WO2011068143A1 - Antibody against influenza virus subtype h5 and use thereof - Google Patents
Antibody against influenza virus subtype h5 and use thereof Download PDFInfo
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- WO2011068143A1 WO2011068143A1 PCT/JP2010/071539 JP2010071539W WO2011068143A1 WO 2011068143 A1 WO2011068143 A1 WO 2011068143A1 JP 2010071539 W JP2010071539 W JP 2010071539W WO 2011068143 A1 WO2011068143 A1 WO 2011068143A1
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- influenza virus
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- 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
-
- 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 an antibody against H5 subtype influenza virus and use thereof. Specifically, the antibody and functional fragment thereof against H5 subtype influenza virus, a hybridoma producing the antibody, and the variable region of the antibody are encoded. Polynucleotides, expression vectors containing the polynucleotides, cells into which the expression vectors have been introduced, methods for detecting H5 subtype influenza viruses using the antibodies, detection kits for H5 subtype influenza viruses containing the antibodies, and the antibodies The present invention relates to a preventive or therapeutic agent for H5 subtype influenza virus infection diseases.
- Influenza viruses are classified into A type, B type and C type according to the difference in antigenicity between nucleoprotein (NP) and membrane protein (M). Among them, influenza A viruses are further classified into 16 types of HA (H1-16) and NA of 9 types (N1-9), depending on the amino acid sequence or antigenicity of hemagglutinin (HA) and neuraminidase (NA). There are as many subtypes of that combination. Among these, the main subtypes that infect humans using humans as hosts are H1-3 and N1-2. Influenza A virus infects not only humans but many mammals and birds.
- An avian infectious disease caused by an influenza A virus infecting birds is “bird flu”, and all subtypes of H1-16 and N1-9 have been confirmed as influenza virus causing the infection. Many of these influenza viruses do not show serious symptoms to birds, but some H5, H7 and H9 subtypes of influenza viruses show severe symptoms when infected with birds. . These are called highly pathogenic avian influenza viruses, and there is concern about the possibility of human infection.
- Antibodies are effective for detection and measurement of influenza virus and prevention or treatment of influenza virus-infected diseases. So far, various monoclonal antibodies having neutralizing activity against H5 subtype influenza virus have been reported (for example, Non-Patent Documents 1 to 4). However, nothing has been put into practical use for the prevention or treatment of H5 subtype influenza virus-infected diseases, and the development of a new antibody having high neutralizing activity against H5 subtype influenza virus is desired.
- Prabakaran M. et al. Combination therapy using chimeric monoclonal antibodies protects mice from lethal H5N1 infection and prevents formation of escape mutants.
- Simmons C.P. et al. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.
- An object of the present invention is to provide a novel antibody having neutralizing activity against H5 subtype influenza virus, and a preventive or therapeutic agent for H5 subtype influenza virus infection disease containing the antibody as an active ingredient.
- the present invention includes the following inventions in order to solve the above problems.
- [8] A polynucleotide encoding the heavy chain variable region of the antibody according to any one of [1] to [5].
- [9] A polynucleotide encoding the light chain variable region of the antibody according to any one of [1] to [5].
- An expression vector comprising the polynucleotide according to [8] and / or [9].
- [11] A cell into which the expression vector according to [10] is introduced.
- Detection of H5 subtype influenza virus comprising the step of bringing the antibody according to any one of [1] to [5] above or the functional fragment according to claim 7 into contact with a test sample Method.
- a detection kit for H5 subtype influenza virus comprising the antibody according to any one of [1] to [5] or the functional fragment according to claim 7.
- a preventive or therapeutic agent for H5 subtype influenza virus infection disease comprising the antibody according to any one of [1] to [5] or the functional fragment according to claim 7 as an active ingredient .
- An H5 subtype comprising a step of administering an effective amount of the antibody according to any one of [1] to [5] or the functional fragment according to claim 7 to an animal.
- a method for preventing or treating influenza virus-infected diseases comprising the antibody according to any one of [1] to [5] or the functional fragment according to claim 7.
- a novel antibody having neutralizing activity against H5 subtype influenza virus can be provided.
- the antibody has been confirmed to have preventive and therapeutic effects in experiments using H5 subtype influenza virus-infected mice, and is useful as an active ingredient for preventive or therapeutic agents for H5 subtype influenza virus-infected diseases.
- FIG. 4 is a diagram comparing the amino acid sequences of the heavy chain variable regions of mouse 36-1 antibody and mouse 61 antibody.
- FIG. 3 is a diagram comparing the amino acid sequences of the light chain variable regions of mouse 36-1 antibody and mouse 61 antibody.
- FIG. 3 is a view showing the structure of a basic vector pDN11-cohCk for light chain expression.
- FIG. 3 is a diagram showing the structure of a heavy chain expression basic vector pUCBR-SPyhCg1c. It is a figure which shows the design of the humanized antibody based on the mouse
- FIG. 1 It is a figure which shows the structure of a recombinant antibody expression vector. It is a figure showing the results of evaluating the binding to H5 subtype influenza virus A / rgViet Num / 1194 ⁇ HA / 2004 (H5N1) strain, (A) shows the results of mouse 36-1 antibody and chimeric 36-1 antibody, (B) shows the results of mouse 61 antibody and chimera 61 antibody. It is a figure which shows the result of having evaluated the neutralization activity with respect to H5 subtype influenza virus A / Hong Kong / 483/1977 (H5N1) stock, (A) shows the result of mouse 36-1 antibody and chimera 36-1 antibody , (B) shows the results of mouse 61 antibody and chimeric 61 antibody.
- the present inventors immunized mice with influenza virus A / Vietnam / 1194/04 (H5N1) as an antigen, prepared hybridomas according to a conventional method, and the reactivity of monoclonal antibodies produced by the obtained hybridomas with various viruses.
- influenza virus A / Vietnam / 1194/04 H5N1
- the neutralizing activity two types of mouse monoclonal antibodies that specifically bind to hemagglutinin of H5 subtype influenza virus and neutralize infectivity of H5 subtype influenza virus were found.
- Research has been completed and the present invention has been completed.
- the mouse monoclonal antibody produced by the hybridoma 36-1 is a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3.
- the mouse monoclonal antibody produced by the hybridoma 61-2-1 has a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 5.
- Hybridoma 36-1 accesion No. NITE BP-832
- Hybridoma 61-2-1 accesion No. NITE BP-833 have been internationally deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation. (Contract date: October 30, 2009).
- the present invention provides an antibody that specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus.
- the antibody of the present invention comprises a lysine at position 177 and lysine at position 205 in the amino acid sequence (SEQ ID NO: 1) of hemagglutinin of influenza virus A / Vietnam / 1194/04 (H5N1) strain, which is a type of H5 subtype influenza virus. Any epitope-recognizing epitope may be used, and the H5 subtype influenza virus hemagglutinin epitope other than the A / Vietnam / 1194/04 (H5N1) strain is not limited.
- the antibody of the present invention can neutralize the infectivity of H5 subtype influenza virus.
- the antibody of the present invention preferably neutralizes H5 subtype influenza virus infectivity by at least 50%, more preferably at least 70% neutralization, and even more preferably at least 90% neutralization.
- Examples of the antibody of the present invention include antibodies including the following (a) and (b).
- the antibody of the present invention is preferably a human chimeric antibody or a humanized antibody.
- a human chimeric antibody refers to an antibody comprising a heavy chain variable region and a light chain variable region of an antibody derived from a non-human animal, and a heavy chain constant region and a light chain constant region of a human antibody.
- a humanized antibody is obtained by grafting a CDR (complementarity determining region) of an antibody derived from a non-human animal into a CDR of a human antibody, and is also referred to as a CDR-grafted antibody, a reconstituted antibody, or the like.
- the FR (framework region) of the humanized antibody is selected so that CDR forms a favorable antigen-binding site. If necessary, the amino acid sequence of the FR in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen-binding site.
- the constant regions of human antibodies are used as the constant regions of chimeric antibodies and humanized antibodies.
- C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4 can be used for the heavy chain
- C ⁇ , C ⁇ can be used for the light chain.
- human antibody constant regions may be modified to improve the stability of the antibody or its production.
- Human antibodies used for humanization may be human antibodies of any isotype such as IgG, IgM, IgA, IgE, IgD, but IgG is preferred, IgG1 or IgG3 is more preferred, and IgG1 is particularly preferred.
- the amino acid sequence of the constant region of a human antibody can be obtained from a known database (Protein Data Bank etc.).
- the human chimeric antibody of the present invention includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or 4, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 or 5, and a human antibody heavy chain. Antibodies consisting of a chain constant region and a light chain constant region are preferred.
- the humanized antibody of the present invention includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 or 8, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 or 9, and a human antibody. An antibody consisting of a heavy chain constant region and a light chain constant region is preferred.
- an antibody having a heavy chain variable region consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, and SEQ ID NO: 3 An antibody having a light chain variable region consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by 5, 7 or 9 is also specific to hemagglutinin of H5 subtype influenza virus.
- One or several amino acids have been deleted, substituted or added means that the number can be deleted, substituted or added by a known mutant peptide production method such as site-directed mutagenesis (preferably 10 or less). , More preferably 7 or less, and even more preferably 5 or less) amino acids are deleted, substituted or added.
- the present invention includes a functional fragment of the antibody of the present invention.
- the “functional fragment” in the present invention is a part (partial fragment) of the antibody of the present invention, which specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus.
- Any hemagglutinin consisting of the amino acid sequence shown in SEQ ID NO: 1 may be used as long as it retains the function of recognizing an epitope containing lysine at position 177 and lysine at position 205.
- Specific examples include Fab, F (ab ′) 2 , Fab ′, Fv, scFv, and the like.
- the antibody of the present invention can be produced by a known method.
- it can be produced as a monoclonal antibody produced by a hybridoma or a recombinant antibody produced using a gene recombination technique.
- the monoclonal antibody-producing hybridoma immunizes a mammal according to a normal immunization method using, for example, H5 subtype influenza virus hemagglutinin as a sensitizing antigen, and fuses the obtained immune cell with a known parent cell by a normal cell fusion method.
- the target monoclonal antibody-producing hybridoma can be obtained by a normal screening method.
- a protein containing lysine at position 177 and lysine at position 205 of hemagglutinin of the influenza virus A / Vietnam / 1194/04 (H5N1) strain consisting of the amino acid sequence represented by SEQ ID NO: 1 Use it.
- a partial fragment containing lysine at position 205 can be used.
- the hemagglutinin or a partial fragment thereof can be produced as a recombinant protein using a known gene recombination technique. Partial fragments can also be produced by chemical synthesis.
- the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.
- mouse, rat, hamster, etc. Rodents, rabbits, monkeys, etc. are used.
- the antigen is mixed with Freund's complete or incomplete adjuvant, or an adjuvant such as potassium alum, and the animal is immunized as an immunogen.
- the administration method of the immunogen is appropriately selected from subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, footpad injection and the like. Immunization can be repeated once or multiple times at appropriate intervals (preferably at intervals of 3 days to 1 week).
- the antibody titer against the antigen in the serum of the immunized animal is measured, and if the animal having a sufficiently high antibody titer is used as a source of antibody-producing cells, the effect of subsequent operations can be enhanced.
- animal-derived antibody-producing cells 3 to 5 days after the final immunization for subsequent cell fusion.
- various known techniques such as radioisotope immunoassay (RIA method), solid-phase enzyme immunoassay (ELISA method), fluorescent antibody method, passive hemagglutination reaction method and the like can be used.
- Mammalian myeloma cells are usually used as parent cells to be fused with antibody-producing cells obtained from immunized mammals.
- a known cell line can be preferably used.
- a cell line obtained from a mouse for example, 8-azaguanine-resistant mouse myeloma line P3X63Ag8U.1 (P3-U1) (Yelton, DE et al., Current Topics in Microbiology and Immunology, 81, 1-7 (1978)), P3 / NSI / 1-Ag4-1 (NS-1) (Kohler, G. et al., European J.
- Antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any part of the individual, generally spleen, lymph nodes, bone marrow, tonsils, peripheral blood, or these as appropriate. Spleen cells are most commonly used, although they can be obtained from a combination or the like. After the final immunization, a site where antibody-producing cells are present, such as the spleen, is removed from a mouse having a predetermined antibody titer, and spleen cells that are antibody-producing cells are prepared. The most commonly used method for fusing these spleen cells and myeloma cells is a method using polyethylene glycol. This method can be performed, for example, by the following procedure.
- Spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, DMEM) or PBS, mixed so that the ratio of the number of spleen cells to myeloma is about 5: 1 to 10: 1, and centrifuged. After removing the supernatant and loosening the precipitated cells, a serum-free medium containing 1 ml of 50% (w / v) polyethylene glycol (molecular weight 1000 to 4000) is added dropwise with stirring. Thereafter, 10 ml of serum-free medium is slowly added and then centrifuged.
- a serum-free medium for example, DMEM
- PBS a serum-free medium containing 1 ml of 50% (w / v) polyethylene glycol (molecular weight 1000 to 4000)
- HAT medium normal medium
- HAT hypoxanthine / aminopterin / thymidine
- IL-2 human interleukin-2
- the myeloma cells are 8-azaguanine resistant strains, that is, hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient strains
- HGPRT hypoxanthine / guanine / phosphoribosyltransferase
- the fused myeloma cells and the fused cells of the myeloma cells cannot survive in the HAT medium.
- non-fused antibody-producing cells and fused cells between antibody-producing cells cannot proliferate. Therefore, by continuing the culture in the HAT medium, only the hybridoma of the antibody producing cell and the myeloma cell grows, and as a result, the hybridoma can be selected.
- a culture medium is changed into the culture medium (HT culture medium) remove
- the hybridoma confirmed to produce a specific antibody against hemagglutinin of H5 subtype influenza virus by antibody titer measurement is transferred to another plate for cloning.
- the cloning method include a limiting dilution method, a soft agar method, a method using a micromanipulator, a method using a cell sorter, etc., but the limiting dilution method is simple and often used. For example, cloning by limiting dilution is repeated 2 to 4 times, hybridomas with stable antibody titers are selected, and the neutralizing activity and epitope of the produced monoclonal antibody are confirmed.
- the neutralizing activity can be confirmed by, for example, the same method as in Example 5 described later. That is, the antibody produced by the selected hybridoma and H5 subtype influenza virus are mixed and incubated, the mixture is added to MDCK cells cultured in a confluent state, the virus is infected, and an agar medium is added. After hardening and culturing for 2 days, the number of plaques is counted. If the number of plaques is reduced as compared with the case where only H5 subtype influenza virus is infected, it can be determined that the antibody has neutralizing activity.
- Epitope identification can be confirmed, for example, by the same method as in Example 7 described later. That is, after reacting the virus with the antibody, infect appropriate cells with the virus to isolate the escape mutant strain, and the sequence analysis of the hemagglutinin gene of the isolated virus strain identifies the amino acid with the mutation. By doing so, the epitope can be identified.
- the medium is changed to a normal medium and cultured.
- Mass culture can be performed by rotary culture or spinner culture using a large culture bottle.
- the antibody of the present invention can be obtained by purifying the supernatant obtained by mass culture using a method well known to those skilled in the art, such as a gel filtration column.
- ascites containing a large amount of the antibody of the present invention can be obtained by growing the hybridoma in the peritoneal cavity of the same strain of mouse or Nu / Nu mouse, rat, guinea pig, hamster or rabbit.
- the antibody of the present invention can be obtained.
- an antibody gene from a hybridoma producing the antibody of the present invention incorporate it into an appropriate vector, and introduce this vector into a host to produce a recombinant antibody (for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
- a recombinant antibody for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990.
- human chimeric antibodies and humanized antibodies can be produced as recombinant antibodies.
- the gene encoding the heavy chain of the human chimeric antibody can be prepared by combining the portion encoding the heavy chain variable region of the antibody gene cloned from the hybridoma with the gene encoding the heavy chain constant region of the human antibody.
- the gene encoding the light chain of the human chimeric antibody is prepared by combining the portion encoding the light chain variable region of the antibody gene cloned from the hybridoma with the gene encoding the light chain constant region of the human antibody. Can do.
- the gene encoding the heavy chain of the humanized antibody In the gene encoding the heavy chain of the humanized antibody, the CDR coding portion of the heavy chain variable region of the antibody gene cloned from the hybridoma is replaced with the corresponding CDR portion of the gene encoding the heavy chain of the human antibody. It can produce by doing. Similarly, the gene encoding the light chain of the humanized antibody is the same as the CDR of the gene encoding the light chain of the human antibody. It can be produced by replacing the portion.
- the obtained gene encoding the heavy chain and the gene encoding the light chain are inserted into an appropriate vector, and a recombinant expression vector for expressing the antibody of the present invention is constructed.
- the gene encoding the heavy chain and the gene encoding the light chain may be incorporated into one vector or into separate vectors.
- the obtained vector is introduced into a host cell to produce a transformant.
- the gene encoding the heavy chain and the gene encoding the light chain are incorporated into separate vectors, the two vectors are co-introduced into the host cell. Details of the vector and the host cell will be described later.
- the host cell (transformant) introduced with the vector can be cultured, and the antibody of the present invention can be collected from the culture.
- the culture include culture supernatant, cultured cells or cultured cells or disrupted products thereof, and secretions of transformants.
- the medium and culture conditions are appropriately selected according to the host cell used.
- the target protein is produced in cells or cells after culturing, antibodies are collected by disrupting the cells or cells.
- the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, the target antibody can be isolated and purified from the culture by appropriately selecting and using a general biochemical method using various chromatographies used for protein isolation and purification.
- the present invention provides a polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region of the antibody of the present invention.
- the polynucleotide of the present invention is useful when the antibody of the present invention is produced as a recombinant antibody. Moreover, it can also be used as a probe for screening an antibody having a function equivalent to that of the antibody of the present invention.
- polynucleotide is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule”.
- the polynucleotides of the present invention can exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA).
- the DNA may be double stranded or single stranded.
- the single-stranded DNA or RNA may be either a coding strand (sense strand) or a non-coding strand (antisense strand).
- the polynucleotide encoding the heavy chain variable region of the present invention includes CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and positions 98 of the amino acid sequence shown in SEQ ID NO: 2 or 4.
- a polynucleotide encoding a heavy chain variable region comprising CDR3 consisting of position 109, more preferably a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 It is.
- Examples of the polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 10, and the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
- polynucleotide encoding the variable region examples include a polynucleotide comprising the base sequence represented by SEQ ID NO: 12, and the polynucleotide encoding the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 includes SEQ ID NO: And a polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 is a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16. However, any of them is not limited.
- the polynucleotide encoding the light chain variable region of the present invention includes CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55, and 88 of the amino acid sequence shown in SEQ ID NO: 3 or 5.
- a polynucleotide encoding a light chain variable region comprising CDR3 consisting of position 96 is preferred, more preferably a polynucleotide encoding a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 3, 5, 7 or 9 It is a nucleotide.
- Examples of the polynucleotide encoding the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 11, and the light chain consisting of the amino acid sequence shown in SEQ ID NO: 5.
- polynucleotide encoding the variable region examples include a polynucleotide comprising the base sequence represented by SEQ ID NO: 13, and the polynucleotide encoding the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 include SEQ ID NO: And a polynucleotide encoding a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9 includes a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17. However, any of them is not limited.
- Examples of the method for obtaining the polynucleotide of the present invention include a method using amplification means such as PCR.
- amplification means such as PCR.
- cDNAs prepared from RNA of hybridoma 36-1 by designing primers based on the 5 ′ side and 3 ′ side sequences (or their complementary sequences) of the nucleotide sequence shown in SEQ ID NO: 10, respectively.
- PCR is performed using the above as a template, and a DNA region sandwiched between both primers is amplified, whereby a large amount of DNA fragments containing a polynucleotide encoding the polypeptide of the present invention can be obtained.
- the present invention provides an expression vector comprising the polynucleotide of the present invention.
- the expression vector of the present invention can be used for producing the antibody of the present invention or a functional fragment of the antibody.
- the expression vector of the present invention is not particularly limited as long as it contains the above-described polynucleotide of the present invention, but a plasmid vector having an RNA polymerase recognition sequence is preferred.
- the polynucleotide encoding the heavy chain variable region in addition to the polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region, the polynucleotide encoding the heavy chain constant region And a polynucleotide encoding the light chain constant region.
- a polynucleotide encoding a heavy chain in which a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a heavy chain constant region are combined, a polynucleotide encoding a light chain variable region, and a light chain constant It is preferable to include a polynucleotide encoding a light chain bound to a polynucleotide encoding a region.
- a method for producing a recombinant expression vector includes, but is not particularly limited to, a method using a plasmid, phage, cosmid or the like.
- the specific type of expression vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, if a promoter sequence is appropriately selected according to the type of host cell to ensure expression of the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good. It is also preferable to include at least one selectable marker.
- Such markers include dihydrofolate reductase or neomycin resistance genes when the host is a eukaryotic cell, and tetracycline resistance genes or ampicillin resistance genes when the host is E. coli or other bacteria.
- the present invention provides a cell into which the expression vector of the present invention has been introduced.
- the cells of the present invention various microorganisms, plant or animal cells known as host cells can be used. Specifically, for example, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), Xenopus oocytes, insect cells, animal cells (eg, CHO cells, COS cells, etc.) Etc. CHO cells are preferred for the purpose of mass expression in animal cells.
- a method for introducing the expression vector of the present invention into a host cell that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method is preferably used. Can do.
- the cell of the present invention preferably stably expresses the protein encoded by the polynucleotide of the present invention, but may be expressed transiently.
- the present invention provides a method for detecting H5 subtype influenza virus.
- the detection method of the present invention only needs to include a step of bringing the antibody of the present invention or a functional fragment thereof (hereinafter referred to as “the antibody of the present invention”) into contact with a test sample.
- the test sample is not particularly limited as long as it may contain H5 subtype influenza virus.
- a sample derived from a living body including biological components of animals and plants is preferable, and a sample derived from an animal including humans is more preferable.
- samples derived from animals including humans include body fluids such as blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, ascites, skin, lung, kidney, mucous membrane, various organs. And a washing solution after washing tissues such as bones, nasal cavity, bronchi, skin, various organs, bones and the like.
- a sample obtained secondarily from the test sample such as a culture solution obtained by culturing cells collected from the body of an organism, is also suitable as the test sample.
- the antibody of the present invention specifically binds to the hemagglutinin of the H5 subtype influenza virus, and the complex Form.
- H5 subtype influenza virus can be detected.
- a known immunological method can be suitably used for detecting the complex. Specific examples include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, and the like, and enzyme immunoassay, particularly ELISA method is preferred.
- the ELISA method can be performed by a method well known to those skilled in the art.
- the antibody of the present invention is bound to a solid phase such as a well of a microplate or a plastic tube, a test sample is added thereto, and after washing, an enzyme-labeled secondary antibody is added.
- control sample in addition to the test sample.
- control sample include a negative control sample not containing H5 subtype influenza virus and a positive control sample containing H5 subtype influenza virus.
- the present invention provides a detection kit for H5 subtype influenza virus.
- the detection kit of the present invention only needs to contain the antibody of the present invention.
