CN107417789A - Neutralize anti-influenza A virus antibody and application thereof - Google Patents

Neutralize anti-influenza A virus antibody and application thereof Download PDF

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CN107417789A
CN107417789A CN201710362549.9A CN201710362549A CN107417789A CN 107417789 A CN107417789 A CN 107417789A CN 201710362549 A CN201710362549 A CN 201710362549A CN 107417789 A CN107417789 A CN 107417789A
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antibody
antigen
binding fragment
amino acid
variants
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A.兰扎韦基亚
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INST RESEARCH IN BIOMEDICINE
Institute for Research in Biomedicine IRB
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INST RESEARCH IN BIOMEDICINE
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Priority claimed from CN201180072356.0A external-priority patent/CN103717617B/en
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Abstract

The present invention relates to the epitope in the stem area of specific binding Flu-A hemagglutinin trimer and the hypotype of neutralization group 1 and the antibody or its antigen-binding fragment of the influenza A virus of 2 hypotypes of group.The invention further relates to the nucleic acid of encoding said antibody or antibody fragment, produce the antibody or the immortalised B-cell of antibody fragment and single thick liquid cell of culture and be related to epitope with reference to the antibody or antibody fragment.In addition, the purposes the present invention relates to the antibody, antibody fragment and epitope in screening technique and in the diagnosis, treatment and prevention of influenza a virus infection.

Description

Neutralize anti-influenza A virus antibody and application thereof
The application is divisional application, and the Chinese application number of its female case is 201180072356.0, and international application no is PCT/ IB2011/002329, the applying date are on July 18th, 2011.
Background technology
It is typically specific to given virus subtype to the Neutralizing antibody response of influenza A virus.There are 16 kinds by them Hemagglutinin (" HA ") albumen limit influenza A subtype.This 16 kinds of HA（H1 – H16）It may be logically divided into two groups.Group 1 by H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 hypotype form, and group 2 includes H3, H4, H7, H10, H14 and H15 hypotype.Although All hypotypes are present in birds, but mainly H1, H2 and H3 hypotype causes disease in people.H5, H7 and H9 hypotype are just Cause sporadic severe infections in people and new be very popular may be produced.H1 and H3 viruses are constantly evolved so as to produce New variant, this phenomenon are referred to as antigenic drift.Therefore, the viral and caused antibody before responding is for new drift disease The protectiveness of poison is poor or without protectiveness.As a result, the new epidemic disease for the estimated H1 occurred the and H3 viruses that must create antagonism every year Seedling, the process are sufficiently expensive and also always ineffective.This is equally applicable to the preparation of H5 influenza vaccines.It is in fact, also unclear Whether existing H5 vaccine of the Chu based on Vietnam or Indonesia's influenza A virus separation strains will can resist the popularity in future H5 viruses.
Because these reasons, will be highly desirable to have can induce can neutralize all subtypes of influenza A virus and they The wide spectrum neutralizing antibody of annual variant（Gerhard et al. is reviewed for 2006）.In addition, in wide spectrum and different subclass antibodies Medicament administration can be used as to prevent or treat influenza A infection.Manufacture for this kind of medicine, select with high titre produce with It is important to reduce the antibody of production cost.
The antibody of identification influenza A virus is characterized.For M2（Express but do not feeling on infected cell Catch an illness a kind of little albumen matter expressed on poison）Antibody have shown that protectiveness effect inside certain, it may be possible to pass through target To destroy by NK cells or cytotoxic T cell to infected cell.It is also possible to target HA albumen with neutralizing antibody.HA conducts Homotrimer premise polypeptide HA0 is synthesized.Each monomer independently can cut upon translation and formed two i.e. HA1 of polypeptide and HA2, pass through single disulfide bond.Larger N-terminal fragment（HA1,320-330 amino acid）Formation contains Binding site of receptor The film distal end globular domain of point and most of determinant by virucidin's identification.HA HA1 polypeptides be responsible for virus with The absorption of cell surface.Less C-terminal part（180 amino acid of HA2, ≈）Formed and the globular domain is anchored into cell Or the bulbous structure of viromembrane.HA2 polypeptides mediate retroviral and cell membrane merge in endosome, are answered so as to discharge ribonucleoprotein Compound is discharged into cytoplasm.
The degree of sequence homology between hypotype is in HA1 polypeptides（34% -59% homology between hypotype）It is more than in HA2 Peptide（51% -80% homology）It is medium and small.Most conservative region is the sequence around cleavage site, particularly 11 ammonia of HA2 N-terminals Base acid, referred to as fusogenic peptide, it is conservative in all subtypes of influenza A virus.Partly the region exposes as HA precursor molecules (HA0) the surface ring region (loop) in, but become non-accessible when HA0 is cut into HA1/HA2.Put it briefly, different HA In hypotype, especially in HA1-HA2 bonding pads and in HA2 areas Zhong Douyou conserved regions.But these regions can for neutralizing antibody Can be unapproachable.
Limited success is only obtained in discriminating and in terms of the antibody of the influenza A virus of more than one hypotype.In addition, The sign of the neutralization of the antibody differentiated so far is narrow and its potency is low.Okuno et al. influenza A virus/Okuda/ 57 (H2N2) are immunized mouse and have separated monoclonal antibody (C179), the conservative comformational epitope in monoclonal antibody combination HA2 And in animal model in vitro and in vivo in and group 1 H2, H1 and H5 hypotype influenza A virus（Okuno et al., 1993； Smirnov et al., 1999；Smirnov et al., 2000）.
Gioia et al. describes has H5N1 diseases in some individual serum for receiving conventional Seasonal Influenza Vaccine Malicious neutralizing antibody（Gioia et al., 2008）.The author proposes that neutralization activity is probably resisting due to neuraminidase (N1) Body.However, monoclonal antibody is not separated and does not characterize target epitope.In addition, not yet understanding whether the serum antibody neutralizes other The influenza A virus of hypotype.
The epitope and energy in the bulbous area that can combine HA have been separated from the memory B cell and thick liquid cell of immune donors The different hypotype human antibody of enough neutralization groups 1 or some subtypes of influenza A virus in group 2.However, not yet find that targeting exists so far In all 16 hypotypes guard HA tripolymers in epitope and can neutralize group 1 and group both 2 hypotypes viral A type Influenza specificity neutralizing antibody, and their separation is still the main target for the treatment of method and vaccine design.
Although having carried out the research of many decades, but without in the wide spectrum to put goods on the market and/or suppress influenza A virus sense Dye or the antibody for alleviating disease caused by influenza A virus.It is then desired to differentiate the influenza A virus for neutralizing a variety of hypotypes And the new antibodies for preventing or treating influenza A infection can be used for as medicine.Also need to differentiate and produced with high titre to reduce The antibody of production cost.
The content of the invention
The present invention is based partially on to isolate from the individual for being vaccinated with Seasonal Influenza Vaccine and can combine HA and neutralize more than one The spontaneous human monoclonal antibodies of infection and the new epitope of antibody binding of the present invention of the influenza A virus of kind hypotype. Therefore, in one aspect of the invention, the present invention includes neutralizing the A type stream of the more than one hypotype selected from 2 hypotypes of group 1 and group The antibody and its antigen-binding fragment of the infection of Influenza Virus.
In one embodiment of the invention, the present invention includes the hypotype of neutralization group 1 and organizes the influenza A virus of 2 hypotypes The antibody of the separation of infection or its antigen-binding fragment.In another embodiment of the present invention, it include the hypotype of neutralization group 1 and Organize the infection of the influenza A virus of 2 hypotypes and specifically bind the table in the stem area of Flu-A hemagglutinin (HA) tripolymer Heavy chain and light chain the contact HA tri- of the antibody or its antigen-binding fragment of the separation of position, the wherein antibody or its antigen-binding fragment First near-end monomer of aggressiveness neutralizes the amino acid in the second Distal Right monomer.
In another embodiment of the present invention, the present invention includes the hypotype of neutralization group 1 and organizes the influenza A virus of 2 hypotypes Infect and specifically bind the antibody or its antigen binding fragment of the separation of the epitope in the stem area of Flu-A HA tripolymers First near-end monomer of section, the wherein heavy chain of antibody or its antigen-binding fragment and light chain contact HA tripolymers neutralizes the second distal end Amino acid in the monomer of right side, and wherein antibody or its antigen-binding fragment is with higher at least than the titre for producing FI6 variants 2 3 times of titre produces in the cell being transfected.
In yet another embodiment of the present invention, the present invention includes the hypotype of neutralization group 1 and organizes the influenza A virus of 2 hypotypes Infect and specifically bind the antibody or its antigen binding fragment of the separation of the epitope in the stem area of Flu-A HA tripolymers First near-end monomer of section, the wherein heavy chain of antibody or its antigen-binding fragment and light chain contact HA tripolymers neutralizes the second distal end Amino acid in the monomer of right side, and wherein antibody or its antigen-binding fragment includes：(i) respectively in SEQ ID NO: 1、 It is being shown in 41 and 43 or respectively in SEQ ID NO:1st, heavy chain CDR1, CDR2 and CDR3 sequence shown in 41 and 42；With (ii) respectively in SEQ ID NO:4th, it is showing in 5 and 6 or respectively in SEQ ID NO:44th, the light chain shown in 5 and 6 CDR1, CDR2 and CDR3 sequence.
In another embodiment of the present invention, the present invention includes the antibody or its antigen-binding fragment of separation, and it is included At least one and SEQ ID NO:The complementation that any one of 1-6,17-22 or 41-44 have at least 95% sequence identity is determined Area (CDR) sequence is determined, wherein in the antibody and influenza A virus.
In another embodiment of the present invention, it includes the antibody or its antigen-binding fragment of separation, and the separation resists In body or its antigen-binding fragment and group 1 hypotype and organize 2 hypotypes influenza A virus infection and comprising：(i) exist respectively SEQ ID NO:1st, it is showing in 41 and 43 or respectively in SEQ ID NO:1st, heavy chain CDR1, CDR2 for showing in 41 and 42 and CDR3 sequences；(ii) is respectively in SEQ ID NO:4th, it is showing in 5 and 6 or respectively in SEQ ID NO:44th, show in 5 and 6 Light chain CDR1, CDR2 and CDR3 sequence gone out.
In another embodiment of the present invention, the present invention includes the antibody or its antigen-binding fragment of separation, the separation Antibody or its antigen-binding fragment include there is SEQ ID NO:1 or SEQ ID NO:The heavy chain of 17 amino acid sequence CDR1；With SEQ ID NO: 2、SEQ ID NO:18 or SEQ ID NO:The heavy chain CDR2 of 41 amino acid sequence；And tool There are SEQ ID NO: 3、SEQ ID NO: 19、SEQ ID NO:42 or SEQ ID NO:The heavy chain of 43 amino acid sequence In CDR3, the wherein antibody and influenza A virus.In another embodiment of the present invention, it include separation antibody or its Antigen-binding fragment, the antibody of the separation or its antigen-binding fragment, which include, has SEQ ID NO: 4、SEQ ID NO:20 or SEQ ID NO:The light chain CDR1 of 44 amino acid sequence；With SEQ ID NO:5 or SEQ ID NO:21 amino acid sequence The light chain CDR2 of row；With with SEQ ID NO:6 or SEQ ID NO:The light chain CDR3 of 22 amino acid sequence, wherein this is anti- In body and influenza A virus.
In yet another embodiment of the present invention, the present invention includes the antibody or its antigen-binding fragment of separation, wherein should Antibody, which includes, has SEQ ID NO:The weight chain variable district of 13 amino acid sequence and there is SEQ ID NO:14 amino acid The light chain variable district of sequence；Or there is SEQ ID NO:The weight chain variable district of 33 amino acid sequence and there is SEQ ID NO: The light chain variable district of 14 amino acid sequence；Or there is SEQ ID NO:The weight chain variable district of 29 amino acid sequence and have SEQ ID NO:The light chain variable district of 30 amino acid sequence；Or there is SEQ ID NO:The heavy chain of 35 amino acid sequence can Become area and there is SEQ ID NO:The light chain variable district of 30 amino acid sequence；Or there is SEQ ID NO:59 amino acid sequence The weight chain variable district of row and there is SEQ ID NO:The light chain variable district of 57 amino acid sequence；Or there is SEQ ID NO: 59 Amino acid sequence weight chain variable district and there is SEQ ID NO:The light chain variable district of 61 amino acid sequence；Or there is SEQ ID NO:The weight chain variable district of 55 amino acid sequence and there is SEQ ID NO:The light chain variable district of 57 amino acid sequence； Or there is SEQ ID NO:The weight chain variable district of 55 amino acid sequence and there is SEQ ID NO:61 amino acid sequence In light chain variable district and the wherein antibody and organize 1 hypotype and organize the influenza A virus of 2 hypotypes.Present invention additionally comprises antibody or Its antigen-binding fragment, the wherein antibody are FI6 variants 1, FI6 variants 2, FI6 variants 3, FI6 variants 4 or FI6 variants 5.
In another embodiment of the present invention, the present invention includes the hypotype of neutralization group 1 and organizes the influenza A virus of 2 hypotypes Infection antibody or its antigen-binding fragment, wherein antibody or its fragment is by immortalised B-cell clonal expression.
In another aspect, the present invention includes the nucleic acid of the polynucleotides of the antibody comprising the coding present invention or antibody fragment Molecule.In yet another aspect, the present invention include comprising the present invention nucleic acid molecules carrier and expression the present invention antibody or its The cell of antigen-binding fragment.In another embodiment, the present invention includes the cell of the carrier comprising the present invention.At another Aspect, the present invention include separation or purifying immunogenic polypeptide, and the immunogenic polypeptide includes the antibody for combining the present invention Or the epitope of antigen-binding fragment.
Present invention additionally comprises pharmaceutical composition, the pharmaceutical composition include the present invention antibody or its antigen-binding fragment, The cell of the nucleic acid molecules of the present invention, the carrier comprising nucleic acid molecules of the invention, expression antibody of the invention or antibody fragment, The cell of carrier comprising the present invention or immunogenic polypeptide and the pharmaceutically acceptable diluent or carrier of the present invention. Present invention additionally comprises pharmaceutical composition, the pharmaceutical composition include first antibody or its antigen-binding fragment and secondary antibody or its Antigen-binding fragment, wherein first antibody are antibody of the invention, and secondary antibody is sick to neutralize Flu-A or influenza B Any antibody or its antigen-binding fragment of poison infection.
The antibody or its antigen-binding fragment of the present invention, the nucleic acid of the present invention, the carrier comprising nucleic acid of the invention, expression The cell of the carrier of the present invention, separation or purifying the immunogene for including the antibody for combining the present invention or the epitope of antibody fragment Property polypeptide or the present invention pharmaceutical composition (i) prepare be used to treat the medicine of influenza a virus infection in, (ii) in epidemic disease The purposes of Miao Zhong or (iii) in the diagnosis of influenza a virus infection is also contemplated within being within the scope of the invention.In addition, this Whether the antibody of invention or its antigen-binding fragment are by checking the antigen of anti influenza A virus vaccine containing having correct structure The specificity epitope of elephant is also contemplated within being within the scope of the invention to monitor the purposes of the quality of the vaccine.
In another aspect, the present invention provides prevention, treatment or mitigates influenza a virus infection or reduce Flu-A The method of the risk of virus infection, including apply the antibody of the invention of therapeutically effective amount to subject in need thereof or resist Former binding antibody fragment.
In another aspect, the present invention includes the antibody of the specific binding present invention or the epitope of its antigen-binding fragment, Its (i) is used in therapy, (ii) is used for the medicine of preparation treatment influenza a virus infection, (iii) is used as vaccine or (iv) is used The part of influenza a virus infection can be neutralized in screening.
Brief description of the drawings
Fig. 1 shows the 293F cells of the carrier transient transfection for the gene for encoding FI6 variants 2,3,4 or 5 with expression Titre caused by antibody.
Fig. 2A and 2B be respectively with FI6 variants 3（It is referred to as FI6 in figure）Compound H1 HA and H3 HA F subdomains Surface display.The side chain for facilitating the hydrophobic groove of conservative selected in HA1 and HA2 is indicated with arrows, and its approximate boundaries is used Black line indicates.Thr (40) and Thr (318) are located in HA1, and Ile (45), Trp (21), Thr (41), Leu (38) and 18-21 turnovers (turn) are located in HA2.
Fig. 3 shows FI6 variants 3（It is referred to as FI6 in figure）And the combination of the F subdomains of HA tripolymers.Fig. 3 A are shown The H3 HA of three antibody of FI6 variants 3 of combination represented with Ribbons representations tripolymer.The HA1 of one of HA monomers is Black, HA2 are Dark grey, and other two HA monomers are light gray.Fig. 3 B and 3C respectively illustrate the compound of H1/FI6 variants 3 With LCDR1 ring regions in the compound of H3/FI6 variants 3 and the scaling view of the interaction of the fusogenic peptide of adjacent HA monomers.Fig. 3 D and 3E shows the compound (pdb of compound (pdb ID 2GBM) (D) and H5 HA and F10 from H5 HA and CR6261 3FKU) the structure of the monomer of (E), they combine similar region compared with FI6 variants 3 but their VH domains are on HA The low 5-10 in position.
Fig. 4 shows FI6 variants 3（It is referred to as FI6-v3 in figure）With the interaction of H1 and H3 HA F subdomains. Which depict HCDR3 the and LCDR1 ring regions of H1 HA and H3 HA F subdomains and the FI6 variants 3 with selected side chain Surface represents.Immediate HA monomers are described with light gray；Distal Right monomer is described with light gray；Tied with H3 HA1 N38 The glycan of conjunction is described with Dark grey spheroid.
Fig. 5 shows the group-specific difference at cross reacting antibody binding site.Fig. 5 A and 5B show Asn-38 The carbohydrate side chain at place combines in structures (apo-structure) (A) of the H3 HA without part and in FI6 variants 3 Positioning in structure (B).Fig. 5 C and 5D show orientations of the HA1 Trp-21 in different antibodies compound.Subgraph C is shown The FI6 variants 3 to be interacted with H1 or H3 HA F subdomains（It is referred to as FI6 in figure）HCDR3 ring regions Phe-100D. Subgraph D shows the HCDR2 ring regions for the F10 and CR6261 antibody for presenting Phe-55 and Phe-54 to H5 HA Trp-21 respectively.
