US20090104208A1 - House Dust Mite Allergen - Google Patents

House Dust Mite Allergen Download PDF

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US20090104208A1
US20090104208A1 US12/298,901 US29890107A US2009104208A1 US 20090104208 A1 US20090104208 A1 US 20090104208A1 US 29890107 A US29890107 A US 29890107A US 2009104208 A1 US2009104208 A1 US 2009104208A1
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amino acid
acid sequence
sequence seq
polypeptide
allergen
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Rudolf Valenta
Margit Weghofer
Susanne Vrtala
Friedrich Horak
Peter Valent
Stefan Florian
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Biomay AG
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Biomay AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43582—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to a polypeptide having allergenic properties.
  • IgE-mediated allergies More than 25% of the population in industrialised countries suffer from IgE-mediated allergies. Allergic patients are characterized by the increased production of IgE antibodies against per se harmless antigens (i.e., allergens). The immediate symptoms of Type I allergy (allergic rhinoconjunctivitis, asthma, dermatitis, anaphylactic shock) are caused by allergen-induced cross-linking of mast cell-bound IgE antibodies and the release of biologically active mediators (e.g., histamine, leukotriens).
  • biologically active mediators e.g., histamine, leukotriens
  • House-dust mites represent one of the most important allergen sources worldwide. Almost 10% of the population and more than 50% of allergic patients are sensitized to mite allergens.
  • the HDM Dermatophagoides pteronyssinus (Der p) is prevalent in Central Europe.
  • the allergens of Der p comprise more than 30 proteins or glycoproteins of which twenty-one allergens have been characterized so far.
  • Group 1 and 2 allergens represent the most important allergens from HDM, which are recognized by more than 80% of Der p allergic patients, but also other HDM allergens (e.g., Der p 5 and Der p 7) were shown to represent important Der p allergens despite a considerably lower IgE-binding frequency.
  • the EP 1 612 219 A1 deals with allergens derived from housedust mites (Der p).
  • the present invention relates to a polypeptide comprising an amino acid sequence having at least 60% identity to the amino acid sequence SEQ ID No. 1 or comprising at least one amino acid fragment of at least 6 consecutive amino acid residues of the amino acid sequence SEQ ID No. 1 or having immunological cross-reactivity to the amino acid sequence SEQ ID No. 1 or fragments thereof, wherein the amino acid sequence SEQ ID No. 1 codes for an allergen and the polypeptide comprises at least one T cell epitope recognized by a T cell receptor specific for a molecule having the amino acid sequence SEQ ID No. 1.
  • the polypeptide with the amino acid sequence SEQ ID No. 1 is a new major Der p allergen which may be useful, e.g., for diagnosis and therapy of Der p allergic patients.
  • This new Der p allergen has a molecular weight of approximately 8 kDa and binds IgE from more than 50% of mite allergic patients.
  • this new mite allergen is also useful for therapy or prevention of house dust mite allergy.
  • the polypeptide according to the present invention induces high titers of specific IgG antibodies in mammals. If these specific IgG antibodies are administered to an individual, the exhibiting house dust mite allergen added to a sample, preferably serum, obtained from a house dust mite allergic individual, comprising IgE molecules of said individual IgE-binding to this new allergen is inhibited. This shows that allergen specific immunotherapy with this new mite allergen will induce blocking IgG antibodies in humans. The importance of the induction of blocking IgG antibodies for a successful immunotherapy has been shown recently in immunotherapy trials with defined allergens and allergen derivatives (Gafvelin, G., et al.
  • Hypoallergenic derivatives may be produced by molecular biological techniques or by the synthesis of peptides derived from the T-cell or B-cell epitopes of the allergen (Kyte, J., and R. F. Doolittle. (1982) J Mol Biol 157:105).
  • polypeptide refers to a molecule comprising at least 6 amino acid residues, preferably at least 8 amino acid residues.
  • identity indicates whether any two (or more) peptide, polypeptide or protein sequences have amino acid sequences that are “identical” to a certain degree (“% identity”) to each other. This degree can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson et al. (1988) PNAS USA 85: 2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) Nucleic Acids Res., 12, 387-395), BLASTP, BLASTN, FASTA (Atschul, S.
