US20060134706A1 - Atopic dermatitis inducer - Google Patents

Atopic dermatitis inducer Download PDF

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US20060134706A1
US20060134706A1 US10/563,795 US56379504A US2006134706A1 US 20060134706 A1 US20060134706 A1 US 20060134706A1 US 56379504 A US56379504 A US 56379504A US 2006134706 A1 US2006134706 A1 US 2006134706A1
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atopic dermatitis
composition
antibody
subject
patient
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Michihiro Hide
Toshihiko Tanaka
Akio Tanaka
Kaori Ishii
Hidenori Suzuki
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Shionogi and Co Ltd
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Shionogi and Co Ltd
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Assigned to SHIONOGI & CO., LTD. reassignment SHIONOGI & CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HIDE, MICHIHIRO, ISHII, KAORI, SUZUKI, HIDENORI, TANAKA, AKIO, TANAKA, TOSHIHIKO
Publication of US20060134706A1 publication Critical patent/US20060134706A1/en
Priority to US12/461,368 priority Critical patent/US8129507B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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 invention of this application relates to an atopic dermatitis inducer (hereinafter sometimes referred to as ‘inducer’) secreted by a patient with atopic dermatitis of his or her own, a method of diagnosing atopic dermatitis using this inducer or an antibody against the inducer, and a drug for desensitization therapy of atopic dermatitis containing this inducer as an active ingredient.
  • inducer atopic dermatitis inducer
  • Atopic dermatitis is one of the atopic diseases caused by a hereditary factor that readily produces an IgE antibody against a common allergen, in addition to various environmental factors.
  • the disease starts in infancy and runs a course of disease chronically with age, and becomes milder before puberty in many cases. However, in some cases, the disease continues to persist after puberty, and also there are some cases where the disease starts after puberty.
  • Non-Patent Document 1 the present inventors have reported on purification and analysis of an antigen contained in sweat in Non-Patent Document 1.
  • the present inventors have filed a patent application for the invention entitled “atopic dermatitis inducing proteins” (Patent document 4).
  • Patent Document 1 JP-A-7-109290
  • Patent Document 2 JP-A-7-109292
  • Patent Document 3 JP-A-9-100236
  • Patent Document 4 WO 03/084991 A1
  • Non-Patent Document 1 Grant-in-Aid for Scientific Research from The Ministry of Health, Labours and Welfare of Japan on 2002, Annual Report of Research Project for Prevention, Treatment, etc. of Immunological Allergic Diseases (Vol. 1, pp. 101 to 103, issued on March 2003 by The Japanese Ministry of Health, Labour and Welfare)
  • a method of administering an inhibitor of production of an IgE antibody against an antigen shows an effect to a certain device, however, it does not essentially block the pathogen, and therefore, although it is useful in alleviating symptoms, it is not an essential treatment.
  • the origins of pathogens have been sought only from food or foreign substances present in the environment, no attention was paid to substances produced in the body.
  • the invention of this application has been made in view of the circumstances as described above, and its object is to provide an atopic dermatitis inducer produced by a patient of his or her own as a causative factor of atopic dermatitis.
  • an object of the invention is to provide an antibody binding to the above-mentioned inducer.
  • an object of the invention is to provide a method of diagnosing atopic dermatitis using the above-mentioned inducer and/or antibody.
  • an object of the invention is to provide a drug for desensitization therapy of atopic dermatitis containing the inducer as an active ingredient.
  • This application provides the following inventions as an invention for solving the above-mentioned problems.
  • a method of diagnosing atopic dermatitis which comprises testing whether or not an IgE antibody binding to the atopic dermatitis inducer of said invention (1) or (2) exists in the serum of a subject and determining that the subject whose serum contains the IgE antibody is a patient with atopic dermatitis or a high-risk individual for atopic dermatitis.
  • a method of diagnosing atopic dermatitis which comprises adding the atopic dermatitis inducer of said invention (1) or (2) to a leukocyte fraction collected from the blood of a subject, and determining that the subject is a patient with atopic dermatitis or a high-risk individual for atopic dermatitis from the degree of histamine release in the leukocyte fraction.