Fig. 6 shows contact surface of the FI6 variants 3 on HA.Four sons have illustrated FI6 variants 3, CR6261 and F10 Trace of the antibody on HA（With black line outlining）；Three HA monomers are described with white, light grey or Dark grey.Fig. 6 A：FI6 Variant 3/ does not cut H1 contact trace；Fig. 6 B：FI6 variants 3/ cut H3；Fig. 6 C：CR6261/H5；Fig. 6 D：F10/H5.With CR6261 and F10 is different, and FI6 variants 3 are formed with two HA monomers and contacted.For the compound of FI6 variants 3, antibody/HA marked The glycosylation site of interface.
Fig. 7 shows the residue that HA is contacted in VH the and VK chains of FI6 variants 3（Runic, Kabat numerical systems）.
Fig. 8 shows that FI6 variants 3 assign protective effect in the mouse model of influenza a virus infection.Fig. 8 A are shown With 10 MLD50（50% lethal dose in mouse）H1N1 reassortant virus is through first three hour of intranasal infection through vein Interior (i.v.) receives the BALB/c mouse of the FI6 variants 3 of various dose（Every kind of experiment condition five）Survival curve and Fig. 8 B Show its body weight loss.Fig. 8 C are shown with 3 × 105Pfu H3N2 HK-x31 viruses infect first three hour through quiet Receive the mouse of the FI6 variants 3 of various dose in arteries and veins（Every kind of experiment condition ten）Body weight loss.Shown is to be carried out Three experiments in a representative experiment.It is also shown that the 0th day after being infected with 10 MLD50 reassortant virus （3 hours before infection）Or the 1st, the 2 and 3 day mouse through intravenously receiving 15mg/kg FI6 variants 3（Every kind of experiment condition five） Survival curve（Fig. 8 D）And body weight loss（Fig. 8 E）.Show a representative experiment in two carried out experiments.Institute It is illustrated that average value ± SD.Fig. 8 F and 8G are shown is receiving 10mg/kg (F) with reassortant virus infection the previous day Or 3mg/kg (G) FI6 variants 2 (FI6-v2), FI6-v2 KA（Lack complement combination）、FI6-v2 LALA（Lack complement With FcR combinations) or control antibodies mouse（Every kind of experiment condition ten）Survival curve.
Fig. 9 shows the Pneumovirinae titre of the mouse handled after H1N1 PR/8/34 lethal challenges through FI6 variants 3. BALB/c mouse（Every kind of four mouse of experiment condition）In first three through intranasal (i.n.) infection of 10 MLD50 H1N1 PR/8/34 Hour（0th day）Or receive within the 1st, 2 or 3 day afterwards intravenous injection 15mg/kg FI6 variants 3 or control antibodies（HJ16, HIV- 1 specificity）.4 days measure virus titers after pulmonary infection.In the brain, virus can not be detected.Data must be schemed with case（box- and-whiskers）Form is presented, and its raising middle flask extends to the 75th hundredths from the 25th hundredths, and centre is horizontal line.Above case With the palpus instruction extreme value of lower section.The result of student t test statisticses analysis is for p<Represented for 0.05 with *, for p<0.001 For represented with * * *.
Figure 10 shows that the FI6 variants 3 combined with HA stems area disturb the HA0 cuttings of proteases mediate.With FI6 variants 3, FE17（Identify the human antibody in the Ca2 sites in HA spherical heads）Or control antibodies incubate the restructuring from H1 NC/99 separation strains HA.Then HA- mixtures of antibodies is made to be exposed to the trypsase handled through TPCK at 37 DEG C 5,10 or 20 minutes.Then becoming Property under the conditions of sample is run on polyacrylamide gel glue and using the HA2 and HA0 that can recognize that all influenza A strains Biotinylation people mAb (FO32) makes Western blotting develop.It is illustrated that HA0 bands.Show a generation in three experiments Table is tested.
Embodiment
The present invention, which is based in part on, to be found and separates from the individual for being vaccinated with seasonal Flu-A vaccine in energy wide spectrum The new epi-position combined with the naturally-produced human antibody of the influenza A virus of different subtype and antibody of the present invention.Wish Such antibody is obtained, because only needing a kind of or a few antibody to neutralize the influenza A virus of different subtype.Separately Outside, produced in wide spectrum with different subclass antibodies with high titre so as to reduce the cost of medicine of the production comprising antibody.In addition, by this kind of The epitope of antibody identification can turn into induce and make for the seasonality of different subtype and the broad spectrum protection of candidate's prevalence separation strains A part for vaccine.
Therefore, in one aspect, the present invention provides the antibody and its antigen-binding fragment of separation, and it can neutralize at least two Influenza A virus in 2 hypotypes of group 1 and group.In one embodiment, the present invention provides antibody or its antigen binding of separation Fragment, its influenza a virus infection that can be neutralized 1 hypotype of group and organize 2 hypotypes.
In another embodiment, there is provided the antibody of separation or its antigen-binding fragment, it can neutralize 1 hypotype of group and group 2 The influenza a virus infection of hypotype and specifically bind the epitope in the stem area of Flu-A HA tripolymers, the wherein antibody Or in the first near-end monomer and the second Distal Right monomer of heavy chain and light chain the contact HA tripolymers of its antigen-binding fragment Amino acid.
As discussed in the early time, HA albumen is as the tripolymer Precursor Peptide HA0 for including three same monomers（Homo-trimeric Body）And synthesize.Each monomer can be cut independently of other two monomers, or may not be such.Cut after each monomer is translated Cut to form two polypeptides, i.e. HA1 and HA2, itself or by single disulfide bond.Heavy chain and light chain the contact HA of antibody of the present invention Two in three monomers of tripolymer.The monomer that antibody of the present invention is contacted can be cutting or not be cut.To be clear For the sake of clear and for the purpose that is schematically shown in accompanying drawing is understood, two antibody that antibody of the present invention is contacted are referred to as near-end Monomer and Distal Right monomer.
As used herein, the interchangeable present invention that is used to refer to of term " antigen-binding fragment ", " fragment " and " antibody fragment " resists Any fragment of the antigen-binding activity of the reservation antibody of body.The example of antibody fragment include but is not limited to single-chain antibody, Fab, Fab ', F (ab') 2, Fv or scFv.In addition, term " antibody " used herein includes antibody and both its antigen-binding fragments.
As used herein, " neutralizing antibody " refers to that one kind can neutralize, that is, prevents, suppresses, reduces, hinders or disturb pathogen Trigger and/or keep the antibody of the ability of infection in host.Term " neutralizing antibody " and " antibody neutralized ... " herein may be used With used interchangeably.As described herein, these antibody can individually or in combination, be used as after appropriate prepare prophylactic or Therapeutic agent, it is used in combination with active vaccine inoculation, as diagnostic tool or as the tool of production.
X-ray crystallography as known in the art with the different subclass antibodies of the specificity of the group of H1 and H5 HA cocrystallization 1 is ground Study carefully and show that the only one monomer of antibody and HA tripolymers interacts.In addition, research show these antibody make HA only with heavy chain CDR contact residues are without the CDR contact residues with light chain.On the contrary, antibody or antigen-binding fragment and non-contact three of the present invention One in individual HA monomers, but contact two.In addition, the antibody of the present invention makes HA and the CDR from both heavy chain and light chain Contact residues.In addition, by the antibody of the present invention or the property of antigen-binding fragment and the HA interactions formed with being resisted by other Body（CR6261 and F10）The interactive property of composition is significantly different.Most significant difference is on antibody of the present invention and HA The interaction of hydrophobic groove is only mediated by the CDR3 (HCDR3) of heavy chain, and for CR6261 and F10, related in the combination And all three HCDR.
In one embodiment, the amino acid in the heavy chain of antibody of the invention or antigen-binding fragment contact near-end monomer Residue, and the near-end monomer and Distal Right monomer two of the light chain of the antibody of the present invention or antigen-binding fragment contact HA tripolymers Amino acid residue in person.The monomer of antibody contact of the present invention（That is, near-end monomer and Distal Right monomer）It can not be cut, or It can be cut and form HA1 and HA2 polypeptides.In one embodiment, near-end monomer and Distal Right monomer are cut.Another In one embodiment, near-end monomer and Distal Right monomer are not cut.
16 kinds of differences of 16 kinds of hypotypes of antibody and antigen-binding fragment the specific binding influenza A virus of the present invention The epitope guarded in HA.In one embodiment, antibody of the invention or antigen-binding fragment, which combine, includes position in HA1 329 The epitope of amino acid residue in the amino acid residue and HA2 at place at position 1,2,3 and 4, wherein HA1 and HA2 are present in HA tri- Aggressiveness is not cut in monomer.
In another embodiment, the heavy chain of antibody of the invention or antigen-binding fragment contact near-end or Distal Right list In amino acid residue and HA2 in the HA1 of body at position 318 at position 18,19,20,21,38,41,42,45,49,53 and 57 Amino acid residue.These monomers can be not cut or cutting.
In yet another embodiment, the near-end list that the contact of the light chain of antibody of the invention or antigen-binding fragment is not cut Position 327 in the HA1 of amino acid residue in the HA2 of body at position 38,39 and 43 and not cut Distal Right monomer, Amino acid residue at 328 and 329 and in HA2 at position 1,2,3 and 4.
In yet another embodiment, antibody of the invention and antigen-binding fragment specific binding are near comprising what is be not cut Hold position 18 in the amino acid residue and HA2 in the HA1 of monomer at position 318,19,20,21,38,39,41,42,43,45, 48th, the epitope of the amino acid residue at 49,53,56 and 57.In addition, these antibody specificities are combined comprising not cut distal end Amino acid in amino acid residue and HA2 polypeptides in the HA1 of right side monomer at position 327,328,329 at position 1,2,3 and 4 The epitope of residue.
In another embodiment, in the HA2 of the light chain of antibody of the invention or antigen-binding fragment contact near-end monomer Amino acid residue at position 38,39,42 and 46 and position 7 in the HA1 of Distal Right monomer at position 321 and 323 and in HA2 With the amino acid residue at 11.In this embodiment, proximally and distally right side both monomers are cut.
In yet another embodiment, antibody of the invention and antigen-binding fragment specific binding include the near-end list of cutting Position 18 in amino acid residue and HA2 in the HA1 of body at position 318,19,20,21,38,39,41,42,45,46,49,52, Amino acid in amino acid residue at 53 and 57, and the HA1 of cut Distal Right monomer at position 321 and 323 is residual The epitope of amino acid residue in base and HA2 at position 7 and 11.
As shown here, antibody of the invention or antigen-binding fragment can specifically bind the A type of all 16 kinds of hypotypes The HA of influenza virus.In one embodiment, antibody specificity combination hypotype H1, H2 of the invention, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 Flu-A HA.
In another embodiment of the present invention, the present invention provides the antibody with high production titre.For example, once Separate two kinds of extremely similar antibody of the present invention（FI6 variants 1 and FI6 variants 2）, just synthesize the several variant of the antibody （The especially variant of FI6 variants 2）To improve the generation in transfectional cell.In one embodiment, antibody of the invention or Antigen-binding fragment is produced with the titre of high at least 1.5 times of the titre than producing FI6 variants 2 in the cell being transfected.Another In one embodiment, antibody of the invention with than produce FI6 variants 2 titre it is high by least 1.8,2,2.2,2.5,2.7,3,3.2, 3.4th, 3.6,3.8,4,4.2,4.4,4.6,4.8,5,5.3,5.6 or 6 times of titre produces.In certain embodiments, it is of the invention Antibody or antigen-binding fragment with than produce FI6 variants 2 high at least 3 times, at least 4 times or at least 4.5 times of titre titre Produced in the cell being transfected.
Therefore, in one embodiment, the present invention provides a kind of antibody or its antigen-binding fragment of separation, its neutralization group The influenza A virus of 2 hypotypes of 1 hypotype and group infects and specifically binds the table in the stem area of Flu-A HA tripolymers First near-end monomer of heavy chain and light chain the contact HA tripolymers of position, the wherein antibody or its antigen-binding fragment and the second distal end Amino acid in the monomer of right side, and wherein antibody or its antigen-binding fragment in transfectional cell with than produce FI6 variants 2 when For example, at least 3 times of titre height titre produce.
As described herein, transfectional cell can be those skilled in the art it is currently known or it is later find be used for express code book Any cell of the nucleotide sequence of invention antibody.The example of such cell includes but is not limited to mammalian host cell, example Such as CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma.In addition, these cells can be transiently transfected or stabilization turns Dye.Transfect type and suitable for transfection cell type in the range of the technical ability of those skilled in the art.
In another embodiment, antibody of the invention or antigen-binding fragment specific binding include SEQ ID NO: 37th, in 38,39 or 40 the amino acid sequence of any one polypeptide.
Human monoclonal antibodies, the immortalised B-cell clone of secretion antibody of the present invention or the host cell and coding of transfection The nucleic acid of antibody of the present invention is intended to be included within the scope of the present invention.As used herein, term " Broadspectrum specificity " is used to refer to tie Close and/or neutralize the antibody of the present invention or anti-of the influenza A virus of 2 hypotypes of 1 hypotype of one or more groups and one or more groups Former binding fragment.
In the antibody or antigen-binding fragment of the present invention and one or more organize 1 hypotype（H1、H2、H5、H6、H8、H9、 H11, H12, H13 and H16 and their variant）2 hypotypes of influenza A virus and one or more group（H3、H4、H7、 H10, H14 and H15 and their variant）Influenza A virus.In one embodiment, the exemplary hypotype of group 1 includes H1, H2, H5, H6 and H9, the exemplary hypotype of group 2 include H3 and H7.
The antibody and antibody fragment of the present invention can neutralize the various combinations of subtypes of influenza A virus.In one embodiment In, antibody can neutralize influenza A virus H1 and H3 hypotype or H2 and H3 hypotypes, or H3 and H5 hypotypes, or H3 and H9 hypotypes, or H1 and H7 hypotypes, or H2 and H7 hypotypes, or H5 and H7 hypotypes, or H7 and H9 hypotypes.
In another embodiment, antibody of the invention and antibody fragment can neutralize influenza A virus H1, H2 and H3 Asia Type, or H1, H3 and H5 hypotype, or H1, H3 and H9 hypotype, or H2, H3 and H5 hypotype, or H2, H3 and H9 hypotype, or H3, H5 and H9 hypotypes, or H1, H2 and H7 hypotype, or H1, H5 and H7 hypotype, or H1, H7 and H9 hypotype, or H2, H5 and H7 hypotype, or H2, H7 and H9 hypotypes, or H5, H7 and H9 hypotype, or H1, H3 and H7 hypotype, or H2, H3 and H7 hypotype, or H3, H5 and H7 hypotype, or H3, H7 and H9 hypotype.
In yet another embodiment, antibody can neutralize influenza A virus H1, H2, H3 and H7 hypotype, or H1, H3, H5 and H7 hypotypes, or H1, H3, H7 and H9 hypotype, or H2, H3, H5 and H7 hypotype, or H2, H3, H7 and H9 hypotype, or H3, H5, H7 and H9 hypotypes, or H1, H2, H3 and H5 hypotype, or H1, H2, H3 and H9 hypotype, or H1, H3, H5 and H9 hypotype, or H2, H3, H5 and H9 hypotypes, or H1, H2, H5 and H7 hypotype, or H1, H2, H7 and H9 hypotype, or H1, H5, H7 and H9 hypotype, or H2, H5, H7 and H9 hypotypes.
In another embodiment, antibody of the invention can neutralize influenza A virus H1, H2, H3, H5 and H7 hypotype, or H1, H2, H3, H7 and H9 hypotype, or H1, H3, H5, H7 and H9 hypotype, or H2, H3, H5, H7 and H9 hypotype, or H1, H2, H3, H5 With H9 hypotypes, or H1, H2, H5, H7 and H9 hypotype, or H1, H2, H3, H5, H7 and H9 hypotype.In yet another embodiment, except Neutralize outside influenza A virus H6 hypotypes, antibody of the invention and antigen-binding fragment also neutralize one of combinations thereof or more Person.
The antibody and antigen-binding fragment of the present invention has high neutralization titer.Required for neutralizing 50% influenza A virus The concentration of antibody of the present invention may be, for example, about 50 μ g/ml or lower.In one embodiment, 50% influenza A virus institute is neutralized The concentration of the antibody of the present invention needed is about 50,45,40,35,30,25,20,17.5,15,12.5,11,10,9,8,7,6,5, 4.5th, 4,3.5,3,2.5,2,1.5 or about 1 μ g/ml or lower.In another embodiment, 50% influenza A virus institute is neutralized The concentration of the antibody of the present invention needed is about 0.9,0.8,0.75,0.7,0.65,0.6,0.55,0.5,0.45,0.4,0.35, 0.3、0.25、0.2、0.15、0.1、0.075、0.05、0.04、0.03、0.02、0.01、0.008、0.006、0.004、0.003、 0.002 or about 0.001 μ g/ml or lower.This means neutralize 50% influenza A virus only to need the antibody of low concentration.It can be used Standard neutralizing mensuration method well known by persons skilled in the art come measure specificity and potency.
The present invention provides has specific Broadspectrum specificity for HA and the influenza A virus of the one or more groups 1 of neutralization is sub- The antibody of the subtypes of influenza A virus of type and one or more groups 2.The present invention antibody binding HA selected from group 1 and group 2 Two or more subtypes of influenza A virus between guard region in epitope.
