US0094.57071B2

(12) United States Patent (10) Patent No.: US 9.457,071 B2 Hide et al. (45) Date of Patent: Oct. 4, 2016

(54) RELEASER CONTAINED IN 2010/0311604 A1 12/2010 Takkinen et al. HUMAN SWEAT 2011/0117103 A1 5, 2011 Hide et al. 2015,0212097 A1 7/2015 Hide et al. (71) Applicant: Hiroshima University, Higashihiroshima-shi, Hiroshima (JP) FOREIGN PATENT DOCUMENTS EP O955366 A1 11, 1999 (72) Inventors: Michihiro Hide, Hiroshima (JP): EP 16O7107 12/2005 EP 1655.307 A1 5, 2006 Takaaki Hiragun, Hiroshima (JP); EP 229.2659 A1 3, 2011 Kaori Ishii, Hiroshima (JP); Shoji JP 3.642340 A 4/2005 Mihara, Hiroshima (JP); Makiko JP 2008-069 118 A 3, 2008 Hiragun, Hiroshim (JP); Jens-M JP 2010-516747 A 5, 2010 WO 03/084991 A1 10, 2003 Schroeder, Kiel (DE) WO 2005.005474 A1 1, 2005 WO 2008/092993 A1 8, 2008 (73) Assignee: Hiroshima University, Hiroshima (JP) WO 2009,133951 A1 11/2009 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. Xu et al., “Malassezia globosa CBS 7966 hypothetical protein MGL 1304 partial mRNA.” Database DDBJ/EMBL/GenBank (21) Appl. No.: 14/409,730 online), XM 001731984.1 (2008). Rasool et al., “Cloning, characterization and expression of complete (22) PCT Filed: Jun. 25, 2013 coding sequences of three IgE binding Malassezia furfur allergens, Mal f7. Mal f8 and Mal f 9...” European Journal of Biochemistry, (86). PCT No.: PCT/UP2013/067396 267: 4355-4361 (2000). Lindberg et al., “Malassezia furfur mRNA coding for potential S 371 (c)(1), allergen, strain ATCC No. 42132,” Database DDBJ/EMBL/ (2) Date: Mar. 10, 2015 GenBank online), AJO 11958.1 (2000). Lindberg et al., “Malassezia sympodialis mRNA for allergen Mals (87) PCT Pub. No.: WO2014/003008 8, strain ATCC No. 42132. Database DDBJ/EMBL/GenBank PCT Pub. Date: Jan. 3, 2014 online), AJO1 1958.2 (2002). Kanbe et al., “ and Malassezia Species: A Study (65) Prior Publication Data of Antigenic Components of Malassezia Species for Immunoglobu lin E of Patients with Atopic Dermatitis,” Japanese Journal of US 2015/O238581 A1 Aug. 27, 2015 Medical Mycology, 44: 71-75 (2003) see English abstract. Shindo et al., “Histamine release-neutralization assay for Sera of (30) Foreign Application Priority Data patients with atopic dermatitis and/or is useful to screen type I against Sweat .” Archives Jun. 28, 2012 (JP) ...... 2012-145814 of Dermatological Research, 304: 647-654 (2012). Hiragun et al., “Fungal protein MGL 1304 in Sweat is an allergen (51) Int. Cl. for atopic dermatitis patients,” Journal of and Clinical GOIN 33/53 (2006.01) Immunology, (2013). A6 IK 39/00 (2006.01) International Search Report issued in corresponding International C07K 16/14 (2006.01) Patent Application No. PCT/JP2013/067396 dated Jul 23, 2013. Steinberger et al., “Construction of a Combinatorial IgE Library GOIN 33/68 (2006.01) from an Allergic Patient.” The Journal of Biological Chemistry, 271: C07K I4/375 (2006.01) 10967-10972 (1996). GOIN 33/569 (2006.01) Tanaka et al., “Cholinergic Urticaria Successfully Treated by A61 K 38/00 (2006.01) Immunotherapy with Partially Purified Sweat .” Allergy, 56: (52) U.S. Cl. 54-57 (2007) (see English abstract on p. 57). CPC ...... A61K 39/0002 (2013.01); C07K 14/375 (Continued) (2013.01); C07K 16/14 (2013.01); G0IN 33/56961 (2013.01); G0IN 33/6854 (2013.01); A6 IK 38/00 (2013.01); C07K 2317/76 Primary Examiner — Jennifer Graser (2013.01); C07K 2319/30 (2013.01); G0IN (74) Attorney, Agent, or Firm — Morgan, Lewis & 2800/202 (2013.01); G0IN 2800/24 (2013.01) Bockius LLP (58) Field of Classification Search None (57) ABSTRACT See application file for complete search history. Provided are a Sweat allergy antigen, an antibody capable of binding to the antigen specifically, and others, which are (56) References Cited produced utilizing a microorganism-originated protein that U.S. PATENT DOCUMENTS exists in Sweat allergy patient in a dissolved state or a partial peptide of the protein. 2006, OO88926 A1 4/2006 Ozawa et al. 2006/01347.06 A1 6, 2006 Hide et al. 12 Claims, 11 Drawing Sheets US 9.457,071 B2 Page 2

(56) References Cited Xu et al., “hypothetical protein MGL 1304 Malassezia globosa CBS 7966).” RefSeq, online), XP 001732036.1 (2008). OTHER PUBLICATIONS Extended European Search Report issued in corresponding Euro pean Patent Application No. 13809354.7 dated Feb. 10, 2016. Tanaka et al., “Semi-purification of the -Sweat Valenta et al., “Autoallergy: A pathogenetic factor in atopic derma antigen acting on mast cells and basophils in atopic dermatitis.” titis?” Journal of Allergy and Clinical Immunology, 105: 432-437 Experimental Dermatology, 15: 283-290 (2006). (2000). Fairley et al., “A Pathogenic Role for IgE in Autoimmunity: Bullous Vilhelmsson, “Structural and functional studies of malassezia Pemphigoid IgE Reproduces the Early Phase of Lesion Develop sympodialis-derived allergens.” Department of Medicine Solna, ment in Human Skin Grafted to nu/nu Mice,” Journal of Investi Clinical Allergy Research Unit, Karolinska Institutet, XP55234699 gative Dermatology, 127: 2605-2611 (2007). Human IgE (non-Immune), BIOPORTO Diagnostics (2011). (2008). International Preliminary Report on Patentability issued in related Partial Supplementary European Search Report issued in related International Patent Application No. PCT/JP2013/071715 dated European Patent Application No. 13879589.3 dated Apr. 20, 2016. Feb. 26, 2015. Ishii et al., “A human monoclonal IgE antibody that binds to International Preliminary Report on Patentability issued in corre MGL 1304, a major allergen in human Sweat, without activation of sponding International Patent Application No. PCT/JP2013/067396 mast cells and basophils.” Biochemical and Biophysical Research dated Dec. 28, 2014. Communications, 468: 99-104 (2015). International Search Report issued in related International Patent “Product Catalog 2010 Bioporto Diagnostics,” XP055257898, Application No. PCT/JP2013/071715 dated Nov. 12, 2013. http://www.ngal.cz/files/bioporto-katalog-2010-en.pdf (2010). U.S. Patent Oct. 4, 2016 Sheet 1 of 11 US 9,457,071 B2

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U.S. Patent Oct. 4, 2016 Sheet 9 of 11 US 9,457,071 B2

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U.S. Patent Oct. 4, 2016 Sheet 10 of 11 US 9,457,071 B2

