EP0669983A1 - Allergene proteine und peptide von pollen der japanischen zeder - Google Patents

Allergene proteine und peptide von pollen der japanischen zeder

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Publication number
EP0669983A1
EP0669983A1 EP94902242A EP94902242A EP0669983A1 EP 0669983 A1 EP0669983 A1 EP 0669983A1 EP 94902242 A EP94902242 A EP 94902242A EP 94902242 A EP94902242 A EP 94902242A EP 0669983 A1 EP0669983 A1 EP 0669983A1
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EP
European Patent Office
Prior art keywords
protein
seq
cryj
fragment
sequence
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP94902242A
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English (en)
French (fr)
Inventor
Mei-Chang Kuo
Siu-Mei Helena Yeung
Andrew W. Brauer
Joanne Pollock
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Immulogic Pharmaceutical Corp
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Immulogic Pharmaceutical Corp
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Publication date
Application filed by Immulogic Pharmaceutical Corp filed Critical Immulogic Pharmaceutical Corp
Publication of EP0669983A1 publication Critical patent/EP0669983A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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. Upon combination of a specific allergen with IgE bound to mast cells or basophils, the IgE may be cross- linked on the cell surface, resulting in the physiological effects of IgE-antigen interaction.
  • Such effects may be systemic or local in nature, depending on the route by which the antigen entered the body and the pattern of deposition of IgE on mast cells or basophils. Local manifestations generally occur on epithelial surfaces at the location at which the allergen entered the body.
  • Systemic effects can include anaphylaxis (anaphy lactic shock), which is the result of an IgE-basophil response to circulating (intravascular) antigen.
  • 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 Cryj I and a sixteen amino acid internal sequence have been determined (Taniai supra).
  • a second allergen has recently been isolated from the pollen of Cryptomeria japonica (Japanese cedar) (Sakaguchi et al. (1990) Allergy 45:309-312).
  • This allergen designated Cryj II
  • Cryj II has been reported to have a molecular weight of approximately 37 kDa and 45 kDa when assayed on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions, respectively (Sukaguchi et al., supra).
  • Cryj II was found to have no immunological cross-reactivity with Cryj I (Sakaguchi (1990) supra: Kawashima et al. (1992) Int. Arch.
  • the present invention also provides purified Cryj II and at least one fragment thereof produced in a host cell transformed with a nucleic acid sequence coding for Cryj II or at least one fragment thereof and fragments of Cryj II prepared synthetically.
  • a fragment of the nucleic acid sequence coding for the entire amino acid sequence of Cryj II refers to a nucleotide sequence having fewer bases than the nucleotide sequence coding for the entire amino acid sequence of Cryj II and/or mature Cryj II.
  • Cryj II 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.
  • Fig. la shows an SDS-PAGE (12%) analysis of Cryj II under non-reducing conditions.
  • Fig. lb shows an SDS-PAGE (12%) analysis of Cryj II under reducing conditions.
  • Fig. 2 shows the results of mono S column chromatography of Cryj II eluted with a step gradient of NaCl in lOmM sodium acetate buffer, pH 5.0.
  • Fig. 3 shows an SDS-PAGE (12%) of purified subfractions of Cryj II analyzed under reducing conditions.
  • Fig. 4 shows the nucleic acid sequence (SEQ ID NO: 1) and the deduced amino acid (SEQ ID NO: 2) coding for Cry j II.
  • Fig. 5 shows the deduced amino acid sequence of Cryj II (SEQ ID NO: 2).
  • Fig. 6 shows the long form (SEQ ID NO: 4) and short form (SEQ ID NO: 5) NH2-terminii amino acid sequences of Cry j II determined by protein sequence analysis as discussed in Example 2 aligned with the ten amino acid sequence of Cry j II (SEQ ID NO: 3) defined by Sakaguchi et al., supra (SEQ ID NO: 6).
  • Fig. 7 is a graphic representation of the results of a direct ELISA assay showing the binding response of the monoclonal antibody 4B11 and seven patients' (Batch 1) plasma IgE to purified Cryj I as the coating antigen.
  • Fig. 8 is a graphic representation of a direct ELISA assay showing the binding response of the monoclonal antibody 4B11, and seven patients' (Batch 1) plasma IgE to purified native Cryj II as the coating antigen.