In one embodiment, the present invention provides antibody, such as the antibody of separation or the antibody of purifying, and it is specifically tied Charge-coupled 1 and group 2 subtypes of influenza A virus HA stem area in conserved epitope and viral interference duplication and propagation.This hair Bright also to provide antibody, such as the antibody of separation or the antibody of purifying, it specifically combines the HA of 2 hypotypes of group 1 and group stem area In conserved epitope and suppress cell entry cell.It is not bound to any theory, it is in one exemplary embodiment, of the invention Conserved epitope in the stem area of antibody or antigen-binding fragment combination influenza A virus is simultaneously suppressed by disturbing fusion steps Cell entry cell.In one embodiment, antibody of the invention or antigen-binding fragment are by raising complement and expressing FcR's Kill cell and mediate antibody dependent cells toxicity (antibody-dependent cell cytotoxicity, ADCC) is come Limiting virus are propagated.The epitope of albumen or antigenic determinant correspond to the binding site that the molecule is antibody（Or paratope）Institute is special Those parts of opposite sex identification.Therefore, epitope is to need complementary paratope to carry out the correlation of their operability identification in fact Body.The epitope guarded between the different variants of albumen refers to identical complementation potential energy by contacting the same sections of these molecules And specifically identify these different variants.
The antibody of the present invention can be monoclonal, such as human monoclonal antibodies, or recombinant antibodies.The present invention also provides this The fragment of invention antibody, especially retain the fragment of the antigen-binding activity of antibody.Although this specification（Including claim Book）In some places, clearly refer to antigen-binding fragment, antibody fragment, variant and/or the derivative of antibody, but should manage Solution, term " antibody " or " antibody of the invention " include all antibody isotypes, the i.e. antigen-binding fragment of antibody, antibody piece Section, variant and derivative.
In one embodiment, two or more A type streams in antibody and antibody fragment of the invention with group 1 and group 2 The combination of Influenza Virus hypotype.Exemplary subtypes of influenza A virus includes but is not limited to H5N1（A/ Vietnam/1203/04）、H1N1 （A/ New Caledonia/20/99）、H1N1（A/ Solomon Islands/3/2006）、H3N2（A/ Wyoming/3/03）And H9N2（A/ Chicken/Hong Kong/G9/97）.In another embodiment, in antibody and 3,4,5,6,7 or more kind groups 1 and group 2 influenza A virus The combination of hypotype and/or the combination tool specificity to 2 subtypes of influenza A virus of 3,4,5,6,7 or more kind groups 1 and group.
In one exemplary embodiment, the present invention is included to subtypes of influenza A virus H1 and H3（Such as H1N1 and H3N2）Or H1, H3, H5 and H9（Such as H1N1, H3N2, H5N1 and H9N2）Have specific antibody or its antibody fragment.Again In one embodiment, antibody or its antibody fragment are to H1, H3, H5, H7 and H9（Such as H1N1, H3N2, H5N1, H7N1, H7N7, H9N2）Tool specificity.Other example combinations of subtypes of influenza A virus are provided in the previous section of the application.
The SEQ of the weight chain variable district (VH) of the exemplary antibodies of the present invention and the amino acid sequence of light chain variable district (VL) The SEQ ID numberings that ID numbered and encoded their nucleotide sequence are listed in Table 1 below.
Table 1：The V of exemplary influenza A virus neutralizing antibodyHAnd VLThe SEQ ID of polypeptide and polynucleotides are numbered
In one embodiment, antibody of the invention or antibody fragment include weight chain variable district, and it has and SEQ ID NO: 13rd, 33,55,59,29 or 35 sequences listed in any one have about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% Or 100% homogeneity amino acid sequence.In another embodiment, antibody of the invention or antibody fragment include light chain variable Area, it has and SEQ ID NO:14th, sequence listed in 57,61 or 30 have about 70%, 75%, 80%, 85%, 90%, 95%, 97%th, the amino acid sequence of 98%, 99% or 100% homogeneity.
In yet another embodiment, the weight chain variable district of antibody of the present invention can be encoded by such nucleic acid, and the nucleic acid has With SEQ ID NO:15th, 34,56,60,31 or 36 sequences listed in any one have about 70%, 75%, 80%, 85%, 90%, 95%th, the sequence of 97%, 98%, 99% or 100% homogeneity.In yet another embodiment, the light chain variable district of antibody of the present invention can be by Such nucleic acid coding, the nucleic acid have and SEQ ID NO:16th, sequence listed in 58,62 or 32 have about 70%, 75%, 80%th, the sequence of 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity.
The CDR of heavy chain of antibody is referred to as CDRH1（Or HCDR1）、CDRH2（Or HCDR2）And CDRH3（Or HCDR3）.Phase As, the CDR of antibody light chain is referred to as CDRL1（Or LCDR1）、CDRL2（Or LCDR1）And CDRL3（Or LCDR1）.CDR ammonia The position of base acid is defined as according to IMGT numbering systems：CDR1-IMGT positions 27 to 38, CDR2-IMGT positions 56 to 65 With CDR3-IMGT positions 105 to 117.
Table 2 each provides the SEQ of the heavy chain of the exemplary antibodies of the present invention and six CDR of light chain amino acid sequence ID is numbered.
Table 2：The SEQ ID numberings of the CDR polypeptides of exemplary influenza A virus neutralizing antibody
In one embodiment, antibody of the invention or antibody fragment are included and at least one had and SEQ ID NO：1-6、41- Any one CDR with least sequence of 95% sequence identity of 44 or 17-22.
In another embodiment, the present invention provides the antibody for including such heavy chain, and the heavy chain includes one or more （I.e. one, two or all three）From FI6 variants 1, FI6 variants 2, FI6 variants 3, FI6 variants 4, FI6 variants 5, FI28 The heavy chain CDR of variant 1 or FI28 variants 2.In one exemplary embodiment, antibody of the invention or antigen-binding fragment include With SEQ ID NO:1 or SEQ ID NO:The heavy chain CDR1 of 17 amino acid sequence；With SEQ ID NO: 2、SEQ ID NO:41 or SEQ ID NO:The heavy chain CDR2 of 18 amino acid sequence；With with SEQ ID NO: 3、SEQ ID NO:42、 SEQ ID NO:43 or SEQ ID NO:The heavy chain CDR3 of 19 amino acid sequence.In certain embodiments, it is presented herein Antibody or antibody fragment include such heavy chain, the heavy chain includes the SEQ ID NO (i) and be used for CDRH1:1st, for CDRH2 SEQ ID NO:The 2 and SEQ ID NO for CDRH3:3, (ii) is used for CDRH1 SEQ ID NO:1st, for CDRH2 SEQ ID NO:The 41 and SEQ ID NO for CDRH3:42, (iii) is used for CDRH1 SEQ ID NO:1st, it is used for CDRH2 SEQ ID NO:The 41 and SEQ ID NO for CDRH3:43, or the SEQ ID NO of (iv) for CDRH1: 17th, the SEQ ID NO for CDRH2:The 18 and SEQ ID NO for CDRH3: 19.
The present invention also provides the antibody for including such light chain, and the light chain includes one or more（I.e. one, two or institute There are three）From FI6 variants 1, FI6 variants 2, FI6 variants 3, FI6 variants 4, FI6 variants 5, FI28 variants 1 or FI28 variants 2 Light chain CDR.In one exemplary embodiment, antibody of the invention or antigen-binding fragment, which include, has SEQ ID NO: 4、SEQ ID NO:44 or SEQ ID NO:The light chain CDR1 of 20 amino acid sequence；With SEQ ID NO:5 or SEQ ID NO:The light chain CDR2 of 21 amino acid sequence；With with SEQ ID NO:6 or SEQ ID NO:22 amino acid sequence Light chain CDR3.In certain embodiments, antibody provided in this article includes such light chain, and the light chain is used for CDRL1 comprising (i) SEQ ID NO:4th, the SEQ ID NO for CDRL2:The 5 and SEQ ID NO for CDRL3:6, or (ii) be used for CDRL1 SEQ ID NO:44th, the SEQ ID NO for CDRL2:The 5 and SEQ ID NO for CDRL3:6, or (iii) SEQ ID NO for CDRL1:20th, the SEQ ID NO for CDRL2:The 21 and SEQ ID NO for CDRL3: 22.
In one embodiment, antibody of the invention or its antigen-binding fragment include the antibody FI6 variants listed in table 2 1 all CDR, and neutralize influenza a virus infection.In another embodiment, antibody of the invention or its antigen binding Fragment includes all CDR for the antibody FI6 variants 2 listed in table 2, and neutralizes influenza a virus infection.In another reality To apply in example, antibody of the invention or its antigen-binding fragment include all CDR for the antibody FI6 variants 3 listed in table 2, and Neutralize influenza a virus infection.In another embodiment, antibody of the invention or its antigen-binding fragment include arranges in table 2 All CDR of the antibody FI6 variants 4 gone out, and neutralize influenza a virus infection.In another embodiment, it is of the invention Antibody or its antigen-binding fragment include all CDR for the antibody FI6 variants 5 listed in table 2, and neutralize influenza A virus Infection.
In yet another embodiment, antibody of the invention or its antigen-binding fragment include the antibody FI28 listed in table 2 All CDR of variant 1, and neutralize influenza a virus infection.In yet another embodiment, antibody of the invention or its antigen Binding fragment includes all CDR for the antibody FI28 variants 2 listed in table 2, and neutralizes influenza a virus infection.
The example of antibody of the present invention includes but is not limited to FI6 variants 1, FI6 variants 2, FI6 variants 3, FI6 variants 4, FI6 and become Body 5, FI28 variants 1 and FI28 variants 2.
Present invention additionally comprises the antibody or its fragment combined with antibody identical epitope of the present invention, or the antibody with the present invention Or the antibody of antigen-binding fragment competition.
The antibody of the present invention also includes heteroantibody molecule, and the heteroantibody molecule includes one or more of antibody of the present invention Individual CDR and another antibody for same epitope one or more CDR.In one embodiment, such hybrid antibody bag Three CDR containing antibody of the present invention and another antibody for same epitope three CDR.Exemplary hybrid antibody includes i) Three light chain CDR of antibody of the present invention and another antibody for same epitope three heavy chain CDR；Or ii) antibody of the present invention Three heavy chain CDR and another antibody for same epitope three light chain CDR.
Variant antibodies are intended to be included within the scope of the present invention.Therefore, the variant for the sequence enumerated in this application also includes Within the scope of the invention.This kind of variant include by during immune response in vivo or culture immortalised B-cell clone When natural variant caused by somatic mutation in vitro.Or, it is also possible to variant is produced because of degenerate, such as It is upper described, or variant is generated due to transcription or translation error.
Antibody sequence has other variants of improved affinity and/or potency can be by using the side being known in the art Method obtains, and is included within the scope of the invention.For example, amino acid replacement can be used for obtaining the affinity with further improving The antibody of power.Or the codon optimization of nucleotide sequence also can be used to improve for produce antibody expression system in turn over Translate efficiency.In addition, comprising by the present invention any nucleotide sequence application orthogenesis method and optimize antibody specificity or in It is also within the scope of the present invention with the polynucleotides of the sequence of activity.
In one embodiment, variant antibodies sequence can have 70% or more with sequence described herein（I.e. 75%, 80%th, 85%, 90%, 95%, 97%, 98%, 99% or more）Amino acid sequence identity.In certain embodiments, such sequence Row homogeneity is relative to reference sequences（Sequence i.e. described herein）Total length calculate.In other embodiment In, percentage identity mentioned herein is to be used using BLAST version 2s .1.3 by NCBI（American National biotechnology Information centre (the National Center for Biotechnology Information)；http:// www.ncbi.nlm.nih.gov/）Default parameters [the matrixes of BIosum 62 specified；Gap open penalty (gap open Penalty)=11 and gap extension penalty (gap extension penalty)=1] determined.
On the other hand, present invention additionally comprises the part or all of of the light chain for encoding antibody of the present invention and heavy chain and CDR Nucleotide sequence.The light chain and heavy chain of the exemplary antibodies provided in this article for being the coding present invention and CDR part or complete The nucleotide sequence in portion.Table 1 provides the SEQ of the heavy chain of the exemplary antibodies of the coding present invention and the nucleotide sequence of light chain variable district ID is numbered.For example, provided herein is nucleotide sequence include SEQ ID NO: 15（Encode the weight chain variable district of FI6 variants 1）、SEQ ID NO: 16（Encode FI6 variants 1 and the light chain variable district of FI6 variants 2）、SEQ ID NO: 34（Encoding the heavy chain of FI6 variants 2 can Become area）、SEQ ID NO: 56（Encode the weight chain variable district of FI6 variants 3）、SEQ ID NO: 58（Encode FI6 variants 3 and FI6 becomes The light chain variable district of body 4）、SEQ ID NO: 60（Encode FI6 variants 4 and the weight chain variable district of FI6 variants 5）、SEQ ID NO: 62 （Encode the light chain variable district of FI6 variants 5）、SEQ ID NO: 31（Encode the weight chain variable district of FI28 variants 1）、SEQ ID NO: 36 （Encode the weight chain variable district of FI28 variants 2) and SEQ ID NO: 32（Encode FI28 variants 1 and the light chain variable district of variant 2).
Table 3 provides the SEQ ID numberings of the CDR of the exemplary antibodies of coding present invention nucleotide sequence.Because heredity is close The redundancy of code, there will be the variant of the coding same amino acid sequence of these sequences.
Table 3：The SEQ ID numberings of the CDR polynucleotides of exemplary influenza A virus neutralizing antibody：
In one embodiment, included according to the nucleotide sequence of the present invention with encoding the heavy chain of antibody of the present invention or the core of light chain Acid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homogeneity Nucleotide sequence.In another embodiment, nucleotide sequence of the invention has the heavy chain or light chain CDR for encoding antibody of the present invention Nucleotide sequence.For example, included and SEQ ID NO according to the nucleotide sequence of the present invention: 7-12、15、16、34、23-28、31、 32nd, 36, the sequence of nucleotide sequence tool at least 75% homogeneity of 45-54,56,58,60 or 62.
Also include the carrier for including the nucleotide sequence according to the present invention, such as expression vector within the scope of the invention.With The cell of such carrier conversion is intended to be included within the scope of the present invention.It is thin that the example of such cell includes but is not limited to eucaryon Born of the same parents, such as yeast cells, zooblast or plant cell.In one embodiment, cell is mammalian cell, such as people is thin Born of the same parents, CHO, HEK293T, PER.C6, NS0, myeloma cell or hybridoma.
The monoclonal antibody of the epitope of antibody of the present invention can be combined the invention further relates to combination, is including but not limited to selected from FI6 variants 1, FI6 variants 2, FI6 variants 3, FI6 variants 4, FI6 variants 5, the monoclonal of FI28 variants 1 and FI28 variants 2 resist Body.
The identification and purifying of monoclonal and recombinant antibodies for its targeted indivedual polypeptide or other antigens are particularly useful. The antibody of the present invention also has other effectiveness, and wherein they can be used as immunoassay, radioimmunoassay (RIA) or enzyme-linked Reagent in immmunosorbent assay (ELISA).In these applications it is possible to such as radio isotope, fluorescence molecule or Detectable reagent carrys out labelled antibody in the analysis of enzyme etc.These antibody can be additionally used in the Molecular Identification and sign of antigen（Table Position mapping）.
The antibody of the present invention can be coupled to contribute to drug coupling for delivery to treatment site or with detectable label Include cell of interest（Such as the cell of influenza a virus infection）Site imaging.Make antibody and medicine and detectable label The method of coupling is well known in the art, and the same method being imaged using detectable label is also in the art Know.Labeled antibody can be used in various measure by using various marks.In antibody of the present invention and epitope of interest （Influenza A virus epitope）Between the detection of Antibody-antigen complex that is formed can be anti-by the way that detectable substance is attached to Body promotes.Suitable detection means is including the use of such as radionuclide, enzyme, coenzyme, fluorescer, chemiluminescence agent, chromogen Body, zymolyte or co-factor, enzyme inhibitor, prothetic group (prosthetic group) compound, free radical, particle, dyestuff etc. it The mark of class.The example of suitable enzymes includes horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholine ester Enzyme；The example of suitable prosthetic group complexes includes streptavidin/biotin and avidin/biotin；Close The example of suitable fluorescent material include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base ammonia fluorescein, Dansyl Cl or phycoerythrin；The example of luminescent material is luminol (luminol）；The example of bioluminescent material includes fluorescence Plain enzyme, fluorescein and aequorin；The example of suitable radioactive material includes 125I, 131I, 35S or 3H.It is such Labelled reagent can be used in a variety of known determination methods, such as radioimmunoassay, enzyme immunoassay（Such as ELISA）, it is glimmering Light immunoassay etc..（Referring to such as US 3,766,162, US 3,791,932, US 3,817,837 and US 4,233,402）.
Can be with such as cytotoxin, therapeutic agent or radioactive metal ion or the same position of radioactivity according to the antibody of the present invention The treatment part of element etc is conjugated.Radioisotopic example includes but is not limited to I-131, I-123, I-125, Y-90, Re- 188th, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111 etc..This kind of antibody conjugates can It is used for the given biological response of modification；Drug moiety should not be understood to be limited to the chemotherapeutant of classics.For example, medicine Part can be protein or polypeptide with required biological activity.For example, this proteinoid may include such as jequirity poison The toxin of element, ricin A, pseudomonad (pseudomonas) exotoxin or diphtheria toxin etc.
Technology by this kind for the treatment of part and antibody conjugate is well-known.See, for example, Arnon et al. (1985) " Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy ", are loaded in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. are edited（Alan R. Liss companies）, The 243-256 pages；" Antibodies for Drug Delivery ", are loaded in Hellstrom et al. editors (1987) Controlled Drug Delivery, Robinson et al. are edited（Second edition；Marcel moral Kerr Corp (Marcel Dekker, Inc.)）, the 623-653 pages；Thorpe (1985)“Antibodies Carriers of Cytotoxic Agents in Cancer Therapy:A Review ", it is loaded in Monoclonal Antibodies ' 84: Biological And Clinical Applications, Pinchera et al. are edited, the 475-506 pages（Editrice Kurtis, Italy Milan (Milano, Italy), 1985）；“Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy ", are loaded in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. are edited（New york academic publishing house (Academic Press, New York), 1985）, 303-316；With Thorpe et al. (1982) Immunol. Rev. （《Immunology is commented on》）62:119-158.