  • BLAST tool of the NCBI database can be used to determine identity.
  • Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison, Wis.)). Percent identity of protein molecules can further be determined, for example, by comparing sequence information using a GAP computer program (e.g.
  • the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter one of the two sequences.
  • Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and for non-identities) and the weighted comparison matrix of Gribskov et al.
  • the amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1.
  • cross-reactivity relates to the ability of an antibody to bind next to the antigen (e.g. peptide, protein, polypeptide), that did stimulate its production in an in vivo system, other antigens.
  • an antibody produced to bind specifically a polypeptide with the amino acid sequence SEQ ID No. 1 or fragments thereof may also show a binding affinity for a polypeptide which is not homologous to SEQ ID No. 1.
  • the binding specificity of an antibody to a polypeptide can be determined by methods known in the art, e.g., ELISA, RIA, immunoblot etc. as described by Valenta et al. J Exp Med. (1992) 175: 377-385.
  • the polypeptide according to the present invention is preferably recombinantly produced by any method known in the art.
  • the host in which said polypeptide may be produced can be of any kind by using corresponding vectors and plasmids (e.g. eukaryotic cells, preferably yeasts, mammalian cells, plant cells and insect cells, and prokaryotic cells, preferably Escherichia coli and Bacillus subtilis ).
  • plasmids e.g. eukaryotic cells, preferably yeasts, mammalian cells, plant cells and insect cells, and prokaryotic cells, preferably Escherichia coli and Bacillus subtilis.
  • polypeptide is hypoallergenic.
  • hypoallergenic refers to the ability of a peptide, polypeptide or protein derived from an allergen with allergenic properties to induce the production of antibodies specifically binding to said allergen and exhibiting reduced or no allergic reactions when administered to an individual.
  • the reduced or missing ability of “hypoallergenic” derivatives of an allergen like SEQ ID No. 1 to induce an allergic reaction in an individual is obtained by removing or destroying the IgE binding epitopes from said allergens, however, by conserving the T cell epitopes present on said allergens.
  • Another method for producing “hypoallergenic” molecules from allergens involves C- and/or N-terminal deletions of the wild type allergen (see e.g. EP 1 224 215).
  • amino acid fragments are fused together in an order differing from the order of said fragments in SEQ ID No. 1.
  • the polypeptide according to the present invention may comprise amino acid fragments derived from SEQ ID No. 1 which may be preferably fused together in an order differing from the order in SEQ ID No. 1.
  • This “shuffling” results in a polypeptide having altered features in respect to the wild type allergen having the amino acid sequence SEQ ID No. 1. For instance, this shuffling will result in an polypeptide comprising intact T cell epitopes and destroyed B cell epitopes. Such a molecule may have hypoallergenic properties.
  • Said at least one amino acid fragment which substantially consists of a T cell epitope, is preferably selected from the group consisting of amino acid molecules comprising amino acids 5 to 13, 9 to 17, 10 to 18, 11 to 19, 12 to 20, 16 to 24, 17 to 25, 43 to 51, 44 to 52, 45 to 53, 47 to 55, 51 to 59 and 60 to 68 of SEQ ID No. 1.
  • T cell epitopes comprise at least 6, preferably at least 7, more preferably at least 8, consecutive amino acid residues of SEQ ID No. 1.
  • Said T cell epitopes may also be bound to a carrier via a or without a linker.
  • Said carrier may be a solid support as used in microarray technology.
  • the presence of T cell epitopes in a polypeptide can be determined by methods known in the art (see e.g. “Epitope Mapping: A practical approach” Ed. O. Westwood and F. Hay, 2001, Oxford University Press). A particularly preferred method is ELISpot (Tobey TW and Caulfield MJ (2004), Methods Mol. Med. 94:121-132).
  • the identified T cell epitopes may be fused N- or C-terminally to other molecules like proteins or be part of a larger fragment obtained from SEQ ID No. 1 by fragmentation.