  • a method of diagnosing atopic dermatitis which comprises testing whether or not a substance binding to an antibody of said invention (3) exists in a biological sample of a subject, and determining that the subject whose sample contains the substance is a patient with atopic dermatitis or a high-risk individual for atopic dermatitis.
  • a reagent for determining a high-risk individual for atopic dermatitis which comprises a patch test material having the atopic dermatitis inducer of said invention (1) or (2).
  • a drug for desensitization therapy of atopic dermatitis which contains the atopic dermatitis inducer of said invention (1) or (2) as an active ingredient.
  • the “atopic dermatitis inducer” of this invention includes a “purified human secretion fraction” (a fraction not adsorbed to a cationic column, which is not adsorbed to a cationic exchange resin or a ConA column) of its own, or an “antigen molecule” or an “antigen determinant” contained in the purified fraction.
  • a purified human secretion fraction a fraction not adsorbed to a cationic column, which is not adsorbed to a cationic exchange resin or a ConA column
  • an antigen molecule or an “antigen determinant” contained in the purified fraction.
  • the one that can become an antigen molecule include constituent molecules of the living body such as proteins, carbohydrates, lipids, complexes or modifications thereof and the like, however, other synthetic compounds, non-natural compounds and the like can become an antigen.
  • what an antibody recognizes is not the entire antigen molecule, but a specific site on its surface.
  • This specific site or structure is called an antigen determinant (epitope).
  • the site (epitope) that an antibody recognizes includes not only a primary structure such as an amino acid sequence of protein, but also a specific tertiary structure constituted by plural portions on the molecule and the like. Accordingly, a high-molecular weight molecule has plural antigen determinants on its surface in many cases.
  • human secretion means secretion from the inside of the human body or from the secretory glands of the body surface, and particularly means saliva, tear, milk and sweat respectively secreted from exocrine glands (salivary glands, tear glands, mammary glands and sweat glands).
  • purified human secretion fraction is sometimes referred to as “Fr.D” in the following description.
  • Non-Patent Document 1 and Patent Document 4 The main; component of a sweat antigen described in Non-Patent Document 1 is adsorbed to a cationic exchange resin.
  • the atopic dermatitis inducing protein (Non-Patent Document 4 is contained in a fraction adsorbed to a cationic exchange resin and a ConA column (fraction adsorbed to a cationic column), and the substances in Non-Patent Document 1 and Patent Document 4 are practically the same.
  • Fr.D according to this invention is a fraction not adsorbed to a cationic column, which is not adsorbed to a cationic exchange resin or a ConA column.
  • Fr.D fraction not adsorbed to a cationic column
  • Patent Document 1 and Patent Document 4 Fr.D according to this invention is different from the fraction adsorbed to a cationic column (Non-Patent Document 1 and Patent Document 4) in terms of the effect as described below.
  • FIG. 1 shows the separation results using an anionic exchange column.
  • the upper graph shows an elution pattern of a fraction not adsorbed to ConA Sepharose using all anionic column, and the lower graph shows the histamine-releasing activities of the respective fractions.
  • FIG. 2 shows the separation results using a reverse phase column.
  • the upper graph shows an elution pattern of a fraction with an activity purified with an anionic column using a reverse phase column, and the lower graph shows the histamine-releasing activities of the respective fractions.
  • FIG. 3 shows the development pattern of Fr.D by a 15% polyacrylamide gel electrophoresis.
  • FIG. 4 shows the histamine-releasing activity of a fraction eluted from a native PAGE using blood cell fractions of 2 patients with atopic dermatitis (AD1 and AD2). The number corresponds to the fraction number in FIG. 3 .
  • antiIgE indicates an anti-IgE antibody and “MW” indicates a molecular weight marker protein used as a negative control.
  • FIG. 5 shows the results of a test of absorbing Fr.D activity by the serum of a patient with atopic dermatitis, and shows a histamine-releasing activity when the serum of a patient with atopic dermatitis or a normal subject was mixed with Fr.D.
  • antiIgE indicates an anti-IgE antibody
  • Fr.D ( ⁇ 500) indicates a 500-fold dilution of Fr.D
  • SE serum
  • Serum (AD) indicates the serum of a patient with atopic dermatitis
  • Serum (N) indicates the serum of a normal subject.