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Incubation period (min) US 9,457,071 B2 1. 2 HISTAMINE RELEASER CONTAINED IN In one aspect, the present invention provides a composi HUMAN SWEAT tion or kit for detecting an antibody binding to a Sweat allergy antigen, or a composition or kit for measuring an A computer readable text file, entitled “SequenceLis amount of a antigen binding to a Sweat allergy antigen, ting..txt, created on or about Mar. 10, 2015 with a file size comprising a Sweat allergy antigen protein that is a protein of about 15 kb contains the sequence listing for this appli derived from a microorganism or a MGL 1304 partial cation and is hereby incorporated by reference in its entirety. peptide. In one aspect, the present invention provides a composi TECHNICAL FIELD tion or kit for diagnosing a Sweat allergy or a disease related 10 to a Sweat allergy antigen, comprising: This application claims priority to Japanese Patent Appli (i) a Sweat allergy antigen protein that is a protein derived cation No. 2012-145814, filed Jun. 28, 2012 and incorpo from a microorganism or a MGL 1304 partial peptide; or rated by reference herein in its entirety. (ii) an antibody or an antibody fragment specifically binding The present invention relates to clinical application of a to the Sweat allergy antigen protein that is a protein derived novel histamine releaser contained in human Sweat (Such as 15 from a microorganism or a MGL 1304 partial peptide. measurement of antigen-specific antibody concentration in a In one aspect, the present invention provides a composi patient serum, preparation of an antibody to a Sweat antigen, tion for treatment of a Sweat allergy or a disease relating to and a specific hyposensitization therapy). a Sweat allergy antigen comprising a Sweat allergy antigen protein that is a protein derived from a microorganism or a BACKGROUND ART MGL 1304 partial peptide. In one aspect, the present invention provides a composi Most of atopic dermatitis patients exhibit an immediate tion for removal or neutralization of a Sweat allergy antigen type allergic reaction to their own Sweat and a roughly or a material for removing a Sweat allergy antigen compris purified Sweat antigen. A roughly purified Sweat antigen and ing an antibody specifically binding to a Sweat allergy an antibody specifically binding thereto have been reported 25 antigen protein that is a protein derived from a microorgan (Patent Document 1 and Patent Document 2). However, a ism or a MGL 1304 partial peptide. real molecule (Sweat antigen) thereof is unknown. In one aspect, the present invention provides a composi tion or kit for determining an effect of a hyposensitization CITATION LIST therapy comprising a Sweat allergy antigen protein that is a 30 protein derived from a microorganism or a MGL 1304 Patent Documents partial peptide. In one aspect, the present invention provides a method for Patent Document 1: WO 2005-005474 manufacturing a Sweat allergy antigen protein comprising a Patent Document 2: WO 2009-133951 step of culturing Malassezia globosa at pH 7 to pH 9, and 35 a step of purifying a Sweat allergy antigen protein. SUMMARY OF THE INVENTION BRIEF DESCRIPTION OF DRAWINGS The present inventers have purified a human Sweat anti gen from human Sweat using a histamine release activity FIG. 1 shows that an amino acid sequence identical to from peripheral blood basophils of atopic dermatitis patient 40 MGL 1304 was detected by mass spectrometry after con as an index and have identified MGL 1304, which is a ducting further purification of partially purified Sweat anti protein derived from Malassezia globosa, as the human gens (hereinafter also referred to as QRX). A recombinant Sweat antigen by mass spectrometry. protein of MGL 1304 was produced by E. coli. In one aspect, the present invention provides a Sweat FIG. 2 shows CBB staining and Western blotting analysis allergy antigen protein that is a protein derived from a 45 of the recombinant protein using an atopic dermatitis patient microorganism. For example, the present invention provides serum and an anti-his tag antibody. a protein consisting of an amino acid sequence represented FIG. 3 shows a histamine release assay of atopic derma by SEQ ID NO: 1. titis patients (AD1, AD2, and AD3) and a healthy person In one aspect, the present invention provides an (HCl) for the recombinant protein of MGL 1304. MGL 1304 partial peptide. For example, the present inven 50 FIG. 4 shows that pretreatment of atopic dermatitis tion provides a peptide consisting of an amino acid sequence patient serum with one of QRX and MGL 1304 Leads to represented by any one of SEQ ID NOS: 2 to 7. neutralization of binding of antibody with the other one, In one aspect, the present invention provides a gene respectively. encoding a Sweat allergy antigen protein that is a protein FIG. 5 shows that when atopic dermatitis patient serum is derived from a microorganism and a gene encoding a 55 pretreated with MGL 1304 in a reaction system where the MGL 1304 partial peptide. atopic dermatitis patient serum is reacted with a rat mast cell In one aspect, the present invention provides an antibody line expressing a human high-affinity IgE receptor to sen or an antibody fragment specifically binding to a Sweat sitize a cell (to provide sensitivity to an antigen), a reactivity allergy antigen protein that is a protein derived from a (ability of histamine release) of the sensitized cell to QRX microorganism or a MGL 1304 partial peptide. 60 disappeared. In one aspect, the present invention provides a composi In FIG. 6. A shows a prepared N-terminal or C-terminal tion or kit for detecting a Sweat allergy antigen, or a truncated protein of MGL 1304. In FIG. 6, B shows a composition or kit for measuring an amount of a Sweat binding ability of the protein truncated at the N-terminal or allergy antigen, comprising an antibody or an antibody the C-terminal of MGL 1304 to atopic dermatitis patient fragment specifically binding to a Sweat allergy antigen 65 serum IgE. In FIG. 6, C and D show histamine release protein that is a protein derived from a microorganism or the activity of the protein truncated at the N-terminal or the MGL 1304 partial peptide. C-terminal of MGL 1304. In FIG. 6, E shows binding of an US 9,457,071 B2 3 4 MGL 1304 recombinant protein and a partial peptide IgE antibody binding to a complex of the mouse monoclonal thereof to a Smith2 antibody, and serums (AD1 and AD2) antibody 8-2 and QRX was detected from the serums. derived from two atopic dermatitis patients. FIG. 14 shows detection of the Sweat antigen-specific IgE FIG. 7 shows that binding of MGL 1304 recombinant antibody in the serums of the atopic dermatitis patients protein with IgE derived from serum of atopic dermatitis 5 (AD1 and AD2) and the healthy volunteer (Nor1). An patients or healthy volunteer was determined by ELISA, ELISA plate was coated with the mouse monoclonal anti indicating that presence or absence of the binding is con body 8-2 prepared by immunization with the MGL 1304 sistent with a reactivity of basophils of the serum donor to recombinant protein as the primary antibody and rMGL was purified Sweat antigens. allowed to bind thereto. The IgE antibody binding to a FIG. 8 shows experimental results when an extract of 10 complex of the mouse monoclonal antibody 8-2 and rMGL Malassezia fungus body (M.G. lysate), Malassezia culture was detected from the serums. supernatant (M.G. Sup), MGL 1304 recombinant protein FIG. 15 shows detection of the Sweat antigen-specific IgE (rMGL) and a partially purified sweat antigen (QRX) were antibody in the serums of the atopic dermatitis patients Subjected to electrophoresis to perform immunoblotting (AD1 and AD2) and the healthy volunteer (Nor1). An using atopic dermatitis patient serum of one patient. In 15 ELISA plate was coated with the Smith2 antibody used as particular, FIG. 8 shows that pretreatment of the serum with the primary antibody and rMGL was allowed to bind thereto. a fusion protein (rTF-MGL) of Trigger Factor and rMGL The IgE antibody binding to a complex of the mouse resulted in disappearance of the binding property of IgE to monoclonal antibody 8-2 and rMGL was detected from the QRX and rMGL. SUS. FIG. 9 shows experimental results when an extract of FIG. 16 shows detection of the Sweat antigen-specific IgE Malassezia fungus body (M.G. lysate), Malassezia culture antibody in the serums of the atopic dermatitis patients supernatant (M.G. Sup), MGL 1304 recombinant protein (AD1 and AD2) and the healthy volunteer (Nor1). An (rMGL) and partially purified sweat antigen (QRX) were ELISA plate was coated with a mouse monoclonal antibody subjected to electrophoresis to perform the immunoblotting 6-3 prepared by immunization with the MGL 1304 recom as performed in FIG. 8 except for using an atopic dermatitis 25 binant protein as the primary antibody and rMGL was patient serum of one person different from the patient of the allowed to bind thereto. The IgE antibody binding to a serum used in FIG. 8. complex of the mouse monoclonal antibody 6-3 and rMGL FIG. 10 shows experimental results when a purified was detected from the serums. extract of Malassezia fungus body (M. lysate (purified)). FIG. 17 shows detection of the Sweat antigen-specific IgE Malassezia culture Supernatant (M. Sup (purified)) and a 30 antibody in the serums of the atopic dermatitis patients partially purified sweat antigen (sweat (QRX)) were sub (AD1 and AD2) and the healthy volunteer (Nor1). An jected to electrophoresis to perform immunoblotting using ELISA plate was coated with the mouse monoclonal anti an atopic dermatitis patient serum of one patient. body 8-2 prepared by immunization with the MGL 1304 In FIG. 11. A shows Western blotting results when an recombinant protein as the primary antibody and rMGL was MGL 1304 gene or a mite antigen gene fused with Myc-tag 35 allowed to bind thereto. The IgE antibody binding to a was expressed in COS7 cells and a culture supernatant complex of the mouse monoclonal antibody 8-2 and rMGL thereof was electrophoresed to perform Western blotting was detected from the serums. using an anti-Myc antibody. In FIG. 11, B shows correlation FIG. 18 shows detection of the Sweat antigen-specific IgE of histamine releasability between a partially purified sweat antibody in serums of patients (allergic antigen (QR) and the MGL 1304 recombinant protein 40 rhinitis 1-5), atopic dermatitis patients (AD1-8) and healthy (rMGL 1304). volunteer (Nor1-3). An ELISA plate was coated with the FIG. 12 shows sites of MGL 1304 protein recognized by MGL 1304 recombinant protein (rMGL) and a binding IgE monoclonal antibodies MGLab6-3, MGLab8-2, MGLab8-4, antibody was detected from the serums. The serums were MGLab9-1, MGLab 9-5, MGLab10-8, MGLab 10-10, diluted 10 times (x10) and 20 times (x20) before being used MGLab22-1, MGLab36-1 and MGLab40-1 created by 45 in the test. The vertical axis indicates absorbance at 450 nm. immunizing mice with a recombinant MGL 1304 protein. FIG. 19 shows detection of the Sweat antigen-specific IgE ELISA was performed for testing whether the antibodies antibody in the serums of the allergic rhinitis patients recognize Trigger Factor (TF), a TF-fused MGL 1304 pro (allergic rhinitis 1-5) and the healthy volunteer (Nor1-3). An tein (TF-MGL), a peptide (TF-P1) corresponding to an ELISA plate was coated with the MGL 1304 recombinant amino acid sequence 1-50 of the TF-fused MGL 1304 50 protein (rMGL) and a binding IgE antibody was detected protein, a peptide (TF-P2) corresponding to an amino acid from the serums. The serums were diluted 10 times (x10) sequence 46-100 of the TF-fused MGL 1304 protein, a before being used in the test. The vertical axis indicates peptide (TF-P3) corresponding to an amino acid sequence absorbance at 450 nm. 96-140 of the TF-fused MGL 1304 protein, and a peptide FIG. 20 shows time course of amount of antibodies (TF-P4) corresponding to an amino acid sequence 136-183 55 binding to rMGL in serum of a patient subjected to a of the TF-fused MGL 1304 protein. The results indicated hyposensitization therapy by repeated Subcutaneous injec that the monoclonal antibodies created by immunizing mice tions of QRX. The blood of the patient was collected three with the recombinant MGL 1304 protein recognize the times (March 2011 (2011.3), December 2011 (2011.12), and amino acid sequence 46-100 of the MGL 1304 protein March 2012 (2012.3)) to prepare the serums, and an rMGL except that MGLab6-3 recognizes the amino acid sequence 60 coated ELISA plate was used for measuring a change in 136-183 of the MGL 1304 protein. amount of anti-MGL human IgE antibodies in the serums. FIG. 13 shows detection of a sweat antigen-specific IgE FIG. 21 shows time course of amount of antibodies antibody in serums of atopic dermatitis patients (AD1 and binding to rMGL in serum of the patient subjected to the AD2) and a healthy volunteer (Nor1). An ELISA plate was hyposensitization therapy by repeated Subcutaneous injec coated with a mouse monoclonal antibody 8-2 prepared by 65 tions of QRX. The blood of the patient was collected three immunization with the MGL 1304 recombinant protein as a times (March 2011 (2011.3), December 2011 (2011.12), and primary antibody and QRX was allowed to bind thereto. The March 2012 (2012.3)) to prepare the serums, and an rMGL US 9,457,071 B2 5 6 coated ELISA plate was used for measuring a change in recognized by those skilled in the art as molecular weights amount of anti-MGL human IgG antibodies in the serums. measured by experiments (SDS-PAGE). For example, FIG. 22 shows time course of amount of antibodies “about 17 kDa and “about 30 kDa described above may be binding to rMGL in serum of the patient subjected to the molecular weights not exceeding ranges of “from 14 kDa to hyposensitization therapy by repeated Subcutaneous injec 20 kDa and “from 27 kDa to 33 kDa, respectively. The tions of QRX. The blood of the patient was collected three protein present in a culture Supernatant or a fungus cell of times (March 2011 (2011.3), December 2011 (2011.12), and Malassezia globosa and binding to serum derived from a March 2012 (2012.3)) to prepare the serums, which were Sweat allergy patient and/or Smith2 antibodies may be a diluted 10 times (x10), 50 times (X50), and 100 times protein having histamine releasing activity for basophils (x100), and an rMGL-coated ELISA plate was used for 10 derived from a Sweat allergy patient and/or a protein which measuring a change in amount of anti-MGL human IgG4 induces histamine release via IgE from a cell expressing an antibodies. IgE receptor. FIG. 23 shows concentration of the MGL 1304 protein As used herein, a Sweat allergy antigen is an antigenic (protein coded by the MGL 1304 gene) produced in culture Substance (hereinafter also referred to as a Sweat antigen) Supernatant when Malassezia globosa was cultured in buffer 15 which is contained in human Sweat and which induces an A (PBS/HEPES/glucose) at pH 4, pH 6, or pH 8 for 2 to 60 allergic reaction to cause diseases such as atopic dermatitis minutes. and cholinergic urticaria, and a Sweat allergy antigen protein is a Sweat allergy antigen that is a protein. In one embodi EMBODIMENT FOR CARRYING OUT THE ment, the Sweat allergy antigen protein is dissolved in Sweat. INVENTION As used herein, the Sweat allergy antigen has histamine release activity. Histamine release activity can be deter 1. Sweat Allergy Antigen Protein mined in accordance with a known method (Koro, O. et al., In a first aspect, the present invention provides a Sweat J. Allergy Clin. Immunol., 103, 663-670, 1999). For allergy antigen protein that is a protein derived from a example, histamine release activity may be determined by microorganism. 25 contacting a Sweat allergy antigen and an IgE antibody with As used herein, microorganisms may be Malassezia glo a cell expressing an IgE receptor on a cell Surface and bosa, and Malassezia globosa may be Malassezia globosa measuring an amount of histamine secreted from the cell. (No. MYA-4612) purchasable from ATCC, for example. The Examples of cells expressing IgE receptors on the cell protein derived from a microorganism may be a protein Surfaces include, basophils, mast cells (mastocytes) and a produced by Malassezia globosa. The protein produced by 30 cell line artificially prepared by expressing IgE receptor Malassezia globosa may be a protein secreted outside a gene which is able to release a chemical transmitter Such as fungus cell or may be a protein present in a fungus cell. histamine. Examples of the proteins secreted outside the Malassezia For example, when an amount of histamine is measured globosa fungus cell include a protein coded by an and an amount of free histamine is within a range of from 3 MGL 1304 gene (e.g., a protein comprising an amino acid 35 to 97% relative to the total amount of histamine, it can be sequence represented by SEQ ID NO: 1) and a protein determined that the histamine release activity is positive present in a culture Supernatant of Malassezia globosa and (Koro, O. et al., J. Allergy Clin. Immunol. 