变的滴度。
F、 在假病毒中和实验中， 中和 5个单个氨基酸的突变和 5个氨基酸的联合突变要达 到 80%的中和所需要的抗体 65C6的浓度， 以及相比之中和原始株病毒抗体浓度的增加 倍数。 红色标记的为对抗体 65C6 中和耐受的单个氨基酸突变或者多个氨基酸的联合突 变。 具体实施方式
本发明人经过广泛而深入的研究， 获得含有独特的 CDR区的抗禽流感病毒的结合分 子， 所述的结合分子对于禽流感病毒具有良好的中和作用。 本发明人还深入研究了其中 一种结合分子在禽流感病毒血球凝集素 (HA)上的结合位点， 获得了所述结合分子的中和 表位。 在此基础上完成了本发明。 结合分子
本发明提供了能特异性结合禽流感病毒的结合分子。 优选地， 所述结合分子结合的 是禽流感的 H5N1病毒。 本发明的结合分子呈现对于禽流感病毒良好的中和活性。
本发明人应用高敏感的 HA和 NA假病毒筛选法及分子克隆技术，成功地从感染 2.3.4 亚亚分支 H5N1病毒的恢复期病人的记忆性 B细胞分离得到了三株人源的抗 H5亚型禽 流感病毒的单克隆抗体 65C6、 100F4和 3C1 1。 三株单克隆抗体都与 HA1具有良好的亲 和力。 其中， 65C6、 100F4能够中和许多种（19中或更多) H5亚型的禽流感病毒， 是广谱 的中和性抗体； 3C11能够中和 4种或多于 4种的 H5亚型的禽流感病毒。
本发明中较为优选的抗体是 65C6抗体， 其对几乎所有的 H5N1病毒的分支均有良好 的中和能力并在动物体内有良好的预防与治疗的效果。 电子显微镜及体外抗体筛选的实 验结果表明， 65C6抗体结合在 H5 HA的头部区域保守的抗原表位， 且体外诱变实验表 明经 1 1代的抗体筛选并没有发现逃逸的突变株，可见 65C6抗体所识别的保守中和表位 位于 HA的头部区域且该表位在所有的 H5N1中都很难发生突变。 因此， 一方面， 65C6 抗体单独使用或与其它抗体或与小分子抑制剂联合使用在治疗 H5N1各种分支引起的感 染方面将有很大的潜力； 另一方面， 利用 H5HA共同的中和表位作为免疫原有可能制备 出针对所有 H5N1分支的广谱抗病毒抗体。
本发明的结合分子可以是完整的免疫球蛋白分子， 所述结合分子可以是抗原结合片 段， 包括但不限于 Fab、 F(ab') F(ab')2 Fv dAb、 Fd、 互补决定区（CDR)片段、 单链 抗体 (scFv)、 二价单链抗体、 单链噬菌体抗体、 双特异双链抗体、 三链抗体、 四链抗体 以及至少含有足以赋予与禽流感病毒毒株的特异性抗原结合的免疫球蛋白的片段的 (多) 肽或其片段。
本发明还提供了所述的结合分子在制备预防、 缓解和 /或治疗禽流感病毒感染的药物 中的应用。 这种感染可以在小群体中发生， 但是也可以以季节流行病方式在世界范围传 播， 或者更严重地在全球蔓延， 数百万个体处于危险之中。 本发明提供了可以中和导致 这种流行病以及潜在的全球性流行病的禽流感病毒毒株的感染的结合分子。 本发明的结 合分子可以大规模地制备和贮存， 因为其提供了针对不同的流行性毒株的保护作用， 且 对于为将来可能发生的禽流感爆发做准备是有利的。
根据本领域公知的技术， 抗体的 CDR区是免疫学感兴趣的蛋白质的序列。 在本发明 的实施方案中， 每一种结合分子可包含本文揭示的二、 三、 四、 五或者所有六个 CDR区。 优选地， 本发明的结合分子包含本文揭示的至少两个 CDR。
本发明还包括所述的结合分子的"功能变体"。 如果变体能与亲代结合分子 (变异前的 结合分子)竞争特异性结合禽流感病毒或其蛋白片段，则认为该变体分子是亲代结合分子 的功能变体。 换句话说， 所述功能变体仍能结合禽流感病毒的 HA1蛋白或其片段， 且与 变异前的结合分子具有相同或相似的结合特性 (例如， 识别的抗原决定区域的相同的)。 功能变体包括但不限于一级结构序列基本相似、 但是含有例如在亲代结合分子中未发现 的体外或体内化学和 /或生物化学修饰的衍生物。 这种修饰包括乙酞化、 酞化、 核苷酸或 者核苷酸衍生物的共价附着、 脂质或者脂质衍生物的共价附着、 交联、 二硫键形成、 糖 基化、 羟基化、 甲基化、 氧化、 聚乙二醇化、 蛋白酶解加工、 磷酸化等。 换句话说， 亲 代结合分子的氨基酸和 /或核苷酸序列中的修饰不显著影响或改变由所述核苷酸序列编 码的或者含有所述氨基酸序列的所述结合分子的结合特性， 即所述结合分子仍能识别并 结合其靶位。
所述功能变体可以具有保守序列修饰， 包括核苷酸和氨基酸取代、 添加和缺失。 这 些修饰可以通过本领域己知的标准技术导入， 例如定向诱变和随机 PCR介导的诱变， 并 且可包含天然以及非天然核苷酸和氨基酸。
保守氨基酸取代包括其中氨基酸残基由具有相似结构或者化学性质的另一氨基酸残 基置换的取代。 具有相似侧链的氨基酸残基的家族己经在本领域中限定。 这些家族包括 具有碱性侧链的氨基酸 (例如赖氨酸、精氨酸、组氨酸)、酸性侧链氨基酸 (例如天冬氨酸、 谷氨酸)、 无电荷极性侧链氨基酸 (例如天冬酞胺、 谷氨酞胺、 丝氨酸、 苏氨酸、 酪氨酸、 半胱氨酸、 色氨酸)、 非极性侧链氨基酸 (例如甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮 氨酸、 脯氨酸、 苯丙氨酸、 甲硫氨酸)、 分支侧链氨基酸 (例如苏氨酸、 缬氨酸、 异亮氨 酸)以及芳香侧链氨基酸 (例如酪氨酸、 苯丙氨酸、 色氨酸)。 本领域技术人员明白也可以 使用除了上述家族之外的其它氨基酸残基家族分类方式。 另外， 变体可具有非保守的氨 基酸取代， 例如氨基酸由具有不同结构或者化学性质的另一氨基酸残基置换。 相似的小 变异也可包括氨基酸缺失和 /或插入。使用本领域熟知的计算机程序可以发现确定哪些氨 基酸残基可以被取代、 插入或者缺失而不消除免疫学活性的指导。 此外， 功能变体可包含氨基酸序列在氨基末端或者羧基末端或者这两端的截短体。 本发明的功能变体与亲代结合分子相比可具有相同或不同的、 更高或更低的结合亲和 性， 但是仍能结合禽流感病毒或其片段。 例如， 本发明的功能变体与亲代结合分子相比 对于禽流感病毒 H5亚型病毒的 HA1或其片段可具有增加或降低的结合亲和性。在本发明 范围内的功能变体与本文所述亲代结合分子具有至少大约 50%至大约 99%、 优选至少大 约 60%至大约 99%、 更优选至少大约 70%至大约 99%、 甚至更优选至少大约 80%至大约 99%、 最优选至少大约 90%至大约 99%、 特别是至少大约 95%至大约 99%， 以及特别是至 少大约 97%至大约 99%的氨基酸序列同源性。 本领域技术人员已知的计算机算法如 Gap 或者 Bestfit可用于最佳地排列氨基酸序列以进行对比以及明确相似或相同的氨基酸残 基。 功能变体可以通过使用本领域己知的普通分子生物学方法改变亲代结合分子或其一 部分而获得， 所述方法包括但不限于易错 PCR、 寡核苷酸指导的诱变、 定点诱变以及重 链和 /或轻链改组法。 因此， 应理解， 当使用术语 (；人)结合分子时， 其也涵盖所述 (人)结 合分子的功能变体。
结合分子的抗原结合特性通常由位于重链和轻链可变区的 3个特定的区域来描述， 称 为互补决定区（complementarity determining region, CDR), 所述的 CDR区将可变区间隔 成 4个框架区域 (FR)， 4个 FR的氨基酸序列相对比较保守，不直接参与结合反应。这些 CDR 形成环状结构， 通过其间的 FR形成的 β折叠在空间结构上相互靠近， 重链上的 CDR和相 应轻链上的 CDR构成了结合分子的抗原结合位点。 可以通过比较同类型的结合分子的氨 基酸序列来确定是哪些氨基酸构成了 FR或 CDR区域。 较佳地， 所述的取代、 插入或者缺 失可以发生在 CDR区以外的区域， 例如抗体重链或轻链的 FR区上； 由于 FR区不参与与 抗原的直接结合， 该区的适当变化是可以的。
作为本发明的优选方式， 所述的结合分子是单克隆抗体， 其包括人源的恒定区 (如人 源恒定区 IgH序列和 IgKappa序列）。所述的抗禽流感病毒单克隆抗体的重链可变区、轻链 可变区以及位于重链可变区和轻链可变区的互补决定区 (CDR)均具有独特的不同于现有 技术的结构， 且它们是全人源的。
作为本发明的优选方式， 本发明包括： 具有所述单克隆抗体的相应氨基酸序列的单 克隆抗体， 具有所述单克隆抗体可变区链的单克隆抗体。 本发明还包括具有含所述的互 补决定区 (CDR)的轻链和重链的任何抗体， 以及 CDR区与本发明的单克隆抗体的 CDR具 有 90%以上 (；更佳地 95%以上)的同源性的任何抗体。
经验证， 本发明的抗禽流感病毒单克隆抗体的 CDR区是全新的， 其针对的是一个独 特的禽流感病毒 HA1蛋白上的抗原表位， 技术构思不同于现有的抗禽流感病毒抗体。
本发明的单克隆抗体可以是全人源的， 其重链、 轻链可变区以及恒定区均来源于人 抗体。 因此， 其在具有特别优异的识别和中和禽流感病毒的作用的同时， 还具有免疫原 性低、 安全性高的特点。
另一方面， 本发明包括免疫缀合物， 即包含本发明所述至少一个结合分子以及进一 步包含至少一个其它治疗分子、 治疗剂或可检测物质。适于治疗和 /或预防的标记可以是 毒素或者其功能部分、 抗生素、 酶、 增强吞噬作用或者免疫剌激作用的其它结合分子。 包含可检测物质的免疫缀合物可诊断性地用于例如评定对象是否己经感染禽流感病毒 毒株或者作为临床实验程序的一部分监测禽流感病毒感染的发生或进展以例如确定指 定治疗方案的功效。 然而， 它们也可以用于其它检测和 /或分析和 /或诊断目的。 可检测 的部分 /物质包括但不限于酶、 辅基、 荧光材料、发光材料， 生物发光材料、放射性材料、 正电子发射金属以及非放射性顺磁性金属离子。 为了检测和 /或分析和 /或诊断目的用于 标记单克隆抗体的标记依赖于使用的特定检测 /分析 /诊断技术和 /或方法例如免疫组织化 学染色 (组织)样品、 流式细胞计量术、 激光扫描细胞计量术检测、 荧光免疫测定、 酶联 免疫吸附测定 (ELISA)、 放射免疫测定 (RIA)、 生物测定 (例如吞噬作用测定）、 蛋白质印 迹应用等。 对于本领域已知的检测 /分析 /诊断技术和 /或方法合适的标记为本领域技术人 员所熟知。
本发明的结合分子可以与一或多个抗原缀合 /附着。 优选地， 这些抗原是由给予了结 合分子-抗原缀合物的对象的免疫系统识别的抗原。所述抗原可以彼此相同， 但也可以是 不同的。使附着抗原和结合分子的缀合方法为本领域所熟知，包括但不限于使用交联剂。