  • FIG. 6 shows the development pattern of Fr.D by a 15% denatured SDS-polyacrylamide gel electrophoresis. The bands shown in the drawing were excised from the gel and used for amino acid sequence determination.
  • FIG. 7 shows the results of a histamine release test when a rat mast cell line was sensitized with purified IgE obtained from the serum of a patient with atopic dermatitis and stimulated by a purified sweat antigen (Fr.D).
  • RBL-2H3 indicates a rat mast cell line
  • RBL-3D4 indicates transformant cell line prepared by using RBL-2H3 as a host and introducing a human high affinity IgE receptor (Fc ⁇ RI) ⁇ -subunit gene into the host
  • anti-IgE antibody indicates an anti-human IgE antibody as a control.
  • FIG. 8 shows the results of a histamine release test when basophils in the peripheral blood of a normal subject were treated with lactic acid and resensitized with IgE obtained from the serum of a patient with atopic dermatitis and then stimulated by a sweat antigen (Fr.D).
  • FIG. 9 shows the results of a histamine release test
  • a human mast cell line LAD2 was sensitized with myeloma IgE or purified IgE obtained from the serum of a patient with atopic dermatitis and stimulated by a purified sweat antigen (Fr.D).
  • anti-IgE antibody indicates on anti-human IgE antibody as a control.
  • “1/5000”, “1/1500” and “1/500” indicate the dilution ratios of the purified sweat antigen (Fr.D).
  • An atopic dermatitis inducer of the invention (1) is a substance which is contained in a human secretion, binds to a human own IgE antibody and activates mast cells and basophils, and is a purified fraction purified through the following method, an antigen molecule or an antigen determinant contained in the purified fraction.
  • insoluble matters are removed by passing a human secretion (saliva, tear or sweat) through a filter.
  • the pore size of the filter can be appropriately selected from about 0.10 to 1.00 ⁇ m depending on the type of the secretion or the like.
  • a filter with a pore size of 0.15 to 0.30 ⁇ m (Bottle Top Filter 431174, Corning) or the like.
  • ConA-affinity carrier a commercially available product (e.g., ConA-Sepharose, Amersham Parmacia, glycoprotein adsorption capacity: 45 mg/ml; ConA-agarose, Calbiochem, glycoprotein adsorption capacity: 3.5 mg/ml; ConA-agarose, Sigma, glycoprotein adsorption capacity 3 to 6 mg/ml and the like) can be used.
  • a required amount of the affinity, carrier should be an amount sufficient to adsorb the total amount of glycoprotein in the filtrate, and adsorption treatment is carried out by mixing and stirring the mixture at around 4° C. to room temperature for several hours to overnight. After the treatment, the supernatant (a fraction not adsorbed to the ConA-affinity carrier) is collected by centrifugation, filtration or the like.
  • a known column chromatography technique e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, partition chromatography, affinity chromatography, slalom chromatography and the like
  • anion exchange chromatography or reverse phase chromatography is used.
  • anion exchange chromatography a commercially available anion exchange column such as MonoQ HR 10/10 (Pharmacia Biotech) or UNO Q12 (BioRad) can be used. As for these columns, one equilibrated with a buffer (e.g., 20 mM Tris-HCl (pH 8.0) or the like) in advance is used.
  • a commercially available reverse phase column such as SOURCE 15RPC ST 4.6/100 (Pharmacia Biotech) can be used. In the case where a fraction obtained by anion exchange chromatography is loaded onto a reverse phase chromatography column, the previous fraction is diluted to around 5- to 20-fold with distilled water or the like, and loaded onto the subsequent chromatography column.
  • histamine-releasing activity can be evaluated by measuring the amount of histamine by a known method described in the report (Koro, O. et al., J. Allergy Clin. Immunol. 103, 663-670, 1999).
  • a purified human secretion fraction (Fr.D) as an atopic dermatitis inducer can be obtained.
  • imparting or Fr.D responsiveness to the blood cells of a normal subject by the serum of a patient with atopic dermatitis is completely inhibited by adding non-specific IgE derived from the serum of a patient with myeloma in advance to the blood cells of a normal subject treated with lactic acid or by heating the serum of a patient with atopic dermatitis at 56° C. for 1 hour.
