WO2014097762A1 - Anticorps dirigé contre la grippe aviaire hautement pathogène - Google Patents

Anticorps dirigé contre la grippe aviaire hautement pathogène Download PDF

Info

Publication number
WO2014097762A1
WO2014097762A1 PCT/JP2013/080060 JP2013080060W WO2014097762A1 WO 2014097762 A1 WO2014097762 A1 WO 2014097762A1 JP 2013080060 W JP2013080060 W JP 2013080060W WO 2014097762 A1 WO2014097762 A1 WO 2014097762A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
virus
influenza
clade
strain
Prior art date
Application number
PCT/JP2013/080060
Other languages
English (en)
Japanese (ja)
Inventor
阪口薫雄
桑原一彦
小原道法
芝▲崎▼太
Original Assignee
国立大学法人熊本大学
公益財団法人東京都医学総合研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人熊本大学, 公益財団法人東京都医学総合研究所 filed Critical 国立大学法人熊本大学
Priority to JP2014553012A priority Critical patent/JPWO2014097762A1/ja
Publication of WO2014097762A1 publication Critical patent/WO2014097762A1/fr

Links

Images

Classifications

    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • 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

Definitions

  • the present invention relates to a specific and anti-affinity antibody against highly pathogenic avian influenza, in particular a monoclonal antibody having a dissociation constant of at least 1 ⁇ 10 ⁇ 10 M.
  • H5N1 highly pathogenic influenza A
  • H5N1 highly pathogenic influenza A
  • Patent Document 1 7,879,326 (Patent Document 1)) produced by cell fusion by immunization with H5N1 antigen, and other monoclonal antibodies against highly pathogenic avian influenza (No. 4,625,485). Patent (Patent) Document 2)) there is.
  • Non-patent Document 1 11 types of antibodies from Simons et al.
  • the antibody of Foun et al. is an antibody using a peptide fragment of H5N1 as an immunogen, and the target epitope in this antibody is limited. The activity of the antibody is shown as a result of measurement by ELISA.
  • An object of the present invention is to provide a specific and high affinity monoclonal antibody against highly pathogenic influenza.
  • the present inventor prepared an antigen using a recombinant vaccinia virus and immunized the GANP (registered trademark) transgenic mouse to obtain a highly pathogenic avian bird.
  • the inventors have found that monoclonal antibodies against influenza virus can be produced and have completed the present invention.
  • the present invention is as follows. (1) An antibody against the HA protein of influenza virus having a dissociation constant of at least 1 ⁇ 10 ⁇ 10 M. (2) The antibody according to (1), wherein the dissociation constant is 1 ⁇ 10 ⁇ 10 M to 1 ⁇ 10 ⁇ 13 M. (3) The antibody according to (1), wherein the influenza virus is an influenza H5 subtype virus. (4) The antibody according to (3), wherein the influenza H5 subtype virus is influenza H5N1 virus.
  • Influenza H5N1 virus is the Indogan Qinghai Lake strain (clade 2.2), Indonesia strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper) (4) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2).
  • the antibody according to (6) which is produced by a hybridoma having a deposit number of NITE BP-01216 or NITE BP-01217, or a reception number of NITE ABP-01737 or NITE ABP-01738.
  • the antibody according to (1) wherein the antibody is a chimeric antibody, a humanized antibody or a humanized antibody.
  • (11) A hybridoma that produces the monoclonal antibody according to (6).
  • a hybridoma whose accession number is NITE BP-01216 or NITE BP-01217, or whose reception number is NITE ABP-01737 or NITE ABP-01738.
  • the method for producing the antibody according to (1) comprising the following steps: (A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus, and (b) collecting the antibody from the non-human mammal. .
  • the method for producing a monoclonal antibody according to (6) or (7) comprising the following steps: (A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus; (B) collecting antibody-producing cells from the immunized non-human mammal; (C) The method comprising: fusing the antibody-producing cells obtained in step (b) and myeloma cells; and (d) collecting the antibody from the fused cells obtained in step (c). (15) The method according to (13) or (14), wherein the non-human mammal is a GANP (registered trademark) transgenic non-human mammal.
  • GANP registered trademark
  • influenza virus characterized by detecting the influenza virus by contacting the antibody according to any one of (1) to (9) or the fragment according to (10) with a biological sample. Detection method.
  • the influenza H5 subtype virus is influenza H5N1 virus.
  • Influenza H5N1 virus has been identified as Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper) (18) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The method described. (20) A reagent for detecting influenza virus comprising the antibody according to any one of (1) to (9) or the fragment according to (10). (21) The reagent according to (20), wherein the influenza virus is an influenza H5 subtype virus.
  • influenza H5 subtype virus is influenza H5N1 virus.
  • Influenza H5N1 virus can be found in Indogan Qinghai Lake strain (clade 2.2), Indonesia strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolia strain (A / whooper) (22) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The reagent described.
  • a pharmaceutical composition for anti-influenza virus comprising the antibody according to any one of (1) to (9) or the fragment according to (10).
  • the pharmaceutical composition according to (24), wherein the influenza virus is an influenza H5 subtype virus.
  • the pharmaceutical composition according to (25), wherein the influenza H5 subtype virus is influenza H5N1 virus.
  • Influenza H5N1 virus has been identified as Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnamese strain (clade 1), Mongolian strain (A / whooper) (26) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2).
  • the pharmaceutical composition as described.
  • the present invention provides an antibody against a highly pathogenic avian influenza virus.
  • the antibody of the present invention showed neutralizing activity against highly pathogenic avian influenza virus. Therefore, the antibody of the present invention is extremely useful as a reagent for detecting highly pathogenic avian influenza viruses or as a pharmaceutical composition for protecting against highly pathogenic avian influenza viruses.
  • the present invention is a high affinity antibody against highly pathogenic avian influenza having a dissociation constant (KD) of at least 1 ⁇ 10 ⁇ 10 M, and a wide range of clade species, such as Indogan Qinghai Lake strain, Indonesian strain, Anhui strain , which can be detected or protected with a single antibody against Vietnamese, Mongolian and Hokkaido strains.
