WO2000068262A1 - Nouvel antigene pollinique - Google Patents

Nouvel antigene pollinique Download PDF

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Publication number
WO2000068262A1
WO2000068262A1 PCT/JP2000/003031 JP0003031W WO0068262A1 WO 2000068262 A1 WO2000068262 A1 WO 2000068262A1 JP 0003031 W JP0003031 W JP 0003031W WO 0068262 A1 WO0068262 A1 WO 0068262A1
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Prior art keywords
protein
antibody
derivative
partial peptide
pollen
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PCT/JP2000/003031
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English (en)
Japanese (ja)
Inventor
Hironao Ueno
Kenya Shitara
Nobuo Hanai
Seiji Sugimoto
Keiichi Yano
Akiko Furuya
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Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU44316/00A priority Critical patent/AU4431600A/en
Publication of WO2000068262A1 publication Critical patent/WO2000068262A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a diagnosis of an allergic disease containing a novel protein having allergen activity in an allergic disease, a partial peptide of the protein or a derivative thereof, the protein, a partial peptide of the protein or a derivative thereof as an active ingredient.
  • Hay fever is one of the most serious allergic diseases in Japan.
  • pollen that cause allergies, such as cedar, cypress, birch, duckweed, oats, rice and ragweed.
  • cedar pollen accounts for about 80% of hay fever.
  • Cedar hay fever cases have increased sharply since the 1970s, accounting for more than 10% of the population.
  • air pollution caused by NOx in vehicle exhaust gas is also considered to be a cause of cedar pollinosis, and the number of cedar pollinosis patients is still increasing.
  • information such as the amount of cedar pollen scattered is taken up in the media every day, and there is a problem that cedar pollinosis can no longer be neglected in public health.
  • Pollinosis is an allergic disease caused mainly by pollen allergen, an antigenic substance in pollen. Allergy is a tissue disorder that occurs locally or systemically when a living body sensitized to a specific antigen comes in contact with the same antigen again. Allergic reactions are classified as type I-IV according to Coombs and Gell Pollinosis is classified as a type I allergy mediated by IgE, which responds specifically to pollen allergens.
  • Its pathological condition is mainly immediate allergic disease with rhinitis and conjunctivitis as the main symptoms, and it causes watery nasal discharge, sneezing, itching, lacrimation, coughing, and sometimes general malaise (atopy Allergy diseases: supervision, supervised by Yuichi Yamamura, Yoshikazu Giri, Nakayama Shoten, 1992, Allergic diseases; edited by Akimasa Motomoto, Takemasa Nakagawa, Yakuhin Yaku Shuppan Co., Ltd., 1985).
  • the pollen allergen eluted from the pollen particles in contact with the mucous membrane of the nasal cavity, mesotrum, and trachea is taken up by antigen presenting cells such as macrophages. Thereafter, upon reaching the antigen-presenting cell campa tissue incorporating the antigen, a specific immunological response to pollen allergen is induced in the lymph tissue, and IgE that specifically reacts with the allergen is produced.
  • the IgE is in the bloodstream Binds to IgE receptors on mast cells and basophils. At this time, when the pollen allergen reenters the living body, IgE specific to the pollen allergen on mast cells and basophils binds to the invading pollen allergen.
  • the mast cells and the basophils are activated, and chemical mediators such as histamine, serotonin, anaphylactic eosinophil chemotactic factor, neutrophil chemotactic factor, prostaglandin, and peptide leukotriene are circulated from within the cell.
  • chemical mediators such as histamine, serotonin, anaphylactic eosinophil chemotactic factor, neutrophil chemotactic factor, prostaglandin, and peptide leukotriene are circulated from within the cell.
  • histamine histamine acts on the sneezing center rather than the sensory terminal via the HI receptor at the sensory nerve terminal, causing sneezing.
  • hay fever is a symptomatic treatment that mainly administers antiallergic agents, anti-inflammatory agents, etc., and desensitization therapy in which an antigen (allergen extract) extracted from pollen is administered little by little as a desensitizing agent. Partially used together.
  • Anti-allergic agents or anti-allergic agents such as anti-allergic agents mainly acting to suppress release of chemical mediators, anti-histamine agents mainly acting to antagonize histamine at HI receptors, and steroid agents mainly acting to suppress the production of inflammatory mediators Inflammatory agents are used for symptomatic treatment to the last of the above-mentioned mechanism of hay fever, so that allergic symptoms caused by hay fever can be temporarily alleviated, but hay fever cannot be cured.
  • allergen extracts used in desensitization therapy are also used for diagnosis and are indispensable for both treatment and diagnosis of hay fever.
  • the currently used allergen extract is a crudely extracted antigen obtained by extracting pollen in an aqueous solvent and appropriately diluting it.Therefore, even if the same extraction method is used, the composition tends to fluctuate greatly depending on the pollen of the raw material. However, there is a problem that it is extremely non-uniform chemically and it is difficult to obtain a certain desensitizing effect. For this reason, antigen administration for a long period of time may be necessary until the desensitizing effect appears, which also has a problem that a heavy burden is imposed on the patient. Furthermore, the fact that allergen extracts currently used contain many impurities also poses a problem in the effectiveness and reliability of treatment and diagnosis.
  • allergenic proteins derived from cedar pollen have been reported as follows. It has been tell.
  • Japanese Patent Application Laid-Open No. 7-188289 reports a polypeptide having a molecular weight of 55,000 Da and a pI of 9.0
  • Japanese Patent Application Laid-Open No. 7-188291 discloses a polypeptide having a molecular weight of 44,000 to 54,000 Da and ⁇ . Glycoproteins have been reported.
  • cedar pollinosis patients are targeted, such as some patients responding to only one of Cry j and Cry j 2, accurate diagnosis or effective treatment of cedar hay fever can be achieved. Is still insufficient with allergens reported so far.
  • the present invention relates to the following (1) to (32).
  • a protein having the following physicochemical properties (1) A protein having the following physicochemical properties.
  • N-terminal amino acid sequence has an amino acid sequence represented by Xaa—Leu—Phe—Pro—Lys—Glu—Ala—Leu—Pro—Thr at the N-terminus (where Xaa is Glu, Asp, His, Gl y, Arg, Trp or Cys).
  • (V) Stability Does not substantially deactivate even when left in an aqueous solution (pH 7.3) at 4 ° C for 2 months.
  • Xaa preferably represents Glu.
  • Protein ⁇ The protein according to the above (1) or (2), which is derived from cedar pollen.
  • a diagnostic agent for an allergic disease comprising the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof as an active ingredient.
  • a therapeutic drug for an allergic disease comprising, as an active ingredient, the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof.
  • An agent for preventing an allergic disease comprising as an active ingredient the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof.
  • An allergic disease desensitizing agent comprising, as an active ingredient, the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof.
  • Desensitization therapy characterized by repeatedly orally or parenterally administering the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof. .
  • An agent for diagnosing an allergic disease comprising the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof, and a known allergen or a derivative thereof as an active ingredient .
  • a therapeutic drug for an allergic disease comprising as an active ingredient the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof, and a known allergen or a derivative thereof as an active ingredient .
  • a prophylactic agent for an allergic disease comprising the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof, and a known allergen or a derivative thereof as an active ingredient .
  • Allergy disease desensitization comprising the protein according to any one of the above (1) to (5), a partial peptide of the protein or a derivative thereof, and a known allergen or a derivative thereof as an active ingredient. Formulation.
  • the protein according to any one of (1) to (5) above, a partial peptide of the protein or a derivative thereof, and a composition containing a known allergen or a derivative thereof are orally or parenterally administered. Desensitization therapy characterized by repeated administration.
