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WO1993001213A1 - Proteines peptides allergeniques provenant du pollen du cedre japonais - Google Patents

Proteines peptides allergeniques provenant du pollen du cedre japonais Download PDF

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Publication number
WO1993001213A1
WO1993001213A1 PCT/US1992/005661 US9205661W WO9301213A1 WO 1993001213 A1 WO1993001213 A1 WO 1993001213A1 US 9205661 W US9205661 W US 9205661W WO 9301213 A1 WO9301213 A1 WO 9301213A1
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WO
WIPO (PCT)
Prior art keywords
cry
cedar pollen
japanese cedar
allergen
fragment
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PCT/US1992/005661
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English (en)
Inventor
Irwin J. Griffith
Joanne Pollock
Julian F. Bond
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immulogic Pharmaceutical Corp
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Immulogic Pharmaceutical Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immulogic Pharmaceutical Corp filed Critical Immulogic Pharmaceutical Corp
Priority to EP92914823A priority Critical patent/EP0595855A1/fr
Priority to JP50237093A priority patent/JP3575802B2/ja
Priority to AU34384/93A priority patent/AU3438493A/en
Priority to PCT/US1993/000139 priority patent/WO1994001560A1/fr
Publication of WO1993001213A1 publication Critical patent/WO1993001213A1/fr
Anticipated expiration legal-status Critical
Priority to AU70569/96A priority patent/AU7056996A/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • allergens are known as allergens.
  • Anaphylaxis or atopy which includes the symptoms of hay fever, asthma, and hives, is one form of immediate allergy. It can be caused by a variety of atopic allergens, such as products of grasses, trees, weeds, animal dander, insects, food, drugs, and chemicals.
  • the antibodies involved in atopic allergy belong primarily to the IgE class of immunoglobulins.
  • IgE binds to mast cells and basophils.
  • the IgE may be cross- linked on the cell surface, resulting in the physiological effects of IgE-antigen interaction.
  • physiological effects include the release of, among other substances, histamine, serotonin, heparin, a chemotactic factor for eosinophilic leukocytes and/or the leukotrienes, C4, D4, and E4, which cause prolonged constriction of bronchial smooth muscle cells (Hood, L.E. et al.
  • Japanese cedar (Sugi; Cryptomeria japonica) pollinosis is one of the most important allergic diseases in Japan. The number of patients suffering from this disease is on the increase and in some areas, more than 10% of the population are affected. Treatment of Japanese cedar pollinosis by administration of Japanese cedar pollen extract to effect hyposensitization to the allergen has been attempted. Hyposensitization using Japanese cedar pollen extract, however, has drawbacks in that it can elicit anaphylaxis if high doses are used, whereas when low doses are used to avoid anaphylaxis, treatment must be continued for several years to build up a tolerance for the extract.
  • SBP Sugi basic protein
  • Cry j I The major allergen from Japanese cedar pollen has been purified and designated as Sugi basic protein (SBP) or Cry j I.
  • SBP Sugi basic protein
  • Cry j I This protein is reported to be a basic protein with a molecular weight of 41-50 kDa and a pi of 8.8.
  • the sequence of the first twenty amino acids at the N-terminal end of Cry j I and a sixteen amino acid internal sequence have been determined (Taniai supra).
  • Cryj II A second allergen from Japanese cedar pollen having a molecular weight of about 37 kDa known as Cryj II has also been reported (Sakaguchi et al. (1990) Allergy 45: 309-312). This allergen was found to have no immunological cross- reactivity with Cry j I. Most patients with Japanese cedar pollinosis were found to have IgE antibodies to both Cry j I and Cry j II, however, sera from some patients reacted with only Cry j I or Cry j II.
  • U.S. patent 4,939,239 issued July 3, 1990 to Matsuhashi et al. discloses a hyposensitization agent comprising a saccharide covalently linked to a Japanese cedar pollen allergen for hyposensitization of persons sensitive to Japanese cedar pollen.
  • This hyposensitization agent is reported to enhance the production of IgG and IgM antibodies, but reduce production of IgE antibodies which are specific to the allergen and responsible for anaphylaxis and allergy.
  • the allergens used in the hyposensitization agent preferably have an NH2-terminal amino acid sequence of Asp-Asn-Pro-Ile-Asp-Ser-X-Trp-Arg-Gly-Asp-Ser-Asn-Trp-Ala-Gln-Asn-Arg-Met- Lys-, wherein X is Ser, Cys, Thr, or His (SEQ ID NO: 18).
  • the Cry j I allergen found in Cryptomeria japonica has also been found to be cross-reactive with allergens in the pollen from other species of trees, including Cupressus sempervirens. Panzani et al. (Annals of Allergy 57: 26-30
  • a 50 kDa allergen isolated from Mountain Cedar has the NH 2 -terminal sequence AspAsnProIleAsp (SEQ ID NO: 25) (Gross et al, (1978) Scand. J. Immunol. & 437-441) which is the same sequence as the first five amino acids of the NH- 2 terminal end of the Cry j I allergen.
  • I allergen has also been found to be allergenically cross-reactive with the following species of trees: Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana, Juniperus communis, Thuya orientalis , and Chamaecyparis obtusa.
  • the present invention provides nucleic acid sequences coding for the Cryptomeria japonica major pollen allergen Cry j I and fragments thereof.
  • the present invention also provides purified Cry j I and at least one fragment thereof produced in a host cell transformed with a nucleic acid sequence coding for Cry j I or at least one fragment thereof and fragments of Cryj I prepared synthetically.
  • a fragment of the nucleic acid sequence coding for the entire amino acid sequence of Cry j I refers to a nucleotide sequence having fewer bases than the nucleotide sequence coding for the entire amino acid sequence of Cry j I and/or mature Cry j I.
  • Cry j I and fragments thereof are useful for diagnosing, treating, and preventing Japanese cedar pollinosis. This invention is more particularly described in the appended claims and is described in its preferred embodiments in the following description. Brief Description of the Drawings
  • Fig. 1a is a graphic representation of affinity purified Cry j I on Superdex 75 (2.6 by 60 cm) equilibrated with 10 mM sodium acetate (pH 5.0) and
  • Fig. 1b shows an SDS-PAGE (12.5%) analysis of the fractions from the major peak shown in Fig la;
  • Fig. 2 shows a Western blot of isoforms of purified native Cry j I proteins separated by SDS-PAGE and probed with mAB CBF2;
  • Fig. 3 is a graphic representation of allergic sera titration of different purified fractions of purified native Cry j I using plasma from a pool of fifteen allergic patients;
  • Figs. 4a-b show the composite nucleic acid sequence from the two overlapping clones JC 71.6 and pUC19JC91A coding for Cry j I.
  • the complete cDNA sequence for Cry j I is composed of 1312 nucleotides, including 66 nucleotides of 5' untranslated sequence, an open reading frame starting with the codon for an initiating methionine, of 1122 nucleotides, and a 3' untranslated region.
  • Figs. 4a-b also show the deduced amino acid sequence of Cry j I;
  • Fig. 5a is a graphic representation of the results of IgE binding reactivity wherein the coating antigen is soluble pollen extract (SPE) from Japanese cedar pollen;
  • SPE soluble pollen extract
  • Fig. 5b is a graphic representation of the results of IgE binding reactivity wherein the coating antigen is purified native Cry j I;
  • Fig. 6 is a graphic representation of the results of a competition
  • SPE soluble pollen extract
  • Fig. 7 is a graphic representation of the results of a competition ELISA using plasma from individual patients (indicated by patient numbers) wherein the coating antigen is soluble pollen extract (SPE) from Japanese cedar pollen and the competing antigen is purified native Cry j I;
  • SPE soluble pollen extract
  • Fig. 8a is a graphic representation of the results from a direct binding ELISA using plasma from seven individual patients (indicated by patient numbers) wherein the coating antigen is soluble pollen extract (SPE) from Japanese cedar pollen:
  • SPE soluble pollen extract
  • Fig. 8b is a graphic representation of the results from a direct binding ELISA using plasma from seven individual patients (indicated by patient numbers) wherein the coating antigen is denatured soluble pollen extract which has been denatured by boiling in the presence of a reducing agent, DTT;
  • Fig. 9 is a graphic representaion of a direct ELISA where the wells were coated with recombinant Cry j I (rCry j I) and IgE binding was assayed on individual patients;
  • Fig. 10a is a graphic representation of the results of a capture ELISA using pooled human plasma from fifteen patients wherein the wells were coated with
  • CBF2 (IgG) mAb PBS was used as a negative antigen control, and the antigen was purified recombinant Cry j I;
  • Fig. 10b is a graphic representation of the results of a capture ELISA using rabbit anti-Amb ⁇ l and II, wherein the wells were coated with 20 ⁇ g/ml CBF2 (IgG), PBS was used as a negative antigen control and the antigen was purified recombinant Cry j I;
  • Fig. 11 is a graphic representation of a histamine release assay performed on one Japanese cedar pollen allergic patient using SPE from Japanese cedar pollen, purified native Cry j I and recombinant Cry j I as the added antigens;
  • Fig. 12 is a graphic representation of the results of a T cell proliferation assay using blood from patient #999 wherein the antigen is recombinant Cry j I protein, purified native Cry j I protein, or recombinant Amb ⁇ 1.1.