  • Another aspect of the present invention relates to a DNA molecule encoding a polypeptide according to the present invention.
  • Yet another aspect of the present invention relates to a vector comprising a DNA molecule according to the present invention.
  • Preferred vectors e.g. plasmids to be used according to the present invention are cloning as well as expression vectors comprising promoters, an origin of replication, regulatory elements, selection markers and/or other vector elements. If the vector is an integration vector able to be integrated into the genome of the host cell corresponding means may be provided on said vector (e.g. insertion sequence elements). The type of vector used and the regulatory elements present on said vector depend also into which host cell said vector will be integrated. If expression vectors are used the DNA molecule according to the present invention and encoding a polypeptide as described above, is operably linked to a promoter region.
  • Another aspect of the present invention relates to a cell transformed with a vector according to the present invention.
  • the cell to be used herein may be a eukaryotic as well as a prokaryotic cell.
  • Preferred eukaryotic cells are yeast cells, in particular Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha , plant cells, in particular tobacco plant cells, insect cells and mammalian cells, like human and animal cells, in particular Chinese hamster ovary cells.
  • Preferred prokaryotic cells are, e.g., Bacillus subtilis and Escherichia coli .
  • a cell comprising a vector or a DNA molecule of the present invention may be used for producing a polypeptide according to the present invention.
  • Another aspect of the present invention relates to an antibody binding to a polypeptide according to the present invention.
  • Antibodies according to the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments and epitope-binding fragments of any of the above. Furthermore, antibodies are considered as being immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention are preferably of the types IgG, IgM, IgD, IgA and IgY, class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • Polyclonal antibodies can be prepared by administering a polypeptide of the invention, preferably using an adjuvant, to a non-human mammal and collecting the resultant antiserum. Improved titres can be obtained by repeated injections over a period of time.
  • a definite amount of immunogen of the invention is e.g. diluted with physiological saline solution to a suitable concentration and the resulting diluted solution is mixed with, e.g.
  • complete Freund's adjuvant to prepare a suspension or with mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • the suspensions and mixtures are administered to mammals, e.g. intraperitoneally, e.g. to a rabbit, using from about 50 ⁇ g to about 2500 ⁇ g polypeptide of the invention per administration.
  • the suspension is preferably administered about every two weeks over a period of up to about 2-3 months, preferably about 1 month, to effect immunization.
  • Antibody is recovered by collecting blood from the immunized animal after the passage of 1 to 2 weeks subsequently to the last administration, centrifuging the blood and isolating serum from the blood.
  • Monoclonal antibodies may e.g. be of human or murine origin.
  • Murine monoclonal antibodies may be prepared by the method of Köhler and Milstein (Köhler, G. and Milstein, C., Nature 256 (1975) 495), e.g. by fusion of spleen cells of hyperimmunized mice with an appropriate mouse myeloma cell line.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. No. 5,807,715; U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modelling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; WO 91/09967; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; and U.S. Pat. No. 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-913 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
  • the antibodies according to the present invention may advantageously be used for passive immunisation of an individual suffering from an allergy, in particular from house dust mite allergy.
  • the antibody is preferably an IgG or a derivative thereof (e.g. chimeric or humanized antibody). Furthermore this antibody may also be used for desensibilisation of an individual.
  • Another aspect of the present invention relates to a vaccine formulation comprising a polypeptide or an antibody according to the present invention.
  • the formulation according to the present invention may also comprise other sub-stances like stabilisers, adjuvants, pharmaceutically acceptable carriers etc.
  • Suitable protocols for the production of vaccine formulations are known to the person skilled in the art and can be found e.g. in “Vaccine Protocols” (A. Robinson, M. P. Cranage, M. Hudson; Humana Press Inc.,U.S.; 2 nd edition 2003).
  • Yet another aspect of the present invention relates to the use of a polypeptide according to the present invention for the diagnosis of an allergy, in particular of house dust mite allergy, in an individual.