  • Fig. 9 is a graphic representation of a direct ELISA assay showing the binding response of the monoclonal antibody, 4B11, and seven patients' (Batch 1) plasma IgE to recombinant Cryj II (rCryj II) as the coating antigen.
  • Fig. 10 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 2) plasma IgE to purified native Cryj I.
  • Fig. 11 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 2) plasma IgE to purified native Cryj II.
  • Fig. 12 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 2) plasma IgE to recombinant Cryj II.
  • Fig. 13 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 3) plasma IgE to purified native Cryj I.
  • Fig. 14 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 3) plasma IgE to purified native Cryj II.
  • Fig. 15 is a graphic representation of a direct ELISA assay showing the binding response of eight patients' (Batch 3) plasma IgE to recombinant Cryj II.
  • Fig. 16 is a table which summarizes both the MAST scores performed on patient's plasma samples (Batch 1-3) and the direct ELISA results shown in Figs. 7- 15; a positive response is indicated by a (+) sign and the number of positive responses for each antigen is shown at the bottom of each column.
  • the present invention provides nucleic acid sequences coding for Cryj II, an allergen found in Japanese cedar pollen.
  • the nucleic acid sequence coding for Cryj II shown in Fig. 4 (SEQ ID NO: 1) encodes a protein of 514 amino acids.
  • the deduced Cryj II amino acid sequence is shown in Figs. 4 and 5 (SEQ ID NO: 2) .
  • the amino acid sequence representing the long form of Cryj II is encoded by the nucleotide sequence extending from bases 177-1586 (SEQ ID NO: 7) as shown in Fig. 4, and the amino acid sequence representing the short form of Cry j II is encoded by the nucleotide sequence extending from 192-1586 (SEQ ID NO: 8) as shown in Fig. 4.
  • a host cell transformed with a vector containing the cDNA insert coding for full- length Cryj II has been deposited with the American Type Culture Collection, ATCC No. 69105.
  • Fragments of the nucleic acid sequence coding for fragments of Cryj II are also within the scope of the invention. Fragments within the scope of the invention include those coding for parts of Cryj II 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 Cryj II 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 Cryj II.
  • a fragment of the nucleic acid sequence coding for Cryj II refers to a nucleotide sequence having fewer bases than the nucleotide sequence coding for the entire amino acid sequence of Cryj II and/or mature Cryj II.
  • the nucleic acid sequence coding for the fragment or fragments of Cryj II 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 Cryj II or fragments thereof.
  • a nucleic acid sequence coding for Cryj II may be obtained from Cryptomeria japonica plants. Applicants have found that fresh pollen and staminate cones are a good source of Cry j II mRNA. It may also be possible to obtain the nucleic acid sequence coding for Cryj II 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 Cryj II 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.
  • Nucleic acid coding for Cryj II, or at least one fragment thereof may be expressed in bacterial cells such as E. coli, insect cells (baculo virus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • bacterial cells such as E. coli, insect cells (baculo virus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • 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 Spring Harbor, New York (1989).
  • Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art.
  • 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 30: 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.
  • baculovirus system is commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commerically available (Pharmacia, Piscataway, NJ) for expression in mammalian cells.
  • suitable expression vectors include, among others, pTRC (Amann et al. (1988) Gene 69: 301-315); pGEX (Amrad Corp. , Melbourne,
  • pMAL N.E. Biolabs, Beverly, MA
  • pRIT5 Pharmacia, Piscataway, NJ
  • pET-lld Novagen, Madison, WI
  • Jameel et al. (1990) J. Virol. 64:3963- 3966
  • pSEM Knapp et al. (1990) BioTechniques 8: 280-281).
  • the use of pTRC, and pET-lld, for example, will lead to the expression of unfused protein.
  • pMAL maltose E binding protein
  • pRIT5 protein A
  • PSEM protein A
  • glutathione S-transferase glutathione S-transferase
  • Cryj II 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 Chemical
  • 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-lld, Novagen, Madison, WI) or temperature induction (pRIT5, Pharmacia, Piscataway, NJ) . It may also be appropriate to express recombinant Cryj II 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 advantageous to alter the nucleic acid sequence to use codons preferentially utilized by E.
  • 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, D ⁇ A ⁇ -dextran-mediated transfection, or electroporation.