  • Fr.D is stable in heating at 85° C. for 15 minutes. In addition, though it shows resistance to DNase and lipase, when Fr.D is treated with a protease, it lose the histamine-releasing activity, accordingly, the inducer has at least a protein or a peptide as one of its constituents.
  • DCD1 A peptide at the C-terminal side of DCD (DCD1) has been reported as an antibacterial peptide secreted from a sweat gland (Nature Immunology, 2, 113-1137, 2001), however, it has been totally unknown that DCD and DCD1 are inducing proteins causing atopic dermatitis by binding to an IgE antibody of the person secreting them and activating mast cells and basophiles.
  • DCD or a partial peptide thereof can be isolated and purified from human sweat by the same method as in Example 1 described later.
  • it can also be synthesized chemically based on a known method of peptide synthesis (shin Seikagaku Jikken Koza 1, Protein IV, Synthesis and Expression, Tokyo Kagaku Dojin, 1992) or the like.
  • it can also be prepared by a method of preparing a protein or a peptide with an in vitro transcription/translation system using a DNA sequence (GenBank No.
  • NM — 053283 encoding the protein or by a known genetic engineering technique using an appropriate host-vector system (a prokaryotic cell such as Bacillus subtilis or a eukaryotic cell such as yeast, an insect cell or a cell of an animal or a plant) (e.g., Shin Seikagaku Jikken Koza 1, Protein IV, Synthesis and Expression, Tokyo Kagaku Dojin, 1992; Shin Seikagaku Jikken Koza 2, Nucleic Acids III, Recombinant DNA Techniques, edited by Japanese Biochemical Society, 1992; Molecular Biology Protocols, The 2nd revised edition, edited by Koike K, Sekiya T and Kondo H., Nankodo, 1999).
  • a prokaryotic cell such as Bacillus subtilis or a eukaryotic cell such as yeast, an insect cell or a cell of an animal or a plant
  • Shin Seikagaku Jikken Koza 1, Protein IV, Synthesis and Expression Tokyo Kagaku Dojin, 1992
  • the inducer of the invention (1) binds to 2 or more antibodies and exerts its physiological activity by allowing it to have 2 or more antigen determinants.
  • the atopic dermatitis inducer of this invention can be an inducer containing 2 or more antigen determinants. Since such an antigen determinant has the same physiological activity (i.e., atopic dermatitis inducing activity) as the inducer, it can be used in a diagnostic method or as an active ingredient of a drug for desensitization therapy of this invention.
  • the antigen determinant of this invention may be one. Such an antigen determinant can be used as an antigen for antibody production, a material for the diagnostic method of the invention (4), or a material for desensitization therapy.
  • the antigen determinant can be used after allowing it to bind to a synthetic polymer or the like such as a protein (e.g., BSA), a polysaccharide or polyacrylamide.
  • a synthetic polymer or the like such as a protein (e.g., BSA), a polysaccharide or polyacrylamide.
  • the invention (3) is an antibody characterized by being prepared by using an atopic dermatitis inducer of the above-mentioned invention (1) as an antigen and specifically binding to the atopic dermatitis inducer of the above-mentioned invention (1).
  • the antibody is a polyclonal antibody or a monoclonal: antibody, and a whole molecule that can bind to the above-mentioned inducer or its epitope site, and Fab, F(ab′) 2 and Fv fragments and the like are all included.
  • Such an antibody can be prepared in accordance with a known method described in a document (Shin Seikagaku Jikken Koza 12, Molecular Immunology III, Antigens, Antibodies and Complements, Tokyo Kagaku Dojin, 1992; “Monoclonal Antibody”, co-authored by Nagamune H. and Terada H., Hirokawa Shoten, 1990; “Monoclonal Antibodies”, James W. Goding, third edition, Academic Press, 1996 or the like).
  • an antibody labeled with a labeling substance is also included.
  • a labeling substance an enzyme, a radioisotope or a fluorescent dye can be used.
  • the enzyme there is no particular limitation on the enzyme as long as it fulfills the requirements such that its turnover number is large, it is stable even upon binding to an antibody and it can specifically stain a substrate, and an enzyme to be used in common EIA such as peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholine esterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase or the like can also be used.