  • Said polypeptide may be used for diagnosis of allergy, in particular house dust mite allergy by exposing, e.g., a sample of an individual comprising histamine releasing cells to said polypeptide (see e.g. Purohit et al., Clin. Exp. Allergy 35 (2005): 186-192).
  • the polypeptide(s) according to the present invention may be immobilised on a surface in order to form a polypeptide array/chip. Such arrays may be used, e.g., in high throughput screening in order to diagnose an allergy in a number of samples taken from a number of individuals.
  • Another aspect of the present invention relates to the use of a polypeptide or antibody according to the present invention for the preparation of a medicament for the immunotherapy of an allergy, in particular of house dust mite allergy.
  • polypeptides and antibodies of the present invention can be used for active vaccination and passive vaccination respectively. Both can be used because the formation of protective IgGs is induced when a polypeptide of the present invention is administered to an individual and the administration of immunoglobulins directed to said polypeptide will lead to competition between IgE and administered protective antibodies which in turn alleviate the symptoms of the allergy.
  • Another aspect of the present invention relates to the use of a polypeptide according to any one of claims 1 to 4 or an antibody according to claim 7 for the preparation of a medicament for the prevention of an allergen sensitisation, in particular of house dust mite allergen sensitisation.
  • the polypeptide used for vaccination of an individual is preferably hypoallergenic.
  • the use of such a polypeptide prevents the binding of IgE to said polypeptide and thus prevents an allergic reaction.
  • said medicament further contains adjuvants, carrier, diluents, preservatives or mixtures thereof.
  • the medicament comprises preferably 10 ng to 1 g, more preferably 100 ng to 10 mg, especially 0.5 ⁇ g to 200 ⁇ g of said polypeptide.
  • FIG. 1 shows the cDNA and amino acid sequence of the clone 30-derived allergen.
  • the start codon and the stop codon are underlined.
  • the signal sequence comprises amino acid residues 1 to 21 with the predicted cleavage site between amino acid residues 21 (A) and 22 (A) in bold.
  • the numbers on the left side of the sequence indicate the nucleotide positions and the numbers on the right side of the sequence the amino acid positions.
  • FIG. 2 shows a Coomassie Blue staining and mass spectroscopy (MS) of the clone 30-derived allergen.
  • A The Coomassie Blue stained SDS-PAGE gel displays a molecular weight marker (M) and 3 ⁇ g of purified clone 30-derived allergen (30).
  • B MS analysis of the purified clone 30-derived allergen shows the mass/charge ratio on the x-axis and the signal intensity on the y-axis as the percentage of the most intense signal obtained in the investigated mass range.
  • the peak at 7979.20 corresponds to the calculated mass of the deduced amino acid sequence of the clone 30-derived allergen.
  • FIG. 3 shows an immunoblot of the clone 30-derived allergen.
  • Samples of the purified clone 30-derived allergen were separated by SDS-PAGE, blotted onto nitrocellulose and incubated with sera from two mite allergic patients (1, 2) and one serum from a non-allergic individual (3).
  • Bound IgE antibodies specific for the clone 30-derived allergen were detected with 125 I -labeled anti-human IgE antibodies.
  • FIG. 4 shows the IgG reactivity of a rabbit anti-clone 30-derived allergen antiserum.
  • the clone 30-derived allergen and the major mite allergen, Der p 2 were dotted onto nitrocellulose strips and incubated with 1:1000-1:1,000,000 diluted rabbit preimmune serum (A) or rabbit anti-clone 30-derived allergen antiserum (B). Bound IgG antibodies were detected with 125 I-labelled anti-rabbit whole antibodies from donkey.
  • FIG. 5 shows the biological activity of the clone 30-derived allergen.
  • Blood samples from a mite allergic patient were exposed to 10 ⁇ g/ml, 1 ⁇ g/ml and 0.1 ⁇ g/ml of clone 30-derived allergen, to 1 ⁇ g/ml anti-IgE antibodies or to PBS as buffer control (Co) (x-axis).
  • CD203c expression was determined by FACS analysis and is displayed as mean fluorescence index (MFI) (y-axis).