  • Highly pathogenic avian influenza mediates chickens and is a highly lethal influenza that infects humans and has a different epidemic from seasonal influenza.
  • infection is being controlled by international containment of highly pathogenic avian influenza and limited vaccine administration.
  • the key to this strategy is to quickly discover and diagnose highly infected flu patients and to take effective treatments and take measures to prevent the spread of infection.
  • hemagglutinin (HA) protein expressed using vaccinia virus is used as an antigen, and a highly pathogenic avian influenza is produced by using a high affinity monoclonal antibody production technique using GANP (registered trademark) transgenic mice.
  • GANP registered trademark
  • Viruses specifically and a wide range of clade species namely, Indangan Qinghai Lake strain (A / Bar-Headed goose / Qinghai / 1A / 05: clade 2.2), Indonesia strain (A / Indonesia / 05/2005: clade 2) .1), Anhui strain (A / Anhui / 1194/2005: clade 2.3), Vietnam strain (A / Vietnam / 1194/2004: clade 1), Mongolian strain (A / whooper swan / Mongolia / 3 / 005: clade 2.2), Hokkaido strain (A / whooper swan / Hokkaido / 1/2008: clade 2.3.2) are prepared with high affinity antibodies capable of detecting all with a single type of antibody. Succeeded.
  • the antibody of the present invention is a molecule that binds to the HA protein via the heavy and light chain variable regions VH and VL.
  • the antibodies of the present invention represent either polyclonal or monoclonal immunoglobulin molecules such as IgG, IgA, IgM, IgE, IgD, or subclasses such as IgG1, IgG2, IgG3 or IgG4.
  • the antibody of the present invention also includes functional fragments or subsequences of immunoglobulin molecules such as Fab, Fab ′, F (ab ′) 2 , Fv, Fd, scFv and sdFv.
  • the antigen used in the present invention is the HA protein of influenza virus.
  • This protein can be expressed using vaccinia virus.
  • vaccinia virus ⁇ Production of recombinant vaccinia virus having HA protein gene of influenza virus>
  • the HA protein gene is registered with a predetermined accession number (Accession No.) in the GenBank database.
  • accession number accession number (Accession No.) in the GenBank database.
  • H5 subtypes for example, genes encoding HA proteins derived from influenza viruses belonging to each clade of H5N1, are registered in the GenBank accession number as follows.
  • Clade 1 _A / Vietnam / 1109 / 2004_EF541040
  • Clade 2.1 _A / Indonesia / 5 / 2005_EF541394
  • Clade 2.2 _A / bar-headed goose / Qinghai / 1A / 2005_DQ659327
  • Clade 2.3 _A / Anhui / 1 / 2005_DQ37171928
  • a gene (cDNA) encoding the HA protein contained in the recombinant vaccinia virus of the present invention in addition to the above genes, a part thereof or a mutant sequence thereof can be used.
  • DNA lacking part of the DNA encoding the HA protein of clade 1 DNA lacking part of the DNA encoding the HA protein of clade 2.1, and HA protein of clade 2.2
  • a DNA in which a part of the DNA is deleted or a DNA in which a part of the DNA encoding the HA protein of clade 2.3 is deleted can be used.
  • These HA protein genes have already been cloned and inserted into plasmids.
  • HA protein gene in addition to the above HA protein gene (in the present specification, the full-length base sequence or the base sequence of the protein coding region.
  • Genes can also be used. For example, it hybridizes under stringent conditions with a sequence consisting of a base sequence complementary to the base sequence shown in EF541040 and is complementary to the base sequence shown in EF541394, which encodes the HA protein of clade 1.
  • Examples of stringent conditions include 0.1 ⁇ SSC to 10 ⁇ SSC, 0.1% to 1.0% SDS, and 20 ° C. to 80 ° C., and more specifically, 37 ° C. to 56 ° C.
  • An example is a condition in which after pre-hybridization at 30 ° C. for 30 minutes or more, washing in 0.1 ⁇ SSC, 0.1% SDS for 10 to 20 minutes at room temperature is performed 1 to 3 times.
  • DNA encoding the HA protein of Clade 2.1 the base sequence shown in DQ659327 and 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or DNA having a homology of 99% or more and encoding the HA protein of clade 2.2, 50% or more, 60% or more, 70% with the base sequence shown in DQ371728 Moreover, 80%, 90%, 95%, have a 98% or 99% or more homology, and it is possible using DNA encoding the
  • the mutant gene is prepared, for example, using a site-directed displacement induction method described in “Molecular cloning: A Laboratory Manual (4th Edition)” or a mutation-introducing kit using a site-specific mutagenesis method. can do.
  • mutagenesis kits include QuickChange TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTaylor TM Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRaectite-Site-Mit-Site-Mit-Site-Mit-Site-Mist
  • Preferred examples include Mutan-Super Express Km and the like (manufactured by Takara Bio Inc.).
  • the HA protein used as an antigen can be obtained by an ordinary genetic engineering technique based on the above gene information.
  • the DNA encoding the HA protein is incorporated into a plasmid for homologous recombination and then introduced into an animal cell, followed by infecting the animal cell with the parent strain, vaccinia virus. After infection, the recombinant vaccinia virus may be cultured to express the HA protein.
  • the HA protein can be expressed in large quantities in the form of complete sugar modification from the early stage to the late stage of vaccinia virus infection.
  • a homologous recombination plasmid can include a promoter, a gene to be expressed, and the like.
  • animal cells include rabbit kidney cells, monkey kidney cells, and human fibroblasts.
  • the expressed HA protein can be purified by a known technique using a His tag or the like. The purification method is not limited, and affinity chromatography, HPLC purification, and the like are employed. Confirmation that the purified HA protein is the target can be performed, for example, by Western blotting, SDS-polyacrylamide gel electrophoresis, or the like.
  • the majority of the HA protein expressed by the recombinant vaccinia virus forms a trimer similar to the HA protein on influenza virus particles and is used as an antigen.
  • the protein produced as described above is immunized by administration to a non-human mammal by itself or together with a carrier and a diluent, and the antibody titer is measured.
  • the type of non-human mammal to be immunized is not particularly limited, and examples include mice, rats, guinea pigs, rabbits, dogs, goats, etc., with mice being preferred.
  • a transgenic non-human mammal called “GANP (registered trademark)” having a high-specificity antibody-producing ability (WO 2004/040971) as an animal to be immunized. Can also be used.