  • an enzyme, inhibitor, a coenzyme or the like can also be used.
  • the conjugation of such an enzyme with the antibody can be carried out by a known method using a crosslinking agent such as a maleimide compound.
  • a crosslinking agent such as a maleimide compound.
  • a known substance can be used according to the type of an enzyme to be used. For example, in the case where peroxidase is used as, the enzyme, 3,3′,5,5′-tetramethylbenzidine can be used, and in the case where alkaline phosphatase is used as the enzyme, p-nitrophenol or the like can be used.
  • the radioisotope those used in a common RIA such as 125I or 3H can be used.
  • fluorescent dye those used in a common fluorescence antibody technique such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) can be used.
  • a test is performed on whether or not an antibody binding to the above-mentioned atopic dermatitis inducer is present in the serum of a test subject and the test subject whose serum contains the antibody is determined to be a patient with atopic dermatitis or a high-risk individual for atopic dermatitis.
  • the serum of a test subject is brought into contact with the atopic dermatitis inducer to react the inducer with the IgE antibody in the serum of the test subject in a liquid phase. Further, a labeled IgE antibody specifically binding to the IgE antibody in the serum is reacted, and the signal of the labeled IgE antibody may be detected.
  • the labeling substance for the labeled IgE antibody an enzyme, a radioisotope or a fluorescent dye as illustrated in the above-mentioned labeled antibody can be used.
  • the activity of the enzyme is obtained by optically measuring the amount of the decomposed substrate, the obtained activity is converted into the amount of bound antibody, and the amount of the antibody is calculated in comparison with the standard value.
  • the amount of radiation emitted by the radioisotope is measured with a scintillation counter or the like.
  • the fluorescent amount may be measured with a measuring apparatus combined with a fluorescence microscope.
  • the detection of the signal for example, Western blot analysis can be adopted.
  • the conjugate of the antigen peptide, the antibody in the serum and the labeled IgE antibody is separated by a known separation method (a chromatography, a salting out method, an alcohol precipitation method, an enzyme method, a solid phase method or the like), and the signal of the labeled IgG antibody may be detected.
  • the diagnostic method of the invention (4) can also be carried out as a method in which the inducer is immobilized on a plate or a membrane, and the binding to the antibody in the serum of a test subject is tested on this substrate. By immobilizing the inducer on a substrate, un-bound labeled binding molecule can be easily removed.
  • the method of this invention (4) permits the determination how much the antibody to be detected is present as well as the diagnosis of atopic dermatitis.
  • a standard standard curve or the like
  • the amount of the antibody obtained from the serum of a test subject is compared with the standard, whereby the accurate amount of the antibody can be measured, and it becomes possible to assess the degree of the disease or the degree of the risk of occurrence of the disease with a high accuracy.
  • the invention (5) is a method of diagnosing a patient with atopic dermatitis or a high-risk patient for atopic dermatitis from the amount of histamine released by adding the above-mentioned inducer to a leukocyte fraction.
  • the amount of histamine may be measured by a method described in the document (Koro, O. et al., J. Allergy Clin. Immunol. 103, 663-670, 1999).
  • a test is performed on whether or not an inducer binding to the antibody of the above-mentioned invention (3) is present in a biological sample (a secretion such as saliva, sweat or tear) of a test subject and determining that the test subject whose sample contains the inducer is a patient with atopic dermatitis or a high-risk individual for atopic dermatitis.
  • a biological sample a secretion such as saliva, sweat or tear
  • One embodiment of the diagnostic method of this invention (6) is a method in which binding of an antibody to an inducer is performed in a liquid phase system. For example, a labeled antibody is brought into contact with a biological sample to bind the labeled antibody to an inducer, and this conjugate is separated in the same manner as in the above-mentioned invention (4) and the labeled signal is detected in the same manner.
  • an antibody in another diagnostic method in the liquid phase system, an antibody (primary antibody) is brought into contact with a biological sample to bind the primary antibody to an inducer, a labeled antibody (secondary antibody) is bound to this conjugate, and the labeled signal in the conjugate of the third party is detected.
  • a non-labeled secondary antibody is bound to the conjugate of an antibody and an inducer, and a labeling substance may be bound to this secondary antibody.