Or antibody or its antibody fragment can be conjugated to be formed such as in US 4,676 with secondary antibody or its antibody fragment, Antibody heteroconjugate (heteroconjugate) described in 980.In addition, it can be used between mark and antibody of the present invention Attachment（Such as US 4,831,175）.Antibody or its antigen-binding fragment can be used directly in radioiodine, indium, yttrium or this area Known other radioactive particles mark（Such as US 5,595,721）.Treatment can be by simultaneously or sequentially applying conjugation of antibodies Formed with the therapeutic combination of unconjugated antibody（Such as WO00/52031；WO00/52473）.
The antibody of the present invention can also be attached to solid support.In addition, the antibody or its functional antibodies piece of the present invention Section can be chemically modified for example to increase its circulating half-life by being covalently conjugated with polymer.The example of polymer and They are attached to the method for peptide in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546 Show.In certain embodiments, these polymer may be selected from oxyethylated polyols and polyethylene glycol (PEG).PEG is in room temperature Under be dissolved in water and there is formula：R (O--CH2--CH2) n O--R, wherein R can be hydrogen or such as alkyl or silane alcohol base etc Blocking group.In one embodiment, blocking group can have 1 to 8 carbon atom.In another embodiment, protection group Group is methyl.Symbol n is positive integer.In one embodiment, n is 1 to 1,000.In another embodiment, n is 2 to 500. In one embodiment, PEG has 1,000 to 40,000 mean molecule quantity.In another embodiment, PEG has 2,000 To 20,000 mean molecule quantity.In yet another embodiment, PEG has 3,000 to 12,000 mean molecule quantity.At one In embodiment, PEG has at least one hydroxyl.In another embodiment, PEG has terminal hydroxyl.In another embodiment In, the terminal hydroxyl is activated reacting with the free amine group on inhibitor.It will be appreciated, however, that reactive base can be changed The type and quantity of group obtain covalently conjugated PEG/ antibody of the present invention.
Water miscible oxyethylated polyols can also be used for the present invention.They include oxyethylated sorbierite, polyoxy Ethylization glucose, oxyethylated glycerol (POG) etc..In one embodiment, using POG.It is without being bound by any theory, Due to the glycerol backbone and the natural backbone of the monoglyceride in such as animal and people, diester and three esters of oxyethylated glycerol It is identical, therefore this branched not necessarily it is counted as foreign substance in vivo.In certain embodiments, POG has and PEG phase homotypes Enclose interior molecular weight.Another kind can be used for the drug delivery system of increase circulating half-life to be liposome.Prepare liposome delivery The method of system has been discussed in Gabizon et al. (1982), Cafiso (1981) and Szoka (1980).Other medicines Delivery system is known in the art, and in Poznansky of such as reference et al. (1980) and Poznansky (1984) It is described.
The antibody of the present invention can be provided with purified form.Generally, antibody will be present in being substantially free of the group of other polypeptides In compound, for example, wherein less than 90 weight %, be typically less than 60 weight % and be by it more commonly less than 50 weight % composition What its polypeptide was formed.
The antibody of the present invention can be inhuman（It is or heterologous）It is immunogenicity in host such as mouse.Particularly, antibody Can have in non-human host is immunogenicity and in human host is the idiotope (idiotope) of non-immunogenic.With Include being not easy the host from such as mouse, goat, rabbit, rat, non-primate mammal or the like in the antibody of the present invention of people Separation simultaneously generally can not pass through humanization or the antibody obtained from xenogenesis-mouse (xeno-mice).
The antibody of the present invention can be any isotype（Such as IgA, IgG, IgM, i.e. α, γ or μ heavy chain）, but typically will For IgG.In IgG isotypes, antibody can be IgG1, IgG2, IgG3 or IgG4 hypotype.The antibody of the present invention can have κ or λ Light chain.
The preparation of antibody
It can be prepared according to the antibody of the present invention with any method as known in the art.Prepared for example with hybridoma technology The common method of monoclonal antibody is known（Kohler, G. and Milstein, C, 1975；Kozbar et al. 1983）. In one embodiment, method is immortalized using the alternative EBV described in WO2004/076677.
Using the method described in WO2004/076677, can be converted in the presence of polyclonal B cell activation thing with EBV Produce the B cell of antibody of the present invention.It is standard technique with EBV conversions, and can be readily adapted to include polyclonal B cell activation Thing.
Optionally, the stimulant of other cell growth and cell differentiation can be added in step of converting further to improve Efficiency.These stimulants can be such as IL-2 and IL-15 etc cell factor.On the one hand, during immortalization step Add IL-2 and efficiency is immortalized with further improve, but it not is required that it, which is used,.Then, can use as known in the art Method is separated antibody to cultivate the immortalised B-cell prepared using these methods therefrom.
Using the method described in UK Patent Application 0819376.5, single thick liquid cell can be cultivated in well plates.Can With the separation antibody from single thick liquid cell culture.Furthermore it is possible to extract RNA from single thick liquid cell culture, and this can be used Known method carries out singe-cell PCR in field.By VH the and VL areas of RT-PCR amplification antibody, sequencing and it can clone into expression In carrier, then the expression vector is transfected into the Τ cells of Η Ε Κ 293 or other host cells.This area skill can be used Any method known to art personnel come complete the clone of the nucleic acid in expression vector, host cell transfection, transfection host it is thin The culture of born of the same parents and the separation of caused antibody.
If desired, filtering, centrifugation and various chromatographies can be used（Such as HPLC or affinity chromatography）It is further purified Antibody.The purification technique of antibody such as monoclonal antibody（Technology including preparing pharmaceutical grade antibody）It is known in the art.
The antibody fragment of the present invention can by including with the enzymic digestion of such as pepsin or papain etc and/or The method that disulfide bond is cut by electronation obtains from antibody.Or antibody fragment can by clone and express heavy chain or The part of the sequence of light chain obtains.Antibody " fragment " may include Fab, Fab ', F (ab') 2 and Fv fragments.The present invention is also contained The Single-Chain Fv Fragment of Murine (scFv) of heavy chain and light chain of the lid from antibody of the present invention, such as the present invention are included containing antibody of the present invention CDR scFv.Also included is heavy chain or light chain monomers and dimer, single domain heavy chain antibody, single domain light chain antibody And single-chain antibody, such as the scFv that wherein heavy chain and light-chain variable domain are engaged by peptide linker.
The antibody fragment of the present invention can assign unit price or multivalence interacts and can be included in above-mentioned various structures. For example, scFv molecules can be synthesized to produce " four chain antibodies " of " three chain antibodies " of trivalent or tetravalence.ScFv molecules may include Cause the domain in the Fc areas of divalence miniantibody (minibody).In addition, the sequence of the present invention can be multispecific molecule Component, wherein the sequence of the present invention targets the epitope of the present invention and other areas of molecule combine other targets.Example molecule Including but not limited to bispecific Fab2, tri-specific Fab3, bispecific scFv and double-chain antibody（Holliger and Hudson, 2005, Nature Biotechnology（《Nature Biotechnol》）9: 1126-1136）.
The standard technique of molecular biology can be used for preparing the antibody of the coding present invention or the DNA sequence dna of antibody fragment. Required DNA sequence dna can completely or partially be synthesized using oligonucleotide synthesis technology.Can also be depended on the circumstances use Direct mutagenesis and polymerase chain reaction (PCR) technology.
Any suitable host cell/carrier system may serve to antibody molecule or its fragment that expression encodes the present invention DNA sequence dna.Bacterium（Such as Escherichia coli）With other microfloras can be partially used for express antibody fragment such as Fab and The fragments of F (ab ') 2, and especially Fv fragments and single chain antibody fragments such as scFv.Eucaryon（Such as mammal）Host cell Expression system can be used for preparing the larger antibody molecule including complete antibody molecule.Suitable mammalian host cell bag Include but be not limited to CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma.
The present invention also provides the method for preparing the antibody molecule according to the present invention, is included in suitable for causing from code book The host cell comprising the carrier for encoding nucleic acid of the present invention is cultivated under conditions of the DNA marking proteins of invention antibody molecule and is divided From antibody molecule.
Antibody molecule can only include heavy chain or light chain polypeptide, in this case, it is only necessary to compile heavy chain or light chain polypeptide Code sequence is used for transfection host cell.Preparation for the product comprising both heavy chain and light chain, it can be transfected with two kinds of carriers Cell line, wherein first vector encode light chain polypeptide and Second support encoding heavy chain polypeptide.Or single carrier can be used, The carrier includes the sequence of coding light chain and heavy chain polypeptide.
Or antibody of the invention can be prepared as follows：I) expressed in host cell according to the present invention Nucleotide sequence, and ii) the expressed antibody products of separation.In addition, this method may also include iii) antibody purification.
The screening of the HEK293T cells of the B cell of conversion, single thick liquid cell of culture and transfection
Those of the antibody with required specificity or function can be produced from the B cell of conversion and single thick liquid cell screening of culture.
Screening step can be completed by the following method：Any immunoassays such as ELISA；To tissue or cell（Including The cell of transfection）Dyeing；Neutralizing mensuration method；Or a variety of other sides known in the art for being used to identify required specificity or function One kind in method.Determination method being easily recognized to choose based on one or more antigens；Or it is also based on required work( It can choose, such as selecting neutralizing antibody to be targeted cell rather than just antigen binding antibody to select to change Characteristic antibody, the characteristic in order to allow they signal cascade, they shape, they growth rate, they influence The ability of other cells, they to from other cells or other reagents or condition change influence response, their differentiation State etc..
Then individually conversion B cell clone can be produced from the conversion B cell culture of the positive.For from positive cell The cloning process that mixture separation is individually cloned can be by using limiting dilution, micrergy, the list by cell sorting Cell deposition or another method known in the art are carried out.
Can be used methods known in the art the nucleic acid of single thick liquid cell from culture is separated, cloned and Expressed in HEK293T cells or other host cells.
The immortalised B-cell clone of the present invention or the HEK293T cells of transfection can use in many ways, such as list The source of clonal antibody, the nucleic acid of the monoclonal antibody of interest as coding（DNA or mRNA）Source, for scientific research etc..
The present invention provides composition, and said composition is different with least two of 2 hypotypes of group selected from group 1 comprising neutralization is produced The immortalization B memory cells of the antibody of subtypes of influenza A virus or the host cell of transfection.
Epitope
As described above, the antibody of the present invention can be used for mapping to the epitope of the antibody binding.In it has been found by the present inventors that The epitope found on HA is directed to the antibody of influenza a virus infection.In one embodiment, antibody is directed to HA stem area One or more of epitope, the epitope one or more influenza A virus groups 1 and group 2 hypotypes in be conservative.This The epitope of invention antibody binding can be linear（Continuously）It is or conforma-tional（It is discontinuous）.In one embodiment, such as Discussed herein, antibody of the invention and antibody fragment combine and include SEQ ID NO:37th, 38,39 or 40 peptide zone.
In another embodiment, the epitope of antibody binding of the present invention includes one or two HA monomer as described above Amino acid residue in HA1 and HA2 polypeptides.HA monomers can be not cut or cut.
The epitope identified by antibody of the present invention can serve many purposes.Purifying or the epitope and its mimic epitope of synthesized form Available for causing immune response（I.e. as vaccine or for preparing the antibody of other purposes）Or for from serum screening and epitope Or the antibody of immune response occurs for its mimic epitope.In one embodiment, such epitope or mimic epitope or comprising so Epitope or mimic epitope antigen be used as vaccine be used for cause immune response.The antibody or antibody fragment of the present invention is also The method that can be used for monitoring vaccine quality.Specifically, whether the antigen that the antibody can be used in detection vaccine, which contains, has The correct immunogenic epitopes of correct conformation.
The epitope can be also used for part of the screening with reference to the epitope.This kind of part includes but is not limited to antibody（Including Those antibody from camel, shark and other species）, antibody fragment, peptide, display technique of bacteriophage product, it is fit, The fragment of adnectin or other viruses or cell protein, epitope can be closed and therefore prevent to infect.These parts are covered In the scope of the present invention.
Recombination expression
The present invention immortalised B-cell clone or culture thick liquid cell be also used as nucleic acid source, the nucleic acid source be used for gram Grand antibody gene is for subsequent recombination expression.During for medicament purpose, ratio is expressed from B cell or hybridoma from recombinant sources Express more conventional, such as because stability, repeatability, the culture reason such as easness.
Therefore, the method that the present invention provides Prepare restructuring cell, comprises the following steps：(i) clone or cultivate from B cell Single thick liquid cell obtains one or more nucleic acid for encoding antibody of interest（For example, heavy chain and/or Light chain mRNA)；(ii) by the core In acid insertion expression vector and the carrier is transfected into host cell to allow to express in the host cell to be closed by (iii) The antibody of note.
Similarly, the method that the present invention provides Prepare restructuring cell, comprises the following steps：(i) to from B cell clone or The nucleic acid sequencing of the coding antibody of interest of single thick liquid cell of culture；And (ii) uses the sequence information system from step (i) It is ready for use in inserting into host cell to allow the nucleic acid that antibody of interest is expressed in the host cell.Can be with（But not being must Need）The nucleic acid is manipulated between step (i) and step (ii) to introduce restriction site, change codon use and/or optimization turn Record and/or translational regulation sequence.
The present invention method that also offer prepares transfection host cell, including the cores with one or more coding antibody of interest The step of sour transfection host cell, the wherein nucleic acid, are derived from immortalised B-cell clone or the single thick liquid cell of culture of the present invention Nucleic acid.Therefore, first prepare nucleic acid and and then with the program of its transfection host cell can in the different time, by different people Member, in different places（For example, in different countries）Come carry out.
Then these recombinant cells of the invention can be used for expressing and cultivating purpose.They are used especially for expression and are used for greatly The antibody of scale medicine preparation.They are also used as the active component of pharmaceutical composition.Any suitable culture can be used Technology, including but not limited to quiescent culture, roller bottle culture, ascites, hollow fiber bioreactor box, Modularized micro fermentation Device, tank diameter, microcarrier culture, ceramic core perfusion etc..
The adaptive immune globulin gene and method that it is sequenced is known in the art from B cell or thick liquid cell （For example, with reference to Kuby Immunology, the 4th chapter of the 4th edition, 2000）.
The host cell of transfection can be eukaryotic, including yeast and zooblast, particularly mammalian cell（Example Such as Chinese hamster ovary celI, such as NS0 cells, PER.C6（Jones et al. 2003）Or HKB-11（Cho et al. 2001；Cho et al. 2003） Etc people's cell, myeloma cell（US 5,807,715；US 6,300,104 etc.））And plant cell.Preferable expression Host can make the antibody glycosylation of the present invention, be especially used in human body the carbohydrate structure of itself and non-immunogenic Glycosylated.In one embodiment, the host cell of transfection be able to can grow in the culture medium of no serum.Another In one embodiment, the host cell of transfection be able to can grow in the culture in the absence of the product of animal derived.It can train The host cell of transfection is supported to produce cell line.
The present invention, which provides, prepares one or more nucleic acid molecules for encoding antibody of interest（For example, heavy chain and light chain base Cause）Method, comprise the following steps：(i) prepare and clone or cultivate thick liquid cell according to the immortalised B-cell of the present invention；(ii) from B Cell clone or single thick liquid cell of culture obtain the nucleic acid for encoding antibody of interest.It is of interest that the present invention also provides acquisition coding Antibody nucleotide sequence method, comprise the following steps：(i) prepare and clone or cultivate according to the immortalised B-cell of the present invention Single thick liquid cell；(ii) nucleic acid of the coding of the thick liquid cell to cloning or cultivating from B cell antibody of interest is sequenced.
The present invention also provides the method for preparing the nucleic acid molecules for encoding antibody of interest, including obtains and turn from the present invention The step of changing the nucleic acid obtained in B cell clone or culture thick liquid cell.Therefore, first obtain B cell clone or culture thick liquid cell, Then from B cell clone or cultivate thick liquid cell obtain nucleic acid program can the different time, by different personnel, not Same place（Such as in different countries）Come carry out.
Offer of the present invention prepares antibody（For example, it is used for medicinal usage）Method, comprise the following steps：(i) from expression institute The selected B cell clone of the antibody of concern or the thick liquid cell of culture obtain one or more nucleic acid（For example, heavy chain and light chain base Cause）And/or it is sequenced；(ii) nucleic acid is inserted in expression vector or prepares expression vector using the nucleotide sequence； (iii) transfection can express the host cell of antibody of interest；(iv) under conditions of antibody of interest is expressed culture or The host cell of Secondary Culture transfection；And optionally, (v) purifies antibody of interest.
The present invention method that also offer prepares antibody, comprises the following steps：Under conditions of antibody of interest is expressed Culture or the host cell population of Secondary Culture transfection；And optionally, antibody of interest is purified, wherein the place of the transfection Chief cell colony is prepared by the following procedure：(i) nucleic acid of the selected antibody of interest of coding is provided, the nucleic acid by making as described above Standby B cell clone or the thick liquid cell of culture produce, and (ii) inserts the nucleic acid into expression vector, and (iii) can express institute Transfection carrier in the host cell of the antibody of concern, and (iv) culture or Secondary Culture include the transfecting host of the nucleic acid of insertion Cell is to produce antibody of interest.Therefore, first Prepare restructuring host cell and then culture recombinant host cell are to express antibody Program can the different time, by different personnel, in different places（Such as in different countries）Come carry out.
Pharmaceutical composition
The present invention provides pharmaceutical composition, and it contains the antibody and/or antibody fragment and/or the core for encoding this antibody-like of the present invention Acid and/or the epitope identified by antibody of the present invention.Pharmaceutical composition can also contain pharmaceutically acceptable carrier to allow to apply. Carrier should not induce in itself produces the antibody harmful to the individual that receives said composition and should not be poisonous.Suitable carrier can be with It is big, metabolism slow macromolecular such as protein, polypeptide, liposome, polysaccharide, PLA, polyglycolic acid, polyaminoacid, ammonia Base acid copolymer and nonactive virion.
Pharmaceutically acceptable salt, such as inorganic acid salt can be used（Such as hydrochloride, hydrobromate, phosphate and sulfuric acid Salt）, or acylate（Such as acetate, propionate, malonate and benzoate）.