- the configuration of the kit other than the antibody and the like of the present invention is not particularly limited, and other necessary reagents and instruments may be appropriately selected to form the kit.
- it when implemented as an ELISA detection kit, it preferably contains a labeled secondary antibody, a coloring reagent, a washing buffer, an ELISA plate, instructions for use, and the like.
- the detection method of the present invention can be carried out simply and rapidly.
- the present invention provides a preventive or therapeutic agent for H5 subtype influenza virus infection diseases comprising the antibody of the present invention as an active ingredient. Since the antibody of the present invention can neutralize the infectivity of H5 subtype influenza virus at a low concentration, it is useful for the prevention or treatment of H5 subtype influenza virus infection diseases.
- the preventive or therapeutic agent of the present invention can be administered orally or parenterally to humans or non-human animals. Moreover, it can be set as a suitable dosage form according to an administration route. Specifically, it can be prepared in various preparation forms such as granules, tablets, pills, capsules, syrups, emulsions, suspensions, injections, drops, external preparations, suppositories and the like. These various preparations are commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives. , And can be produced by conventional methods using colorants, flavoring agents, stabilizers and the like.
- excipient examples include lactose, fructose, glucose, corn starch, sorbit, crystalline cellulose, sterilized water, ethanol, glycerol, physiological saline, and buffer solution.
- disintegrant examples include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate.
- binder examples include methyl cellulose, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like.
- lubricant examples include talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, and the like.
- stabilizer examples include amino acids such as arginine, histidine, lysine, and methionine, human serum albumin, gelatin, dextran 40, methylcellulose, sodium sulfite, and sodium metasulfite.
- other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite, and sodium phosphate.
- a preferred route of administration is parenteral administration, and preferred dosage forms include injections, nasal administration agents, pulmonary administration agents, transdermal administration agents, and the like.
- the injection can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose of the prophylactic or therapeutic agent of the present invention can be selected, for example, within the range of 0.0001 mg to 1000 mg per kg of body weight at one time, but is not limited.
- the dose and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
- the present invention further includes a method for preventing or treating an H5 subtype influenza virus-infected disease using the antibody of the present invention.
- the method for preventing or treating an H5 subtype influenza virus-infected disease of the present invention includes a step of administering a pharmaceutically effective amount of the preventive or therapeutic agent of the present invention.
- Example 1 Preparation of anti-influenza virus H5 subtype mouse monoclonal antibody
- Mice were immunized using the influenza virus A / Vietnam / 1194/04 (H5N1) strain as an antigen, and a hybridoma producing a monoclonal antibody against the antigen was prepared according to a standard method. That is, the virus solution and an equal volume of Freund's complete adjuvant were mixed, and the mouse (BALB / c) was immunized three times, and the splenocytes were used for cell fusion.
- the collected spleen cells and mouse myeloma cells Sp2 / 0 were cell-fused by the polyethylene glycol method and cultured in a HAT medium to obtain a hybridoma producing a monoclonal antibody against the antigen virus.
- mouse monoclonal antibodies derived from four types of hybridomas (clone names: 36-1, 61-2-1, 25, 64) (hereinafter referred to as “mouse 36-1 antibody” and “mouse 61 antibody”, respectively) , “Mouse 25 antibody” and “mouse 64 antibody”) had binding activity on A / swan / Hokaido / 67 (H5N3).
- mouse 36-1 antibody and mouse 61 antibody showed high reactivity to the A / rgViet Num / 1194 ⁇ HA / 2004 (H5N1) strain attenuated from the virus isolated from the patient, while the mouse 25 antibody and mouse 64 antibody were The reactivity to the same strain was found to be low. It was also confirmed that the mouse 36-1 antibody and mouse 61 antibody can neutralize the A / Hong / Kong / 483 / K / (H5N1) strain at a relatively low concentration (see Example 5).
- hybridoma 36-1 has been deposited internationally with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation of Product Evaluation Technology under the accession number NITE BP-832 and the hybridoma 61-2-1 has the accession number NITE BP-833. (Contract date: October 30, 2009).
- Sense primer CGACTGGAGCACGAGGACACTGA (SEQ ID NO: 18, attached to GeneRacer kit)
- -Antisense primer for heavy chain TTGGGCAGCAGATCCAGGGGCC (SEQ ID NO: 19)
- -Antisense primer for light chain CACCTCCAGATGTTAACTGCTCAC (SEQ ID NO: 20)
- the obtained gene fragment was cloned into pT7Blue T-Vector (manufactured by Novagen), and the base sequence was determined according to a conventional method.
- the nucleotide sequence of the heavy chain variable region cDNA of the mouse 36-1 antibody is shown in SEQ ID NO: 10
- the nucleotide sequence of the light chain variable region cDNA is shown in SEQ ID NO: 11.
- the nucleotide sequence of the heavy chain variable region cDNA of mouse 61 antibody is shown in SEQ ID NO: 12
- the nucleotide sequence of the light chain variable region cDNA is shown in SEQ ID NO: 13.
- nucleotide sequences were converted into amino acid sequences based on mammalian codons, and divided into framework regions (FWR1 to 4) and complementarity determining regions (CDR1 to 3) with reference to the domain structure of the mouse antibody.
- the amino acid sequence of the heavy chain variable region of the mouse 36-1 antibody is shown in SEQ ID NO: 2
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3
- the amino acid sequence of the heavy chain variable region of the mouse 61 antibody is shown in SEQ ID NO: 4
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5.
- FIG. 1 shows a comparison of the amino acid sequences of the heavy chain variable regions of mouse 36-1 antibody and mouse 61 antibody.
- FIG. 2 the amino acid sequences of the light chain variable regions of mouse 36-1 antibody and mouse 61 antibody are shown in comparison.
- the framework regions of the H5HA-specific mouse 36-1 antibody and mouse 61 antibody were very similar and were thought to be derived from the same germline sequence.
- both the amino acid sequence of the light chain and the amino acid sequence of the heavy chain were very well conserved, so that one cell is the somatic cell that produces these two antibodies. There was a possibility that it was differentiated by mutation.
- Example 3 Preparation of chimeric antibody or humanized antibody expression vector and production cell
- a DNA fragment containing a human ⁇ chain constant region gene whose codon was optimized for CHO cells was artificially synthesized by linking synthetic oligonucleotides having the base sequences shown in Table 2 to form double-stranded DNA.
- the obtained gene fragment was inserted into a high expression vector for CHO cells to complete a basic vector pDN11-cohCk for light chain expression (see FIG. 3).
- the base sequence of human ⁇ chain constant region cDNA in the synthesized DNA fragment is shown in SEQ ID NO: 21, and the encoded amino acid sequence is shown in SEQ ID NO: 22.
- DNA fragments containing human IgG1 chain constant region genes whose codons are optimized for CHO cells are artificially synthesized by linking synthetic oligonucleotides having the base sequences shown in Table 3 to form double-stranded DNA.
- the obtained gene fragment was inserted into a high expression vector for CHO cells to complete a heavy chain expression basic vector pUCBR-SPyhCg1c (see FIG. 4).
- the nucleotide sequence of human ⁇ chain constant region cDNA in the synthesized DNA fragment is shown in SEQ ID NO: 23, and the encoded amino acid sequence is shown in SEQ ID NO: 24.
- amino acid sequences in which the light chain variable region and the heavy chain variable region were humanized were designed based on the mouse 36-1 antibody and mouse 61 antibody by structure prediction using a computer.
- the amino acid sequences most similar to FWR1, 2, 3, 4 were individually extracted from the human antibody germline sequence database. This was designed by embedding CDRs 1, 2 and 3 of the respective antibodies.
- amino acids near the CDRs in the human antibody FWR were substituted with amino acids of the mouse antibody.
- humanized 36-1 antibody The design of a humanized antibody based on the mouse 36-1 antibody (hereinafter referred to as “humanized 36-1 antibody”) is shown in FIG.
- humanized 61 antibody The design of a humanized antibody based on the mouse 61 antibody (hereinafter referred to as “humanized 61 antibody”) is shown in FIG. 5 and 6, the upper part shows the amino acid sequence of the mouse antibody, the middle part shows the amino acid sequence of the human antibody FWR extracted from the human antibody germline sequence database based on the amino acid sequence of the mouse antibody, and the lower part shows Frankenstein. -The amino acid sequence of the humanized antibody designed by the protocol is shown. In the amino acid sequence of the lower humanized antibody, the light letter indicates the amino acid derived from the mouse antibody sequence.
- a DNA sequence with optimized codons was designed in mammalian cells, and 6 types of synthetic oligonucleotides shown in Table 4 were joined together. PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody.
- hz36-1VH-F TCTTAAGTCAGGTGCAGCTGGTG (SEQ ID NO: 48)
- hz36-1VH-R TGCTAGCGCTGCTCACGGTC (SEQ ID NO: 49)
- a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 5 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
- hz36-1VL-F TCCGCGGTGCTAGACAGATC (SEQ ID NO: 56)
- hz36-1VL-R CCGTACGCTTGATCTCCAGC (SEQ ID NO: 57)
- hz61VH-F TCTTAAGTCAGGTGCAGCTGGTG (same as hz36-1VH-F, SEQ ID NO: 48)
- hz61VH-R TGCTAGCGCTGCTCACGGTC (same as hz36-1VH-R, SEQ ID NO: 49)
- a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 7 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
- hz61VL-F TCCGCGGTGCTAGACAGATC (same as hz36-1VL-F, SEQ ID NO: 56)
- hz61VL-R CCGTACGCTTGATCTCCAG (SEQ ID NO: 70)
- the DNA fragment encoding the light chain variable region of the mouse 36-1 antibody was inserted into the basic vector pDN11-cohCk for light chain expression to complete the light chain expression vector of the chimeric 36-1 antibody.
- a DNA fragment encoding the heavy chain variable region of mouse 36-1 antibody was inserted into basic vector for heavy chain expression pUCBR-SPyhCg1c to complete the heavy chain expression vector of chimeric 36-1 antibody.
- a chimeric 61 antibody light chain expression vector and a chimeric 61 antibody heavy chain expression vector were completed.
- DNA fragment encoding the light chain variable region of the humanized 36-1 antibody was inserted into the light chain expression basic vector pDN11-cohCk to complete the light chain expression vector of the humanized 36-1 antibody.
- a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody was inserted into the basic vector pUCBR-SPyhCg1c for heavy chain expression to complete the heavy chain expression vector of the humanized 36-1 antibody.
- a humanized 61 antibody light chain expression vector and a humanized 61 antibody heavy chain expression vector were completed.
- Each light chain expression vector was treated with SmaI and EcoRI to obtain fragments.
- Each heavy chain expression vector was treated with EcoRV and EcoRI to obtain fragments.
- an antibody expression vector was constructed by ligating the corresponding light and heavy chain fragments. According to the instructions of LipofectAmine2000 (Invitrogen), each antibody expression vector was transfected into CHO cells to obtain high expression clones of each antibody-producing cell.
- Example 4. Evaluation of binding to H5 subtype influenza virus The binding of H5 subtype influenza virus to A / rgViet Num / 1194 ⁇ HA / 2004 (H5N1) strain was evaluated by ELISA.
- Four types of antibodies were used: chimeric 36-1 antibody and chimeric 61 antibody, and the corresponding mouse 36-1 antibody and mouse 61 antibody, respectively.
- As each chimeric antibody the chimeric 36-1 antibody-producing cells and chimeric 61 antibody-producing cells prepared in Example 3 were cultured in a protein-free medium and then purified using a protein G agarose column. Specifically, the experiment was performed according to the following procedure.
- a secondary antibody peroxidase-labeled anti-human constant region antibody or peroxidase-labeled anti-mouse constant region antibody
- PBST peroxidase-labeled anti-human constant region antibody or peroxidase-labeled anti-mouse constant region antibody
- FIG. (A) shows the results of the mouse 36-1 antibody and the chimeric 36-1 antibody
- (B) shows the results of the mouse 61 antibody and the chimeric 61 antibody. Due to the difference in reactivity of the secondary antibody (anti-human constant region antibody or anti-mouse constant region antibody), the mouse antibody shows a lower absorbance in all results, but the affinity (dissociation constant (Kd)) As a result, the mouse 36-1 antibody was about 0.4 nM, the chimera 36-1 antibody was about 0.3 nM, the mouse 61 antibody was about 0.3 nM, and the chimera 61 antibody was about 0.2 nM. Almost the same numerical value was shown.
- the chimeric 36-1 antibody and the chimeric 61 antibody have high affinity for the H5 subtype influenza virus. Judging from the fact that the range of affinity for antigen in therapeutic antibodies currently in practical use is 0.1 nM to 10 nM, chimeric 36-1 antibody and chimeric 61 antibody are effective in preventing against H5 subtype influenza virus infection or It was thought that it could be applied as a therapeutic agent.
- Example 5 Evaluation of neutralizing activity against H5 subtype influenza virus
- H5N1 influenza virus isolated from a human infected subject using four types of chimeric 36-1 antibody and chimeric 61 antibody and corresponding mouse 36-1 antibody and mouse 61 antibody, respectively.
- Strains A / Hong Kong / 483/1977 (H5N1), A / Viet Nam / 1194/2004 (H5N1)) were selected, and experiments were performed according to the following procedure.
- (i) MDCK cells were seeded on a 12-well plate and cultured in a DMEM medium containing 10% serum at 37 ° C. for 2 days to obtain a confluent state.
- the results for the A / Hong Kong / 483/1977 (H5N1) strain are shown in FIG. 10, and the results for the A / Viet Nam / 1194/2004 (H5N1) strain are shown in FIG.
- (A) shows the results of the mouse 36-1 antibody and the chimeric 36-1 antibody
- (B) shows the results of the mouse 61 antibody and the chimeric 61 antibody.
- the chimeric 36-1 antibody and the chimeric 61 antibody had an activity to neutralize the infectious titer of the target virus in the same manner as the mouse 36-1 antibody and the mouse 61 antibody.
- the antibody concentration required to reduce the number of plaques by 50% was different depending on the antibody and virus, but both antibodies showed sufficient neutralizing activity at a low concentration of 0.5 ⁇ g / mL or less. From these results, it was considered that both the chimeric 36-1 antibody and the chimeric 61 antibody are promising as preventive or therapeutic agents for the H5 subtype influenza virus.
- Example 6 Evaluation of efficacy using infected mice Since the infectious titer neutralizing activity was sufficiently observed in both the chimeric 36-1 antibody and the chimeric 61 antibody in the culture test, the prevention and treatment effect in the actual infected animal was expected.
- the effectiveness of chimeric 36-1 antibody and chimeric 61 antibody was evaluated by infection experiments. The mice used were BALB / c, female, and 6 weeks old (at the start of the experiment). The A / Hong Kong / 483/1977 (H5N1) strain was used as the H5 subtype influenza virus.
- the antibody of the present invention is effective for the prevention and treatment of H5 subtype influenza virus infection.
- Example 7 The epitopes of mouse 36-1 antibody and mouse 61 antibody were determined by the following procedure. As a virus, chicken chorioallantoic fluid infected with A / Viet Nam / 1194/2004 (H5N1) was used. (i) A 10-fold serial dilution series of virus was prepared. (ii) Purified antibodies of mouse 36-1 antibody and mouse 61 antibody were each diluted with a medium (MEM) to 20 ⁇ g / mL, and dispensed in 100 ⁇ L each into a small test tube. As a control, 100 ⁇ L of the medium alone was dispensed into small test tubes.
- MEM medium
- acetyltrypsin was added immediately before the overlay so that the concentration was 0.0005%.
- ascites obtained from BALB / c mice inoculated intraperitoneally with hybridoma 36-1 or hybridoma 61-2-1 was added to a final concentration of 1: 1000.
- the solution of (v) was layered on the MDCK cells of (iv) and cultured at 35 ° C. in the presence of 5% CO 2 for 2 to 3 days, and it was observed that plaques could be confirmed.
- Plaque was collected and added to 1 mL of MEM / BSA and mixed well.
- the escape mutant strain for the mouse 36-1 antibody and the escape mutant strain for the mouse 61 antibody are both lysine at position 177 in the amino acid sequence of hemagglutinin (SEQ ID NO: 1) of the A / Viet Nam / 1194/2004 (H5N1) strain. And at least one of lysine at position 205 was mutated to another amino acid. From this result, the mouse 36-1 antibody and the mouse 61 antibody were prepared by lysing the lysine at position 177 and lysine at position 205 in the amino acid sequence (SEQ ID NO: 1) of hemagglutinin of the A / Viet Nam / 1194/2004 (H5N1) strain. It was revealed that the antibody recognizes the contained epitope.
Abstract
Disclosed is a novel antibody which specifically binds to influenza virus subtype H5 hemagglutinin and neutralizes the infectiousness of influenza virus subtype H5. The antibody recognizes an epitope that includes the lysine at position 177 and the lysine at position 205 of a hemagglutinin comprising the amino acid sequence represented by SEQ ID NO: 1. Also disclosed is a prophylactic or therapeutic agent for influenza virus subtype H5 infectious diseases, which contains the antibody as an active ingredient.
Description
本発明は、Ｈ５亜型インフルエンザウイルスに対する抗体およびその利用に関するものであり、詳細には、Ｈ５亜型インフルエンザウイルスに対する抗体およびその機能的断片、当該抗体を産生するハイブリドーマ、当該抗体の可変領域をコードするポリヌクレオチド、当該ポリヌクレオチドを含む発現ベクター、当該発現ベクターが導入された細胞、当該抗体を用いるＨ５亜型インフルエンザウイルスの検出方法、当該抗体を含むＨ５亜型インフルエンザウイルスの検出キット、当該抗体を含むＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬に関するものである。
The present invention relates to an antibody against H5 subtype influenza virus and use thereof. Specifically, the antibody and functional fragment thereof against H5 subtype influenza virus, a hybridoma producing the antibody, and the variable region of the antibody are encoded. Polynucleotides, expression vectors containing the polynucleotides, cells into which the expression vectors have been introduced, methods for detecting H5 subtype influenza viruses using the antibodies, detection kits for H5 subtype influenza viruses containing the antibodies, and the antibodies The present invention relates to a preventive or therapeutic agent for H5 subtype influenza virus infection diseases.
インフルエンザウイルスは、核タンパク質（ＮＰ）と膜タンパク質（Ｍ）の抗原性の違いによりＡ型、Ｂ型およびＣ型に分類される。このうちのインフルエンザＡ型ウイルスは、さらにヘマグルチニン（ＨＡ）とノイラミニダーゼ（ＮＡ）のアミノ酸配列または抗原性の違いにより、ＨＡはＨ１～１６の１６種類、ＮＡはＮ１～９の９種類に分類され、その組み合わせの数の亜型が存在する。この中で、ヒトを宿主としてヒトに感染する主な亜型は、Ｈ１～３、Ｎ１～２である。インフルエンザＡ型ウイルスはヒトだけでなく、多くの哺乳動物および鳥類にも感染する。インフルエンザＡ型ウイルスが鳥類に感染して起きる鳥類の感染症が「鳥インフルエンザ」であり、その原因となるインフルエンザウイルスには、Ｈ１～１６、Ｎ１～９のすべての亜型が確認されている。これらのインフルエンザウイルスの多くは鳥類に対して重篤な症状を示さないが、Ｈ５、Ｈ７およびＨ９亜型のインフルエンザウイルスの中には、鳥類に感染した場合に重篤な症状を示すものもある。これらは高病原性鳥インフルエンザウイルスと呼ばれるものであり、ヒトへの感染の可能性が危惧されている。
Influenza viruses are classified into A type, B type and C type according to the difference in antigenicity between nucleoprotein (NP) and membrane protein (M). Among them, influenza A viruses are further classified into 16 types of HA (H1-16) and NA of 9 types (N1-9), depending on the amino acid sequence or antigenicity of hemagglutinin (HA) and neuraminidase (NA). There are as many subtypes of that combination. Among these, the main subtypes that infect humans using humans as hosts are H1-3 and N1-2. Influenza A virus infects not only humans but many mammals and birds. An avian infectious disease caused by an influenza A virus infecting birds is “bird flu”, and all subtypes of H1-16 and N1-9 have been confirmed as influenza virus causing the infection. Many of these influenza viruses do not show serious symptoms to birds, but some H5, H7 and H9 subtypes of influenza viruses show severe symptoms when infected with birds. . These are called highly pathogenic avian influenza viruses, and there is concern about the possibility of human infection.
インフルエンザウイルスの検出および測定、インフルエンザウイルス感染疾患の予防または治療には抗体が有効である。これまでに、Ｈ５亜型インフルエンザウイルスに対して中和活性を有する種々のモノクローナル抗体が報告されている（例えば、非特許文献１～４）。しかし、未だＨ５亜型インフルエンザウイルス感染疾患の予防または治療に実用化されているものはなく、Ｈ５亜型インフルエンザウイルスに対して高い中和活性を有する新たな抗体の開発が望まれている。
Antibodies are effective for detection and measurement of influenza virus and prevention or treatment of influenza virus-infected diseases. So far, various monoclonal antibodies having neutralizing activity against H5 subtype influenza virus have been reported (for example, Non-Patent Documents 1 to 4). However, nothing has been put into practical use for the prevention or treatment of H5 subtype influenza virus-infected diseases, and the development of a new antibody having high neutralizing activity against H5 subtype influenza virus is desired.
本発明は、Ｈ５亜型インフルエンザウイルスに対して中和活性を有する新規な抗体、および当該抗体を有効成分とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬を提供することを目的とする。
An object of the present invention is to provide a novel antibody having neutralizing activity against H5 subtype influenza virus, and a preventive or therapeutic agent for H5 subtype influenza virus infection disease containing the antibody as an active ingredient.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する抗体であって、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識することを特徴とする抗体。
［２］以下の（ａ）および（ｂ）を含むことを特徴とする前記［１］に記載の抗体。
（ａ）配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域
（ｂ）配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域
［３］配列番号２または４で示されるアミノ酸配列からなる重鎖可変領域と、配列番号３または５で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする前記［２］に記載の抗体。
［４］配列番号６または８で示されるアミノ酸配列からなる重鎖可変領域と、配列番号７または９で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする前記［２］に記載の抗体。
［５］ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）によって産生されることを特徴とする前記［３］に記載の抗体。
［６］ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）。
［７］前記［１］～［５］のいずれかに記載の抗体の機能的断片。
［８］前記［１］～［５］のいずれかに記載の抗体の重鎖可変領域をコードするポリヌクレオチド。
［９］前記［１］～［５］のいずれかに記載の抗体の軽鎖可変領域をコードするポリヌクレオチド。
［１０］前記［８］および／または［９］に記載のポリヌクレオチドを含む発現ベクター。
［１１］前記［１０］に記載の発現ベクターが導入された細胞。
［１２］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を被験試料に接触させる工程を包含することを特徴とするＨ５亜型インフルエンザウイルスの検出方法。
［１３］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を含むことを特徴とするＨ５亜型インフルエンザウイルスの検出キット。
［１４］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を有効成分として含むことを特徴とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬。
［１５］動物に対して、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片の有効量を投与する工程を含むことを特徴とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療方法。
［１６］Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療薬を製造するための、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片の使用。
［１７］Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療に使用するための、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片。 The present invention includes the following inventions in order to solve the above problems.