  • conjugation of the labeling substance to the secondary antibody can be carried out by, for example, biotinylating the secondary antibody and avidinylating the labeling substance.
  • an antibody that recognizes a partial region of the secondary antibody (e.g., Fc region) is labeled, and the tertiary antibody may be bound to the secondary antibody.
  • monoclonal antibodies can be used for both antibodies, or a polyclonal antibody can be used for either of them.
  • the separation of the conjugate from the liquid phase or the detection of the signal can be carried out in the same manner as in the above-mentioned invention (4).
  • Another embodiment of the diagnostic method of the invention (6) is a method in which the binding of an antibody to an inducer is tested in a solid phase system.
  • This method in the solid phase system is a preferred method because of the detection of a very little amount of the inducer and the convenience of the operation. More specifically, this method in the solid phase system is a method in which an antibody (primary antibody) is immobilized on a resin plate, membrane or the like, an inducer is bound to this immobilized antibody, a non-bound molecule is washed out, a labeled antibody (secondary antibody) is bound to the conjugate of the antibody and the inducer remaining on the plate, then the signal of this secondary antibody is detected.
  • This method is what is called a “sandwich method”, and in the case of using an enzyme as a marker, it is a widely used method as “ELISA (enzyme linked immunosorbent assay)”.
  • ELISA enzyme linked immunosorbent assay
  • monoclonal antibodies can be used for both antibodies, or a polyclonal antibody can be used for either of them.
  • the detection of the signal can be carried out in the same manner as in the above-mentioned invention (6).
  • an ELISA kit it is possible to carry out such a diagnostic method conveniently and extensively.
  • the method of this invention (6) permits the determination how much the atopic dermatitis inducer is present as well as the diagnosis of atopic dermatitis.
  • a standard standard curve or the like
  • the amount of the protein obtained from the biological sample of a test subject is compared with the standard, whereby the accurate amount of the protein can be measured, and it becomes possible to assess the degree of the disease or the degree of the risk of occurrence of the disease with a high accuracy.
  • the diagnosis of atopic dermatitis can be carried out by what is called a “patch test” using the inducer of the above-mentioned invention (1).
  • the patch test is carried out widely in the dermatology field as a convenient test method for contact allergy.
  • contact allergy is present, not only the area of dermatitis but also the skin throughout the body is sensitized, therefore, the cause of the contact dermatitis can be determined by artificially reproducing allergic contact dermatitis on the healthy skin.
  • the above-mentioned inducer is dropped or coated onto an adhesive patch and placed on the upper back, upper arm, thigh or the like. Determination is carried out according to the ICDRG (International Contact Dermatitis Research Group) standard after 2 days, 3 days and 1 week.
  • ICDRG International Contact Dermatitis Research Group
  • the condition where there is no skin reaction is determined to be ( ⁇ )
  • the condition where the area exposed to the agent on the patch has erythema and edema is determined to be (+) (++) or (+++) depending on the degree of the reaction.
  • This application provides a determination reagent of the invention (7) as a means that can diagnose atopic dermatitis by such a patch test conveniently, uniformly and extensively at low cost.
  • the invention (8) is a drug for desensitization therapy of atopic dermatitis characterized by containing the above-mentioned atopic dermatitis inducer as an active ingredient.
  • “Desensitization therapy” is a therapeutic method in which as for an allergy associated with an IgE antibody, a small amount of therapeutic allergen is administered and the dose is gradually increased at intervals of predetermined number of days, so that even if a causative allergen invades the body, an allergic reaction does not occur. Since the atopic dermatitis inducer of this invention has an atopic dermatitis inducing activity as described above, it can be a therapeutic allergen for desensitization.
  • Example 9 when desensitization therapy using Fr.D was attempted in patients with atopic dermatitis who actually show an allergic reaction to sweat, improvement was observed in the intraderinal test and in the clinical findings.
  • This drug for desensitization therapy can be formulated by uniformly tiling the above-mentioned inducer with a pharmacologically acceptable carrier.
  • the carrier can be appropriately selected from a wide range depending on the dosage form of the drug, however, it is preferred that the drug of this invention is in a unit dosage form that can be administered orally or by injection.