  • the antigen is recombinant Cry j I protein, purified native Cry j I protein, or recombinant Amb ⁇ 1.1.
  • the present invention provides nucleic acid sequences coding for Cry j I, the major allergen found in Japanese cedar pollen.
  • the nucleic acid sequence coding for Cry j I preferably has the sequence shown in Figs. 4a and 4b (SEQ ID NO: 1).
  • the nucleic acid sequence coding for Cry j I shown in Figs. 4a and 4b (SEQ ID NO: 1) contains a 21 amino acid leader sequence from base 66 through base 128. This leader sequence is cleaved from the mature protein which is encoded by bases 129 through 1187.
  • the deduced amino acid sequence of Cry j I is also shown in Figs. 4a and 4b (SEQ ID NO: 2).
  • the nucleic acid sequence of the invention codes for a protein having a predicted molecular weight of 38.5 kDa. with a pI of 7.8. and five potential N-linked glycosylation sites. Utilization of these glycosylation sites will increase the molecular weight and affect the pi of the mature protein.
  • the deduced amino acid sequence for the mature protein encoded by the nucleic acid sequence of the invention is identical with the known NH2-terminal and internal amino acid sequences reported by Taniai et al., supra.
  • the NH2-terminal end of Cry j I reported by Taniai et al., supra has the sequence shown in SEQ ID NO: 18.
  • the internal sequence reported by Taniai et al., supra has the sequence GluAlaPheAsnValGluAsnGlyAsnAlaThrProGlnLeuThrLys (SEQ ID NO: 19).
  • sequence polymorphisms observed in the nucleic acid sequence of the invention. For example, single independent nuleotide substitutions at the codons encoding amino acids 38, 51 and 74 (GGA vs. GAA, GTG vs. GCG, and GGG vs. GAG, respectively) of SEQ ID #1 may result in amino acid polymorphisms (G vs. E, V vs. A, and G vs. E. respectively) at these sites.
  • a single nucleotide substitution has been detected in one cDNA clone derived from Cryptomeria japonica pollen collected in Japan. This substitution in the codon for amino acid 60
  • nucleic acid sequence coding for Cry j I may vary among individual Cryptomeria japonica plants due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention.
  • family members of Cry j I are defined as proteins related in function and amino acid sequence to Cry j I but encoded by genes at separate genetic loci.
  • Fragments of the nucleic acid sequence coding for fragments of Cry j I are also within the scope of the invention. Fragments within the scope of the invention include those coding for parts of Cry j I which induce an immune response in mammals, preferably humans, such as stimulation of minimal amounts of IgE: binding of IgE; eliciting the production of IgG and IgM antibodies: or the eliciting of a T cell response such as proliferation and/or lymphokine secretion and/or the induction of T cell anergy.
  • the foregoing fragments of Cry j I are referred to herein as antigenic fragments.
  • Fragments within the scope of the invention also include those capable of hybridizing with nucleic acid from other plant species for use in screening protocols to detect allergens that are cross-reactive with Cry j I.
  • a fragment of the nucleic acid sequence coding for Cry j I refers to a nucleotide sequence having fewer bases than the nucleotide sequence coding for the entire amino acid sequence of Cry j I and/or mature Cry j I.
  • nucleic acid sequence coding for the fragment or fragments of Cry j I will be selected from the bases coding for the mature protein, however, in some instances it may be desirable to select all or a part of a fragment or fragments from the leader sequence portion of the nucleic acid sequence of the invention.
  • the nucleic acid sequence of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for cloning, expression or purification of Cry j I or fragments thereof.
  • a nucleic acid sequence coding for Cry j I may be obtained from Cryptomeria japonica plants. However, Applicants have found that mRNA coding for Cry j I could not be obtained from commercially available Cryptomeria japonica pollen. This inability to obtain mRNA from the pollen may be due to problems with storage or transportation of commercially available pollen. Applicants have found that fresh pollen and staminate cones are a good source of Cry j I mRNA. It may also be possible to obtain the nucleic acid sequence coding for Cry j I from genomic DNA. Cryptomeria japonica is a well-known species of cedar, and plant material may be obtained from wild, cultivated, or ornamental plants. The nucleic acid sequence coding for Cry j I may be obtained using the method disclosed herein or any other suitable techniques for isolation and cloning of genes. The nucleic acid sequence of the invention may be DNA or RNA.
  • the present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the invention.
  • I or at least one fragment thereof may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • baculovirus insect cells
  • yeast or mammalian cells
  • CHO Chinese hamster ovary cells
  • Suitable expression vectors, promoters, enhancers, and other expression control elements may be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, second edition. Cold Spring Harbor Laboratory Press. Cold
  • yeast or insect cells leads to partial or complete glycosylation of the recombinant material and formation of any inter- or intra-chain disulfide bonds.
  • Suitable vectors for expression in yeast include YepSecl (Baldari et al. (1987) Embo J.6: 229-234); pMFa (Kurjan and Herskowitz (1982) Cell M: 933-943); JRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). These vectors are freely available.
  • Baculovirus and mammalian expression systems are also available. For example, a baculovirus system is commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commerically available (Pharmacia,
  • suitable expression vectors include, among others, pTRC (Amann et al. (1988) Gene 62: 301-315); pGEX (Amrad Corp., Melbourne,
  • pMAL N.E. Biolabs, Beverly, MA
  • pRIT5 Pharmacia, Piscataway, NJ
  • pET-11d Novagen, Madison, WD Jameel et al., (1990) J. Virol. 64:3963-3966
  • pSEM Knapp et al. (1990) BioTechniques &: 280-281).
  • the use of pTRC, and pET-1 Id, for example, will lead to the expression of unfused protein.
  • pMAL maltose E binding protein
  • pRlT5 protein A
  • PSEM protein A
  • glutathione S-transferase pGEX
  • Cry j I or fragment thereof may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using conventional techniques for purification of proteins and peptides.
  • Suitable enzymatic cleavage sites include those for blood clotting Factor Xa or thrombin for which the appropriate enzymes and protocols for cleavage are commercially available from for example Sigma
  • the different vectors also have different promoter regions allowing constitutive or inducible expression with, for example, IPTG induction (PRTC, Amann et al., (1988) supra: pET-1 Id. Novagen, Madison. WI) or temperature induction (pRIT5, Pharmacia. Piscataway, NJ) . It may also be appropriate to express recombinant Cry j I in different E. coli hosts that have an altered capacity to degrade recombinantly expressed proteins (e.g. U.S. patent 4,758,512). Alternatively, it may be
  • nucleic acid sequence advantageous to alter the nucleic acid sequence to use codons preferentially utilized bv E. coli. where such nucleic acid alteration would not affect the amino acid sequence of the expressed protein.
  • Host cells can be transformed to express the nucleic acid sequences of the invention using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming the host cells may be found in Sam brook et al. supra, and other laboratory textbooks.
  • nucleic acid sequences of the invention may also be synthesized using standard techniques.
  • the present invention also provides a method of producing purified Japanese cedar pollen allergen Cry j I or at least one fragment thereof comprising the steps of culturing a host cell transformed with a DNA sequence encoding Japanese cedar pollen allergen Cry j I or at least one fragment thereof in an appropriate medium to produce a mixture of cells and medium containing said Japanese cedar pollen allergen Cry j I or at least one fragment thereof; and purifying the mixture to produce substantially pure Japanese cedar pollen allergen Cry j I or at least one fragment thereof.
  • Host cells transformed with an expression vector containing DNA coding for Cry j I or at least one fragment thereof are cultured in a suitable medium for the host cell.
  • Cry j I protein and peptides can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and immunopurification with antibodies specific for Cry j I or fragments thereof.
  • the terms isolated and purified are used interchangeably herein and refer to peptides, protein, protein fragments, and nucleic acid sequences substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when synthesized chemically.
  • Another aspect of the invention provides preparations comprising Japanese cedar pollen allergen Cry j I or at least one fragment thereof synthesized in a host cell transformed with a DNA sequence encoding all or a portion of Japanese cedar pollen allergen Cry j I, or chemically synthesized, and purified Japanese cedar pollen allergen Cry j I protein , or at least one antigenic fragment thereof produced in a host cell transformed with a nucleic acid sequence of the invention, or chemically synthesized.
  • the Cry j I protein is produced in a host cell transformed with the nucleic acid sequence coding for at least the mature Cry j I protein.
  • Fragments of an allergen from Japanese cedar pollen, preferably Cry j I, eliciting a desired antigenic response may be obtained, for example, by screening peptides recombinantly produced from the corresponding fragment of me nucleic acid sequence of the invention coding for such peptides, synthesized chemically using techniques known in the art, or produced by chemical cleavage of the allergen, the allergen may be arbitrarily divided into fragments of a desired length with no overlap of the peptides, or preferably divided into fragments of a desired length with no overlap of the peptides, or preferably divided into overlapping fragments of a desired length. The fragments are tested to determine their antigenicity (e.g.