除了通过直接或间接 (例如通过接头)缀合而化学产生免疫缀合物之外，所述免疫缀合 物可以作为融合蛋白而产生， 所述融合蛋白包含本发明的结合分子及合适的治疗分子、 治疗剂或可检测物质。 融合蛋白可以通过本领域已知方法产生， 例如通过构建核酸分子 以及随后表达所述核酸分子而重组产生， 所述核酸分子包含符合读框的编码结合分子的 核苷酸序列以及编码合适标记的核苷酸序列。
本发明另一方面提供了编码本发明的至少一种结合分子、 其功能变体或者免疫缀合 物的核酸分子。 这种核酸分子可以用作中间物以进行克隆。 在一个优选的实施方案中， 所述核酸分子是分离或纯化的。 DNA分子的序列可以用常规技术， 或利用杂交瘤技术获 得。
本领域技术人员可以理解， 这些核酸分子的变体也是本发明的一部分。 核酸分子的 变体是这样的核酸序列， 通过使用标准遗传密码可以将其直接翻译以提供与从亲代核酸 分子中翻译的序列相同的氨基酸序列。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通 过先合成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来得到编码本发明的结合分子 （或其片段， 或其衍 生物)的 DNA序列。然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 (或如 载体)和细胞中。 此外， 还可通过化学合成将突变引入本发明的结合分子的序列中。
本发明还涉及包含上述的适当 DNA序列以及适当启动子或者控制序列的载体。 这些 载体可以用于转化适当的宿主细胞， 以使其能够表达蛋白质。 优选的， 本发明的载体是 例如含病毒启动子的质粒表达载体， 且在所述表达载体中分别插入了抗禽流感病毒结合 分子重链可变区 (VH)与恒定区的 I gH (来自人源 I gH的恒定区）融合序列和轻链可变区 VL 与人体 Ig kappa (来自人源 Ig kappa的恒定区）融合序列。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高 等真核细胞， 如哺乳动物细胞。 代表性例子有： 细菌细胞如大肠杆菌， 链霉菌属； 鼠伤 寒沙门氏菌； 真菌细胞如酵母； 植物细胞； 昆虫细胞如果蝇 S2或 Sf ; 动物细胞如 CHO、 COS7、 NSO或 Bowes黑素瘤细胞等。 特别适用于本发明的宿主细胞是真核宿主细胞， 如 果蝇 S2细胞。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原核 生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可用电穿 孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 或 常规机械方法如显微注射、 电穿孔、 脂质体包装等。
获得的转化子可以用常规方法培养， 表达本发明的结合分子。 根据所用的宿主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 (如温度转换或化学诱导)诱导选择 的启动子， 将细胞再培养一段时间。
如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组 的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规 的复性处理、 用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、 超声处理、 超离心、 分 子筛层析 (凝胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相 层析技术及这些方法的结合。
本发明的结合分子也可以在转基因非人哺乳动物如兔、 山羊或者牛中产生， 并且分 泌进例如其乳中。 中和表位
本发明人的深入研究发现， 本发明的一种中和分子 （65C6) 能够识别一个位于 HA上 远膜区的球形末端上保守的中和表位， 该抗体可以很好地中 H5N1病毒。 因此， 可以设 计基于抗体 65C6的表位的免疫原，从而诱导出可以中和各种 (亚)型的 H5N1病毒的免疫反 应。
所述的免疫原较好地包含以下抗原表位： 相对于血球凝集素的氨基酸序列第 121位 的 Ser; 和相对于血球凝集素的氨基酸序列第 162位的 Arg， 上述表位是与所述结合分 子结合的表位。
所述的免疫原更好地还包含以下抗原表位： 相对于血球凝集素的氨基酸序列第 117 位的 lie; 相对于血球凝集素的氨基酸序列第 118位的 Pro; 相对于血球凝集素的氨基酸 序列第 161位的 Lys; 相对于血球凝集素的氨基酸序列第 164位的 Tyr; 或相对于血球 凝集素的氨基酸序列第 167位的 Thr。
基于上述所示的表位可设计出合适的免疫原， 以诱导产生一些新的广谱中和性结合 分子 (如抗体)。 所述的免疫原的设计可以参照本领域已知的一些技术， 其原则是将上述 的中和性表位暴露于其空间结构的表面上。 药物组合物
本发明的结合分子可用于制备抑制禽流感病毒的组合物。
基于本发明的新发现， 还提供了一种可抑制禽流感病毒或禽流感病毒感染相关疾病 的组合物， 其包含： 有效量的本发明所述的结合分子； 以及药学上可接受的载体。
本文所用的术语"药学上可接受的"是指当分子本体和组合物适当地给予动物或人 时， 它们不会产生不利的、 过敏的或其它不良反应。 本文所用的"药学上可接受的载体" 应当与本发明的结合分子相容， 即能与其共混而不会在通常情况下大幅度降低组合物的 效果。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类， 如乳糖、 葡萄 糖和蔗糖； 淀粉， 如玉米淀粉和土豆淀粉； 纤维素及其衍生物， 如羧甲基纤维素钠、 乙 基纤维素和甲基纤维素； 西黄蓍胶粉末； 麦芽； 明胶； 滑石； 固体润滑剂， 如硬脂酸和 硬脂酸镁； 硫酸钙； 植物油， 如花生油、 棉籽油、 芝麻油、 橄榄油、 玉米油和可可油； 多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如 Tween®; 润湿剂， 如月桂基硫酸钠； 着色剂； 调味剂； 压片剂、 稳定剂； 抗氧化剂； 防腐剂； 无 热原水； 等渗盐溶液； 和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型， 并可由医师根据患者种类、 年龄、 体重 和大致疾病状况、 给药方式等因素确定对病人有益的剂量进行施用。 给药方式例如可以 采用注射或其它治疗方式。
本发明的结合分子可以以未分离的或者分离的形式使用。 此外， 本发明的结合分子 可以单独应用或者于包含至少一种本发明的结合分子 (或其变体或片段)的混合物中应 用。 换句话说， 所述结合分子可以组合应用， 例如作为包含两或更多种本发明的结合分 子、 其变体或片段的药物组合物。 例如， 具有不同但互补活性的结合分子可以组合在一 个治疗方案中以达到希望的预防、 治疗或诊断作用， 但是或者也可以将具有相同活性的 结合分子组合在一个治疗方案中以达到希望的预防、 治疗或诊断作用。 任选地， 所述混 合物进一步包含至少一种其它治疗剂。
所述药物组合物可包含两或多个对于禽流感病毒具有中和活性的结合分子。 在一个 实施方案中， 当组合应用时， 所述结合分子呈现协同中和活性。 换句话说， 所述组合物 包含至少两种具有中和活性的结合分子， 特征在于所述结合分子在中和禽流感病毒中起 协同作用。 如本文所用， 术语 "协同"是指当组合应用时， 结合分子的组合作用高于单 独应用时的加合作用。 所述协同作用的结合分子可以结合禽流感病毒的相同或不同片段 上的不同结构。 计算协同作用的方式是通过组合指数计算。 组合指数 (CI)的概念己经由 Chou and Talalay (1984)描述。所述组合物也可包含具有中和活性的一种结合分子以及一 种非中和性禽流感病毒特异性结合分子。 所述非中和性及中和性禽流感病毒特异性结合 分子在中和禽流感病毒 H5亚型中也可以协同作用。
本发明的结合分子或药物组合在用于人体之前可以在合适的动物模型系统中检测。 这种动物模型系统包括但不限于小鼠、 雪貂 (ferret)和猴。
本发明的结合分子还可与其它抗流感病毒的药物联合用药， 所述的抗流感药物例如 但不限于： 烷胺类药物 (金刚烷胺和金刚乙胺)； 2)流感病毒神经氨酸酶抑制剂 (奧司他韦 和扎那米韦）。因此本发明还提供了包含本发明的结合分子以及上述抗流感药物的药物组 合物。
可以调整给药方案以提供最佳的所需应答 (例如治疗应答)。合适的剂量范围可例如是 0.01-500mg/kg体重， 优选 0.1-50mg/kg体重。 此外， 例如可以给予一次推注、 随时间给予 多次分开剂量或者根据治疗情况的紧急性而可以按比例降低或增加剂量。 本发明的分子 和组合物优选是无菌的。使得这些分子和组合物无菌的方法为本领域所熟知。用于诊断、 预防和 /或治疗的其它分子可以以与本发明的结合分子相似的给药方案给予。如果单独给 予其它分子， 则可以在给予本发明的一或多种人结合分子或药物组合物之前、 同时或者 之后给予患者。 对于人患者的精确给药方案通常在临床实验期间挑选出。 检测试剂和试剂盒
本发明的结合分子可用于制备检测流感病毒的试剂或试剂盒。
如本文所用， 术语 "待检测样品" 或 "待测样品" 涵盖了多种样品类型， 包括生物 学来源的血液及其它体液样品， 实体组织样品如活检组织样品或者组织培养物， 或者衍 生自其中的细胞或者其后代。 该术语还包括在获得后已经通过任何方式处理的样品， 例 如用试剂处理、 溶解、 或者富集某些成分如蛋白质或者多核苷酸。 该术语涵盖了得自任 何物种的各种临床样品， 也包括培养的细胞、 细胞上清和细胞溶解产物。
以所述的结合分子为基础， 可制备方便、 快速且准确地检测禽流感病毒 (如 H5N1)的 试剂盒。
因此， 本发明提供了一种用于检测样品中是否存在禽流感病毒的检测试剂盒， 该试 剂盒中含有本发明的结合分子。
在获得了本发明提供的结合分子后， 可以方便地制备出用于特异性检测禽流感病毒 的检测试剂盒。
作为本发明的一种检测方式， 采用间接 ELISA法， 将待测的抗原包被于固相载体上， 利用本发明的结合分子进行检测。 作为本发明的一种优选方式， 所述的结合分子是抗体， 可根据双抗夹心法的原理来 检测。 双抗夹心法常规的做法是将一抗 (如本发明的单克隆抗体)固定于载体， 然后使一 抗与抗原反应，洗涤后再与二抗反应 (所述的二抗携带可检测信号， 或可与携带可检测信 号的物质结合)， 最后进行化学发光或酶联显色反应检测信号。双抗夹心法特别适用于具 有两个或两个以上表位的抗原的检测。