  • GANP registered trademark
  • transgenic non-human mammal is a non-human mammal into which a gene encoding a germinal center-associated nuclear protein has been introduced, and the animal is immunized with a predetermined antigen.
  • it is an animal that can produce a high affinity or high specificity antibody against the antigen (WO 00/50611, Sakaguchi N. et al., J Immunol. 2005 Apr 15; 174 (8 ): 4485-94.).
  • a GANP transgenic non-human mammal for example, a mouse
  • the total dose of the antigen per animal is 10 to 2000 ⁇ g.
  • an adjuvant and an antigen solution are generally mixed.
  • the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Aluminum hydroxide adjuvant examples include aluminum hydroxide adjuvant.
  • Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, intramuscularly, subcutaneously into the footpad, or the like.
  • the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 4 weeks.
  • the antibody titer can be examined using blood collected from the immunized animal.
  • the obtained serum is serially diluted, and the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA (radioimmunoassay)) and the like.
  • ELISA enzyme immunoassay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • an animal having a high antibody titer against the HA protein is subjected to final immunization.
  • the immunity of the antigen and the measurement of the antibody titer are not limited to the above measurement methods.
  • immunocompetent cells such as spleen cells
  • regional lymph nodes are removed several days after the final immunization day, preferably 2 to 5 days later.
  • the interval between blood collections is 1 to 4 weeks, preferably 1 to 2 weeks after immunization.
  • polyclonal antibody when obtaining a polyclonal antibody, blood is collected on the day when the desired antibody titer is shown to obtain antiserum.
  • antibody purification is required, purify by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or a combination thereof. Can do. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.
  • the HANP transgenic non-human mammal is immunized with the HA protein, antibody-producing cells (for example, B cells) are collected from the immunized animal, and this antibody production is performed. Cells and myeloma cells are fused to produce a hybridoma (fusion cell line). And the target monoclonal antibody can be obtained by extract
  • antibody-producing cells for example, B cells
  • Antibody-producing cells are prepared from spleen cells or lymph node cells or spleen or lymph nodes of animals such as immunized non-human mammals. Even when a GANP transgenic non-human mammal is used, antibody-producing cells can be collected from spleen cells, lymph node cells, spleen, lymph nodes, or the like in the same manner as described above. Examples of lymph nodes include inguinal lymph nodes and mediastinal lymph nodes. Although it is not necessary to separate the antibody-producing cells from the collected cell population, it is desirable to separate only antibody-producing cells from the cell population. It is preferable to remove tissue debris and red blood cells as much as possible.
  • myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
  • the cell line to be used is preferably one that has the property that it cannot survive in a HAT selection medium (medium containing hypoxanthine, aminopterin, and thymidine) and can survive only in a state fused with antibody-producing cells.
  • HAT selection medium medium containing hypoxanthine, aminopterin, and thymidine
  • myeloma cells include P3X63-Ag. 8).
  • U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1), etc. are mentioned.
  • Cell fusion is a commercially available medium such as DMEM or RPMI 1640 medium that does not contain fetal calf serum (FCS) and the like, and 1 ⁇ 10 6 to 1 ⁇ 10 7 cells / mL of spleen cells and / or lymph node cells.
  • 1 ⁇ 10 5 to 1 ⁇ 10 6 cells / mL of myeloma cells are mixed (the ratio of spleen cells and / or lymph node cells to myeloma cells is preferably 5: 1), and in the presence of a cell fusion promoter.
  • the cell fusion promoter polyethylene glycol having an average molecular weight of 200 to 20,000 daltons can be used.
  • cells can be fused using Sendai virus.
  • a person skilled in the art can fuse the antibody-producing cells and myeloma cells using a known cell fusion method. After the fusion, the cells are diluted with a HAT medium prepared with RPMI 1640 medium containing 10 to 20% FCS, for example, and then 0.5 to 3 ⁇ 10 5 cells are seeded in each well of a 96-well culture plate, and a CO 2 incubator. Incubate at
  • a hybridoma producing the target antibody is selected from the cells after cell fusion treatment. Ten to 14 days after cell fusion, the cells selected in the HAT medium as described above form colonies. The culture supernatant of each well of the colony positive 96-well culture plate is collected and the antibody titer against HA is confirmed. As a confirmation method, an enzyme immunoassay (ELISA), a radioimmunoassay (RIA) or the like is performed. After confirming antibody production positive wells against HA, the cells are transferred to a 24-well or 12-well culture plate.
  • ELISA enzyme immunoassay
  • RIA radioimmunoassay
  • the medium is preferably replaced with an HT medium (a medium containing hypoxanthine and thymidine) excluding aminopterin.
  • HT medium is used as a recovery medium for hybridomas that continue to supply purine and pyrimidine precursors to the salvage pathway during the effect of residual aminopterin in the cells.
  • the antibody titer in the culture supernatant is confirmed again. Since the hybridoma is unstable because it is a fused cell, and it is highly likely that the antibody producing ability is lost immediately, it is preferable to confirm the antibody titer for the second time.
  • Cloning is performed to make the cells in the finally selected well into a single cell.
  • the cell suspension is appropriately diluted with RPMI 1640 medium containing 10 to 20% FCS, and then seeded with 0.3 to 2 cells in each well of a 96-well culture plate.
  • the number of cells in each well of the 96-well culture plate is preferably seeded so that there is one cell in each well so that there is a high probability of one cell in each well.
  • the culture supernatant of the colony-positive well is collected 7 to 10 days later. At this time, it is preferable to confirm that it is a single colony after 3 to 5 days.
  • the collected culture supernatant is checked for antibody titer.
  • clones having high specificity for HA and low cross-reactivity for SARS S protein are selected.
  • the number of cells in the selected well is increased to some extent to establish a hybridoma strain. Cloning may be performed several times as necessary.