  • FIG. 6 shows an analysis of the hydrophobicity (Kyte & Doolittle) of the mature clone 30-derived allergen using ProtScale.
  • the amino acid positions of the mature protein including an N-terminal methionin are displayed on the x-axis.
  • the present examples describe the identification of a new major Der p allergen which may be useful, e.g., for diagnosis and therapy of Der p allergic patients.
  • the cDNA coding for this new mite allergen was isolated from a Der p expression cDNA library and expressed in Escherichia coli ( E. coli ) as recombinant allergen.
  • the new allergen has a molecular weight of approximately 8 kDa and binds IgE from more than 50% of mite allergic patients, thus representing a major allergen.
  • the cDNA sequence of clone 30 ( FIG. 1 ) coding for the pre-dicted mature clone 30-derived allergen (nucleotides 89-295 with an additional ATG at the N-terminus) was subcloned into the expression vector pET-17b (Novagene, Wis.) and expressed in Escherichia coli BL21 (DE3) cells (Stratagene, Calif.). The bacterial cells were grown overnight in LB-medium containing 100 mg/L ampicillin at 27° C. and expression of the recombinant protein was induced by adding isopropyl- ⁇ -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the clone 30-derived allergen was eluted by a 500-0 mM ammonium sulphate gradient and fractions containing the clone 30-derived allergen were pooled. After dialysis against 20 mM Tris-Cl pH 8.0/10 mg/L PMSF, the sample was applied to a HiTrap DEAE Sepharose FF column (Amersham Biosciences). The clone 30-derived allergen was eluted by a 0-500 mM NaCl gradient and fractions containing more than 90% pure clone 30-derived allergen were pooled. The clone 30-derived allergen was dialysed against 20 mM Tris-Cl pH 8.0 and stored at ⁇ 20° C.
  • FIG. 2 A A protein sample was analyzed for purity by 14% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue protein staining ( FIG. 2 A).
  • SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis
  • FIG. 2 B Molecular mass analysis of the mature protein depicted a mass of 7.98 kDa ( FIG. 2 B) which corresponds to the calculated mass of the deduced amino acid sequence of the clone 30-derived allergen although in SDS-PAGE the protein runs at approximately 14 kDa ( FIG. 2A ).
  • the IgE binding capacity of the clone 30-derived allergen was demonstrated by immunoblot analysis using two sera of Demmatophagoides pteronyssinus sensitized individuals ( FIG. 3 ).
  • Samples of the clone 30-derived allergen were separated by SDS-PAGE and blotted onto nitrocellulose. Nitrocellulose strips were incubated with 1:10 diluted human sera (1-3) and bound IgE antibodies were detected with 1:10 diluted 125 I-labelled anti-human IgE antibodies.
  • the sera of the mite allergic patients (1, 2) reacted specifically with the clone 30-derived allergen.
  • the control serum from a non-allergic individual (3) did not react with the clone 30-derived allergen.
  • the frequency of IgE binding was determined in an ELISA assay with sera from 53 mite allergic individuals with perennial symptoms indicative for mite allergy, positive SPT and D. pteronyssinus specific IgE-RAST.
  • An ELISA plate (Nunc, Denmark) was coated with 5 ⁇ g/ml clone 30-derived allergen and incubated with 1:10 diluted sera from mite allergic patients.
  • Human IgE binding was detected with 1:1000 diluted AKP-conjugated anti-human IgE antibodies (BD Biosciences-Pharmingen, N.J.).
  • a rabbit was immunized with the new allergen using Freund's adjuvant.
  • the rabbit was immunized 3 times with 200 ⁇ g protein/injection using once Freund's complete and twice incomplete adjuvants (Charles River, Germany).
  • IgG antibodies were studied by dot blot experiments. Recombinant Der p 2 and the clone 30-derived allergen were dotted onto nitrocellulose strips (0.5 ⁇ g/dot) and the strips were incubated with 1:1000, 1:10,000, 1:100,000 and 1:1,000,000 diluted rabbit preimmune serum and anti-clone 30-derived allergen antiserum. Bound IgG antibodies were detected with 125 I-labelled anti-rabbit whole antibodies from donkey (Amersham).