为了在检测时更方便， 所述试剂盒中除了含有本发明的结合分子以外， 还可以包含 其它检测试剂或辅助试剂， 所述的辅助试剂例如是 ELISA 试剂盒中常规使用的一些试 剂， 这些试剂的特性以及它们的配制方法均是本领域技术人员所熟知的， 如显色剂、 标 记物、 二抗、 抗抗体、 增敏剂等。 本领域人员应理解， 各种变化形式的检测试剂盒均是 包含在本发明中的， 只要在其中利用了本发明的结合分子作为识别禽流感病毒的试剂。
此外， 在所述试剂盒中还可包含使用说明书， 用于说明其中装载的试剂的使用方法。 在获得了本发明提供的结合分子和 /或试剂盒后， 可以利用多种免疫学相关方法来检 测样品中 HA蛋白或其含量， 从而得知待测样品的供体是否感染禽流感病毒， 这些方法 均被包含在本发明中。 较佳地， 所述的方法是以非疾病诊断为目的的。
作为一种优选方式， 本发明提供一种体外 (非诊断或治疗性地)检测禽流感病毒的方 法， 包括以下步骤：
(al) 将待测样品包被于固相载体；
(a2) 将本发明的结合分子加样于 (al)的固相载体，从而使待测样品中的禽流感病毒与 结合分子结合， 形成带有 "禽流感病毒-本发明的结合分子" 二元复合物的固相载体； (a3) 将特异性结合本发明的结合分子的检测物加样于 (a2)的固相载体， 形成带有"禽 流感病毒-本发明的结合分子-检测物" 三元复合物的固相载体； 所述的检测物上携带一 标记物；
(a4) 检测三元复合物中的标记物， 确定待检测样品中禽流感病毒的存在与否以或存 在的量。
按照上述方法， 只要设置已知浓度的抗原对照， 制作浓度标准曲线， 通过比照浓度 标准曲线就可以得出待测样品中的流感病毒含量。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002中所述的条件， 或 按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。 I. 方法材料
人 H5N1病例
该深圳病人于 2006年 6月被诊断出感染了高致病性 H5N1 禽流感病毒，通过输入感 染过高致病性 H5N1 禽流感病毒的康复期病人血浆， 该病人被治愈了。 血液样品在该病 人康复 6个月后采集， 通过 Ficoll 密度梯度离心， 分离出外周血单核细胞。 血浆和外周 血单核细胞样品于 -80°C储存 31。 动物
实验小鼠为 6-8周雌性 BALB/c小鼠购自 Charles River Laboratories (L'Arbresle, France)伺养在负压， 微生物隔离装置中， 空气通过 HEPA过滤装置过滤。 12 小时光照 和 12小时黑暗周期。 攻毒实验在柬埔寨巴斯德研究所生物安全 3级实验室中进行。 小 鼠在接种前通过腹腔注射 75mg/kg的戊巴比妥钠先麻醉小鼠。 细胞系
培养病毒包装细胞系 293FT购自 Invitrogen)的培养液为完全 DMEM培养液 [高糖，
10%胎牛血清， 2mM L-谷氨酸， 1 mM 丙酮酸钠， 青霉素 （100 U/ml))和链霉素 （100 g/ml); Invitrogen Life Technologies] 含有 0.5 mg/ml of G418。 MDCK细胞（购自美国组 织培养公司）的培养液为完全 DMEM培养液， 果蝇 S2 细胞 (Invitrogen)的培养液为完全 SFM含有 10%(v/v) FBS， 50 U/ml青霉素， 50 g/ml链霉素和 2 mM L-谷氨酸]， S2细胞 培养温度为 28 °C。 病毒
高致病性 H5N1病毒 A/Shenzhen/406H/06和 A/Cambodia/P0322095/05分别获自深圳 东湖医院、 柬埔寨巴斯德研究所。 病毒在 MDCK细胞中繁殖， 含有病毒的上清最后分 装后储存于 -80°C32。
半数组织感染剂量的计算： 通过系列稀释病毒， 并感染 MDCK细胞， 通过 Reed and Muench公式计算出半数组织感染剂量 33。
半数致死量的计算： 每组 5只老鼠鼻腔滴注 50ul的 10倍系列稀释的病毒， 观察 14 天， 体重下降超过 35%的老鼠被安乐死。 最后通过 Reed and Muench公式计算出半数致 死量。
所有高致病性 H5N1禽流感病毒相关实验都在生物安全 3级实验室中完成。 HA/NA假病毒的制备
H5病毒包括 10个分枝以及 5个分枝 2的亚分枝， 其中分枝 0、 1、 2.1、 2.2、 2.3禾口 7分离自人，其余分离自禽类。构建密码子优化的 H5病毒和 HI HA及 flag标签的 Nl NA 的方法以及生产流感 HA/NA假病毒的方法参考以前发表的文章中的描述 34' 35。
VSV-G 包埋假病毒： VSV-G 病毒包膜蛋白所包埋的假病毒， 其包埋方法请参照 Vaccine 27: 6777-6790 (2009) 文章所述的方法。
用于包装 HA和 NA假病毒所用的 HA基因的原始病毒株来源及其 Accession Number 见表 1。 HA是通过常规的合成方法获得的。
表 1
Figure imgf000017_0001
基于假病毒的中和试验
基于假病毒中和试验筛选康复期血清中和抗体的方法以及筛选果蝇 S2转染细胞系的 上清中和抗体的方法如前所述 3ό。简要讲，将上清和 ΗΑ和 ΝΑ (如 A/Shenzhen/406H/06) 包被的假病毒一起在 37°C孵育 1小时， 然后加入到 MDCK细胞中。 过夜孵育后， 细胞 用 PBS洗一遍并补充完全培养液， 48小时后按照 BrightGlo Luciferase试剂盒的说明书 中的操作步骤去测定荧光素酶活性。
抑制百分数的计算为： （完全培养液中的假病毒的荧光素酶相对数值-含有系列稀释抗 体的完全培养液中的假病毒的荧光素酶相对数值 )/完全培养液中的假病毒的荧光素酶相 对数值 X I 00%。
为了检测纯化的人单克隆抗体的中和能力， 3倍系列稀释的抗体 65C6， 100F4和 3C11 与假病毒在 37°C孵育 1小时， 然后加入到 MDCK细胞中。过夜培养后， 用 PBS洗细胞， 并置换新鲜完全 DMEM培养液。 荧光素酶活性测定如前所述。 95%抑制浓度 (即中和率 95%； IC95)通过 Graph Pad Prism软件对系列稀释的抗体抑制曲线的拟合计算得出。 血凝抑制试验
病毒与等体积系列稀释的人单克隆抗体 65C6于室温孵育。 然后加入等体积的 0.5% 鸡红细胞， 室温孵育 30分钟。 红细胞于孔底呈小圆点， 边缘光滑整齐， 认为血凝抑制。 构建含有人免疫球蛋白重链和轻链恒定区并能在果蝇 S2细胞稳定表达的载体 为了方便人单克隆抗体的克隆。 EB病毒转化的 B细胞总 RNA被抽提出来， 反转录 成 cDNA， 编码抗体 κ1， λΐ和 γΐ恒定区的片段通过 PCR扩增出来连接到 ΤΑ克隆载体 上测序， 正确的抗体 κΐ和 γΐ 恒定区的片段经 Bglll和 Pmel酶切后连接到经同样酶切 的 pMT/Bip空载体 (购自 Invitrogen)上，得到 MT/Bip/κΙ constant,和 pMT/Bip/γΙ constant 质粒。正确的抗体 λΐ恒定区的片段经 Xhol和 Pmel酶切后连接到经同样酶切的 pMT/Bip 空载体上， 得到 pMT/Bip/λΙ constant载体。
扩增各恒定区片段的引物序列如下:
Pmt-γΐ -恒定区 -正向 GATACC^G^ TCrACA GGTACC
CCTCCACCAAGGGCCCATC (SEQ I D NO: 25)
Pmt-γΐ -恒定区 -反向 CAGCGG TCTAGA GTTTAAACTT AT C A
TTTACCCGGAGACAGGGAGAGG(SEQ I D NO: 26)
Pmt-Kl -恒定区 -正向 GATACCAGA rCrCAC GGTACC GTACGG
TGGCTGCACCATCTGTC(SEQ I D NO: 27)
Pmt-Kl -恒定区 -反向 CAGCGG TCTAGA GTTTAAACJJAJCA
ACACTCTCCCCTGTTGAAGCTC(SEQ I D NO: 28)
pmt-λ 1 -恒定区 -正向 GTTCCCGCC CTCGAG TGAGGAGCTCC(SEQ I D NO:
29)
pmt-λ 1 -恒定区 -反向 GG TCTAGA GTTTAAAC JJAJCA
TGAACATTCTGTAGGGGCCACTG(SEQ I D NO: 30) 建立能产生人单克隆抗体的稳定转染的果蝇 S2细胞
通过 Milteny公司的耦连抗人 CD22+抗体的磁珠分离 CD22+阳性的细胞， 具体步骤参 照该产品的说明书。 分离得到的 CD22+细胞每 30个铺一个 96孔板的孔， 在 RPMI1640 细胞培养液中补充 10%的胎牛血清， CpG 2006， EB病毒和作为滋养层细胞的辐射过的 异体外周血单核细胞。 两周后收集细胞上清， 通过假病毒中和实验来筛选阳性克隆。 经 过一轮亚克隆， 抽提出阳性亚克隆的细胞 RNA， 反转录成 cDNA， 通过 PCR扩增出抗 体的轻链和重链 38。 扩增出的 PCR产物被连接到 T-载体上， 经过 Sfil， BsiWI (κ链）， Sffl Xhol (λ链)和 Sfil， Apal (γ链） 酶切后连接到经过同样酶切的 PMT/Bip载体上。
因为一轮亚克隆后， 亚克隆中依然混有分泌其它不相关抗体的 B细胞。 为了分离出 正确的抗体基因， 本发明人将包含抗体的重链的混合质粒转化大肠杆菌后， 涂板， 随机 挑出单个菌克隆， 抽提出质粒。 将单个的抗体的重链与轻链的混合质粒一起瞬转 S2细 胞， 经 CdCl2诱导 3天后， 收集上清通过假病毒的中和实验来筛选出正确的重链的质粒 克隆， 然后把正确的克隆质粒送去测序得到抗体的重链的序列。 用同样的方法， 本发明 人鉴定得到了抗体的轻链的基因序列。
为了得到稳转的 S2 细胞系， 含有抗体的重链和轻链的质粒和 pCoBlast (购自 Invitrogen; 带有 blasticidin抗性基因） 一起共转 S2细胞。 72小时后， 加入 25ug/ml的 blasticidin 进行筛选， 两周后可得到稳转细胞系。 将稳转细胞进行有限稀释， 通过测定 每个亚克隆细胞的上清中的抗体的量来筛选出高产的单克隆稳转细胞系。 产生和纯化由稳转果蝇 S2细胞产生人单克隆抗体