  • 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 Cryj II or at least one fragment thereof comprising the steps of culturing a host cell transformed with a DNA sequence encoding Japanese cedar pollen allergen Cryj II or at least one fragment thereof in an appropriate medium to produce a mixture of cells and medium containing said Japanese cedar pollen allergen Cryj II or at least one fragment thereof; and purifying the mixture to produce substantially pure Japanese cedar pollen allergen Cryj II or at least one fragment thereof.
  • Host cells transformed with an expression vector containing DNA coding for Cryj II or at least one fragment thereof are cultured in a suitable medium for the host cell.
  • Cryj II 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 Cryj II 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.
  • Cryj II protein may also be isolated from Japanese cedar pollen as described in Example 1.
  • Cry j II isolated directly from Japanese cedar pollen is referred to herein as "purified native" Cryj II. It is preferable that purified native Cry j II of the invention be at least 80% pure, and more preferably at least 90% pure and even more preferably be purified to homogeneity (at least 99% pure).
  • Another aspect of the invention provides preparations comprising Japanese cedar pollen allergen Cryj II 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 Cryj II, or chemically synthesized, and purified Japanese cedar pollen allergen Cryj II 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 Cryj II protein is produced in a host cell transformed with the nucleic acid sequence coding for at least the mature Cryj II protein.
  • antigenic fragments Fragments of an allergen from Cry j II, eliciting a desired antigenic response (referred to herein as antigenic fragments) are defined herein as any protein fragment or peptide which can be derived from the Cryj II proteins, but does not include the ten amino acid fragments which extends from amino acid residues 55-64, as shown in Figs. 4, 5 and 6, but may include any portion of that ten amino acid fragment in conjunction with another fragment derived from Cryj II.
  • Antigenic fragments of Cryj II may be obtained, for example, by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid sequence of the invention coding for such peptides, or by screening peptides which have been synthesized chemically using techniques known in the art, or by screening peptides 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. the ability of the fragment to induce an immune response such as T cell proliferation as discussed in Example 7).
  • Antigenic fragments may also be predicted using an algorithm such as that discussed in a paper by Hill et al, Journal of Immunology, 147:184-197 (1991).
  • Algorithms for predicting peptides which elicit T cell activity such as the algorithm discussed by Hill et al. are based on the protein's sequence wherein certain patterns within the sequence are likely to bind MHC and therefore may contain T cell epitopes.
  • the peptides predicted by the algorithm such as Cry j II A and Cry j IIB discussed in Example 7 may be produced recombinantly or synthetically and tested for T cell activity as discussed in Example 7.
  • fragments of Japanese cedar pollen allergen e.g. Cryj II 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.
  • purified Japanese cedar pollen allergens, e.g. Cryj II, 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.
  • Minimal IgE stimulating activity refers to IgE stimulating activity that is less than the amount of IgE production stimulated by the native Cryj II protein.
  • Isolated antigenic fragments or peptides 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. These events lead to 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.
  • An epitope is 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 particularly T cell epitopes and which modify the allergic response to protein allergens including those capable of down regulating allergic response to Cry j II, are within the scope of this invention.
  • human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to Japanese cedar pollen allergen, (i.e., an individual who has an IgE mediated immune response to Japanese cedar pollen allergen) with a peptide derived from the allergen and determining whether proliferation of T cells occurs in response to the peptide as measured, e.g., by cellular uptake of tritiated thymidine.
  • Stimulation indices for responses by T cells to peptides can be calculated as the maximum CPM in response to a peptide divided by the control CPM.
  • a stimulation index (S.I.) equal to or greater than two times the background level is considered "positive".
  • Preferred peptides of this invention comprise at least one T cell epitope and have a mean T cell stimulation index of greater than or equal to 2.0.
  • a peptide having a mean T cell stimulation index of greater than or equal to 2.0 is considered useful as a therapeutic agent.
  • Cryj II peptides Cry j II A and Cry j IIB have mean stimulation indexes of at least two and therefore comprise at least one T cell epitope as predicted.
  • 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, 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.
  • a diminution in symptoms includes any reduction in allergic response of an individual to the allergen after the individual has completed a treatment regimen with a peptide or protein of the invention. This diminution may be subjective (i.e. the patient feels more comfortable in the presence of the allergen). Diminution in symptoms can be determined clinically as well, using standard skin tests as is known in the art.