  • fragments of Japanese cedar pollen allergen e.g.Cry j, I are to be used for therapeutic purposes, then the fragments of Japanese cedar pollen allergen which are capable of eliciting a T cell response such as stimulation (i.e., proliferation or lymphokine secretion) and/or are capable of inducing T cell anergy are particularly desirable and fragments of Japanese cedar pollen which have minimal IgE stimulating activity are also desirable.
  • purifed Japanese cedar pollen allergens e.g.
  • Cry j I, and fragments thereof preferably do not bind IgE specific for Japanese cedar pollen or bind such IgE to a substantially lesser extent than the purified native Japanese cedar pollen allergen binds such IgE. If the purified Japanese cedar pollen allergen or fragment or fragments thereof bind IgE, it is preferable that such binding does not result in the release of mediators (e.g. histamines) from mast cells or basophils.
  • mediators e.g. histamines
  • Minimal IgE stimulating activity refers to IgE stimulating activity that is less than the amount of IgE production stimulated by the native Cry j I protein.
  • Purified protein allergens from Japanese cedar pollen or preferred antigenic fragments thereof when administered to a Japanese cedar pollen-sensitive individual, or an individual allergic to an allergen cross-reactive with Japanese cedar pollen allergen, such as allergen from the pollen of Cupressus sempervirens or Juniperus sabinoides etc. (discussed previously) are capable of modifying the allergic response of the individual to Japanese cedar pollen or such cross-reactive allergen of the individual, and preferably are capable of modifying the B-cell response, T-cell response or both the B-cell and the T-cell response of the individual to the allergen.
  • modification of the allergic response of an individual sensitive to a Japanese cedar pollen allergen can be defined as non- responsiveness or diminution in symptoms to the allergen, as determined by standard clinical procedures (See e.g. Varney et al, British Medical Journal, 302:265-269 (1990)).
  • the purified Cry j I protein or fragments thereof are preferably tested in mammalian models of Japanese cedar pollinosis such as the mouse model disclosed in Tamura et al. (1986) Microbiol. Immunol. 30: 883-896, or U.S. patent 4,939,239; or the primate model disclosed in Chiba et al. (1990) Int. Arch. Allergy Immunol. 93: 83-88.
  • Initial screening for IgE binding to the protein or fragments thereof may be performed by scratch tests or intradermal skin tests on laboratory animals or human volunteers, or in in vitro systems such as RAST (radioallergosorbent test), RAST inhibition, ELISA assay, radioimmunoassay (RIA), or histamine release (see Examples 7 and 8).
  • RAST radioallergosorbent test
  • ELISA assay ELISA assay
  • RIA radioimmunoassay
  • histamine release see Examples 7 and 8).
  • Antigenic fragments of the present invention which have T cell stimulating activity, and thus comprise at least one T cell epitope are particularly desirable.
  • T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation.
  • T cell proliferation lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies.
  • IgE is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted.
  • a T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition. Amino acid sequences which mimic those of the T cell epitopes and which modify the allergic response to protein allergens are within the scope of this invention.
  • Exposure of patients to purified protein allergens of the present invention or to the antigenic fragments of the present invention which comprise at least one T cell epitope and are derived from protein allergens may tolerize or anergize appropriate T cell subpopulations such that they become unresponsive to the protein allergen and do not participate in stimulating an immune response upon such exposure.
  • administration of the protein allergen of the invention or an antigenic fragment of the present invention which comprises at least one T cell epitope may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring protein allergen or portion thereof (e.g. result in a decrease of IL-4 and/or an increase in IL-2).
  • T cell subpopulations which normally participate in the response to the allergen such that these T cells are drawn away from the site(s) of normal exposure to the allergen (e.g., nasal mucosa, skin, and lung) towards the site(s) of therapeutic administration of the fragment or protein allergen.
  • This redistribution of T cell subpopulations may ameliorate or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal exposure to the allergen, resulting in a dimunution in allergic symptoms.
  • the purified Cry j I protein, and fragments or portions derived therefrom (peptides) can be used in methods of diagnosing, treating and preventing allergic reactions to Japanese cedar pollen allergen or a cross reactive protein allergen.
  • the present invention provides therapeutic compositions comprising purified Japanese cedar pollen allergen Cry j I or at least one fragment thereof produced in a host cell transformed to express Cry j I or at least one fragment thereof, and a pharmaceutically acceptable carrier or diluent.
  • the therapeutic compositions of the invention may also comprise synthetically prepared Cry j I or at least one fragment thereof and a pharmaceutically acceptable carrier or diluent.
  • Administration of the therapeutic compositions of the present invention to an individual to be desensitized can be carried out using known techniques.
  • Cry j I protein or at least one fragment thereof may be administered to an individual in combination with, for example, an appropriate diluent, a carrier and/or an adjuvant.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al. (1981) Int. Arch. Allergy Appl. Immunol. £4:84-99) and liposomes (Strejan et al. (1984) J.
  • the therapeutic composition is preferably administered in nonimmunogenic form, e.g. it does not contain adjuvant.
  • Such compositions will generally be administered by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application or rectal administration.
  • the therapeutic compositions of the invention are administered to Japanese cedar pollen-sensitive individuals at dosages and for lengths of time effective to reduce sensitivity (i.e. reduce the allergic response) of the individual to Japanese cedar pollen.
  • Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to Japanese cedar pollen, the age, sex, and weight of the individual, and the ability of the Cry j I protein or fragment thereof to elicit an antigenic response in the individual.
  • the Cry j I cDNA (or the mRNA from which it was transcribed) or a portion thereof can be used to identify similar sequences in any variety or type of plant and thus, to identify or "pull out" sequences which have sufficient homology to hybridize to the Cry j I cDNA or mRNA or portion thereof, for example, DNA from allergens of Cupressus sempervirens, Juniperus sabinoides etc., under conditions of low stringency. Those sequences which have sufficient homology (generally greater than 40%) can be selected for further assessment using the method described herein.
  • DNA of the present invention can be used to identify, in other types of plants, preferably related families, genera, or species such as Juniperus, or Cupressus, sequences encoding polypeptides having amino acid sequences similar to that of Japanese cedar pollen allergen Cry j I, and thus to identify allergens in other species.
  • the present invention includes not only Cry j I, but also other allergens encoded by DNA which hybridizes to DNA of the present invention.
  • the invention further includes isolated allergenic proteins or fragments thereof that are immunologically related to Cry j I or fragments thereof, such as by antibody cross-reactivity wherein the isolated allergenic proteins or fragments thereof are capable of binding to antibodies specific for the protein and peptides of the invention, or by T cell cross-reactivity wherein the isolated allergenic proteins or fragments thereof are capable of stimulating T cells specific for the protein and peptides of this invention.
  • Proteins or peptides encoded by the cDNA of the present invention can be used, for example as "purified” allergens. Such purified allergens are useful in the standardization of allergen extracts which are key reagents for the diagnosis and treatment of Japanese cedar pollinosis. Furthermore, by using peptides based on the nucleic acid sequences of Cry j I, anti-peptide antisera or monoclonal antibodies can be made using standard methods. These sera or monoclonal antibodies can be used to standardize allergen extracts.
  • compositions and biological activity can be made and administered for therapeutic purposes (e.g. to modify the allergic response of a Japanese cedar sensitive individual to pollen of such trees).
  • Administration of such peptides or protein may, for example, modify B-cell response to Cry j I allergen, T-cell response to Cry j I allergen or both responses.
  • Purified peptides can also be used to study the mechanism of immunotherapy of Cryptomeria japonica allergy and to design modified derivatives or analogues useful in immunotherapy.
  • Modification of naturally-occurring allergens can be designed in such a manner that modified peptides or modified allergens which have the same or enhanced therapeutic properties as the corresponding naturally-occurring allergen but have reduced side effects (especially anaphylactic reactions) can be produced.
  • modified peptides or modified allergens which have the same or enhanced therapeutic properties as the corresponding naturally-occurring allergen but have reduced side effects (especially anaphylactic reactions) can be produced.
  • These can be, for example, a protein or peptide of the present invention (e.g., one having all or a portion of the amino acid sequence of Cry j I), or a modified protein or peptide, or protein or peptide analogue.
  • a modified protein or peptide of the invention can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify immunogenicity and/or reduce allergenicity, or to which a component has been added for the same purpose.
  • the amino acid residues essential to T cell epitope function can be determined using known techniques (e.g., substitution of each residue and determination of the presence or absence of T cell reactivity).
  • Those residues shown to be essential can be modified (e.g., replaced by another amino acid whose presence is shown to enhance T cell reactivity), as can those which are not required for T cell reactivity (e.g., by being replaced by another amino acid whose incorporation enhances T cell reactivity but does not diminish binding to relevant MHC).
  • Another example of a modification of protein or peptides is substitution of cysteine residues preferably with alanine, serine. threonine, leucine or glutamic acid to minimize dimerization via disulfide linkages.
  • Another example of modification of the peptides of the invention is by chemical modification of amino acid side chains or cyclization of the peptide.
  • the protein or peptides of the invention can also be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein allergen resulting from natural allelic variation.
  • D-amino acids, non-natural amino acids or non-amino acid analogues can be substituted or added to produce a modified protein or peptide within the scope of this invention.
  • proteins or peptides of the present invention can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al. supra) to produce a protein or peptide conjugated with PEG.