波浪生物反应器 20/50EHT带有一个 WAVEPOD控制单元 （GE, Healthcare)被用来生产人 单克隆抗体。 简要讲， 150ml表达人单克隆抗体 (1到 2百万每毫升)稳转 S2细胞加到 1-L的 细胞袋中。波浪生物反应器起始转速设定为 22 rpm最大角度 8°在第三天时调整为 26 rpm角 度 9度。过滤后的空气以 0.15 L/分钟的速度流经细胞培养袋，溶液 PH在 6.0和 6.3之间。 起 始培养 6天后， 灌注开始进行， 灌注速率逐渐从 0.3到 1.5个培养体积 (CV)/天， 以保持葡萄 糖浓度不低于 4克 /升。 10天后， 5uM的氯化铬被加入到细胞培液中。 诱导 5天后， 收集上 清。
收集好的上清用 12,000xg的转速 4°C离心 10分钟， 通过 0.45 μιη的滤器过滤。 过滤后的 上清用分子量 50KD的 Hollow Fiber Cartridge (Model UFP-50-C-4MA)在 QuixStand Benchtop 系统中浓缩 5倍。 浓缩后的上清用 12， 000xg的转速 4度离心 10分钟， 通过 0.45 μιη的滤 器过滤。 加入 1 mM of PMSF上样 5ml 的 pre-packed Protein G柱子。 洗脱后的组分通过 HiTrap desalting柱子脱盐处理， 最后抗体溶解于 PBS中。 抗体浓度通过 BCA法测定。
ELISA
用来检测人抗体 IgG的酶联免疫试剂盒购自 Mabtech AB (瑞典）。 具体步骤参考生产 商的试剂说明书， 简要地讲， 将抗人 IgG的抗体在 pH 7.4 的 PBS中稀释成 lug/ml然后 加到 96孔酶连免疫板上于 4°C过夜。第二天用 PBS洗板再用含有 0.1% BSA的 PBST室 温封闭 1小时。 一定比例稀释后的细胞培养上清或者纯化的人单克隆抗体加入到孔中， 同时从 0.1到 500ng/ml的人抗体标准品也加到孔里室温孵育 2小时。 然后用 PBST 洗 4 次， 加入按 1 : 1000 稀释的 ALP-conjugated 抗人 IgG-antibody 室温孵育 1小时， PBST 洗四遍后加入 NPD底物显色一定时间后， 加入中止液于 405 纳米波长度数。 Western Blot
病毒样颗粒 (VLP)样品的制备：
HA/NA VLP为表达流感病毒 HA/NA的病毒样颗粒； HIV-1 VLP为表达 HIV-1包膜 蛋白的病毒样颗粒； 它们的制备方法参照 2009年发表在 Vaccine 27: 6777-6790上的文 章所述的方法。
为了检测人单克隆抗体的结合的特异性， 在 HIV-1和 H5N1 的病毒样颗粒样品加入 SDS上样缓冲液含有 0.6M DTT然后在 90 °C水浴中煮 15分钟，上样 12%的 SDS-PAGE, 然后转到 PVDF膜上， 用 0.1% Tween 20 (TBST) and 5% 脱脂奶粉室温封闭 1小时。 然 后与 3 ml 含有 TG15，3C1 1， 100 F4和 65C6 (0.5 g/ml)的抗体室温孵育 2小时， PBST 洗 两次后，用 AP-耦连的 羊抗兔 IgG 抗体 （Southern Biotech, USA)室温孵育 1小时， PBST 洗两遍后 AP底物显色。 膜表面共振 (SPR)分析
膜表面共振 (SPR)分析按照制造商的说明在 BIAcore T100 (Biacore AB， Sweden)仪器 上进行， 抗体 3C11， 65C6, 100F4和一个不相关的 TG15抗体 (为不识别禽流感病毒的 抗体)分别用氨基耦连试剂盒被固定于 CM5的芯片上，系列稀释的 (从 2090 nM到 84 nM) 可溶性重组 A/Anhui/05/Ol 株的 HA蛋白在 25 °C。 以恒定速度 50 μΐ/minute流经芯片表 面 180s。 数据经过 BIAcore T100 evaluation software (版本 3.2)， 处理分析。 用负染电镜检测 65C6 抗体和 HA复合体
按照以前描述的病毒提纯与 Bromelian的酶促反应的方法将可溶性血凝素从 H5N1病 毒 (A/Shenzhen/406H/06)上消化下来 39。 消化下来的可溶性血凝素按照以前描述的方法同 65C6抗体形成免疫复合物 39。 简单的描述为可溶性血凝素先用 PBS (PH7.2) 稀释到 50 μ§/ηι1并将其涂到碳胶片上。抗体 65C6逐步地加入到涂有可溶性血凝素的碳胶片上直到 所有的可溶性血凝素都与抗体形成复合物。 然后通过印迹的方法将其转移到另一个薄碳 片上并在空气中干燥。 为了在镜下有最佳的观察效果， 抗体 65C6 的量选择在能使其形 成复合物的最少量。 抗体 65C6在小鼠体内对高致病禽流感的预防和治疗作用
为了检测 65C6的预防效果， 8组雌性 BALB/c小鼠 (6个每组， 6到 8周， 平均体重 20克)腹腔注射 50ul的 PBS含有 15 mg/kg, 5 mg/kg和 lmg/kg的 65C6或者 15mg/kg的 对照抗体 TG15。 在 4个小时后， 24只小鼠被鼻腔滴注 50ul的 PBS含有 5 MLD50的 A/Shenzhen/406H/06 另外 24只小鼠被鼻腔滴注 50ul的 PBS含有 5 MLD5。的 HPAI H5N1 A/Cambodia/P0322095/05。 在此后的 14天里， 每天称重小鼠， 记录生存情况， 体重下降 超过 35%的小鼠被安乐死掉。 在第四天时， 每组一只老鼠被用来取组织做组织病理切片 分析。
为了检测 65C6的治疗效果， 4组雌性 BALB/c小鼠 (6个每组， 6到 8周， 平均体重 20克）鼻腔注射 50ul的 PBS含有 5 MLD5。的 A/Shenzhen/406H/06 另外 4组雌性 BALB/c 小鼠被鼻腔滴注 50ul的 PBS含有 5 MLD50的 HPAI H5N1 A/Cambodia/P0322095/05。 在 接种病毒 24， 48， 72 小时后分别腹腔注射 1ml 的 PBS 含有 40 mg/kg 的 65C6或者 40mg/kg的对照抗体 TG15。 在此后的 14天里， 每天称重小鼠， 记录生存情况， 小鼠体 重下降超过 35%的小鼠被安乐死掉。 在第 4天时， 每组- 只老鼠取其肺组织做组织病理 切片分析。 病理学分析
取下的肺部组织经过- -定的处理， 进行切片。切片经固定进行 HE染色为病理学分析 提供依据。 病毒变异株的研究
为了产生中和抗体逃逸突变病毒， 进而定位抗体识别表位， 本发明人将 2 微升的 A/Shenzhen/406H/06病毒原液做 5倍系列稀释再与 2ug/ml 的 65C6和 7.8ug/ml的 100F4分 别在 37°C孵育一小时， 再加到 MDCK细胞上去。 在接下来的 72到 96小时里观察细胞的细 胞病变 (CPE)。 将病毒最高稀释度下出现 CPE的孔中的病毒上清收集起来按照前面的方法重 复下一轮的传代。经过 11代的抗体选择性的筛选， 65C6没有产生明显的逃逸突变，而 100F4 明显产生了逃逸突变， 通过空斑实验得到了两株 100F4的逃逸突变株。
II. 实施例
实施例 1、 制备人单抗 65C6、 100F4和 3C11
血液样本来自于 H5N1感染恢复期六个月的个体。 实验证明其血清对 H5N12.3.4和 1 分支有高度中和活性。 然后将记忆性 B细胞分选出来接种到 96孔上， 每孔含大约 30个 细胞， 然后用 EB病毒和 CpG按照 Traggiai et al的方法 36将 B细胞永生化。并对收集的 上清的中和活性进行筛选。 一开始就观察到由 EBV转染的 B细胞分泌抗体并不稳定。 经过两轮亚克隆上清的中和活性显著下降。 因此在随后的实验中一旦发现有中和活性的 孔就对其细胞进行一轮亚克隆并可以将 R A 从阳性细胞中分离出来。 重链可变区、 κ 链可变区、 λ链可变区的基因片段经 RT-PCR扩增后插入到前述构建的带有重链恒定区、 κ链恒定区、 λ链恒定区基因表达系统中； 构建示意图见图 la。
扩增重链可变区、 κ链可变区、 λ链可变区的基因片段的引物如下 (其中以加黑斜体表 示酶切位点）：
扩職链" ¾¾区 (SEQ ID NO: )
primary PCR 正向 5' L-VH 1 ACAGGTGCCCACTCCCAGGTGCAG (31)
L-VH AAGGTGTCCAGTGTGARGTGCAG (32)
5' L-VH 4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG (33)
5' L-VH 5 CAAGGAGTCTGTTCCGAGGTGCAG (34)
反向 (35)
(36)
nest PCR 正向 5' Sfil VH1 TACACTGGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG GTACATTCC CAGGTGCAGCTGGTGCAG (37)
5' Sfil VH1/5 TACACTGGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG GTACATTCC GAGGTGCAGCTGGTGCAG (38)
5' Sfil VH3 TACACTGGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG GTACATTCT GAGGTGCAGCTGGTGGAG (39)
5' Sfil VH3-23 TACACTGGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG GTACATTCT GAGGTGCAGCTGTTGGAG (40) (49) (51) 扩增 K链破区 (SEQ ID NO: ) 正向 (52)
(53) 反向
正向
(56)
反向
正向
TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATTCTGACATCCAGATGACCCAGTC (58) GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG (59)
TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATTGTGCCATCCGGATGACCCAGTC (60)
TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATGGGGATATTGTGATGACCCAG^C (61)
TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATGGGGATATTGTGATGACTCAGTC (62)
TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATGGGGATGTTGTGATGACTCAGTC (63)
GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG (66) TCGCCT GGCCG7CG7GGCCrTTGTTGGCCTCTCGCTCGGG GTACATTCGGACATCGTGATGACCCAGTC (67) 反向 (68)
(69)
扩增 λ链破区 (SEQ ID NO: ) 正向
反向
正向 5'SfilVA2 CCT GGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG TCCTGGGCCCAGTCTGCCCTG^CTCAG (81)
5'SfilVA3 CCT GGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG TCTGTGACCTCCTATGAGCTGACWCAG (82)
5'SfilVA45 CCT GGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG TCTCTCTCSCAGCYTGTGCTGACTCA (83)
5'SfilVA6 CCT GGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG TCTTGGGCCAATTTTATGCTGACTCAG (84)
5'SfilVA7/8 CCT GGCCG7CG7GGCCTTTGTTGGCCTCTCGCTCGGG TCCAATTCYCAGRCTGTGGTGACYCAG (85) 反向 3'Xhol CA CTCCTCAC71CGAGGGYGGGAACAGAGTG (86) 这些引物的应用根据已发表的文章 Tiller T， Meffre E， Yurasov S， Tsuiji M， Nussenzweig MC， et al. (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329: 112-124。
然后在果蝇 S2细胞中进行一系列抗体重链和轻链配对共转染实验， 来找出产生有效 中和抗体的重链和轻链对。 从大约 16000个 EB病毒转染的 B细胞的上清中发现了其中 6个上清具有 99%的中和活性。从几百个重轻链配对转染的果蝇 S2细胞株鉴定出三株能 够分泌 65C6、 100F4和 3C1 1三种人的单克隆抗体。
另外， 表达抗 HIV-lgp41的 TG15人单克隆抗体的果蝇 S2细胞株也被制备出来用于 阴性对照。 其制备方法与制备表达 65C6的果蝇 S2细胞株相同。
65C6、100F4和 3C11三种抗体的重链可变区分别为 5-a*03 5-a*03和 4-61 *03， 65C6、 100F4和 3C11三种抗体的轻链可变区分别为 VK3D-15*01 , VK2D-28*01和 νλ1-40*01 , 抗体的 VH和 VL链蛋白序列见表 2。其中， 65C6的重链 (VH)的氨基酸序列为 SEQ ID NO: 1； 轻链 (VL)的氨基酸序列为 SEQ ID NO: 2。 100F4的重链 (VH)的氨基酸序列为 SEQ ID NO: 3； 轻链 (VL)的氨基酸序列为 SEQ ID NO: 4。 3C11的重链 (VH)的氨基酸序列为 SEQ
ID NO: 5； 轻链 (VL)的氨基酸序列为 SEQ ID NO: 6。 它们的 CDR区的序列编号见表 2。
表 2
Germline Usage F 1 CD 1 FR2 CDR2
VL
QSVLTQPPSVSGAPGQ VTISC TGGSSNIGAGYSVH WYQQLPGTAPKLLIY GSNSRPS
100F4 IGLV140*01
SEQ ID NO: 16 SEQ ID NO: 17
EIVMTQSPLTLPVTPGAPASISC RSSQSLLHSDGYNYLD WYLQKPGQSPQLLIY LGSHRAS
3C1 1 IGKV2D-28*01
SEQ ID NO: 22 SEQ ID NO: 23
EIVLTQSPLTLSVSPGERATLSC RASQSVSSNLA WYQQMPGQAPRLLIY GASTRAT
65C6 IGKV3D-15*01
SEQ ID NO: 10 SEQ ID NO: 11
FR3 CDR3 F 4
GVPDRFSGSKSGTSASLATTGLRPEDEADYYC QSYDSSLSGSQV FGAGT VTVL
100F4
SEQ ID NO: 18
GVPDRFSGSGSGTDFTLKIS VEAEDVGVYYC MQALQTPD FGQGTRLEIK
3C1 1
SEQ ID NO: 24
GIPARLSGSASGTEFTraSSLQSEDFAVYYC QQYNNWPYT FGQGTKLEIK
65C6
SEQ ID NO: 12
VH Gamline Usage FR1 CD 1 FR2 CDR2
SGSYYWS NMHGSGHTNYNPSLKS
100F4 IGHV4-61*03 QLQLQESGLGLVKPSETLSLTCTVSGDSVS WL QPPGKGLEWIG
SEQ ID NO: 13 SEQ ID NO: 14
SYYIS RIDPSDSDTNYRPSFQG
3C11 IGHV5-a*03 QVQLVQSGAEVKETGESLNISCKVSGNNFP WV QMPGNGLEWMG
SEQ ID NO: 19 SEQ ID NO: 20
YFWIS RIDPTDSY1NYSPSFQG
65C6 IGHV5-a*03 EVQLVQSGAEVKKPGESLRISCKGFAYSST WV QMPGKGPEWMG
SEQ ID NO: 7 SEQ ID NO: 8 FR3 CDR3 FR4 ALLTTVTTFEY
RVnTPDTSKNHFSLRLSSVTAADTAVYYCAR
SEQ IDNO: 15
RATYYYGSGSYFDAFD:
HVHSADKSTSTAYLQWRSLKASDTAMYYCAR
SEQ IDNO: 21
YHRRGHFYGSGSAWDWi
HVTISVDRSISTVYLQWSSLKASDTAMYYCA SEQ IDNO: 9 图 2为 65C6、 100F4、3C11和 TG15纯化抗体的台盼蓝染色结果。抗体的重链 (；50 kDa) 和轻链 (24-26 kDa)的染色条带清晰可见， 且有很高的纯度。 实施例 2、 人单抗 65C6， 100F4和 3C11抗原特异性和亲和力实验
用免疫印迹方法来检测人单克隆抗体的抗原特异性实验， 首先 HIV-1、 HA 和 NA 病毒样颗粒经 SDS/PAGE电泳再用 PVDF膜进行转膜， 然后与抗体 65C6、 100F4、 3C11 和 TG15进行反应，根据印迹可以分析抗体的特异性。如图 lb所示阴性对照的抗体 TG15 能特异的与 HIV-1类病毒上的包膜蛋白结合但不能与流感类病毒上的 HA和 NA结合。 阳性对照所用的小鼠的免疫血清 (Immune sera)能特异的与流感类病毒上的 HA。、 HAj和 HA2结合但不能与 HIV-1类病毒上的包膜蛋白结合。 抗体 65C6、 100F4和 3C11能特异 的与 HA。和 HAi结合但不能与 HA2和 HIV-1的包膜蛋白结合。 由此提示， 抗体 65C6、 100F4和 3C11 所识别的抗原表位是在流感血凝素蛋白的 HAi区域中。
应用表面等离子体共振的方法来测定抗原抗体的亲和力。 结果如图 l c， 显示不同浓 度的血凝素与抗体 100F4、 65C6和 3C11的结合和游离曲线。 由此估算出 100F4、 65C6 和 3C11抗体与血凝素的亲和力（KD)分别为 2.42 Χ 1(Γ9、 4.14 X 1(Γ8和 7.02 X 1(Γ8， 见表 3。 由此， 本发明人得出结论， 100F4、 65C6和 3C11抗体与血凝素都有良好的亲和力。
表 3
Abs Immob U ka (1/Ms) kd (1/s) KD (M) Chi2 (RU2) U- value
100F4 573 7.30E+03 1.76E-05 2.42E-09 0.924 20
3C11 363.3 6.86E+03 4.82E-04 7.02E-08 1.56 2
65C6 301.6 8.86E+03 3.67E-04 4.14E-08 0.861 2 实施例 3、 体外验证人单抗 65C6, 100F4和 3C11的中和广度、 效力
图 3和表 4是抗体 100F4、 65C6、 3C11禾 Π TG15对 19个所有 H5N1禾 Π 1个 H1N1 亚类的假病毒以及 VSV-G 包埋假病毒的中和活性测试的结果， 阴性对照的抗体 TG15 对 19个 H5N1和 1个 H1N1亚类的假病毒以及 VSV-G包埋假病毒都没有中和活性。 抗 体 3C11 对四种 H5N1 假病毒（A/Hong Kong/ 156/97 , A/Turkey/65-595/2006 , A/Xingjiang/1/2006和 A/Beijing/01/2003)有良好的中和活性 (IC95值分别在 0.516， 4.04， 5.612和 3.465 g/ml)。 相反， 抗体 100F4可以很好地中和所有 19个 H5N1亚类的假病 毒。 在小于 0.5 g/ml 浓度时抗体 100F4对其中的 6个 H5N1的假病毒的中和率就可达 到 95% ; 在小于 l g/ml浓度时抗体 100F4对其中 13个 H5N1的假病毒的中和率就可达 到 95%，而对余下 6个 H5N1假病毒中和率要达到 95%所需的浓度为 1.022到 8.122 g/ml 之间。 出乎意料的是虽然抗体 65C6与血凝素的结合率低于抗体 100F4 (；如表 4) 但其中 和活性却高于抗体 100F4。 在小于 0.5 g/ml 浓度时抗体 65C6对其中的 16个 H5N1 的 假病毒的中和率就可达到 95%; 在小于 l g/ml 浓度时抗体 65C6对其中的 17个 H5N1 的假病毒的中和率就可达到 95%， 而对余下 2个 H5N1假病毒的中和率要达到 95%所需 的浓度也仅为 1.085 g/ml和 1.528 g/ml (表 4)。 由此本发明人得出结论： 抗体 65C6可以 高效地中和所有 19个 H5N1亚类的假病毒。