103, 663-670, binding to serum derived from a Sweat allergy patient and/or 1999). Smith2 antibodies (antibody produced by a hybridoma of 'sweat allergy antigen protein that is a protein derived Accession No. FERM BP-11111). The protein encoded by 40 from microorganisms' provided by the present invention the MGL 1304 gene (e.g., a protein consisting of the amino may be prepared by purification from a secretion of a Sweat acid sequence represented by SEQ ID NO: 1) may be a gland using the histamine release activity as an index. For protein expressed in an appropriate host Such as E. coli, example, the Sweat allergy antigen protein is prepared by a COS7 cells and Malassezia globosa. The amino acid method comprising a step of concentrating human Sweat, a sequence represented by SEQ ID NO: 1 is as follows: 45 step of purification using anion-exchange column chroma tography, a step of purification using reverse-phase column chromatography, and a step of purification using gel filtra SEO ID NO 1: tion column chromatography. Therefore, in one embodi MWSLNIFSAAFWASLASAWFAAPSALERRAAPDNTWWWTSWADHCL ment, the present invention provides a Sweat allergy antigen ILPRHKMSVGDSESPGNMRSFCTKPYSSKOGOLASDFWTKAHFKKT 50 protein that is a protein derived from a microorganism and that is prepared by a method comprising a step of concen DKYVOITGCINPNVOSTLLSNDEGGOYDSNGGEGGRGNPAGSWCLG trating human Sweat, a step of purification using anion exchange column chromatography, a step of purification YSSYWELVEPAGNRACIRCCYDPSDCDVSODEAGCETVIPGKYDC. using reverse-phase column chromatography, and a step of Examples of a protein present in the Malassezia globosa 55 purification using gel filtration column chromatography. In fungus cell include a protein present in a fungus cell of one embodiment, the present invention provides a method of Malassezia globosa and binding to serum derived from a manufacturing a Sweat allergy antigen protein that is a Sweat allergy patient and/or Smith2 antibodies. protein derived from a microorganism comprising a step of The protein present in a culture Supernatant of Malassezia concentrating human Sweat, a step of purification using globosa and binding to serum derived from a Sweat allergy 60 anion-exchange column chromatography, a step of purifica patient and/or Smith2 antibodies may be a protein having a tion using reverse-phase column chromatography, and a step molecular weight of about 17 kDa measured by SDS-PAGE. of purification using gel filtration column chromatography. The protein present in a cell of Malassezia globosa and 'sweat allergy antigen protein that is a protein derived binding to serum derived from a Sweat allergy patient and/or from a microorganism’ provided by the present invention Smith2 antibodies may be a protein having a molecular 65 may be prepared from a culture of Malassezia globosa. weight of about 30 kDa measured by SDS-PAGE. “About 17 Condition and method for the culture may be appropriately kDa and “about 30 kDa described above are reasonably selected by those skilled in the art and those are not limited. US 9,457,071 B2 7 8 For example, “Sweat allergy antigen protein that is a protein gene (e.g., a protein consisting of the amino acid sequence derived from a microorganism’ provided by the present represented by SEQ ID NO: 1) in a suitable host such as a invention may be prepared by culturing Malassezia globosa microorganism and a cell (e.g., E. coli. COS7 cells, and (No. MYA-4612) available from ATCC in a 2693 mDixon Malassezia globosa). medium (2693 mDixon medium is prepared by mixing Malt In one embodiment, the present invention provides an Extract (36 g): Desiccated Oxbile (20g); Tween 40 (10 ml); analog of the protein encoded by the MGL 1304 gene (e.g., Peptone (6.0 g); Glycerol (2.0 ml); Oleic Acid (2.0 ml); and a protein consisting of the amino acid sequence represented DI Water (1.0 L) to acquire a solution adjusted to pH 6 by by SEQID NO: 1). The analog is not particularly limited as using HCl and autoclaving the solution put into a Suitable long as the analog can be prepared based on the protein container at 121°C.) at 32°C. for four days, centrifuging the 10 encoded by the MGL 1304 gene or the protein consisting of culture (at 2000 rpm for 10 minutes), and purifying an the amino acid sequence represented by SEQ ID NO: 1. acquired Supernatant or lysate (cell lysate) of fungus cells Examples of the analog of the protein consisting of the dissolved in a phosphate buffer solution (PBS) using binding amino acid sequence represented by SEQID NO: 1 include to the Smith2 antibody as an index. a protein consisting of an amino acid sequence having 60, Therefore, in one embodiment, the present invention 15 70, 80, 90, or 95% or more identity to the amino acid provides a Sweat allergy antigen protein that is a protein sequence represented by SEQ ID NO: 1, and a protein derived from a microorganism and that is prepared by a step consisting of an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, of culturing Malassezia globosa and a step of purifying a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids culture Supernatant or fungus cell lysate using binding to the deleted, Substituted, or added in the amino acid sequence Smith2 antibody as an index. In one embodiment, the represented by SEQ ID NO: 1. present invention provides a method of manufacturing a Another example of the analog of the protein encoded by Sweat allergy antigen protein that is a protein derived from the MGL 1304 gene (e.g., a protein consisting of the amino a microorganism comprising a step of culturing Malassezia acid sequence represented by SEQ ID NO: 1) may be a globosa and a step of purifying a culture Supernatant or protein encoded by the MGL 1304 gene (e.g., a protein fungus cell lysate using binding to the Smith2 antibody as an 25 consisting of the amino acid sequence represented by SEQ index. ID NO: 1) to which a tag is added. As used herein, a tag The production of the MGL 1304 protein (protein means a portion added to a protein or a polypeptide for encoded by the MGL 1304 gene), i.e., the sweat allergy purification, detection, etc. of the protein or polypeptide and antigen protein from Malassezia globosa, is increased when is exemplified by histidine (His), glutathione-S-transferase Malassezia globosa is cultured at pH 8 as compared to when 30 (GST), a maltose-binding protein (MBP), myc, a FLAG tag, Malassezia globosa is cultured at pH 4 or pH 6. Therefore, Trigger Factor (TF), etc. A polypeptide having a tag added Malassezia globosa may be cultured at pH 7 to 10, prefer thereto is obtained by expressing the polypeptide by using ably pH 7 to 9, more preferably pH 8. an expression vector, for example, pET30a (manufactured The purification of the Sweat allergy antigen protein from by Novagen) (for His tags), pGEX (manufactured by GE a culture solution of Malassezia globosa is performed as a 35 Healthcare Bio-Sciences Corp.) (for GST tags), and a pCold more simple operation when the culture solution contains no TF vector (manufactured by TAKARA BIO INC.), in a protein other than the Sweat allergy antigen protein. There Suitable host Such as a microorganism and a cell (e.g., E. fore, after Malassezia globosa is cultured at pH 4 to 6. coli, COS7 cells, and Malassezia globosa). preferably pH 4 to increase a fungus cell amount, a culture The analog of the protein encoded by the MGL 1304 Supernatant is discarded and Malassezia globosa is Subse 40 gene (e.g., a protein consisting of the amino acid sequence quently cultured by using a buffer solution (e.g., PBS/ represented by SEQ ID NO: 1) is able to bind to the IgE HEPES/glucose buffer solution) of pH 7 to 10, preferably antibody present in a serum of a Sweat allergy patient. pH 7 to 9, more preferably pH 8, so as to produce a sweat The analog of the protein encoded by the MGL 1304 allergy antigen protein, and the Sweat allergy antigen protein gene (e.g., a protein consisting of the amino acid sequence may be purified from the buffer solution containing the 45 represented by SEQ ID NO: 1) may have the histamine obtained Sweat allergy antigen protein. The purification may release activity. be performed by column chromatography (e.g., ion-ex The protein encoded by the MGL 1304 gene (e.g., a change column chromatography, reverse-phase column protein consisting of the amino acid sequence represented by chromatography, and gel filtration column chromatography). SEQ ID NO: 1) and the analog thereof provided by the The “Sweat allergy antigen protein that is a protein 50 preset invention can be manufactured by a conventional derived from a microorganism’ provided by the present genetic engineering method or a method used in peptide invention may be prepared by expressing a protein encoded synthesis. The manufactured proteins and the analogs by the MGL 1304 gene (e.g., a protein comprising the thereof may be used as the Sweat allergy antigen. Therefore, amino acid sequence represented by SEQ ID NO: 1) in a in one embodiment, the present invention provides a use of Suitable host Such as a microorganism and a cell (e.g., E. 55 a protein derived from a microorganism or an analog thereof coli, COS7 cells, and Malassezia globosa). (e.g., a protein consisting of the amino acid sequence Therefore, in one embodiment, the present invention represented by SEQ ID NO: 1 and an analog thereof, and a provides a Sweat allergy antigen protein that is a protein protein present in a culture Supernatant of Malassezia glo derived from a microorganism and that is prepared by bosa and binding to serum derived from a Sweat allergy expressing a protein encoded by the MGL 1304 gene (e.g., 60 patient and/or Smith2 antibodies) in manufacturing of the a protein comprising the amino acid sequence represented Sweat allergy antigen. by SEQ ID NO: 1) in a suitable host such as a microorgan The protein or polypeptide provided by the present inven ism and a cell (e.g., E. coli. COS7 cells, and Malassezia tion binds to the IgE antibody and/or the IgG antibody globosa). In one embodiment, the present invention provides binding to a Sweat allergy antigen present in a serum of a a method of manufacturing a Sweat allergy antigen protein 65 Sweat allergy patient, and therefore can be used for detect that is a protein derived from a microorganism comprising ing, quantifying, or removing IgE antibody and/or IgG a step of expressing a protein encoded by the MGL 1304 antibody binding to the Sweat allergy antigen present in a US 9,457,071 B2 10 serum of a Sweat allergy patient. Thus, in one embodiment, thione-S-transferase (GST), a maltose-binding protein the present invention provides use of a protein derived from (MBP), myc, a FLAG tag, Trigger Factor (TF), etc. A a microorganism (e.g., the proteins encoded by the polypeptide having a tag added thereto is obtained by MGL 1304 gene (e.g., a protein consisting of the amino acid expressing the polypeptide by using an expression vector, sequence represented by SEQ ID NO: 1) and the analogs for example, pFT30a (manufactured by Novagen) (for His thereof) in detection, quantification, neutralization, or tags), pGEX (manufactured by GE Healthcare Bio-Sciences removal of IgE antibody and/or IgG antibody binding to a Corp.) (for GST tags), and a pCold TF vector (manufactured Sweat allergy antigen. by TAKARA BIO INC.), in a suitable host cell such as a The protein or polypeptide provided by the present inven microorganism and a cell. tion may be an isolated protein or polypeptide. 10 These analogs of the MGL 1304 partial peptide can be 2. MGL 1304 Partial Peptide analogs of peptides binding to IgE antibody present in a In a second aspect, the present invention provides an serum of a Sweat allergy patient. MGL 1304 partial peptide. The analogs of the MGL 1304 partial peptide may release The MGL 1304 partial peptide may be a sweat allergy histamine via IgE from a cell expressing IgE receptor. antigen. Therefore, in one embodiment, the present inven 15 The analogs of the MGL 1304 partial peptide provided tion provides use of the MGL 1304 partial peptide in by the present invention may be an isolated protein or manufacturing of a Sweat allergy antigen. polypeptide. As used herein, the MGL 1304 partial peptide may be a The MGL 1304 partial peptide and the analog thereof peptide corresponding to a portion of a protein encoded by provided by the preset invention can be manufactured by a the MGL 1304 gene. The MGL 1304 partial peptide may conventional genetic engineering method or a method used be a peptide consisting of a portion of the amino acid in peptide synthesis. For example, the MGL 1304 partial sequence represented by SEQ ID NO: 1. Examples of the peptide and the analog thereof may be manufactured by the MGL 1304 partial peptide include a peptide corresponding following method: to a residue 32-173 of the polypeptide represented by SEQ (i) the MGL 1304 partial peptide provided by the preset ID NO: 1 (SEQ ID NO: 2), a peptide corresponding to a 25 invention is expressed in E. coli or cells using an expression residue 32-183 of the polypeptide represented by SEQ ID vector into which a nucleotide encoding an amino sequence NO: 1 (SEQID NO:3), a peptide corresponding to a residue represented by any of SEQ ID NOs: 2 to 7 is inserted; or 27-173 of the polypeptide represented by SEQ ID NO: 1 (ii) the MGL 1304 partial peptide provided by the preset (SEQ ID NO: 4), a peptide corresponding to a residue invention is expressed in E. coli or cells using an expression 27-183 of the polypeptide represented by SEQ ID NO: 1 30 vector inserted into which a nucleotide encoding an amino (SEQ ID NO. 5), a peptide corresponding to a residue sequence having 60, 70, 80, 90, or 95% or more identity to 22-173 of the polypeptide represented by SEQ ID NO: 1 an amino acid sequence represented by any of SEQID NOs: (SEQ ID NO: 6) or a peptide corresponding to a residue 2 to 7 is inserted; or 22-183 of the polypeptide represented by SEQ ID NO: 1 (iii) the MGL 1304 partial peptide provided by the preset (SEQ ID NO: 7). 35 invention is expressed in E. coli or cells using an expression Therefore, in one embodiment, the present invention vector into which a nucleotide encoding an amino sequence provides a peptide comprising an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, represented by any of SEQ ID NOS: 2 to 7. 18, 19, or 20 amino acids deleted, substituted, or added in an The MGL 1304 partial peptide may be a peptide binding amino acid sequence represented by any of SEQ ID NOS: 2 to the IgE antibody present in a serum of a Sweat allergy 40 to 7 is inserted. patient. The MGL 1304 partial peptide and the analog thereof In one embodiment, the MGL 1304 partial peptide may provided by the preset invention bind to IgE antibody and/or release histamine via IgE from a cell expressing the IgE IgG antibody binding to a Sweat allergy antigen present in a receptor. serum of a Sweat allergy patient and therefore can be used The MGL 1304 partial peptide provided by the present 45 for detecting, quantifying, neutralizing, or removing IgE invention may be an isolated protein or polypeptide. antibody and/or IgG antibody binding to a Sweat allergy In one embodiment, the present invention provides an antigen present in a serum of a Sweat allergy patient. Thus, analog of the MGL 1304 partial peptide. The analog is not in one embodiment, the present invention provides use of the particularly limited as long as the analog can be prepared MGL 1304 partial peptide and the analog thereof (e.g., the based on a peptide consisting of a portion of the amino acid 50 proteins consisting of an amino acid sequence represented sequence represented by SEQ ID NO: 1. Examples of the by any of SEQ ID NOS: 2 to 7 and the analogs thereof) in analog of the peptide consisting of a portion of the amino detection, quantification, neutralization, or removal of IgE acid sequence represented by SEQ ID NO: 1 include a antibody and/or IgG antibody binding to a Sweat allergy peptide consisting of an amino acid sequence having 60, 70. antigen. 80, 90, or 95% or more identity to an amino acid sequence 55 3. Gene, Vector, and Transformant represented by any of SEQID NOs: 2 to 7, and a polypeptide In a third aspect, the present invention provides a gene consisting of an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, encoding a Sweat allergy antigen protein that is a protein 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids derived from a microorganism, the MGL 1304 partial pep deleted, Substituted, or added in an amino acid sequence tide, or an analog thereof. represented by any of SEQ ID NOS: 2 to 7. 60 In one embodiment, the present invention provides a Another example of the analog of the peptide consisting polynucleotide encoding an amino acid sequence repre of a portion of the amino acid sequence represented by SEQ sented by any of SEQ ID NOs: 1 to 7. ID NO: 1 may be a peptide consisting of an amino acid In one embodiment, the present invention provides a sequence represented by any of SEQID NOs: 2 to 7 to which polynucleotide consisting of a nucleotide sequence having a tag is added. As used herein, a tag means a portion added 65 60, 70, 80, 90, or 95% or more identity to a polynucleotide to a polypeptide for purification, detection, etc. of the encoding an amino acid sequence represented by any of polypeptide and is exemplified by histidine (His), gluta SEQ ID NOs: 1 to 7, and a polynucleotide consisting of a US 9,457,071 B2 11 12 nucleic acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, derived from a sweat allergy patient and/or smith2 antibod 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, ies, to a mammal (e.g., a mouse or a rabbit). 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, As used herein, antibodies include polyclonal antibodies 45, 46, 47, 48, 49, or 50 bases deleted, substituted, or added and monoclonal antibodies. in a polynucleotide encoding an amino acid sequence rep- 5 As used herein, monoclonal antibodies include genetic resented by any of SEQ ID NOs: 1 to 7. recombinant monoclonal antibodies artificially modified for The polynucleotide provided by the present invention the purpose of reducing heterologous antigenicity to a may be an isolated polynucleotide. human, for example, chimera monoclonal antibodies, The polynucleotide provided by the present invention can humanized monoclonal antibodies and human monoclonal 10 antibodies. be introduced into a vector and expressed in a host cell as A fragment of an antibody is a portion of an antibody needed. specifically binding to an antigen. A fragment of an antibody Therefore, in one embodiment, the present invention may be Fab (fragment of antigen binding), F(ab')2, Fab', a provides a vector containing the polynucleotide provided by single-chain antibody (single chain Fv; hereinafter referred the present invention, and a host cell (transformant) in which 15 to as schv), a disulfide stabilized antibody (disulfide stabi the polynucleotide provided by the present invention is lized FV; hereinafter referred to as dsEv), a dimerized introduced. V-region fragment (hereinafter referred to as a diabody), and The polynucleotide, the vector, and the host cell provided a peptide comprising CDR (Expert Opinion on Therapeutic by the present invention can be used in manufacturing of the Patents, Vol. 6, No. 5, pp. 441-456, 1996). The antibodies protein or peptide consisting of an amino sequence repre- 20 and the fragments of antibodies can be prepared by a sented by any of any of SEQID NOs: 2 to 7 or the analog well-known method in the industry (see, e.g., Antibodies: A thereof. Therefore, in one embodiment, the present inven Laboratory Manual, Cold Spring Harbor Laboratory Press, tion provides use of the polynucleotide, the vector, and the 1988: http://www.gene.mie-u.ac.jp/Protocol/Original/Anti host cell provided by the present invention in manufacturing body.html; U.S. Pat. Nos. 6,331415, 5,693,761, 5,225,539, of a Sweat allergy antigen. 25 5,981,175, 5,612.205, 5,814,318, 5,545,806, 7,145,056, 4. Antibody 6,492,160, 5,871,907, and 5,733,743). In a fourth aspect, the present invention provides an The antibodies provided by the present invention may be antibody or an antibody fragment specifically binding to a used as a tool recognizing a protein consisting of the amino Sweat allergy antigen protein that is a protein derived from acid sequence represented by SEQ ID NO: 1 present in a a microorganism or the MGL 1304 partial peptide. 30 sample derived from a human, for example, human Sweat or In one embodiment, the present invention provides an skin, to analyze an amount, distribution, a function, etc. of antibody or an antibody fragment specifically binding to a the protein. protein encoded by the MGL 1304 gene (e.g., a protein The antibodies provided by the present invention may consisting of the amino acid sequence represented by SEQ have a label bound thereto. Examples of the label include an ID NO: 1), a peptide consisting of an amino acid sequence 35 enzyme, a fluorescent Substance, a radioisotope, biotin, etc. represented by any of SEQID NOs: 2 to 7 and SEQID NOs: Examples of the enzyme include alkaline phosphatase, 10 to 13, or an analog thereof. For example, an antibody or peroxidase, glucose oxidase, tyrosinase, acid phosphatase, an antibody fragment specifically binding to a protein etc. encoded by the MGL 1304 gene (e.g., a protein consisting Examples of the fluorescent substance include fluorescein of the amino acid sequence represented by SEQ ID NO: 1) 40 isothiocyanate (FITC), GFP. luciferin, etc. can be prepared by administering a protein or a peptide Examples of the radioisotope include 'I, C, ‘P, etc. consisting of an amino acid sequence represented by any of Examples of an antibody specifically binding to a Sweat SEQ ID NOs: 1 to 7 and 10 to 13 or an analog thereof allergy antigen protein, i.e., a protein derived from a micro expressed in E. coli, COS7 cells, or Malassezia globosa as organism, provided by the present invention include, but not an antigen to a mammal (e.g., a mouse or a rabbit). 