  • a diagnostic agent for an allergic disease comprising the antibody according to any one of (22) to (26) above as an active ingredient.
  • a remedy for allergic disease comprising the antibody according to (1) or (22) to (26) as an active ingredient.
  • a prophylactic agent for allergic diseases which comprises the antibody described in the item (1) of (22) to (26) above as an active ingredient.
  • the present inventors have found that an unknown cedar pollen-derived protein has allergen activity and has properties and properties different from those of known cedar pollen-derived proteins. Further, compared to known cedar pollen-derived proteins, the protein of the present invention shows a stronger binding activity to serum IgE of cedar pollinosis patients, and furthermore, serum IgE antibodies against the purified protein of the present invention. The titer showed a high correlation with the IgE antibody titer against cedar pollen-derived allergen extract used in the existing in vitro diagnosis.
  • the allergic disease of the present invention includes all allergic diseases such as an allergic disease caused by one or more homologous allergens and an allergic disease caused by one or more different allergens.
  • insects such as pollen, house dust and mite, and allergens such as food.
  • -Allergen extracts which are crude extracts from allergens and allergens, and major allergens purified from the allergen extract.
  • Major allergens include Cry j 1 and Cry j 2 derived from cedar pollen, Cha 0 KSuzeki derived from hinoki pollen, et al. Molecular Immunology, Vol. 33, 451, 1996), derived from cypress pollen Bet v Bet v 2, Amb a 1, Amb a 2, derived from ragweed pollen, Der f 1, Der f 2, Der pl, Derp 2, derived from mite bodies, Fel d 1, derived from cat hair, etc. Can be
  • Allergic diseases caused by pollen allergens include allergic diseases caused by multiple types of pollen, and allergic diseases caused by allergens other than pollen in addition to pollen.
  • an allergic disease caused by multiple types of pollen Japanese cedar pollinosis patients have an allergic reaction to S-cypress pollen (Science of hay fever, Yozo Saito 'Tidetake Ide, Doujin Kagaku, 1994
  • An example of an allergic disease caused by allergens other than pollen in addition to pollen is an example in which a cedar pollinosis patient shows an allergic reaction to unrelated allergens other than pollen such as mites.
  • the main symptoms of allergic diseases include allergic rhinitis, allergic conjunctivitis, allergic dermatitis and bronchial asthma.
  • the protein of the present invention is obtained by isolating and purifying from a male flower or a flower bud that forms pollen or pollen. Any pollen can be used as long as the protein of the present invention can be isolated and purified.
  • Any pollen can be used as long as the protein of the present invention can be isolated and purified.
  • Japanese cedar (Cryptomeria japonica) pollen which belongs to the cedar family of the cedar family, may be mentioned, for example.
  • Proteins homologous to the protein of the present invention can also be isolated from pollen that causes allergies other than cedar pollen.For example, pollen such as hinoki, birch, duckweed, ryegrass, rice, and grasshopper can be isolated. can give.
  • the protein of the present invention can be produced by extracting the above-mentioned pollen with a solvent and purifying the resulting extract.
  • pollen collected from a male flower is suspended and suspended in a solvent, and immersed while stirring as necessary.
  • Extraction solvents include buffers or water commonly used by those skilled in the art of protein chemistry.
  • the buffer include a phosphate buffer, a citrate buffer, a Tris-HCl buffer, an acetate buffer and the like.
  • An appropriate salt or hydrophilic organic solvent may be added to the above-mentioned solvent for the purpose of increasing the protein extraction efficiency.
  • the salt include sodium chloride and potassium chloride.
  • the hydrophilic organic solvent include methanol, ethanol, butanol, acetone and the like.
  • the suspension obtained above is centrifuged, and the supernatant is collected. Furthermore, the amount of protein recovered can be increased by repeatedly performing the above-described immersion and centrifugation extraction operations on the residue. Usually, most of the protein can be extracted by repeating this extraction operation 1 to 5 times.
  • Purification means usually used for protein separation and purification include salting out, dialysis, filtration, concentration, centrifugation, various types of chromatography, and electrophoresis.
  • Examples of various types of chromatography include ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
  • Examples of the electrophoresis include polyacrylamide gel electrophoresis, isoelectric focusing, and the like.
  • the allergen activity of the protein obtained above can be detected using the presence and reactivity of specific IgE or specific lymphocytes derived from serum or blood cells of hay fever patients as indicators.
  • Methods for detecting specific IgE or a reaction mediated by specific IgE include a stress test method and an in vitro test method.
  • the stress test method is a test method in which the obtained protein is loaded on a hay fever patient and an allergic reaction is observed.
  • In vitro test methods include enzyme immunoassay and radioimmunoassay using serum from hay fever patients, and leukocytes from hay fever patients.
  • a histamine release test that detects histamine release by a full cell, basophil) force.
  • Means for detecting specific lymphocytes include a proliferative response test using lymphocytes from hay fever patients. (Atopy's allergic disease; supervised by Yuichi Yamamura, Yoshikazu Giri, Nakayama Shoten, 1992)
  • serum obtained from peripheral blood collected from hay fever patients is reacted with the obtained protein immobilized on an appropriate carrier, and the serum for the obtained protein is used.
  • This is a method for detecting specific IgE in the medium, such as ELISA (enzyme-linked immunosorbent assay) and RA f (radio-allergosorbent test).
  • the histamine release test is performed using the obtained protein and the leukocyte fraction containing fertilized cells and basophils prepared from the peripheral blood of a hay fever patient, or the peripheral blood of a healthy human subject to the serum of a hay fever patient. This is a method for detecting histamine released by reacting the obtained leukocyte fraction and activating mast cells or basophils via IgE specific to the obtained protein.
  • the obtained protein is reacted with lymphocytes prepared from the peripheral blood of a hay fever patient, and the T cell or the B cell specific activation reaction or proliferation to the obtained protein is performed. This is a method of detecting a response.
  • partially purified proteins contain pollen antigens such as Cry j 1. Therefore, in order to obtain a higher purity protein, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc.
  • the purified protein of the present invention can be obtained by applying a combination of methods such as reverse phase chromatography, hydrophobic chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing.
  • a partial peptide having allergen activity can be obtained from the protein of the present invention.
  • the partial peptide of the present invention can be produced from the protein separated and purified by the method (1). That is, the protein can be limitedly degraded by a proteolytic enzyme or a chemical technique, and a partial peptide can be obtained by a method similar to the method for purifying the protein from the obtained reaction mixture.
  • proteolytic enzymes include trypsin, chymotrypsin, pepsin, papain, and V8 protease.
  • the chemical method include a decomposition reaction with bromomethane under acidic conditions of formic acid.
  • a peptide having a partial amino acid sequence of the protein can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • Fmoc method fluorenylmethyloxycarbonyl method
  • tBoc method t-butyloxycarbonyl method.
  • Kuwawa Trading Advanced ChemTech, USA
  • Perkin-Elmer Japan US Perkin-Elmer
  • Aroca US Protein Technology Instrument
  • Kurabo US SyntheceU-Vega
  • Nippon Perceptive 'Limited US PerSeptive
  • Shimadzu Corporation It can also be produced using a peptide synthesizer such as
  • the partial peptide having an allergen activity of the present invention can be obtained by screening a plurality of peptides having a partial sequence of the amino acid sequence of the protein by a method similar to the method for detecting the allergen activity of a protein.
  • Examples of the method include the above-mentioned 1 (1) enzyme immunoassay using sera from hay fever patients and a proliferation response test using lymphocytes from hay fever patients.