Pharmaceutically acceptable carrier in therapeutic combination can contain liquid such as water, salt solution, glycerine and ethanol in addition. In addition, there may also be auxiliary substance in such composition, such as wetting agent or emulsifying agent or pH buffer substance.Such carrier Pharmaceutical composition is enabled to be made into the tablet for subject's intake, pill, dragee, capsule, liquid agent, gel, sugar Starch agent, paste and supensoid agent.
Within the scope of the invention, administration form may include to be applied to parenteral administration（Such as by injecting or being transfused, example Such as bolus injection or Continuous Perfusion）Form.When product is used to inject or be transfused, can use in oiliness or aqueous vehicles In suspension, the form of solution or emulsion, and blender can be contained, such as suspending agent, preservative, stabilizer and/or point Powder.Or antibody molecule can be dry powder form, for being compounded before use with appropriate sterile liquid.
Once preparing, composition can of the invention is administered directly to subject.In one embodiment, combination is made Thing is suitable for administration to people experimenter.
The pharmaceutical composition of the present invention can be applied by number of ways, including but not limited to oral, intravenous, intramuscular, Intra-arterial, marrow are interior, intraperitoneal, intrathecal, intra-ventricle, transdermal, percutaneous, local, subcutaneous, intranasal, enteron aisle, sublingual, intravaginal or straight Intestines approach.Needleless injection agent (Hyposprays) may also be used for the pharmaceutical composition using the present invention.Generally, therapeutic combination Liquid solution or the injectable agent of form of suspension can be made into.It can also prepare and be adapted to dissolve before the injection or be suspended in liquid Solid form in medium.
The direct delivering of composition is typically completed by subcutaneous, intraperitoneal, intravenous or intramuscular injection, or is delivered to group The space between cells knitted.Composition can also be applied diseased region.Dosage treatment can be single dose dosage regimen or multi-agent Measure dosage regimen.The known medicine based on antibody can provide on for example whether should daily, weekly, the delivering medicine such as monthly Frequency of administration guidance.Frequency and dosage additionally depend on the order of severity of symptom.
Various forms can be made in the composition of the present invention.For example, liquid solution or suspension shape can be made in composition The injectable agent of formula.The solid form for being adapted to dissolve or be suspended in liquid vehicle before the injection can also be prepared（Such as freeze Dry composition, such as Synagis™And Herceptin™, for being compounded with the sterilized water containing preservative）.Composition can be made Into such as ointment, emulsifiable paste or powder type for local application.Composition can be made such as tablet or capsule, spray or Syrup（Optionally through seasoning）Form is for orally administering.Using fine powder or spray, composition can be made for example Inhalant form is for pulmonary administration.Suppository or pessary form can be made in composition.Composition, which can be made, for example to drip Dosage form formula is for nose, ear or ocular administration.Composition can use kit form, it is designed so that The composition to subject using preceding compounded combination can faced.For example, lyophilized antibody can be with sterilized water or sterile buffer Liquid is provided in the form of kit together.
It should be appreciated that the active component in composition would is that antibody molecule, antibody fragment or its variant and derivative.Cause This, composition is readily able to degrade in the gastrointestinal tract.Therefore, if composition is applied by using the approach of intestines and stomach, Composition will be needed containing protection antibody from degrading but being discharged once antibody is absorbed from intestines and stomach the material of antibody.
Deep discussion on pharmaceutically acceptable carrier refers to Gennaro (2000) Remington: The Science and Practice of Pharmacy（《Remington：Pharmaceutical science and practice》）, the 20th edition, ISBN: 0683306472。
The pharmaceutical composition of the present invention typically has 5.5 to 8.5 pH, and in certain embodiments, the pH can be 6 to 8, It is about 7 in other embodiments.PH can be maintained by using buffer solution.Composition can be sterile and/or without heat Former.For people, composition can be isotonic.In one embodiment, pharmaceutical composition of the invention is closed Container in provide.
Pharmaceutical composition is by the antibody of the one or more present invention comprising effective dose and/or comprising with reference to antibody of the present invention Epitope polypeptide, be enough the desired disease for the treatment of, ameliorating or preventing or illness or be enough to show detectable curative effect Amount.Curative effect also includes the alleviation of physical symptom.For any particular subject, accurate effective dose is by depending on theirs The therapeutic agent or therapeutic agent that body weight is applied with health status, the property of symptom and the order of severity and selection combine.Given situation Under effective dose determined and within the judgement of clinician by normal experiment.For the purpose of the present invention, to individual institute The effective dose of the present composition of administration is typically about 0.01mg/kg to about 50mg/kg or about 0.05mg/kg to about 10mg/kg.The known medicine based on antibody provides the guidance on this respect, such as Herceptin™It is to pass through venoclysis 21mg/ml solution is applied, and initial dosages are 4mg/kg body weight, maintenance dose is 2mg/kg body weight weekly；Rituxan™It is weekly with 375mg/m2 administrations etc..
In one embodiment, composition may include more than one（Such as, 2 kinds, 3 kinds etc.）The antibody of the present invention is tired to provide Add or coordinating effect.In another embodiment, composition can include one or more（Such as, 2 kinds, 3 kinds etc.）The present invention's is anti- Body and one or more（Such as, 2 kinds, 3 kinds etc.）To anti-influenza A virus or the other antibody of influenza B virus.For example, A kind of antibody can combine HA epitopes, and another antibody can combine the different epitopes on HA, or combine neuraminidase and/or Epitope on stromatin.In addition, antibody and the Flu-A vaccine of the present invention or with the virus to non-influenza A virus（Example Such as influenza B virus）Having specific antibody, administration is within together.The antibody of the present invention can be with stream Influenza vaccine or with having specific Antibody Combination/to non-influenza A virus while or applying in different time.
In another embodiment, the present invention provides the pharmaceutical composition for including two or more antibody, wherein first Antibody is the antibody of the present invention and has specificity to HA epitopes, and secondary antibody is for neuraminidase epitope, the 2nd HA tables Position and/or matrix epitope have specificity.For example, the present invention provides the pharmaceutical composition for including two or more antibody, its Middle first antibody has specificity for the epitope in influenza A virus HA stem area, and secondary antibody is for neuraminidase Epitope, the 2nd HA epitopes（For example, the second epitope in the stem area of epitope, HA in HA spherical head）And/or matrix epitope With specificity.The epitope in the second epitope or spherical head in influenza A virus HA stem area can be with（But it is not necessarily to） It is conservative in more than one subtypes of influenza A virus.
In yet another embodiment, the present invention provides the pharmaceutical composition for including two or more antibody, wherein first Antibody for neuraminidase epitope have specificity, and secondary antibody for nervus opticus propylhomoserin enzyme epitope, HA epitopes and/or Matrix epitope has specificity.
In yet another embodiment, the present invention provides the pharmaceutical composition for including two or more antibody, wherein first Antibody has specificity for matrix epitope, and secondary antibody is for the epitope and/or neuraminic acid on the second matrix epitope, HA Epitope on enzyme has specificity.
For influenza A virus target proteinses there are specific exemplary antibodies of the invention to include but is not limited to FI6 Variant 1, FI6 variants 2, FI6 variants 3, FI6 variants 4, FI6 variants 5, FI28 variants 1 or FI28 variants 2.
In one embodiment, the present invention provides pharmaceutical composition, and it includes antibody FI6 variants 1 or its antigen binding fragment Section and pharmaceutically acceptable carrier.In another embodiment, the present invention provides pharmaceutical composition, and it includes antibody FI6 Variant 2 or its antigen-binding fragment and pharmaceutically acceptable carrier.In another embodiment, the present invention provides medicine group Compound, it includes antibody FI6 variants 3 or its antigen-binding fragment and pharmaceutically acceptable carrier.In another embodiment In, the present invention provides pharmaceutical composition, and it includes antibody FI6 variants 4 or its antigen-binding fragment and pharmaceutically acceptable Carrier.In another embodiment, the present invention provides pharmaceutical composition, and it includes antibody FI6 variants 5 or its antigen-binding fragment And pharmaceutically acceptable carrier.In yet another embodiment, the present invention provides pharmaceutical composition, and it becomes comprising antibody FI28 Body 1 or its antigen-binding fragment and pharmaceutically acceptable carrier.In yet another embodiment, the present invention provides drug regimen Thing, it includes antibody FI28 variants 2 or its antigen-binding fragment and pharmaceutically acceptable carrier.
The antibody of the present invention can be applied together with other therapeutic agents such as chemotherapy compound, radiotherapy（It is administered in combination or only On the spot apply）.In one embodiment, therapeutic compounds includes such as Tamiflu™Etc antiviral compound.Relative to For the single therapy agent being administered alone, such combination treatment can provide the effect of cumulative or collaboration improves.Term " collaboration " It is bigger than the summation of each each independent effect of activating agent for describing the combined effect of two or more activating agents.Therefore, If the combined effect of two or more agents causes " collaboration suppresses " of activity or process, refer to activity or process Suppression than it is each each activating agent inhibition summation it is big.Term " coordinating effect " refers to two or more treatments The effect of method is observed when combining, the wherein curative effect（As measured by any one in multiple parameters）It is more independent than each The summation of observed independent curative effect is big during therapy.
Antibody can be administered to the no response for the treatment of previously for influenza a virus infection（Show for treatment against influenza Show tool tolerance）Those subjects.This treatment may include the treatment previously carried out with antivirotic.This is probably because example Antiviral resistance bacterial strain as infected influenza A virus.
In one embodiment, composition of the invention can include the antibody of the present invention, and wherein antibody can account for combination At least 50 weight % of gross protein in thing（Such as 60 weight %, 70 weight %, 75 weight %, 80 weight %, 85 weight %, 90 weights Measure %, 95 weight %, 97 weight %, 98 weight %, 99 weight % or more）.In such composition, antibody is purified form.
The method that offer of the present invention prepares medicine, comprises the following steps：(i) antibody of the present invention is prepared；And (ii) will The antibody of purifying mixes with one or more pharmaceutically acceptable carriers.
The present invention method that also offer prepares medicine, including antibody and one or more pharmaceutically acceptable carriers are mixed The step of conjunction, wherein the antibody is the monoclonal antibody obtained from the conversion B cell of the present invention or the thick liquid cell of culture.Cause This, first obtain monoclonal antibody then prepare medicine program can the different time, by different personnel, on different ground Point（Such as in different countries）Come carry out.
As delivering antibody or B cell for the alternative of therapeutic purposes, coding can be delivered to subject and be derived from B cell Or the monoclonal antibody of interest of the thick liquid cell of culture（Or its active fragment）Nucleic acid（Usually DNA）, to cause nucleic acid Expression in situ can be carried out in subject's body so as to provide the effect of required.Suitable gene therapy and nucleic acid delivery vector It is known in the art.
The present invention composition can be immunogenic composition, and in certain embodiments can be include contain by The vaccine combination of the antigen for the epitope that the antibody of the present invention is identified.Vaccine according to the present invention can be preventative（I.e. Prevention infection）It is either curative（That is treatment infection）.In one embodiment, the present invention, which provides to include, has SEQ ID NO:37th, the vaccine of the polypeptide of 38,39 or 40 amino acid sequence.In another embodiment, the present invention is provided to include and had The vaccine of the polypeptide of amino acid residue in HA1 the and HA2 polypeptides of one or two kinds of HA monomers as described above.HA monomers can be with It is not cut or cut.
Composition can include antimicrobial, particularly when being packed with multiple dose form.They can include de-sludging Agent, such as Tween（Polysorbate）, such as Tween 80.Detergent generally exists with low-level, such as<0.01%.Composition Sodium salt can also be included（Such as sodium chloride）To provide tonicity.NaCl typical concentration is 10+2mg/ml.
In addition, composition can include such as 15-30mg/ml or so（Such as 25mg/ml）Sugar alcohol（Such as mannitol）Or disaccharides （Such as sucrose or trehalose）, particularly these compositions will be lyophilized or they include the material compounded via freeze-dried material When.It will can be adjusted for the pH of lyophilized composition before lyophilized to about 6.1.
The composition of the present invention can also include one or more immunomodulators.In one embodiment, it is a kind of or more Kind immunomodulator includes adjuvant.
The epitope composition of the present invention can trigger cell-mediated immune response and humoral immune response with effectively right Anti-influenza A virus infects.It is lasting that this immune response can induce（As neutralized）Antibody and exposed to Flu-A disease Can be with the cell-mediated immunity of rapid answer when malicious.
Therapeutic treatment and purposes
Antibody and antibody fragment of the present invention or derivatives thereof and variant can be used for the treatment of influenza a virus infection, A type stream The prevention of Influenza Virus infection or the diagnosis of influenza a virus infection.
Diagnostic method can include making antibody or antibody fragment contact with sample.These samples can be led to from such as nasal cavity Road, sinus cavities, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary canal, heart, ovary, hypophysis, adrenal gland, first shape The tissue sample of gland, brain or skin extraction.The method of diagnosis may also include the detection of antigen/antibody compound.
Therefore, the present invention provides (i) antibody, antibody fragment or its variant and derivative according to the present invention；(ii) basis The immortalised B-cell clone of the present invention；(iii) epitope or (iv) part of antibody of the present invention can be combined, is preferably capable tying The antibody of epitope as conjunction, the epitope combine the antibody of the present invention being used in therapy.
The method that the present invention also provides treatment subject, including apply the antibody according to the present invention, antibody piece to subject Section or its variant and derivative, or part, are preferably able to combine the antibody of such epitope, and the epitope combines the anti-of the present invention Body.In one embodiment, this method causes the infection of influenza A virus in subject to reduce.In another embodiment, This method prevention, reduce subject in influenza a virus infection risk or delay subject in influenza A virus sense Dye.
The present invention also provides (i) antibody, antibody fragment or its variant and derivative according to the present invention；(ii) according to this hair Bright immortalised B-cell clone；(iii) epitope of antibody of the present invention can be combined；Or (iv) part, preferably in combination with can combine The antibody of the epitope of antibody of the present invention, manufacturing the purposes in being used to prevent or treat the medicine of influenza a virus infection.
The present invention provides the composition of the invention of the medicine as prevention or treatment influenza a virus infection.The present invention Also provide the antibody of the present invention and/or the protein of epitope comprising the antibody binding prepare be used to treating subject and/or Diagnose the purposes in the medicine of subject.The method that the present invention also provides treatment subject, including apply the present invention to subject The step of composition.In certain embodiments, subject can be people.A kind of method for the effect of checking treatment processing is included in Using monitoring of diseases symptom after the present composition.Treatment can use single dose dosage regimen or multiple dose administration scheme.
In one embodiment, applied to the subject for needing this treatment according to the antibody of the present invention, antibody fragment, forever Biochemical B cell clone, epitope or composition.Such subject includes but is not limited to especially in the presence of influenza a virus infection Risk is easy to by the subject of influenza a virus infection, includes the subject of such as immunologic hypofunction.The present invention's is anti- Body or antibody fragment can also be used for passive immunity or active immunity.
Heretofore described antibody and its fragment can be used in the kit of diagnosis influenza a virus infection.This Outside, the epitope that can combine antibody of the present invention can be used in kit to by detecting protectiveness anti-influenza A virus antibody Presence to monitor vaccination program the effect of.Antibody, antibody fragment or its variant and derivative of the present invention also may be used There is the production of vaccine of required immunogenicity for being monitored in kit.
The present invention method that also offer prepares medicine, including monoclonal antibody is pharmaceutically acceptable with one or more The step of carrier mixes, wherein the monoclonal antibody is the monoclonal antibody obtained from the transfection host cell of the present invention. Therefore, first obtain（For example, by expressing and/or purifying）Monoclonal antibody and then monoclonal antibody is mixed with pharmaceutical carrier Program can the different time, by different people, in different places（For example, in different countries）Come carry out.
Since the present invention conversion B cell or culture thick liquid cell, can be cultivated, Secondary Culture, clone, Ya Ke Multiple steps that grand, sequencing, nucleic acid are prepared etc. so that the antibody expressed by conversion B cell or the thick liquid cell of culture immortalizes, its In optionally optimized in each step.In a preferred embodiment, the above method also includes the core applied to encoding antibody The optimisation technique of acid（For example, affinity maturation or optimization）.The present invention cover use and prepare during these steps it is all Cell, nucleic acid, carrier, sequence, antibody etc..
In all these methods, nucleic acid used in expressive host can be manipulated to insert, lack or modify some nucleic acid Sequence.Change caused by this manipulation includes but is not limited to introducing restriction site, Modify password uses, increase or optimization are transcribed And/or the change of translational control sequence etc..It is also possible to change nucleic acid to change coded amino acid.It is for example, it may be possible to useful Be introduced in the amino acid sequence of antibody it is one or more（For example, 1,2,3,4,5,6,7,8,9,10 etc.）Amino acid Displacement, missing and/or insertion.Such point mutation can modify effector function, antigen-binding affinity, posttranslational modification, exempt from Epidemic focus etc.；Amino acid can be introduced to attach covalent groups（Such as mark）Or label can be introduced（Such as it is used to purify purpose）.Mutation Specific site can be introduced into or can be randomly incorporated into, but selected（Such as molecular evolution）.For example, one or more can be made Encode in CDR region, weight chain variable district or the light chain variable district of antibody of the present invention that the nucleic acid of any one is random or orthomutation with Different characteristics is introduced in coded amino acid.These changes can be the result of iterative process, wherein retaining initial change And introduce new change in other nucleotide positions.Furthermore, it is possible to combine the change realized in a separate step.Coded by introducing Different qualities in amino acid may include but be not limited to the affinity of enhancing.
Universal
Term "comprising" covers " comprising " and " Consists of ", such as "comprising" X composition only can be made up of X or can be with Including other some things, such as X+Y.
Word " substantial " is not excluded for " complete ", such as " being substantially free of " Y composition can be entirely free of Y.It is necessary When, word " substantial " can omit from the definition of the present invention.
Term " about " on numerical value x means such as x+10%.
Term " disease " used herein is synonymous in a general sense, and can be with term " imbalance " and " illness "（Such as In medical condition）Used interchangeably, wherein all human or animal's bodies for all reflecting infringement normal function or one position Unusual condition, generally showed by distinguishing symptom and symptom, and make human or animal the lost of life or life quality decline.