45 limited to, the following (i) to (iii): In one embodiment, the present invention provides an (i) an antibody produced by the hybridoma (Mouse-Mouse antibody or an antibody fragment specifically binding to a hybridoma Smith-1) of Accession No. FERM BP-11110 protein present in a culture Supernatant of Malassezia glo (transferred from FERMP-21439) deposited to the Interna bosa and binding to serum derived from a Sweat allergy tional Patent Organism Depositary of the National Institute patient and/or Smith2 antibodies. For example, an antibody 50 of Advanced Industrial Science and Technology on Apr. 1, or an antibody fragment specifically binding to a protein 2009; present in a culture Supernatant of Malassezia globosa and (ii) an antibody produced by the hybridoma (Mouse-Mouse binding to serum derived from a Sweat allergy patient and/or hybridoma Smith-2) of Accession No. FERM BP-11111 Smith2 antibodies can be prepared by administering a pro (transferred from FERMP-21440) deposited to the Interna tein present in a culture Supernatant of Malassezia globosa 55 tional Patent Organism Depositary of the National Institute and binding to serum derived from a Sweat allergy patient of Advanced Industrial Science and Technology on Apr. 1, and/or Smith2 antibodies to a mammal (e.g., a mouse or a 2009; and rabbit) as an antigen. (iii) an antibody produced by the hybridoma (Mouse-Mouse In one embodiment, the present invention provides a hybridoma Smith-8) of Accession No. FERM BP-11112 method of manufacturing an antibody or an antibody frag- 60 (transferred from FERMP-21697) deposited to the Interna ment specifically binding to a protein encoded by the tional Patent Organism Depositary of the National Institute MGL 1304 gene (e.g., a protein consisting of the amino acid of Advanced Industrial Science and Technology on Apr. 1, sequence represented by SEQID NO: 1) comprising admin 2009. istering a protein or a peptide consisting of an amino acid 5. Composition or Kit for Detecting or Quantifying Sweat sequence represented by any of SEQID NOs: 1 to 7 and 10 65 Allergy Antigen to 13 or an analog thereof, or a protein present in a culture In a fifth aspect, the present invention provides a com Supernatant of Malassezia globosa and binding to serum position or kit for detecting a Sweat allergy antigen, or a US 9,457,071 B2 13 14 composition or kit for measuring an amount of a Sweat mouse IgG antibody provided in the fourth aspect of the allergy antigen, comprising an antibody or an antibody present invention, followed by an enzyme-labeled anti fragment specifically binding to a Sweat allergy antigen mouse IgG antibody; and, Subsequently, the Sweat allergy protein that is a protein derived from a microorganism or the antigen can be detected or quantified through the enzyme MGL 1304 partial peptide. activity. The detection of a Sweat allergy antigen and the measure For example, in the case of ELISA, a sample containing ment of an amount of a Sweat allergy antigen are measured a Sweat allergy antigen is coated on a plate by adding the by any method. For example, the detection of Sweat allergy sample to the plate; the coated sample is reacted with an antigens and the measurement of an amount of a Sweat antibody against the Sweat allergy antigens, i.e., the mouse allergy antigen can be performed using Western blotting or 10 IgG antibody provided in the fourth aspect of the present ELISA. invention, followed by an enzyme-labeled anti-mouse IgG Therefore, in one embodiment, the composition for antibody; and, Subsequently, the Sweat allergy antigen can detecting a Sweat allergy antigen or the composition for be detected or quantified through the enzyme activity. In measuring an amount of a Sweat allergy antigen may be a another example, a Sweat allergy antigen may be detected or composition comprising an antibody or an antibody frag 15 quantified by sandwich ELISA where two types of the ment specifically binding to a Sweat allergy antigen protein antibodies against the Sweat allergy antigen provided in the that is a protein derived from a microorganism or the fourth aspect of the present invention are used. Among these MGL 1304 partial peptide for use in Western blotting or reactions, the reaction with the enzyme-labeled anti-mouse ELISA. IgG antibody may be omitted when the mouse IgG antibody In one embodiment, the kit for detecting a Sweat allergy provided in the fourth aspect of the present invention, which antigen or the kit for measuring an amount of a Sweat allergy is enzymatically labeled, is used. antigen may be a kit comprising a reagent necessary for In one embodiment, the present invention provides a Western blotting or ELISA. Examples of the reagents nec method of detecting a Sweat allergy antigen or a method of essary for Western blotting include an SDS-PAGE gel, a measuring an amount of a Sweat allergy antigen comprising nitrocellulose membrane or PVDF membrane, the antibody 25 subjecting the antibody provided in the fourth aspect of the provided in the fourth aspect of the present invention, a present invention to reacting with a sample derived from a blocking solution (e.g., a BSA solution, a milk protein human. Examples of a sample derived from a human Solution), wash Solution (phosphate buffer containing a include, but not limited to, a human Sweat, human skin Surfactant (e.g., PBS containing Tween20)), a luminescence washings, a solution of an extract of human skin, a human detection reagent, etc. Examples of the reagents necessary 30 serum, a human plasma, etc. for ELISA include a plate (e.g., a 96-well plate), the anti In one embodiment, the present invention provides use of body provided in the fourth aspect of the present invention, an antibody or an antibody fragment specifically binding to a blocking solution (e.g., a BSA Solution, a milk protein a Sweat allergy antigen protein that is a protein derived from Solution), wash Solution (phosphate buffer containing a a microorganism or the MGL 1304 partial peptide provided Surfactant (e.g., PBS containing Tween20)), a chromogenic 35 in the fourth aspect of the present invention, for manufac substrate (e.g., TMB), etc. turing a composition or kit for detecting a Sweat allergy The kit for detecting a Sweat allergy antigen or the kit for antigen, or a composition or kit for measuring an amount of measuring an amount of a Sweat allergy antigen may include a Sweat allergy antigen. as a standard a protein encoded by the MGL 1304 gene 6. Composition or Kit for Detecting or Quantifying Anti (e.g., a protein consisting of the amino acid sequence 40 body binding to Sweat Allergy Antigen represented by SEQ ID NO: 1) or a protein present in a In a sixth aspect, the present invention provides a com culture Supernatant of Malassezia globosa and binding to position or kit for detecting an antibody binding to a Sweat serum derived from a Sweat allergy patient and/or Smith2 allergy antigen, or a composition or kit for measuring an antibodies. Using a solution at a known concentration of a amount of an antibody binding to a Sweat allergy antigen, protein encoded by the MGL 1304 gene (e.g., a protein 45 comprising a Sweat allergy antigen protein that is a protein consisting of the amino acid sequence represented by SEQ derived from a microorganism or the MGL 1304 partial ID NO: 1) or a protein present in a culture supernatant of peptide. Examples of the detected or quantified antibodies Malassezia globosa and binding to serum derived from a may be, but not limited to, human IgE antibodies or human Sweat allergy patient and/or Smith2 antibodies, an amount of IgG antibodies (e.g., all the human IgG antibodies binding a Sweat allergy antigen can accurately be quantified. For 50 to Sweat allergy antigens, or human IgG4 antibodies binding example, using a plurality of Solutions at known concentra to Sweat allergy antigens). tions of a protein present in a culture Supernatant of Mal The detection of an antibody binding to a Sweat allergy assezia globosa and binding to serum derived from a Sweat antigen and the measurement of an amount of an antibody allergy patient and/or Smith2 antibodies, an amount of a binding to Sweat allergy antigens can be measured by any Sweat allergy antigen can be accurately quantified. The 55 method. For example, the detection of an antibody binding protein encoded by the MGL 1304 gene (e.g., a protein to a Sweat allergy antigen and the measurement of an amount consisting of the amino acid sequence represented by SEQ of an antibody binding to a Sweat allergy antigen can be ID NO: 1) may be prepared by expressing the gene provided performed using Western blotting or ELISA. in the third aspect of the present invention in a host such as Therefore, in one embodiment, the composition for E. coli and COS7 cells. 60 detecting an antibody binding to a Sweat allergy antigen or Western blotting and ELISA can be conducted as needed the composition for measuring an amount of an antibody by those skilled in the art. binding to a Sweat allergy antigen may be a composition For example, in the case of Western blotting, a sample comprising the Sweat allergy antigen protein that is a protein containing a Sweat allergy antigen is electrophoresed using derived from a microorganism or the MGL 1304 partial the SDS-PAGE gel; the electrophoresed sample is trans 65 peptide for use in Western blotting or ELISA. ferred to the PVDF membrane; the membrane is reacted In one embodiment, the kit for detecting an antibody with an antibody against the Sweat allergy antigen, i.e., the binding to a Sweat allergy antigen or the kit for measuring US 9,457,071 B2 15 16 an amount of an antibody binding to a Sweat allergy antigen enzyme-labeled anti-human antibody (e.g., enzyme-labeled may be a kit comprising a reagent necessary for Western anti-human IgG antibody, enzyme-labeled anti-human IgM blotting or ELISA. Examples of the reagents necessary for antibody, enzyme-labeled anti-human IgG1 antibody); and Western blotting include an SDS-PAGE gel, a nitrocellulose the enzyme activity may be measured to detect or quantify membrane or PVDF membrane, the Sweat allergy antigen 5 the Sweat allergy antigen. provided in the first aspect of the present invention (e.g., a In one embodiment, the present invention provides a protein encoded by the MGL 1304 gene (e.g., a protein method of detecting an antibody binding to a Sweat allergy consisting of the amino acid sequence represented by SEQ antigen or a method of measuring an amount of an antibody ID NO: 1), a protein present in a culture supernatant of binding to a Sweat allergy antigen comprising Subjecting the Malassezia globosa and binding to serum derived from a 10 Sweat allergy antigen provided in the first aspect of the Sweat allergy patient and/or Smith2 antibodies) or the present invention or the MGL 1304 partial peptide provided MGL 1304 partial peptide provided in the second aspect of in the second aspect of the present invention to reacting with the present invention (e.g., a peptide indicated by any of a sample derived from a human. The method may be SEQ ID NOS: 2 to 7), a blocking solution (e.g., a BSA performed in vitro. Examples of the sample derived from a Solution, a milk protein solution), wash solution (phosphate 15 human include, but not limited to, a human Sweat, human buffer Solution containing a surfactant (e.g., PBS containing skin washings, a solution of an extract of human skin, a Tween20)), a luminescence detection reagent, etc. Examples human serum, a human plasma, etc. of the reagents necessary for ELISA include a plate (e.g., a In one embodiment, the present invention provides use of 96-well plate), the sweat allergy antigen provided in the first the Sweat allergy antigen protein provided in the first aspect aspect of the present invention (e.g., a protein coded by the of the present invention or the MGL 1304 partial peptide MGL 1304 gene (e.g., a protein consisting of the amino acid provided in the second aspect of the present invention, for sequence represented by SEQ ID NO: 1), a protein present manufacturing a composition or kit for detecting an antibody in a culture Supernatant of Malassezia globosa and binding binding to a Sweat allergy antigen, or a composition or kit for to serum derived from a Sweat allergy patient and/or Smith2 measuring an amount of an antibody binding to a Sweat antibodies (Accession No. FERM BP-11111)) or the 25 allergy antigen. MGL 1304 partial peptide provided in the second aspect of 7. Composition or Kit for Diagnosing Sweat Allergy or the present invention (e.g., a peptide indicated by any of Disease Related to Sweat Allergy Antigen SEQ ID NOS: 2 to 7), a blocking solution (e.g., a BSA In a seventh aspect, the present invention provides a Solution, a milk protein solution), wash solution (phosphate composition or kit for diagnosing a Sweat allergy or a buffer Solution containing a surfactant (e.g., PBS containing 30 disease related to a Sweat allergy antigen, comprising: Tween20)), etc. (i) the Sweat allergy antigen protein that is a protein derived The kit for detecting an antibody binding to a sweat from a microorganism or the MGL 1304 partial peptide; or allergy antigen or the kit for measuring an amount of an (ii) an antibody or an antibody fragment specifically binding antibody binding to a Sweat allergy antigen may comprise as to the Sweat allergy antigen protein that is a protein derived a standard a protein encoded by the MGL 1304 gene (e.g., 35 from a microorganism or the MGL 1304 partial peptide. a protein consisting of the amino acid sequence represented As used herein, a disease related to a Sweat allergy antigen by SEQ ID NO: 1) or a protein present in a culture may be a disease accompanied with a Sweat allergy induced Supernatant of Malassezia globosa and binding to serum by an antigenic Substance contained in Sweat and may be, for derived from a sweat allergy patient and/or smith2 antibod example, atopic dermatitis, urticaria (e.g., cholinergic urti ies. Using a solution at a known concentration of these 40 caria), allergic rhinitis, etc. proteins, an antibody binding to the protein can accurately The composition or kit for diagnosing a Sweat allergy or be quantified. For example, using a plurality of Solutions at a disease related to a Sweat allergy antigen provided in the known concentrations of a protein present in a culture seventh aspect of the present invention can diagnose, or Supernatant of Malassezia globosa and binding to serum assist the diagnosis of a Sweat allergy or a disease related to derived from a sweat allergy patient and/or smith2 antibod 45 a Sweat allergy antigen through detection or quantification of ies, the antibody binding to a Sweat allergy antigen can the antibody binding to a Sweat allergy antigen or the Sweat accurately be quantified based on an amount of the antigen. allergy antigen. Western blotting and ELISA can be conducted as needed In one embodiment, the composition or kit for detecting by those skilled in the art. or quantifying an antibody binding to a Sweat allergy antigen For example, in the case of ELISA, the Sweat allergy 50 provided in the sixth aspect of the present invention is antigen protein provided in the first aspect of the present available for the composition or kit, relating to the seventh invention or the MGL 1304 partial peptide provided in the aspect of the present invention, for diagnosing a Sweat second aspect of the present invention is coated; a sample allergy or a disease relating to a Sweat allergy antigen containing an antibody binding to a Sweat allergy antigen is comprising (i) the Sweat allergy antigen protein that is a added thereto; the sample is reacted with an enzyme-labeled 55 protein derived from a microorganism or the MGL 1304 anti-human antibody (e.g., enzyme-labeled anti-human IgG partial peptide. For example, an amount of an antibody (e.g., antibody, enzyme-labeled anti-human IgM antibody, IgE and/or IgG (e.g., all the IgGs and/or IgG4)) binding to enzyme-labeled anti-human IgG4 antibody); and the a Sweat allergy antigen in a sample (e.g., serum or plasma) enzyme activity can be measured to detect or quantify the derived from blood of a human Subject is measured by using Sweat allergy antigen. In another example, the antibody 60 the kit provided in the sixth aspect of the present invention, against a Sweat allergy antigen provided in the fourth aspect and is compared with an amount of an antibody binding to of the present invention is coated; the antibody is reacted a Sweat allergy antigen in a sample derived from blood of a with the Sweat allergy antigen protein provided in the first healthy person, and if the amount of an antibody binding to aspect of the present invention or the MGL 1304 partial the Sweat allergy antigens in the sample derived from blood peptide provided in the second aspect of the present inven 65 of the Subject is larger than the amount of an antibody tion; a sample containing an antibody binding to a Sweat binding to the Sweat allergy antigen in the sample derived allergy antigen is added thereto; the sample is reacted with from blood of a healthy person, the Subject can be diagnosed US 9,457,071 B2 17 18 with a Sweat allergy or a disease related to a Sweat allergy The determination is indicated by (-) when no reaction antigen, or having a risk of a Sweat allergy or a disease exists, (+) when a reagent portion of the adhesive tape is related to a Sweat allergy antigen. Alternatively, this mea associated with erythema and edema, or (++) or (+++) Surement result of an amount of an antibody binding to the depending on a degree of the reaction. Sweat allergy antigen can be used for assisting a diagnosis of 5 The Scratch test and the prick test are examinations a Sweat allergy or a disease relating to a Sweat allergy performed by dropping an antigen Solution onto a skin antigen from clinical finding. Surface, making a scratch on the skin with a needle tip Such In one embodiment, the composition or kit for detecting that bleeding does not occur, and examining a reaction after a Sweat allergy antigen, or the composition or kit for 15 to 20 minutes. If the antigen-specific IgE antibody exists measuring an amount of a Sweat allergy antigen, provided in 10 the fifth aspect of the present invention is available for the on a surface of a mast cell in an upper layer of the dermis, composition or kit, relating to the seventh aspect of the the antibody reacts with the dropped antigen, and histamine present invention, for diagnosing a Sweat allergy or a disease and chemical transmitters in the mast cell are released, related to a Sweat allergy antigen comprising (ii) an antibody resulting in