  • the present invention includes derivatives of the protein of the present invention or partial peptides thereof.
  • Derivatives refer to modified forms and modified forms aimed at eliminating or attenuating the reactivity with IgE in a living body.
  • Derivatives can be used alone or in combination with existing protein chemistry or protein chemistry techniques [Biochemistry Experiment Course 1 Protein Chemistry IV, edited by the Biochemical Society of Japan, Neogene Chemistry Experiment Course 1 Protein IV, Biochemistry Society], etc. Can be obtained. For example, after cleaving the disulfide bond of the protein of the present invention in the presence of a reducing agent such as dithiothreitol, the SH group of the resulting cysteine residue is blocked with odoacetic acid or the like to modify the higher-order structure.
  • a reducing agent such as dithiothreitol
  • the protein of the present invention a partial peptide of the protein and a derivative thereof can be used alone or in combination as a diagnostic, preventive or therapeutic agent for allergic diseases.
  • At least one of the protein of the present invention, a partial peptide of the protein and a derivative thereof is mixed with the above-mentioned known allergen to be used as a diagnostic, preventive or therapeutic drug for allergic diseases. You can also.
  • the method for preparing a diagnostic agent for pollen allergic disease using the protein of the present invention, a partial peptide of the protein and a derivative thereof as an active ingredient is not particularly limited, but the protein and the partial protein purified according to the above-mentioned method are used. Peptides and their derivatives can be concentrated and collected as a liquid, or further freeze-dried and collected as a solid, and can be prepared and used alone or as an appropriate combination agent.
  • the diagnostic agent used in the stress test is not only a single form, but also a physiologically acceptable carrier, excipient, diluent, stabilizer, solubilizer, emulsifier, buffer Agent, soothing agent, preservative, coloring agent and the like.
  • a diagnostic agent used in an in vitro test which is one of the diagnostic methods described later, not only a single form, but also a physiologically, immunologically or immunochemically acceptable carrier, excipient, and dilution can be used. It can be used as a compounding agent to which agents, stabilizers, dissolution aids, emulsifiers, buffers, preservatives, coloring agents and the like are added.
  • the method for preparing a therapeutic or prophylactic agent comprising the protein of the present invention, a partial peptide of the protein and a derivative thereof as an active ingredient is not particularly limited.
  • a protein purified according to the above-described method, a protein purified from the protein, The partial peptides and their derivatives are concentrated and prepared as a liquid, or further freeze-dried and prepared as a solid, and prepared alone or as an appropriate combination, and used as a therapeutic or prophylactic agent for hay fever. Can be used.
  • prophylactic or therapeutic agent for hay fever can be administered by a usual administration route, for example, an oral, subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, etc. administration method.
  • Dosage forms include, for example, injections, powders, fine granules, granules, tablets, capsules, sprays, eye drops, nasal drops, cataplasms, lotions, troches, sublingual tablets and the like.
  • the desensitizer containing the protein of the present invention, a partial peptide of the protein and a derivative thereof as an active ingredient is prepared in the same manner as the above-mentioned drugs, and is used in the same manner as a general desensitizer containing an allergen. be able to.
  • the use example is shown below, but it is not limited to this.
  • a diagnostic agent containing the above-mentioned protein of the present invention, a partial peptide of the protein or a derivative thereof as an active ingredient, or a protein of the present invention, a partial peptide of the protein or a derivative thereof and a known allergen or the allergen Appropriate drugs, dosages and usages are determined based on the results of skin tests using a diagnostic drug or the like whose derivative is the active ingredient.
  • an appropriate amount of the diagnostic agent is administered, and if the wheal diameter or redness diameter at the administration site after 15 to 30 minutes is a certain value or more, the result is considered positive.
  • the protein of the present invention For a patient determined to be positive, the protein of the present invention, a partial peptide of the protein Or a derivative thereof, or the protein of the present invention, or a partial peptide of the protein, orally or parenterally administer the derivative and a known allergen or a desensitizer containing the allergen derivative as an active ingredient. .
  • the dose varies depending on the patient's symptoms and the route of administration.In general, the dose should be about 100 // g or less per adult, preferably 10 zg or less, while observing the course after administration. It is appropriately selected [JP-A-7-179494, JP-A-7-133229], and is administered repeatedly once a week or once a month.
  • desensitization containing 1/10 of the minimum concentration of allergen that is positive in the intradermal test, which is the most sensitive skin test
  • the initial dose is 0.05 ml of the drug. While observing the course of the repeated administration described above, repeat the administration once a week to once a month while gradually increasing the dose so that the local swelling of the administration is within about 5 cm in diameter. Repeated administration is continued up to the upper limit of the dose at which the swelling of the administration site is about 5 cm in diameter.
  • a desensitizer containing a partial peptide of the protein having such properties or a derivative thereof as an active ingredient has a desensitizing effect similar to a normal desensitizer, and further has the advantage of avoiding an anaphylactic reaction. It is also useful as a sensitizer with low side effects.
  • Methods for diagnosing allergic diseases include a stress test in which a subject is directly loaded with an allergen to observe an allergic reaction, and an in vitro test in which a sample prepared from the subject's tissue is used to detect reactivity with the allergen. can give.
  • Samples prepared from the subject's tissue include serum separated from peripheral blood, leukocytes such as mast cells and basophils, and lymphocytes.
  • Examples of the load test include a skin test and a provocation test.
  • In vitro tests include enzyme immunoassays and radioimmunoassays that detect allergen-specific IgE, histamine release tests that detect histamine release of mast cells and basophils by activation through allergen-specific IgE, Proliferation response tests that detect allergen-specific lymphocyte proliferation responses are examples.
  • Allergen-loaded tissues such as skin tests and challenge tests include skin, nose, ocular mucosa, and respiratory tract.
  • Skin testing methods include prick test, scratch test and intradermal test. I can do it. In the brick test and scratch test, an allergen is loaded on the subject's epithelium, and in the intradermal test, the allergen is loaded on the subject's skin.
  • the provocation test includes a nasal mucosal provocation test, an ocular mucosal provocation test, and an inhalation provocation test. Allergens are loaded into the nasal mucosa of the subject in the nasal mucosa provocation test, to the subject's eye mucosa in the ocular mucosa provocation test, and to the subject's airway in the inhalation provocation test.
  • Stress is the reaction of an allergen while scratching or pecking the epithelium in the case of a brick test or a scratch test.
  • the allergen is injected intradermally.
  • a provocation test Spraying, applying, and inhaling an allergen solution, and sticking an allergen-adhered paper piece to a mucous membrane.
  • test in a test tube is described below.
  • Enzyme immunoassay and radioimmunoassay are methods for detecting IgE bound to allergen by reacting serum in the blood collected from the subject with an allergen immobilized on a suitable carrier.
  • RAST radio-allergosorbent est
  • the histamine release test involves reacting an allergen with a leukocyte fraction, such as mast cells and basophils, prepared from the peripheral blood of a subject, or a leukocyte fraction prepared from the peripheral blood of a healthy subject to which the serum of the subject has been reacted. This is a method for detecting histamine released by the method.
  • the lymphocyte proliferation test is a method for detecting the activation or proliferative response of T cells or B cells that occurs when allergen is added to lymphocytes separated from the peripheral blood of a subject.
  • antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, and antisera containing them.
  • Examples of the monoclonal antibody of the present invention include antibodies produced by hybridomas, humanized antibodies, human antibodies, and the like.