  • PEG can be added during chemical synthesis of a protein or peptide of the invention. Modifications of proteins or peptides or portions thereof can also include reduction/ alyklation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed. Humana Press, Clifton, NJ, pp 155-194
  • reporter group(s) to the peptide backbone.
  • poly-histidine can be added to a peptide to purify the peptide on immobilized metal ion affinity chromatography (Hochuli, E. et al., Bio/Technology, 6:1321-1325 (1988)).
  • specific endoprotease cleavage sites can be introduced, if desired, between a reporter group and amino acid sequences of a peptide to facilitate isolation of peptides free of irrelevant sequences.
  • canonical protease sensitive sites can be recombinantiy or synthetically engineered between regions, each comprising at least one T cell epitope.
  • charged amino acid pairs such as KK or RR
  • the resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate portions of the peptide containing one or more T cell epitopes.
  • such charged amino acid residues can result in an increase in solubility of a peptide.
  • Site-directed mutagenesis of DNA encoding a peptide or protein of the invention can be used to modify the structure of the peptide or protein by methods known in the art. Such methods may, among others, include PCR with degenerate oligonucleotides (Ho et al., Gene, 77:51-59
  • Cry j I peptides which, when administered to a Japanese cedar pollen sensitive individual in sufficient quantities, will modify the individual's allergic response to Japanese cedar pollen. This can be done, for example, by examining the structure of Cry j I, producing peptides (via an expression system, synthetically or otherwise) to be examined for their ability to influence B-cell and/or T-cell responses in Japanese cedar pollen sensitive individuals and selecting appropriate peptides which contain epitopes recognized by the cells.
  • the epitope will be the basic element or smallest unit of recognition by a receptor, particularly immunoglobulins, histocompatibility antigens and T cell receptors where the epitope comprises amino acids essential to receptor recognition. Amino acid sequences which mimic those of the epitopes and which are capable of down regulating allergic response to Cry j I can also be used.
  • an agent or a drug capable of blocking or inhibiting the ability of Japanese cedar pollen allergen to induce an allergic reaction in Japanese cedar pollen sensitive individuals.
  • agents could be designed, for example, in such a manner that they would bind to relevant anti-Cry j I IgEs. thus preventing IgE-allergen binding and subsequent mast cell degranulation.
  • agents could bind to cellular components of the immune system, resulting in suppression or desensitization of the allergic response to Cryptomeria japonica pollen allergens.
  • a non-restrictive example of this is the use of appropriate B- and T-cell epitope peptides, or modifications thereof, based on the cDNA/protein structures of the present invention to suppress the allergic response to Japanese cedar pollen. This can be carried out by defining the structures of B- and T-cell epitope peptides which affect B- and T-cell function in in vitro studies with blood components from Japanese cedar pollen sensitive individuals.
  • Protein, peptides or antibodies of the present invention can also be used for detecting and diagnosing Japanese cedar pollinosis. For example, this could be done by combining blood or blood products obtained from an individual to be assessed for sensitivity to Japanese cedar pollen with an isolated antigenic peptide or peptides of Cry j I, or isolated Cry j I protein, under conditions appropriate for binding of components in the blood (e.g., antibodies, T-cells, B- cells) with the peptide(s) or protein and determining the extent to which such binding occurs.
  • components in the blood e.g., antibodies, T-cells, B- cells
  • the present invention also provides a method of producing Cryj I or fragment thereof comprising culturing a host cell containing an expression vector which contains DNA encoding all or at least one fragment of Cry j I under conditions appropriate for expression of Cry j I or at least one fragment.
  • the expressed product is then recovered, using known techniques.
  • Cry j I or fragment thereof can be synthesized using known mechanical or chemical techniques.
  • the DNA used in any embodiment of this invention can be cDNA obtained as described herein, or alternatively, can be any oligodeoxynucleotide sequence having all or a portion of a sequence represented herein, or their functional equivalents. Such oligodeoxynucleotide sequences can be produced chemically or enzymatically, using known techniques.
  • a functional equivalent of an oligonucleoti.de sequence is one which is 1) a sequence capable of hybridizing to a complementary oligonucleotide to which the sequence (or corresponding sequence portions) of SEQ ID NO: 1 or fragments thereof hybridizes, or 2) the sequence (or corresponding sequence portion) complementary to SEQ ID NO: 1, and/or 3) a sequence which encodes a product (e.g., a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) of SEQ ID NO: 1.
  • a product e.g., a polypeptide or peptide
  • a functional equivalent must meet one or both criteria will depend on its use (e.g., if it is to be used only as an oligoprobe, it need meet only the first or second criteria and if it is to be used to produce a Cryj I allergen, it need only meet the third criterion).
  • the defatted pollen was extracted at 4°C overnight in 2 L extraction buffer containing 50 mM tris-HCI, pH 7.8, 0.2 M NaCl and protease inhibitors in final concentrations: soybean trypsin inhibitor (2 ⁇ g/ml), leupeptin (1 ⁇ g/ml), pepstatin A (1 ⁇ g/ml) and phenyl methyl sulfonyl fluoride (0.17 mg/ml).
  • soybean trypsin inhibitor (2 ⁇ g/ml
  • leupeptin (1 ⁇ g/ml
  • pepstatin A (1 ⁇ g/ml
  • phenyl methyl sulfonyl fluoride (0.17 mg/ml).
  • the insoluble material was reextracted with 1.2 L extraction buffer at 4°C overnight and both extracts were combined together and depigmented by batch absorption with
  • the depigmented material was then fractionated by ammonium sulfate precipitation at 80% saturation (4°C), which removed much of the lower molecular weight material.
  • the resultant partially purified Cry j I was either dialyzed in PBS buffer and used in T cell studies (see Example 6) or subjected to further purification as described below.
  • the enriched Cry j I material was then dialyzed against 50 mM Na-acetate, pH 5.0 at 4°C with 50 mM Na-acetate, pH 5.0 with protease inhibitors.
  • the unbound material (basic proteins) was then applied to a 50 ml cation exchange column (Whatman
  • Cry j I was eluted in the early fractions of a linear gradient 0.3 M NaCl.
  • the enriched Cry j I material was lyophilized and was then purified by FPLC over a 300 ml Superdex 75 column (Pharmacia) at a flow rate of 30 ml/h in 10 mM Na-acetate, pH 5.0 at 25°C.
  • the purified Cry j I was further applied to FPLC S-Sepharose 16/10 column chromatography (Pharmacia) with a linear gradient of 0 - 1 M NaCl at 25°C.
  • Cryj I eluted as the major peak was subjected to a second gel filtration chromatography.
  • FPLC Superdex 75 column (2.6 by 60 cm)(Pharmacia, Piscataway, NJ) was eluted with a downward flow of 10 mM Na-acetate, pH 5.0 with 0.15 M NaCl at a flow rate of 30 ml/h at 25°C.
  • Fig. 1a shows the chromatography on gel filtration. Only Cry; I was detected (Fig. 1b, lane 2 to lane 8).
  • Cryj I was fractionated into 3 bands as analyzed by SDS-PAGE using silver staining (Fig. 1b) As shown in Fig. 1b, SDS PAGE (12.5%) analysis of the fractions from the major peak shown in Fig. 1a was performed under reducing conditions. The gel was silver stained using the silver staining kit from Bio-Rad.
  • Lane 1 prestained standard proteins (Gibco BRL) including ovalbumin (43,000 kD), carbonic anhydrase (29,000 kD), and ⁇ -lactoglobulin (18,400 kD); lane 2, fraction 36 ; lane 3 fraction 37; lane 4 fraction 38; lane 5 fraction 39 ; lane 6 fraction 41, lane 7 fraction 43; and lane 8 fraction 44. All fractions are shown in Fig. 1a.
  • phosphorylation or lipid content might be different in these isoforms.
  • the other possibility could be due to polymorphism in the gene or alternate splicing in the mRNA though only one major form of Cry j I protein has been detected in cDNA cloning studies (see Example 4).
  • Another approach which may be used to purify native Cry j I or recombinant Cryj I is immunoaffinity chromatography. This technique provides a very selective protein purification due to the specificity of the interaction between monoclonal antibodies and antigen.
  • Cry j I-reactive monoclonal antibodies female Balbl/c mice were obtained from Jackson Labs. Each mouse was initially immunized intraperitoneally with 70-100 ⁇ g purified native Cryj I, (>99% purity lower band, as shown in Fig. 1b), emulsified in Freund's complete adjuvant
  • One further intravenous injection of 10 ⁇ g purified native Cry j I in PBS was given 54 days after the initial injection.
  • the spleen was removed 3 days later and myeloma fusion was conducted as described (Current Protocols in Immunology, 1991, Coligan et al, eds.) using the myeloma line SP2.0.
  • the cells were cultured in 10% fetal calf serum (Hybrimax), hypoxanthine and azaserine and wells containing colonies of hybridoma cells were screened for antibody production using antigen-binding ELISA.
  • Capture ELISA (see Example 7) was used for secondary and tertiary screening. This assay offers the advantage that a clone that recognizes the native protein may be selected and thus may be useful for immunoaffinity purification. Thus, the mAbs will provide a useful tool in purification of Cryj I from pollen extracts.