[1] An antibody that specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus, which is located at position 177 of hemagglutinin consisting of the amino acid sequence represented by SEQ ID NO: 1. An antibody characterized by recognizing an epitope containing lysine and lysine at position 205.
[2] The antibody according to [1] above, comprising the following (a) and (b):
(A) Heavy chain variable comprising CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and CDR3 consisting of positions 98 to 109 of the amino acid sequence shown in SEQ ID NO: 2 or 4 Region (b) a light chain comprising CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55 and CDR3 consisting of positions 88 to 96 of the amino acid sequence shown in SEQ ID NO: 3 or 5 Chain variable region [3] comprising the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or 4 and the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 or 5 2].
[4] In the above [2], comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 or 8, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 or 9 The antibody described.
[5] The antibody according to [3] above, which is produced by hybridoma 36-1 (Accession No. NITE BP-832) or hybridoma 61-2-1 (Accession No. NITE BP-833).
[6] Hybridoma 36-1 (Accession number NITE BP-832) or Hybridoma 61-2-1 (Accession number NITE BP-833).
[7] A functional fragment of the antibody according to any one of [1] to [5].
[8] A polynucleotide encoding the heavy chain variable region of the antibody according to any one of [1] to [5].
[9] A polynucleotide encoding the light chain variable region of the antibody according to any one of [1] to [5].
[10] An expression vector comprising the polynucleotide according to [8] and / or [9].
[11] A cell into which the expression vector according to [10] is introduced.
[12] Detection of H5 subtype influenza virus comprising the step of bringing the antibody according to any one of [1] to [5] above or the functional fragment according toclaim 7 into contact with a test sample Method.
[13] A detection kit for H5 subtype influenza virus comprising the antibody according to any one of [1] to [5] or the functional fragment according toclaim 7.
[14] A preventive or therapeutic agent for H5 subtype influenza virus infection disease comprising the antibody according to any one of [1] to [5] or the functional fragment according toclaim 7 as an active ingredient .
[15] An H5 subtype comprising a step of administering an effective amount of the antibody according to any one of [1] to [5] or the functional fragment according toclaim 7 to an animal. A method for preventing or treating influenza virus-infected diseases.
[16] Use of the antibody according to any one of [1] to [5] above or the functional fragment according toclaim 7 for producing a prophylactic or therapeutic agent for H5 subtype influenza virus infection disease.
[17] The antibody according to any one of [1] to [5] or the functional fragment according toclaim 7, for use in the prophylaxis or treatment of H5 subtype influenza virus-infected diseases.
［１］Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する抗体であって、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識することを特徴とする抗体。
［２］以下の（ａ）および（ｂ）を含むことを特徴とする前記［１］に記載の抗体。
（ａ）配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域
（ｂ）配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域
［３］配列番号２または４で示されるアミノ酸配列からなる重鎖可変領域と、配列番号３または５で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする前記［２］に記載の抗体。
［４］配列番号６または８で示されるアミノ酸配列からなる重鎖可変領域と、配列番号７または９で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする前記［２］に記載の抗体。
［５］ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）によって産生されることを特徴とする前記［３］に記載の抗体。
［６］ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）。
［７］前記［１］～［５］のいずれかに記載の抗体の機能的断片。
［８］前記［１］～［５］のいずれかに記載の抗体の重鎖可変領域をコードするポリヌクレオチド。
［９］前記［１］～［５］のいずれかに記載の抗体の軽鎖可変領域をコードするポリヌクレオチド。
［１０］前記［８］および／または［９］に記載のポリヌクレオチドを含む発現ベクター。
［１１］前記［１０］に記載の発現ベクターが導入された細胞。
［１２］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を被験試料に接触させる工程を包含することを特徴とするＨ５亜型インフルエンザウイルスの検出方法。
［１３］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を含むことを特徴とするＨ５亜型インフルエンザウイルスの検出キット。
［１４］前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片を有効成分として含むことを特徴とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬。
［１５］動物に対して、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片の有効量を投与する工程を含むことを特徴とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療方法。
［１６］Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療薬を製造するための、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片の使用。
［１７］Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療に使用するための、前記［１］～［５］のいずれかに記載の抗体または請求項７に記載の機能的断片。 The present invention includes the following inventions in order to solve the above problems.
[1] An antibody that specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus, which is located at position 177 of hemagglutinin consisting of the amino acid sequence represented by SEQ ID NO: 1. An antibody characterized by recognizing an epitope containing lysine and lysine at position 205.
[2] The antibody according to [1] above, comprising the following (a) and (b):
(A) Heavy chain variable comprising CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and CDR3 consisting of positions 98 to 109 of the amino acid sequence shown in SEQ ID NO: 2 or 4 Region (b) a light chain comprising CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55 and CDR3 consisting of positions 88 to 96 of the amino acid sequence shown in SEQ ID NO: 3 or 5 Chain variable region [3] comprising the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or 4 and the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 or 5 2].
[4] In the above [2], comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 or 8, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 or 9 The antibody described.
[5] The antibody according to [3] above, which is produced by hybridoma 36-1 (Accession No. NITE BP-832) or hybridoma 61-2-1 (Accession No. NITE BP-833).
[6] Hybridoma 36-1 (Accession number NITE BP-832) or Hybridoma 61-2-1 (Accession number NITE BP-833).
[7] A functional fragment of the antibody according to any one of [1] to [5].
[8] A polynucleotide encoding the heavy chain variable region of the antibody according to any one of [1] to [5].
[9] A polynucleotide encoding the light chain variable region of the antibody according to any one of [1] to [5].
[10] An expression vector comprising the polynucleotide according to [8] and / or [9].
[11] A cell into which the expression vector according to [10] is introduced.
[12] Detection of H5 subtype influenza virus comprising the step of bringing the antibody according to any one of [1] to [5] above or the functional fragment according to
[13] A detection kit for H5 subtype influenza virus comprising the antibody according to any one of [1] to [5] or the functional fragment according to
[14] A preventive or therapeutic agent for H5 subtype influenza virus infection disease comprising the antibody according to any one of [1] to [5] or the functional fragment according to
[15] An H5 subtype comprising a step of administering an effective amount of the antibody according to any one of [1] to [5] or the functional fragment according to
[16] Use of the antibody according to any one of [1] to [5] above or the functional fragment according to
[17] The antibody according to any one of [1] to [5] or the functional fragment according to
本発明により、Ｈ５亜型インフルエンザウイルスに対して中和活性を有する新規な抗体を提供することができる。当該抗体は、Ｈ５亜型インフルエンザウイルス感染マウスを用いた実験において予防効果および治療効果を有することが確認されており、Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療薬の有効成分として有用である。
According to the present invention, a novel antibody having neutralizing activity against H5 subtype influenza virus can be provided. The antibody has been confirmed to have preventive and therapeutic effects in experiments using H5 subtype influenza virus-infected mice, and is useful as an active ingredient for preventive or therapeutic agents for H5 subtype influenza virus-infected diseases.
〔抗体〕
本発明者らは、インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株を抗原としてマウスを免疫し、定法に従ってハイブリドーマを作製し、得られたハイブリドーマが産生するモノクローナル抗体と各種ウイルスとの反応性、中和活性を評価した結果、Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する２種類のマウスモノクローナル抗体を見出し、この知見に基づいてさらに研究を重ね、本発明を完成させるに至った。 〔antibody〕
The present inventors immunized mice with influenza virus A / Vietnam / 1194/04 (H5N1) as an antigen, prepared hybridomas according to a conventional method, and the reactivity of monoclonal antibodies produced by the obtained hybridomas with various viruses. As a result of evaluating the neutralizing activity, two types of mouse monoclonal antibodies that specifically bind to hemagglutinin of H5 subtype influenza virus and neutralize infectivity of H5 subtype influenza virus were found. Research has been completed and the present invention has been completed.
本発明者らは、インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株を抗原としてマウスを免疫し、定法に従ってハイブリドーマを作製し、得られたハイブリドーマが産生するモノクローナル抗体と各種ウイルスとの反応性、中和活性を評価した結果、Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する２種類のマウスモノクローナル抗体を見出し、この知見に基づいてさらに研究を重ね、本発明を完成させるに至った。 〔antibody〕
The present inventors immunized mice with influenza virus A / Vietnam / 1194/04 (H5N1) as an antigen, prepared hybridomas according to a conventional method, and the reactivity of monoclonal antibodies produced by the obtained hybridomas with various viruses. As a result of evaluating the neutralizing activity, two types of mouse monoclonal antibodies that specifically bind to hemagglutinin of H5 subtype influenza virus and neutralize infectivity of H5 subtype influenza virus were found. Research has been completed and the present invention has been completed.
ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）によって産生されるマウスモノクローナル抗体は、配列番号２で示されるアミノ酸配列からなる重鎖可変領域および配列番号３で示されるアミノ酸配列からなる軽鎖可変領域を有する。ハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）によって産生されるマウスモノクローナル抗体は、配列番号４で示されるアミノ酸配列からなる重鎖可変領域および配列番号５で示されるアミノ酸配列からなる軽鎖可変領域を有する。なお、ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）、ハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）は、それぞれ独立行政法人製品評価技術基盤機構特許微生物寄託センターに国際寄託済みである（受託日：２００９年１０月３０日）。
The mouse monoclonal antibody produced by the hybridoma 36-1 (Accession No. NITE BP-832) is a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3. Have The mouse monoclonal antibody produced by the hybridoma 61-2-1 (Accession No. NITE BP-833) has a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 5. Has a variable region. Hybridoma 36-1 (Accession No. NITE BP-832) and Hybridoma 61-2-1 (Accession No. NITE BP-833) have been internationally deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation. (Contract date: October 30, 2009).
本発明は、Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する抗体を提供する。本発明の抗体は、Ｈ５亜型インフルエンザウイルス一種であるインフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株のヘマグルチニンのアミノ酸配列（配列番号１）における第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識する抗体であればよく、Ａ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株以外のＨ５亜型インフルエンザウイルスのヘマグルチニンのエピトープについては限定されない。インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株のヘマグルチニンのアミノ酸配列における第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識する抗体であることは、第１７７位のリジンおよび第２０５位のリジンのいずれか一方が他のアミノ酸に置換されたヘマグルチニンに対する親和性がなくなることにより確認できる。
The present invention provides an antibody that specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus. The antibody of the present invention comprises a lysine at position 177 and lysine at position 205 in the amino acid sequence (SEQ ID NO: 1) of hemagglutinin of influenza virus A / Vietnam / 1194/04 (H5N1) strain, which is a type of H5 subtype influenza virus. Any epitope-recognizing epitope may be used, and the H5 subtype influenza virus hemagglutinin epitope other than the A / Vietnam / 1194/04 (H5N1) strain is not limited. An antibody that recognizes an epitope containing the lysine at position 177 and the position lysine at position 205 in the amino acid sequence of the hemagglutinin of influenza virus A / Vietnam / 1194/04 (H5N1) strain This can be confirmed by the loss of affinity for hemagglutinin in which either one of the lysines is substituted with another amino acid.
本発明の抗体は、Ｈ５亜型インフルエンザウイルスの感染性を中和することができる。本発明の抗体は、Ｈ５亜型インフルエンザウイルスの感染性を少なくとも５０％中和することが好ましく、少なくとも７０％中和することがより好ましく、少なくとも９０％中和することがさらに好ましい。
The antibody of the present invention can neutralize the infectivity of H5 subtype influenza virus. The antibody of the present invention preferably neutralizes H5 subtype influenza virus infectivity by at least 50%, more preferably at least 70% neutralization, and even more preferably at least 90% neutralization.
本発明の抗体としては、以下の（ａ）および（ｂ）を含む抗体が挙げられる。
（ａ）配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域
（ｂ）配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域 Examples of the antibody of the present invention include antibodies including the following (a) and (b).
(A) Heavy chain variable comprising CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and CDR3 consisting of positions 98 to 109 of the amino acid sequence shown in SEQ ID NO: 2 or 4 Region (b) a light chain comprising CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55 and CDR3 consisting of positions 88 to 96 of the amino acid sequence shown in SEQ ID NO: 3 or 5 Chain variable region
（ａ）配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域
（ｂ）配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域 Examples of the antibody of the present invention include antibodies including the following (a) and (b).
(A) Heavy chain variable comprising CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and CDR3 consisting of positions 98 to 109 of the amino acid sequence shown in SEQ ID NO: 2 or 4 Region (b) a light chain comprising CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55 and CDR3 consisting of positions 88 to 96 of the amino acid sequence shown in SEQ ID NO: 3 or 5 Chain variable region
本発明の抗体としては、ヒト型キメラ抗体またはヒト化抗体が好ましい。ヒト型キメラ抗体は、ヒト以外の動物由来の抗体の重鎖可変領域および軽鎖可変領域と、ヒト抗体の重鎖定常領域および軽鎖定常領域からなる抗体をいう。ヒト化抗体は、ヒト以外の動物由来の抗体のＣＤＲ（相補性決定領域：complementarity determining region）をヒト抗体のＣＤＲへ移植したものをいい、ＣＤＲ移植抗体、再構成抗体などとも称される。ヒト化抗体のＦＲ（フレームワーク領域：framework region）は、ＣＤＲが良好な抗原結合部位を形成するものが選択される。必要に応じ、ヒト化抗体のＣＤＲが適切な抗原結合部位を形成するように、抗体の可変領域におけるＦＲのアミノ酸配列を置換してもよい。
The antibody of the present invention is preferably a human chimeric antibody or a humanized antibody. A human chimeric antibody refers to an antibody comprising a heavy chain variable region and a light chain variable region of an antibody derived from a non-human animal, and a heavy chain constant region and a light chain constant region of a human antibody. A humanized antibody is obtained by grafting a CDR (complementarity determining region) of an antibody derived from a non-human animal into a CDR of a human antibody, and is also referred to as a CDR-grafted antibody, a reconstituted antibody, or the like. The FR (framework region) of the humanized antibody is selected so that CDR forms a favorable antigen-binding site. If necessary, the amino acid sequence of the FR in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen-binding site.
キメラ抗体およびヒト化抗体の定常領域には、ヒト抗体の定常領域が使用され、例えば重鎖では、Ｃγ１、Ｃγ２、Ｃγ３、Ｃγ４を、軽鎖ではＣκ、Ｃλを使用することができる。また、抗体またはその産生の安定性を改善するために、ヒト抗体定常領域を修飾してもよい。ヒト化の際に用いられるヒト抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、ＩｇＤなどいずれのアイソタイプのヒト抗体でもよいが、ＩｇＧを用いることが好ましく、さらにＩｇＧ１またはＩｇＧ３が好ましく、特にＩｇＧ１が好ましい。ヒト抗体の定常領域のアミノ酸配列は、公知のデータベース（Ｐｒｏｔｅｉｎ Ｄａｔａ Ｂａｎｋ等）から取得することができる。
The constant regions of human antibodies are used as the constant regions of chimeric antibodies and humanized antibodies. For example, Cγ1, Cγ2, Cγ3, Cγ4 can be used for the heavy chain, and Cκ, Cλ can be used for the light chain. In addition, human antibody constant regions may be modified to improve the stability of the antibody or its production. Human antibodies used for humanization may be human antibodies of any isotype such as IgG, IgM, IgA, IgE, IgD, but IgG is preferred, IgG1 or IgG3 is more preferred, and IgG1 is particularly preferred. The amino acid sequence of the constant region of a human antibody can be obtained from a known database (Protein Data Bank etc.).
本発明のヒト型キメラ抗体としては、配列番号２または４で示されるアミノ酸配列からなる重鎖可変領域と、配列番号３または５で示されるアミノ酸配列からなる軽鎖可変領域と、ヒト抗体の重鎖定常領域および軽鎖定常領域からなる抗体が好ましい。また、本発明のヒト化抗体としては、配列番号６または８で示されるアミノ酸配列からなる重鎖可変領域と、配列番号７または９で示されるアミノ酸配列からなる軽鎖可変領域と、ヒト抗体の重鎖定常領域および軽鎖定常領域からなる抗体が好ましい。
The human chimeric antibody of the present invention includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or 4, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 or 5, and a human antibody heavy chain. Antibodies consisting of a chain constant region and a light chain constant region are preferred. The humanized antibody of the present invention includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 or 8, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 or 9, and a human antibody. An antibody consisting of a heavy chain constant region and a light chain constant region is preferred.
また、配列番号２、４、６または８で示されるアミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されたアミノ酸配列からなる重鎖可変領域を有する抗体、ならびに、配列番号３、５、７または９で示されるアミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されたアミノ酸配列からなる軽鎖可変領域を有する抗体も、Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和し、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識する限り、本発明の抗体として好適である。「１もしくは数個のアミノ酸が欠失、置換もしくは付加された」とは、部位特異的変異法等の公知の変異ペプチド作製法により欠失、置換もしくは付加できる程度の数（好ましくは１０個以下、より好ましくは７個以下、さらに好ましくは５個以下）のアミノ酸が欠失、置換もしくは付加されることを意味する。
In addition, an antibody having a heavy chain variable region consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, and SEQ ID NO: 3, An antibody having a light chain variable region consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by 5, 7 or 9 is also specific to hemagglutinin of H5 subtype influenza virus. As long as it binds, neutralizes the infectivity of H5 subtype influenza virus, and recognizes an epitope containing lysine at position 177 and lysine at position 205 of hemagglutinin consisting of the amino acid sequence represented by SEQ ID NO: 1, Suitable as an antibody. “One or several amino acids have been deleted, substituted or added” means that the number can be deleted, substituted or added by a known mutant peptide production method such as site-directed mutagenesis (preferably 10 or less). , More preferably 7 or less, and even more preferably 5 or less) amino acids are deleted, substituted or added.
本発明には、上記本発明の抗体の機能的断片が含まれる。本発明における「機能的断片」は、上記本発明の抗体の一部分（部分断片）であって、Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和し、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識する機能を維持しているものであればいかなるものでもよい。具体的には、例えば、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆｖ、ｓｃＦｖなどが挙げられる。
The present invention includes a functional fragment of the antibody of the present invention. The “functional fragment” in the present invention is a part (partial fragment) of the antibody of the present invention, which specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus. Any hemagglutinin consisting of the amino acid sequence shown in SEQ ID NO: 1 may be used as long as it retains the function of recognizing an epitope containing lysine at position 177 and lysine at position 205. Specific examples include Fab, F (ab ′) 2 , Fab ′, Fv, scFv, and the like.
本発明の抗体は、公知の方法により製造することができる。例えば、ハイブリドーマが産生するモノクローナル抗体、または、遺伝子組換え技術を用いて産生させる組換え抗体として製造することができる。モノクローナル抗体産生ハイブリドーマは、例えば、Ｈ５亜型インフルエンザウイルスのヘマグルチニンを感作抗原として通常の免疫方法に従って哺乳動物を免疫し、得られる免疫細胞を通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により、目的のモノクローナル抗体産生ハイブリドーマを取得することができる。
The antibody of the present invention can be produced by a known method. For example, it can be produced as a monoclonal antibody produced by a hybridoma or a recombinant antibody produced using a gene recombination technique. The monoclonal antibody-producing hybridoma immunizes a mammal according to a normal immunization method using, for example, H5 subtype influenza virus hemagglutinin as a sensitizing antigen, and fuses the obtained immune cell with a known parent cell by a normal cell fusion method. The target monoclonal antibody-producing hybridoma can be obtained by a normal screening method.
抗体取得の感作抗原としては、配列番号１で示されるアミノ酸配列からなるインフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株のヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むタンパク質を用いればよい。具体的には、インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの全長、または、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含む部分断片を用いることができる。当該ヘマグルチニンまたはその部分断片は、公知の遺伝子組換え技術を用いて組換えタンパク質として作製することができる。また、部分断片は化学合成により作製することも可能である。
As a sensitizing antigen for obtaining an antibody, a protein containing lysine at position 177 and lysine at position 205 of hemagglutinin of the influenza virus A / Vietnam / 1194/04 (H5N1) strain consisting of the amino acid sequence represented by SEQ ID NO: 1 Use it. Specifically, the influenza virus A / Vietnam / 1194/04 (H5N1) strain, the full length of hemagglutinin consisting of the amino acid sequence represented by SEQ ID NO: 1, or the 177th position of hemagglutinin consisting of the amino acid sequence represented by SEQ ID NO: 1 And a partial fragment containing lysine at position 205 can be used. The hemagglutinin or a partial fragment thereof can be produced as a recombinant protein using a known gene recombination technique. Partial fragments can also be produced by chemical synthesis.
感作抗原で免疫される哺乳動物は、特に限定されるものではないが、細胞融合に使用する親細胞との適合性を考慮して選択するのが好ましく、例えば、マウス、ラット、ハムスター等のげっ歯類、ウサギ、サル等が使用される。抗原と、フロイントの完全もしくは不完全アジュバント、またはカリミョウバンのような助剤とを混合し、免疫原として動物に免疫する。免疫原の投与方法は、皮下注射、腹腔内注射、静脈内注射、皮内注射、筋肉内注射、足蹠注射などから適宜選択される。免疫は、一回または適当な間隔で（好ましくは３日間から１週間間隔で）複数回繰返し行なうことができる。その後、免疫した動物の血清中の抗原に対する抗体価を測定し、抗体価が十分高くなった動物を抗体産生細胞の供給源として用いれば、以後の操作の効果を高めることができる。一般的には、最終免疫後３～５日後の動物由来の抗体産生細胞を、後の細胞融合に用いることが好ましい。抗体価の測定法としては、放射性同位元素免疫定量法（ＲＩＡ法）、固相酵素免疫定量法（ＥＬＩＳＡ法）、蛍光抗体法、受身血球凝集反応法など種々の公知技術を用いることができる。
The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. For example, mouse, rat, hamster, etc. Rodents, rabbits, monkeys, etc. are used. The antigen is mixed with Freund's complete or incomplete adjuvant, or an adjuvant such as potassium alum, and the animal is immunized as an immunogen. The administration method of the immunogen is appropriately selected from subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, footpad injection and the like. Immunization can be repeated once or multiple times at appropriate intervals (preferably at intervals of 3 days to 1 week). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and if the animal having a sufficiently high antibody titer is used as a source of antibody-producing cells, the effect of subsequent operations can be enhanced. In general, it is preferable to use animal-derived antibody-producing cells 3 to 5 days after the final immunization for subsequent cell fusion. As a method for measuring the antibody titer, various known techniques such as radioisotope immunoassay (RIA method), solid-phase enzyme immunoassay (ELISA method), fluorescent antibody method, passive hemagglutination reaction method and the like can be used.