  • HA-specific monoclonal antibody is purified and collected from the established hybridoma strain by the following method. That is, a method of preparing an antibody from a culture supernatant cultured in a medium with reduced serum concentration, a method of preparing an antibody from a culture supernatant cultured in a commercially available serum-free medium, or injecting a hybridoma into the abdominal cavity of an animal. There is a method of collecting ascites and preparing antibodies from the ascites. The culture supernatant is collected from cells prepared at 0.1 to 4 ⁇ 10 5 cells / mL and cultured for 1 to 2 weeks.
  • hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. Ascites fluid is collected after 1 to 2 weeks.
  • Examples of the culture method include a method using a culture flask, a method using a spinner flask, a method using a shaker flask, and a method using a bioreactor.
  • Antibody purification methods include HA affinity column purification method, ammonium sulfate salting-out fraction purification method by gel filtration chromatography, ion exchange chromatography purification method, etc., selecting these known methods as appropriate, Or it can refine
  • the cell lines (hybridomas) producing the monoclonal antibodies of the present invention are referred to as “3E10 (rVV cl2.2)” and “7G10 (rVV cl2.2)”, both of which are independent administrative corporation products. Deposited internationally based on the Budapest Treaty at the Japan Institute for Evaluation Technology Patent Microorganisms Deposit Center (2-5-8 KITESA Kamashita, Kisarazu City, Chiba Prefecture 292-0818) based on the Budapest Treaty (original deposit date: February 2012) 1 day).
  • accession numbers are NITE BP-01216 for “3E10 (rVV cl2.2)” and NITE BP-01217 for “7G10 (rVV cl2.2)”.
  • the cell lines (hybridomas) that produce the monoclonal antibodies of the present invention are also referred to as “8C1 (rVV cl2.2)” and “14A7 (rVV cl2.2)”, both of which are listed in the Budapest Microorganism Depositary Center. (The original deposit date: October 18, 2013).
  • the receipt number is NITE ABP-01737 for “8C1 (rVV cl2.2)” and NITE ABP-01738 for “14A7 (rVV cl2.2)”.
  • the antibody of the present invention specifically binds to HA and exhibits high affinity. “Specific” means that it does not cross-react with subtypes other than the HA protein subtype used for immunization, and reacts only with the subtype protein used for immunization (binding / neutralization). For example, when H5N1 HA protein is used as an antigen as a subtype, the antibody of the present invention only reacts (binding / neutralizes) with H5 subtype protein or H5 subtype virus, and other H1N1, H1N2, etc. This means that there is no cross-reactivity with the subtype of.
  • binding affinity can be determined by association constant (KA) and dissociation constant (KD).
  • KD affinity equilibrium constant
  • Its binding affinity can be detected as follows.
  • the binding affinity has a dissociation constant (KD) of at least 1 ⁇ 10 ⁇ 10 M.
  • KD dissociation constant
  • the dissociation constant is 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times.
  • it has an affinity 1000 to 10,000 times higher.
  • the dissociation constant for the binding affinity of the HA protein is, 1 ⁇ 10 -10 M, 5 ⁇ 10 -11 M, 1 ⁇ 10 -11 M, 5 ⁇ 10 - 12 M, 1 ⁇ 10 ⁇ 12 M, 5 ⁇ 10 ⁇ 13 M, 1 ⁇ 10 ⁇ 13 M, 5 ⁇ 10 ⁇ 14 M, 1 ⁇ 10 ⁇ 14 M, 5 ⁇ 10 ⁇ 15 M, or 1 ⁇ 10 ⁇ 15 M, and more preferably 1 ⁇ 10 ⁇ 10 M to 1 ⁇ 10 ⁇ 13 M.
  • the value may be lower than these KDs and may have high affinity.
  • the dissociation constant (KD) of the antibody to be measured for affinity is within about 1 to 100 times the KD of the antibody of the present invention, the antibody is substantially the same as the antibody of the present invention. Is included in the present invention.
  • the association constant (KA) and dissociation constant (KD) can be measured using surface plasmon resonance (SPR), and it is possible to employ a known device and method for detecting the binding rate in real time and further monitoring it.
  • SPR surface plasmon resonance
  • Biacore registered trademark
  • T200 GE Healthcare
  • ProteON XPR36 Bio-Rad
  • the KD value of a typical antibody as defined herein is sufficient to saturate half (50%) of the binding site of the HA antigen of the immobilized purified HA protein (A / Anhui / 1/2005). Defined as the concentration of antibody required.
  • the antibody of the present invention can specifically bind to multiple types of virus strains. Examples of the virus strains to be bound include A / whooper swan / Monglia / 03/2005, A / whooper swan / Hokaido / 1/2008, A / duck / Hokaido / Vac-3 / 2007.
  • the antibody fragment of the present invention is an antibody against HA and has the above KD value.
  • the antibody fragment means a partial region of the antibody of the present invention.
  • Fab, Fab ′, F (ab ′) 2 Fv, diabodies (dibodies), dsFv (disulphide-stabilized Fv), scFv (single chain Fv) and the like.
  • the antibody fragment can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes according to the purpose.
  • Fab can be obtained by treating antibody molecules with papain, and F (ab ′) 2 can be obtained by treating antibody molecules with pepsin.
  • Fab ′ can be obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 .
  • scFv cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and DNA encoding scFv is constructed. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, scFv can be produced.
  • diabody cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and a DNA encoding scFv is constructed so that the length of the amino acid sequence of the peptide linker is 8 residues or less.
  • a diabody can be produced by inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression.
  • dsFv cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and a DNA encoding dsFv is constructed. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, dsFv can be produced.
  • One preferred embodiment of the antibody of the present invention is a recombinant antibody.
  • the recombinant antibody include, but are not limited to, chimeric antibodies, humanized antibodies, and humanized antibodies.
  • human antibodies or consensus residues thereof, or amino acid residues present in CDR regions are known (Kabat, Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human. Services.Public Health Service (1987)).
  • human antibodies include antibodies in which one or more amino acid residues are replaced with amino acids present in any of the other human antibodies.