  • An oral liquid preparation such as a suspension or a syrup can be prepared by using water, a saccharide such as sucrose, sorbitol or fructose, a glycol such as polyethylene glycol, an oil such as sesame oil or soybean oil, a preservative such as alkyl p-hydroxybenzoate, a flavor such as strawberry flavor or peppermint, or the like.
  • a saccharide such as sucrose, sorbitol or fructose
  • a glycol such as polyethylene glycol
  • an oil such as sesame oil or soybean oil
  • a preservative such as alkyl p-hydroxybenzoate
  • a flavor such as strawberry flavor or peppermint, or the like.
  • a powder, a pill, a capsule and a tablet can be formulated by using a excipient such as lactose, glucose, sucrose or mannitol, a disintegrator such as potato starch or sodium alginate, a lubricant such as magnesium stearate or talc, a binder such as polyvinyl alcohol, hydroxypropyl cellulose or gelatin, a surface active agent such as a fatty acid ester, a plasticizer such as glycerin or the like.
  • a tablet and a capsule is a preferred unit dosage form in the preparation of this invention in terms of easiness of administration. When a tablet or a capsule is prepared, a solid pharmaceutical carrier is used.
  • a solution for injection can be formulated by using a salt solution, a glucose solution or, a mixture of a salt solution and a glucose solution, a carrier comprising a variety of buffers or the like.
  • it is formulated into a powder and an injectable solution may be prepared by mixing the powder with the above-mentioned liquid carrier at the time of use.
  • the administration schedule for the drug for desensitization therapy of this invention varies depending on the patient's age and body weight, and the symptoms, the administration route and the like.
  • a threshold is determined on an individual basis and the first dose is set to the amount around the threshold.
  • the dosing interval after the first dose and the increasing level of the allergen can be appropriately determined depending on the degree or the presence of allergic reaction. Eventually, the allergen in an amount around 10,000-fold threshold can be administered.
  • the drug for desensitization therapy of this invention when the drug for desensitization therapy of this invention is formulated such that the first dose of allergen is set at 1 ng/ml ⁇ 0.05 ml, the drug for the final administration can be formulated at a dose of 10 mg/ml ⁇ 0.05 ml.
  • ConA-affinity carrier ConA-Sepharose, Amersham Pharmacia; glycoprotein adsorption capacity: 45 mg/ml
  • the ratio of the amount of histamine in the supernatant to the total amount of histamine is defined as a histamine-releasing activity.
  • a fraction having an activity eluted at a NaCl concentration of 0.05 M to 0.34 M was collected ( FIG. 1 ).
  • the amount of histamine was measured according to the method described in the document (Koro, O. et al., J. Allergy Clin. Immunol. 103, 663-670, 1999).
  • the collected fraction with the anionic column was diluted to 10-fold with 0.1% TFA in distilled water and loaded onto a reverse phase column (SOURCE 15RPC ST 4.6/100; Pharmacia Biotech), and elution was carried out with a concentration gradient from 0.1% TFA in distilled: water to 0.1% TFA in CH3CN. After 2 ⁇ l of each of the eluted fractions war lyophilized to evaporate TFA or CH3CN, a histamine release test was carried out, and a fraction having a releasing activity (CH3CN with a concentration of 25% to 40%) was collected ( FIG. 2 ).
  • FIG. 3 A pattern when Fr.D was developed by a native 15% PAGE is shown in FIG. 3 . As shown in FIG. 3 , when the band was divided into 6 bands, and the histamine-releasing activity was measured for each band, the activity was observed from band 2 to band 6 ( FIG. 4 ).
  • Fr.D was collected, lyophilized, redissolved in PBS at a concentration of 1 mg/ml and used as an Fr.D standard.
  • 10 ⁇ l of a 50-fold dilution of the Fr.D standard was added to 40 ⁇ l of the serum of a patient with atopic dermatitis and preincubation was carried out at 37° C. for 30 minutes, the histamine-releasing activity was completely lost. On the contrary, when the serum of a normal subject was added, the histamine-releasing activity was not lost ( FIG. 5 ).
  • antiIgE and Fr.D ( ⁇ 500) are a positive control
  • serum (AD) serum of a patient
  • serum (N) serum of a normal subject
  • FIG. 6 A pattern when Fr.D was developed by a 15% SDS-PAGE is shown in FIG. 6 (the band number does not necessarily correspond to the number of FIG. 3 ).