  • monoclonal antibodies that bind to recombinant Cryj I can also be used for immunoaffinity chromatography.
  • the monoclonal antibodies generated may be useful for diagnostic purposes. It may also be possible to raise different mAbs that show some specificity towards these different isoforms of Cry j I and thus would provide a useful tool to characterize these isoforms.
  • RNA from commercially-available, non-defatted, Cryptomeria japonica (Japanese cedar) pollen (Hollister
  • RNA pellet was obtained from defatted Ambrosia artemisiifolia (ragweed) pollen (Greer Laboratories, Lenior, NC) using this protocol, defatting the Cryptomeria japonica pollen with acetone before guanidine extraction also did not yield any RNA, as determined by absorbance at A 260 .
  • Cryptomeria japonica pollen was suspended in 10 ml extraction buffer (50 mM Tris, pH 9.0, 0.2 M NaCl, 10 mM Mg acetate and diethylpyrocarbonate (DEPC) to 0.1%), ground in a mortar and pestle on dry ice, transferred to a centrifuge tube with 1% SDS, 10 mM EDTA and 0.5% N- lauroyl sarcosine, and the mixture was extracted five times with warm phenol. The aqueous phase was recovered after the final centrifugation, 2.5 vol. absolute ethanol was added, and the mixture was incubated overnight at 4°C.
  • 10 ml extraction buffer 50 mM Tris, pH 9.0, 0.2 M NaCl, 10 mM Mg acetate and diethylpyrocarbonate (DEPC) to 0.1%)
  • EPC diethylpyrocarbonate
  • the pellet was recovered by centrifugation, resuspended in 1 ml dH 2 O by heating to 65°C, and reprecipitated by the addition of 0.1 vol. 3 M Na acetate and 2.0 vol. of ethanol. No detectable RNA was recovered in the pellet as judged by absorbance at A 260 and gel electrophoresis.
  • RNA recovered as determined by absorbance at A 260 and gel electrophoresis.
  • RNA was precipitated from the aqueous phase with 0.1 volume 2 M sodium acetate and 2 volumes ethanol.
  • the pellets were recovered by centrifugation, resuspended in dH2 ⁇ and heated to 65°C for 5 min.
  • Two ml of 4 M lithium chloride were added to the RNA preparations and they were incubated overnight at 0°C.
  • the RNA pellets were recovered by centrifugation, resuspended in 1 ml dH 2 O, and again precipitated with 3 M sodium acetate and ethanol overnight.
  • the final pellets were resuspended in 100 ⁇ l dH2 ⁇ and stored at -80°C.
  • First strand cDNA was synthesized from 8 ⁇ g flowerhead and 4 ⁇ g pollen RNA using a commercially available kit (cDNA synthesis systems kit, BRL, Gaithersburg, MD) with oligo dT priming according to the method of Gubler and Hoffman (1983) Gene 25: 263-269.
  • Primer ED has the sequence 5'-GGAATTCTCTAGACTGCAGGT-3' (SEQ ID NO: 23).
  • CP-1 is the degenerate oligonucleotide sequence encoding the first six amino acids of the amino terminus (AspAsnProIleAspSer, amino acids 1-6 of SEQ ID NO: 1) of Cryj I. EDT will hybridize with the poly A tail of the gene.
  • oligonucleotides were synthesized by Research Genetics, Inc. Huntsville, AL. Polymerase chain reactions (PCR) were carried out using a commercially available kit (GeneAmp DNA Amplification kit, Perkin Elmer Cetus, Norwalk, CT) whereby 10 ⁇ l 10x buffer containing dNTPs was mixed with 1 ⁇ g of CP- 1 and 1 ⁇ g of ED/EDT primers (ED:EDT in a 3:1 M ratio), cDNA (3-5 ⁇ l of a 20 ⁇ l first strand cDNA reaction mix), 0.5 ⁇ l Amplitaq DNA polymerase, and distilled water to 100 ⁇ l.
  • GeneAmp DNA Amplification kit Perkin Elmer Cetus, Norwalk, CT
  • the samples were amplified with a programmable thermal controller (MJ Research, Inc., Cambridge, MA).
  • the first 5 rounds of amplification consisted of denaturation at 94°C for 1 minute, annealing of primers to the template at 45 °C for 1.5 minutes, and chain elongation at 70°C for 2 minutes.
  • the final 20 rounds of amplification consisted of denaturation as above, annealing at 55°C for 1.5 minutes, and elongation as above.
  • sequence 5'-GGGAATTC-3' (bases 1 through 8 of SEQ ID NO: 4) in primer CP-2 represents' an Eco Rl site added for cloning purposes; the remaining degenerate oligonucleotide sequence encodes amino acids 13-18 of Cryj I (AsnTrpAlaGlnAsnArg, amino acids 13 through 18 of SEQ ID NO: 1). Multiple DNA bands were resolved on a 1% GTG agarose gel (FMC, Rockport, ME), none of which hybridized with 32 P end- labeled probe CP-3 (SEQ ID NO: 5) in a Southern blot performed according to the method in Sambrook et al. supra.
  • CP-3 has the sequence 5'- CTGCAGCCATTTTCIACATTAAA-3' wherein A at position 9 can also be G; T at position 12 can also be C; A at position 18 can also be G; and A at position 21 can also be G) (SEQ ID NO: 5). Inosine (I) is used at position 15 in place of G or A or
  • a primary PCR was also performed on first-strand cDNA using CP-1 (SEQ ID NO: 3) and CP-3 (SEQ ID NO: 5), as above.
  • a secondary PCR was performed using 5% of the primary reaction using CP-2 (SEQ ID NO: 4) and CP-3 (SEQ ID NO: 5). Again, multiple bands were observed, none of which could be specifically identified in a Southern blot as Cry j I, and this approach was also not pursued.
  • Double-stranded cDNA was then synthesized from approximately 4 ⁇ g (pollen) or 8 ⁇ g (flowerhead) RNA using a commercially available kit (cDNA Synthesis System kit BRL, Gaithersburg, MD). After a phenol extraction and ethanol precipitation, the cDNA was blunted with T4 DNA polymerase (Promega, Madison, WI), and ligated to ethanol precipitated, self-annealed, AT (SEQ ID NO: 20) and AL (SEQ ID NO: 22) oligonucleotides for use in a modified Anchored PCR reaction, according to the method in Rafnar et al. (1991) J Biol. Chem. 266: 1229- 1236; Frohman et al.
  • Oligonucleotide AT has the sequence 5'- GGGTCTAGAGGTACCGTCCGATCGATCATT-3'(SEQ ID NO: 20) (Rafnar et al. supra ).
  • Oligonucleotide AL has the sequence 5 -AATGATCGATGCT-3' (SEQ ID NO: 22) (Rafnar et al. Supra.
  • Cryj I The amino terminus of Cryj I was amplified from the linkered cDNA (3 ul from a 20 ⁇ l reaction) with 1 ⁇ g each of oligonucleotides AP (SEQ ID NO: 21) and degenerate Cry j I primer CP-7 (which has the sequence 5'- TTCATICGATTCTGGGCCCA-3' wherein G at position 8 can also be T; A at position 9 can also be G; C at position 12 can also be T; and G at position 15 can also be A, T, or C)(SEQ ID NO: 6).
  • Inosine (I) is used at position 6 in place of G or
  • oligonucleotide CP-7 (SEQ ID NO: 6) is the non-coding strand sequence corresponding to coding strand sequence encoding amino acids 14-20 (TrpAlaGlnAsnArgMetLys) from the amino terminus of Cry j I (amino acids 14-20 of SEQ ID NO: 1).
  • Oligonucleotide AP has the sequence 5'-
  • the primary PCR reaction was carried out as described herein. Five percent (5 ⁇ l) of this initial amplification was then used in a secondary amplification with 1 ⁇ g each of AP (SEQ ID NO: 21) and degenerate Cryj I primer CP-8 (SEQ ID NO: 7) an internally nested Cry j I oligonucleotide primer, as described herein.
  • Primer CP-8 has the sequence 5'-CCTCCAGCGATCCTGGGCCCAAATT-3' wherein G at position 9 can also be T; A at position 10 can also be G; C at position 13 can also be T; G at position 16 can also be A, T, or C; and A at position 23 can also be G)(SEQ ID NO: 7).
  • the nucleotides 5'-CCTGCAG-3' (bases 1 through 7 of SEQ ID NO: 7) represent a Pst I restriction site added for cloning purposes.
  • the remaining degenerate oligonucleotide sequence is the non-coding strand sequence corresponding to coding strand sequence encoding amino acids 13-18 of Cry j I (AsnTrpAlaGlnAsnArg, amino acids 13-18 of SEQ ID NO: 1) from the amino terminus of Cry j I.
  • the dominant amplified product was a DNA band of approximately 193 base pairs, as visualized on an ethidium bromide (EtBr)-stained
  • Amplified DNA was recovered by sequential chloroform, phenol, and chloroform extractions, followed by precipitation at -20°C with 0.5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washing with 70% ethanol, the DNA was simultaneously digested with Xba I and Pst I in a 15 ⁇ l reaction and electrophoresed through a preparative 3% GTG NuSieve low melt gel (FMC, Rockport ME). The appropriate sized DNA band was visualized by EtBr staining, excised, and -ligated into appropriately digested M13mp18 for sequencing by the dideoxy chain termination method (Sanger et al. (1977) Proc. Natl Acad Sci.