  • ELISA-inhibition assays The ability of rabbit antibodies specific for the clone 30-derived allergen to block the binding of patients' IgE to the allergen was examined by ELISA-inhibition assays.
  • ELISA plate-bound clone 30-derived allergen (5 ⁇ g/ml) was preincubated with 1:100 in PBST/0.5% (w/v) BSA diluted rabbit anti-clone 30-derived allergen antibodies or rabbit preimmune serum and incubated at 4° C. overnight. Subsequently, the plate was exposed to 1:5 in PBST/0.5% (w/v) BSA diluted sera from 14 mite allergic patients overnight at 4° C.
  • the Clone 30-Derived Allergen is Biologically Active
  • the upregulation of CD203c on basophils can be used as marker for induced activation and subsequent degranulation of basophils and therefore for the determination of the allergenic activity of an allergen.
  • Heparinized blood samples 100 ⁇ l
  • a monoclonal anti-IgE antibody Immunotech, France
  • PBS PBS for 15 minutes at 37° C.
  • CD203c expression was determined by two-color flow cytometry on a FACScan (Becton Dickinson, Calif.).
  • the clone 30-derived allergen induced upregulation of CD203c expression on basophils of a mite allergic patient at a concentration of 10 ⁇ g/ml ( FIG. 5 ).
  • the hydrophilic regions of a protein are likely to be exposed on the surface of the molecule and may potentially be antigenic. Therefore, the hydrophilic regions on the surface of the clone 30-derived allergen may represent potential B-cell epitopes.
  • ProtScale http://www.expasy.org/tools/protscale.html
  • the ProtScale output of the mature clone 30-derived allergen shows a protein with lots of negative peaks representing hydrophilic segments ( FIG. 6 A).
  • B-cell epitopes of the clone 30-derived allergen are located between amino acids 3-12, 15-28, 34-43 and 49-68 of the mature protein.
  • T-cells of the human immune system recognize allergens as short peptide fragments (T-cell epitopes) derived from the degradation of the allergens.
  • MULTIPRED http://antigen.i2r.astar.edu.sg/multipred/
  • HLAs human leukocyte antigen
  • FIG. 6 B The predicted results for individual 9 mer peptides with a ‘Sum’ (the sum of the individual binding scores of the peptide to the MHC molecules) over 40 are shown in FIG. 6 B.
  • T-cell epitopes are located near the N- and the C-terminus of the mature clone 30-derived allergen.

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WO2013007706A1 (fr) 2011-07-08 2013-01-17 Biomay Ag Polysaccharide lyases
EP3269730A1 (fr) 2016-07-12 2018-01-17 Universität Salzburg Variante d'allergène
US11312966B2 (en) 2018-05-11 2022-04-26 Astellas Pharma Inc. Nucleic acid for treating mite allergy

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CO6260019A1 (es) * 2009-01-30 2011-03-22 Fundacion Universidad Del Norte Metodo y composicion inmunoquimica para la deteccion de alergenos de acaros usando anticuerpos policlonales igy
CN103687617B (zh) 2011-06-09 2017-09-15 碧欧美公司 作为过敏症疫苗的肽载体融合蛋白
CN108892715B (zh) * 2018-06-12 2021-11-12 刘志刚 屋尘螨变应原Der p 33及其制备方法和应用
CN108840917B (zh) * 2018-06-12 2021-11-12 刘志刚 屋尘螨变应原Der p 30及其制备方法和应用
CN108892716B (zh) * 2018-06-12 2021-11-12 刘志刚 屋尘螨变应原Der p 29及其制备方法和应用

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WO2013007706A1 (fr) 2011-07-08 2013-01-17 Biomay Ag Polysaccharide lyases
EP3269730A1 (fr) 2016-07-12 2018-01-17 Universität Salzburg Variante d'allergène
US11312966B2 (en) 2018-05-11 2022-04-26 Astellas Pharma Inc. Nucleic acid for treating mite allergy

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