As used herein, refer to that " treatment " of subject or patient is intended to include prevention, preventing and treating and therapy.Term is " tested Person " or " patient " are used interchangeably herein to refer to all mammals including people.The example of subject include people, Milk cow, dog, cat, horse, goat, sheep, pig and rabbit.In one embodiment, patient is people.
Example
The exemplary embodiment of the present invention is provided in following example.Following instance is given only by way of example and contributed to general Logical technical staff uses the present invention.The example is not intended in any way to limit the scope of the present invention in addition.
Example 1：The generation of influenza A virus wide spectrum neutralizing antibody from thick liquid cell and sign
In order to determine that response Seasonal Influenza Vaccine can be produced（Contain H1 and H3 HA）Different subclass antibodies individual, we Combined vaccine or unrelated H5 HA (A/ are secreted for it to the circulation thick liquid cell collected for the 7th day afterwards in booster shot (boost) VN/1203/04 the ability of antibody), it is screened by ELISPOT.But, it is surprising that at tested 5 H5- specific plasma cells are not detected in 4 in donor, 14% IgG secretory thick liquid cells generate in a donor H5 antibody is resisted, and 57% generates the antibody for resisting vaccine.By using magnetic microballon, then carried out using FACSAria instrument Cell sorting, the 7th day PMBC (PBMC) collected has separated CD138+ thick liquid cells after vaccine inoculation.Will be limited In the thick liquid cell inoculation of number and well plates.Made using H5 or H9 HA and incoherent antigen tetanus toxoid is recombinated For antigen culture supernatants are tested in three parallel ELISA.In the culture supernatants of the screening at 4,928 parts, have 12 parts combine H5 without combining H9 HA, 25 parts of combination H9 without combining H5 HA, and 54 parts had not only combined H5 but also combined H9.Have to 54 parts There is some progress RT-PCR in the culture of highest OD signals, obtain VH the and VL genes of two pairings.
VH and VL gene clonings are entered in expression vector and produce recombinant antibodies by transfecting HEK293T cells.Two kinds of lists Clonal antibody FI6 variants 2 and FI28 share most V, D and J genetic fragment（IGHV3-30*01、IGHD3-9*01、 IGHJ4*02 and IGKV4-1*01）, but N areas, IGKJ using and somatic mutation pattern for the use of have difference and therefore on clone It is incoherent.
Use the specificity for belonging to one group of HA of different subtype by ELISA and studying recombinant antibodies.It is apparent that FI6 is tied Conjunction includes group 1（H1, H5 and H9）With group 2（H3 and H7）All tested Flu-A HA hypotypes, without combine come from second The HA of type influenza virus.By contrast, HAs of the FI28 only in conjunction with 3 groups 1（H1, H5 and H9）.
Table 4
In view of the homology of the VH and VL sequences of both antibody, FI6 variants 2, FI28 variants 1 and 7I13 H and L are utilized Chain carries out shuffling experiments, and 7I13 is the hCMV specific antibodies for identical V, D and J element for using H chains.Although being combined with H7 needs H chains and the pairing of L chains of FI6 variants 2 are wanted, but when the L chains of FI6 variants 2 and FI28 variants 1 are reorganized, holding is combined with H5.This Outside, when the H chains of FI6 variants 2 match with incoherent 7I13 L chains, also observe that H5 is combined.By contrast, when homologous When 7I13 H chains match with the L chains of FI6 variants 2, H5 combinations are not observed.Inventionwithout being bound to any specific theory, these results Show that the main contributions that H5 is combined come from H chains, and H7 combines the perfect match then needed between the H chains of FI6 variants 2 and L chains.
Then pseudotype virus is used（Table 5）And infectious virus（Table 6）To test in FI6 variants 2 and FI28 variants 1 With the ability of 2 subtypes of influenza A virus of group 1 and group.It is apparent that FI6 variants 2 have neutralized all tested pseudotype virus, bag Include six H5 separation strains for belonging to antigen divergence branch (clade) 0,1,2.1,2.2 and 2.3 and two H7 fowl separation strains.This Outside, FI6 variants 2 have neutralized all tested infective virus, including two kinds of H3N2 viruses of existing many decades and four kinds H1N1 viruses and H1N1 until recently are very popular separation strains A/CA/04/09（Table 6）.FI28 variants 1 have neutralized all H5 pseudotype virus, but without neutralization H7 pseudotype virus and all tested infective virus.Neutralization to pseudotype virus The titre that titre compares infective virus is high.
Table 5
Nd, do not carry out
Example 2：FI6 variants 2 and the antigen binding site of FI28 variants 1
In order to identify the antigen site of antibody FI6 variants 2 and the combination of FI28 variants 1, we test it and suppress C179 knots first The ability of conjunction, C179 are a kind of mouse monoclonal antibodies of the conserved region by mapping to HA stems area（Y. Okuno et al.,J Virol（《Journal of Virology》）67, 2552 (1993)）.Both FI6 variants 2 and FI28 variants 1 all completely inhibit C179 with H5 VN/1203/04 HA combination is recombinated, so as to show that they identify overlapping epitope.On the contrary, FI6 variants 2 and FI28 variants 1 The H5 specific antibodies competition of different epitopes in spherical head do not separated with one group from H5N1 immune donors, identification HA（C. P. Simmons et al., PLoS Med 4, e178 (2007)；S. Khurana et al., PLoS Med 6, e1000049 (2009)）.Failed by the trial for selecting escape mutants (escape mutant) to map the epitope of FI6 variants 2 , so as to show that, in the case where not endangering viral adaptability, its epitope will not be mutated easily.
Then, we using the linear of HA A/VN/1194/04 and cyclisation peptide library carried out based on the mapping of peptide with And use Pepscan Presto BV（Lelystad, Holland (Lelystad, The Netherlands)）System carry out Helical scanning.The analysis determines the land of FI6 variants 2, and the land includes HA2 fusogenic peptides FGAIAG（Amino acid 3-8, According to H3 numberings；SEQ ID NO: 37）, HA2 spiral A PEPDs GVTNKVNS（Amino acid 46-54；SEQ ID NO: 38）、 HA2 spiral B peptides MENERTLDFHDSNVK（Amino acid/11 02-116；SEQ ID NO: 39）With HA1 C- terminal peptides LVLATGLRNSP（Amino acid 315-325；SEQ ID NO: 40）.Due to FI28 variants 1 not with HA1 C- terminal peptides and HA2 spiral shells B reactive polypeptides are revolved, so the land of FI28 variants 1 is different from FI6 land.
Example 3：Generation and sign with the FI6 variants 3,4 and 5 for improving productivity ratio
The VH and VL of synthesis FI6 variants 2 some variants come improve the production in mammalian cell and remove it is unnecessary Somatic mutation and unfavorable feature.VH and VL genes are cloned to the expression vector of the constant region and C κ into coding IgG1 respectively In.The Germline sequences of FI6 variants 2 are determined according to IMGT databases.Pass through synthesis（New Jersey Pi Sikatewei Jin Sirui is public Take charge of (Genscript, Piscatawy, NJ)）Or produced by rite-directed mutagenesis Pu Luomaige companies (Promega) wherein single Or multiple germ line mutations are restored to the antibody variants of germline and confirmed by being sequenced.All variant sequence thereofs all use Jin Sirui The OptimumGeneTM systems of company carry out codon optimization to be expressed in people's cell.The training that suspends is transiently transfected by using PEI 293 foster Freestyle cells（Hero company (Invitrogen)）To produce monoclonal antibody.From transfection after cultivating 7 days Cell collects supernatant and passes through protein A chromatography（General Electric's medical company (GE Healthcare)）Affinity IgG purification And with PBS desalinations.With several productivity ratio of the independent experimental evaluation in transient expression system.Average value figure 1 illustrates. As shown in fig. 1, FI6 variants 2 are produced with 13.5 μ g/ml titre；FI6 variants 3 are produced with 46.3 μ g/ml titre；FI6 becomes Body 4 is produced with 60.7 μ g/ml titre；And FI6 variants 5 are produced with 61.6 μ g/ml titre.Therefore, compared with FI6 variants 2, The titre that we can produce FI6 variants 3,4 and 5 increases by 3.4 times, 4.5 times and 4.6 times respectively.
Compared with initial FI6 variants 2IgG, also it is directed to by ELISA and H5 and H7 HA combination and false to H5 and H7 The neutralization of virus（Table 7）The recombinant antibodies are characterized.The restructuring HA of baculoviral is derived from 1 μ g/ml of the 5 μ l in PBS （Protein Sciences Corp. (Protein Sciences Corp.)）It is coated with Half-area elisa plates（Corning Incorporated (Corning)）.After being closed with 1% PBS/BSA, the goats of F (ab ') 2 for adding antibody and being conjugated using alkaline phosphatase are resisted Human IgG（Southern biotech company (Southern Biotech)）To disclose combination.Plate is washed out, adds substrate（p- NPP, Sigma (Sigma)）And the read plate under 405nm.The antibody concentration required for 50% maximum combined is realized by measurement (EC50), the relative affinity that antibody is combined with HA is determined with ELISA.For pseudovirus neutralizing mensuration method, by the company of antibody The culture supernatants containing pseudovirus of continuous dilution and fixed concentration incubate 1 hour at 37 DEG C.Then mixture is added Incubated 3 days to HEK 293T/17 cells and at 37 DEG C.Then cell is dissolved with Britelite reagents (Perkin Elmer), And the list of light relatively in cell dissolved matter is determined in photometer microplate (Veritas, Turner Biosystems) Position (RLU).By compare in the presence of and RLU during in the absence of antibody determine infective reduction, and be expressed as percent neutralization. 50% suppression dosage (IC50) is defined as after the background RLU in the cell in deducting the only hole of control and virus control wells Sample concentration during compared to RLU reductions 50%.Table 7 shows that the combination for retaining or improving is combined based on improved sequence characteristic is lived Property and the FI6 variants 3-5 selected.
Table 7
What FI6 variants 2 and the combination of FI6 variants 3 were tested belongs to group 1（H1, H2, H5, H6, H8 and H9）With group 2（H3, H4, H7 and H10）All restructuring or purifying HA, wherein EC50 values are in the range of 10 to 270ng/ml（Table 8）.In addition, FI6 variants 2 Have with the transfection of the staining cell of FI6 variants 3 and belong to group 1（H11, H12, H13 and H16）With group 2（H4, H10, H14 and H15）HA bases Cause（Table 8）.
Table 8
(1) the EC50 values (ng/ml) measured by ELISA
(2)+refer to HA transfection 293 cells positive staining
Example 4：The structural characterization of the epitope of FI6 variants 3 on H1 and H3 HA
In order to differentiate between the epitope of the identification of FI6 variants 3 on 2 HA of group 1 and group and following description antibody and its target antigen Interaction of molecules, we make the Fab fragments of FI6 variants 3 and H1（Group 1）And H3（Group 2）The compound knot of HA homotrimers It is brilliant.To make FI6 variant 3/H1 homotrimer HA complex crystallizations, H1 HA0 extracellular structure is expressed in Sf9 insect cells Domain.Residue 11-329 (HA1) and 1-176 (HA2) will be corresponded to（Based on H3 numberings）CDNA clone enter mixed with GP67 In the BioFocus expression vectors of secretion signal, the secretion signal enables expressed protein to be secreted into culture medium.Gram Grand cDNA folds sub (foldon) sequence with C-terminal trimerizing and merged to make it possible to be formed the H1 HA0 of trimeric form. Fold includes blood coagulation cleavage sequences so that obtain to mark except unfolding is sub before crystallization between subsequence and HA2 C-terminal Label, and 6-His labels are mixed for affinity purification in the C- least significant ends of expressed peptide sequence.
With recombinate shape virus infection Sf9 insect cells, and by through Ni-NTA resins (Qiagen) and gel filtration （S200 posts）The H1 HA0 with 6-His labels are reclaimed from culture medium.By corresponding to tripolymer H1 HA0 elution protein compression To 1mg/ml, then by using fibrin ferment（The fibrin ferment of 5 units is used per mg HA0）Two hours are handled at 20 DEG C to remove C End tag.The albumen H1 HA0 of purified cutting are finally classified to separation on Mono Q anion-exchange columns.For shape The compound of Cheng Qiyu Fab-FI6 variants 3, by 0.5 to purifying H1 HA0 and twice of molar excess between 1mg/ml purifying Fab-FI6 variants 3 mix.Compound is formed by incubating three hours at 4 DEG C, then by S200 solvent resistant columns Classification separation and from the excessive isolated complex of Fab-FI6 variants 3.
The purifying compound of Fab-FI6 variants 3 and unprocessed tripolymer H1 HA0 is concentrated into 12mg/ml to be used to tie It is brilliant.By being steamed on the hole solution (well solution) being made up of 0.1M Bis Tris propane pH 7.0,2.2M ammonium sulfate Gas spreads, and the crystal of Fab-FI6 variants 3 and H1 HA0 compound is grown with hanging drop.Crystal grows surrounding simultaneously at 20 DEG C 1 into hole solution and 3.4M sodium malonates pH 7.0 is harvested from drop:Low-temperature protection is carried out in 1 mixture, then in liquid nitrogen Flash freezing.Data set is collected under Diamond Light Source light beam lines IO3, and is carried out respectively using MOSFLM and SCALA Indexing (indexed), integration and normalization (scaled).
Statistical analysis to unit cell parameters and molecular weight of albumen shows that each asymmetry unit has a hemagglutinin list Body and a Fab fragment；Therefore molecular replacement is carried out using the search model of free state.Use monomer H1 HA (PDB ID Coordinate 1RD8) obtains initial phase as search model with CCP4 programs PHASER.Use automodel fit procedure FFFEAR, the variable domain of heavy chain are successfully fitted with density, and then to place compared with HA- antibody structures 3FKU and 3GBN Light variable domains.The alternate cycles of model foundation and refine are carried out using COOT and REFMAC5 respectively.It is straight to repeat this operation To fitting electron-density map as much as possible and flatten out R-work values and R-free.
The amino acid in final PDB files is numbered according to Kabat conventions.Final mask contains whole H1 HA With heavy chain and light-chain variable domain.Crystallization for FI6 variant 3/H3 abnormal shape tripolymer HA compounds, purifying X-31 (H3N2) diseases The HA (BHA) of poison and bromelain enzyme r e lease, and Fab fragments are prepared by papain digestion.Use Protein A sepharose Affinity chromatography (HiTrap Protein A HP, 1ml) and then the Fab of use S-200 size exclusions post purifying FI6 variants 3. 3.5mg Fab are mixed with 3mg X-31 BHA and are incubated overnight at 4 DEG C to form compound, use the SEC of superose 6 Post purifies compound.Collect the peak value fraction corresponding to Fab-HA compounds and concentrate for crystallizing.
Pass through the steam in the sitting drop by crystallizing robot distribution from the Oryx-6 of Douglas Instruments companies Diffusion grows FI6 variant 3-H3 compound crystals.Low temperature guarantor is carried out to crystal by adding 25% glycerine into storage tank solution Shield.Data set is collected in Diamond Light Source light beam lines IO3, enters row index using Denzo and Scalepack Change, integrate and normalize.Parsed using Amore by molecular replacement in asymmetry unit containing one and three FI6 variants 3 The crystal of H3 HA tripolymers compound Fab.Use the coordinate of 2 structures of H3 HA tripolymers, compound from FI6 variants 3/H1 The heavy chain and the coordinate of light-chain variable domain and constant domain (PDB ID 3HC0.pdb) of the FI6 variants 3 of thing are as independent search Target carries out molecular replacement calculating.Using Refmac5 and Pheonix and counted being manually adjusted with what Coot was carried out for wheel Carry out refine molecular replacement analytic structure.Using DM electron-density map is averagely substantially improved by noncrystal.
X-ray crystallography shows that FI6 variants 3 combine the conserved epitope in F subdomains.Although both HA are in system Developmentally with it is different in structure, and compound is crystallized in a manner of different stacked arrangements, but finds interactive surfaces extremely It is similar（Fig. 2, A and B）.In both cases, a molecule of each monomer combination FI6 variants 3 of HA tripolymers（Fig. 3）. The HCDR3 ring regions of FI6 variants 3 combine in the shallow trench on HA F subdomains, and wherein the side of groove is by the A from HA2 The part of the residue of spiral and two gangs of HA1（38-42 and 318-320）Formed, and bottom is transferred by the HA2 comprising residue 18-21 Formed（Fig. 2, A and B）.
HCDR3 ring regions are intersected with about 45 ° of angle with spiral A so that Leu-100A, Tyr-100C, Phe-100D and Trp-100F produces hydrophobic contact with the residue in groove（Fig. 4）.Tyr-100C and Trp-100F also Thr- with HA1 respectively The backbone carbonyl of 318 side chain and HA1 residue 19 forms potential hydrogen bond.By the main chain carbonyl of HCDR3 residue 98 and 99 Base forms two extra polar interactions with the Asn-53 on spiral A and Thr-49.HCDR3 and HA（H1 and H3）It is mutual Effect has embedded about 750 2 antibody surface together, and about the 2/3 of the interaction is drawn by the interaction with HA2 chains Rise.
In a word, FI6 variants 3 are obvious similar to interaction caused by the hydrophobic groove on H1 and H3.FI6 variants 3 LCDR1 ring regions form two with side relative with facilitating the side of hydrophobic groove on spiral A and contacted；Phe-27D and Lys-39 Aliphatic part form hydrophobic contact and Asn-28 and form hydrogen bond with Asn-43, produce about 190 for both H1 and H3 together 2 embedding surface area.By with the H1 HA, LCDR1 of non-cutting form cocrystallization also with adjacent Distal Right HA monomers " fusogenic peptide " is not cut forms contact extensively（Fig. 3, B and C and Fig. 4, A and B）, cause the FI6 variants 3 of other 320 2 to wrap Bury surface.