a red Swollen spot. For example, the Sweat or an antibody fragment specifically binding to the Sweat 15 allergy antigen protein or the MGL 1304 partial peptide allergy antigen protein that is a protein derived from a (e.g., a protein consisting of an amino acid sequence repre microorganism or the MGL 1304 partial peptide. For sented by any of SEQ ID NOs: 1 to 7) provided in the first example, an amount of a Sweat allergy antigen in a sample or second aspect of the present invention can be used as an of Sweat of a human subject is measured by the kit provided antigen for the Scratch test and the prick test. in the fifth aspect of the present invention, and is compared The test for intracutaneous reactivity is a test performed with an amount of a Sweat allergy antigen in a sample of by injecting an extremely small amount of antigen Solution Sweat of a healthy person, and if the amount of a Sweat inside a thin skin and observing whether the injected portion allergy antigen in the Sweat of the Subject is larger than the becomes red and Swollen after a certain time so as to amount of an Sweat allergy antigen in the Sweat of a healthy determine the presence of an allergy. For example, the Sweat person, the Subject can be diagnosed with a Sweat allergy or 25 allergy antigen protein or the MGL 1304 partial peptide a disease related to a Sweat allergy antigen, or having a risk (e.g., a protein consisting of an amino acid sequence repre of a Sweat allergy or a disease related to a Sweat allergy sented by any of SEQ ID NOs: 1 to 7) provided in the first antigen. Alternatively, this measurement result of an amount or second aspect of the present invention can be used as the of a Sweat allergy antigen can be used for assisting a antigen for the intradermal test. diagnosis of a Sweat allergy or a disease relating to a Sweat 30 allergy antigen from clinical finding. In another embodiment, the present invention provides a The kit for diagnosing a Sweat allergy or a disease relating kit or composition for a histamine release test comprising (i) to a Sweat allergy antigen provided in the seventh aspect of the Sweat allergy antigen protein that is a protein derived the present invention may comprise as a standard a protein from a microorganism or the MGL 1304 partial peptide. In encoded by the MGL 1304 gene (e.g., a protein consisting 35 the histamine release test, a cell reaction is used to measure of the amino acid sequence represented by SEQ ID NO: 1) an amount histamine release from blood cells (basophils) or a protein present in a culture Supernatant of Malassezia due to antigen stimulation. For example, an amount of globosa and binding to serum derived from a Sweat allergy histamine released from blood cells (basophils) due to patient and/or Smith2 antibodies. Use of this standard antigen stimulation may allow a diagnosis of a Sweat allergy enables accurate quantification and enables an accurate 40 or a disease relating to a Sweat allergy or having a risk diagnosis. thereof, or may provide data assisting the diagnosis. In one embodiment, the present invention provides a kit The kit for a histamine release test may further comprise or composition for a skin test comprising (i) a Sweat allergy an anti-histamine antibody and histamine as a standard. antigen protein that is a protein derived from a microorgan In one embodiment, the present invention provides a ism or the MGL 1304 partial peptide. The skin test may be, 45 method of diagnosing, or assisting diagnosis of a Sweat for example, a patch test, a scratch test, a prick test, or an allergy or a disease relating to a Sweat allergy antigen, intradermal test. The skin test may enable to diagnose a comprising contacting Subject with a Sweat allergy or a disease relating to a Sweat (i) the Sweat allergy antigen protein that is a protein derived allergy or having a risk thereof, or the skin test may provide from a microorganism or the MGL 1304 partial peptide data for assisting the diagnosis. 50 provided in the first or second aspect of the present inven The kit for a skin test may further comprise a saline used tion, or as a comparative control. (ii) an antibody or an antibody fragment specifically binding The patch test is widely performed as a simple method of to the Sweat allergy antigen protein that is a protein derived examination for a contact allergy in the dermatological field. from a microorganism or the MGL 1304 partial peptide Since the presence of a contact allergy leads to sensitization 55 provided in the fourth aspect of the present invention of not only a portion of dermatitis but also the whole body with a sample derived from a human (a blood sample (e.g., skin, a cause of can be determined by plasma, serum), Sweat, a sample derived from skin (e.g., artificially reproducing allergic contact dermatitis on a skin washings)). The method may be performed in vitro. healthy skin. For example, the Sweat allergy antigen protein In one embodiment, the present invention provides use of or the MGL 1304 partial peptide (e.g., a protein consisting 60 (i) the Sweat allergy antigen protein that is a protein derived of an amino acid sequence represented by any of SEQ ID from a microorganism or the MGL 1304 partial peptide NOs: 1 to 7) provided in the first or second aspect of the provided in the first or second aspect of the present inven present invention is dripped or applied to an adhesive tape, tion, or and the adhesive tape is affixed to the back, the upper arm, (ii) an antibody or an antibody fragment specifically binding the thigh, etc. The determination is made after two days, 65 to the Sweat allergy antigen protein that is a protein derived three days, and one weak in accordance with the ICDRG from a microorganism or the MGL 1304 partial peptide (International Contact Dermatitis Research Group) scale. provided in the fourth aspect of the present invention for US 9,457,071 B2 19 20 manufacturing the composition for diagnosing, or assisting contacted with the affected part to neutralize the sweat diagnosis of a Sweat allergy or a disease relating to a Sweat allergy antigen and/or to achieve removable of the Sweat allergy antigen. allergy antigen. 8. Composition for Treating Sweat Allergy or Disease Relat The antibody specifically binding to the sweat allergy ing to Sweat Allergy Antigen antigen protein or the MGL 1304 partial peptide may be In an eighth aspect, the present invention provides a immobilized to fibers and used as a material for removing therapeutic composition for a Sweat allergy or a disease the Sweat allergy antigen from the affected part. An example relating to a Sweat allergy antigen comprising a Sweat of the material for removal is a wiping sheet. The material allergy antigen protein that is a protein derived from a for removal can appropriately be manufactured by those 10 skilled in the art. microorganism or the MGL 1304 partial peptide. For example, Japanese Patent No. 3642340 discloses a The therapeutic composition for a Sweat allergy or a method of manufacturing a material for removal Such as a disease relating to a Sweat allergy antigen may be a com wiping sheet comprising immobilizing an antibody to fibers position for a hyposensitization therapy. The "hyposensiti (woven fabric or nonwoven fabric) with an official moisture zation therapy” is a method of treatment performed by 15 regain of 7% or more. administering a slight amount of a therapeuticallergen for an Examples of a method of immobilizing an antibody to a allergy associated with IgE antibody in a gradually increas carrier such as fibers include a method in which after the ing amount at intervals of a certain number of days so as to carrier is silanized using Y-aminopropyltriethoxysilane etc., avoid occurrence of an allergic reaction even when a caus an aldehyde group is introduced to a carrier Surface by ative allergen enters. The protein or the peptide provided in means of glutaraldehyde etc. to covalently bind the aldehyde the first or second aspect of the present invention may be group and the antibody; a method in which an untreated used as the therapeutic allergen in the hyposensitization carrier is immersed in an aqueous solution of the antibody to therapy. immobilize the antibody to the carrier though ion binding; a The therapeutic composition provided by the present method in which an aldehyde group is introduced to a carrier invention is appropriately formulated using the protein or 25 having a certain functional group to covalently bind the the peptide provided by the present invention. For example, aldehyde group and the antibody; a method in which the the therapeutic composition provided by the present inven antibody is subjected to ion-binding to a carrier having a tion can be formulated with pharmaceutically acceptable certain functional group; and a method in which after a carriers (including additives). The pharmaceutically accept carrier is coated with a polymer having a certain functional able carriers include, but not limited to, excipients (e.g., 30 group, an aldehyde group is introduced to covalently bind dextrin, hydroxypropyl cellulose, and polyvinylpyrroli the aldehyde group and the antibody. The certain functional done), disintegrators (e.g., carboxymethyl cellulose), lubri group as described above may be an NHR group (R is an cants (e.g., magnesium Stearate), Surfactants (e.g., sodium alkyl group of any of methyl, ethyl, propyl, and butyl other lauryl Sulfate), solvents (e.g., water, saline, and soybean oil), than H), an NH group, a CH-NH group, a CHO group, a and preservatives (e.g., p-hydroxybenzoic acid ester). 35 COOH group, and an OH group. The antibody may be A dosage and an administration method of the therapeutic Supported via a linker on the carrier, and examples of the composition may appropriately be selected by those skilled linker used include maleimide, NHS (N-Hydroxysuccinim in the art depending on an age, a body weight, and a health idyl) ester, imidoester, EDC (1-Etyl-3-3-dimetylaminopro condition of a subject to be administered. pylcarbodiimido) and PMPI (N-p-Maleimidophenyliso In one embodiment, the present invention provides a 40 cyanete). method of treating a Sweat allergy or a disease relating to a The material for removal may be impregnated with water Sweat allergy antigen comprising administering the Sweat containing glycerol to be stored. allergy antigen protein that is a protein derived from a In one embodiment, the present invention provides a microorganism or the MGL 1304 partial peptide provided method of removing or neutralizing a Sweat allergy antigen in the first or second aspect of the present invention. 45 comprising contacting the antibody specifically binding to In one embodiment, the present invention provides use of the sweat allergy antigen protein or the MGL 1304 partial the Sweat allergy antigen protein that is a protein derived peptide provided in the fourth aspect of the present invention from a microorganism or the MGL 1304 partial peptide with the Sweat allergy antigen. provided in the first or second aspect of the present invention In one embodiment, the present invention provides use of for manufacturing a therapeutic composition for a Sweat 50 the antibody specifically binding to the Sweat allergy antigen allergy or a disease relating to a Sweat allergy antigen. protein or the MGL 1304 partial peptide provided in the 9. Composition for Removing or Neutralizing Sweat Allergy fourth aspect of the present invention for manufacturing a Antigen and Material for Removing Sweat Allergy Antigen composition for removing or neutralizing a Sweat allergy In a ninth aspect, the present invention provides a com antigen or a material for removing a Sweat allergy antigen. position for removing or neutralizing a Sweat allergy antigen 55 10. Composition or Kit for Determining Effect of Hypos and a material for removing a Sweat allergy antigen com ensitization Therapy prising a Sweat allergy antigen protein that is a protein In a tenth aspect, the present invention provides a com derived from a microorganism or the MGL 1304 partial position or kit for determining an effect of a hyposensitiza peptide. tion therapy comprising a Sweat allergy antigen protein that An antibody specifically binding to the Sweat allergy 60 is a protein derived from a microorganism or the MGL 1304 antigen protein or the MGL 1304 partial peptide can be partial peptide. used for neutralizing the antigen activity of the Sweat allergy The hyposensitization therapy can be performed by antigen. The antibody can be also used for removing the administering a composition comprising a Sweat allergy Sweat allergy antigen from an affected part. antigen (e.g., the protein provided in the first aspect or the For example, an isotonic Solution Such as a saline com 65 peptide provided in the second aspect of the present inven prising an antibody specifically binding to the Sweat allergy tion) in a gradually increasing amount as described in the antigen protein or the MGL 1304 partial peptide may be eighth aspect of the present invention. US 9,457,071 B2 21 22 In general, it is known that the blood concentration of EXAMPLES IgG4 against the antigen is increased while the blood concentration of IgE against the antigen is decreased in the Example 1 course of treatment in the hyposensitization therapy. Also in the hyposensitization therapy performed by 5 Purification of Partially Purified Sweat Antigen administering a composition comprising a Sweat allergy (QRX) antigen (e.g., the protein provided in the first aspect or the peptide provided in the second aspect of the present inven 1-1. Preparation of Concentrated Sweat tion) in a gradually increasing amount at intervals of a After insolubles were removed from a human sweat 10 through 100-um and 70-um mesh filters (Nylon Cell Strain certain number of days, if the administration shows thera ers, Falcon), precipitates were further removed by a 0.22-um peutic effects, the blood concentration of IgG4 against the filter (Bottle Top Filter, 1 L, Corning). Four litters of the Sweat allergy antigen may be increased, and the blood sweat filtrated by the filters were concentrated by ultrafil concentrations of IgE antibody and total IgG antibody tration (3000 M.W.cut) to about 150 mL and used as a against the Sweat allergy antigen may be decreased in the 15 material for the Sweat antigen purification. course of treatment. 1-2. Separation by Means of Anion-Exchange Column Therefore, in one embodiment, the present invention To an anion-exchange column MonoQ 10/100 GT (GE provides a method of determining an effect of a hyposensi Healthcare Bio-Sciences) preliminarily equilibrated by 10 tization therapy comprising measuring an amount of a mmol/L Tris-HCl (pH 8.0), 75 mL of the concentrated sweat human IgG4 antibody binding to a Sweat allergy antigen prepared at pH 8.0 was loaded, and was eluted through 0 to (e.g., the protein provided in the first aspect or the peptide 1.0 M NaCl concentration gradient in 10 mmol/L Tris-HCl provided in the second aspect of the present invention) in a (pH 8.0). AKTA Explorer (GE Healthcare Bio-Sciences) human blood sample (e.g., human serum or plasma). In one was used as a chromatographic device for purification. embodiment, the present invention provides a method of To select a fraction containing a substance inducing the determining an effect of a hyposensitization therapy com 25 histamine release activity, a histamine release test using prising comparing an amount of a human IgG4 antibody basophils of an atopic dermatitis patient was performed for binding to a Sweat allergy antigen (e.g., the protein provided each of fractions. in the first aspect or the peptide provided in the second First, each of the appropriately diluted fractions was aspect of the present invention) in a blood sample collected mixed at 1:1 with a basophil fraction of an atopic dermatitis before performing the hyposensitization therapy with a 30 patient prepared in a HEPES buffer containing 5 mmol/L blood sample collected after performing the hyposensitiza glucose, 0.03 w/v '% HSA, 2 mmol/L CaCl, and 1 mmol/L tion therapy. In these methods of determining an effect of a MgCl, and was incubated at 37° C. for 40 minutes. After hyposensitization therapy, if the concentration of IgG4 anti Supernatant and sedimented blood cells were separated by body binding to a Sweat allergy antigen in blood of a patient centrifugation and were respectively denatured by adding increases, it can be determined that the hyposensitization 35 0.2 mol/L perchloric acid, a histamine concentration in the therapy exerts an effect. The methods of determining an Supernatant obtained by the centrifugation was measured by effect of a hyposensitization therapy may further comprise HPLC (Shimadzu LC solution). A rate of the histamine measuring the amounts of IgE antibody and/or total IgG amount of the Supernatant relative to the total histamine antibody in the blood to the Sweat allergy antigens, and/or amount was defined as the histamine release activity. comparing the amounts of the IgE antibody and/or total IgG 40 The histamine amount was measured in accordance with antibody binding to the Sweat allergy antigen in a blood a method described in a literature (Koro, O. et al., J. Allergy sample collected before performing the hyposensitization Clin. Immunol., 103,663-670, 1999). therapy with a blood sample collected after performing the As a result, a fraction eluted within a salt concentration hyposensitization therapy. The methods of determining an range of 0.25 to 0.3 mol/L NaCl having the histamine release effect of a hyposensitization therapy may be performed in 45 activity higher than the other fractions was recovered as a vitro. fraction exhibiting the histamine release activity. The composition or kit for detecting or quantifying an 1-3. Separation by Means of Reverse-Phase Column antibody binding to a Sweat allergy antigen provided in the After diluting 18 mL of the fraction acquired in Example sixth aspect of the present invention can be utilized in the 1-2 with pure water 10 times, TFA was added at a final measurement of an amount of an antibody binding to the 50 concentration of 0.1 Viv 96. This was loaded to a reverse Sweat allergy. phase column (SOURCE 15RPC ST 4.6/100 (GE Health Therefore, the composition or kit comprising a Sweat care Bio-Sciences)) and was eluted through concentration allergy antigen protein that is a protein derived from a gradient from 0.1 v/v % TFA/distilled water to 0.1 v/v % microorganism or the MGL 1304 partial peptide provided TFA/acetonitrile. AKTA Explorer (GE Healthcare Bio-Sci in the sixth aspect of the present invention can be used as a 55 ences) was used as a chromatographic device for purifica composition or kit for determining an effect of a hyposen tion. sitization therapy. After volatilizing TFA and acetonitrile of the eluted In one embodiment, the present invention provides use of fractions, the histamine release test was performed as in a Sweat allergy antigen protein that is a protein derived from Example 1-2. a microorganism provided in the first aspect of the present 60 As a result, a fraction within a range of about 30 to 35 V/v invention or the MGL 1304 partial peptide provided in the % acetonitrile having the histamine release activity higher second aspect of the present invention for manufacturing a than the other fractions was recovered as the fraction exhib composition or kit for determining an effect of a hyposen iting the histamine release activity (4 mL). sitization therapy. 