为了进一步验证抗体 65C6中和活性的广度和强度， 本发明人还进行了血凝抑制试验 (；表 5)，结果表明：抗体 65C6在 0.3 g/ml和 2.7 g/ml 浓度间能够完全抑制所有 6个 H5N1 病毒的血凝活性； 然而该抗体对 H1N1、 H2N2和 H3N2 病毒的血凝活性却没有抑制作 用。 由此可以得出结论： 抗体 65C6 所识别中和表位是所有 H5亚型的 HA共有的， 但 在 Hl、 H2和 H3亚型的 HA中是没有的。
表 4
Figure imgf000025_0001
* 抗体 65C6、 100F4和 3C1 1的引起 H5N1假病 IC95的剂量 （ug/ml) ；
¾φ n.d..代表未测到；
表
亚型 病毒 Sub)CIades !gG (fig.ml 1}
HS 1 A Viet am 1194/2004 1
A/indorsesia/5/2005 2.1 2.7
A Bar headed goose/Qii^hai 1 A/2D05 2.2 1 3
AW ooper swan*'Moogo!ia/244/2005 2.2 0,66
A Tiiri ey/ieOOS 2.2 0.33
A/Anhui/1/2005 2.3 0.33
H1N1 A Cafifomsa/7QD09 >170
H2N1 A^Singapore 1/1957 >170
Η¾ί1 A Aich 1/2/1968 >170 实施例 4、 逃逸株筛选
为了确定抗体 65C6和 100F4所识别的氨基酸位点， 本发明人利用抗体筛选逃逸的 突变株。 经过一两代的 100F4抗体的筛选， 逃逸株便可以检测到； 随着传代次数的增加 抗 100F4抗体的突变株的活性越来越强； 传至 1 1代以后在抗体浓度为 1600μ§/ιη1 的情 况下突变株也可以逃逸。 随后应用空斑方法克隆了两株突变株并对其完整 ΗΑ序列进行 了测试与比对，发现克隆的两个突变株在抗体浓度为 1600μ§/ηι1 的情况下突变株也可以 逃逸。 其中一株变异株的 ΗΑ 序列存在 8个单一氨基酸突变位， 其中 6个突变在 HA1 区域。 另一个突变株有 10个单一氨基酸突变， 其中 8个突变在 HA1区域。 两个突变株 共同的突变序列在 HA1区域有 6个氨基酸分别在 68、 120、 127、 195、 209和 313位点。 由此本发明人推测， 100F4抗体所识别的中和表位与该六个位点相关。
与 100F4相反， 经过 1 1代传代的抗体 65C6筛选却没有检测到任何逃逸株。 另外， 上述两个对 100F4抗体的突变株对 65C6抗体还是敏感的 (；表 6)。由此本发明人得出结论， 上述六个位点与 100F4抗体所识别的中和表位有关， 而与 65C6抗体所识别的中和表位 无关， 并且 65C6抗体所识别的中和表位很难突变， 该表位一旦突变将会影响其生存。
两个 100F4抗体的逃逸株的 ΗΑ序列分析发现， 逃逸株 1含有 8个单一的氨基酸突 变其中 6个在 HA1区域； 逃逸株 2有 10个氨基酸突变， 其中 8个在 HA1区域。在 HA1 中有这两个逃逸株共有的 6个氨基酸突变， 分别在第 68、 120， 127， 195， 209和 313 位， 由此提示， 上述 6个突变点与 100F4的识别有关 (见图 4)。
表 6、 65C6、 100F4抗体的抑制 50%由 100TCID50病毒引起的 CPE所需浓度 抗体浓^ { iifl/mlj 野生型和逃逸变体 gSCfi
Figure imgf000027_0001
8Ζ-6506-Ρ11·* ND
SZ-100F4-P11***
SZ-1 (M3F4 Resislant variant-A > 6i
SZ-10€F42 Resistant variant-B W,
* SZ表示 ' A S.lienzheii/4 H 06 H5M1病
·* SZ「fi5C(S-Pl 1表示 A/Sl™lieii/406H/06 H5M1病毒， 经 1 1代传代
*** SZ-iCK3(F -Pi 1表示 A/Sh£nzhmi fl6H¾6 Η5ΝΪ病毒， 经 1 1代传代 实施例 5、 抗体 65C6的体内预防效果
为了验证抗体 65C6在体内的预防效果，雌性的 BALB/c小鼠经腹腔注射将 15 mg/kg、 5mg/kg禾 Π lmg/kg的 65C6抗体 (浓度 200mg/ml，纯度大于 95%)和 15 mg/kg对照抗体 TG15 注入小鼠体内， 4小时后再将 5个 MLD5。高致病性禽流感 H5N1 A/Shenzhen/406H/06和 ：
HPAI H5N1 A/Cambodia/P0322095/05经鼻腔滴液注入小 ^ f 鼠上呼吸道。选用 5个 MLD5o 高 致病性禽流感 H5N1 A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/P0322095/05是经预 实验证明该剂量对对照组小鼠的致死率可达 100%。
图 5a和 b显示 HPAI H5N1 A/Shenzhen/406H/06病毒接种后的 14天内小鼠的体重改 变和存活率， 图 5c和 d显示 HPAI H5N1 A/Cambodia/P0322095/05病毒接种后的 14天 内小鼠的体重改变和存活率。 注入对照抗体 TG15组小鼠经 H5N1 A/Shenzhen/406H/06 感染的自 3天起出现明显的疾病症状和体重下降， 其中在 8-1 1天所有 5只小鼠均死亡。 相反， 注入 lmg/kg 65C6抗体的小鼠组 4-6天出现明显的疾病症状和体重下降， 且在 11 和 13天有 2只小鼠死亡， 三只存活。注入 5mg/kg 65C6抗体的小鼠在 5-7天出现疾病症 状，但体重减轻不明显，其中 1只小鼠在 11天死亡，余下的 4个存活。然而注入 15mg/kg 65C6抗体的小鼠未出现疾病症状和体重减轻， 且全部存活。
注入对照抗体 TG15组小鼠经 H5N1 A/Cambodia/P0322095/05感染的自 3天起出现 明显的疾病症状和体重下降， 其中在 8-11天所有 5只小鼠均死亡。 相反， 注入 lmg/kg 65C6抗体的小鼠组 4-6天出现明显的疾病症状和体重下降，且在 10天有 1只小鼠死亡， 4只存活。 然而注入 5mg/kg和 15mg/kg 65C6抗体的小鼠未出现疾病症状和体重减轻， 且全部存活。
为了进一步研究抗体 65C6 体内的预防作用， 本发明人将感染 H5N1 A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/P0322095/054天后的肺部组织进行病理 切片观察， 如图 6所示， 与临床观察的症状相吻合， TG15抗体处理的小鼠在感染 4天 后出现明显的肺部炎症病理改变包括肺泡壁增厚、 炎症细胞浸润和血管扩张充血 (见图 6d和 6h)。 注入 lmg/kg 65C6抗体的经 H5N1 A/Shenzhen/406H/06感染的小鼠组有少量 的炎症反应，肺泡壁增厚、炎症细胞浸润和血管扩张充血均不明显 (6c)。相反，注入 5mg/kg 和 15mg/kg 65C6 抗体经 H5N1 A/Shenzhen/406H/06 组未出现任何炎症反应。 注入 lmg/kg 、 5mg/kg禾 B 15mg/kg 65C6抗体经 HPAI H5N1 A/Cambodia/P0322095/05感染组 未出现任何炎症反应。 实施例 6、 抗体 65C6 的体内治疗效果
确定了抗体 65C6的预防作用后， 本发明人进一步验证其治疗作用。 雌性的 BALB/c 小鼠经鼻腔滴液将 5 个 MLD5。 高致病性禽流感 H5N1 A/Shenzhen/406H/06 禾 Π HPAI H5N1 A/Cambodia/P0322095/05注入小鼠上呼吸道， 24、 48禾 Π 72小时后再将 40mg/kg 的 65C6抗体、 40mg/kg和对照抗体 TG15注入小鼠腹腔。
图 7a和 b显示 HPAI H5N1 A/Shenzhen/406H/06病毒接种后的 14天内小鼠的体重改 变和存活率， 图 7c和 d显示 HPAI H5N1 A/Cambodia/P0322095/05病毒接种后的 14天 内小鼠的体重改变和存活率。 经 HPAI H5N1 A/Shenzhen/406H/06 禾 Π HPAI H5N1 A/Cambodia/P0322095/05感染后注入对照抗体 TG15组小鼠出现明显的疾病症状、 体重 下降， 且在 8-10天所有小鼠均死亡。 相反， 经 HPAI H5N1 A/Cambodia/P0322095/05感 染后 24、 48和 72小时后注入 65C6抗体的小鼠均无明显的疾病症状和体重减轻， 且所 有小鼠均存活。经 HPAI H5N1 A/Shenzhen/406H/06感染后 72小时后注入 65C6抗体除 1 只小鼠死亡外其他小鼠均存活， 且不出现任何疾病症状和体重减轻。
为了进一步研究抗体 65C6 体内的治疗作用， 本发明人将感染 H5N1
A/Shenzhen/406H/06和 HPAI H5N1 A/Cambodia/P0322095/05 4天后的肺部组织进行病 理切片观察； 感染后 24小时经 TG15抗体处理的小鼠在感染 4天后出现明显的肺部炎 症病理改变包括肺泡壁增厚、 炎症细胞浸润和血管扩张充血 (见图 8b和 d)。 相反， 感染 后 24小时经 65C6抗体处理的小鼠组未出现任何明显的炎症反应 (见图 8a和 c)。