免疫された哺乳動物から得られる抗体産生細胞と融合する親細胞には、通常哺乳動物のミエローマ細胞が用いられる。ミエローマ細胞としては、公知の細胞株を好適に用いることができるが、一般的にはマウスから得られた株化細胞、例えば８－アザグアニン耐性マウスミエローマ株Ｐ３Ｘ６３Ａｇ８Ｕ.１（Ｐ３－Ｕ１）（Yelton, D.E. et al., Current Topics in Microbiology and Immunology, 81, 1-7（1978））、Ｐ３／ＮＳＩ／１－Ａｇ４－１（ＮＳ－１）（Kohler, G. et al., European J. Immunology, 6, 511-519 （1976））、Ｓｐ２／Ｏ－Ａｇ１４（ＳＰ－２）（Shulman, M. et al., Nature, 276, 269-270（1978））、Ｐ３Ｘ６３Ａｇ８．６５３（６５３）（Kearney, J. F. et al., J. Immunology, 123, 1548-1550（1979））、Ｐ３Ｘ６３Ａｇ８（Ｘ６３）（Horibata, K. and Harris, A. W. Nature, 256, 495-497 （1975））などを用いることが好ましい。
Mammalian myeloma cells are usually used as parent cells to be fused with antibody-producing cells obtained from immunized mammals. As the myeloma cell, a known cell line can be preferably used. Generally, a cell line obtained from a mouse, for example, 8-azaguanine-resistant mouse myeloma line P3X63Ag8U.1 (P3-U1) (Yelton, DE et al., Current Topics in Microbiology and Immunology, 81, 1-7 (1978)), P3 / NSI / 1-Ag4-1 (NS-1) (Kohler, G. et al., European J. Immunology, 6, 511-519 (1976)), Sp2 / O-Ag14 (SP-2) (Shulman, M. et al., Nature, 276, 269-270 (1978)), P3X63Ag8.653 (653) (Kearney, J. F. et al., J. Immunology, 123, 1548-1550 (1979)), P3X63Ag8 (X63) (Horibata, K. and Harris, A. W. Nature, 256, 495-497 (1975)) Is preferably used.
抗体産生細胞は、形質細胞、およびその前駆細胞であるリンパ球であり、これは個体のいずれの部位から得てもよく、一般には脾、リンパ節、骨髄、扁桃、末梢血、またはこれらを適宜組み合わせたもの等から得ることができるが、脾細胞が最も一般的に用いられる。最終免疫後、所定の抗体価が得られたマウスから抗体産生細胞が存在する部位、例えば脾臓を摘出し、抗体産生細胞である脾細胞を調製する。この脾細胞とミエローマ細胞とを融合させる手段として現在最も一般的に行われているのは、ポリエチレングリコールを用いる方法である。この方法は、例えば以下の手順で行うことができる。
Antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any part of the individual, generally spleen, lymph nodes, bone marrow, tonsils, peripheral blood, or these as appropriate. Spleen cells are most commonly used, although they can be obtained from a combination or the like. After the final immunization, a site where antibody-producing cells are present, such as the spleen, is removed from a mouse having a predetermined antibody titer, and spleen cells that are antibody-producing cells are prepared. The most commonly used method for fusing these spleen cells and myeloma cells is a method using polyethylene glycol. This method can be performed, for example, by the following procedure.
脾細胞とミエローマとを無血清培地（例えばＤＭＥＭ）、またはＰＢＳでよく洗浄し、脾細胞とミエローマの細胞数の比が５：１～１０：１程度になるように混合し、遠心分離する。上清を除去し、沈澱した細胞群をよくほぐした後、撹拌しながら１ｍｌの５０％（ｗ／ｖ）ポリエチレングリコール（分子量１０００～４０００）を含む無血清培地を滴下する。その後、１０ｍｌの無血清培地をゆっくりと加えた後遠心分離する。再び上清を捨て、沈澱した細胞を適量のヒポキサンチン・アミノプテリン・チミジン（ＨＡＴ）液およびヒトインターロイキン－２（ＩＬ－２）を含む正常培地（ＨＡＴ培地）中に懸濁して培養用プレートの各ウェルに分注し、５％炭酸ガス存在下、３７℃で２週間程度培養する。途中適宜ＨＡＴ培地を補う。
Spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, DMEM) or PBS, mixed so that the ratio of the number of spleen cells to myeloma is about 5: 1 to 10: 1, and centrifuged. After removing the supernatant and loosening the precipitated cells, a serum-free medium containing 1 ml of 50% (w / v) polyethylene glycol (molecular weight 1000 to 4000) is added dropwise with stirring. Thereafter, 10 ml of serum-free medium is slowly added and then centrifuged. The supernatant was discarded again, and the precipitated cells were suspended in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (HAT) solution and human interleukin-2 (IL-2). Dispense into each well and incubate at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide gas. HAT medium is supplemented as needed.
ミエローマ細胞が８－アザグアニン耐性株、すなわちヒポキサンチン・グアニン・ホスホリボシルトランスフェラーゼ（ＨＧＰＲＴ）欠損株である場合、融合しなかったミエローマ細胞およびミエローマ細胞どうしの融合細胞は、ＨＡＴ培地中では生存できない。また、融合しなかった抗体産生細胞および抗体産生細胞同士の融合細胞は増殖できない。したがって、ＨＡＴ培地での培養を続けることによって、抗体産生細胞とミエローマ細胞とのハイブリドーマのみが増殖し、結果的にハイブリドーマを選択することができる。コロニー状に生育してきたハイブリドーマについて、培地をＨＡＴ培地からアミノプテリンを除いた培地（ＨＴ培地）に変更する。以後、培養上清の一部を採取し、例えば、ＥＬＩＳＡ法によりＨ５亜型インフルエンザウイルスのヘマグルチニンに対する抗体価を測定する。
When the myeloma cells are 8-azaguanine resistant strains, that is, hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient strains, the fused myeloma cells and the fused cells of the myeloma cells cannot survive in the HAT medium. In addition, non-fused antibody-producing cells and fused cells between antibody-producing cells cannot proliferate. Therefore, by continuing the culture in the HAT medium, only the hybridoma of the antibody producing cell and the myeloma cell grows, and as a result, the hybridoma can be selected. About the hybridoma which grew to colony form, a culture medium is changed into the culture medium (HT culture medium) remove | excluding aminopterin from the HAT culture medium. Thereafter, a part of the culture supernatant is collected, and the antibody titer against hemagglutinin of H5 subtype influenza virus is measured, for example, by ELISA.
抗体価測定により、Ｈ５亜型インフルエンザウイルスのヘマグルチニンに対する特異的抗体を産生することが確認されたハイブリドーマを別のプレートに移しクローニングを行う。クローニング法としては、限界希釈法、軟寒天法、マイクロマニュピレーターによる方法、セルソーターによる方法などが挙げられるが、限界希釈法が簡便であり、よく用いられる。例えば限界希釈法によるクローニングを２～４回繰返し、安定して抗体価の認められたハイブリドーマを選択し、産生されるモノクローナル抗体の中和活性およびエピトープを確認する。
The hybridoma confirmed to produce a specific antibody against hemagglutinin of H5 subtype influenza virus by antibody titer measurement is transferred to another plate for cloning. Examples of the cloning method include a limiting dilution method, a soft agar method, a method using a micromanipulator, a method using a cell sorter, etc., but the limiting dilution method is simple and often used. For example, cloning by limiting dilution is repeated 2 to 4 times, hybridomas with stable antibody titers are selected, and the neutralizing activity and epitope of the produced monoclonal antibody are confirmed.
中和活性は、例えば後述する実施例５と同様の方法で確認することができる。すなわち、選択したハイブリドーマにより産生された抗体とＨ５亜型インフルエンザウイルスとを混合してインキュベーションし、コンフルエントな状態に培養したＭＤＣＫ細胞に当該混合液を添加してウイルスを感染させ、寒天培地を入れて固め、２日間培養した後プラークの数を計測する。Ｈ５亜型インフルエンザウイルスのみを感染させた場合よりプラーク数が減少していれば、当該抗体は中和活性を有すると判定できる。
The neutralizing activity can be confirmed by, for example, the same method as in Example 5 described later. That is, the antibody produced by the selected hybridoma and H5 subtype influenza virus are mixed and incubated, the mixture is added to MDCK cells cultured in a confluent state, the virus is infected, and an agar medium is added. After hardening and culturing for 2 days, the number of plaques is counted. If the number of plaques is reduced as compared with the case where only H5 subtype influenza virus is infected, it can be determined that the antibody has neutralizing activity.
エピトープの同定は、例えば後述する実施例７と同様の方法で確認することができる。すなわち、ウイルスと抗体とを反応させた後に、適当な細胞にウイルスを感染させてエスケープミュータント株を単離し、単離したウイルス株のヘマグルチニン遺伝子をシークエンス解析した結果から、変異が生じたアミノ酸を同定することでエピトープを同定することができる。
Epitope identification can be confirmed, for example, by the same method as in Example 7 described later. That is, after reacting the virus with the antibody, infect appropriate cells with the virus to isolate the escape mutant strain, and the sequence analysis of the hemagglutinin gene of the isolated virus strain identifies the amino acid with the mutation. By doing so, the epitope can be identified.
本発明の抗体を産生するハイブリドーマが選択できれば、培地を正常培地に換えて培養する。大量培養は、大型培養瓶を用いた回転培養またはスピナー培養で行うことができる。大量培養により得られる上清を、ゲル濾過カラム等、当業者に周知の方法を用いて精製することにより、本発明の抗体を得ることができる。また、同系統のマウスもしくはＮｕ／Ｎｕマウス、ラット、モルモット、ハムスターまたはウサギ等の腹腔内で当該ハイブリドーマを増殖させることにより、本発明の抗体を大量含む腹水を得ることができ、これを精製して本発明の抗体を得ることができる。
If the hybridoma producing the antibody of the present invention can be selected, the medium is changed to a normal medium and cultured. Mass culture can be performed by rotary culture or spinner culture using a large culture bottle. The antibody of the present invention can be obtained by purifying the supernatant obtained by mass culture using a method well known to those skilled in the art, such as a gel filtration column. In addition, ascites containing a large amount of the antibody of the present invention can be obtained by growing the hybridoma in the peritoneal cavity of the same strain of mouse or Nu / Nu mouse, rat, guinea pig, hamster or rabbit. Thus, the antibody of the present invention can be obtained.
本発明の抗体を産生するハイブリドーマから抗体遺伝子をクローニングし、適当なベクターに組み込んで、このベクターを宿主に導入して組換え抗体を製造することも可能である（例えば、Vandamme, A. M. et al., Eur. J. Biochem.（1990）192, 767-775, 1990参照）。本発明の抗体のうち、ヒト型キメラ抗体およびヒト化抗体は、組換え抗体として製造することができる。
It is also possible to clone an antibody gene from a hybridoma producing the antibody of the present invention, incorporate it into an appropriate vector, and introduce this vector into a host to produce a recombinant antibody (for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990). Of the antibodies of the present invention, human chimeric antibodies and humanized antibodies can be produced as recombinant antibodies.
ヒト型キメラ抗体の重鎖をコードする遺伝子は、ハイブリドーマからクローニングした抗体遺伝子の重鎖可変領域をコードする部分をヒト抗体の重鎖定常領域をコードする遺伝子と結合して作製することができる。同様に、ヒト型キメラ抗体の軽鎖をコードする遺伝子は、ハイブリドーマからクローニングした抗体遺伝子の軽鎖可変領域をコードする部分をヒト抗体の軽鎖定常領域をコードする遺伝子と結合して作製することができる。
The gene encoding the heavy chain of the human chimeric antibody can be prepared by combining the portion encoding the heavy chain variable region of the antibody gene cloned from the hybridoma with the gene encoding the heavy chain constant region of the human antibody. Similarly, the gene encoding the light chain of the human chimeric antibody is prepared by combining the portion encoding the light chain variable region of the antibody gene cloned from the hybridoma with the gene encoding the light chain constant region of the human antibody. Can do.
ヒト化抗体の重鎖をコードする遺伝子は、ハイブリドーマからクローニングした抗体遺伝子の重鎖可変領域のＣＤＲ１、２および３をコードする部分をヒト抗体の重鎖をコードする遺伝子の対応するＣＤＲ部分と置換することで作製することができる。同様に、ヒト化抗体の軽鎖をコードする遺伝子は、ハイブリドーマからクローニングした抗体遺伝子の軽鎖可変領域のＣＤＲ１、２および３をコードする部分をヒト抗体の軽鎖をコードする遺伝子の対応するＣＤＲ部分と置換することで作製することができる。
In the gene encoding the heavy chain of the humanized antibody, the CDR coding portion of the heavy chain variable region of the antibody gene cloned from the hybridoma is replaced with the corresponding CDR portion of the gene encoding the heavy chain of the human antibody. It can produce by doing. Similarly, the gene encoding the light chain of the humanized antibody is the same as the CDR of the gene encoding the light chain of the human antibody. It can be produced by replacing the portion.
得られた重鎖をコードする遺伝子と軽鎖をコードする遺伝子を適切なベクターに挿入し、本発明の抗体を発現するための組換え発現ベクターを構築する。重鎖をコードする遺伝子と軽鎖をコードする遺伝子は、１つのベクターに組み込んでもよく、別々のベクターに組み込んでもよい。次に、得られたベクターを宿主細胞に導入し形質転換体を作製する。重鎖をコードする遺伝子と軽鎖をコードする遺伝子を別々のベクターに組み込んだ場合には、２つのベクターを宿主細胞に共導入する。ベクターおよび宿主細胞の詳細については後述する。
The obtained gene encoding the heavy chain and the gene encoding the light chain are inserted into an appropriate vector, and a recombinant expression vector for expressing the antibody of the present invention is constructed. The gene encoding the heavy chain and the gene encoding the light chain may be incorporated into one vector or into separate vectors. Next, the obtained vector is introduced into a host cell to produce a transformant. When the gene encoding the heavy chain and the gene encoding the light chain are incorporated into separate vectors, the two vectors are co-introduced into the host cell. Details of the vector and the host cell will be described later.
ベクターが導入された宿主細胞（形質転換体）を培養し、その培養物から本発明の抗体を採取することができる。培養物としては、培養上清、培養細胞もしくは培養菌体またはその破砕物、形質転換体の分泌物等が挙げられる。培地および培養条件は、使用する宿主細胞に応じて適宜選択する。培養後、目的タンパク質が菌体内または細胞内に生産される場合には、菌体または細胞を破砕することにより抗体を採取する。また、目的抗体が菌体外または細胞外に生産される場合には、培養液をそのまま使用するか、遠心分離等により菌体または細胞を除去する。その後、タンパク質の単離精製に用いられる各種クロマトグラフィーを用いた一般的な生化学的方法を適宜選択して用いることにより、培養物中から目的の抗体を単離精製することができる。
The host cell (transformant) introduced with the vector can be cultured, and the antibody of the present invention can be collected from the culture. Examples of the culture include culture supernatant, cultured cells or cultured cells or disrupted products thereof, and secretions of transformants. The medium and culture conditions are appropriately selected according to the host cell used. When the target protein is produced in cells or cells after culturing, antibodies are collected by disrupting the cells or cells. When the target antibody is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, the target antibody can be isolated and purified from the culture by appropriately selecting and using a general biochemical method using various chromatographies used for protein isolation and purification.
〔ポリヌクレオチド〕
本発明は、本発明の抗体の重鎖可変領域をコードするポリヌクレオチドおよび軽鎖可変領域をコードするポリヌクレオチドを提供する。本発明のポリヌクレオチドは、本発明の抗体を組換え抗体として製造する際に有用である。また、本発明の抗体と同等の機能を有する抗体をスクリーニングするために、プローブとして用いることもできる。 [Polynucleotide]
The present invention provides a polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region of the antibody of the present invention. The polynucleotide of the present invention is useful when the antibody of the present invention is produced as a recombinant antibody. Moreover, it can also be used as a probe for screening an antibody having a function equivalent to that of the antibody of the present invention.
本発明は、本発明の抗体の重鎖可変領域をコードするポリヌクレオチドおよび軽鎖可変領域をコードするポリヌクレオチドを提供する。本発明のポリヌクレオチドは、本発明の抗体を組換え抗体として製造する際に有用である。また、本発明の抗体と同等の機能を有する抗体をスクリーニングするために、プローブとして用いることもできる。 [Polynucleotide]
The present invention provides a polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region of the antibody of the present invention. The polynucleotide of the present invention is useful when the antibody of the present invention is produced as a recombinant antibody. Moreover, it can also be used as a probe for screening an antibody having a function equivalent to that of the antibody of the present invention.
本明細書において「ポリヌクレオチド」は、「遺伝子」、「核酸」または「核酸分子」と交換可能に使用される。本発明のポリヌクレオチドは、ＲＮＡ（例えば、ｍＲＮＡ）の形態、またはＤＮＡの形態（例えば、ｃＤＮＡまたはゲノムＤＮＡ）で存在することができる。ＤＮＡは、二本鎖でもよく一本鎖でもよい。一本鎖ＤＮＡまたはＲＮＡは、コード鎖（センス鎖）、または、非コード鎖（アンチセンス鎖）のいずれであってもよい。
In the present specification, “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule”. The polynucleotides of the present invention can exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). The DNA may be double stranded or single stranded. The single-stranded DNA or RNA may be either a coding strand (sense strand) or a non-coding strand (antisense strand).
本発明の重鎖可変領域をコードするポリヌクレオチドとしては、配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域をコードするポリヌクレオチドが好ましく、より好ましくは、配列番号２、４、６または８で示されるアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチドである。
The polynucleotide encoding the heavy chain variable region of the present invention includes CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and positions 98 of the amino acid sequence shown in SEQ ID NO: 2 or 4. A polynucleotide encoding a heavy chain variable region comprising CDR3 consisting of position 109, more preferably a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 It is.
配列番号２で示されるアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチドとしては、配列番号１０で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号４で示されるアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチドとしては、配列番号１２で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号６で示されるアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチドとしては、配列番号１４で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号８で示されるアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチドとしては、配列番号１６で示される塩基配列からなるポリヌクレオチドが挙げられるが、いずれも限定されるものではない。
Examples of the polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 10, and the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 4. Examples of the polynucleotide encoding the variable region include a polynucleotide comprising the base sequence represented by SEQ ID NO: 12, and the polynucleotide encoding the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 6 includes SEQ ID NO: And a polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 is a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16. However, any of them is not limited.
本発明の軽鎖可変領域をコードするポリヌクレオチドとしては、配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域をコードするポリヌクレオチドが好ましく、より好ましくは、配列番号３、５、７または９で示されるアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチドである。
The polynucleotide encoding the light chain variable region of the present invention includes CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55, and 88 of the amino acid sequence shown in SEQ ID NO: 3 or 5. A polynucleotide encoding a light chain variable region comprising CDR3 consisting of position 96 is preferred, more preferably a polynucleotide encoding a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 3, 5, 7 or 9 It is a nucleotide.
配列番号３で示されるアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチドとしては、配列番号１１で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号５で示されるアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチドとしては、配列番号１３で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号７で示されるアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチドとしては、配列番号１５で示される塩基配列からなるポリヌクレオチドが挙げられ、配列番号９で示されるアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチドとしては、配列番号１７で示される塩基配列からなるポリヌクレオチドが挙げられるが、いずれも限定されるものではない。
Examples of the polynucleotide encoding the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 11, and the light chain consisting of the amino acid sequence shown in SEQ ID NO: 5. Examples of the polynucleotide encoding the variable region include a polynucleotide comprising the base sequence represented by SEQ ID NO: 13, and the polynucleotide encoding the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 include SEQ ID NO: And a polynucleotide encoding a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 9 includes a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17. However, any of them is not limited.
本発明のポリヌクレオチドを取得する方法としては、ＰＣＲ等の増幅手段を用いる方法を挙げることができる。例えば、配列番号１０で示される塩基配列の５’側および３’側の配列（またはその相補配列）に基づいてそれぞれプライマーを設計し、これらプライマーを用いてハイブリドーマ３６－１のＲＮＡから調製したｃＤＮＡ等を鋳型にしてＰＣＲ等を行い、両プライマー間に挟まれるＤＮＡ領域を増幅することで、本発明のポリペプチドをコードするポリヌクレオチドを含むＤＮＡ断片を大量に取得することができる。
Examples of the method for obtaining the polynucleotide of the present invention include a method using amplification means such as PCR. For example, cDNAs prepared from RNA of hybridoma 36-1 by designing primers based on the 5 ′ side and 3 ′ side sequences (or their complementary sequences) of the nucleotide sequence shown in SEQ ID NO: 10, respectively. PCR is performed using the above as a template, and a DNA region sandwiched between both primers is amplified, whereby a large amount of DNA fragments containing a polynucleotide encoding the polypeptide of the present invention can be obtained.
〔発現ベクター〕
本発明は、上記本発明のポリヌクレオチドを含む発現ベクターを提供する。本発明の発現ベクターは、上記本発明の抗体や当該抗体の機能的断片を製造するために使用することができる。本発明の発現ベクターは、上述した本発明のポリヌクレオチドを含むものであれば特に限定されないが、ＲＮＡポリメラーゼの認識配列を有するプラスミドベクターが好ましい。また、本発明の抗体を製造するために使用する発現ベクターの場合、重鎖可変領域をコードするポリヌクレオチドおよび軽鎖可変領域をコードするポリヌクレオチドの他に、重鎖定常領域をコードするポリヌクレオチドおよび軽鎖定常領域をコードするポリヌクレオチドを含むことが好ましい。具体的には、重鎖可変領域をコードするポリヌクレオチドと重鎖定常領域をコードするポリヌクレオチドとが結合した重鎖をコードするポリヌクレオチドと、軽鎖可変領域をコードするポリヌクレオチドと軽鎖定常領域をコードするポリヌクレオチドとが結合した軽鎖をコードするポリヌクレオチドとを含むことが好ましい。 [Expression vector]
The present invention provides an expression vector comprising the polynucleotide of the present invention. The expression vector of the present invention can be used for producing the antibody of the present invention or a functional fragment of the antibody. The expression vector of the present invention is not particularly limited as long as it contains the above-described polynucleotide of the present invention, but a plasmid vector having an RNA polymerase recognition sequence is preferred. In the case of the expression vector used for producing the antibody of the present invention, in addition to the polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region, the polynucleotide encoding the heavy chain constant region And a polynucleotide encoding the light chain constant region. Specifically, a polynucleotide encoding a heavy chain in which a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a heavy chain constant region are combined, a polynucleotide encoding a light chain variable region, and a light chain constant It is preferable to include a polynucleotide encoding a light chain bound to a polynucleotide encoding a region.
本発明は、上記本発明のポリヌクレオチドを含む発現ベクターを提供する。本発明の発現ベクターは、上記本発明の抗体や当該抗体の機能的断片を製造するために使用することができる。本発明の発現ベクターは、上述した本発明のポリヌクレオチドを含むものであれば特に限定されないが、ＲＮＡポリメラーゼの認識配列を有するプラスミドベクターが好ましい。また、本発明の抗体を製造するために使用する発現ベクターの場合、重鎖可変領域をコードするポリヌクレオチドおよび軽鎖可変領域をコードするポリヌクレオチドの他に、重鎖定常領域をコードするポリヌクレオチドおよび軽鎖定常領域をコードするポリヌクレオチドを含むことが好ましい。具体的には、重鎖可変領域をコードするポリヌクレオチドと重鎖定常領域をコードするポリヌクレオチドとが結合した重鎖をコードするポリヌクレオチドと、軽鎖可変領域をコードするポリヌクレオチドと軽鎖定常領域をコードするポリヌクレオチドとが結合した軽鎖をコードするポリヌクレオチドとを含むことが好ましい。 [Expression vector]
The present invention provides an expression vector comprising the polynucleotide of the present invention. The expression vector of the present invention can be used for producing the antibody of the present invention or a functional fragment of the antibody. The expression vector of the present invention is not particularly limited as long as it contains the above-described polynucleotide of the present invention, but a plasmid vector having an RNA polymerase recognition sequence is preferred. In the case of the expression vector used for producing the antibody of the present invention, in addition to the polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region, the polynucleotide encoding the heavy chain constant region And a polynucleotide encoding the light chain constant region. Specifically, a polynucleotide encoding a heavy chain in which a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a heavy chain constant region are combined, a polynucleotide encoding a light chain variable region, and a light chain constant It is preferable to include a polynucleotide encoding a light chain bound to a polynucleotide encoding a region.