  • a hybridoma is a monoclonal antibody having a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing a non-human mammal with an antigen and myeloma cells derived from a mouse or the like. Means a cell that produces
  • humanized antibody examples include a human-type chimeric antibody, a human-type homology determining region (hereinafter, abbreviated as CDR) -grafted antibody, and the like.
  • CDR human-type homology determining region
  • the human chimeric antibody is composed of an antibody heavy chain variable region (hereinafter, the heavy chain is referred to as an H chain, the variable region is referred to as HV or VH as a V region) and an antibody light chain variable region (hereinafter, referred to as a light chain,
  • the L chain is also called LV or VL, and the heavy chain constant region of a human antibody And the light chain constant region of a human antibody (hereinafter also referred to as CL).
  • any animal can be used as long as hybridomas can be produced, such as mice, rats, hamsters, and rabbits.
  • the human chimeric antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma that produces a monoclonal antibody that specifically reacts with the pollen antigen of the present invention, and obtains human antibody CH and human antibody CL.
  • a human-type chimeric antibody expression vector can be constructed by inserting each into an expression vector having a gene to be encoded, and can be expressed and produced by introducing into a host cell.
  • any CH can be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg).
  • the hlgG class is preferable, and the higGl and hIH belong to the hlgG class.
  • g G2, hIgG3, any subclass such hIgG4 can be used.
  • the CL of the human chimeric antibody any CL belonging to hlg can be used, which can be of ⁇ class or ⁇ class.
  • the human CDR-grafted antibody means an antibody obtained by grafting the amino acid sequences of CDRs of VH and VL of an antibody of a non-human animal at appropriate positions of VH and VL of a human antibody.
  • the human CDR-grafted antibody of the present invention is obtained by grafting VH and VL CDR sequences of a non-human animal antibody specifically reacting with the pollen antigen of the present invention into VH and VL CDR sequences of any human antibody.
  • CDNA encoding the region is constructed and inserted into an expression vector having genes encoding human antibody CH and human antibody CL, respectively, to construct a human CDR-grafted antibody expression vector, and the expression vector Can be expressed into a host cell to express and produce a human CDR-grafted antibody.
  • the CH of human CDR-grafted antibody may be any so long as it belongs to the hlg, are preferred those hlgG class, further hI g Gl belonging to hlgG class, hIgG2, hIgG3, any subclass such hIgG4 Can be used.
  • the CL of the human-type CDR-grafted antibody any CL can be used as long as it belongs to hlg, or a ⁇ class or ⁇ class.
  • Human antibodies originally mean antibodies naturally occurring in the human body, but human antibody phage libraries and human antibody production produced by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Antibodies and the like obtained from transgenic animals are also included.
  • Antibodies present in the human body can be isolated, for example, by isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibodies.
  • the antibody can be purified.
  • the human antibody phage library is a phage gene encoding antibody gene prepared from human B cells. This is a library in which antibody fragments, such as Fab and single-chain antibodies, are expressed on the phage surface by insertion into a phage. From the library, phages expressing antibody fragments having the desired antigen-binding activity can be recovered using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted to a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.
  • a human antibody-producing transgenic animal means an animal in which a human antibody gene has been integrated into cells.
  • a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of another mouse, and then developing the embryo.
  • Production of human antibodies from human antibody-producing transgenic animals can be performed in a culture by obtaining human antibody-producing antibodies and ipridomas by the usual method for producing hybridomas in mammals other than humans, and culturing them. It can be performed by producing and accumulating human antibodies.
  • the antibodies of the present invention also include the antibody fragments described below.
  • antibody fragments include Fab (abbreviation of fragment of antigen binding), Fab ′, F (ab ′) 2 , single chain antibody (hereinafter abbreviated as scFv), disulfide stabilized antibody (disulfide sta bilized). Fv; dsFv), peptides including CDRs, and the like.
  • Fab is a fragment obtained by treating IgG with proteolytic enzyme papain (which is cleaved at amino acid residue 224 of H chain). It is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is linked by a disulfide bond.
  • the Fab of the present invention can be obtained by treating an antibody that specifically reacts with the pollen antigen of the present invention with the proteolytic enzyme papain.
  • a DNA encoding the Fab of the antibody is inserted into an expression vector, and the vector is expressed by introducing the vector into a host cell to produce a Fab;
  • F (ab ') 2 is a fragment obtained by treating IgG with the protease pepsin (which is cleaved at the 234th amino acid residue in the H chain).
  • Fab binds via a disulfide bond in the hinge region. This is an antibody fragment having a molecular weight of about 100,000 and having antigen-binding activity, which is slightly larger than that obtained.
  • the F (ab ') 2 of the present invention can be obtained by treating an antibody specifically reacting with the pollen antigen of the present invention with the protease pepsin.
  • the following Fa has a thioether bond! /, Is a disulfide bond and can be made S.
  • Fab ' is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity in which a disulfide bond in the hinge region of F (ab') 2 is cleaved.
  • Fab ′ of the present invention is a reducing agent dithios that reduces F (ab ′) 2 which specifically reacts with the pollen antigen of the present invention. It can be obtained by late processing. Alternatively, Fab ′ can be expressed and produced by inserting a DNA encoding the Fab ′ fragment of the antibody into an expression vector and introducing the vector into a host cell.
  • scFv refers to a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter, referred to as P).
  • VH and VL contained in the scFv of the present invention can be used for the antibody, humanized antibody, and human antibody produced by the hybridoma of the present invention.
  • the scFv of the present invention obtains cDNA encoding VH and VL of an antibody that specifically reacts with the pollen antigen of the present invention, constructs a scFv-encoding DNA, and inserts the DNA into an expression vector.
  • the vector can be expressed by introducing the vector into a host cell to produce an scFv.
  • dsFv is obtained by binding a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted for the cystine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697 (1994)].
  • the VH and VL contained in the dsFv of the present invention any of the antibodies, humanized antibodies, and human antibodies produced by the hybridoma of the present invention can be used.
  • the dsFv of the present invention obtains cDNA encoding VH and VL of an antibody that specifically reacts with the pollen antigen of the present invention, constructs a DNA encoding dsFv, and inserts the DNA into an expression vector. Then, the vector can be expressed by introducing the vector into a host cell to produce dsFv.
  • the peptide containing the CDR comprises at least one region of the H chain or L chain CDR. Multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide containing the CDR of the present invention can be obtained by obtaining cDNA encoding VH and VH of an antibody specifically reacting with the pollen antigen of the present invention, constructing a DNA encoding the CDR, and expressing the DNA.
  • the peptide can be expressed by introducing the expression vector into a host cell to produce a peptide containing CDR.
  • the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the t Boc method (t-butyloxycarbonyl method).
  • Fmoc method fluorenylmethyloxycarbonyl method
  • t Boc method t-butyloxycarbonyl method
  • the monoclonal antibody of the present invention is prepared by preparing an antigen, immunizing an animal, and immunizing the animal.
  • a hybridoma is prepared by fusing cells with myeloma cells, and the hybridoma is cultured or administered to an animal to cause the animal to undergo ascites carcinoma, and the culture solution or ascites is collected.
  • polyclonal antibodies can be prepared by collecting immune serum from immunized animals.
  • the animals are immunized as follows. That is, an antigen prepared as described below is immunized subcutaneously, intravenously, or intraperitoneally in a non-human mammal, such as a mouse, rat, or hamster, and spleen cells, lymph nodes, and peripheral blood of the animal are immunized. The antibody producing cells are collected.