  • CDR grafting refers to transplanting a complementarity determining region (CDR) from a variable region of a mouse antibody into a human variable region, wherein the framework region (FR) is derived from a human and the CDR is derived from a mouse. This is a method for producing a configured variable region. These humanized reshaped human variable regions are then linked to human constant regions. Methods for producing such humanized antibodies are well known in the art (see, for example, Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987)). .
  • Humanized antibodies (fully human antibodies) generally have the same structure as that of a human antibody in the hypervariable region, which is the antigen-binding site of the V region, the rest of the V region, and the constant region. It is. However, the hypervariable site may be derived from another animal. Techniques for producing humanized antibodies are also known, and methods for producing gene sequences common to humans by genetic engineering techniques have been established.
  • the humanized antibody can be obtained by, for example, a method using a human antibody-producing mouse having a human chromosome fragment containing H and L chain genes of human antibody (Tomizuka, K. et al., Nat. Genet., (1977) 16). 133-143; Kuroiwa, Y.
  • the hybridoma of the present invention for example, a hybridoma whose accession number is NITE BP-01216 or NITE BP-01217, or whose reception number is NITE ABP-01737 or NITE ABP-01738), or extracted from the hybridoma Chimeric antibodies, humanized antibodies, and humanized antibodies can be prepared using DNA or RNA as a raw material according to the well-known methods described above.
  • the anti-HA antibody of the present invention includes, for example, an antibody that binds to a site (for example, an epitope) to which the antibody of the present invention binds (recognizes), preferably the accession number is NITE BP- Examples include an antibody that binds to a site (eg, an epitope) to which a monoclonal antibody produced by a hybridoma of 01216 or NITE BP-01217 or a receipt number of NITE ABP-01737 or NITE ABP-01738 binds.
  • the site to which the anti-HA antibody of the present invention binds is not limited as long as it is at least a part of the antigen HA.
  • the present invention provides a method for detecting influenza virus, which comprises detecting an influenza virus by bringing the antibody or fragment thereof of the present invention into contact with a biological sample.
  • a biological sample to be examined is collected from a subject such as an influenza patient, a patient suspected of influenza, or a health checkup examinee, and an HA measurement sample is prepared.
  • the influenza virus to be detected is an influenza A virus or subtype, and examples include Indogan Qinghai Lake strain, Indonesia strain, Anhui strain, and Vietnam strain.
  • Subtypes include, for example, H1N1, H1N2, H2N1, H2N2, H2N3, H3N2, H3N8, H4N6, H4N8, H5N1, H5N2, H5N3, H5N5, H5N8, H6N1, H6N2, H7N1, H7N9, H7N1, H7N9 H10N3, H11N1, H11N2, H11N9, H12N5, H13N6, H13N8, H15N8 or H16N3, and one or more of these are selected.
  • an influenza H5 subtype influenza virus for example, H5N1 is preferable.
  • the biological sample examples include blood, sputum, saliva, pharyngeal swab, nasal discharge, nasal discharge, and nasal aspirate.
  • the antibody is contacted with a biological sample.
  • Contact means that the antibody of the present invention or a fragment thereof (also referred to as an antibody or the like) and a biological sample to be tested are placed in the same environment so that they can be bound or reacted. For example, adding a biological sample to a container (well) containing an antibody or the like, adding an antibody or the like to a container (well) containing a biological sample, or culturing the antibody or the like and the biological sample in the same container It means to do.
  • the measurement of HA can be performed by ELISA or EIA that is generally performed, and is not particularly limited.
  • the antibody of the present invention that specifically binds to this amino acid sequence detects H5N1 influenza virus. Is particularly preferred. In the case of detection by ELISA, when the signal intensity is stronger than that of the negative control, it can be determined that influenza virus is contained in the test sample (ie, positive).
  • kits and Reagents Containing Antibodies of the Present Invention antibodies against HA can be used as reagents or kits for detecting influenza viruses.
  • the antibody of the present invention for example, a monoclonal antibody
  • the monoclonal antibody can be combined with another solvent or solute to form a composition.
  • distilled water, pH buffer reagent, salt, protein, surfactant and the like can be combined.
  • Monoclonal antibodies can also be enzyme-labeled.
  • labeling enzyme in addition to HRP (horseradish peroxidase), alkaline phosphatase, malate dehydrase, ⁇ -glucosidase, ⁇ -galactosidase, colloidal gold and the like can be used.
  • the present invention when used in a kit, in addition to the antibody of the present invention, the above solvent, solute, enzyme labeling reagent, antigen-immobilized microplate, antibody dilution solution, OPD (orthophenylenediamine) tablet, substrate solution, A reaction stop solution, a concentrated washing solution, instructions for use, and the like can be included.
  • a reaction medium such as a buffer solution that gives optimum conditions for the reaction, a buffer solution that is useful for stabilizing the reaction product, and a stabilizer for the reaction material may be included in the kit of the present invention.
  • a PCR method can also be employed to detect HA mRNA or DNA.
  • Primers used for PCR can also be included in the reagents or kits of the present invention.
  • the region on which the primer is designed is not particularly limited as long as the HA protein can be detected.
  • the length of the primer is 18 to 24 bases, preferably 20 to 22 bases. The following can be illustrated as a base sequence of a primer.
  • the pharmaceutical composition of the present invention contains the antibody of the present invention as an active ingredient, is used for anti-influenza virus, and is effective for the prevention or treatment of influenza.
  • influenza virus that is the target of prevention or treatment of influenza is an influenza A virus or subtype, and examples include Indogan Qinghai Lake strain, Indonesia strain, Anhui strain, and Vietnam strain.
  • Subtypes are, for example, H1N1, H1N2, H2N1, H2N2, H2N3, H3N2, H3N8, H4N6, H4N8, H5N1, H5N2, H5N3, H5N5, H5N8, H6N1, H6N2, H7N1, H7N1, H7N1, H7N2, H7N1, H7N2, H7N , H11N1, H11N2, H11N9, H12N5, H13N6, H13N8, H15N8 or H16N3 are selected from one or more.
  • H5 subtype influenza virus for example, H5N1 is preferable.