組換え発現ベクターの作製方法としては、プラスミド、ファージ、またはコスミドなどを用いる方法が挙げられるが特に限定されない。発現ベクターの具体的な種類は特に限定されず、宿主細胞中で発現可能なベクターを適宜選択することができる。すなわち、宿主細胞の種類に応じて、確実に本発明のポリヌクレオチドを発現させるために適宜プロモーター配列を選択し、これと本発明のポリヌクレオチドを各種プラスミド等に組み込んだベクターを発現ベクターとして用いればよい。また、少なくとも１つの選択マーカーを含むことが好ましい。このようなマーカーとしては、宿主が真核生物細胞の場合はジヒドロ葉酸レダクターゼまたはネオマイシン耐性遺伝子、宿主が大腸菌または他の細菌の場合はテトラサイクリン耐性遺伝子またはアンピシリン耐性遺伝子が挙げられる。上記選択マーカーを用いれば、本発明に係るポリヌクレオチドが宿主細胞に導入されたか否か、さらには宿主細胞中で確実に発現しているか否かを確認することができる。
A method for producing a recombinant expression vector includes, but is not particularly limited to, a method using a plasmid, phage, cosmid or the like. The specific type of expression vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, if a promoter sequence is appropriately selected according to the type of host cell to ensure expression of the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good. It is also preferable to include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes when the host is a eukaryotic cell, and tetracycline resistance genes or ampicillin resistance genes when the host is E. coli or other bacteria. By using the above selection marker, it can be confirmed whether or not the polynucleotide according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell.
〔細胞〕
本発明は、上記本発明の発現ベクターが導入された細胞を提供する。本発明の細胞としては、宿主細胞として公知の各種微生物、植物または動物の細胞を用いることができる。具体的には、例えば、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等の細菌、酵母（出芽酵母Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、***酵母Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）、アフリカツメガエル卵母細胞、昆虫細胞、動物細胞（例えば、ＣＨＯ細胞、ＣＯＳ細胞等）などが挙げられる。動物細胞において大量発現を目的とする場合にはＣＨＯ細胞が好ましい。 〔cell〕
The present invention provides a cell into which the expression vector of the present invention has been introduced. As the cells of the present invention, various microorganisms, plant or animal cells known as host cells can be used. Specifically, for example, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), Xenopus oocytes, insect cells, animal cells (eg, CHO cells, COS cells, etc.) Etc. CHO cells are preferred for the purpose of mass expression in animal cells.
本発明は、上記本発明の発現ベクターが導入された細胞を提供する。本発明の細胞としては、宿主細胞として公知の各種微生物、植物または動物の細胞を用いることができる。具体的には、例えば、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等の細菌、酵母（出芽酵母Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、***酵母Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）、アフリカツメガエル卵母細胞、昆虫細胞、動物細胞（例えば、ＣＨＯ細胞、ＣＯＳ細胞等）などが挙げられる。動物細胞において大量発現を目的とする場合にはＣＨＯ細胞が好ましい。 〔cell〕
The present invention provides a cell into which the expression vector of the present invention has been introduced. As the cells of the present invention, various microorganisms, plant or animal cells known as host cells can be used. Specifically, for example, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), Xenopus oocytes, insect cells, animal cells (eg, CHO cells, COS cells, etc.) Etc. CHO cells are preferred for the purpose of mass expression in animal cells.
本発明の発現ベクターを宿主細胞に導入する方法、すなわち形質転換法も特に限定されるものではなく、電気穿孔法、リン酸カルシウム法、リポソーム法、ＤＥＡＥデキストラン法等の従来公知の方法を好適に用いることができる。本発明の細胞は、本発明のポリヌクレオチドがコードするタンパク質を安定的に発現することが好ましいが、一過性に発現してもよい。
A method for introducing the expression vector of the present invention into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method is preferably used. Can do. The cell of the present invention preferably stably expresses the protein encoded by the polynucleotide of the present invention, but may be expressed transiently.
〔検出方法〕
本発明は、Ｈ５亜型インフルエンザウイルスの検出方法を提供する。本発明の検出方法は、上記本発明の抗体またはその機能的断片（以下「本発明の抗体等」という。）を被験試料に接触させる工程を包含するものであればよい。被験試料は、Ｈ５亜型インフルエンザウイルスが含まれる可能性のある試料であればよく、特に限定されない。動物および植物の生体構成成分を含む生体由来試料であることが好ましく、ヒトを含む動物由来の試料であることがより好ましい。ヒトを含む動物由来の試料としては、例えば、血液、組織液、リンパ液、脳脊髄液、膿、粘液、鼻水、喀痰、尿、糞便、腹水等の体液類、皮膚、肺、腎、粘膜、各種臓器、骨等の組織、鼻腔、気管支、皮膚、各種臓器、骨等を洗浄した後の洗浄液などが挙げられる。また、生物の体から採取した細胞を培養した際の培養液など、被検試料から二次的に得られる試料も被検試料として好適である。 [Detection method]
The present invention provides a method for detecting H5 subtype influenza virus. The detection method of the present invention only needs to include a step of bringing the antibody of the present invention or a functional fragment thereof (hereinafter referred to as “the antibody of the present invention”) into contact with a test sample. The test sample is not particularly limited as long as it may contain H5 subtype influenza virus. A sample derived from a living body including biological components of animals and plants is preferable, and a sample derived from an animal including humans is more preferable. Examples of samples derived from animals including humans include body fluids such as blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, ascites, skin, lung, kidney, mucous membrane, various organs. And a washing solution after washing tissues such as bones, nasal cavity, bronchi, skin, various organs, bones and the like. A sample obtained secondarily from the test sample, such as a culture solution obtained by culturing cells collected from the body of an organism, is also suitable as the test sample.
本発明は、Ｈ５亜型インフルエンザウイルスの検出方法を提供する。本発明の検出方法は、上記本発明の抗体またはその機能的断片（以下「本発明の抗体等」という。）を被験試料に接触させる工程を包含するものであればよい。被験試料は、Ｈ５亜型インフルエンザウイルスが含まれる可能性のある試料であればよく、特に限定されない。動物および植物の生体構成成分を含む生体由来試料であることが好ましく、ヒトを含む動物由来の試料であることがより好ましい。ヒトを含む動物由来の試料としては、例えば、血液、組織液、リンパ液、脳脊髄液、膿、粘液、鼻水、喀痰、尿、糞便、腹水等の体液類、皮膚、肺、腎、粘膜、各種臓器、骨等の組織、鼻腔、気管支、皮膚、各種臓器、骨等を洗浄した後の洗浄液などが挙げられる。また、生物の体から採取した細胞を培養した際の培養液など、被検試料から二次的に得られる試料も被検試料として好適である。 [Detection method]
The present invention provides a method for detecting H5 subtype influenza virus. The detection method of the present invention only needs to include a step of bringing the antibody of the present invention or a functional fragment thereof (hereinafter referred to as “the antibody of the present invention”) into contact with a test sample. The test sample is not particularly limited as long as it may contain H5 subtype influenza virus. A sample derived from a living body including biological components of animals and plants is preferable, and a sample derived from an animal including humans is more preferable. Examples of samples derived from animals including humans include body fluids such as blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, ascites, skin, lung, kidney, mucous membrane, various organs. And a washing solution after washing tissues such as bones, nasal cavity, bronchi, skin, various organs, bones and the like. A sample obtained secondarily from the test sample, such as a culture solution obtained by culturing cells collected from the body of an organism, is also suitable as the test sample.
本発明の抗体等を被験試料に接触させることにより、被験試料中にＨ５亜型インフルエンザウイルスが存在すれば、本発明の抗体等がＨ５亜型インフルエンザウイルスのヘマグルチニンに特異的に結合し、複合体を形成する。この複合体を検出することにより、Ｈ５亜型インフルエンザウイルスを検出することができる。当該複合体の検出には、公知の免疫学的方法を好適に用いることができる。具体的には、例えば、ラジオイムノアッセイ、エンザイムイムノアッセイ、蛍光イムノアッセイ、発光イムノアッセイ、免疫沈降法、免疫比濁法などが挙げられ、エンザイムイムノアッセイ、特にＥＬＩＳＡ法が好ましい。
When the H5 subtype influenza virus is present in the test sample by bringing the antibody of the present invention into contact with the test sample, the antibody of the present invention specifically binds to the hemagglutinin of the H5 subtype influenza virus, and the complex Form. By detecting this complex, H5 subtype influenza virus can be detected. A known immunological method can be suitably used for detecting the complex. Specific examples include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, and the like, and enzyme immunoassay, particularly ELISA method is preferred.
ＥＬＩＳＡ法は、当業者に周知の方法で実施することができる。例えば、本発明の抗体等をマイクロプレートのウェルやプラスチックチューブなどの固相に結合させ、これに被験試料を添加し、洗浄後、酵素標識した二次抗体を添加する。未結合の二次抗体を洗浄、除去したのち、発色基質を添加して生成した発色物質の量を分光光度計で吸光度を測定する方法が挙げられる。
The ELISA method can be performed by a method well known to those skilled in the art. For example, the antibody of the present invention is bound to a solid phase such as a well of a microplate or a plastic tube, a test sample is added thereto, and after washing, an enzyme-labeled secondary antibody is added. An example is a method in which after the unbound secondary antibody is washed and removed, the absorbance is measured with a spectrophotometer for the amount of the chromogenic material produced by adding a chromogenic substrate.
本発明の検出方法において、被験試料以外に対照試料を用いることが好ましい。対照試料としては、Ｈ５亜型インフルエンザウイルスを含まない陰性対照試料、Ｈ５亜型インフルエンザウイルスを含む陽性対照試料が挙げられる。被験試料で得られた結果を陰性対照試料および陽性対照試料で得られた結果と比較することにより、被験試料中のＨ５亜型インフルエンザウイルスの存在を正確に検出することができる。また、被験試料の結果および対照試料の結果を数値データとして取得すれば、被験試料中のＨ５亜型インフルエンザウイルスを定量的に検出することが可能となる。
In the detection method of the present invention, it is preferable to use a control sample in addition to the test sample. Examples of the control sample include a negative control sample not containing H5 subtype influenza virus and a positive control sample containing H5 subtype influenza virus. By comparing the results obtained with the test sample with the results obtained with the negative control sample and the positive control sample, the presence of the H5 subtype influenza virus in the test sample can be accurately detected. Moreover, if the result of the test sample and the result of the control sample are acquired as numerical data, it becomes possible to quantitatively detect the H5 subtype influenza virus in the test sample.
〔検出キット〕
本発明は、Ｈ５亜型インフルエンザウイルスの検出キットを提供する。本発明の検出キットは、本発明の抗体等を含むものであればよい。本発明の抗体等以外のキットの構成については特に限定されるものではなく、他に必要な試薬や器具等を適宜選択してキットの構成とすればよい。例えば、ＥＬＩＳＡ用検出キットとして実施する場合には、標識された二次抗体、発色用試薬、洗浄用緩衝液、ＥＬＩＳＡ用プレート、使用説明書などを含むことが好ましい。本発明のキットを用いることにより、上記本発明の検出方法を簡便かつ迅速に実施することができる。 [Detection kit]
The present invention provides a detection kit for H5 subtype influenza virus. The detection kit of the present invention only needs to contain the antibody of the present invention. The configuration of the kit other than the antibody and the like of the present invention is not particularly limited, and other necessary reagents and instruments may be appropriately selected to form the kit. For example, when implemented as an ELISA detection kit, it preferably contains a labeled secondary antibody, a coloring reagent, a washing buffer, an ELISA plate, instructions for use, and the like. By using the kit of the present invention, the detection method of the present invention can be carried out simply and rapidly.
本発明は、Ｈ５亜型インフルエンザウイルスの検出キットを提供する。本発明の検出キットは、本発明の抗体等を含むものであればよい。本発明の抗体等以外のキットの構成については特に限定されるものではなく、他に必要な試薬や器具等を適宜選択してキットの構成とすればよい。例えば、ＥＬＩＳＡ用検出キットとして実施する場合には、標識された二次抗体、発色用試薬、洗浄用緩衝液、ＥＬＩＳＡ用プレート、使用説明書などを含むことが好ましい。本発明のキットを用いることにより、上記本発明の検出方法を簡便かつ迅速に実施することができる。 [Detection kit]
The present invention provides a detection kit for H5 subtype influenza virus. The detection kit of the present invention only needs to contain the antibody of the present invention. The configuration of the kit other than the antibody and the like of the present invention is not particularly limited, and other necessary reagents and instruments may be appropriately selected to form the kit. For example, when implemented as an ELISA detection kit, it preferably contains a labeled secondary antibody, a coloring reagent, a washing buffer, an ELISA plate, instructions for use, and the like. By using the kit of the present invention, the detection method of the present invention can be carried out simply and rapidly.
〔Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療薬〕
本発明は、本発明の抗体等を有効成分として含むＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬を提供する。本発明の抗体等はＨ５亜型インフルエンザウイルスの感染性を低濃度で中和することができるので、Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療に有用である。 [Preventive or therapeutic agent for H5 influenza virus infection]
The present invention provides a preventive or therapeutic agent for H5 subtype influenza virus infection diseases comprising the antibody of the present invention as an active ingredient. Since the antibody of the present invention can neutralize the infectivity of H5 subtype influenza virus at a low concentration, it is useful for the prevention or treatment of H5 subtype influenza virus infection diseases.
本発明は、本発明の抗体等を有効成分として含むＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬を提供する。本発明の抗体等はＨ５亜型インフルエンザウイルスの感染性を低濃度で中和することができるので、Ｈ５亜型インフルエンザウイルス感染疾患の予防または治療に有用である。 [Preventive or therapeutic agent for H5 influenza virus infection]
The present invention provides a preventive or therapeutic agent for H5 subtype influenza virus infection diseases comprising the antibody of the present invention as an active ingredient. Since the antibody of the present invention can neutralize the infectivity of H5 subtype influenza virus at a low concentration, it is useful for the prevention or treatment of H5 subtype influenza virus infection diseases.
本発明の予防または治療薬は、経口投与または非経口投与で、ヒトまたはヒト以外の動物に投与することができる。また、投与経路に応じて適当な剤形とすることができる。具体的には顆粒剤、錠剤、丸剤、カプセル剤、シロップ剤、乳剤、懸濁剤、注射剤、点滴剤、外用剤、坐剤などの各種製剤形態に調製することができる。これらの各種製剤は、通常用いられている賦形剤、増量剤、結合剤、浸潤剤、崩壊剤、表面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、着色料、香味剤、安定化剤などを用いて常法により製造することができる。
The preventive or therapeutic agent of the present invention can be administered orally or parenterally to humans or non-human animals. Moreover, it can be set as a suitable dosage form according to an administration route. Specifically, it can be prepared in various preparation forms such as granules, tablets, pills, capsules, syrups, emulsions, suspensions, injections, drops, external preparations, suppositories and the like. These various preparations are commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives. , And can be produced by conventional methods using colorants, flavoring agents, stabilizers and the like.
賦形剤としては、例えば乳糖、果糖、ブドウ糖、コーンスターチ、ソルビット、結晶セルロース、滅菌水、エタノール、グリセロール、生理食塩水、緩衝液などが挙げられる。崩壊剤としては、例えば澱粉、アルギン酸ナトリウム、ゼラチン、炭酸カルシウム、クエン酸カルシウム、デキストリン、炭酸マグネシウム、合成ケイ酸マグネシウムなどが挙げられる。結合剤としては、例えばメチルセルロース、エチルセルロース、アラビアゴム、ゼラチン、ヒドロキシプロピルセルロース、ポリビニルピロリドンなどが挙げられる。滑沢剤としては、タルク、ステアリン酸マグネシウム、ポリエチレングリコール、硬化植物油などが挙げられる。安定化剤としては、例えばアルギニン、ヒスチジン、リジン、メチオニンなどのアミノ酸、ヒト血清アルブミン、ゼラチン、デキストラン４０、メチルセルロース、亜硫酸ナトリウム、メタ亜硫酸ナトリウムなどが挙げられる。その他の添加剤としては、シロップ、ワセリン、グリセリン、エタノール、プロピレングリコール、クエン酸、塩化ナトリウム、亜硝酸ソーダ、リン酸ナトリウムなどが挙げられる。
Examples of the excipient include lactose, fructose, glucose, corn starch, sorbit, crystalline cellulose, sterilized water, ethanol, glycerol, physiological saline, and buffer solution. Examples of the disintegrant include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate. Examples of the binder include methyl cellulose, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like. Examples of the lubricant include talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, and the like. Examples of the stabilizer include amino acids such as arginine, histidine, lysine, and methionine, human serum albumin, gelatin, dextran 40, methylcellulose, sodium sulfite, and sodium metasulfite. Examples of other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite, and sodium phosphate.
好ましい投与経路は非経口投与であり、好ましい剤形としては、注射剤、経鼻投与剤、経肺投与剤、経皮投与剤などが挙げられる。注射剤は、例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより全身または局部的に投与することができる。また、患者の年齢、症状により適宜投与方法を選択することができる。本発明の予防または治療薬の投与量としては、例えば、１回につき体重１ｋｇあたり０．０００１ｍｇから１０００ｍｇの範囲で選択することが可能であるが、限定されるものでははい。投与量、投与方法は、患者の体重や年齢、症状などにより変動し、当業者は適宜選択することが可能である。
A preferred route of administration is parenteral administration, and preferred dosage forms include injections, nasal administration agents, pulmonary administration agents, transdermal administration agents, and the like. The injection can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose of the prophylactic or therapeutic agent of the present invention can be selected, for example, within the range of 0.0001 mg to 1000 mg per kg of body weight at one time, but is not limited. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
本発明は、さらに上記本発明の抗体等を用いるＨ５亜型インフルエンザウイルス感染疾患の予防または治療方法を包含する。本発明のＨ５亜型インフルエンザウイルス感染疾患の予防または治療方法は、上記本発明の予防または治療薬の薬学的有効量を投与する工程を含むものである。
The present invention further includes a method for preventing or treating an H5 subtype influenza virus-infected disease using the antibody of the present invention. The method for preventing or treating an H5 subtype influenza virus-infected disease of the present invention includes a step of administering a pharmaceutically effective amount of the preventive or therapeutic agent of the present invention.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。
Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.
〔実施例１．抗インフルエンザウイルスＨ５亜型マウスモノクローナル抗体の作製〕
インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株を抗原としてマウスを免疫し、定法に従って当該抗原に対するモノクローナル抗体を産生するハイブリドーマを作製した。すなわち、ウイルス液と等量のフロイント完全アジュバントとを混合し、マウス（ＢＡＬＢ/ｃ）に３回免疫し、その脾細胞を細胞融合に用いた。採取した脾細胞とマウスミエローマ細胞Ｓｐ２／０とを、ポリエチレングリコール法にて細胞融合し、ＨＡＴ培地で培養することにより、上記抗原ウイルスに対するモノクローナル抗体を産生するハイブリドーマを得た。 [Example 1. Preparation of anti-influenza virus H5 subtype mouse monoclonal antibody]
Mice were immunized using the influenza virus A / Vietnam / 1194/04 (H5N1) strain as an antigen, and a hybridoma producing a monoclonal antibody against the antigen was prepared according to a standard method. That is, the virus solution and an equal volume of Freund's complete adjuvant were mixed, and the mouse (BALB / c) was immunized three times, and the splenocytes were used for cell fusion. The collected spleen cells and mouse myeloma cells Sp2 / 0 were cell-fused by the polyethylene glycol method and cultured in a HAT medium to obtain a hybridoma producing a monoclonal antibody against the antigen virus.
インフルエンザウイルスＡ／Ｖｉｅｔｎａｍ／１１９４／０４（Ｈ５Ｎ１）株を抗原としてマウスを免疫し、定法に従って当該抗原に対するモノクローナル抗体を産生するハイブリドーマを作製した。すなわち、ウイルス液と等量のフロイント完全アジュバントとを混合し、マウス（ＢＡＬＢ/ｃ）に３回免疫し、その脾細胞を細胞融合に用いた。採取した脾細胞とマウスミエローマ細胞Ｓｐ２／０とを、ポリエチレングリコール法にて細胞融合し、ＨＡＴ培地で培養することにより、上記抗原ウイルスに対するモノクローナル抗体を産生するハイブリドーマを得た。 [Example 1. Preparation of anti-influenza virus H5 subtype mouse monoclonal antibody]
Mice were immunized using the influenza virus A / Vietnam / 1194/04 (H5N1) strain as an antigen, and a hybridoma producing a monoclonal antibody against the antigen was prepared according to a standard method. That is, the virus solution and an equal volume of Freund's complete adjuvant were mixed, and the mouse (BALB / c) was immunized three times, and the splenocytes were used for cell fusion. The collected spleen cells and mouse myeloma cells Sp2 / 0 were cell-fused by the polyethylene glycol method and cultured in a HAT medium to obtain a hybridoma producing a monoclonal antibody against the antigen virus.
得られた１５種類のハイブリドーマ由来のモノクローナル抗体と各種ウイルスとの反応性をＥＬＩＳＡ法で評価した。表１に示すように、４種類ハイブリドーマ（クローン名：３６－１、６１－２－１、２５、６４）由来のマウスモノクローナル抗体（以下、それぞれ「マウス３６－１抗体」、「マウス６１抗体」、「マウス２５抗体」および「マウス６４抗体」という。）がＡ／ｓｗａｎ／Ｈｏｋｋａｉｄｏ／６７（Ｈ５Ｎ３）に結合活性を有していた。さらに、マウス３６－１抗体およびマウス６１抗体は患者から分離したウイルスを弱毒化したＡ／ｒｇＶｉｅｔ Ｎｕｍ／１１９４ΔＨＡ／２００４（Ｈ５Ｎ１）株に高い反応性を示したが、マウス２５抗体およびマウス６４抗体は同株に対する反応性が低いことが判明した。また、マウス３６－１抗体およびマウス６１抗体が比較的低濃度でＡ／Ｈｏｎｇ Ｋｏｎｇ／４８３／Ｋ／（Ｈ５Ｎ１）株を中和できることを確認した（実施例５参照）。なお、ハイブリドーマ３６－１は受託番号ＮＩＴＥ ＢＰ－８３２として、ハイブリドーマ６１－２－１は受託番号ＮＩＴＥ ＢＰ－８３３として、それぞれ独立行政法人製品評価技術基盤機構特許微生物寄託センターに国際寄託済みである（受託日：２００９年１０月３０日）。
The reactivity of the obtained 15 types of hybridoma-derived monoclonal antibodies with various viruses was evaluated by ELISA. As shown in Table 1, mouse monoclonal antibodies derived from four types of hybridomas (clone names: 36-1, 61-2-1, 25, 64) (hereinafter referred to as “mouse 36-1 antibody” and “mouse 61 antibody”, respectively) , "Mouse 25 antibody" and "mouse 64 antibody") had binding activity on A / swan / Hokaido / 67 (H5N3). Furthermore, the mouse 36-1 antibody and mouse 61 antibody showed high reactivity to the A / rgViet Num / 1194ΔHA / 2004 (H5N1) strain attenuated from the virus isolated from the patient, while the mouse 25 antibody and mouse 64 antibody were The reactivity to the same strain was found to be low. It was also confirmed that the mouse 36-1 antibody and mouse 61 antibody can neutralize the A / Hong ／ Kong / 483 / K / (H5N1) strain at a relatively low concentration (see Example 5). The hybridoma 36-1 has been deposited internationally with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation of Product Evaluation Technology under the accession number NITE BP-832 and the hybridoma 61-2-1 has the accession number NITE BP-833. (Contract date: October 30, 2009).