  • Each band was excised from the gel and subjected to in-gel trypsin digestion and inner amino acid sequence was determined by MS/MS measurement of the eluted fraction.
  • peptides having amino acid sequences of LGKDAVEDLESVGK and DAVEDLESVGK were detected. From the database search, it was found that these sequences correspond to amino acid number 83 to 96 and 86 to 96 of Dermcidin.
  • the Fr.D standard was added (at a final concentration of 2 ⁇ g/ml) to a leukocyte fraction derived from a normal subject or a patient with atopic dermatitis, and the amount of released histamine was measured.
  • a cut-off value was set to be 5% based on the result and determination was carried out, 3 out of 33 normal subjects (9.1%) and 36 out of 40 patients with atopic dermatitis (90%) were determined to be positive.
  • IgE was removed by lactic acid treatment from the surface of basophils in the peripheral blood of 3 normal subjects who, do not have sensitivity, to sweat, and these basophils were sensitized with IgE purified from the serum of a patient with atopic dermatitis and then stimulated by the purified sweat antigen Fr.D.
  • a human mast cell line (LAD2: Arnold S. et al; Leukemia Research 27 (2003) 677-682) was sensitized with myeloma IgE or patent IgE and stimulated by a purified sweat antigen.
  • FIG. 8 when sensitization was carried out with non-specific myeloma IgE as a control of human IgE, degranulation due to the stimulation by the purified sweat antigen (the degree was indicated by the release of ⁇ -hexosaminidase which is an enzyme released at the same time and at the same degree as histamine) was not observed.
  • the threshold after the 75th day after the initiation of therapy became a high value, however, the sensitivity of the patient for histamine in the intradermal test which was carried out simultaneously a; a control did not change from the first treatment, therefore, it is assumed to be due to a high potency of the antigen of Lot 2. Even after changing to Lot 2, the threshold on the 125th day quadrupled, and it could bc conned that the therapeutic effect wan further increased.
  • an atopic dermatitis inducer produced by a patient of his or her own can be provided as a causative factor of atopic dermatitis. This makes it possible to achieve an accurate diagnosis of atopic dermatitis and a treatment of atopic dermatitis.

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US20110117103A1 (en) * 2008-05-02 2011-05-19 Hiroshima University Anti-sweat antigen monoclonal antibody
US9457071B2 (en) 2012-06-28 2016-10-04 Hiroshima University Histamine releaser contained in human sweat
US9528998B2 (en) 2010-04-16 2016-12-27 Abbott Laboratories Methods and reagents for diagnosing rheumatoid arthrtis
US9989538B2 (en) 2012-08-17 2018-06-05 Hiroshima University Monoclonal IgE antibody that binds to sweat allergy antigen protein

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EP2382982A3 (en) * 2001-08-24 2012-02-22 Neuren Pharmaceuticals Limited Neural regeneration peptide and methods for their use in treatment of brain damage
WO2003084991A1 (fr) 2002-04-08 2003-10-16 Japan Science And Technology Agency Proteines inductrices de dermatites atopiques
JP2006513700A (ja) * 2002-09-25 2006-04-27 ジェネンテック・インコーポレーテッド 乾癬の治療のための新規組成物と方法
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Cited By (6)

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Publication number Priority date Publication date Assignee Title
US20110117103A1 (en) * 2008-05-02 2011-05-19 Hiroshima University Anti-sweat antigen monoclonal antibody
US8546542B2 (en) * 2008-05-02 2013-10-01 Hiroshima University Anti-sweat antigen monoclonal antibody
US9528998B2 (en) 2010-04-16 2016-12-27 Abbott Laboratories Methods and reagents for diagnosing rheumatoid arthrtis
US9457071B2 (en) 2012-06-28 2016-10-04 Hiroshima University Histamine releaser contained in human sweat
US9709576B2 (en) 2012-06-28 2017-07-18 Hiroshima University Histamine releaser contained in human sweat
US9989538B2 (en) 2012-08-17 2018-06-05 Hiroshima University Monoclonal IgE antibody that binds to sweat allergy antigen protein

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