  • the antibody of the present invention can prevent a subject from being infected with one or more influenza viruses (isolate or subtype) or reduce the sensitivity of the subject to the infection. Also, one or more symptoms or complications associated with influenza infection, such as fatigue, chills, fever, body pain, headache, cough, sore throat, nasal congestion, sinus closure, pneumonia, bronchitis, ear infection, ear Reduce or eliminate pain, etc., or prevent criticism of these symptoms. Furthermore, it is possible to shorten the period of suffering from these symptoms.
  • the antibody of the present invention can be administered alone or together with a pharmaceutically acceptable carrier or diluent as a pharmaceutical composition, and the administration can be performed once or divided into several times.
  • pharmaceutically acceptable carrier refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners. , Flavoring agents, solubilizers or other additives.
  • a composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally.
  • various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate and glycine can be used together with a disintegrant, a binder and the like.
  • the disintegrant include starch, alginic acid, and certain types of silicate double salts.
  • the binder include polyvinylpyrrolidone, sucrose, gelatin, and gum arabic.
  • lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are very effective for tablet formation.
  • an emulsifier and a suspending agent may be used in combination, if necessary, and used with water, ethanol, propylene glycol, glycerin, etc., and diluents that combine them.
  • Other forms for parenteral administration include injections that contain one or more active substances and are prescribed by conventional methods.
  • injections for example, by dissolving or suspending in an pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection to an antibody concentration of 1 ⁇ g / ml to 100 mg / ml.
  • an pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water
  • the injection thus produced is a ratio of 1 ⁇ g / kg to 100 mg / kg, preferably a ratio of 100 ⁇ g / kg to 15 mg / kg, in a single administration to a subject (eg, human influenza patient). And can be administered once to several times per day.
  • the dosage is not limited to this range, and may vary depending on the weight and symptoms of the patient and individual administration routes.
  • the dose may vary depending on the sensitivity of the patient to be treated, the way the drug is prescribed, the period of administration, and the interval between doses. In some cases, a dose lower than the lower limit of the above range may be appropriate. .
  • injections can be prepared as non-aqueous diluents (for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions, or emulsions.
  • non-aqueous diluents for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol
  • suspensions or emulsions.
  • sterilization of the injection can be performed by filtration sterilization through a bacteria-retaining filter, blending of a bactericide, or irradiation.
  • the injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid or powder composition by lyophilization, etc., and dissolved in sterile water for injection or other solvent before use.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • H5N1 antigen was prepared by recombinant vaccinia virus (rVV).
  • rVV recombinant vaccinia virus
  • a recombinant plasmid in which the H5N1 influenza HA gene was introduced into the homologous recombination vector was introduced into rabbit kidney cells RK13 and infected with the parent strain vaccinia virus to obtain a recombinant vaccinia virus (rVV) expressing HA.
  • purification of His-Tag protein by a conventional method using TARON column chromatography was performed.
  • the HA protein gene of H5N1 HPAIV has already been cloned and inserted into a plasmid.
  • the HA protein gene of H5N1 HPAIV HA gene (GenBank accession numbers: EF541040, EF541394, DQ65937, DQ371937) inserted into the plasmid was used as a PCR template. That is, H5N1 HPAIV HA protein gene region was prepared by performing PCR using each gene-specific primer using cDNA of H5N1 HPAIV HA protein gene as a template. The resulting fragment was cloned into the plasmid pBMSF7C (Arch. Virol. Vol. 138, p.
  • the expression promoter contained in the recombinant vaccinia virus of the present invention is inserted into the hemagglutinin (HA) gene region of vaccinia virus, and includes a poxvirus A type inclusion body (ATI) promoter and a plurality of repetitive vaccinia viruses.
  • HA hemagglutinin
  • ATI poxvirus A type inclusion body
  • a recombinant vaccinia virus was produced by introducing the plasmid vector into a host vaccinia virus.
  • the plasmid vector was introduced into the host by introducing plasmid vectors pBMSF7C-mCl1, pBMSF7C-mCl2.1, pBMSF7C-mCl2.2, and pBMSF7C-mCl2.3 into animal cells infected with the attenuated vaccinia virus LC16m8 strain, respectively.
  • Recombinant vaccinia viruses (RVV-Flu HA (H5-mCl1), RVV-Flu HA (H5-mCl2), which cause homologous recombination in the hemagglutinin (HA) gene region of vaccinia virus and express the HA protein gene of H5N1 HPAIV, respectively.
  • RVV-Flu HA H5-mCl2.2
  • RVV-Flu HA H5-mCl2.3
  • the HA protein was expressed by infecting the produced recombinant vaccinia virus into RK-13 cells, which are a rabbit kidney cell line. Subsequently, purification of HA protein to which His-Tag was added by a conventional method using TARON column chromatography was performed.
  • HA protein The purification results of HA protein (Western blotting and CBB staining) are shown in FIG.
  • the obtained purified HA protein was designated as “H5mcl2.2 antigen”.
  • FIG. 2 the left panel is a diagram showing the HA protein constituting the influenza virus particle, the middle panel is a diagram showing the purified HA protein expressed by rVV, and the right panel is the diagram showing the purified HA protein. It is a figure which shows having received sugar chain modification.
  • the purified protein yield was 0.5-2 mg.
  • Mouse thymocytes of 2 ⁇ 10 5 cells / well were used as feeder cells, and the feeder cells and recombinant IL-6 (0.25 unit / ml) were added to the wells and cultured.
  • HAT hypoxanthine, aminopterin, thymidine
  • Cloning was performed according to a conventional method, and cell culture was performed in the presence of recombinant IL-6 0.25 unit / ml in 96 wells in the presence of 4 ⁇ 10 5 mouse thymocytes feeder cells. A subclone having the same activity as that of the original cell was established and stored in the proliferating clone, and used for the subsequent analysis. The above operation was performed using RPMI-1640, 10% -heat-immobilized fetal bovine serum, and HT (hypoxanthine, thymidine) medium. The obtained clones are shown in Table 1.