The backbone carbonyl shape of LCDR1 residue 28 and 29 and HA1 residues 329 and adjacent second residue HA2 Leu-2 Into backbone amide hydrogen bond, therefore cross over cleavage site.LCDR1 Phe-27D and adjacent Distal Right HA2 Leu-2 are formed Hydrophobic contact, and the backbone carbonyl of Tyr-29 pendant hydroxyl group and the residue 325 of adjacent Distal Right HA1 chains forms hydrogen bond. Compared to extremely similar interactions of both H1 and H3 HA between HCDR3, LCDR1 and from the distal end of adjacent cutting The interaction of " fusogenic peptide " of right side H3 HA monomers is significantly too late extensive with the interaction of not cut H1 HA formation （Fig. 3, illustration B）.Although aliphatic parts of the Phe-27D again with HA2 Lys-39 contacts, Tyr-29 is right with adjacent distal end Side HA2 Ala-7 backbone carbonyl forms potential hydrogen bond contact（It is opposite with the residue 329 in not cut H1 HA）.
On the contrary, being touched between LCDR1 ring regions and the H3 HA of cutting " fusogenic peptide " without main link, 114 smaller is caused to connect Contacting surface is accumulated（Reference in H1 HA 320 2）.Also appear to the cutting of HA precursors and produce H3 HA, cause FI6 variant 3/H3 compounds It is slightly different relative to HA orientation with FI6 variants 3 in FI6 variant 3/H1 compounds.Positioned at VH the and VL chains of FI6 variants 3 with The contact residues of interface are given in Table 9 between the H3 homotrimers HA of cutting.Positioned at VH the and VL chains of FI6 variants 3 and not The contact residues of interface are given in Table 10 between cut H1 homotrimers HA.
Table 9
The contact residues of interface between the VH of FI6 variants 3 and VL and the H3-HA tripolymers of cutting
Table 9（It is continuous）
The contact residues of interface between the VH of FI6 variants 3 and VL and the H3-HA tripolymers of cutting
Table 10
The contact residues of interface between positioned at the VH of FI6 variants 3 and VL and not cut H1-HA tripolymers
Table 10（It is continuous）
The contact residues of interface between positioned at FI6 variant 3VH and VL and not cut H1-HA tripolymers
Tool group 1 specific two kind of cross reacting antibody CR6261 and F10 structure had previously been answered as with H5 and H1 HA Solvate form is reported.CR6261 the and F10 antibody combined with HA is only mediated by its VH domain, and these VH domains are relative to HA Substantially mutually the same orientation, but two kinds of antibody are substantially rotated relative to FI6 variants 3 and closer HA film near-end 5-10（Fig. 3, D and E）.
FI6 variants 3/H1 and FI6 variant 3/H3 given herein structure also discloses, although the knot on the HA of three kinds of antibody It is overlapping extensively to close site, but those are mutual caused by interactive property and CR6261 and F10 antibody as caused by FI6 variants 3 Interaction property is significantly different.Most significant difference is the interaction of the hydrophobic groove on FI6 variants 3 and HA only by long HCDR3 is mediated, and for CR6261 and F10, with reference to being related to all three HCDR.
Important difference between FI6 variant 3/H1 compounds and FI6 variant 3/H3 compounds is H3 HA such as group 2 H7, H10 are as H15 HA in Asn-38（HA1）Place is glycosylated, and H1 is the same with all groups of 1 HA does not glycosylate.H3's In uncombined structure, the carbohydrate side chain is from the HA1 β stock protrusions containing Asn-38 residues, towards identical HA subunits HA2 spiral A so that it is overlapping with the trace of FI6 variants 3（Fig. 5 A）.Known carbohydrate side chain can influence the sugared egg of virus White antigenicity, therefore have pointed out overlapping other group 1 cross reacting antibodies shortages caused with targeting HA film proximal end region And organize 2 HA combination.However, the reorientation for passing through oligosaccharides（From HA surfaces, rotation opens about 80 °）So that FI6 variants 3 and H3 HA is combined, so as to form new contact with the Asp-53 and Asn-55 of HCDR2 ring regions（Fig. 5 B）.
Make it be suitable for FI6 variants 3 to be combined with H3 HA in view of the flexibility of carbohydrate side chain at Asn-38, therefore We suspect whether the glycosylation site is probably the reason for H3 HA do not combine CR6261 or F10.Simply modeling shows, carbon water Compounds in side chain orientation identical change by and CR6261 combination it is compatible, but not with F10（Cross reacting antibody）Knot Close compatible.VH residues 73-77 comprising F10 β turnover will with the orientation adopted in FI6 variant 3/H3 compounds The carbohydrate of Asn-38 connections clash, but whether the carbohydrate can freely further rotate out bound site Point is still not clear with adapting to F10 combinations.However, CR6261 or F10, which can not be neutralized, wherein removes glycosylation site (Asn- 38) H7 pseudovirus（A/ chickens/Italy/99）(IC50 >50 μ g/ml), show the glycan steric hindrance and non-blocking CR6261 Limited with F10 antibody with 2 HA of the group exclusive architectures combined.
In addition to Asn-38 glycosylation, the most significant difference between 2 HA of group 1 and group in F subdomain structures is related to Different environment and HA2 Trp-21 orientation between group.In 1 HA is organized, Trp-21 and F subdomains surface is almost parallel, and In 2 HA are organized, it is roughly perpendicular to surface orientation（Fig. 5, C and D）.All three antibody（FI6 variants 3, CR6261 and F10） Mainly pass through phenylalanine side-chain（The Phe-55 on the Phe-54 and F10 on Phe-100D, CR6261 on FI6 variants 3）With Trp-21 forms contact（Fig. 5, C and D）.For FI6 variants 3, local reset in HCDR3 ring regions means Phe-100D positions Compare in hydrophobic groove of the point in H1 compounds substantially deep by 2 in H3 compounds；Therefore kept in both cases with Trp-21 Similar contact distance.
The Phe-54 (CR6261) and Phe-55 (F10) of two kinds of specific antibodies of group 1 position are similar to and answered with H1 HA The FI6 variants 3 of conjunction.However, because Phe-54 (CR6261) and Phe-55 (F10) is adjacent antiparallel positioned at connection two On the becate area HCDR2 of stock, it appears that than being moved further out hydrophobic groove for Phenylalanine in FI6 variants 3 to adapt to The flexibility that Trp-21 group 2 is orientated is small.Therefore, 2 HA of CR6261 and F10 and group combination may be by HCDR2 phenylalanines The obstruction of Steric clashes between Trp-21.
Sum it up, the structured data obtained shows, although the core epitope on spiral A identifies with CR6261 and F10 Epitope it is similar, but cut and non-cutting form in, FI6 variants 3 combined with different angle, apart from the remote 5-10 in film distal end and Contact covers spiral A and extends to the more large area of the fusogenic peptide of adjacent Distal Right monomer（Fig. 6）.FI6 variants 3 combine Mediated by VH and VL CDR, main contributions are long HCDR3（Its adapt to group-specific Trp-21 ring regions tripe systems as）With The LCDR1 being seriously mutated.HCDR3 using VH and VK chains and length is the feature of the antibody naturally selected, and with only using VH chains With reference to the antibody from bacteriophage（Such as CR6261 and F10）Characteristic it is completely different.The VH of FI6 variants 3 contacts with VK are residual Base is described in the figure 7.
Example 5：Prevention effect inside FI6 variants 3
Internal test protectiveness effect of FI6 variants 3 in the mouse model of influenza a virus infection.Big to 6 to 8 weeks Intravenous antibody purification of (i.v.) injection concentration in the range of 1 to 16mg/kg of female BAl BIc/c mouse group.After three hours, make Mouse deep anaesthesia and with 10MLD50（50% mouse lethal dose）H1N1 A/PR/8/34 attacked through intranasal (i.n.).One In kind treatment setting, mouse receives antibody in 1,2 or 3 day after injection.The survival condition of monitoring mouse and body weight loss are straight daily (p.i.) the 14th day after to infection.According to animal research protocol, more than 25% animal euthanasia of original body mass will be lost.
To assess influence of the FI6 variants 3 to virus replication, existed with the 10MLD50 H1N1 A/PR/8/34 mouse attacked Different time points receives antibody and put to death after four days to collect lung and brain.It is being supplemented with antibiotic-antimycotic solution（English Outstanding company）Leibovitz L-15 culture mediums（Hero company）Middle homogenized tissues are to obtain 10% w/v organ suspension. Organ homogenate is titrated on mdck cell and determines virus titer.In preventative setting, FI6 variants 3 are applied to 4mg/kg Have complete protectiveness during by the mouse that 1 H1N1 reassortant virus of group infects, have partial protection when being applied with 2mg/kg （80% survival rate）（Fig. 8）.After infection the 0th day or the 1st day with FI6 variants 3 handle mouse in, the tuberculosis of the 4th day after infection Malicious titre reduces about 100 times（Fig. 9）.In addition, FI6 variants 3 prevent the body of the mouse with group 2 H3N2 HK-x31 virus infection Lose again（Fig. 8）.
Example 6：The viral neutralization mechanism of FI6 variants 3
Tested inside protectiveness effect for being intended to determine FI6 antibody, we produce the FI6 variants for lacking complement combination The Fc mutant (FI6-v2 LALA) for the FI6 variants 2 that 2 Fc mutant (FI6-v2 KA) or missing complement and FcR combines.This A little antibody show combined with the identical of FI6 variants 2 and external neutralization characteristic and it is suitable inside half-life period（FI6-v2、 FI6-v2 KA and FI6-v2 LALA average value are respectively 3.3,3.4 and 3.5 days）.With A/Puerto Rico/8/34 (H1N1) their protectiveness effect is tested in the mouse of viral lethal infection.FI6.v2 is protected completely when being applied with 4mg/kg Mouse is protected from death, the mouse of protection 80% when being applied with 2mg/kg（Fig. 8 F）.When being applied with 10mg/kg, FI6-v2 and FI6-v2 KA have complete protectiveness, and activity is largely lost at FI6-v2 LALA displays, can only protect 40% animal（Figure 8F）.It is especially apparent when mutant antibodies are applied with 3mg/kg Finite Concentration, the effect of the reduction（Fig. 8 G）.
The mechanism of the neutralization activity of FI6 variants 3 is produced for research, by the HA from NC/99 baculovirals（Protein science Company (Protein Sciences Corporation)）With FI6 variants 3, FE17, the non-specific mAb of high 15 times of moles (HBD85) or the PBS solution without mAb incubates 40 minutes at 37 DEG C.The trypsase handled to every part of sample addition through TPCK To 2.5 μ g/ml ultimate density, and digestion 5,10 and 20 minutes is carried out at 37 DEG C.At each time point, contained by adding SDS and DTT buffer solution and stop digesting by making it seethe with excitement at 95 DEG C 5 minutes.Then by sample loading to 12% On Tris- Glycine polyacrylamide gels.Albumen is carried out on pvdf membrane with the iBlot blotting systems from hero company to turn Move.Pvdf membrane is closed 30 minutes with 10% skimmed milk power in TBS-Tween.With 0.5 μ g/ in TBS-Tween at 4 DEG C Ml confrontation HA0 primary antibody（The FO32 of biotinylation mark prepared by inside）It is incubated overnight.PVDF is washed with TBS-Tween The streptavidin being conjugated three times and under room temperature (RT) with HRP（Sigma）Incubate 1 hour.
Pvdf membrane is washed three times and using ECL Plus with TBS-Tween™Western blotting detection reagent（General Electric Medical company）Positive band is detected with LAS4000 CCD camera systems.Data in Figure 10 show that FI6 variants 3 can suppress TPCK- trypsase cut HA0, so as to show that the antibody light chain combined with unprocessed HA0 can hinder infectivity, at least for For extracellular those viruses cut.
Bibliography
Okuno et al. (1993) Journal of Virology（《Journal of Virology》）67: 2552
Gerhard et al. (2006) Emerging Infectious Diseases（《Emerging infectious disease》）12: 569
Gioia et al. (2008) Emerging Infectious Diseases（《Emerging infectious disease》）14: 121
US 3,766,162
US 3,791,932
US 3,817,837
US 4,233,402
US 4,676,980
US 4,831,175
US 5,595,721
WO00/52031
WO00/52473
US 4,766,106
US 4,179,337
US 4,495,285
US 4,609,546
Gabizon et al. (1982) Cancer Research（《Cancer research》）42:4734
Cafiso (1981) Biochem Biophys Acta（《Biochemistry and biophysics journal》）649:129
Szoka (1980) Ann. Rev. Biophys. Eng.（《Biophysics is commented with bioengineering year》）9:467
Poznansky et al. (1980) Drug Delivery Systems（《Drug delivery system》）（R.L. Juliano is compiled Volume, New York Oxford (Oxford, N.Y.)）The 253-315 pages
Poznansky (1984) Pharm Revs《Pharmacological review》）36:277
Kohler, G. and Milstein, C, 1975, Nature（《It is natural》）256:495-497.
Kozbar et al. 1983, Immunology Today（《ImmunoL Today》）4:72.
WO2004/076677
Kuby Immunology chapter 4（4th edition, 2000；ASIN: 0716733315）
Jones et al., Biotechnol Prog（《Biotechnological Advances》）2003,19(1):163-8
Cho et al., Cytotechnology（《Cell technology》）2001,37:23-30
Cho et al., Biotechnol Prog（《Biotechnological Advances》）2003,19:229-32
US 5,807,715
US 6,300,104
Rowe et al. (1999) J Clin Microbiol（《Clinical microbiology magazine》）37(4):937-43.
Temperton et al. (2005) Emerg Infect Dis（《Emerging infectious disease》）11, 411-416.
Smirnov et al. (2000) Arch Virol（《Virology document》）145, 1733-1741.
Smirnov et al. (1999) Acta Virol（《Virology journal》）43, 237-244.
Simmons et al. (2007) PLoS Med 4, e178.
Traggiai et al. (2004) Nat Med（《Nature-medical science》）10, 871-875.