  • the protein of the present invention is purified by the above-described method, or a recombinant vector containing a DNA encoding the protein of the present invention, a partial peptide of the protein or a derivative thereof is transformed into Escherichia coli or yeast. And a method of expressing a protein or polypeptide by introducing it into a host such as an animal cell or an insect cell. Alternatively, a polypeptide having a partial sequence can also be obtained by synthesizing using an amino acid synthesizer.
  • the obtained antigen is combined with a carrier protein having high antigenicity (keyhole limpet hemocyanin, bovine serum albumin, etc.) by chemical modification or the like, or administered, or an appropriate adjuvant (eg, Freund's). Complete adjuvant (Freund's Complex Adjuvant), aluminum hydroxide gel and B. pertussis vaccine.
  • the antigen is administered after the first dose: 5 to 10 times every 2 weeks. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen is determined by an enzyme immunoassay [Antibodidies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988J! I will. A non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization is provided as a source of the antibody-producing cells.
  • the polyclonal antibody is obtained by separating and purifying serum obtained from the animal.
  • myeloma cells cell lines obtained from mice are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • myeloma cell line P3-X63Ag8-U1 P3-U1
  • P3-U1 Current Topics in Microbiology and Immunology, 18: 1-7, 1978
  • P3_NS1 / 1-Ag41 NS_l
  • SP2 / 0-Agl4 SP2 / 0-Agl4
  • SP-2 SP2 / 0-Agl4
  • P3-X63- Ag8653 6
  • J. Immunology, 123: 1548-1550, 1979 P3-X63-Ag8 (X63) (Nature, 256: 495-497, 1975) and the like
  • P3-X63Ag8 X63
  • the antibody-producing cells and myeloma cells obtained above are mixed, suspended in a HAT medium (a medium in which hypoxanthin, thymidine and aminopterin are added to a normal medium), and cultured for 7 to 14 days. After the culture, a part of the culture supernatant is taken and selected by enzyme immunoassay or the like. Then, the clones were cloned by limiting dilution, and those with a stable high antibody titer determined by enzyme immunoassay were selected as anti-pollen antigen monoclonal antibody-producing hybridoma strains. Select.
  • HAT medium a medium in which hypoxanthin, thymidine and aminopterin are added to a normal medium
  • a monoclonal antibody can be produced by culturing the above-described hybridoma cells or administering the hybridoma cells into the peritoneal cavity of an animal to cause the animal to develop ascites cancer, and collecting and purifying the culture solution or ascites.
  • Methods for purifying polyclonal and monoclonal antibodies include centrifugation, ammonium sulfate precipitation, and hydroprilic acid precipitation, or DEAE-Sepharose column, anion exchange column, protein A or G-column, or gel filtration. Use a column! /, And a method of combining chromatography and the like.
  • the monoclonal antibody of the present invention obtained by the above-mentioned method includes the monoclonal antibody KM2715.
  • the hybridoma KM2715 which produces the monoclonal antibody KM2715, was contacted by the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (1-3 1-3 Tsukuba East, Ibaraki, Japan) based on the Budapest Treaty. Deposited as FERM BP-7149.
  • the antibody or antibody fragment thereof obtained in the above 5 can be used as a diagnostic, therapeutic or prophylactic agent for allergic diseases.
  • Diagnostic, therapeutic or prophylactic agents comprising the antibody of the present invention or an antibody fragment thereof as an active ingredient are allergic diseases comprising the protein of the present invention described in 2 above, a partial peptide of the protein and a derivative thereof as an active ingredient. It can be prepared using the same method as that for preparing diagnostic, therapeutic and prophylactic agents.
  • a composition comprising the antibody or an antibody fragment thereof of the present invention may be used in a method for treating nose of an allergic disease patient. It can be administered or administered directly to mucosal tissues such as the mouth and respiratory tract, the skin, the eyes, etc., to prevent allergens from entering the body, thereby treating or preventing allergic diseases. Further, it is possible to treat or prevent an allergic disease by removing the allergen that has entered the body by intravenously administering a composition comprising the antibody or the antibody fragment thereof of the present invention to a patient with an allergic disease. it can.
  • Examples of a method for diagnosing an allergic disease using an antibody against the pollen antigen of the present invention or an antibody fragment thereof include a method for immunologically quantifying or detecting the pollen antigen of the present invention.
  • Methods include fluorescent antibody method, immunoenzymatic antibody method (ELISA), radioactive substance-labeled immunological antibody method (RIA), immunocytostaining method, western blotting method, immunoprecipitation method, Enzyme immunoassay, sandwich EL1SA method [Monoclonal antibody experiment manual (Kodansha Scientific, 1987), Seismic Chemistry Laboratory Course 5 Immunobiochemical research method (Tokyo Kagaku Dojin, 1986)] Is raised.
  • the antibody antibody method should be performed using the method described in the literature [Monoclonal Antibodies: Principles and practice, Third edition (Academic Press, 1996), Monoclonal antibody experiment manual (Kodansha Scientific, 1987)], etc. Can be.
  • the antibody of the present invention is applied to tissues exposed to pollen antigens such as nasal mucosa, palatal mucosa, respiratory mucosa, and skin isolated from a living body, or body fluids such as tears and nasal secretions, blood and blood cells, and the like.
  • the fluorescent dye is measured with a fluorometer or flow cytometer. This is a method for measuring or measuring pollen antigens at a time.
  • the immunoenzyme-linked immunosorbent assay is applied to tissues exposed to pollen antigens such as nasal mucosa, palate mucosa, airway mucosa, and skin isolated from the body, or body fluids such as tears and nasal secretions, blood, and blood cells.
  • pollen antigens such as nasal mucosa, palate mucosa, airway mucosa, and skin isolated from the body, or body fluids such as tears and nasal secretions, blood, and blood cells.
  • pollen antigens such as nasal mucosa, palate mucosa, airway mucosa, and skin isolated from the body, or body fluids such as tears and nasal secretions, blood, and blood cells.
  • an enzyme such as peroxidase or biotin
  • Radiolabeled immunoassay is applied to tissues exposed to pollen antigens such as nasal mucosa, palate mucosa, respiratory mucosa and skin isolated from the body, or body fluids such as tears and nasal secretions, blood and blood cells, etc. After reacting the antibody of the present invention or an antibody fragment thereof, and further reacting with an anti-immunoglobulin antibody which has been subjected to radiolabeling, measurement is performed using a scintillation counter or the like to quantify or detect pollen antigens. is there.
  • the antibody of the present invention or an antibody fragment thereof is reacted with intranasally separated nasal mucosa, palate mucosa, respiratory mucosa, skin and tissue exposed to ivy pollen antigen, blood cells, and the like.
  • a fluorescent substance such as fluorescin'isothiosinate (FITC), an anti-immunoglobulin antibody labeled with an enzyme such as peroxidase, and the like are reacted, and then observed using a microscope.
  • the Western blotting method uses SDS-polysaccharide to isolate tissues exposed to pollen antigens, such as nasal mucosa, palate mucosa, respiratory mucosa, and skin, or body fluids such as tears and nasal secretions, blood, and blood cells.
  • pollen antigens such as nasal mucosa, palate mucosa, respiratory mucosa, and skin
  • body fluids such as tears and nasal secretions, blood, and blood cells.
  • acrylamide gel electrophoresis Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988
  • the gel was blotted onto a PVDF membrane or a nitrocellulose membrane, and the antibody of the present invention or its antibody was applied to the membrane.
  • Antibody fragment And then reacting with a fluorescent substance such as FITC, an anti-IgG antibody labeled with an enzyme such as peroxidase, and the like, followed by confirmation.