1-4. Separation by means of Gel Filtration Chromatography The present invention will hereinafter further be described 65 After lyophilization, the fraction obtained in Example 1-3 in examples and these examples are not intended to limit the was re-dissolved in PBS, loaded to Superdex 75 PC 3.2/30 present invention. (GE Healthcare Bio-Sciences), and fractionated and eluted US 9,457,071 B2 23 24 in PBS (-). Smart System (GE Healthcare Bio-Sciences) phils (FIG. 3, HCl) to perform the histamine release test. The was used as a chromatographic device for purification. results indicated that MGL 1304 induces histamine release The histamine release test was performed for the eluted specifically in the atopic dermatitis patients. fractions as in Example 1-2. Additionally, cDNA encoding the MGL 1304 described As a result, a fraction within a range of elution positions above or mite cDNA is inserted into pSecTag2/Hygro vector from 15 to 60 kD was recovered as the fraction exhibiting (manufactured by Invitrogen) containing Myc-tag to trans the histamine release activity (1.2 mL) and Subsequently fect COS7 cells. A culture supernatant of the COS7 cells defined as a QRX fraction. transfected with these DNAs was subjected to acrylamide gel electrophoresis and trasferred to a PVDF membrane for Example 2 10 immunoblotting with anti-Myc tag antibodies. The results indicated that the culture Supernatant contains proteins cor Purification and Mass Spectrometry of Partially responding to respective cDNAs (In FIG. 11, A). The culture Purified Sweat Antigen (QRX) Supernatant was further reacted with atopic dermatitis patient peripheral blood basophils to perform the histamine The partially purified sweat antigen (QRX) was purified 15 release test. The same basophils were reacted with sweat by using an Aqua 5u-C18-200A HPLC column (manufac antigens (QR) partially purified from the concentrated tured by Phenomenex) (eluted through concentration gradi human Sweat by the anion-exchange column chromatogra ent from 0.1 V/v '% TFA? distilled water to 0.1 v/v '% phy and the reverse-phase column chromatography to per TFA/100% acetonitrile). The fraction exhibiting the hista form the histamine release test for comparison with the mine release activity was recovered and further purified by histamine release rate from the culture supernatant (In FIG. a Jupiter 5u-C18-300A HPLC column (manufactured by 11, B). Phenomenex) (eluted through concentration gradient from The results indicated that MGL 1304 protein 0.1 VfV 96 TFA? distilled water to 0.1 W/V 9% TFA/80% (rMGL 1304) produced by the COST cells induces hista acetonitrile), and a mass spectrometry (TOF-MS) was per mine release like the partially purified human Sweat antigens formed for the fraction exhibiting the histamine release 25 (QR). activity (fraction of an arrow of FIG. 1). Although a sample is normally cationized in TOF-MS, the mass measurement Example 4 was performed through anionization in this experiment. The histamine release activity was measured in accordance with Reactivity Between Recombinant Protein of a method described in Koro, O. et al., J. Allergy Clin. 30 MGL 1304 and Atopic Dermatitis Patient IgE Immunol., 103, 663-670, 1999. The detected amino acid sequence matched MGL 1304. Experiments were conducted to study whether MGL 1304 had substantially the same property as the Example 3 partially purified sweat antigen (QRX) used so far. 35 (1) Preparation of Recombinant Protein of MGL 1304 Recombinant mite antigens (Der f1), QRX, and and Reactivity to Atopic Dermatitis Patient IgE MGL 1304 were electrophoresed and transferred to PVDF membranes to prepare a plurality of membranes. An atopic Malassezia globosa was purchased from ATCC (MYA patient serum (AD serum) pretreated with QRX or 4612). Reverse transcription of mRNA extracted from Mal 40 MGL 1304 prepared in Example 3 or AD serum without assezia globosa to cDNA was performed and the cDNA pretreatment was used for immunoblotting. The atopic der encoding MGL 1304 was amplified by PCR (sense primer: matitis patient serum used was obtained from three patients. 5'-GGGGTACCGTATCCCTCA ACATTTTCTCAGCTGC The pretreatment with MGL 1304 inhibited the binding of 3' (SEQ ID NO: 8); antisense primer: 5'-CCCAAGCTTT IgE to QRX and the pretreatment with QRX inhibited the TAGCAGTCGTACTTGCCGGGGATG-3' (SEQ ID NO: 45 binding of IgE to MGL 1304 (FIG. 4). 9), (94°C., 5 min/60° C., 1 min/72°C., 1 min)x1 cycle, (94° (2) C., 1 min/60° C., 1 min/72° C., 1 min)x30 cycles, (94° C. The atopic dermatitis patient serums (AD1 to AD4) were 5 min/60° C., 1 min/72° C., 10 min)x1 cycle) and the pretreated with TF or TF-MGL 1304 and used for sensitiz amplified cDNA was inserted into the p Cold TF vectors ing a rat cell line expressing human IGE receptors (Sub-unit (manufactured by TAKARA BIO INC.) followed by trans 50 A) so as to measure degranulation when being stimulated fection of E. coli JM109 with the obtained vector. After with anti-IgE, QRX, and a mite extract (Mite-Df). The culturing at 15° C. for 24 hours, the resulting E. coli was results indicated that when MGL 1304 specific IgE is dissolved in xTractor buffer and a recombinant protein was removed by pretreatment with MGL 1304 prepared in purified by a cobalt column. A protein with Trigger Factor Example 3, the reactivity to QRX stimulation disappears only (TF), a TF-MGL 1304 fusion protein (TF 55 (FIG. 5). MGL 1304), and a protein obtained by removing TF from the fusion protein through enzyme treatment (rMGL 1304) Example 5 were prepared. The obtained proteins were subjected to acrylamide gel electrophoresis and were directly CBB Structure Necessary for IgE Binding in MGL 1304 stained (left side of FIG. 2) or transferred to PVDF mem 60 branes for immunoblotting with an anti-His tag antibody MGL 1304 proteins (183 amino acids) truncated at the (center of FIG. 2) and an atopic dermatitis patient serum N-terminal, truncated at the C-terminal, and corresponding (right side of FIG. 2). The results indicate that the atopic to 1-50 (P1), 46-100 (P2), 96-140 (P3), and 146-183 (P4) of dermatitis patient IgE binds to rMGL 1304 (FIG. 2). the polypeptide represented by SEQ ID NO: 1 were The prepared rMGL 1304 was reacted with atopic der 65 expressed in E. coli to be prepared, and immunoblotted with matitis patient peripheral blood basophils (FIG. 3, AD1, anti-His tag antibodies, atopic dermatitis patient serums (In AD2, and AD3) and healthy person peripheral blood baso FIG. 6, B: AD1, AD2, AD3, and AD4), and/or the Smith2 US 9,457,071 B2 25 26 antibodies, which are antibodies produced by the hybridoma tant, the MGL 1304 protein (rMGL) prepared in Example3, (Mouse-Mouse hybridoma Smith-2) of Accession No. and QRX are electrophoresed and immunoblotted with each FERM BP-11111 (transferred from FERMP-21440) depos atopic dermatitis patient serum from two patients (in FIGS. ited to the International Patent Organism Depositary of the 8 and 9, the atopic dermatitis patient serums used are National Institute of Advanced Industrial Science and Tech different). nology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305 After purification of the Malassezia fungus body extract 8566) on Apr. 1, 2009. For peptide fragments, the histamine and the Malassezia culture Supernatant, the purified Mal release test was performed using the atopic dermatitis assezia fungus body extract, the purified Malassezia culture patient basophils (In FIG. 6, C and D). Numbers denote the Supernatant, and QRX were electrophoresed and immuno numbers of the amino acid sequences. 10 blotted with the Smith2 antibody (FIG. 10). The Malassezia The results indicated that the protein with the N-terminal fungus body extract and the Malassezia culture Supernatant shortened by 32 or more amino acids or with the C-terminal were purified as follows. M. Globosa was cultured in a 2693 deleted by 10 or more amino acids loses the binding ability mDixon medium at 32° C. for four days. The culture to the atopic dermatitis patient IgE. Neither the binding medium was centrifuged (2000 rpm) and the supernatant ability to the atopic dermatitis patient IgE nor the histamine 15 release activity was shown by polypeptides P1 to P4 pre was recovered to prepare the Malassezia culture Supernatant pared by fragmenting MGL 1304 polypeptide into four through a 0.22-um filter. The culture Supernatant was frac pieces having a short overlap with each other (P1: a poly tionated by the ion-exchange column chromatography and peptide corresponding to the amino acid sequence 1-50 of the reverse-phase column chromatography described in the MGL 1304 protein (SEQID NO: 10); P2: a polypeptide Example 1 using the histamine release as an index, so as to corresponding to the amino acid sequence 46-100 of the recover a fraction having the histamine release activity. MGL 1304 protein (SEQ ID NO: 11); P3: a polypeptide Although rMGL exhibited a molecular weight (about 23 corresponding to the amino acid sequence 96-140 of the kDa) expected from the DNA sequence (FIG. 2), a protein MGL 1304 protein (SEQ ID NO: 12); P4: a polypeptide detected in the atopic dermatitis patient serum, which is corresponding to the amino acid sequence 136-183 of the 25 present in the lysate of the Malassezia fungus body, exhib MGL 1304 protein (SEQ ID NO: 13). On the other hand, ited about 30 kDa, indicating that MGL is subjected to the Smith-2 antibody exhibits the binding property to both post-translational modification (FIGS. 8, 9, and 10). On the the MGL 1304 protein and the polypeptide (P1) corre other hand, a protein detected in the atopic dermatitis patient sponding to the amino acid sequence 1-50 thereof (In FIG. serum, which is present in the culture Supernatant of the 6, E). These results indicated that the atopic dermatitis 30 Malassezia fungus body, exhibited about 17 kDa, which is patient IgE has a high possibility of recognizing a higher the same as QRX (FIG. 10). Therefore, it was revealed that order structure rather than a short peptide constituting the MGL is further modified when it is secreted from the fungus MGL 1304 protein (In FIG. 6, A). The results also indicated body. A band of about 30 kDa present in the lysate of the that the P1 (MGL 1304 protein and the polypeptide corre 35 Malassezia fungus body, a band of about 17 kDa present in sponding to the amino acid sequence 1-50 thereof) region the culture serum of the Malassezia fungus body, and a band may be a recognition site of an anti-MGL 1304 protein of QRX disappeared when the atopic dermatitis patient antibody in the MGL 1304 protein. serum was pretreated with rMGL (FIGS. 8 and 9). In terms Example 6 of the histamine release activity, the purified antigen from 40 the culture Supernatant secreted from the Malassezia fungus ELISA Using MGL 1304 body shows higher activity compared to rMGL (full-length rMGL obtained by an enzyme treatment to cleave TF bound rMGL 1304 prepared in Example 3 was coated onto a to rMGL for the protein expression). 96-well microplate, followed by blocking using bovine serum albumin (BSA), and subsequently, serums of 4AD 45 Example 8 patients (including AD2 who is HRT-negative) and a healthy volunteer (Normal) were added thereto. The results of Binding of Antibody Raised Against Recombinant detecting IgE binding to rMGL 1304 with the anti-human MGL 1304 Protein as Immunogen to QRX and IgE antibody are shown (FIG. 7). It was revealed that use of Recombinant MGL 1304 Protein a recombinant MGL 1304 protein allows measurements of 50 MGL 1304-specific IgE in serum (FIG. 7). The recombinant MGL 1304 protein (rMGL 1304) pre pared in Example 3 was used for immunization of Balb/c Example 7 mice, and Screening was performed by using the binding property to rMGL as an index so as to prepare a monoclonal Immunoreactivity of Malassezia Fungus Body (M. 55 antibody producing strain (MGLab). The binding properties Globosa) Extract, Malassezia Fungus Body Culture of these antibodies to the polypeptides (P1 to 4) prepared in Supernatant, QRX, and Recombinant MGL 1304 Example 5 having the sequences obtained by fragmenting Protein the MGL 1304 protein into four pieces were examined by ELISA. M. Globosa was cultured in a 2693 mDixon medium at 60 P1 to P4 fused with TF (TF-P1 to 4) dissolved in PBS 32° C. for four days. The culture medium was centrifuged resulting in 3 Jug/ml Solution were coated on a 96-well (2000 rpm) to separate a culture Supernatant and a fungus microplate (50 uL/well) and blocked by BSA, and the body. The fungus body was dissolved in PBS, fragmented monoclonal antibodies were added to detect mouse IgG with ultrasonic waves, and centrifuged (2000 rpm), and the binding to the polypeptides using enzyme-labeled anti Supernatant was recovered to prepare a fungus body extract 65 mouse IgG antibodies. through a 0.22-um filter. The Malassezia fungus body As a result, MGLab6-3 bound to P4 while MGLab8-2, extract, the medium only, the Malassezia culture Superna 8-4, 9-1, 9-5, 10-8, 10-10, 22-1, 36-1, and 40-1 bound to P2, US 9,457,071 B2 27 28 indicating preparation of monoclonal antibodies which rec perature, one hour), and then washed twice. The serum ognize epitopes different from Smith-2 described in diluted by 1% BSA was added at 100 ul/well and left for one Example 3 (FIG. 12). hour at room temperature. After the plate was washed three The results indicated that the P2 (the polypeptide corre times, a solution containing HRP-labeled anti-human IgE sponding to the amino acid sequence 46-100 (Seq ID NO: antibodies is added at 100 ul/well, left for one hour, and 11) of the MGL 1304 protein) and P4 (P4: the polypeptide washed three times, and a color was developed by using corresponding to the amino acid sequence 136-183 (Seq ID TMB to measure absorbance (450 nm). NO: 13) of the MGL 1304 protein) regions may be recog As a result, IgE binding to the Sweat antigen (MGL) was nition sites of an anti-MGL 1304 protein antibody in the detected in the serums of the atopic dermatitis patients and MGL 1304 protein. 10 the allergic rhinitis patients by ELISA coating rMGL (FIGS. 18 and 19). On the other hand, IgE binding to the Sweat Example 9 antigen (MGL) was not detected in the serum of the healthy Measurement of Anti-Sweat Antigen-Specific IgE person (FIGS. 18 and 19). 15 These results indicated that a Sweat allergy can be diag Antibody in Patient Serum nosed in atopic dermatitis and allergic rhinitis patients by Monoclonal antibodies MGLab8-2 (8–2) and MGLab6-3 ELISA coating rMGL. (6-3) prepared by immunizing mice with rVGL prepared in Example 3 were used for studying whether IgE antibodies in Example 11 serums of atopic dermatitis patients (AD1 and AD2) can be detected. Measurement of Anti-MGL Antibody in Patient A 96-well ELISA plate was coated with the antibodies at Subjected to Hyposensitization Therapy 10 ug/ml and 50 uL/well and was left overnight at 4°C. The plate was washed twice, blocked by 2% BSA (one hour), and QRX was repeatedly Subcutaneously injected to a Sweat then washed twice. After 100-fold diluted QRX or 3 ug/ml 25 allergy patient at concentration gradually increased from a of TF-MGL prepared in Example 3 was added at 100 low concentration. Serums were collected over time and ul/well, left for 90 minutes, and washed three times, the changes in the anti-MGL antibody amount in the collected serum was added at 100 ul/well and left for 90 minutes. After serums were measured by ELISA using the anti-human IgE the plate was washed three times, a solution containing antibody, the anti-human IgG antibody, or the anti-human HRP-labeled anti-human IgE antibody is added at 100 30 IgG4 antibody, where rMGL prepared in Example 3 was ul/well, left for one hour, and washed three times, and a coated. color was developed by using TMB to measure absorbance As a result, changes in respective antibody amounts of the (450 nm). anti-MGL-IgE antibody, the anti-MGL-IgG antibody, and As a result, the monoclonal antibody MGLab8-2 prepared the anti-MGL-IgG4 antibody were measured in patient by immunizing mice with rMGL detected the IgE antibody 35 serums (FIGS. 20 to 22). Although the concentrations of the in the serums of the atopic dermatitis patients via binding to anti-MGL-IgE and the anti-MGL-IgG (including sub-types QRX or rMGL. On the other hand, the Smith2 antibody was 1 to 4) was decreased in the course of treatment, the notable to detect the IgE antibody in the serums of the atopic anti-MGL-IgG4 was increased in the course of treatment. dermatitis patients via rMGL (FIGS. 13 to 15). This phenomenon is consistent with a general knowledge Then, experiments were conducted to study whether the 40 that IgE is slightly decreased while the concentration of IgE antibody in the serum of the atopic dermatitis patient can IgG4 against the antigen thereof at first rises in the hypos be quantified by means of the monoclonal antibodies ensitization therapy. MGLab8-2 and MGLab6-3 prepared by immunizing mice According to a clinical diagnosis by a physician, DLOI. with rMGL. As a result, the absorbance was decreased in which is an index indicative of quality of life, was improved proportion to a decrease in an IgE antibody amount due to 45 by due to the hyposensitization therapy applied to the dilution of the serum of the atopic dermatitis patient. It was patient. revealed that the both antibodies (monoclonal antibodies These results indicated that rMGL is useful as an antigen MGLab8-2 and MGLab6-3) enable quantification of the IgE for the hyposensitization therapy as is the case with QRX, antibodies in the serum of the atopic dermatitis patient using and also indicated that rMGL is useful for determination of rMGL (FIGS. 16 and 17). 50 a therapeutic effect of the hyposensitization therapy. These results indicated that a Sweat allergy in atopic dermatitis can be diagnosed by utilizing the monoclonal Example 12 antibody prepared by immunizing mice with rMGL. Study on Production Amount of MGL 1304 Example 10 55 Protein in Culturing Using Buffer Solution of M. Globosa Measurement of Anti-Sweat Antigen-Specific IgE Antibody in Patient Serum (2) M. Globosa was cultured in a 2693 mDixon medium at 32° C. for four days. The culture supernatant was removed Experiments were conducted to study whether IgE anti 60 and the fungus body was washed with a buffer solution body specifically binding to a Sweat antigen can be detected (buffer A: PBS/HEPES/glucose buffer solution) at pH 4, pH in serums of atopic dermatitis (AD), allergic rhinitis, and a 6, or pH 8. The buffer solutions were subsequently used in healthy person (Normal) by using ELISA coating rTF-MGL culturing for 2 to 60 minutes and a concentration of the prepared in Example 3. produced MGL 1304 protein was measured by ELISA. As A 96-well ELISA plate was coated with rTF or rTF-MGL 65 a result, the production of the MGL 1304 protein was at 3 ug/ml and 50 ul/well and was left overnight at 4°C. The increased when the culture was performed using the buffer plate was washed twice, blocked by 2% BSA (room tem solution at pH 8 (FIG. 23). US 9,457,071 B2 29 30