Sequence table
<110>Biomedical research institute
<120>Neutralize anti-influenza A virus antibody and application thereof
<130> HMB0012-401-PC
<150> 61/083,838
<151> 2008-07-25
<150> 61/181,582
<151> 2009-05-27
<150> 12/509,731
<151> 2009-07-27
<160> 62
<170>PatentIn version 3s .5
<210> 1
<211> 8
<212> PRT
<213>Homo sapiens
<400> 1
Gly Phe Thr Phe Ser Thr Tyr Ala
1 5
<210> 2
<211> 8
<212> PRT
<213>Homo sapiens
<400> 2
Ile Ser Tyr Asp Gly Asn Tyr Lys
1 5
<210> 3
<211> 22
<212> PRT
<213>Homo sapiens
<400> 3
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
1 5 10 15
Gln Gly Tyr Phe Asp Pro
20
<210> 4
<211> 10
<212> PRT
<213>Homo sapiens
<400> 4
Gln Ser Val Thr Phe Asn Tyr Lys Asn Tyr
1 5 10
<210> 5
<211> 3
<212> PRT
<213>Homo sapiens
<400> 5
Trp Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213>Homo sapiens
<400> 6
Gln Gln His Tyr Arg Thr Pro Pro Thr
1 5
<210> 7
<211> 24
<212> DNA
<213>Homo sapiens
<400> 7
ggattcacgt tcagtaccta tgcc 24
<210> 8
<211> 24
<212> DNA
<213>Homo sapiens
<400> 8
atctcatacg atggaaatta taaa 24
<210> 9
<211> 66
<212> DNA
<213>Homo sapiens
<400> 9
gcgaaagact cccaactgcg atcactcctc tattttgaat ggttatccca gggatatttt 60
gacccc 66
<210> 10
<211> 30
<212> DNA
<213>Homo sapiens
<400> 10
cagagtgtca ccttcaacta taagaactac 30
<210> 11
<211> 9
<212> DNA
<213>Homo sapiens
<400> 11
tgggcatct 9
<210> 12
<211> 27
<212> DNA
<213>Homo sapiens
<400> 12
cagcaacatt ataggactcc tccgacg 27
<210> 13
<211> 129
<212> PRT
<213>Homo sapiens
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Asn Ser Thr Leu His
65 70 75 80
Leu Glu Met Asn Thr Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
100 105 110
Gln Gly Tyr Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Thr
115 120 125
Ser
<210> 14
<211> 111
<212> PRT
<213>Homo sapiens
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Ala Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Thr Phe Asn
20 25 30
Tyr Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Val Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr
85 90 95
Arg Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 15
<211> 388
<212> DNA
<213>Homo sapiens
<400> 15
caggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgtag cctctggatt cacgttcagt acctatgcca tgcactgggt ccgtcaggct 120
ccaggcaggg ggctggagtg ggtggcagtt atctcatacg atggaaatta taaatactat 180
gcagactctg tgaagggccg attctccatc tccagagaca attccaacag cacgctgcat 240
ctagaaatga acaccctgag aactgaggac acggctttat attactgtgc gaaagactcc 300
caactgcgat cactcctcta ttttgaatgg ttatcccagg gatattttga cccctggggc 360
cagggaaccc ttgtcaccgt cacctcag 388
<210> 16
<211> 334
<212> DNA
<213>Homo sapiens
<400> 16
gacatccaga tgacccagtc tccagactcc ctggctgtat ctctgggcgc gagggccacc 60
atcaactgca agtccagcca gagtgtcacc ttcaactata agaactactt agcttggtac 120
cagcagaaac caggacagcc tcctaaagtg ctcatttact gggcatctgc ccgggaatca 180
ggggtccctg accgattcag tggcagcggg tctgggacag atttcactct caccatcagc 240
agcctgcagg ctgaagatgt ggctgtttat tactgtcagc aacattatag gactcctccg 300
acgttcggcc aagggaccaa ggtggagatc aaac 334
<210> 17
<211> 8
<212> PRT
<213>Homo sapiens
<400> 17
Gly Phe Thr Phe Ser Asn Tyr Gly
1 5
<210> 18
<211> 8
<212> PRT
<213>Homo sapiens
<400> 18
Ile Ser Tyr Asp Gly Ser Asn Lys
1 5
<210> 19
<211> 22
<212> PRT
<213>Homo sapiens
<400> 19
Ala Lys Glu Arg Pro Leu Arg Leu Leu Arg Tyr Phe Asp Trp Leu Ser
1 5 10 15
Gly Gly Ala Asn Asp Tyr
20
<210> 20
<211> 12
<212> PRT
<213>Homo sapiens
<400> 20
Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 21
<211> 3
<212> PRT
<213>Homo sapiens
<400> 21
Trp Ala Ser
1
<210> 22
<211> 8
<212> PRT
<213>Homo sapiens
<400> 22
Gln Gln Tyr Tyr Arg Ser Pro Ser
1 5
<210> 23
<211> 24
<212> DNA
<213>Homo sapiens
<400> 23
ggattcacct tcagtaacta tggc 24
<210> 24
<211> 24
<212> DNA
<213>Homo sapiens
<400> 24
atatcatatg atggatctaa taag 24
<210> 25
<211> 66
<212> DNA
<213>Homo sapiens
<400> 25
gcgaaagaga gaccccttcg cctattacga tattttgact ggttatcggg gggggcgaat 60
gactac 66
<210> 26
<211> 36
<212> DNA
<213>Homo sapiens
<400> 26
cagagtgttt tatacagctc caacaataag aactac 36
<210> 27
<211> 9
<212> DNA
<213>Homo sapiens
<400> 27
tgggcatct 9
<210> 28
<211> 24
<212> DNA
<213>Homo sapiens
<400> 28
cagcagtatt atagaagtcc gtcc 24
<210> 29
<211> 129
<212> PRT
<213>Homo sapiens
<400> 29
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Phe Tyr Cys
85 90 95
Ala Lys Glu Arg Pro Leu Arg Leu Leu Arg Tyr Phe Asp Trp Leu Ser
100 105 110
Gly Gly Ala Asn Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 30
<211> 112
<212> PRT
<213>Homo sapiens
<400> 30
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Asp Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Asn Leu Gln Val Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Arg Ser Pro Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 31
<211> 388
<212> DNA
<213>Homo sapiens
<400> 31
gaggtgcagc tggtggagtc tgggggaggc gcggtccagc ctggggagtc cctgaaactc 60
tcctgtgcag cctctggatt caccttcagt aactatggca tgcactgggt ccgccaggct 120
ccaggcaagg gactggagtg ggtggcagtc atatcatatg atggatctaa taagtactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagga cacgctgtat 240
ctgcaaatga acagcctgag agctgaggac acggctctgt tttactgtgc gaaagagaga 300
ccccttcgcc tattacgata ttttgactgg ttatcggggg gggcgaatga ctactggggc 360
cagggaaccc tggtcaccgt ctcctcag 388
<210> 32
<211> 337
<212> DNA
<213>Homo sapiens
<400> 32
gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60
atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120
tggtaccagc agaaaccagg acagcctcct aagttgctca ttgactgggc atctacccgg 180
gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240
atcagcaatc tgcaggttga agatgtggcc gtttattact gtcagcagta ttatagaagt 300
ccgtcctttg gccaggggac caagctggag atcaaac 337
<210> 33
<211> 129
<212> PRT
<213>Homo sapiens
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Asn Asn Thr Leu His
65 70 75 80
Leu Glu Met Asn Thr Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
100 105 110
Gln Gly Tyr Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Thr
115 120 125
Ser
<210> 34
<211> 388
<212> DNA
<213>Homo sapiens
<400> 34
caggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgtag cctctggatt cacgttcagt acctatgcca tgcactgggt ccgtcaggct 120
ccaggcaggg ggctggagtg ggtggcagtt atctcatacg atggaaatta taaatactat 180
gcagactctg tgaagggccg attctccatc tccagagaca attccaacaa cacgctgcat 240
ctagaaatga acaccctgag aactgaggac acggctttat attactgtgc gaaagactcc 300
caactgcgat cactcctcta ttttgaatgg ttatcccagg gatattttga cccctggggc 360
cagggaaccc tggtcaccgt cacctcag 388
<210> 35
<211> 129
<212> PRT
<213>Homo sapiens
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Phe Tyr Cys
85 90 95
Ala Lys Glu Arg Pro Leu Arg Leu Leu Arg Tyr Phe Asp Trp Leu Ser
100 105 110
Gly Gly Ala Asn Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 36
<211> 388
<212> DNA
<213>Homo sapiens
<400> 36
gaggtgcagc tggtggagtc tgggggaggc gcggtccagc ctggggagtc cctgaaactc 60
ccctgtgcag cctctggatt caccttcagt aactatggca tgcactgggt ccgccaggct 120
ccaggcaagg gactggagtg ggtggcagtc atatcatatg atggatctaa taagtactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagga cacgctgtat 240
ctgcaaatga acagcctgag agctgaggac acggctctgt tttactgtgc gaaagagaga 300
ccccttcgcc tattacgata ttttgactgg ttatcggggg gggcgaatga ctactggggc 360
cagggaaccc tggtcaccgt ctcctcag 388
<210> 37
<211> 6
<212> PRT
<213>Homo sapiens
<400> 37
Phe Gly Ala Ile Ala Gly
1 5
<210> 38
<211> 9
<212> PRT
<213>Homo sapiens
<400> 38
Asp Gly Val Thr Asn Lys Val Asn Ser
1 5
<210> 39
<211> 15
<212> PRT
<213>Homo sapiens
<400> 39
Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys
1 5 10 15
<210> 40
<211> 11
<212> PRT
<213>Homo sapiens
<400> 40
Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro
1 5 10
<210> 41
<211> 8
<212> PRT
<213>Homo sapiens
<400> 41
Ile Ser Tyr Asp Ala Asn Tyr Lys
1 5
<210> 42
<211> 22
<212> PRT
<213>Homo sapiens
<400> 42
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
1 5 10 15
Gln Gly Tyr Phe Asp Tyr
20
<210> 43
<211> 22
<212> PRT
<213>Homo sapiens
<400> 43
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
1 5 10 15
Gln Gly Tyr Phe Glu Pro
20
<210> 44
<211> 10
<212> PRT
<213>Homo sapiens
<400> 44
Gln Ser Val Thr Phe Asn Asn Lys Asn Tyr
1 5 10
<210> 45
<211> 24
<212> DNA
<213>Homo sapiens
<400> 45
ggattcacct tttctacata cgct 24
<210> 46
<211> 24
<212> DNA
<213>Homo sapiens
<400> 46
atctcatacg acgctaacta taag 24
<210> 47
<211> 66
<212> DNA
<213>Homo sapiens
<400> 47
gccaaagatt ctcagctgag gagtctgctg tatttcgaat ggctgagcca ggggtacttt 60
gattat 66
<210> 48
<211> 30
<212> DNA
<213>Homo sapiens
<400> 48
cagtctgtga ctttcaacta caaaaattat 30
<210> 49
<211> 9
<212> DNA
<213>Homo sapiens
<400> 49
tgggcttca 9
<210> 50
<211> 27
<212> DNA
<213>Homo sapiens
<400> 50
cagcagcact accggactcc acccacc 27
<210> 51
<211> 24
<212> DNA
<213>Homo sapiens
<400> 51
ggattcactt tttccaccta cgca 24
<210> 52
<211> 24
<212> DNA
<213>Homo sapiens
<400> 52
atctcatacg acgccaacta taag 24
<210> 53
<211> 66
<212> DNA
<213>Homo sapiens
<400> 53
gctaaggatt ctcagctgag aagtctgctg tattttgaat ggctgtctca ggggtatttt 60
gaacct 66
<210> 54
<211> 30
<212> DNA
<213>Homo sapiens
<400> 54
cagtctgtga ctttcaacaa caaaaattat 30
<210> 55
<211> 129
<212> PRT
<213>Homo sapiens
<400> 55
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Ala Asn Tyr Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
100 105 110
Gln Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 56
<211> 387
<212> DNA
<213>Homo sapiens
<400> 56
caggtgcagc tggtggagtc cggaggagga gtggtgcagc cagggcggtc tctgagactg 60
agttgcgccg cttcaggatt caccttttct acatacgcta tgcactgggt gcggcaggct 120
cctggcaagg gactggaatg ggtggccgtg atctcatacg acgctaacta taagtactat 180
gccgatagcg tgaaaggcag gttcacaatt agccgcgaca actccaagaa tactctgtac 240
ctgcagatga attccctgag ggctgaggac accgccgtgt actattgtgc caaagattct 300
cagctgagga gtctgctgta tttcgaatgg ctgagccagg ggtactttga ttattgggga 360
cagggcactc tggtgaccgt gagctcc 387
<210> 57
<211> 111
<212> PRT
<213>Homo sapiens
<400> 57
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Thr Phe Asn
20 25 30
Tyr Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr
85 90 95
Arg Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 58
<211> 333
<212> DNA
<213>Homo sapiens
<400> 58
gacatcgtga tgactcagtc tcccgatagt ctggccgtgt ccctgggcga gagggctaca 60
attaactgca agagctccca gtctgtgact ttcaactaca aaaattatct ggcctggtac 120
cagcagaagc ctggacagcc ccctaaactg ctgatctatt gggcttcaac ccgggaaagc 180
ggcgtgccag acagattctc aggcagcggg tccggaacag acttcaccct gacaatttct 240
agtctgcagg ccgaggacgt ggccgtgtac tattgtcagc agcactaccg gactccaccc 300
acctttggcc aggggacaaa ggtggaaatc aaa 333
<210> 59
<211> 129
<212> PRT
<213>Homo sapiens
<400> 59
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Ala Asn Tyr Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu His
65 70 75 80
Leu Glu Met Asn Thr Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Gln Leu Arg Ser Leu Leu Tyr Phe Glu Trp Leu Ser
100 105 110
Gln Gly Tyr Phe Glu Pro Trp Gly Gln Gly Thr Leu Val Thr Val Thr
115 120 125
Ser
<210> 60
<211> 387
<212> DNA
<213>Homo sapiens
<400> 60
caggtccagc tggtccagag cggcggcggc gtggtccagc cagggaggtc actgagactg 60
tcatgcgtcg cttcaggatt cactttttcc acctacgcaa tgcactgggt gcggcaggca 120
cctggaagag gactggagtg ggtggcagtc atctcatacg acgccaacta taagtactat 180
gctgatagcg tcaaaggcag gttcagcatt tcccgcgaca acagtcagaa tacactgcat 240
ctggagatga ataccctgcg aacagaagac actgccctgt actattgcgc taaggattct 300
cagctgagaa gtctgctgta ttttgaatgg ctgtctcagg ggtattttga accttggggg 360
cagggcactc tggtcaccgt cacttcc 387
<210> 61
<211> 111
<212> PRT
<213>Homo sapiens
<400> 61
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Thr Phe Asn
20 25 30
Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr
85 90 95
Arg Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 62
<211> 333
<212> DNA
<213>Homo sapiens
<400> 62
gacatcgtga tgactcagtc tcccgatagt ctggccgtgt ccctgggcga gagggctaca 60
attaactgca agagctccca gtctgtgact ttcaacaaca aaaattatct ggcctggtac 120
cagcagaagc ctggacagcc ccctaaactg ctgatctatt gggcttcaac ccgggaaagc 180
ggcgtgccag acagattctc aggcagcggg tccggaacag acttcaccct gacaatttct 240
agtctgcagg ccgaggacgt ggccgtgtac tattgtcagc agcactaccg gactccaccc 300
acctttggcc aggggacaaa ggtggaaatc aaa 333

Claims (14)

1. a kind of antibody of separation or its antigen-binding fragment, the sense of the influenza A virus of 2 hypotypes of its hypotype of neutralization group 1 and group Contaminate and specifically bind the epitope in the stem area of Flu-A hemagglutinin (HA) tripolymer, wherein the antibody or its antigen binding The heavy chain and light chain of fragment contact the amino acid in the first near-end monomer and the second Distal Right monomer of the HA tripolymers, and And wherein described antibody or its antigen-binding fragment are being transfected with the titre of high at least 3 times of the titre than producing FI6 variants 2 Produced in cell.
2. antibody according to claim 1 or its antigen-binding fragment, are selected from
A. antibody or its antigen-binding fragment, wherein the heavy chain of the antibody or its antigen-binding fragment contacts the near-end monomer In amino acid residue, and the light chain of wherein described antibody or its antigen-binding fragment contacts the amino in the near-end monomer Amino acid residue in the Distal Right monomer of sour residue and the HA tripolymers;With
B. antibody or its antigen-binding fragment, it specifically binds the epitope in the stem area of Flu-A HA tripolymers, and its Described in described first or second comonomer in HA tripolymers be not cut or cutting.
3. antibody according to claim 1 or 2 or its antigen-binding fragment, are selected from：
A. antibody or its antigen-binding fragment, wherein the heavy chain of the antibody or its antigen-binding fragment contact described first or the In amino acid and HA2 in the HA1 of two monomers at position 318 at position 18,19,20,21,38,41,42,45,49,53 and 57 Amino acid residue, and wherein described monomer be it is not cut or be cut；
B. antibody or its antigen-binding fragment, wherein the light chain of the antibody or its antigen-binding fragment contacts the near-end monomer HA2 in the HA1 of amino acid residue at position 38,39 and 43 and the Distal Right monomer position 327,328 and 329 with And the amino acid residue in HA2 at position 1,2,3 and 4, and wherein described near-end monomer and the Distal Right monomer are not Cut;
C. antibody or its antigen-binding fragment, wherein the light chain of the antibody or its antigen-binding fragment contacts the near-end monomer HA2 in position 321 and 323 in the HA1 of amino acid residue at position 38,39,42 and 46 and the Distal Right monomer And the amino acid residue in HA2 at position 7 and 11, and wherein described near-end monomer and the Distal Right monomer are to be cut Cut;
D. antibody or its antigen-binding fragment, it specifically binds the ammonia at position 318 in the HA1 comprising the near-end monomer Base acid and the amino acid residue at position 18,19,20,21,38,39,41,42,43,45,48,49,53,56 and 57 in HA2, with And at amino acid residue in the HA1 of the Distal Right monomer at position 327,328,329 and HA2 polypeptides position 1,2,3 and 4 Amino acid residue epitope, wherein the near-end monomer and the Distal Right monomer are not cut;
E. antibody or its antigen-binding fragment, it specifically binds the amino at position 318 in the HA1 comprising the near-end monomer Acid and the amino acid residue at position 18,19,20,21,38,39,41,42,45,46,49,52,53 and 57 in HA2, Yi Jisuo State the amino acid residue at position 7 and 11 in the amino acid residue and HA2 in the HA1 of Distal Right monomer at position 321 and 323 Epitope, wherein the near-end monomer and the Distal Right monomer are cut;With
F. antibody or its antigen-binding fragment, it is specifically bound comprising position in the amino acid and HA2 at position 329 in HA1 1st, the epitope of the amino acid residue at 2,3 and 4, wherein the HA1 and HA2 are present in the not cut list of the HA tripolymers In body.
4. antibody or its antigen-binding fragment according to any one of preceding claims, wherein the antibody be human antibody, Monoclonal antibody, the antibody of purifying, single-chain antibody, Fab, Fab ', F (ab') 2, Fv or scFv.
5. antibody or its antigen-binding fragment according to any one of preceding claims, wherein the antibody or its antigen knot Close fragments specific combination hypotype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 Flu-A HA.
6. antibody or its antigen-binding fragment according to any one of preceding claims, it is used to treat influenza A virus Infection.
7. a kind of nucleic acid molecules, it includes the antibody or its antigen-binding fragment encoded any one of preceding claims Polynucleotides.
8. a kind of carrier, it includes nucleic acid molecules according to claim 7.
9. a kind of cell, it expresses the antibody or its antigen-binding fragment according to any one of claim 1-6；Or bag Containing carrier according to claim 8.
10. a kind of separation or purifying immunogenic polypeptide, it is included with reference to according to any one of claim 1-6 The epitope of antibody or its antigen-binding fragment.
11. a kind of pharmaceutical composition, it includes the antibody or its antigen binding fragment according to any one of claim 1-6 Section, nucleic acid according to claim 7, carrier according to claim 8, cell according to claim 9 or Immunogenic polypeptide according to claim 10 and pharmaceutically acceptable diluent or carrier.
It is 12. antibody or its antigen-binding fragment according to any one of claim 1-6, according to claim 7 Nucleic acid, carrier according to claim 8, cell according to claim 9 according to claim 10 are exempted from The purposes of epidemic disease antigenic polypeptide or pharmaceutical composition according to claim 11, (i), which is used to manufacture, to be used for treating Flu-A Medicine, (ii) of virus infection are used for vaccine or (iii) is used to diagnose influenza a virus infection.
13. the purposes of the antibody or its antigen-binding fragment according to any one of claim 1-6, for anti-by checking The antigen of influenza A virus vaccine contains the specificity epitope with correct conformation to monitor the quality of the vaccine.
14. the antibody or its antigen-binding fragment any one of claim 1-6 are being prepared for reducing Flu-A disease Purposes in the medicine of the risk of poison infection or reduction influenza a virus infection.
CN201710362549.9A 2011-07-18 2011-07-18 Neutralize anti-influenza A virus antibody and application thereof Pending CN107417789A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181303A (en) * 2020-09-14 2022-03-15 东莞市朋志生物科技有限公司 Anti-influenza a virus antibodies and kits

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495511A (en) * 2006-05-15 2009-07-29 航道生物技术有限责任公司 Neutralizing antibodies to influenza viruses
WO2010010466A2 (en) * 2008-07-25 2010-01-28 Institute For Research In Biomedicine Neutralizing anti-influenza a virus antibodies and uses thereof
CN102046656A (en) * 2008-03-28 2011-05-04 航道生物技术有限责任公司 Neutralizing molecules to viral antigens
CN103717617B (en) * 2011-07-18 2017-06-20 生物医学学会 Neutralize anti-influenza A virus antibody and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495511A (en) * 2006-05-15 2009-07-29 航道生物技术有限责任公司 Neutralizing antibodies to influenza viruses
CN102046656A (en) * 2008-03-28 2011-05-04 航道生物技术有限责任公司 Neutralizing molecules to viral antigens
WO2010010466A2 (en) * 2008-07-25 2010-01-28 Institute For Research In Biomedicine Neutralizing anti-influenza a virus antibodies and uses thereof
CN103717617B (en) * 2011-07-18 2017-06-20 生物医学学会 Neutralize anti-influenza A virus antibody and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181303A (en) * 2020-09-14 2022-03-15 东莞市朋志生物科技有限公司 Anti-influenza a virus antibodies and kits

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