  • ligatable material could only be derived from staminate cone-derived RNA. However, upon subsequent examination, it was shown that ligatable material could be recovered from PCR product generated from pollen-derived RNA, and from staminate cone- derived RNA.
  • the clone designated JC71.6 was found to contain a partial sequence of Cry j I. This was confirmed as an authentic clone of Cry j I by having complete identity to the disclosed NH2-terminal sequence of Cry j I (Taniai et al. supra).
  • the amino acid at position 7 was determined to be cysteine (Cys) in agreement with the sequence disclosed in U.S. patent 4, 939,239.
  • Amino acid numbering is based on the sequence of the mature protein; amino acid 1 corresponds to the aspartic acid (Asp) disclosed as the NH2-terminus of Cry j I (Taniai et al. supra)
  • the initiating methionine was found to be amino acid -21 relative to the first amino acid of the mature protein.
  • the position of the initiating methionine was supported by the presence of upstream in-frame-stop codons and by 78% homology of the surrounding nucleotide sequence with the plant consensus sequence that encompasses the initiating methionine, as reported by Lutcke et al. (1987) EMBO J.
  • cDNA encoding the remainder of Cry j I gene was cloned from the linkered cDNA by using oligonucleotides CP-9 (which has the sequence 5'- ATGGATTCCCCTTGCTTA-3')(SEQ ID NO: 8) and AP (SEQ ID NO: 21) in the primary PCR reaction.
  • Oligonucleotide CP-9 (SEQ ID NO: 8) encodes amino acids
  • MetAspSerProCysLeu of Cryj I (amino acids -21 through -16 of SEQ ID NO: 1) from the leader sequence of Cry j I, and is based on the nucleotide sequence determined for the partial Cryj I clone JC76.1.
  • a secondary PCR reaction was performed on 5% of the initial amplification mixture, with 1 ⁇ g each of AP (SEQ ID NO: 21) and CP-10 (which has the sequence 5'- GG GAATTCGATAATCCCATAGACAGC-3 , )(SEQ ID NO: 9). the nested primer.
  • the nucleotide sequence 5'-GGGAATTC-3' of primer CP- 10 (bases 1 through 8 of SEQ ID NO: 9) represent an Eco Rl restriction site added for cloning purposes.
  • the remaining oligonucleotide sequence encodes amino acids 1 -6 of Cryj I (AspAsnProIleAspSer) (amino acids 1 through 6 of SEQ ID NO: 1), and is based on the nucleotide sequence determined for the partial Cry j I clone JC76.1.
  • the amplified DNA product was purified and precipitated as above, followed by digestion with Eco Rl and Xba I and electrophoresis through a preparative 1% low melt gel. The dominant DNA band was excised and ligated into M13mp19 and pUC19 for sequencing. Again, ligatable material was recovered from cDNA generated from pollen-derived RNA, and from staminate cone-derived RNA. Two clones, designated pUC19JC91a and pUC19JC91d, were selected for full-length sequencing. They were subsequently found to have identical sequences.
  • DNA was sequenced by the dideoxy chain termination method (Sanger et al. supra) using a commercially available kit (sequenase kit (U.S. Biochemicals, Cleveland, OH). Both strands were completely sequenced using M13 forward and reverse primers (N.E. Biolabs, Beverly, MA) and internal sequencing primers CP-13 (SEQ ID NO: 10), CP-14 (SEQ ID NO: 11), CP-15 (SEQ ID NO: 12), CP-16 (SEQ ID NO: 13), CP-18 (SEQ ID NO: 15), CP-19 (SEQ ID NO: 16), and CP-20 (SEQ ID NO: 17).
  • CP-13 has the sequence 5'-ATGCCTATGTACATTGC-3' (SEQ ID NO: 10).
  • CP-13 (SEQ ID NO: 10) encodes amino acids 82-87 of Cry j I (MetProMetTyrIleAla, amino acids 82 through 87 of SEQ ID NO: 1).
  • CP-14 has the sequence 5'-GCAATGTACATAGGCAT-3' (SEQ ID NO: 11) and corresponds to the non-coding strand sequence of CP-13 SEQ ID NO: 10).
  • CP-15 has the sequence 5'- TCCAATTCTTCTGATGGT-3' ((SEQ ID NO: 12) which encodes amino acids 169-174 of Cry j I (SerAsnSerSerAspGly, amino acids 169 through 174 of SEQ ID NO: 1).
  • CP-16 has the sequence 5'- TTTTGTCAATTGAGGAGT-3 , (SEQ ID NO: 13) which is the non-coding strand sequence which corresponds to coding strand sequence encoding amino acids 335- 340 of Cry j I (ThrProGlnLeuThrLys, amino acids 335 through 340 of SEQ ID NO: 1).
  • CP-18 has the sequence 5'-TAGCAACTCCAGTCGAAGT-3' (SEQ ID NO: 15) which is the non-coding strand sequence which substantially corresponds to coding strand sequence encoding amino acids 181 through 186 of Cry j I (ThrSerThrGlyValThr, amino acids 181 through 186 of SEQ ID NO: 1) except that the fourth nucleotide of CP-18 (SEQ ID NO: 15) was synthesized as a C rather than the correct nucleotide, T. CP-19 which has the sequence 5'-TAGCAACTCCAGTCGAAGT-3' (SEQ ID NO: 15) which is the non-coding strand sequence which substantially corresponds to coding strand sequence encoding amino acids 181 through 186 of Cry j I (ThrSerThrGlyValThr, amino acids 181 through 186 of SEQ ID NO: 1) except that the fourth nucleotide of CP-18 (SEQ ID NO: 15) was synthesized as a C rather than the correct nucleo
  • TAGCTCTCATTTGGTGC-3' (SEQ ID NO: 16) is the non-coding strand sequence which corresponds to coding strand sequence encoding amino acids 270 through 275 of Cry j I (AlaProAsnGluSerTyr, amino acids 270 through 275 of SEQ ID NO: 1).
  • CP-20 has the sequence 5'- TATGCAATTGGTGGGAGT-3' (SEQ ID NO: 17) which is the coding strand sequence for amino acids 251-256 of Cry j I (TyrAlaHeGlyGlySer, amino acids 251 through 256 of SEQ ID NO: 1).
  • the sequenced DNA was found to have the sequence shown in Figs. 4a and 4b (SEQ ID NO: 1).
  • the complete cDNA sequence for Cry j I is composed of 1312 nucleotides, including 66 nucleotides of 5' untranslated sequence, an open reading frame starting with the codon for an initiating methionine, of 1122 nucleotides, and a 3' untranslated region. There is a consensus polyadenylation signal sequence in the 3' untranslated region 25 nucleotides 5' to the poly A tail.
  • the position of the initiating methionine is confirmed by the presence of in- frame upstream stop codons and by 78% homology with the plant consensus sequence that encompasses the initiating methionine (AAAAAUGGA (bases 62 through 70 of SEQ ID NO: 1) found in Cry j I compared with the AACAAUGGC consensus sequence for plants, Lutcke et al. (1987) EMBO J. 6: 43-48).
  • the open reading frame encodes a protein of 374 amino acids of which the first 21 amino acids comprise a leader sequence that is cleaved from the mature protein. The amino terminus of the mature protein was identified by comparison with the published NH2-terminal sequence (Taniai et al.
  • the deduced amino acid sequence of the mature protein comprised of 353 amino acids has complete sequence identity with the published protein sequence for Cry j I (Taniai et al. supra), including the first twenty amino acids for the NH 2 -terminal and sixteen contiguous internal amino acids.
  • the mature protein also contains five potential N-linked glycosylation sites corresponding to the consensus sequence N-X-S/T.
  • RNA was precipitated from the aqueous phase with 0.1 volume 3 M sodium acetate and 2 volumes ethanol.
  • the pellets were recovered by centrifugation, resuspended in dH 2 O and heated to 65°C for 5 minutes.
  • Two ml of 4 M lithium chloride were added to the RNA preparations and they were incubated overnight at 9°C.
  • the RNA pellets were recovered by centrifugation, resuspended in 1 ml dH 2 O, and again precipitated with
  • Double stranded cDNA was synthesized from 8 ⁇ g pollen RNA using the cDNA Synthesis Systems kit (BRL) with oligo dT priming according to the method of Gubler and Hoffman (1983) Gene 25:263-269.
  • Polymerase chain reactions (PCR) were carried out using the GeneAmp DNA Amplification kit (Perkin Elmer Cetus) whereby 10 ⁇ l 10x buffer containing dNTPs was mixed with 100 pmol each of a sense oligonucleotide and an anti-sense oligonucleotide, (10 ⁇ l of a 400 ⁇ l double stranded cDNA reaction mix), 0.5 ⁇ l Amplitaq DNA polymerase, and distilled water to 100 ⁇ l.
  • the samples were amplified with a programmable thermal controller from MJ Research, Inc. (Cambridge, MA).
  • the first 5 rounds of amplification consisted of denaturation at 94°C for 1 minute, annealing of primers to the template at 45°C for 1 minute, and chain elongation at 72°C for 1 minute.