  • clones indicated as “3E10”, “7G10”, “8C1”, and “14A7” represent deposit clones having the following deposit numbers or receipt numbers, respectively.
  • Table 6 shows the evaluation results of the crossability using the candidate immunochromatography kit.
  • Labeling method 0.3 mg of antibody was mixed with 1.3 ml of 0.4% fluorescent bead suspension and allowed to react at room temperature for 1 hour. After discarding the supernatant by centrifugation, 1% BSA solution was added and blocked for 1 hour. After discarding the supernatant by centrifugation, it was washed with 0.1% Trion X-100 solution at pH 7.7, suspended in 1% BSA solution at pH 7.7, and incubated at 37 ° C. for 48 hours. The fluorescent bead labeled antibody thus produced was used in place of the colloidal gold labeled antibody.
  • influenza virus H5N1 (1 ⁇ 10 3 PFU / ml) virus solution diluted with MEM medium containing 1% BSA and 0.1 ml of hybridoma culture supernatant diluted 100 times, 1000 times, and 10000 times Were mixed and neutralized at 37 ° C. for 1 hour.
  • 0.1 ml of a mixture of influenza virus solution and hybridoma culture supernatant was added to each well and spread throughout.
  • FIG. 3 shows a photograph of the plate after culture. Compared to the negative control (no addition), 3E10, 10B9, 14A7 and 3B9 showed a clear difference in plaque size and number. The amount of virus used was confirmed by performing back titration (no addition), but it was a predetermined amount of virus.
  • FIG. 4 The results are shown in FIG. In FIG. 4, the numbers in the upper left of each plate indicate the number of plaques when the hybridoma culture supernatant (No. 1 to 4) is diluted 10 ⁇ 2 , 10 ⁇ 3 , and 10 ⁇ 4 .
  • the control group was Back titration (Back), and the virus solution used for neutralization value measurement was added in stock solution (1 time) and diluted 10 times.
  • Relenza (GlaxoSmithKline) was added at concentrations of 1 ⁇ M, 0.1 ⁇ M, and 0.01 ⁇ M, and it was confirmed that the appearance of plaques was possible. As shown in FIG.
  • 4 animals / group were necropsied 3 days after the infection, and 4 animals / group were necropsied at the end of the experiment.
  • Non-infected (diluted solution) inoculated group (4 mice per group) was used for monitoring body weight change.
  • the antibody amount was 15 mg / kg / mouse, and were administered to Day 0 (immediately before virus infection) and Day 2 from the orbital vein nest. The method is shown in FIG.
  • FIG. 6 Panel B shows the change in body weight after challenge infection with influenza virus.
  • ⁇ 14A7-3-21-1 clone
  • ⁇ 14A7-3-21-1 clone
  • ⁇ 14A7-3-21-1 clone
  • ⁇ 14A7-3-21-1 clone
  • ⁇ Vehicle
  • ⁇ no decrease in body weight
  • the infected mouse group administered with 14A7-3-21-1 clone ( ⁇ ) had a survival rate of 25%, and the infected mouse group administered with Vehicle ( ⁇ ) had a survival rate of 50%.
  • the infected mouse group ( ⁇ ) and the non-infected mouse group ( ⁇ ) to which the 3B9-11-6-9 clone was administered no decrease in the survival rate was observed. From the above results, the 3B9-11-6-9 clone having a wide binding ability to H5N1 highly pathogenic avian influenza virus showed a remarkable therapeutic effect even against highly pathogenic avian influenza virus infection.
  • H3N1 A / whooper swan / Hokaido in 3B9-11-6-9 clone / 1/08 strain showed a therapeutic effect on highly pathogenic avian influenza virus.
  • the antibody was administered to examine the therapeutic effect on mice (in vivo test).
  • FIG. 7 is a diagram showing the results of studying the therapeutic effect of anti-HA monoclonal antibody administration (BALB / crice (Female, 9-week-old), H5N1 HPAIV 300 PFU, 15 mg / kg 3B9 administration group).
  • FIG. 7 panel A shows the change in body weight after challenge infection with influenza virus. In the infected mouse group ( ⁇ ) in which the 3B9-11-6-9 clone was administered to Day 8 and the non-administered mouse group ( ⁇ ), significant weight loss was observed after 6 days of infection.
  • FIG. 7 panel B shows the change in survival rate after challenge infection. On the 14th day of infection, an infected mouse group ( ⁇ ) administered to the infected mouse group Day0 ( ⁇ ), Day4 ((), Day6 ( ⁇ ), and Day8 administered with the 3B9-11-6-9 clone is shown.
  • the survival rates were Day 4 (() 75%, Day 6 (() 50%, and Day 8 ( ⁇ ) 0%.
  • the infected mouse group ( ⁇ ) and the non-infected mouse group ( ⁇ ) in which the 3B9-11-6-9 clone was administered to Day 0 ( ⁇ ) no decrease in the survival rate was observed. From the above results, the 3B9-11-6-9 clone having a wide binding ability to H5N1 highly pathogenic avian influenza virus shows a remarkable therapeutic effect even after administration after infection with highly pathogenic avian influenza virus. It was.
  • the antibody of the present invention makes it possible to produce an ultrasensitive diagnostic kit. Further, since it is effective not only for detection of viruses of various clades but also for inhibition of virus growth, it is extremely useful for medical and clinical examinations.
  • hybridomas “3E10 (rVV cl2.2)” and “7G10 (rVV cl2.2)” are both registered by the National Institute of Technology and Evaluation for Microorganisms (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 292-0818). -5-8 Deposited internationally at NITE Biotechnology Headquarters (Patent Microorganism Deposit Center) based on the Budapest Treaty (original deposit date: February 1, 2012). The accession number is NITE BP-01216 for “3E10 (rVV cl2.2)” and NITE BP-01217 for “7G10 (rVV cl2.2)”.
  • Hybridomas “8C1 (rVV cl2.2)” and “14A7 (rVV cl2.2)” were both internationally deposited at the above-mentioned patent microorganism deposit center based on the Budapest Treaty (original deposit date: October 18, 2013) .
  • the receipt number is NITE ABP-01737 for “8C1 (rVV cl2.2)” and NITE ABP-01738 for “14A7 (rVV cl2.2)”.
  • SEQ ID NO: 1 synthetic DNA
  • SEQ ID NO: 2 Synthetic DNA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un anticorps dirigé contre la protéine HA d'un virus de la grippe ayant une constante de dissociation d'au moins 1×10-10 M, une méthode de production dudit anticorps, une méthode de détection du virus de la grippe consistant à faire réagir un anticorps et un échantillon biologique, et une composition pharmaceutique contenant ledit anticorps dirigée contre le virus de la grippe.
PCT/JP2013/080060 2012-12-20 2013-10-30 Anticorps dirigé contre la grippe aviaire hautement pathogène WO2014097762A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014553012A JPWO2014097762A1 (ja) 2012-12-20 2013-10-30 高病原性トリインフルエンザに対する抗体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012278228 2012-12-20
JP2012-278228 2012-12-20