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS : 13

<210s, SEQ ID NO 1 &211s LENGTH: 183 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 1

Met Wal Ser Lieu. Asn. Ile Phe Ser Ala Ala Phe Wall Ala Ser Lieu Ala 1. 5 1O 15 Ser Ala Val Phe Ala Ala Pro Ser Ala Lieu. Glu Arg Arg Ala Ala Pro 2O 25 3O Asp Asn Thr Val Trp Val Thir Ser Val Ala Asp His Cys Lieu. Ile Lieu 35 4 O 45 Pro Arg His Llys Met Ser Val Gly Asp Ser Glu Ser Pro Gly Asn Met SO 55 6 O Arg Ser Phe Cys Thr Lys Pro Tyr Ser Ser Lys Glin Gly Glin Leu Ala 65 70 7s 8O Ser Asp Phe Trp Thir Lys Ala His Phe Llys Llys Thr Asp Llys Tyr Val 85 90 95 Glin Ile Thr Gly Cys Ile Asn Pro Asn Val Glin Ser Thr Lieu. Leu Ser 1OO 105 11 O Asn Asp Glu Gly Gly Glin Tyr Asp Ser Asn Gly Gly Glu Gly Gly Arg 115 12 O 125 Gly Asn Pro Ala Gly Ser Val Cys Lieu. Gly Tyr Ser Ser Tyr Val Glu 13 O 135 14 O Lieu Val Glu Pro Ala Gly Asn Arg Ala Cys Ile Arg Cys Cys Tyr Asp 145 150 155 160 Pro Ser Asp Cys Asp Val Ser Glin Asp Glu Ala Gly Cys Glu Thr Val 1.65 17O 17s Ile Pro Gly Lys Tyr Asp Cys 18O