〔実施例２．抗体遺伝子の解析〕
ハイブリドーマ３６－１および６１－２－１から、ＲＮｅａｓｙ（登録商標）Ｍｉｎｉ（QIAGEN）を用いてトータルＲＮＡを抽出した。このトータルＲＮＡを鋳型として、ＧｅｎｅＲａｃｅｒキット（Invitrogen）を用いて逆転写反応を行い、ｃＤＮＡを得た。得られたｃＤＮＡを鋳型として、以下のプライマーを用いてＰＣＲを行い、それぞれの重鎖および軽鎖をコードする遺伝子断片を増幅させた。
・センスプライマー：CGACTGGAGCACGAGGACACTGA（配列番号１８、GeneRacerキットに付属）
・重鎖用アンチセンスプライマー：TTGGGCAGCAGATCCAGGGGCC（配列番号１９）
・軽鎖用アンチセンスプライマー：CACCTCCAGATGTTAACTGCTCAC（配列番号２０） [Example 2. Analysis of antibody genes)
Total RNA was extracted from the hybridomas 36-1 and 61-2-1 using RNeasy (registered trademark) Mini (QIAGEN). Using this total RNA as a template, reverse transcription reaction was performed using GeneRacer kit (Invitrogen) to obtain cDNA. PCR was carried out using the obtained cDNA as a template and the following primers, and gene fragments encoding the respective heavy and light chains were amplified.
Sense primer: CGACTGGAGCACGAGGACACTGA (SEQ ID NO: 18, attached to GeneRacer kit)
-Antisense primer for heavy chain: TTGGGCAGCAGATCCAGGGGCC (SEQ ID NO: 19)
-Antisense primer for light chain: CACCTCCAGATGTTAACTGCTCAC (SEQ ID NO: 20)
ハイブリドーマ３６－１および６１－２－１から、ＲＮｅａｓｙ（登録商標）Ｍｉｎｉ（QIAGEN）を用いてトータルＲＮＡを抽出した。このトータルＲＮＡを鋳型として、ＧｅｎｅＲａｃｅｒキット（Invitrogen）を用いて逆転写反応を行い、ｃＤＮＡを得た。得られたｃＤＮＡを鋳型として、以下のプライマーを用いてＰＣＲを行い、それぞれの重鎖および軽鎖をコードする遺伝子断片を増幅させた。
・センスプライマー：CGACTGGAGCACGAGGACACTGA（配列番号１８、GeneRacerキットに付属）
・重鎖用アンチセンスプライマー：TTGGGCAGCAGATCCAGGGGCC（配列番号１９）
・軽鎖用アンチセンスプライマー：CACCTCCAGATGTTAACTGCTCAC（配列番号２０） [Example 2. Analysis of antibody genes)
Total RNA was extracted from the hybridomas 36-1 and 61-2-1 using RNeasy (registered trademark) Mini (QIAGEN). Using this total RNA as a template, reverse transcription reaction was performed using GeneRacer kit (Invitrogen) to obtain cDNA. PCR was carried out using the obtained cDNA as a template and the following primers, and gene fragments encoding the respective heavy and light chains were amplified.
Sense primer: CGACTGGAGCACGAGGACACTGA (SEQ ID NO: 18, attached to GeneRacer kit)
-Antisense primer for heavy chain: TTGGGCAGCAGATCCAGGGGCC (SEQ ID NO: 19)
-Antisense primer for light chain: CACCTCCAGATGTTAACTGCTCAC (SEQ ID NO: 20)
得られた遺伝子断片を、ｐＴ７Ｂｌｕｅ Ｔ－Ｖｅｃｔｏｒ（Ｎｏｖａｇｅｎ社製）にクローニングし、定法に従い塩基配列を決定した。マウス３６－１抗体の重鎖可変領域のｃＤＮＡの塩基配列を配列番号１０に、軽鎖可変領域のｃＤＮＡの塩基配列を配列番号１１に示した。また、マウス６１抗体の重鎖可変領域のｃＤＮＡの塩基配列を配列番号１２に、軽鎖可変領域のｃＤＮＡの塩基配列を配列番号１３に示した。これらの塩基配列を哺乳動物のコドンを基準にアミノ酸配列に変換し、マウス抗体のドメイン構造を参考にしてフレームワーク領域（ＦＷＲ１～４）と相補性決定領域（ＣＤＲ１～３）に区分した。マウス３６－１抗体の重鎖可変領域のアミノ酸配列を配列番号２に、軽鎖可変領域のアミノ酸配列を配列番号３に示し、マウス６１抗体の重鎖可変領域のアミノ酸配列を配列番号４に、軽鎖可変領域のアミノ酸配列を配列番号５に示した。
The obtained gene fragment was cloned into pT7Blue T-Vector (manufactured by Novagen), and the base sequence was determined according to a conventional method. The nucleotide sequence of the heavy chain variable region cDNA of the mouse 36-1 antibody is shown in SEQ ID NO: 10, and the nucleotide sequence of the light chain variable region cDNA is shown in SEQ ID NO: 11. The nucleotide sequence of the heavy chain variable region cDNA of mouse 61 antibody is shown in SEQ ID NO: 12, and the nucleotide sequence of the light chain variable region cDNA is shown in SEQ ID NO: 13. These nucleotide sequences were converted into amino acid sequences based on mammalian codons, and divided into framework regions (FWR1 to 4) and complementarity determining regions (CDR1 to 3) with reference to the domain structure of the mouse antibody. The amino acid sequence of the heavy chain variable region of the mouse 36-1 antibody is shown in SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3, the amino acid sequence of the heavy chain variable region of the mouse 61 antibody is shown in SEQ ID NO: 4, The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5.
図１に、マウス３６－１抗体およびマウス６１抗体の重鎖可変領域のアミノ酸配列を対比して示した。図２に、マウス３６－１抗体およびマウス６１抗体の軽鎖可変領域のアミノ酸配列を対比して示した。Ｈ５ＨＡ特異的なマウス３６－１抗体とマウス６１抗体のフレームワーク領域は非常に類似性が高く、同一のジャームライン配列に由来するものと考えられた。また、マウス３６－１抗体とマウス６１抗体では、軽鎖のアミノ酸配列および重鎖のアミノ酸配列ともに非常によく保存されていたことから、これら２つの抗体を産生する細胞は一つの細胞が体細胞突然変異により分化したものである可能性が考えられた。
FIG. 1 shows a comparison of the amino acid sequences of the heavy chain variable regions of mouse 36-1 antibody and mouse 61 antibody. In FIG. 2, the amino acid sequences of the light chain variable regions of mouse 36-1 antibody and mouse 61 antibody are shown in comparison. The framework regions of the H5HA-specific mouse 36-1 antibody and mouse 61 antibody were very similar and were thought to be derived from the same germline sequence. In addition, in the mouse 36-1 antibody and the mouse 61 antibody, both the amino acid sequence of the light chain and the amino acid sequence of the heavy chain were very well conserved, so that one cell is the somatic cell that produces these two antibodies. There was a possibility that it was differentiated by mutation.
〔実施例３．キメラ抗体またはヒト化抗体発現ベクターおよび産生細胞の作製〕
表２に記載の塩基配列からなる合成オリゴヌクレオチドを連結して二本鎖ＤＮＡを形成させることにより、コドンをＣＨＯ細胞に最適化したヒトκ鎖の定常領域遺伝子を含むＤＮＡ断片を人工合成した。得られた遺伝子断片をＣＨＯ細胞用高発現ベクターに挿入することにより、軽鎖発現用基本ベクターｐＤＮ１１－ｃｏｈＣｋを完成させた（図３参照）。合成したＤＮＡ断片中のヒトκ鎖定常領域ｃＤＮＡの塩基配列を配列番号２１に示し、コードするアミノ酸配列を配列番号２２に示した。 [Example 3. Preparation of chimeric antibody or humanized antibody expression vector and production cell]
A DNA fragment containing a human κ chain constant region gene whose codon was optimized for CHO cells was artificially synthesized by linking synthetic oligonucleotides having the base sequences shown in Table 2 to form double-stranded DNA. The obtained gene fragment was inserted into a high expression vector for CHO cells to complete a basic vector pDN11-cohCk for light chain expression (see FIG. 3). The base sequence of human κ chain constant region cDNA in the synthesized DNA fragment is shown in SEQ ID NO: 21, and the encoded amino acid sequence is shown in SEQ ID NO: 22.
表２に記載の塩基配列からなる合成オリゴヌクレオチドを連結して二本鎖ＤＮＡを形成させることにより、コドンをＣＨＯ細胞に最適化したヒトκ鎖の定常領域遺伝子を含むＤＮＡ断片を人工合成した。得られた遺伝子断片をＣＨＯ細胞用高発現ベクターに挿入することにより、軽鎖発現用基本ベクターｐＤＮ１１－ｃｏｈＣｋを完成させた（図３参照）。合成したＤＮＡ断片中のヒトκ鎖定常領域ｃＤＮＡの塩基配列を配列番号２１に示し、コードするアミノ酸配列を配列番号２２に示した。 [Example 3. Preparation of chimeric antibody or humanized antibody expression vector and production cell]
A DNA fragment containing a human κ chain constant region gene whose codon was optimized for CHO cells was artificially synthesized by linking synthetic oligonucleotides having the base sequences shown in Table 2 to form double-stranded DNA. The obtained gene fragment was inserted into a high expression vector for CHO cells to complete a basic vector pDN11-cohCk for light chain expression (see FIG. 3). The base sequence of human κ chain constant region cDNA in the synthesized DNA fragment is shown in SEQ ID NO: 21, and the encoded amino acid sequence is shown in SEQ ID NO: 22.
同様に、表３に記載の塩基配列からなる合成オリゴヌクレオチドを連結して二本鎖ＤＮＡを形成させることにより、コドンをＣＨＯ細胞に最適化したヒトＩｇＧ１鎖定常領域遺伝子を含むＤＮＡ断片を人工合成した。得られた遺伝子断片をＣＨＯ細胞用高発現ベクターに挿入することにより、重鎖発現用基本ベクターｐＵＣＢＲ－ＳＰｙｈＣｇ１ｃを完成させた（図４参照）。合成したＤＮＡ断片中のヒトκ鎖定常領域ｃＤＮＡの塩基配列を配列番号２３に示し、コードするアミノ酸配列を配列番号２４に示した。
Similarly, DNA fragments containing human IgG1 chain constant region genes whose codons are optimized for CHO cells are artificially synthesized by linking synthetic oligonucleotides having the base sequences shown in Table 3 to form double-stranded DNA. did. The obtained gene fragment was inserted into a high expression vector for CHO cells to complete a heavy chain expression basic vector pUCBR-SPyhCg1c (see FIG. 4). The nucleotide sequence of human κ chain constant region cDNA in the synthesized DNA fragment is shown in SEQ ID NO: 23, and the encoded amino acid sequence is shown in SEQ ID NO: 24.
次に、マウス３６－１抗体、マウス６１抗体の重鎖および軽鎖の可変領域をコードするＤＮＡ断片をＰＣＲにより増幅した。ＰＣＲには以下のプライマーを用いた。
・重鎖可変領域用プライマー（両抗体共通）
m36-1VH-F：TCTTAAGCCAGGTCCAATTGCAGCAGCC（配列番号４４）
m36-1VH-R：TGCTAGCTGAGGAGACTGTGAGAGTGG（配列番号４５）
・軽鎖可変領域用プライマー（両抗体共通）
m36-1VL-F：TCCGCGGCGCCCGCCAAATTGTTCTCTCCCAGTC（配列番号４６）
m36-1VL-R：CCGTACGTTTGATTTCCAGCTTGGTGC（配列番号４７） Next, DNA fragments encoding the variable regions of the heavy and light chains of mouse 36-1 antibody and mouse 61 antibody were amplified by PCR. The following primers were used for PCR.
-Primer for heavy chain variable region (common to both antibodies)
m36-1VH-F: TCTTAAGCCAGGTCCAATTGCAGCAGCC (SEQ ID NO: 44)
m36-1VH-R: TGCTAGCTGAGGAGACTGTGAGAGTGG (SEQ ID NO: 45)
・ Light chain variable region primer (common to both antibodies)
m36-1VL-F: TCCGCGGCGCCCGCCAAATTGTTCTCTCCCAGTC (SEQ ID NO: 46)
m36-1VL-R: CCGTACGTTTGATTTCCAGCTTGGTGC (SEQ ID NO: 47)
・重鎖可変領域用プライマー（両抗体共通）
m36-1VH-F：TCTTAAGCCAGGTCCAATTGCAGCAGCC（配列番号４４）
m36-1VH-R：TGCTAGCTGAGGAGACTGTGAGAGTGG（配列番号４５）
・軽鎖可変領域用プライマー（両抗体共通）
m36-1VL-F：TCCGCGGCGCCCGCCAAATTGTTCTCTCCCAGTC（配列番号４６）
m36-1VL-R：CCGTACGTTTGATTTCCAGCTTGGTGC（配列番号４７） Next, DNA fragments encoding the variable regions of the heavy and light chains of mouse 36-1 antibody and mouse 61 antibody were amplified by PCR. The following primers were used for PCR.
-Primer for heavy chain variable region (common to both antibodies)
m36-1VH-F: TCTTAAGCCAGGTCCAATTGCAGCAGCC (SEQ ID NO: 44)
m36-1VH-R: TGCTAGCTGAGGAGACTGTGAGAGTGG (SEQ ID NO: 45)
・ Light chain variable region primer (common to both antibodies)
m36-1VL-F: TCCGCGGCGCCCGCCAAATTGTTCTCTCCCAGTC (SEQ ID NO: 46)
m36-1VL-R: CCGTACGTTTGATTTCCAGCTTGGTGC (SEQ ID NO: 47)
さらに、コンピューターを用いた構造予測により、マウス３６－１抗体およびマウス６１抗体に基づいて、軽鎖可変領域および重鎖可変領域をそれぞれヒト化したアミノ酸配列をデザインした。デザインに当たっては、Ａ．パドランらのフランケンシュタイン・プロトコールを採用した。具体的には、マウス３６－１抗体およびマウス６１抗体の可変領域のアミノ酸配列のうち、ＦＷＲ１、２、３、４に最も類似したアミノ酸配列をそれぞれ個別にヒト抗体ジャームライン配列データベースより抽出して、これにそれぞれの抗体のＣＤＲ１、２、３を埋め込んでデザインした。また、抗原との反応性の低下を押さえるためにヒト抗体ＦＷＲのうちＣＤＲ近傍のアミノ酸を当該マウス抗体のアミノ酸に置換した。
Furthermore, amino acid sequences in which the light chain variable region and the heavy chain variable region were humanized were designed based on the mouse 36-1 antibody and mouse 61 antibody by structure prediction using a computer. In designing, A. Adopted the Frankenstein protocol by Padran et al. Specifically, among the amino acid sequences of the variable regions of mouse 36-1 antibody and mouse 61 antibody, the amino acid sequences most similar to FWR1, 2, 3, 4 were individually extracted from the human antibody germline sequence database. This was designed by embedding CDRs 1, 2 and 3 of the respective antibodies. Further, in order to suppress a decrease in reactivity with the antigen, amino acids near the CDRs in the human antibody FWR were substituted with amino acids of the mouse antibody.
マウス３６－１抗体に基づくヒト化抗体（以下、「ヒト化３６－１抗体」という。）のデザインを図５に示した。また、マウス６１抗体に基づくヒト化抗体（以下、「ヒト化６１抗体」という。）のデザインを図６に示した。図５および図６において、上段にマウス抗体のアミノ酸配列を示し、中段にマウス抗体のアミノ酸配列に基づいてヒト抗体ジャームライン配列データベースより抽出されたヒト抗体ＦＷＲのアミノ酸配列を示し、下段にフランケンシュタイン・プロトコールにてデザインされたヒト化抗体のアミノ酸配列を示した。下段のヒト化抗体のアミノ酸配列中、薄い文字がマウス抗体配列由来のアミノ酸を示す。
The design of a humanized antibody based on the mouse 36-1 antibody (hereinafter referred to as “humanized 36-1 antibody”) is shown in FIG. The design of a humanized antibody based on the mouse 61 antibody (hereinafter referred to as “humanized 61 antibody”) is shown in FIG. 5 and 6, the upper part shows the amino acid sequence of the mouse antibody, the middle part shows the amino acid sequence of the human antibody FWR extracted from the human antibody germline sequence database based on the amino acid sequence of the mouse antibody, and the lower part shows Frankenstein. -The amino acid sequence of the humanized antibody designed by the protocol is shown. In the amino acid sequence of the lower humanized antibody, the light letter indicates the amino acid derived from the mouse antibody sequence.
ヒト化３６－１抗体の重鎖可変領域のアミノ酸配列に基づいて、哺乳動物細胞にコドンを最適化したＤＮＡ配列をデザインし、表４に示す６種類の合成オリゴヌクレオチドをつなぎ合わせ、得られたＤＮＡ断片を鋳型として以下のプライマーを用いてＰＣＲを行い、ヒト化３６－１抗体の重鎖可変領域をコードするＤＮＡ断片を合成した。
hz36-1VH-F：TCTTAAGTCAGGTGCAGCTGGTG（配列番号４８）
hz36-1VH-R：TGCTAGCGCTGCTCACGGTC（配列番号４９） Based on the amino acid sequence of the heavy chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed in mammalian cells, and 6 types of synthetic oligonucleotides shown in Table 4 were joined together. PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody.
hz36-1VH-F: TCTTAAGTCAGGTGCAGCTGGTG (SEQ ID NO: 48)
hz36-1VH-R: TGCTAGCGCTGCTCACGGTC (SEQ ID NO: 49)
hz36-1VH-F：TCTTAAGTCAGGTGCAGCTGGTG（配列番号４８）
hz36-1VH-R：TGCTAGCGCTGCTCACGGTC（配列番号４９） Based on the amino acid sequence of the heavy chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed in mammalian cells, and 6 types of synthetic oligonucleotides shown in Table 4 were joined together. PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody.
hz36-1VH-F: TCTTAAGTCAGGTGCAGCTGGTG (SEQ ID NO: 48)
hz36-1VH-R: TGCTAGCGCTGCTCACGGTC (SEQ ID NO: 49)
ヒト化３６－１抗体の軽鎖可変領域のアミノ酸配列に基づいて、哺乳動物細胞にコドンを最適化したＤＮＡ配列をデザインし、表５に示す６種類の合成オリゴヌクレオチドをつなぎ合わせ、得られたＤＮＡ断片を鋳型として以下のプライマーを用いてＰＣＲを行い、ヒト化３６－１抗体の軽鎖可変領域をコードするＤＮＡ断片を合成した。
hz36-1VL-F：TCCGCGGTGCTAGACAGATC（配列番号５６）
hz36-1VL-R：CCGTACGCTTGATCTCCAGC（配列番号５７） Based on the amino acid sequence of the light chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 5 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
hz36-1VL-F: TCCGCGGTGCTAGACAGATC (SEQ ID NO: 56)
hz36-1VL-R: CCGTACGCTTGATCTCCAGC (SEQ ID NO: 57)
hz36-1VL-F：TCCGCGGTGCTAGACAGATC（配列番号５６）
hz36-1VL-R：CCGTACGCTTGATCTCCAGC（配列番号５７） Based on the amino acid sequence of the light chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 5 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
hz36-1VL-F: TCCGCGGTGCTAGACAGATC (SEQ ID NO: 56)
hz36-1VL-R: CCGTACGCTTGATCTCCAGC (SEQ ID NO: 57)
ヒト化６１抗体の重鎖可変領域のアミノ酸配列に基づいて、哺乳動物細胞にコドンを最適化したＤＮＡ配列をデザインし、表６に示す６種類の合成オリゴヌクレオチドをつなぎ合わせ、得られたＤＮＡ断片を鋳型として以下のプライマーを用いてＰＣＲを行い、ヒト化３６－１抗体の重鎖可変領域をコードするＤＮＡ断片を合成した。
hz61VH-F：TCTTAAGTCAGGTGCAGCTGGTG（hz36-1VH-Fと同じ、配列番号４８）
hz61VH-R：TGCTAGCGCTGCTCACGGTC（hz36-1VH-Rと同じ、配列番号４９） Based on the amino acid sequence of the heavy chain variable region of the humanized 61 antibody, a DNA sequence with a codon optimized for mammalian cells was designed, and the six synthetic oligonucleotides shown in Table 6 were joined together, and the resulting DNA fragment PCR was carried out using the following primers as a template to synthesize a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody.
hz61VH-F: TCTTAAGTCAGGTGCAGCTGGTG (same as hz36-1VH-F, SEQ ID NO: 48)
hz61VH-R: TGCTAGCGCTGCTCACGGTC (same as hz36-1VH-R, SEQ ID NO: 49)
hz61VH-F：TCTTAAGTCAGGTGCAGCTGGTG（hz36-1VH-Fと同じ、配列番号４８）
hz61VH-R：TGCTAGCGCTGCTCACGGTC（hz36-1VH-Rと同じ、配列番号４９） Based on the amino acid sequence of the heavy chain variable region of the humanized 61 antibody, a DNA sequence with a codon optimized for mammalian cells was designed, and the six synthetic oligonucleotides shown in Table 6 were joined together, and the resulting DNA fragment PCR was carried out using the following primers as a template to synthesize a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody.