Publications (1)

Publication Number Publication Date
WO2014097762A1 true WO2014097762A1 (fr) 2014-06-26

Family

ID=50978108

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/080060 WO2014097762A1 (fr) 2012-12-20 2013-10-30 Anticorps dirigé contre la grippe aviaire hautement pathogène

Country Status (2)

Country Link
JP (1) JPWO2014097762A1 (fr)
WO (1) WO2014097762A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647610A (zh) * 2020-06-02 2020-09-11 扬州大学 一种互换ha和ns1缺失基因包装信号的h9n2亚型禽流感病毒及其构建方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63245670A (ja) * 1986-09-23 1988-10-12 アプライド　バイオテクノロジー，インコーポレーテッド 偽狂犬病ワクチン
JP2006115845A (ja) * 1994-05-20 2006-05-11 Genelabs Technol Inc Ｇ型肝炎ウイルスおよびその分子クローニング
WO2011068143A1 (fr) * 2009-12-03 2011-06-09 独立行政法人科学技術振興機構 Anticorps dirigé contre le sous-type h5 du virus de la grippe et son utilisation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6165460A (en) * 1995-07-10 2000-12-26 Therion Biologics Corporation Generation of immune responses to prostate-specific antigen (PSA)
EP1613736A4 (fr) * 2003-03-11 2007-04-11 Dana Farber Cancer Inst Inc Inhibition de la pathogenese virale
MX2011005171A (es) * 2008-11-21 2011-05-30 Bavarian Nordic As Vector que comprende multiples secuencias de nucleotidos homologas.
JP5716257B2 (ja) * 2009-02-06 2015-05-13 国立大学法人 熊本大学 胆管癌特異的糖鎖エピトープを認識するモノクローナル抗体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63245670A (ja) * 1986-09-23 1988-10-12 アプライド　バイオテクノロジー，インコーポレーテッド 偽狂犬病ワクチン
JP2006115845A (ja) * 1994-05-20 2006-05-11 Genelabs Technol Inc Ｇ型肝炎ウイルスおよびその分子クローニング
WO2011068143A1 (fr) * 2009-12-03 2011-06-09 独立行政法人科学技術振興機構 Anticorps dirigé contre le sous-type h5 du virus de la grippe et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DU ANARIWA ET AL.: "Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 378, 2009, pages 197 - 202 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647610A (zh) * 2020-06-02 2020-09-11 扬州大学 一种互换ha和ns1缺失基因包装信号的h9n2亚型禽流感病毒及其构建方法和应用

Also Published As

Publication number Publication date
JPWO2014097762A1 (ja) 2017-01-12

Similar Documents

Publication Publication Date Title
EP3011968B1 (fr) Composition comprenant au moins deux molécules de liaison de neutralisation du virus de la grippe a
US9340603B2 (en) Neutralizing anti-influenza A virus antibodies and uses thereof
CA2841551C (fr) Anticorps neutralisant le virus de la grippe a, et leurs utilisations
US8871207B2 (en) Neutralizing anti-influenza A virus antibodies and uses thereof
JP6018916B2 (ja) 抗インフルエンザウイルス中和抗体およびそのスクリーニング方法
JP5998222B2 (ja) ヒトｂ細胞で生産されたインフルエンザａウイルス中和活性を持つ結合分子
WO2012045001A2 (fr) Anticorps dirigés contre le virus de la grippe, immunogènes et leurs utilisations
EP3134111A1 (fr) Anticorps neutralisant le coronavirus du syndrome respiratoire du moyen-orient et procédés d'utilisation de ceux-ci
JP6423550B2 (ja) 広域スペクトルモノクローナル抗ＦｌｕＢ抗体およびその使用
CN114025785B (zh) 慢性炎症和病毒感染的诊断和治疗
JP2022531556A (ja) Ａ型インフルエンザ感染の抗体及び治療法
US20230250157A1 (en) Antibodies for coronavirus and uses thereof
JPWO2014115893A1 (ja) ヒト・メタニューモウイルスに特異的なヒト抗体もしくはその抗原結合性断片
WO2014097762A1 (fr) Anticorps dirigé contre la grippe aviaire hautement pathogène
US20240209065A1 (en) Secretory iga antibodies against covid infection
TW202309069A (zh) 糖鏈修飾rbd及其用途
WO2022266546A2 (fr) Anticorps anti-coronavirus à réactivité croisée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13864834

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2014553012

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13864834

Country of ref document: EP

Kind code of ref document: A1