本发明抗体可被用于治疗广谱的 H5N1 的病毒感染， 本文只是举例说明本发明抗体 对各 H5N1 分支 (dade)的病毒感染治疗效果 所属技术领域人员了解本发明抗体亦可被 应用于同类 H5N1 分支的其他病毒的感染， 包括但不限于 0 分支的 A/Chicken/Hong Kong/317.5/2001 A/Chicken/Hong Kong/728/97 A/chicken/Hubei/wf/2002等， 1分支的 A/chicken/Kohn Kaen/NIAH330/2004 A/chicken/Phichit/NIAH6-4-0001/2006等， 2.1分支 的 A/Chicken/West Java/GARUT-MAY/2006 、 A/Duck/Bufeleng/BPP V 1/2005 、 A/Duck/Pali/BB VW 1358/2005 等 ， 2.2 分 支 的 A/duck/Romania/TL/nov/2007 、 A/duck/Switzerland V389/2006 、 A/eagle owl/Sweden/V1218/2006 等 ， 2.3.2 分支 的 A/bar-headed goose/Mongolia/X25/2009、 A/bean goose/Tyva/10/2009、 A/black-crowned night heron/Hong Kong/659/2008等， 2.3.4分支的 A/blue magpie/Hong Kong/ 1993/2007 A/chestnut munia/Hong Kong/2442/2007 A/chicken/Thailand/NP- 172/2006等， 2.4分支 的 A/chicken/China/1204/04、 A/Ck/YN/1 15/2004、 A/duck/Yunnan/485/2004等， 2.5分支 的 A/blow fly/Kyoto/93/2004 A/chicken/Guangdong/ 174/04 A/chicken/Jiangxi/25/2004 等， 3 分支的 A/Chicken/Hong Kong/SF219/01、 A/Chicken/HongKong/FY 150/01、 A/chicken/Xinjiang/16/2005 等 ， 4 分 支 的 A/duck/Guangdong/22/2002 、 A/duck/Shantou/700/2002等， 5分支的 A/duck/Shantou/5526/2001等， 6分支的 A/black bulbul/Fujian/439/04 等 ， 8 分 支 的 A/chicken/Hong Kongi/86.3/2002 、 A/chicken/Vietnam/G62/2005 、 A/Ck/HK/YU777/02 等 ， 9 分 支 的 A/chicken/Henan/210/2004 、 A/chicken/Hubei/ 14/2004 等 ， 更 多 的 病 毒 株详见 http ://h5nl .flugenome.org/ show_subtyp e s . p hp。 实施例 7、 65C6抗体识别部位
图 9显示电镜下观察到的经负染的 HA和抗体 65C6的复合物及其示意图。 每个抗体 分子与两个 HA结合。 每个抗体的 Fab段跟 HA的末端相结合并于 HA形成固定的 1 10 度的角度。 其中图 9d显示 5个 HA分子末端相接形成一个聚合体， 抗体分子与聚合体 中两个 HA的另一个末端结合。
已有报道显示 HA分子近膜端相互结合形成聚合体， 由此提示被抗体结合的 N端是 HA的头部区。 实施例 8、 鉴定抗体 65C6的表位
为了鉴定出抗体 65c6的中和表位， 基于区域层次的以及精细表位层次的酵母展示被 应用于抗体 65c6 的中和表位的鉴定实验， 精细表位层次的酵母展示方法在以前已有报 道 (Zuo T， Shi X , Liu Z， Guo L， Zhao Q, et al. (201 1) Comprehensive analysis of pathogen-specific antibody response in vivo based on an antigen library displayed on the surface of yeast. J Biol Chem)。 简要的讲， 诱导的酿酒酵母细胞 （106 -107)通过离心收集 (12,000 转 /秒， 1分钟）， 用 PBS 洗一次， 加上 500ng的抗体 65C6在冰上孵育 1小时。 然后用冷的 PBS洗两次， 接着与 PE-标记的抗 -人的 IgG (1 :200 稀释)在冰上孵育 45分 钟。 细胞再用冷的 PBS 洗两次， 接着用 Aria II (BD， USA)的流式细胞仪进行分析分选， PE-阳性的酵母克隆被分选出来并且测序。 为了进行精细的表位鉴定， 通过低保真 PCR 技术在 HA的 51-260位置的氨基酸上引进了一系列的随机突变，这些随机突变的片段通 过胶纯化并用 Qiaquick胶回收试剂盒 （Qiagen)来回收。具体的构建、 生长并在酵母表面 表达该文库的方法已在以前的文献中有过描述。
通过对 PE-染色阴性的酵母克隆的测序分析， 本发明人鉴定出 23个单个氨基酸的突 变能够破坏抗体 65C6对 HA的结合。 从 HA的三维结构来看， 其中 13个位于第 1 16， 117， 1 18, 121， 147， 152， 160, 161， 162， 163 , 164， 167和 187位位置的氨基酸突变在 HA蛋白的表面上， 另外 10个 氨基酸的突变则埋在 HA蛋白里面， 意味这这些埋在蛋白里面的氨基酸不会直接和抗体 65C6直接接触。 见图 10A。
为了鉴定这 13个 HA蛋白表面的氨基酸突变是否会影响抗体 65C6的中和活性。 13 个基于 H5N1的 7.1亚型的 A/Beijing/01/2003株的 HA骨架的单点突变被用来构建 H5N1 的假病毒。
抗体对于这些带有各个单点突变的假病毒的中和性通过中和试验来确定。 结果显示， 与原始株的 7.1 亚型的 H5N1假病毒相比， H5N1 的 HA在 116， 147， 152， 160， 163 或者 187位置突变形成的假病毒更容易被抗体 65C6所中和。 而带有 117， 1 18， 121， 161， 162， 164和 167位置的 HA突变形成的假病毒则对抗体 65C6相比之原始株更加耐 受 （在 IC95的抑制率下， HA第 117和 162位突变的假病毒对抗体的耐受提高了 2倍， 而 HA第 121和 161位突变的假病毒对抗体的耐受提高了超过 8倍)， 见图 10B。 有趣的 是所有耐受提高的假病毒相应的 HA突变氨基酸的位置在 HA蛋白的三维结构上看都是 相邻的， 见图 1(^和1)。
通 过 比 较 65C6 敏 感 株 A/Beijing/01/2003 与 65C6 耐 受 株 A/Chicken/Vietnam/NCVD-016/08 的 HA 的氨基酸序列， 本发明人发现在 117-121 和 159-167这两段位置上， 这两株的 HA在第 121， 159， 162， 163和 165位的 5个氨基酸 是不同的。
为了鉴定这 5个氨基酸在 65C6的中和表位中的作用， 本发明人将 A/Beijing/01/2003
7.1亚型 HA中相应的这 5个氨基酸分别替换成了 A/Chicken/Vietnam/NCVD-016/08 7.2 亚型 HA中相应的的氨基酸， 同时构建了一替换了 7.2亚型的所有 5个氨基酸的突变并 用这些突变去包装假病毒。
图 10E 和 F 显示了抗体 65C6 对这些假病毒突变的中和活力。 与原始株的 A/Beijing/01/2003 7.1亚型相比， 带有 HA上第 159， 163或 165位单个氨基酸突变的假 病毒显得更易于被抗体 65C6中和。
相对的，带有 HA上第 162或 121位突变的假病毒则对于 65C6的中和更加耐受了 (在 IC80的抑制率下， 抗体浓度分别增加了 1.26倍和 3.37倍)。 值得注意的是， 当 5个氨基 酸一起突变后对于抗体 65C6的耐受性相比之在 121和 162位置的单个点突变的耐受性 有了极大的增强（图 10F)。
纵观这些结果， 可以得出结论， HA上第 121和 162位位置的氨基酸是在抗体 65C6 识别的表位里面的， 而且这两个氨基酸在 7.2亚型的骨架中能够更好地被抗体 65C6所 识别。
电镜及表位鉴定的结果显示， 抗体 65C6结合的表位包含氨基酸 1 17， 1 18， 121， 161， 162， 164和 167， 这样一个构象表位位于 HA 的远离膜端的球形区域的末端。
除了 7.2亚型， 这些氨基酸在各个型和各个亚型的 H5N1里面是高度保守的， 但是这 些氨基酸在不同亚类的 HA里面是很不一样的， 这与中和实验的结果是相吻合的。 这些 结果提示， 这些氨基酸所在的区域在自然感染的过程中是一个主要的产生免疫反应的区 域，这样一个包含了一个环形结构和一个反向平行的 β 折叠结构可以被用来设计免疫原 从而诱导产生亚类特异性的广谱中和抗体。 讨论
对于人畜共患的高致病性禽流感 H5N1病毒感染虽有一些具体的治疗方案，但因其持 续的 60%的死亡率使其仍然是一个公众健康的重大威胁。 本发明中， 发明人成功的从感 染分支 2.3.4 H5N1病毒的恢复期病人的记忆性 Β细胞中分离得到了 65C6、100F4和 3C1 1 三株人抗 H5HA的单克隆抗体。
本发明有两个重要发现， 一个是 65C6抗体对高致病性禽流感 H5N1病毒所有 10个 分支和 5个亚类都有高度的中和效力，且经过 11代的体外 65C6抗体筛选并没有发现逃 逸的突变株。 这些结果表明 65C6抗体所识别的中和表位包括所有 H5N1病毒株， 而且 65C6 抗体所识别的中和表位很难突变， 可能因为该表位一旦突变将会影响病毒株本身 的生存。 因为 65C6抗体是从感染 H5N1病毒的恢复期病人的记忆性 B细胞中分离得到 的， 因此 65C6抗体的这种中和反应是基于人类自然感染并获得免疫的中和抗体反应的， 因此针对这种中和表位产生的疫苗不仅对正在人类中传播的 H5N1病毒株有中和活性且 对目前在禽类传播将来有可能传播到人类的 H5N1病毒株也将有很好的中和活性。
另一个重要发现是 65C6抗体对高致病性禽流感有很好的预防和治疗效果。腹腔注射 5mg/kg的 65C6抗体可以保护小鼠免受致死剂量的高致病性禽流感 H5N1病毒的感染， 即使在小鼠被高致病性禽流感 H5N1 病毒感染 72小时后腹腔注射该抗体也可以使小鼠 存活且不发生体重减轻。因此， 65C6抗体在治疗人类或人畜共患 H5N1病毒感染方面有 很大的潜力。