hz61VH-F: TCTTAAGTCAGGTGCAGCTGGTG (same as hz36-1VH-F, SEQ ID NO: 48)
hz61VH-R: TGCTAGCGCTGCTCACGGTC (same as hz36-1VH-R, SEQ ID NO: 49)
ヒト化３６－１抗体の軽鎖可変領域のアミノ酸配列に基づいて、哺乳動物細胞にコドンを最適化したＤＮＡ配列をデザインし、表７に示す６種類の合成オリゴヌクレオチドをつなぎ合わせ、得られたＤＮＡ断片を鋳型として以下のプライマーを用いてＰＣＲを行い、ヒト化３６－１抗体の軽鎖可変領域をコードするＤＮＡ断片を合成した。
hz61VL-F：TCCGCGGTGCTAGACAGATC（hz36-1VL-Fと同じ、配列番号５６）
hz61VL-R：CCGTACGCTTGATCTCCAG（配列番号７０） Based on the amino acid sequence of the light chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 7 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
hz61VL-F: TCCGCGGTGCTAGACAGATC (same as hz36-1VL-F, SEQ ID NO: 56)
hz61VL-R: CCGTACGCTTGATCTCCAG (SEQ ID NO: 70)
hz61VL-F：TCCGCGGTGCTAGACAGATC（hz36-1VL-Fと同じ、配列番号５６）
hz61VL-R：CCGTACGCTTGATCTCCAG（配列番号７０） Based on the amino acid sequence of the light chain variable region of the humanized 36-1 antibody, a DNA sequence with optimized codons was designed for mammalian cells and obtained by joining the 6 synthetic oligonucleotides shown in Table 7 PCR was performed using the following primers with the DNA fragment as a template to synthesize a DNA fragment encoding the light chain variable region of the humanized 36-1 antibody.
hz61VL-F: TCCGCGGTGCTAGACAGATC (same as hz36-1VL-F, SEQ ID NO: 56)
hz61VL-R: CCGTACGCTTGATCTCCAG (SEQ ID NO: 70)
図７に示すように、マウス３６－１抗体の軽鎖可変領域をコードするＤＮＡ断片を軽鎖発現用基本ベクターｐＤＮ１１－ｃｏｈＣｋに挿入し、キメラ３６－１抗体の軽鎖発現ベクターを完成させた。また、マウス３６－１抗体の重鎖可変領域をコードするＤＮＡ断片を重鎖発現用基本ベクターｐＵＣＢＲ－ＳＰｙｈＣｇ１ｃに挿入し、キメラ３６－１抗体の重鎖発現ベクターを完成させた。同様に、キメラ６１抗体の軽鎖発現ベクターおよびキメラ６１抗体の重鎖発現ベクターを完成させた。さらに、ヒト化３６－１抗体の軽鎖可変領域をコードするＤＮＡ断片を軽鎖発現用基本ベクターｐＤＮ１１－ｃｏｈＣｋに挿入し、ヒト化３６－１抗体の軽鎖発現ベクターを完成させた。また、ヒト化３６－１抗体の重鎖可変領域をコードするＤＮＡ断片を重鎖発現用基本ベクターｐＵＣＢＲ－ＳＰｙｈＣｇ１ｃに挿入し、ヒト化３６－１抗体の重鎖発現ベクターを完成させた。同様に、ヒト化６１抗体の軽鎖発現ベクターおよびヒト化６１抗体の重鎖発現ベクターを完成させた。
As shown in FIG. 7, the DNA fragment encoding the light chain variable region of the mouse 36-1 antibody was inserted into the basic vector pDN11-cohCk for light chain expression to complete the light chain expression vector of the chimeric 36-1 antibody. . In addition, a DNA fragment encoding the heavy chain variable region of mouse 36-1 antibody was inserted into basic vector for heavy chain expression pUCBR-SPyhCg1c to complete the heavy chain expression vector of chimeric 36-1 antibody. Similarly, a chimeric 61 antibody light chain expression vector and a chimeric 61 antibody heavy chain expression vector were completed. Furthermore, the DNA fragment encoding the light chain variable region of the humanized 36-1 antibody was inserted into the light chain expression basic vector pDN11-cohCk to complete the light chain expression vector of the humanized 36-1 antibody. In addition, a DNA fragment encoding the heavy chain variable region of the humanized 36-1 antibody was inserted into the basic vector pUCBR-SPyhCg1c for heavy chain expression to complete the heavy chain expression vector of the humanized 36-1 antibody. Similarly, a humanized 61 antibody light chain expression vector and a humanized 61 antibody heavy chain expression vector were completed.
各軽鎖発現ベクターをＳｍａIおよびＥｃｏＲＩで処理し断片を得た。また、各重鎖発現ベクターをＥｃｏＲＶおよびＥｃｏＲＩで処理し断片を得た。図８に示すように、対応する軽鎖および重鎖の断片を連結して抗体発現ベクターを構築した。ＬｉｐｏｆｅｃｔＡｍｉｎｅ２０００（Invitrogen）の説明書に従い、各抗体発現ベクターをＣＨＯ細胞にトランスフェクションし、各抗体産生細胞の高発現クローンを得た。
Each light chain expression vector was treated with SmaI and EcoRI to obtain fragments. Each heavy chain expression vector was treated with EcoRV and EcoRI to obtain fragments. As shown in FIG. 8, an antibody expression vector was constructed by ligating the corresponding light and heavy chain fragments. According to the instructions of LipofectAmine2000 (Invitrogen), each antibody expression vector was transfected into CHO cells to obtain high expression clones of each antibody-producing cell.
〔実施例４．Ｈ５亜型インフルエンザウイルスに対する結合性の評価〕
Ｈ５亜型インフルエンザウイルスのＡ／ｒｇＶｉｅｔ Ｎｕｍ／１１９４ΔＨＡ／２００４（Ｈ５Ｎ１）株に対する結合性をＥＬＩＳＡ法で評価した。抗体には、キメラ３６－１抗体およびキメラ６１抗体と、それぞれ対応するマウス３６－１抗体およびマウス６１抗体の４種類を用いた。各キメラ抗体は、実施例３で作製したキメラ３６－１抗体産生細胞およびキメラ６１抗体産生細胞を無タンパク培地で培養した後、プロテインＧアガロースカラムにて精製したものを使用した。具体的には、以下の手順で実験を行った。
(i) ２０μｇ／ｍＬ（in PBS）に希釈したＡ／ｒｇＶｉｅｔ Ｎｕｍ／１１９４ΔＨＡ／２００４（Ｈ５Ｎ１）のウイルス液を、９６ｗｅｌｌのプレートに５０μＬずつ分注し、４℃で２０時間インキュベーションした。
(ii) ＰＢＳＴ（３００μＬ／ｗｅｌｌ）で３回ｗｅｌｌを洗浄した後、ＰＢＳＴを用いて各濃度（0、0.015625、0.03125、0.0625、0.125、0.25、0.5、1 μg/mL）に希釈した各抗体を１００μＬ／ｗｅｌｌずつ分注し、室温で１時間インキュベーションした。
(iii) ＰＢＳＴ（３００μＬ／ｗｅｌｌ）で３回ｗｅｌｌを洗浄した後、ＰＢＳＴを用いて０．１μｇ／ｍＬに希釈した２次抗体（ペルオキシダーゼ標識抗ヒト定常領域抗体、またはペルオキシダーゼ標識抗マウス定常領域抗体）を１００μＬ／ｗｅｌｌずつ分注し、室温で１時間インキュベーションした。 [Example 4. Evaluation of binding to H5 subtype influenza virus]
The binding of H5 subtype influenza virus to A / rgViet Num / 1194ΔHA / 2004 (H5N1) strain was evaluated by ELISA. Four types of antibodies were used: chimeric 36-1 antibody and chimeric 61 antibody, and the corresponding mouse 36-1 antibody and mouse 61 antibody, respectively. As each chimeric antibody, the chimeric 36-1 antibody-producing cells and chimeric 61 antibody-producing cells prepared in Example 3 were cultured in a protein-free medium and then purified using a protein G agarose column. Specifically, the experiment was performed according to the following procedure.
(i) A / rgViet Num / 1194ΔHA / 2004 (H5N1) virus solution diluted to 20 μg / mL (in PBS) was dispensed in 50 μL aliquots onto a 96-well plate and incubated at 4 ° C. for 20 hours.
(ii) After washing the well three times with PBST (300 μL / well), each antibody diluted to each concentration (0, 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL) usingPBST 100 μL / well was dispensed and incubated at room temperature for 1 hour.
(iii) A secondary antibody (peroxidase-labeled anti-human constant region antibody or peroxidase-labeled anti-mouse constant region antibody) diluted with PBST (300 μL / well) three times and then diluted to 0.1 μg / mL with PBST ) Was dispensed at 100 μL / well and incubated at room temperature for 1 hour.
Ｈ５亜型インフルエンザウイルスのＡ／ｒｇＶｉｅｔ Ｎｕｍ／１１９４ΔＨＡ／２００４（Ｈ５Ｎ１）株に対する結合性をＥＬＩＳＡ法で評価した。抗体には、キメラ３６－１抗体およびキメラ６１抗体と、それぞれ対応するマウス３６－１抗体およびマウス６１抗体の４種類を用いた。各キメラ抗体は、実施例３で作製したキメラ３６－１抗体産生細胞およびキメラ６１抗体産生細胞を無タンパク培地で培養した後、プロテインＧアガロースカラムにて精製したものを使用した。具体的には、以下の手順で実験を行った。
(i) ２０μｇ／ｍＬ（in PBS）に希釈したＡ／ｒｇＶｉｅｔ Ｎｕｍ／１１９４ΔＨＡ／２００４（Ｈ５Ｎ１）のウイルス液を、９６ｗｅｌｌのプレートに５０μＬずつ分注し、４℃で２０時間インキュベーションした。
(ii) ＰＢＳＴ（３００μＬ／ｗｅｌｌ）で３回ｗｅｌｌを洗浄した後、ＰＢＳＴを用いて各濃度（0、0.015625、0.03125、0.0625、0.125、0.25、0.5、1 μg/mL）に希釈した各抗体を１００μＬ／ｗｅｌｌずつ分注し、室温で１時間インキュベーションした。
(iii) ＰＢＳＴ（３００μＬ／ｗｅｌｌ）で３回ｗｅｌｌを洗浄した後、ＰＢＳＴを用いて０．１μｇ／ｍＬに希釈した２次抗体（ペルオキシダーゼ標識抗ヒト定常領域抗体、またはペルオキシダーゼ標識抗マウス定常領域抗体）を１００μＬ／ｗｅｌｌずつ分注し、室温で１時間インキュベーションした。 [Example 4. Evaluation of binding to H5 subtype influenza virus]
The binding of H5 subtype influenza virus to A / rgViet Num / 1194ΔHA / 2004 (H5N1) strain was evaluated by ELISA. Four types of antibodies were used: chimeric 36-1 antibody and chimeric 61 antibody, and the corresponding mouse 36-1 antibody and mouse 61 antibody, respectively. As each chimeric antibody, the chimeric 36-1 antibody-producing cells and chimeric 61 antibody-producing cells prepared in Example 3 were cultured in a protein-free medium and then purified using a protein G agarose column. Specifically, the experiment was performed according to the following procedure.
(i) A / rgViet Num / 1194ΔHA / 2004 (H5N1) virus solution diluted to 20 μg / mL (in PBS) was dispensed in 50 μL aliquots onto a 96-well plate and incubated at 4 ° C. for 20 hours.
(ii) After washing the well three times with PBST (300 μL / well), each antibody diluted to each concentration (0, 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL) using
(iii) A secondary antibody (peroxidase-labeled anti-human constant region antibody or peroxidase-labeled anti-mouse constant region antibody) diluted with PBST (300 μL / well) three times and then diluted to 0.1 μg / mL with PBST ) Was dispensed at 100 μL / well and incubated at room temperature for 1 hour.
結果を図９に示した。（Ａ）はマウス３６－１抗体およびキメラ３６－１抗体の結果であり、（Ｂ）はマウス６１抗体およびキメラ６１抗体の結果である。２次抗体（抗ヒト定常領域抗体または抗マウス定常領域抗体）の反応性の違いにより、いずれの結果もマウス抗体の方が低い吸光度を示しているが、親和性（解離常数（Ｋｄ））を計算したところ、マウス３６－１抗体が約０．４ｎＭ、キメラ３６－１抗体が約０．３ｎＭ、マウス６１抗体が約０．３ｎＭ、キメラ６１抗体が約０．２ｎＭであり、いずれの抗体もほぼ同一の数値を示した。これらの結果から、キメラ３６－１抗体およびキメラ６１抗体は、抗体はＨ５亜型インフルエンザウイルスに対して高い親和性を有することが明らかとなった。現在実用化されている治療用抗体における抗原との親和性の範囲が０．１ｎＭから１０ｎＭであることから判断して、キメラ３６－１抗体およびキメラ６１抗体はＨ５亜型インフルエンザウイルス感染に対する予防または治療薬として応用できるものと考えられた。
The results are shown in FIG. (A) shows the results of the mouse 36-1 antibody and the chimeric 36-1 antibody, and (B) shows the results of the mouse 61 antibody and the chimeric 61 antibody. Due to the difference in reactivity of the secondary antibody (anti-human constant region antibody or anti-mouse constant region antibody), the mouse antibody shows a lower absorbance in all results, but the affinity (dissociation constant (Kd)) As a result, the mouse 36-1 antibody was about 0.4 nM, the chimera 36-1 antibody was about 0.3 nM, the mouse 61 antibody was about 0.3 nM, and the chimera 61 antibody was about 0.2 nM. Almost the same numerical value was shown. From these results, it was revealed that the chimeric 36-1 antibody and the chimeric 61 antibody have high affinity for the H5 subtype influenza virus. Judging from the fact that the range of affinity for antigen in therapeutic antibodies currently in practical use is 0.1 nM to 10 nM, chimeric 36-1 antibody and chimeric 61 antibody are effective in preventing against H5 subtype influenza virus infection or It was thought that it could be applied as a therapeutic agent.
〔実施例５．Ｈ５亜型インフルエンザウイルスに対する中和活性の評価〕
実施例４と同様に、キメラ３６－１抗体およびキメラ６１抗体と、それぞれ対応するマウス３６－１抗体およびマウス６１抗体の４種類を用い、対象ウイルスにヒト感染者から分離されたＨ５Ｎ１型インフルエンザウイルス株（Ａ／Ｈｏｎｇ Ｋｏｎｇ／４８３／１９７７（Ｈ５Ｎ１）、Ａ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１））を選定して、以下の手順で実験を行った。
(i) ＭＤＣＫ細胞を１２ｗｅｌｌプレートに播き、１０％血清入りＤＭＥＭ培地で３７℃、２日間培養してコンフルエントな状態にした。
(ii) 各濃度（0、0.125、0.25、0.5、1、2 μg/mL）に希釈した各抗体と、２００ＰＦＵ／０．１ｍＬに希釈した各ウイルス溶液を混合し、室温で１時間中和反応を行った。
(iii) 抗体とウイルスの混合溶液をＭＤＣＫ細胞に感染させた後、寒天培地を入れて固めた。
(iv) ３５℃で２日間培養した後、プラークの数をカウントした。 [Example 5. Evaluation of neutralizing activity against H5 subtype influenza virus]
In the same manner as in Example 4, H5N1 influenza virus isolated from a human infected subject using four types of chimeric 36-1 antibody and chimeric 61 antibody and corresponding mouse 36-1 antibody and mouse 61 antibody, respectively. Strains (A / Hong Kong / 483/1977 (H5N1), A / Viet Nam / 1194/2004 (H5N1)) were selected, and experiments were performed according to the following procedure.
(i) MDCK cells were seeded on a 12-well plate and cultured in a DMEM medium containing 10% serum at 37 ° C. for 2 days to obtain a confluent state.
(ii) Each antibody diluted to each concentration (0, 0.125, 0.25, 0.5, 1, 2 μg / mL) and each virus solution diluted to 200 PFU / 0.1 mL were mixed and neutralized for 1 hour at room temperature. Went.
(iii) MDCK cells were infected with a mixed solution of antibody and virus, and then agar medium was added and hardened.
(iv) After culturing at 35 ° C. for 2 days, the number of plaques was counted.
実施例４と同様に、キメラ３６－１抗体およびキメラ６１抗体と、それぞれ対応するマウス３６－１抗体およびマウス６１抗体の４種類を用い、対象ウイルスにヒト感染者から分離されたＨ５Ｎ１型インフルエンザウイルス株（Ａ／Ｈｏｎｇ Ｋｏｎｇ／４８３／１９７７（Ｈ５Ｎ１）、Ａ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１））を選定して、以下の手順で実験を行った。
(i) ＭＤＣＫ細胞を１２ｗｅｌｌプレートに播き、１０％血清入りＤＭＥＭ培地で３７℃、２日間培養してコンフルエントな状態にした。
(ii) 各濃度（0、0.125、0.25、0.5、1、2 μg/mL）に希釈した各抗体と、２００ＰＦＵ／０．１ｍＬに希釈した各ウイルス溶液を混合し、室温で１時間中和反応を行った。
(iii) 抗体とウイルスの混合溶液をＭＤＣＫ細胞に感染させた後、寒天培地を入れて固めた。
(iv) ３５℃で２日間培養した後、プラークの数をカウントした。 [Example 5. Evaluation of neutralizing activity against H5 subtype influenza virus]
In the same manner as in Example 4, H5N1 influenza virus isolated from a human infected subject using four types of chimeric 36-1 antibody and chimeric 61 antibody and corresponding mouse 36-1 antibody and mouse 61 antibody, respectively. Strains (A / Hong Kong / 483/1977 (H5N1), A / Viet Nam / 1194/2004 (H5N1)) were selected, and experiments were performed according to the following procedure.
(i) MDCK cells were seeded on a 12-well plate and cultured in a DMEM medium containing 10% serum at 37 ° C. for 2 days to obtain a confluent state.
(ii) Each antibody diluted to each concentration (0, 0.125, 0.25, 0.5, 1, 2 μg / mL) and each virus solution diluted to 200 PFU / 0.1 mL were mixed and neutralized for 1 hour at room temperature. Went.
(iii) MDCK cells were infected with a mixed solution of antibody and virus, and then agar medium was added and hardened.
(iv) After culturing at 35 ° C. for 2 days, the number of plaques was counted.
Ａ／Ｈｏｎｇ Ｋｏｎｇ／４８３／１９７７（Ｈ５Ｎ１）株の結果を図１０に示し、Ａ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１）株の結果を図１１に示した。いずれも（Ａ）はマウス３６－１抗体およびキメラ３６－１抗体の結果であり、（Ｂ）はマウス６１抗体およびキメラ６１抗体の結果である。図１０および図１１から明らかなように、キメラ３６－１抗体およびキメラ６１抗体は、マウス３６－１抗体およびマウス６１抗体と同様に対象ウイルスの感染価を中和する活性を有していた。プラーク数を５０％減少させるのに必要な抗体濃度は、抗体およびウイルスにより異なっていたが、いずれの抗体も０．５μｇ／ｍＬ以下の低濃度で十分な中和活性を示していた。これらの結果から、キメラ３６－１抗体およびキメラ６１抗体のどちらもＨ５亜型インフルエンザウイルス予防または治療薬として有望であるものと考えられた。
The results for the A / Hong Kong / 483/1977 (H5N1) strain are shown in FIG. 10, and the results for the A / Viet Nam / 1194/2004 (H5N1) strain are shown in FIG. In both cases, (A) shows the results of the mouse 36-1 antibody and the chimeric 36-1 antibody, and (B) shows the results of the mouse 61 antibody and the chimeric 61 antibody. As is clear from FIGS. 10 and 11, the chimeric 36-1 antibody and the chimeric 61 antibody had an activity to neutralize the infectious titer of the target virus in the same manner as the mouse 36-1 antibody and the mouse 61 antibody. The antibody concentration required to reduce the number of plaques by 50% was different depending on the antibody and virus, but both antibodies showed sufficient neutralizing activity at a low concentration of 0.5 μg / mL or less. From these results, it was considered that both the chimeric 36-1 antibody and the chimeric 61 antibody are promising as preventive or therapeutic agents for the H5 subtype influenza virus.
〔実施例６．感染マウスを用いた有効性の評価〕
培養試験により十分にキメラ３６－１抗体、キメラ６１抗体のどちらにも高い感染価中和活性が認められたことから、実際の感染動物における予防・治療効果が期待されたため、マウスを対象とした感染実験によりキメラ３６－１抗体およびキメラ６１抗体の有効性を評価した。マウスは、ＢＡＬＢ／ｃ、雌、６週齢（実験開始時）を用いた。Ｈ５亜型インフルエンザウイルスとして、Ａ／Ｈｏｎｇ Ｋｏｎｇ／４８３／１９７７（Ｈ５Ｎ１）株を用いた。 [Example 6. Evaluation of efficacy using infected mice)
Since the infectious titer neutralizing activity was sufficiently observed in both the chimeric 36-1 antibody and the chimeric 61 antibody in the culture test, the prevention and treatment effect in the actual infected animal was expected. The effectiveness of chimeric 36-1 antibody and chimeric 61 antibody was evaluated by infection experiments. The mice used were BALB / c, female, and 6 weeks old (at the start of the experiment). The A / Hong Kong / 483/1977 (H5N1) strain was used as the H5 subtype influenza virus.
培養試験により十分にキメラ３６－１抗体、キメラ６１抗体のどちらにも高い感染価中和活性が認められたことから、実際の感染動物における予防・治療効果が期待されたため、マウスを対象とした感染実験によりキメラ３６－１抗体およびキメラ６１抗体の有効性を評価した。マウスは、ＢＡＬＢ／ｃ、雌、６週齢（実験開始時）を用いた。Ｈ５亜型インフルエンザウイルスとして、Ａ／Ｈｏｎｇ Ｋｏｎｇ／４８３／１９７７（Ｈ５Ｎ１）株を用いた。 [Example 6. Evaluation of efficacy using infected mice)
Since the infectious titer neutralizing activity was sufficiently observed in both the chimeric 36-1 antibody and the chimeric 61 antibody in the culture test, the prevention and treatment effect in the actual infected animal was expected. The effectiveness of chimeric 36-1 antibody and chimeric 61 antibody was evaluated by infection experiments. The mice used were BALB / c, female, and 6 weeks old (at the start of the experiment). The A / Hong Kong / 483/1977 (H5N1) strain was used as the H5 subtype influenza virus.
（６－１）予防効果の評価
第１の実験として、ウイルス感染１日前に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与した後、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１２に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１２（Ａ）および（Ｂ）から明らかなように、抗体を投与した４群のマウスは全例が生存し、体重減少も観察されなかった。一方、陰性対照群のマウスは９日目までに全例が死亡した。この結果から、キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体はいずれもインフルエンザ感染予防効果を有することが明らかとなった。 (6-1) Evaluation of preventive effect As a first experiment, 100 μg of antibody (chimera 36-1, antibody 36-1, antibody 61, mouse 61) or PBS (negative control group) one day before virus infection ) Was administered intraperitoneally, the virus was infected nasally with 50 PFU / mouse (0 days after infection), and the increase and decrease in survival rate and body weight were monitored until 19 days after infection.
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 12A and 12B, all of the four groups of mice administered with the antibody survived and no weight loss was observed. On the other hand, all mice in the negative control group died byday 9. From these results, it was clarified that the chimeric 36-1 antibody, mouse 36-1 antibody, chimeric 61 antibody, and mouse 61 antibody all have the effect of preventing influenza infection.