  • the final 20 rounds of amplification consisted of denaturation as above, annealing at 55°C for 1 minute, and elongation as above.
  • nucleotide sequence 5'-CCTGCAGAAGCTT-3' (bases 1 through 13 of SEQ. ID # 14) represents Pst I and Hin dIII restriction sites added for cloning purposes.
  • the nucleotide sequence 5'-TCA-3' (bases 13 through 15 of SEQ. ID # 14) correspond to the non-coding strand sequence of a stop codon.
  • Amplified DNA was recovered by sequential chloroform, phenol, and chloroform extractions, followed by precipitation at -20°C with 0.5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washing with 70% ethanol, the DNA was blunted with T4 polymerase followed by digestion with Eco RI, in the case of JC130 , or simultaneously digested with Eco Rl and Pst I, in the case of JC135, in a 15 ⁇ l reaction and electrophoresed through a preparative 1% SeaPlaque low melt gel (FMC). Appropriate sized DNA bands were visualized by EtBr staining, excised, and ligated into appropriately digested pUC19 for dideoxy DNA sequencing by the dideoxy chain termination method (Sanger et al. (1977)
  • This nucleotide change results in a predicted amino acid change from a Tyr to a His at amino acid 60 of the mature Cryj I protein.
  • This polymorphism has not yet been confirmed in an independently-derived PCR clone or by direct amino acid sequencing. However, such polymorphisms in primary nucleotide and amino acid sequences are expected.
  • Cry j I was performed as follows. Ten ⁇ g of pUC19JC91a was digested with Xba I, precipitated, then blunted with T4 polymerase. Bam HI linkers
  • the linkered cDNA was then digested simultaneously with Eco Rl and Bam HI.
  • the Cry j I insert (extending from the nucleotides encoding the amino terminus of the mature protein through the stop codon) was isolated by electrophoresis of this digest through a 1% SeaPlaque low melt agarose gel.
  • the insert was then ligated into the appropriately digested expression vector pET-11d (Novagen, Madison, WI; Jameel et al. (1990) J. Virol. (64:3963-3966) modified to contain a sequence encoding 6 histidines (His 6) immediately 3' of the ATG initiation codon followed by a unique Eco Rl
  • a recombinant clone was used to transform Escherichia coli strain BL21-DE3 which harbors a plasmid that has an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible promoter preceding the gene encoding T7 polymerase. Induction with IPTG leads to high levels of T7 polymerase expression, which is necessary for expression of the recombinant protein in pET-11d, which has a T7 promoter. Clone pET-11d ⁇ HRhis 6 JC91a.d was confirmed by dideoxy sequencing (Sanger et al. Supra) with
  • Recombinant protein expression was visualized as a band with the predicted molecular weight of approximately 38 kDa on a Coomassie blue-stained SDS-PAGE gel, according to the method in Sambrook et al., supra, on which 40 ⁇ l of the crude lysate was loaded.
  • a negative control consisted of crude lysates from uninduced bacteria containing the plasmid with Cry j I and an induced lysate from bacteria carrying no plasmid.
  • the pET-11d ⁇ HRhis 6 JC91a.d clone was then grown on a large scale for recombinant protein expression and purification.
  • a 2 ml culture bacteria containing the recombinant plasmid was grown for 8 hr, then streaked onto solid media (e.g. 6 petri plates (100 ⁇ 15 mm) with 1.5% agarose in LB medium (Gibco-BRL,
  • IPTG added (1 mM final concentration), and the culture grown for an additional 2 hours.
  • Bacteria was recovered by centrifugation (7,930 ⁇ g, 10 min), and lysed in 90 ml of 6M Guanidine-HCl, 0.1M Na 2 HPO 4 , pH 8.0 for 1 hour with vigorous shaking. Insoluble material was removed by centrifugation (11,000 x g, 10 min, 4°
  • the pH of the lysate was adjusted to pH 8.0, and the lysate applied to an 80 ml Nickel NTA agarose column (Qiagen) that had been equilibrated with 6 M
  • Guanidine HCl 100 mM Na 2 HPO 4 , pH 8.0.
  • the column was sequentially washed with 6 M Guanidine HCl, 100 mM Na2HP ⁇ 4, 10 mM Tris-HCl, pH 8.0, then 8 M urea, 100 mM Na2HPO4, pH 8.0, and finally 8 M urea, 100 mM sodium acetate, 10 mM Tris-HCl, pH 6.3.
  • the column was washed with each buffer until the flow through has an A280 ⁇ 0.05.
  • the recombinant protein, Cry j I was eluted with 8 M urea, 100 mM sodium acetate, 10 mM Tris-HCl, pH 4.5, and collected in 10 ml aliquots. The protein concentration of each fraction was determined by A280 and the peak fractions pooled. An aliquot of the collected recombinant protein was analyzed on SDS-PAGE according to the method in Sambrook et al., supra.
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • T cells proliferate to recombinant Cry j I (rCry j I), purified native Cry j I, or recombinant Amb a l.l ( ⁇ Amb al ⁇ ) was then assessed.
  • 2 X 10 4 rested cells were restimulated in the presence of 4 X 10 ⁇ autologous Epstein-Barr virus (EBV)-transfor ⁇ ned B cells (prepared as described below) (gamma-irradiated with 25,000 RADS) with 2-50 ⁇ g/ml of rCryj L purified native Cryj I or rAmb a I.1, in a volume of 200 ⁇ l complete medium in duplicate or triplicate wells in 96-well round bottom plates for 2-4 days.
  • EBV Epstein-Barr virus
  • Fig. 12 shows the effect of varying antigen dose in assays with recombinant Cryj I, purified native Cryj I, and recombinant Amb a I.1.
  • the results shown in Fig. 12 demonstrate that patient #999 responds well to recombinant Cry j I, and purified native Cry j I, but not to recombinant Amb a I.1. This indicates that Cry j I T cell epitopes are recognized by T cells from this particular allergic patient and that rCry j I contains such T cell epitopes.
  • CRL1612 American Type Culture Collection, Rockville, MD
  • PMA phorbol 12-myristate 13-acetate
  • SPE Japanese cedar pollen or purified native Cry j I (assayed at 90% purity by protein sequencing) and human IgE antibody binding to these antigens was analyzed.
  • Pooled human plasma consisting of an equal volume of plasma from 15 patients with a Japanese cedar pollen MAST score of 2.5 or greater, and two individual patient plasma samples were compared in this assay.
  • Fig. 5 shows the results of the binding reactivity with these two antigens. The overall pattern of binding is very similar whether the coating antigen is SPE (Fig. 5a) or purified native Cry j I (Fig. 5b).
  • ELISA wells were coated with Japanese cedar pollen SPE and then allergic patient IgE binding was measured in the presence of competing purified native Cry j I in solution.
  • the source of allergic IgE in these assays was either the pool of plasma from 15 patients (denoted PHP) or seven individual plasma samples from patients with a Japanese cedar MAST score of 2.5 or greater.
  • the competition assay using the pooled human plasma samples compares the competitive binding capacity of purified native Cry j I to Japanese cedar pollen
  • Fig. 6 shows the graphed results of the competition ELISA with pooled human plasma.
  • the concentration of protein present in the Japanese cedar pollen SPE is approximately 170 times greater at each competing point than is the purified native Cry j I . From this analysis it is clear that the purified native Cry j I competes very well for IgE binding to the whole range of proteins present in the Japanese cedar pollen soluble pollen extract. This implies that most of the anti-Cry j I IgE reactivity is directed against purified native Cry j I .
  • the negative control shows no specific competitive activity and the competing SPE in solution can completely remove binding to the coated wells.
  • Fig. 8a shows the direct binding assay to the SPE with seven individual plasma samples.
  • a capture ELISA Another method of analysis that was applied to the examination of IgE reactivity to the recombinant Cry j I was a capture ELISA.
  • This analysis relies on the use of a defined anitibody, in this case CBF2 to bind the antigen and allow for the binding of antibodies to other epitope sites.
  • the format of this capture ELISA is 1) wells are coated with MAb CBF2, 2) antigen or PBS (as one type of negative control) is added and captured by specific interaction with the coated MAb, 3) either the control antibody anti- ⁇ mb a I & II (Fig. 10b) or human allergic plasma (Fig. 10a) is added as the detecting antibody, and 4) detection of antibody binding is assayed.
  • Figs. 10a and 10b are the graphed results of these assays.
  • the pooled human plasma (15 patients) was used. The conclusion from these results is that there is no indication of any specific binding of human allergic
  • a histamine release assay was performed on one Japanese cedar pollen allergic patient using Japanese cedar pollen SPE, purified native Cry j I and rCry j I as the added antigens. This assay is a measure of IgE reactivity through human basophil mediator release. The results of this assay, shown in Fig. 11, demonstrate strong histamine release with both purified native Cry j I and the Japanese cedar pollen SPE over a wide concentration range. The only point where there is any measurable histamine release with the Cry j I is at the highest concentration, 50 ⁇ g/ml.
  • coli expressed material has T cell reactivity (Example 6), but does not appear to bind IgE from Crytpomeria japonica atopes nor cause histamine release from the mast cells and basophils of such atopes in vitro.