尽管基于抗体的治疗方法并不是一种新的治疗策略， 但对于流感病例该疗法还是很 有代表性的。通过特异性抗体的后天免疫也可以使婴儿获得针对流感病毒的免疫力 1(M3。 从 1918 年西班牙流感大流行的幸存者体内所分离得到的单克隆抗体可以有效的将流感 的死亡率降到 50% 甚至是到 37〜16%14。输入感染过 H5N1的康复病人的血浆可以有效 的降低 H5N1病毒感染病人的病毒载量并可以完全康复 15。 这些临床现象的生理机制是 血浆中的中和抗体可以调节病毒感染的过程和减缓急性呼吸窘迫综合症及其他并发症 的发病速度 14。 因此， 本发明及其他相关研究开发的人单克隆抗体在治疗流感病毒感染 方面比感染流感病毒康复病人的血浆有更好的疗效 2' 21' 24' 4Q' 41。 此外， 人单克隆抗体 还有两个好处， 一个是可以大批量生产， 事实上在本发明中就发现超过 lg/L/d的人单克 隆抗体就可以利用波浪式生物反应器和灌注培养的方法用果蝇 S2 细胞产生， 另一个是 该抗体对于人血浆中存在的外在抗原不发生反应， 且自应用人源抗体替代其它物质来源 抗体治疗疾病以来免疫排斥反应也大大降低了。
尽管目前还未确切地获得 65C6抗体所识别的中和表位的氨基酸残基的序列， 但负染 电镜对 65C6抗体和 HA复合体的观察表明， 65C6抗体结合在 HA1头部区域保守的抗 原表位。这个结果与本发明人的中和试验结果相一致， 即抗体 65C6是 H5亚型特异性抗 体。 虽然 65C6抗体可以中和所有 H5HA(亚类)分支， 但其对亚型 1、 2和 3流感病毒却 没有中和作用。 由此 65C6抗体所识别的表位与最近发现的群特异性 (group specific)抗体 C17942、CR626124'42和 F10 23所识别的表位不同，后三个抗体所识别的抗原表位位于 HA2 的颈部区域 23' 24' 42。
综上所述， 本发明人利用 H5N1 假病毒株中和试验技术与分子克隆技术成功的从 H5N1康复的病人的记忆性 B细胞筛选出 3个有效的人源单克隆抗体 15。 其中 65C6抗 体所识别的中和表位位于 HA1的头部区域且该抗体对所有的 H5N1病毒的分支均有良好 的中和能力并在小鼠体内具有良好的预防与治疗的效果， 同时本发明人的体外实验证明 该表位很难发生突变 43' 44。 因此， 一方面 65C6抗体单独使用或与其它小分子抑制剂联 合使用在治疗 H5N1各种分支引起的感染方面将有很大的潜力， 另一方面， 利用 H5HA 共同的中和表位作为免疫原有可能制备出针对所有 H5N1分支的广谱抗病毒抗体。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。
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Claims

m ^
1. 一种结合分子， 其识别禽流感病毒的血球凝集素 HA1， 并结合于血球凝集素 N端 区域上的表位上， 该表位包含以下位点：
血球凝集素氨基酸序列第 121位的 Ser; 和
血球凝集素氨基酸序列第 162位的 Arg。
2. 如权利要求 1所述的结合分子， 其特征在于， 所述的表位还包含以下位点： 血球凝集素氨基酸序列第 1 17位的 lie;
血球凝集素氨基酸序列第 1 18位的 Pro ;
血球凝集素氨基酸序列第 161位的 Lys;
血球凝集素氨基酸序列第 164位的 Tyr; 或
血球凝集素氨基酸序列第 167位的 Thr。
3. 如权利要求 1所述的结合分子， 其特征在于， 所述结合分子包含 SEQ ID NO: 7 所示的重链 CDR1， SEQ ID NO: 8所示的重链 CDR2， SEQ ID NO: 9所示的重链 CDR3。
4. 如权利要求 1所述的结合分子， 其特征在于， 所述结合分子包含 SEQ ID NO: 10 所示的轻链 CDR1，SEQ ID NO: 1 1所示的轻链 CDR2， SEQ ID NO: 12所示的轻链 CDR3。
5. 如权利要求 1所述的结合分子， 其特征在于， 所述结合分子包含 SEQ ID NO: 7 所示的重链 CDR1， SEQ ID NO: 8所示的重链 CDR2， SEQ ID NO: 9所示的重链 CDR3 ; 以及 SEQ ID NO: 10所示的轻链 CDR1， SEQ ID NO: 1 1所示的轻链 CDR2， SEQ ID NO: 12所示的轻链 CDR3。
6. 如权利要求 1所述的结合分子， 其特征在于， 所述的结合分子包含重链可变区， 该重链可变区具有 SEQ ID NO: 1所示的氨基酸序列。
7. 如权利要求 1所述的结合分子， 其特征在于， 所述的结合分子包含轻链可变区， 该轻链可变区具有 SEQ ID NO: 2所示的氨基酸序列。
8. 如权利要求 1所述的结合分子， 其特征在于， 所述的结合分子包含：
重链可变区， 该重链可变区具有 SEQ ID NO: 1所示的氨基酸序列； 以及
轻链可变区， 该轻链可变区具有 SEQ ID NO: 2所示的氨基酸序列。
9. 如权利要求 1所述的结合分子， 其特征在于， 所述结合分子包含 SEQ ID NO: 13 所示的重链 CDR1，SEQ ID NO: 14所示的重链 CDR2， SEQ ID NO: 15所示的重链 CDR3 ; 禾口 /或
包含 SEQ ID NO: 16所示的轻链 CDR1， SEQ ID NO: 17所示的轻链 CDR2， SEQ ID NO: 18所示的轻链 CDR3。
10. 如权利要求 9所述的结合分子， 其特征在于， 所述的结合分子包含重链可变区， 该重链可变区具有 SEQ ID NO: 3所示的氨基酸序列。
11. 如权利要求 9所述的结合分子， 其特征在于， 所述的结合分子包含轻链可变区， 该轻链可变区具有 SEQ ID NO: 4所示的氨基酸序列。
12. 如权利要求 9所述的结合分子， 其特征在于， 所述的结合分子包含：
重链可变区， 该重链可变区具有 SEQ ID NO: 3所示的氨基酸序列； 以及
轻链可变区， 该轻链可变区具有 SEQ ID NO: 4所示的氨基酸序列。
13. 如权利要求 1所述的结合分子， 其特征在于， 所述结合分子包含 SEQ ID NO: 19 所示的重链 CDR1， SEQ ID NO: 20所示的重链 CDR2， SEQ ID NO: 21所示的重链 CDR3 ; 禾口 /或
包含 SEQ ID NO: 22所示的轻链 CDR1， SEQ ID NO: 23所示的轻链 CDR2， SEQ ID NO: 24所示的轻链 CDR3。
14. 如权利要求 13所述的结合分子，其特征在于， 所述的结合分子包含重链可变区， 该重链可变区具有 SEQ ID NO: 5所示的氨基酸序列。
15. 如权利要求 13所述的结合分子，其特征在于， 所述的结合分子包含轻链可变区， 该轻链可变区具有 SEQ ID NO: 6所示的氨基酸序列。
16. 如权利要求 13所述的结合分子， 其特征在于， 所述的结合分子包含： 重链可变区， 该重链可变区具有 SEQ ID NO: 5所示的氨基酸序列； 以及
轻链可变区， 该轻链可变区具有 SEQ ID NO: 6所示的氨基酸序列。
17. 如权利要求 1-16任一所述的结合分子， 其特征在于， 所述的结合分子是人单克 隆抗体、 Fab、 F(ab' )、 F(ab' )2、 Fv、 dAb、 Fd、互补决定区 (CDR)片段、单链抗体 (scFv)、 二价单链抗体、 单链噬菌体抗体、 双特异双链抗体、 三链抗体、 四链抗体；
优选的， 所述的结合分子是人单克隆抗体； 更优选的， 所述的人单克隆抗体其重链恒定区选择下组中重链类型之一的恒定区： IgGl、 IgG2a、 IgG2b和 IgG3， 和其轻链恒定区选择下组轻链类型的恒定区之一： κ链和 λ链；
更优选的， 所述的人单克隆抗体其重链恒定区和轻链恒定区分别具有 Genebank号 ACK87036和 ACK87038所示的氨基酸序列。
18. 一种多核苷酸， 其特征在于， 它编码权利要求 1-17任一所述的结合分子。
19. 一种表达载体， 其特征在于， 所述表达载体中含有：
编码权利要求 3-17任一所述的结合分子的重链的多核苷酸； 和 /或
编码权利要求 3-17任一所述的结合分子的轻链的多核苷酸。
20. 一种宿主细胞， 其特征在于， 所述宿主细胞中含有权利要求 19所述的表达载体; 或其基因组中整合有权利要求 18所述的多核苷酸。
21. 如权利要求 20所述的宿主细胞， 其特征在于， 所述的宿主细胞是果蝇 S2细胞。
22. 权利要求 1-17任一所述的结合分子的用途， 用于制备预防、 缓解或治疗禽流感 病毒感染的组合物。
23. 如权利要求 22所述的用途， 其特征在于， 所述的禽流感病毒是 H5亚型的病毒。
24. 如权利要求 23所述的用途， 其特征在于， 所述的禽流感病毒是 H5N1病毒。
25. —种药物组合物， 其特征在于， 它含有有效量的权利要求 1-17任一所述的合分 子， 以及药学上可接受的载体。
26. 如权利要求 25所述的药物组合物， 其特征在于， 所述的药物组合物还含有有效 量的其它抗流感药物， 选自： 烷胺类药物或流感病毒神经氨酸酶抑制剂。
27. 如权利要求 26所述的药物， 其特征在于， 所述的烷胺类药物包括金刚烷胺或金 刚乙胺； 或
所述的流感病毒神经氨酸酶抑制剂包括： 奧司他韦或扎那米韦。
28. 权利要求 1-17任一所述的结合分子的用途， 用于制备鉴定禽流感病毒的试剂或 试剂盒。
29. 一种预防、 缓解或治疗禽流感病毒感染的方法， 其特征在于， 所述的方法包括给 予患者有效量的权利要求 1-17任一所述的结合分子。
30. 一种鉴定禽流感病毒的方法， 所述方法包括： 将权利要求 1-17任一所述的结合 分子与待检测样品接触， 观察所述的结合分子与待检测样品的结合情况， 若所述的结合 分子与待检测样品发生结合， 则该样品中存在禽流感病毒。
31. 一种抗禽流感病毒的免疫原， 其包含有一段能与权利要求 1-17任一所述的结合 分子结合的抗原表位。
32. 如权利要求 31所述的免疫原， 其特征在于， 所述的抗原表位包含：
相对于血球凝集素的氨基酸序列第 121位的 Ser; 和
相对于血球凝集素的氨基酸序列第 162位的 Arg。
33. 如权利要求 32所述的免疫原， 其特征在于， 所述的抗原表位还包含： 相对于血球凝集素的氨基酸序列第 117位的 lie;
相对于血球凝集素的氨基酸序列第 118位的 Pro;
相对于血球凝集素的氨基酸序列第 161位的 Lys;
相对于血球凝集素的氨基酸序列第 164位的 Tyr; 或
相对于血球凝集素的氨基酸序列第 167位的 Thr。
PCT/CN2012/082277 2011-09-30 2012-09-28 高致病性禽流感的中和分子及其制备方法 WO2013044840A1 (zh)

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