  • the immunoprecipitation method refers to the nasal mucosa, palatal mucosa, airway mucosa, tissues exposed to pollen antigens such as skin, or body fluids such as tears and nasal secretions, blood and blood cells, etc. of the present invention.
  • a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to precipitate an antigen-antibody complex.
  • the sandwich ELISA method is a method using two kinds of antibodies having different antigen recognition sites in the antibody of the present invention or an antibody fragment thereof. Specifically, one antibody or antibody fragment is adsorbed on a plate in advance, and the other antibody or antibody fragment is labeled with a fluorescent substance such as FITC or an enzyme such as peroxidase in advance. Tissues exposed to pollen antigens such as nasal mucosa, palate mucosa, respiratory mucosa and skin isolated from the body, body fluids such as tears and nasal secretions, blood and blood cells, etc. were reacted on the antibody adsorption plate and labeled. This is a method in which an antibody or an antibody fragment thereof is reacted and a reaction is performed according to the labeling substance.
  • a fluorescent substance such as FITC or an enzyme such as peroxidase
  • the protein of the present invention, the partial peptide of the protein, and their derivatives can be detected or quantified from the pollen extract using the immunological method described in 8 above.
  • the diagnostic, therapeutic, prophylactic and desensitizing agents for allergic diseases consisting of the allergen extract described in 2 or 3 above must be kept constant in activity and quality.
  • allergen extracts are extracts obtained using a plant-based solvent, it is difficult to maintain constant activity and quality.
  • the novel pollen antigen of the present invention is considered to be a major allergen because it has high allergen activity and excellent storage stability. Therefore, keeping the content and properties of the pollen antigen in the allergen extract constant results in an allergen extract having constant activity and quality.
  • an antibody capable of specifically detecting and quantifying the pollen antigen can be a simple and useful tool for examining the pollen antigen.
  • Examples of the method for quantifying the pollen antigen include the immunological measurement method described in the above item 8, preferably a sandwich ELISA.
  • Examples of a method for measuring the stability of the pollen antigen in an allergen extract include the Western blotting method described in the above item 8 and the like.
  • the antibody of the present invention or the antibody fragment thereof is obtained by extracting the protein from the pollen protein extract. Can be used to isolate and purify.
  • FIG. 1 shows the results obtained by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) (7.5% polyacrylamide gel concentration) for the purpose of measuring the molecular weight of the protein of the present invention. It is a figure showing the result of having detected the protein component by staining.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • FIG. 2 is a diagram showing the results of analysis of the reactivity of IgE in the sera of 12 cedar pollinosis patients and 4 healthy subjects with the protein of the present invention by enzyme immunoassay.
  • IgE bound to the antigen was removed. Detected by anti-human IgE antibody.
  • CE unpurified total protein fraction
  • Fig. 3 shows the IgE antibody titer specific to the cedar pollen antigen by the cap RAST FEIA and the antigens of the protein (New), unpurified total protein fraction (CE), Cry j 1 and Cry j 2 of the present invention. It is a scatter diagram showing a correlation with a specific IgE antibody titer.
  • FIG. 4 is a diagram showing the results of analyzing the reaction specificity of the monoclonal antibody KM2715 of the present invention by an enzyme immunoassay.
  • FIG. 3 is a diagram comparing the reactivity of the monoclonal antibody with the antigen peptide-THY conjugate immobilized at 5 / ig / ml and the control peptide THY conjugate.
  • FIG. 5 shows the results of Western blotting analysis of the specific detection of the protein in the unpurified total protein fraction (CE) by the monoclonal antibody KM2715 of the present invention.
  • FIG. 2 is a diagram showing 2 ⁇ g of CE subjected to SDS-PAGE and Western blotting. BEST MODE FOR CARRYING OUT THE INVENTION
  • PBS phosphate-buffered salin; hereinafter, referred to as PBS
  • soluble components are eluted while stirring at 4 ° C for 4 hours, and centrifuged.
  • the supernatant was recovered.
  • the residue was further washed with 2 L of PBS, the same elution procedure was performed, and the supernatant was recovered and combined with the supernatant obtained the first time to obtain a crude extract fraction.
  • ammonium sulfate was added to the crude extract to 80% saturation, and the mixture was allowed to stand at 4 ° C for 2 hours to salt out protein components.
  • the salted out product collected by centrifugation is dissolved in 100 ml of 50 mM Tris-HC1 buffer (pH 7.8) and dialyzed twice for 4 to 12 hours against 2 L of the same buffer at 4 ° C. After equilibration with 50 mM Tris-HC1 buffer ( ⁇ 7.8), the mixture was passed through 10 ml of DEAE-Sepharose CL6B (Pharmacia). Further, 30 ml of a 50 mM Tris-HC1 buffer (pH 7, 8) was passed, and 130 ml of the non-adsorbed fraction was collected.
  • This non-adsorbed fraction was dialyzed twice at 4 ° C against a 10 mM acetic acid aqueous solution containing 200 mM NaCl (NaC triacetic acid aqueous solution: ⁇ 3.4) for 4 to 12 hours, and then subjected to NaC triacetic acid aqueous solution in advance.
  • the column was passed through 10 ml of equilibrated CM-Sepharose CL6B (Pharmacia).
  • the adsorbed components are separated under a continuous NaCl concentration gradient from 0 to 1 M using an aqueous solution of NaC-to-acetic acid and an aqueous solution of 10 mM acetic acid (PH3.4) containing 1 M NaCl. I let it.
  • the absorbance at 280 nm of each fraction was measured, and the gel was stained with SDS-PAGE to detect protein components.
  • the protein of the present invention was recovered at a NaCl concentration of around 0.45 M. Was. This eluted fraction contained Cry j 1, unidentified protein and the like in addition to the protein.
  • this crude fraction is concentrated by centrifugation using an ultrafiltration membrane Centricon 10 (manufactured by Amicon), equilibrated with PBS in advance, and passed through a column of Super Loose 12 (manufactured by Pharmacia) to gel. Filtration was performed. The fraction having a maximum molecular weight of 110,000 ⁇ 15,000 Da was collected as a fraction containing the protein. This fraction contained the protein and one unidentified protein.
  • the molecular weight markers for gel filtration include glutamate dehydrogenase (290,000 Da), lactate dehydrogenase (140,000 Da;), enolase (67,000 Da), adenylate kinase (32,000 Da), and cytochrome c. (12,400 Da) was used.
  • ion exchange chromatography was performed by a batch method. That is, the fraction collected by gel filtration was dialyzed twice against 10 mM acetate buffer (pH 5.0) containing 200 mM NaCl for 4 to 12 hours, and then equilibrated with the same buffer in a microcentrifuge tube in advance. The mixture was reacted with 50 ⁇ l of CM-Sepharose CL6B (Pharmacia) placed for 30 minutes. After the reaction, the non-adsorbed fraction was collected as a purified sample of the protein. The purified sample was used in the following tests after buffer replacement with ⁇ S as necessary. The yield of the protein was 0.4 ⁇ / g of cedar pollen used as a raw material, as determined by converting it into serum albumin using a BCA kit (Pierce).
  • a major band was detected at a position corresponding to a molecular weight of 51,000 soils and 5,000 Da under reducing conditions of 2-mercaptoethanol-added calo. Under non-reducing conditions, a major band was detected at a position corresponding to a molecular weight of 48,000 ⁇ 5,000 Da.
  • the molecular weight markers of SDS-PAGE include myosin (200,000 Da),] -galactosidase (116,300 Da), phosphorylase b (97,400 Da), and serum albumin (66,300 Da). Aldolase (42,400 Da;), carbonic anhydrase (30,000 Da), trypsin inhibitor (20,100 Da), and lysozyme (14,400 Da) were used.