<210s, SEQ ID NO 2 &211s LENGTH: 142 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 2 Pro Asp Asn Thr Val Trp Val Thr Ser Val Ala Asp His Cys Lieu. Ile 1. 5 1O 15 Lieu Pro Arg His Llys Met Ser Val Gly Asp Ser Glu Ser Pro Gly Asn 2O 25 3O Met Arg Ser Phe Cys Thr Lys Pro Tyr Ser Ser Lys Glin Gly Glin Leu 35 4 O 45 Ala Ser Asp Phe Trp Thir Lys Ala His Phe Llys Llys Thr Asp Llys Tyr SO 55 6 O Val Glin Ile Thr Gly Cys Ile Asin Pro Asn Val Glin Ser Thr Lieu. Leu 65 70 7s 8O Ser Asn Asp Glu Gly Gly Glin Tyr Asp Ser Asn Gly Gly Glu Gly Gly 85 90 95 Arg Gly Asn Pro Ala Gly Ser Val Cys Lieu. Gly Tyr Ser Ser Tyr Val 1OO 105 11 O Glu Lieu Val Glu Pro Ala Gly Asn Arg Ala Cys Ile Arg Cys Cys Tyr 115 12 O 125

Asp Pro Ser Asp Cys Asp Val Ser Glin Asp Glu Ala Gly Cys US 9,457,071 B2 31 32 - Continued

13 O 135 14 O

<210s, SEQ ID NO 3 &211s LENGTH: 152 212. TYPE : PRT &213s ORGANISM: Malassezia globosa

<4 OOs, SEQUENCE: 3

Pro Asp Asn. Thir Wall Trp Wall Thir Ser Wall Ala Asp His Luell Ile 1. 15

Lell Pro Arg His Lys Met Ser Wall Gly Asp Ser Glu Ser Pro Gly Asn 25

Met Arg Ser Phe Cys Thir Pro Ser Ser Glin Gly Glin Luell 35 4 O 45

Ala Ser Asp Phe Trp Thir Lys Ala His Phe Lys Thir Asp SO 55 6 O

Wall Glin Ile Thir Gly Cys Ile Asn Pro Asn Wall Glin Ser Thir Luell Luell 65 70 7s 8O

Ser Asn Asp Glu Gly Gly Glin Asp Ser ASn Gly Gly Glu Gly Gly 85 90 95

Arg Gly Asn Pro Ala Gly Ser Wall Cys Luell Gly Ser Ser Wall 105 11 O

Glu Luell Wall Glu Pro Ala Gly Asn Arg Ala Ile Arg 115 12 O 125

Asp Pro Ser Asp Cys Asp Wall Ser Glin Asp Glu Ala Gly Glu Thir 13 O 135 14 O

Wall Ile Pro Gly Lys Tyr Asp 145 150

<210s, SEQ ID NO 4 &211s LENGTH: 147 212. TYPE : PRT <213> ORGANISM: Malassezia globosa

<4 OOs, SEQUENCE: 4.

Glu Arg Arg Ala Ala Pro Asp Asn Thir Wall Trp Wall Thir Ser Wall Ala 1. 5 1O 15

Asp His Cys Luell Ile Lell Pro Arg His Met Ser Wall Gly Asp Ser 2O 25

Glu Ser Pro Gly Asn Met Arg Ser Phe Thir Pro Ser Ser 35 4 O 45

Glin Gly Glin Lell Ala Ser Asp Phe Trp Thir Lys Ala His Phe SO 55 6 O

Lys Thir Asp Tyr Wall Glin Ile Thir Gly Cys Ile Asn Pro Asn Wall 65 70 8O

Glin Ser Thir Luell Lell Ser Asn Asp Glu Gly Gly Glin Asp Ser Asn 85 90 95

Gly Gly Glu Gly Gly Arg Gly Asn Pro Ala Gly Ser Wall Cys Luell Gly 1OO 105 11 O

Ser Ser Wall Glu Lell Wall Glu Pro Ala Gly Asn Arg Ala 115 12 O 125

Ile Arg Tyr Asp Pro Ser Asp Asp Wall Ser Glin Asp Glu 13 O 135 14 O

Ala Gly 145 US 9,457,071 B2 33 34 - Continued

<210s, SEQ ID NO 5 &211s LENGTH: 157 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 5 Glu Arg Arg Ala Ala Pro Asp Asn Thr Val Trp Val Thir Ser Val Ala 1. 5 1O 15 Asp His Cys Lieu. Ile Lieu Pro Arg His Llys Met Ser Val Gly Asp Ser 2O 25 3O Glu Ser Pro Gly Asn Met Arg Ser Phe Cys Thr Lys Pro Tyr Ser Ser 35 4 O 45 Lys Glin Gly Glin Lieu Ala Ser Asp Phe Trp Thir Lys Ala His Phe Lys SO 55 6 O Lys Thr Asp Llys Tyr Val Glin Ile Thr Gly Cys Ile Asn Pro Asn Val 65 70 7s 8O Glin Ser Thr Lieu Lleu Ser Asn Asp Glu Gly Gly Glin Tyr Asp Ser Asn 85 90 95 Gly Gly Glu Gly Gly Arg Gly Asn Pro Ala Gly Ser Val Cys Lieu. Gly 1OO 105 11 O Tyr Ser Ser Tyr Val Glu Lieu Val Glu Pro Ala Gly Asn Arg Ala Cys 115 12 O 125 Ile Arg Cys Cys Tyr Asp Pro Ser Asp Cys Asp Val Ser Glin Asp Glu 13 O 135 14 O Ala Gly Cys Glu Thr Val Ile Pro Gly Llys Tyr Asp Cys 145 150 155

<210s, SEQ ID NO 6 &211s LENGTH: 152 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 6 Ala Pro Ser Ala Lieu. Glu Arg Arg Ala Ala Pro Asp Asn. Thr Val Trip 1. 5 1O 15 Val Thir Ser Val Ala Asp His Cys Lieu. Ile Lieu Pro Arg His Llys Met 2O 25 3O Ser Val Gly Asp Ser Glu Ser Pro Gly Asn Met Arg Ser Phe Cys Thr 35 4 O 45 Llys Pro Tyr Ser Ser Lys Glin Gly Glin Leu Ala Ser Asp Phe Trp Thr SO 55 6 O Lys Ala His Phe Llys Llys Thr Asp Llys Tyr Val Glin Ile Thr Gly Cys 65 70 7s 8O Ile Asin Pro Asn Val Glin Ser Thir Lieu. Lieu. Ser Asn Asp Glu Gly Gly 85 90 95 Glin Tyr Asp Ser Asn Gly Gly Glu Gly Gly Arg Gly Asn Pro Ala Gly 1OO 105 11 O

Ser Val Cys Lieu. Gly Tyr Ser Ser Tyr Val Glu Lieu Val Glu Pro Ala 115 12 O 125

Gly Asn Arg Ala Cys Ile Arg Cys Cys Tyr Asp Pro Ser Asp Cys Asp 13 O 135 14 O

Val Ser Glin Asp Glu Ala Gly Cys 145 150

<210s, SEQ ID NO 7 &211s LENGTH: 162 212. TYPE: PRT US 9,457,071 B2 35 36 - Continued

<213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide fragment 22-183 of MGL 1304

<4 OO > SEQUENCE: 7 Ala Pro Ser Ala Lieu. Glu Arg Arg Ala Ala Pro Asp Asn. Thr Val Trip 1. 5 1O 15 Val Thir Ser Val Ala Asp His Cys Lieu. Ile Lieu Pro Arg His Llys Met 2O 25 3O Ser Val Gly Asp Ser Glu Ser Pro Gly Asn Met Arg Ser Phe Cys Thr 35 4 O 45 Llys Pro Tyr Ser Ser Lys Glin Gly Glin Leu Ala Ser Asp Phe Trp Thr SO 55 6 O Lys Ala His Phe Llys Llys Thr Asp Llys Tyr Val Glin Ile Thr Gly Cys 65 70 7s 8O Ile Asin Pro Asn Val Glin Ser Thir Lieu. Lieu. Ser Asn Asp Glu Gly Gly 85 90 95 Glin Tyr Asp Ser Asn Gly Gly Glu Gly Gly Arg Gly Asn Pro Ala Gly 1OO 105 11 O Ser Val Cys Lieu. Gly Tyr Ser Ser Tyr Val Glu Lieu Val Glu Pro Ala 115 12 O 125 Gly Asn Arg Ala Cys Ile Arg Cys Cys Tyr Asp Pro Ser Asp Cys Asp 13 O 135 14 O Val Ser Glin Asp Glu Ala Gly Cys Glu Thr Val Ile Pro Gly Lys Tyr 145 150 155 160 Asp Cys

<210s, SEQ ID NO 8 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs, SEQUENCE: 8 gggg taccgt atc cct caac attitt ct cag ctgc

<210s, SEQ ID NO 9 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs, SEQUENCE: 9 Cccaagcttt tag cagtcgt acttgc.cggg gatg

<210s, SEQ ID NO 10 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Malassezia globosa

<4 OOs, SEQUENCE: 10

Met Wal Ser Lieu. Asn. Ile Phe Ser Ala Ala Phe Wall Ala Ser Lieu Ala 1. 5 1O 15

Ser Ala Val Phe Ala Ala Pro Ser Ala Lieu. Glu Arg Arg Ala Ala Pro 2O 25 3O Asp Asn Thr Val Trp Val Thir Ser Val Ala Asp His Cys Lieu. Ile Lieu 35 4 O 45 US 9,457,071 B2 38 - Continued

Pro Arg SO

<210s, SEQ ID NO 11 &211s LENGTH: 55 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 11 Lieu. Ile Lieu Pro Arg His Llys Met Ser Val Gly Asp Ser Glu Ser Pro 1. 5 1O 15 Gly Asn Met Arg Ser Phe Cys Thr Llys Pro Tyr Ser Ser Lys Glin Gly 2O 25 3O Glin Lieu Ala Ser Asp Phe Trp Thir Lys Ala His Phe Llys Llys Thr Asp 35 4 O 45 Lys Tyr Val Glin Ile Thr Gly SO 55

<210s, SEQ ID NO 12 &211s LENGTH: 45 212. TYPE: PRT <213> ORGANISM: Malassezia globosa <4 OOs, SEQUENCE: 12 Val Glin Ile Thr Gly Cys Ile Asin Pro Asn Val Glin Ser Thir Lieu Lleu. 1. 5 1O 15 Ser Asn Asp Glu Gly Gly Glin Tyr Asp Ser Asn Gly Gly Glu Gly Gly 2O 25 3O

Arg Gly Asn. Pro Ala Gly Ser Val Cys Lieu. Gly Tyr Ser 35 4 O 45

SEQ ID NO 13 LENGTH: 43 TYPE PRT ORGANISM: Malassezia globosa

< 4 OOs SEQUENCE: 13 Ser Tyr Val Glu Lieu Val Glu Pro Ala Gly Asn Arg Ala Cys Ile Arg 1. 5 1O 15 Cys Cys Tyr Asp Pro Ser Asp Cys Asp Wal Ser Glin Asp Glu Ala Gly 2O 25 3O Cys Glu Thr Val Ile Pro Gly Lys Tyr Asp Cys 35 4 O

The invention claimed is: 50 (iii) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid 1. A method of measuring an amount of a Sweat allergy sequence represented by SEQ ID NO: 1; and antigen protein in a sample from a human, comprising (iv) a peptide comprising an amino acid sequence contacting the sample with an antibody that specifically having 90% or more identity to the amino acid binds to a Sweat allergy antigen protein selected from 55 sequence represented by any of SEQID NO: 2 to 7: the group consisting of: detecting the standard antibody that binds to the standard (i) a protein consisting of an amino acid sequence Sweat allergy antigen protein; and having 90% or more identity to the amino acid determining the amount of the Sweat allergy antigen sequence represented by SEQ ID NO: 1; and protein in the sample based on results from detecting 60 the standard antibody that binds to the standard sweat (ii) a peptide comprising an amino acid sequence allergy antigen protein. having 90% or more identity to the amino acid 2. The method of claim 1, further comprising preparing sequence represented by any of SEQ ID NO: 2 to 7: the standard Sweat allergy antigen protein, wherein detecting the antibody that binds to the sample: the standard Sweat allergy antigen protein is a protein contacting a standard antibody that has the same sequence 65 present in a fungus body of Malassezia globosa, as said antibody with a standard Sweat allergy antigen the standard Sweat allergy antigen protein has a molecular protein selected from the group consisting of: weight of about 30 kDa measured by SDS-PAGE, and US 9,457,071 B2 39 40 the standard Sweat allergy antigen protein binds to (i) 7. The method of claim 1, wherein the sample from a serum derived from a Sweat allergy patient and/or (ii) human is a human Sweat, human skin washings, a solution Smith2 antibody. of an extract of human skin, a human serum, or a human 3. The method of claim 1, further comprising manufac plasma. turing the standard Sweat allergy antigen protein comprising: culturing Malassezia globosa at pH 7 to 9; and 8. The method of claim 1, wherein the standard Sweat isolating the Sweat allergy antigen protein. allergy antigen protein is (iii) the protein consisting of an 4. The method of claim 1, further comprising preparing amino acid sequence having 90% or more identity to the the standard Sweat allergy antigen protein, wherein amino acid sequence represented by SEQ ID NO: 1. the standard Sweat allergy antigen protein is a protein 9. The method of claim 1, wherein standard sweat allergy Secreted from a fungus body of Malassezia globosa, 10 antigen protein is a protein consisting of the amino acid the standard Sweat allergy antigen protein has a molecular sequence represented by SEQ ID NO: 1. weight of about 17 kDa measured by SDS-PAGE, and the standard Sweat allergy antigen protein binds to (i) 10. The method of claim 1, wherein the standard Sweat serum derived from a Sweat allergy patient and/or (ii) allergy antigen protein is peptide comprising the amino acid Smith2 antibody. 15 sequence represented by any of SEQ ID NO: 2 to 7. 5. A method of diagnosing a Sweat allergy or a disease 11. The method of claim 1, wherein the standard Sweat relating to a Sweat allergy antigen protein, comprising allergy antigen protein is peptide consisting of the amino measuring an amount of a Sweat allergy antigen protein in acid sequence represented by any of SEQ ID NO: 2 to 7. a sample from a human according to claim 1. 6. The method of claim 1, wherein the sweat allergy 12. The method of claim 1, wherein the detecting the antigen protein to be measured consists of the amino acid antibody is performed using Western blotting or ELISA. sequence represented by SEQ ID NO: 1. k k k k k