第１の実験として、ウイルス感染１日前に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与した後、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１２に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１２（Ａ）および（Ｂ）から明らかなように、抗体を投与した４群のマウスは全例が生存し、体重減少も観察されなかった。一方、陰性対照群のマウスは９日目までに全例が死亡した。この結果から、キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体はいずれもインフルエンザ感染予防効果を有することが明らかとなった。 (6-1) Evaluation of preventive effect As a first experiment, 100 μg of antibody (chimera 36-1, antibody 36-1, antibody 61, mouse 61) or PBS (negative control group) one day before virus infection ) Was administered intraperitoneally, the virus was infected nasally with 50 PFU / mouse (0 days after infection), and the increase and decrease in survival rate and body weight were monitored until 19 days after infection.
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 12A and 12B, all of the four groups of mice administered with the antibody survived and no weight loss was observed. On the other hand, all mice in the negative control group died by
（６－２）治療効果の評価１
第２の実験として、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、ウイルス感染１日後に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与し、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１３に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１３（Ａ）および（Ｂ）から明らかなように、抗体を投与した４群のマウスは、わずかな体重減少が観察されたが全例が生存した。一方、陰性対照群のマウスは９日目までに全例が死亡した。この結果から、キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体は、いずれもインフルエンザ感染の初期に投与すれば、症状の緩和に役立つことが示唆された。 (6-2) Evaluation oftherapeutic effect 1
As a second experiment, the virus was infected nasally with 50 PFU / mouse (day 0 after infection), and 100 μg of antibody (chimeric 36-1, antibody 36-1, antibody 61, mouse 61) one day after virus infection. Antibody) or PBS (negative control group) was administered intraperitoneally, and the increase and decrease in survival rate and body weight were monitored until 19 days after infection.
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 13 (A) and 13 (B), in the four groups of mice administered with the antibody, a slight weight loss was observed, but all cases survived. On the other hand, all mice in the negative control group died byday 9. From these results, it was suggested that the chimeric 36-1 antibody, mouse 36-1 antibody, chimeric 61 antibody, and mouse 61 antibody are all useful for alleviating symptoms if administered at the early stage of influenza infection.
第２の実験として、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、ウイルス感染１日後に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与し、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１３に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１３（Ａ）および（Ｂ）から明らかなように、抗体を投与した４群のマウスは、わずかな体重減少が観察されたが全例が生存した。一方、陰性対照群のマウスは９日目までに全例が死亡した。この結果から、キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体は、いずれもインフルエンザ感染の初期に投与すれば、症状の緩和に役立つことが示唆された。 (6-2) Evaluation of
As a second experiment, the virus was infected nasally with 50 PFU / mouse (
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 13 (A) and 13 (B), in the four groups of mice administered with the antibody, a slight weight loss was observed, but all cases survived. On the other hand, all mice in the negative control group died by
（６－３）治療効果の評価２
第３の実験として、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、ウイルス感染３日後に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与し、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１４に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１４（Ａ）および（Ｂ）から明らかなように、いずれの群のマウスも著しい体重減少が観察され、陰性対照群は１２日目までに全例が死亡した。抗体投与群の生存率は、マウス３６－１抗体投与群が４０％、キメラ３６－１抗体投与群が２０％、マウス６１抗体投与群が４０％であったが、キメラ６１抗体投与群は８０％と良好であった。 (6-3) Evaluation oftherapeutic effect 2
In a third experiment, the virus was infected nasally with 50 PFU / mouse (0 days after infection), and 100 μg of antibody (chimeric 36-1, antibody 36-1, antibody 61,mouse 61, 3 days after virus infection). Antibody) or PBS (negative control group) was administered intraperitoneally, and the increase and decrease in survival rate and body weight were monitored until 19 days after infection.
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 14 (A) and (B), significant weight loss was observed in all groups of mice, and all cases in the negative control group died byday 12. The survival rate of the antibody administration group was 40% for the mouse 36-1 antibody administration group, 20% for the chimera 36-1 antibody administration group, and 40% for the mouse 61 antibody administration group. % And good.
第３の実験として、ウイルスを５０ＰＦＵ／マウスで経鼻感染させ（感染後０日）、ウイルス感染３日後に１００μｇの抗体（キメラ３６－１抗体、マウス３６－１抗体、キメラ６１抗体、マウス６１抗体）またはＰＢＳ（陰性対照群）を腹腔内に投与し、感染後１９日まで生存率と体重の増減をモニターした。
結果を図１４に示した。（Ａ）は生存率の結果であり、（Ｂ）は平均体重の結果である。図１４（Ａ）および（Ｂ）から明らかなように、いずれの群のマウスも著しい体重減少が観察され、陰性対照群は１２日目までに全例が死亡した。抗体投与群の生存率は、マウス３６－１抗体投与群が４０％、キメラ３６－１抗体投与群が２０％、マウス６１抗体投与群が４０％であったが、キメラ６１抗体投与群は８０％と良好であった。 (6-3) Evaluation of
In a third experiment, the virus was infected nasally with 50 PFU / mouse (0 days after infection), and 100 μg of antibody (chimeric 36-1, antibody 36-1, antibody 61,
The results are shown in FIG. (A) is the result of survival rate, and (B) is the result of average body weight. As is clear from FIGS. 14 (A) and (B), significant weight loss was observed in all groups of mice, and all cases in the negative control group died by
以上の結果から、本発明の抗体は、Ｈ５亜型インフルエンザウイルス感染の予防および治療に有効であることが明らかとなった。
From the above results, it has been clarified that the antibody of the present invention is effective for the prevention and treatment of H5 subtype influenza virus infection.
〔実施例７．エピトープの同定〕
マウス３６－１抗体およびマウス６１抗体のエピトープを以下の手順で決定した。ウイルスとしてＡ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１）を感染させた鶏漿尿液を用いた。
(i) ウイルスの１０倍階段希釈列を作製した。
(ii) マウス３６－１抗体およびマウス６１抗体の精製抗体をそれぞれ培地（ＭＥＭ）で２０μｇ／ｍＬに希釈し、小試験管に１００μＬずつ分注した。コントロールとして培地のみを小試験管に１００μＬずつ分注した。
(iii) 抗体を分注した小試験管に１０－１～１０－６までのウイルスを１００μＬずつ加え、時々混和しながら室温で１時間反応させた。
(iv) ６ｗｅｌｌプレートでコンフルエントな状態のＭＤＣＫ細胞を、血清を含まないＭＥＭ培地で洗浄し、(iii)の反応液を０．２ｍＬずつ各ウェルに接種して、プレートを１０～１５分ごとに振とうしながら３５℃で１時間吸着させた。
(v) ２×ＭＥＭ／ＢＳＡと２％Ｂａｃｔ Ａｇａｒ液をウォーターバスで約４２℃に温めてから、両液を等量混合した。これにアセチルトリプシン０．０００５％濃度になるように重層直前に添加した。また、ハイブリドーマ３６－１またはハイブリドーマ６１－２－１を腹腔内に接種したＢＡＬＢ／ｃマウスから得た腹水を、最終濃度が１：１０００になるように添加した。
(vi) (v)の溶液を(iv)のＭＤＣＫ細胞に重層し、３５℃、５％ＣＯ２存在下で２～３日間培養し、プラークが確認できることを観察した。
(vii) プラークを採取し、１ｍＬのＭＥＭ／ＢＳＡに加え、よく混和した。
(viii) (vii)のウイルス液を用いて(i)～(vii）の操作を行い、エスケープミュータント株であることを確認した。
(ix) エスケープミュータントのウイルス培養液からＶｉｒａｌ ＲＮＡ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（QIAGEN）を用いてＲＮＡを抽出し、ＲＴ－ＰＣＲによりヘマグルチニン遺伝子を増幅した。
(x) ヘマグルチニン遺伝子のシークエンスを行い、マウス３６－１抗体およびマウス６１抗体の結合に必須のアミノ酸を同定した。シークエンスには、マウス３６－１抗体に対するエスケープミュータント１０株、マウス６１抗体に対するエスケープミュータント１０株を供した。 [Example 7. (Identification of epitope)
The epitopes of mouse 36-1 antibody and mouse 61 antibody were determined by the following procedure. As a virus, chicken chorioallantoic fluid infected with A / Viet Nam / 1194/2004 (H5N1) was used.
(i) A 10-fold serial dilution series of virus was prepared.
(ii) Purified antibodies of mouse 36-1 antibody and mouse 61 antibody were each diluted with a medium (MEM) to 20 μg / mL, and dispensed in 100 μL each into a small test tube. As a control, 100 μL of the medium alone was dispensed into small test tubes.
(iii) 100 μL of 10 −1 to 10 −6 virus was added to each small test tube into which the antibody was dispensed, and allowed to react at room temperature for 1 hour with occasional mixing.
(iv) MDCK cells in a confluent state on a 6-well plate are washed with MEM medium without serum, and 0.2 ml of the reaction solution of (iii) is inoculated into each well, and the plate is inoculated every 10 to 15 minutes. The sample was adsorbed at 35 ° C. for 1 hour with shaking.
(v) 2 × MEM / BSA and 2% Bact Agar solution were warmed to about 42 ° C. in a water bath, and then equal amounts of both solutions were mixed. To this, acetyltrypsin was added immediately before the overlay so that the concentration was 0.0005%. In addition, ascites obtained from BALB / c mice inoculated intraperitoneally with hybridoma 36-1 or hybridoma 61-2-1 was added to a final concentration of 1: 1000.
(vi) The solution of (v) was layered on the MDCK cells of (iv) and cultured at 35 ° C. in the presence of 5% CO 2 for 2 to 3 days, and it was observed that plaques could be confirmed.
(vii) Plaque was collected and added to 1 mL of MEM / BSA and mixed well.
(viii) The operations of (i) to (vii) were performed using the virus solution of (vii) to confirm that it was an escape mutant strain.
(ix) RNA was extracted from the escape mutant virus culture solution using the Virtual RNA Extraction Kit (QIAGEN), and the hemagglutinin gene was amplified by RT-PCR.
(x) The hemagglutinin gene was sequenced to identify amino acids essential for the binding of mouse 36-1 antibody and mouse 61 antibody. For the sequence, 10 strains of escape mutant against mouse 36-1 antibody and 10 strains of escape mutant against mouse 61 antibody were provided.
マウス３６－１抗体およびマウス６１抗体のエピトープを以下の手順で決定した。ウイルスとしてＡ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１）を感染させた鶏漿尿液を用いた。
(i) ウイルスの１０倍階段希釈列を作製した。
(ii) マウス３６－１抗体およびマウス６１抗体の精製抗体をそれぞれ培地（ＭＥＭ）で２０μｇ／ｍＬに希釈し、小試験管に１００μＬずつ分注した。コントロールとして培地のみを小試験管に１００μＬずつ分注した。
(iii) 抗体を分注した小試験管に１０－１～１０－６までのウイルスを１００μＬずつ加え、時々混和しながら室温で１時間反応させた。
(iv) ６ｗｅｌｌプレートでコンフルエントな状態のＭＤＣＫ細胞を、血清を含まないＭＥＭ培地で洗浄し、(iii)の反応液を０．２ｍＬずつ各ウェルに接種して、プレートを１０～１５分ごとに振とうしながら３５℃で１時間吸着させた。
(v) ２×ＭＥＭ／ＢＳＡと２％Ｂａｃｔ Ａｇａｒ液をウォーターバスで約４２℃に温めてから、両液を等量混合した。これにアセチルトリプシン０．０００５％濃度になるように重層直前に添加した。また、ハイブリドーマ３６－１またはハイブリドーマ６１－２－１を腹腔内に接種したＢＡＬＢ／ｃマウスから得た腹水を、最終濃度が１：１０００になるように添加した。
(vi) (v)の溶液を(iv)のＭＤＣＫ細胞に重層し、３５℃、５％ＣＯ２存在下で２～３日間培養し、プラークが確認できることを観察した。
(vii) プラークを採取し、１ｍＬのＭＥＭ／ＢＳＡに加え、よく混和した。
(viii) (vii)のウイルス液を用いて(i)～(vii）の操作を行い、エスケープミュータント株であることを確認した。
(ix) エスケープミュータントのウイルス培養液からＶｉｒａｌ ＲＮＡ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（QIAGEN）を用いてＲＮＡを抽出し、ＲＴ－ＰＣＲによりヘマグルチニン遺伝子を増幅した。
(x) ヘマグルチニン遺伝子のシークエンスを行い、マウス３６－１抗体およびマウス６１抗体の結合に必須のアミノ酸を同定した。シークエンスには、マウス３６－１抗体に対するエスケープミュータント１０株、マウス６１抗体に対するエスケープミュータント１０株を供した。 [Example 7. (Identification of epitope)
The epitopes of mouse 36-1 antibody and mouse 61 antibody were determined by the following procedure. As a virus, chicken chorioallantoic fluid infected with A / Viet Nam / 1194/2004 (H5N1) was used.
(i) A 10-fold serial dilution series of virus was prepared.
(ii) Purified antibodies of mouse 36-1 antibody and mouse 61 antibody were each diluted with a medium (MEM) to 20 μg / mL, and dispensed in 100 μL each into a small test tube. As a control, 100 μL of the medium alone was dispensed into small test tubes.
(iii) 100 μL of 10 −1 to 10 −6 virus was added to each small test tube into which the antibody was dispensed, and allowed to react at room temperature for 1 hour with occasional mixing.
(iv) MDCK cells in a confluent state on a 6-well plate are washed with MEM medium without serum, and 0.2 ml of the reaction solution of (iii) is inoculated into each well, and the plate is inoculated every 10 to 15 minutes. The sample was adsorbed at 35 ° C. for 1 hour with shaking.
(v) 2 × MEM / BSA and 2% Bact Agar solution were warmed to about 42 ° C. in a water bath, and then equal amounts of both solutions were mixed. To this, acetyltrypsin was added immediately before the overlay so that the concentration was 0.0005%. In addition, ascites obtained from BALB / c mice inoculated intraperitoneally with hybridoma 36-1 or hybridoma 61-2-1 was added to a final concentration of 1: 1000.
(vi) The solution of (v) was layered on the MDCK cells of (iv) and cultured at 35 ° C. in the presence of 5% CO 2 for 2 to 3 days, and it was observed that plaques could be confirmed.
(vii) Plaque was collected and added to 1 mL of MEM / BSA and mixed well.
(viii) The operations of (i) to (vii) were performed using the virus solution of (vii) to confirm that it was an escape mutant strain.
(ix) RNA was extracted from the escape mutant virus culture solution using the Virtual RNA Extraction Kit (QIAGEN), and the hemagglutinin gene was amplified by RT-PCR.
(x) The hemagglutinin gene was sequenced to identify amino acids essential for the binding of mouse 36-1 antibody and mouse 61 antibody. For the sequence, 10 strains of escape mutant against mouse 36-1 antibody and 10 strains of escape mutant against mouse 61 antibody were provided.
マウス３６－１抗体に対するエスケープミュータント株、およびマウス６１抗体に対するエスケープミュータント株は、いずれもＡ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１）株のヘマグルチニンのアミノ酸配列（配列番号１）における第１７７位のリジンおよび第２０５位のリジンの少なくとも一方が他のアミノ酸に変異していた。この結果から、マウス３６－１抗体およびマウス６１抗体は、Ａ／Ｖｉｅｔ Ｎａｍ／１１９４／２００４（Ｈ５Ｎ１）株のヘマグルチニンのアミノ酸配列（配列番号１）における第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識する抗体であることが明らかとなった。
The escape mutant strain for the mouse 36-1 antibody and the escape mutant strain for the mouse 61 antibody are both lysine at position 177 in the amino acid sequence of hemagglutinin (SEQ ID NO: 1) of the A / Viet Nam / 1194/2004 (H5N1) strain. And at least one of lysine at position 205 was mutated to another amino acid. From this result, the mouse 36-1 antibody and the mouse 61 antibody were prepared by lysing the lysine at position 177 and lysine at position 205 in the amino acid sequence (SEQ ID NO: 1) of hemagglutinin of the A / Viet Nam / 1194/2004 (H5N1) strain. It was revealed that the antibody recognizes the contained epitope.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。
The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.
[規則26に基づく補充 10.02.2011]
[Supplement under rule 26 10.02.2011]
Claims (14)
- Ｈ５亜型インフルエンザウイルスのヘマグルチニンと特異的に結合し、Ｈ５亜型インフルエンザウイルスの感染性を中和する抗体であって、配列番号１で示されるアミノ酸配列からなるヘマグルチニンの第１７７位のリジンおよび第２０５位のリジンを含むエピトープを認識することを特徴とする抗体。 An antibody that specifically binds to hemagglutinin of H5 subtype influenza virus and neutralizes infectivity of H5 subtype influenza virus, the lysine at position 177 of hemagglutinin consisting of the amino acid sequence shown in SEQ ID NO: 1 and An antibody characterized by recognizing an epitope containing lysine at position 205.
- 以下の（ａ）および（ｂ）を含むことを特徴とする請求項１に記載の抗体。
（ａ）配列番号２または４で示されるアミノ酸配列の第３１位－第３５位からなるＣＤＲ１、第５０位－第６６位からなるＣＤＲ２および９８位－第１０９位からなるＣＤＲ３を含む重鎖可変領域
（ｂ）配列番号３または５で示されるアミノ酸配列の第２４位－第３３位からなるＣＤＲ１、第４９位－第５５位からなるＣＤＲ２および第８８位－第９６位からなるＣＤＲ３を含む軽鎖可変領域 The antibody according to claim 1, comprising the following (a) and (b):
(A) Heavy chain variable comprising CDR1 consisting of positions 31 to 35, CDR2 consisting of positions 50 to 66, and CDR3 consisting of positions 98 to 109 of the amino acid sequence shown in SEQ ID NO: 2 or 4 Region (b) a light chain comprising CDR1 consisting of positions 24 to 33, CDR2 consisting of positions 49 to 55 and CDR3 consisting of positions 88 to 96 of the amino acid sequence shown in SEQ ID NO: 3 or 5 Chain variable region - 配列番号２または４で示されるアミノ酸配列からなる重鎖可変領域と、配列番号３または５で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする請求項２に記載の抗体。 The antibody according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or 4, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 or 5.
- 配列番号６または８で示されるアミノ酸配列からなる重鎖可変領域と、配列番号７または９で示されるアミノ酸配列からなる軽鎖可変領域とを含むことを特徴とする請求項２に記載の抗体。 The antibody according to claim 2, comprising a heavy chain variable region consisting of the amino acid sequence shown by SEQ ID NO: 6 or 8, and a light chain variable region consisting of the amino acid sequence shown by SEQ ID NO: 7 or 9.
- ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）によって産生されることを特徴とする請求項３に記載の抗体。 4. The antibody according to claim 3, which is produced by hybridoma 36-1 (Accession No. NITE BP-832) or hybridoma 61-2-1 (Accession No. NITE BP-833).
- ハイブリドーマ３６－１（受託番号ＮＩＴＥ ＢＰ－８３２）またはハイブリドーマ６１－２－１（受託番号ＮＩＴＥ ＢＰ－８３３）。 Hybridoma 36-1 (Accession Number NITE BP-832) or Hybridoma 61-2-1 (Accession Number NITE BP-833).
- 請求項１～５のいずれかに記載の抗体の機能的断片。 A functional fragment of the antibody according to any one of claims 1 to 5.
- 請求項１～５のいずれかに記載の抗体の重鎖可変領域をコードするポリヌクレオチド。 A polynucleotide encoding the heavy chain variable region of the antibody according to any one of claims 1 to 5.
- 請求項１～５のいずれかに記載の抗体の軽鎖可変領域をコードするポリヌクレオチド。 A polynucleotide encoding the light chain variable region of the antibody according to any one of claims 1 to 5.
- 請求項８および／または９に記載のポリヌクレオチドを含む発現ベクター。 An expression vector comprising the polynucleotide according to claim 8 and / or 9.
- 請求項１０に記載の発現ベクターが導入された細胞。 A cell into which the expression vector according to claim 10 has been introduced.
- 請求項１～５のいずれかに記載の抗体または請求項７に記載の機能的断片を被験試料に接触させる工程を包含することを特徴とするＨ５亜型インフルエンザウイルスの検出方法。 A method for detecting an H5 subtype influenza virus, comprising a step of bringing the antibody according to any one of claims 1 to 5 or the functional fragment according to claim 7 into contact with a test sample.
- 請求項１～５のいずれかに記載の抗体または請求項７に記載の機能的断片を含むことを特徴とするＨ５亜型インフルエンザウイルスの検出キット。 A detection kit for an H5 subtype influenza virus comprising the antibody according to any one of claims 1 to 5 or the functional fragment according to claim 7.
- 請求項１～５のいずれかに記載の抗体または請求項７に記載の機能的断片を有効成分として含むことを特徴とするＨ５亜型インフルエンザウイルス感染疾患の予防または治療薬。 A preventive or therapeutic agent for an H5 subtype influenza virus infection disease comprising the antibody according to any one of claims 1 to 5 or the functional fragment according to claim 7 as an active ingredient.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013087069A (en) * | 2011-10-17 | 2013-05-13 | Toyobo Co Ltd | Monoclonal antibody specifically recognizing h5 subtype influenza virus |
| WO2014097762A1 (en) * | 2012-12-20 | 2014-06-26 | 国立大学法人熊本大学 | Antibody to highly pathogenic avian influenza |
| US8877200B2 (en) | 2012-05-10 | 2014-11-04 | Visterra, Inc. | HA binding agents |
| US10513553B2 (en) | 2015-11-13 | 2019-12-24 | Visterra, Inc. | Compositions and methods for treating and preventing influenza |
| US11230593B2 (en) | 2019-03-25 | 2022-01-25 | Visterra, Inc. | Compositions and methods for treating and preventing influenza |
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| JP2008104450A (en) * | 2006-09-29 | 2008-05-08 | Osaka Industrial Promotion Organization | Monoclonal antibody to highly pathogenic avian influenza virus |
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| JP2008104450A (en) * | 2006-09-29 | 2008-05-08 | Osaka Industrial Promotion Organization | Monoclonal antibody to highly pathogenic avian influenza virus |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013087069A (en) * | 2011-10-17 | 2013-05-13 | Toyobo Co Ltd | Monoclonal antibody specifically recognizing h5 subtype influenza virus |
| US8877200B2 (en) | 2012-05-10 | 2014-11-04 | Visterra, Inc. | HA binding agents |
| US9096657B2 (en) | 2012-05-10 | 2015-08-04 | Visterra, Inc. | HA binding agents |
| US9969794B2 (en) | 2012-05-10 | 2018-05-15 | Visterra, Inc. | HA binding agents |
| US10800835B2 (en) | 2012-05-10 | 2020-10-13 | Visterra, Inc. | HA binding agents |
| WO2014097762A1 (en) * | 2012-12-20 | 2014-06-26 | 国立大学法人熊本大学 | Antibody to highly pathogenic avian influenza |
| JPWO2014097762A1 (en) * | 2012-12-20 | 2017-01-12 | 国立大学法人 熊本大学 | Antibodies against highly pathogenic avian influenza |
| US10513553B2 (en) | 2015-11-13 | 2019-12-24 | Visterra, Inc. | Compositions and methods for treating and preventing influenza |
| US11230593B2 (en) | 2019-03-25 | 2022-01-25 | Visterra, Inc. | Compositions and methods for treating and preventing influenza |
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