  • the N-terminal amino acid sequence of the purified protein was analyzed by an ordinary protein chemistry method using an automated protein sequencer (Shimadzu, PPSQ-10) utilizing the Edman degradation method.
  • a sequence represented by Xaa—Leu_Phe_Pro—Lys—Glu—Ala—Leu—Pro—Thr was confirmed.
  • Xaa indicates any of Glu, Asp, His, Gly, Arg, Trp, or Cys
  • the protein having the binding activity to IgE in the serum of patients with cedar pollinosis of the present invention was analyzed by the following IgE-ELISA.
  • the cedar pollinosis patients were those who showed high reactivity (+4 grade) to sugya allergen by testing with a cap RAST FEIA allergen test diagnostic reagent manufactured by Pharmacia Upjohn. It is.
  • the purified protein was adjusted to 3 g / ml in PBS, added to a 96-well mic opening plate at 50/1 / well, allowed to stand at 4 ° C for 16 hours, and immobilized on the plate. After washing each well of the plate with PBS, PBS (B-PBS) containing l% (w / v) BSA was added at 100 1 / well, and the plate was allowed to stand at room temperature for 2 hours. After washing each well with PBS, add 50 ⁇ l / well of cedar pollinosis patient and healthy human serum diluted 2-fold with B-PBS, and react with the immobilized protein at 4 ° C for 16 hours. I let it.
  • T-PBS After washing with PBS containing 0.05% (v / v) Tween20 (T-PBS), 50 ⁇ l of biotin-labeled goat anti-HgE antibody (AmericanQualex, code: A116 BN) diluted 1,000-fold with B-PBS was used. 1 / well was added thereto, and the reaction was further performed at room temperature for 1 hour. After washing with T-PBS, avidin D-conjugated horseradish peroxidase (VEC) diluted 4,000-fold with B-PBS TOR Co., Code; A-2004) was added at 50 / l / well, and left at room temperature for 30 minutes.
  • VEC horseradish peroxidase
  • the solubility of the protein of the present invention in various solvents was examined. It was soluble in water, saline and phosphate buffer.
  • the aqueous solution dissolved in (5) was stored in a refrigerator at 4 ° C. Two months later, when the experiment (4) was performed using the aqueous solution, the same result as that obtained two months ago was obtained.
  • the protein having the above physicochemical properties was not known until now, and was determined to be a novel substance.
  • the purified proteins Cry j 1 and Cry j 2 were prepared in PBS at 3 ⁇ g / ml and CE at 30 / ig / ml in PBS. The solution was added in 50-well portions, and allowed to stand at 4 ° C for 16 hours to immobilize on a plate. After each well was washed with PBS, B-PBS was added at 100 / i1 / well and left at room temperature for 2 hours. After washing each well with PBS again, 50 ⁇ l / well of serum of a Japanese cedar pollinosis patient diluted 2-fold with B-PBS was used. And reacted at 4 ° C. for 16 hours.
  • the ABTS chromogenic substrate solution was added at 100 ⁇ l / ⁇ l at room temperature and reacted at room temperature. After color development, the reaction was stopped by adding a 5% SDS aqueous solution at 100 ⁇ l / well. The absorbance of the developed substrate was measured at a wavelength of 415 wavelengths and used as an index of the antibody titer of IgE specific to the antigen protein.
  • the diagnostic reagent used for the diagnosis of cedar pollinosis uses the total protein fraction extracted from cedar pollen as an antigen and measures the antibody titer of serum IgE specific to cedar pollen allergen Things.
  • a cap RAST FEIA manufactured by Pharmacia's Upjohn Co., Ltd. (a spear allergen binding cap; code No.CT17, an enzyme-labeled antibody reagent, and a reference reagent), which is generally used as this kind of diagnostic method, is used. ) was used as a control.
  • Cap RAST FEIA was performed according to the method recommended by Pharmacia's Upjohn.
  • the sample serum was reacted with the cap on which the total cedar pollen allergen (unpurified total protein fraction) had been previously immobilized, washed, and then reacted with an enzyme-labeled anti-IgE antibody. After further washing, the fluorescent substance generated by adding the substrate solution was detected by a fluorometer, and IgE specifically bound to the allergen was quantified.
  • Fig. 2 shows the results of IgE-ELISA.
  • a specific IgE antibody against the protein was detected, indicating that the protein was an allergen in cedar pollinosis.
  • no specific IgE antibody against the protein was detected in healthy subjects.
  • Anti-New indicates an antibody against the protein of the present invention.
  • a comparison of the ratio obtained by dividing the specific IgE antibody titer against the protein by the specific IgE antibody titer against Cry j 1 (the protein ZCry j 1) showed that 9 out of 24 patients showed 1.3 or more.
  • Fig. 3 shows a two-variable scatter plot of 12 cedar pollinosis patients and 4 healthy subjects.
  • Cap RAST FEIA and IgE-ELISA use different antigen immobilization methods, detection methods, and enzymes / substrates for detection, but specific IgE antibody titers with cap RAST FEIA and unpurified IgE-EL1SA
  • the correlation coefficient with the total protein-specific IgE antibody titer is 0.921. In other words, the evaluation of the specific IgE antibody titer performed this time was not affected by differences in the measurement system.
  • the specific IgE antibody titer against the protein in the serum of cedar pollinosis patients is equivalent to the specific IgE antibody titer against Cry j1 and Cry j2, even though the ratio of the protein to the total protein of cedar pollen is extremely low. It was more expensive. A single protein exhibiting such high allergen activity is not yet known. Therefore, it can be said that the protein has extremely high antigenicity and is a very important causative substance in the onset of cedar pollinosis.
  • a peptide (SEQ ID NO: 2) with Cys added to the C-terminal Thr of the N-terminal amino acid sequence shown in SEQ ID NO: 1 obtained in 2 (2) above for binding to a carrier protein (SEQ ID NO: 2) Tide synthesis was performed.
  • an automated peptide synthesizer P SSM-8 manufactured by Shimadzu Corporation
  • Synthetic peptides and KLH to enhance immunogenicity was used as an immunogen.
  • KLH is dissolved in PBS to adjust to 10 mg / ml, 25 mg / ml MBS [N- (maleimidobenzoyloxy) succini mide, manufactured by Nacalai Tesque, Inc.] of 1/10 volume of the KLH solution is added dropwise and stirred for 30 minutes. I let it. Free MBS was removed using a Sephadex G-25 column (Pharmacia), a gel filtration column equilibrated with PBS in advance, to obtain 2.5 mg of a KLH-MBS conjugate. Furthermore, it was mixed with 1 mg of peptide dissolved in 0.1 M sodium phosphate buffer (PH 7.0), and reacted by stirring at room temperature for 3 hours. After the reaction, what was dialyzed against PBS-0.5 MN & C1 was obtained as an antigen peptide-KLH conjugate, which was used as an immunogen.
  • MBS N- (maleimidobenzoyloxy) succini mide, manufactured by Nacalai Tesque
  • antigen was prepared by the method shown in the peptide - KLH Konjiyuge M00 / g to Mizusani ⁇ Honoré mini ⁇ Moore Ho cement (Antibodies - A Laboratory Manual, Cold Sprin g Harbor Laboratory, P 99, 1988 ) 2 mg and pertussis vaccine (manufactured by Chiba Prefectural Serum Research Institute) were administered to 5-week-old female mice (Balb / c) together with 1 ⁇ 10 9 cells, and after 2 weeks, 100 // g of antigen ⁇ peptide KLH conjugate was administered. It was administered once a week for a total of four doses.