  • Expression of rCry j I which is capable of binding IgE could be achieved in yeast insect (baculovirus) or mammalian cells (e.g. CHO, human and mouse).
  • a rCry j I capable of actively binding IgE may be important for the use of recombinant material for diagnostic purposes.
  • ORGANISM Crytpomeria japonica

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Abstract

L'invention décrit des séquences d'acide nucléique codant l'allergène principal du pollen de Cryptomeria japonica, Cry j I, ainsi que ses fragments. L'invention décrit également Cry j I purifié, ainsi qu'au moins un de ses fragments produits dans une cellule hôte transformée par une séquence d'acide nucléique codant Cry j I ou au moins un de ses fragments et des fragments de Cry j préparés synthétiquement. Cry j I et ses fragments sont efficaces dans le diagnostic, le traitement et la prévention de la pollinose provoquée par le cèdre japonais.
PCT/US1992/005661 1991-07-12 1992-07-10 Proteines peptides allergeniques provenant du pollen du cedre japonais Ceased WO1993001213A1 (fr)

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EP92914823A EP0595855A1 (fr) 1991-07-12 1992-07-10 Proteines peptides allergeniques provenant du pollen du cedre japonais
JP50237093A JP3575802B2 (ja) 1991-07-12 1992-07-10 日本杉花粉からのアレルゲン性蛋白質及びペプチド
AU34384/93A AU3438493A (en) 1992-07-10 1993-01-15 Allergenic proteins and peptides from japanese cedar pollen
PCT/US1993/000139 WO1994001560A1 (fr) 1991-07-12 1993-01-15 Proteines et peptides allergenes tires du pollen du cedre du japon
AU70569/96A AU7056996A (en) 1992-07-10 1996-11-04 Allergenic proteins and peptides from Japanese cedar pollen

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US72913491A 1991-07-12 1991-07-12
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001560A1 (fr) * 1991-07-12 1994-01-20 Immulogic Pharmaceutical Corporation Proteines et peptides allergenes tires du pollen du cedre du japon
JPH07118295A (ja) * 1993-10-20 1995-05-09 Meiji Milk Prod Co Ltd スギ花粉アレルゲンのｔ細胞エピトープペプチド及びそのアナログペプチド
EP0661294A1 (fr) * 1993-12-27 1995-07-05 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Protéine du pollen de cèdre et son utilisation
EP0661295A1 (fr) * 1993-12-21 1995-07-05 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Protéine du pollen de cèdre et son utilisation
US5691167A (en) * 1992-10-30 1997-11-25 The University Of Melbourne DNA encoding allergenic proteins and peptides from Johnson grass pollen
US6090386A (en) * 1991-07-12 2000-07-18 Griffith; Irwin J. T cell peptides of the CRX JII allergen
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
US6982326B1 (en) 1991-07-12 2006-01-03 Immulogic Pharmaceutical Corporation Allergenic proteins and peptides from Japanese cedar pollen
US7025964B1 (en) 1996-11-13 2006-04-11 Meiji Dairies Corporation Peptide-based immunotherapeutic agent
US7112329B1 (en) 1996-06-14 2006-09-26 Meiji Milk Products Co. Ltd. T cell epitope peptide
WO2011137420A1 (fr) 2010-04-30 2011-11-03 Apellis Pharmaceuticals, Inc. Procédés, articles et trousses pour la désensibilisation allergique par l'intermédiaire des muqueuses orales
US9260491B2 (en) 2009-10-30 2016-02-16 Nippon Paper Industries Co., Ltd. Protein having immunogenicity of cedar pollen, polynucleotide encoding the protein, and use thereof
US9724271B2 (en) 2010-04-30 2017-08-08 Allovate, Llc Methods and articles for preventing or reducing risk of developing a hyperallergenic immune system
WO2020147985A1 (fr) * 2019-01-17 2020-07-23 Laboratorios Leti Slu Procédés de purification d'extraits d'allergènes
US10967059B2 (en) 2013-09-19 2021-04-06 Allovate, Llc Toothpaste for delivery of allergens to oral mucosa

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JP3707817B2 (ja) * 1994-12-21 2005-10-19 明治乳業株式会社 ヒノキ花粉アレルゲン
JP4176750B2 (ja) * 1996-06-14 2008-11-05 明治乳業株式会社 Ｔ細胞エピトープペプチド
WO2000068262A1 (fr) * 1999-05-11 2000-11-16 Kyowa Hakko Kogyo Co., Ltd. Nouvel antigene pollinique
JP2003116556A (ja) * 2001-10-09 2003-04-22 Nippon Zenyaku Kogyo Kk アレルギー性皮膚炎治療剤
JP4686712B2 (ja) * 2005-03-31 2011-05-25 国立大学法人広島大学 スギ花粉アレルゲンの高次構造ＩｇＥエピトープを含むペプチドおよびその利用

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EP0416816A1 (fr) * 1989-09-02 1991-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Agent d'hyposensibilisation

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EP0416816A1 (fr) * 1989-09-02 1991-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Agent d'hyposensibilisation

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001560A1 (fr) * 1991-07-12 1994-01-20 Immulogic Pharmaceutical Corporation Proteines et peptides allergenes tires du pollen du cedre du japon
US6090386A (en) * 1991-07-12 2000-07-18 Griffith; Irwin J. T cell peptides of the CRX JII allergen
US6982326B1 (en) 1991-07-12 2006-01-03 Immulogic Pharmaceutical Corporation Allergenic proteins and peptides from Japanese cedar pollen
US8540999B2 (en) 1991-07-12 2013-09-24 Merck Patent Gmbh Allergenic proteins and peptides from Japanese cedar pollen
US5691167A (en) * 1992-10-30 1997-11-25 The University Of Melbourne DNA encoding allergenic proteins and peptides from Johnson grass pollen
JPH07118295A (ja) * 1993-10-20 1995-05-09 Meiji Milk Prod Co Ltd スギ花粉アレルゲンのｔ細胞エピトープペプチド及びそのアナログペプチド
EP0661295A1 (fr) * 1993-12-21 1995-07-05 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Protéine du pollen de cèdre et son utilisation
US5631221A (en) * 1993-12-21 1997-05-20 Kabushiki Kaisha Hayashibara Sibutsu Kagaku Kenkyujo Pollenosis-inducing polypeptide, process for preparing the same, and uses thereof
EP0661294A1 (fr) * 1993-12-27 1995-07-05 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Protéine du pollen de cèdre et son utilisation
JPH07188291A (ja) * 1993-12-27 1995-07-25 Hayashibara Biochem Lab Inc 蛋白質とその製造方法並びに用途
US5874401A (en) * 1993-12-27 1999-02-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Cedar pollen protein and use thereof in treating, preventing, and diagnosing pollenosis
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
US7547440B2 (en) 1996-06-14 2009-06-16 Meiji Dairies Corporation T-cell epitope peptides
US7112329B1 (en) 1996-06-14 2006-09-26 Meiji Milk Products Co. Ltd. T cell epitope peptide
US7407657B2 (en) 1996-06-14 2008-08-05 Meiji Dairies Corporation T-cell epitope peptides
US7025964B1 (en) 1996-11-13 2006-04-11 Meiji Dairies Corporation Peptide-based immunotherapeutic agent
US9260491B2 (en) 2009-10-30 2016-02-16 Nippon Paper Industries Co., Ltd. Protein having immunogenicity of cedar pollen, polynucleotide encoding the protein, and use thereof
US9724271B2 (en) 2010-04-30 2017-08-08 Allovate, Llc Methods and articles for preventing or reducing risk of developing a hyperallergenic immune system
US9271899B2 (en) 2010-04-30 2016-03-01 Allovate, Llc Methods, articles and kits for allergic desensitization, via the oral mucosa
WO2011137420A1 (fr) 2010-04-30 2011-11-03 Apellis Pharmaceuticals, Inc. Procédés, articles et trousses pour la désensibilisation allergique par l'intermédiaire des muqueuses orales
EP3305319A1 (fr) 2010-04-30 2018-04-11 Allovate, LLC Procédés et articles permettant de prévenir ou de réduire le risque de développer un système immunitaire hyperallergénique
EP3524259A1 (fr) 2010-04-30 2019-08-14 Allovate, LLC Procédés, articles et kits de désensibilisation allergique par l'intermédiaire de la muqueuse orale
EP4011362A1 (fr) 2010-04-30 2022-06-15 Allovate, LLC Procédés, articles et kits de désensibilisation allergique par l'intermédiaire de la muqueuse orale
US10967059B2 (en) 2013-09-19 2021-04-06 Allovate, Llc Toothpaste for delivery of allergens to oral mucosa
US11980664B2 (en) 2013-09-19 2024-05-14 Allovate, Llc Toothpaste for delivering allergens to oral mucosa
WO2020147985A1 (fr) * 2019-01-17 2020-07-23 Laboratorios Leti Slu Procédés de purification d'extraits d'allergènes
CN113825518A (zh) * 2019-01-17 2021-12-21 莱蒂生物制药公司 纯化过敏原提取物的方法
US12478648B2 (en) 2019-01-17 2025-11-25 LETI Pharma S.L. Methods of purifying an allergen extract

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