  • Blood was collected from the fundus venous plexus of the mouse, and its serum antibody titer was examined by the enzyme immunoassay described in 4 (3). Three days after the final immunization, spleens were removed from mice with sufficiently high serum antibody titers.
  • the spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and centrifuged (OO rpm, 5 minutes). ! Resulting tris precipitate fraction - was added Anmoniumu buffer chloride solution (P H7.6),: erythrocytes were removed by treating 1-2 minutes. The resulting precipitate fraction (cell fraction) was washed three times with MEM medium and used for cell fusion.
  • MEM Minimum Essential Medium
  • Anmoniumu buffer chloride solution P H7.6
  • whole blood is collected at the time of spleen extraction, and the serum obtained after standing at room temperature for 2 hours is a peptide-specific antiserum, and the antiserum is specific for the protein of the present invention obtained in the above 1). It was an antiserum.
  • THY Thiroglobulin; thyroglobulin, Sigma
  • the above conjugate prepared at 5 ⁇ g / ml was dispensed at 50 / Z 1 / well onto an EIA plate (Grainer Co., Ltd.) and left at 4 ° C for adsorption. .
  • PBS containing l% (w / v) BSA (B-PBS) was added at 100 ⁇ l / well to block the remaining active groups, and the mixture was reacted at room temperature for 1 hour.
  • the B-PBS was discarded, and the antiserum of the immunized mouse, the supernatant of the hybridoma culture or the purified monoclonal antibody was dispensed at 50 ⁇ l / well and allowed to react for 2 hours.
  • T-PBS peroxidase-labeled ⁇ sagi anti-mouse immunoglobulin
  • P3X63Ag8U.l 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.l (P3-U1: purchased from ATCC) was cultured in normal medium (RPI medium supplemented with 10% fetal calf serum), and 2 x 10 7 or more cells were fused at the time of cell fusion. And used for cell fusion as a parent strain.
  • mice spleen cells obtained in 4 (2) and the myeloma cells obtained in 4 (4) were mixed at a ratio of 10: 1, and centrifuged (1, 200 rpm, 5 minutes). Was loosened well resulting precipitated fraction of cell groups, while stirring, at 37 ° C, polyethylene glycol one 1000 (PEG- 1000) 2g, mixture 10 8 of MEM medium 2ml and dimethyl sulfoxide 0.7ml 0.5 ml was added per mouse splenocytes. Further, lml of MEM medium was added several times to the suspension every 1 to 2 minutes, and then MEM medium was added so that the total volume became 50ml.
  • PEG- 1000 polyethylene glycol one 1000
  • the cells in the precipitate fraction obtained are gently loosened by tapping, and 100 ml of HAT medium [10. % Hat fetal serum-supplemented RPMI medium supplemented with HAT Media Supplement (Boehringer Mannheim).
  • HAT medium 10. % Hat fetal serum-supplemented RPMI medium supplemented with HAT Media Supplement (Boehringer Mannheim).
  • the suspension was dispensed into a 96-well culture plate at a volume of 200 ml / well and cultured at 37 ° C. for 10 to 14 days in a 5% CO 2 incubator.
  • the culture supernatant was examined by the enzyme immunoassay described in 4 (3), and cells that reacted with the antigen peptide but did not react with the control peptide were selected. Further, the cells contained in the well were repeatedly cloned twice by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma KM2715 recognizing a partial sequence of the protein of the present invention.
  • the monoclonal The results of analyzing the reactivity of the antibody KM2715 with the antigen peptide and the control peptide by an enzyme immunoassay are shown in FIG.
  • the monoclonal antibody KM2715 showed specific reactivity to an antigen peptide consisting of a partial sequence of the protein of the present invention.
  • Monoclonal antibodies were purified from mouse ascites.
  • An 8-week-old nude female mouse (BALB / c) treated with pristane was intraperitoneally injected with 5 to 20 ⁇ 10 6 cells / animal of the hybridoma and the hybridoma strain obtained in 4 (5). 10 to 21 days later, ascites was collected from mice in which the ascitic carcinoma of the hybridoma was changed (1 to 8 mlZ mice). After the ascites was centrifuged (1, 200 ⁇ g, 5 minutes) to remove solids, the monoclonal antibody was purified by the caprinoleic acid precipitation method [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988]. The subclass of the monoclonal antibody was determined to be IgGl by ELISA using a subcluster typing kit.
  • the monoclonal antibody KM2715 specifically reacted with a band around 51 kDa corresponding to the molecular weight of the protein in CE.
  • KM511 Japanese Unexamined Patent Publication No. 8-165300
  • KM511 Japanese Unexamined Patent Publication No. 8-165300
  • the monoclonal antibody can specifically detect and quantify the protein of the present invention in pollen allergen extract, confirming its usefulness.
  • allergen immunotherapy a vaccine for the treatment of allergic diseases
  • Allergy 696 (1998)
  • the protein has extremely high antigenicity and can be said to be a very important causative substance in the onset of cedar pollinosis. Therefore, quantification of the allergen extract using the monoclonal antibody, standardization of the allergen extract, and a method thereof are considered to be extremely useful in promoting efficient desensitization therapy.
  • the protein of the present invention has the property of binding to IgE in the serum of hay fever patients, it is useful not only as an antigen in immunochemical diagnostic methods, but also has the property of inducing allergic disease, and It is also useful as a desensitizing agent for diagnosing, treating, or preventing.

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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une nouvelle protéine possédant une activité allergène dans la pollinose ; des fragments peptidiques de cette protéine ou ses dérivés ; des méthodes diagnostiques, des médicaments et des traitements prophylactiques de l'allergie au pollen contenant, comme ingrédient actif, la protéine selon l'invention, ses fragments peptidiques ou ses dérivés. L'invention concerne également une méthode de production de la protéine selon l'invention, ses fragments peptidiques ou ses dérivés ; ainsi que des méthodes diagnostiques et des médicaments contenant cet anticorps comme ingrédient actif.
PCT/JP2000/003031 1999-05-11 2000-05-11 Nouvel antigene pollinique WO2000068262A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU44316/00A AU4431600A (en) 1999-05-11 2000-05-11 Novel pollen antigen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP11/129363 1999-05-11
JP12936399 1999-05-11

Publications (1)

Publication Number Publication Date
WO2000068262A1 true WO2000068262A1 (fr) 2000-11-16

Family

ID=15007745

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2000/003031 WO2000068262A1 (fr) 1999-05-11 2000-05-11 Nouvel antigene pollinique

Country Status (2)

Country Link
AU (1) AU4431600A (fr)
WO (1) WO2000068262A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0308147A1 (fr) * 1987-09-12 1989-03-22 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Agent d'hyposensibilisation
EP0595855A1 (fr) * 1991-07-12 1994-05-11 Immulogic Pharmaceutical Corporation Proteines peptides allergeniques provenant du pollen du cedre japonais
JPH10259198A (ja) * 1996-12-24 1998-09-29 Sankyo Co Ltd 連結したｔ細胞エピトープとその用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0308147A1 (fr) * 1987-09-12 1989-03-22 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Agent d'hyposensibilisation
EP0595855A1 (fr) * 1991-07-12 1994-05-11 Immulogic Pharmaceutical Corporation Proteines peptides allergeniques provenant du pollen du cedre japonais
JPH10259198A (ja) * 1996-12-24 1998-09-29 Sankyo Co Ltd 連結したｔ細胞エピトープとその用途

Also Published As

Publication number Publication date
AU4431600A (en) 2000-11-21

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