  • the purified Cryj II 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
  • Exposure of allergic individuals 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 isolated Cryj II protein, and fragments or portions derived therefrom can be used in methods of diagnosing, treating and preventing allergic reactions to
  • the present invention provides therapeutic compositions comprising purified Japanese cedar pollen allergen Cryj II or at least one fragment thereof produced in a host cell transformed to express Cryj II 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 II 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.
  • Cryj II 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.
  • compositions include saline and aqueous buffer solutions.
  • Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al. (1981) Int. Arch. Allergy Appl. Immunol. 64:84-99) and liposomes (Strejan et al. (1984) J. Neuroimmunol 7: 27).
  • 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 Cryj II protein or fragment thereof to elicit an antigenic response in the individual.
  • the Cryj II 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 Cryj II 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. Alternatively, high stringency conditions can be used.
  • 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 Cryj II, and thus to identify allergens in other species.
  • the present invention includes not only Cryj II, but also other allergens encoded by DNA which hybridizes to DNA of the present invention.
  • the invention further includes previously unidentified isolated allergenic proteins or fragments thereof that are immunologically related to Cryj II 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.
  • 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 II allergen, modify T-cell response to Cryj II allergen or modify both B-cell and T-cell 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 anaphy lactic 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 anaphy lactic 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 II), 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).
  • a peptide can be modified so that it maintains the ability to induce T cell anergy and bind MHC proteins without the ability to induce a strong proliferative response or possibly any proliferative response when administered in immunogenic form.
  • critical binding residues for the T cell receptor can be determined using known techniques (e.g., substitution of each residue and determination of the presence or absence of T cell reactivity).
  • residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance, diminish but not eliminate binding to relevant MHC.
  • peptides of the invention can be modified by replacing an amino acid shown to be essential to interact with the MHC protein complex with another, preferably similar amino acid residue (conservative substitution) whose presence is shown to enhance, diminish but not eliminate or not effect T cell activity.
  • amino acid residues which are not essential for interaction with the MHC protein complex but which still bind the MHC protein complex can be modified by being replaced by another amino acid whose incorporation may enhance, not effect, or diminish but not eliminate T cell reactivity.
  • Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with alanine, glutamic acid, or a methyl amino acid.
  • 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 gly col (PEG) method of A. Sehon and co-workers (Wie et al. supra) to produce a protein or peptide conjugated with PEG.
  • PEG polyethylene gly col
  • 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 (1986)); acylation (Tarr, supra): chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980); U.S. Patent
  • 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 recombinantly or synthetically engineered between regions, each comprising at least one T cell epitope.
  • charged amino acid pairs such as KK or RR, can be introduced between regions within a peptide during recombinant construction of the peptide.
  • 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
  • Cryj II 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 Cryj II, 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. It is now also possible to design 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.
  • Such agents could be designed, for example, in such a manner that they would bind to relevant anti- Cry j II Ig ⁇ s, thus preventing Ig ⁇ -allergen binding and subsequent mast cell degranulation.
  • such 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.
  • 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 Cryj II, or isolated Cryj II 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 detennining the extent to which such binding occurs.
  • components in the blood e.g., antibodies, T-cells, B-cells
  • RAST radio-allergosorbent test
  • PRIST paper radioimmunosorbent test
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassays
  • IRMA immuno-radiometric assays
  • LIA luminescence immunoassays
  • the presence in individuals of IgE specific for Cryj II at least one protein allergen and the ability of T cells of the individuals to respond to T cell epitope(s) of Cryj II protein allergen can be determined by administering to the individuals an Immediate Type Hypersensitivity test and a Delayed Type Hypersensitivity test.
  • the individuals are administered an Immediate Type Hypersensitivity test (see e.g. Immunology (1985) Roitt, I.M., Brostoff, J., Male, D.K. (eds), CN. Mosby Co., Gower Medical Publishing, London, ⁇ Y, pp. 19.2-19.18; pp.
  • the Delayed Type Hypersensitivity test would be given to those individuals exhibiting a specific Immediate Type Hypersensitivity reaction.
  • the Delayed Type Hypersensitivity test utilizes a modified form of the protein allergen or a portion thereof, the protein allergen produced recombinantly, or a recombitope peptide derived from the protein allergen, each of which has human T cell stimulating activity and each of which does not bind IgE specific for the allergen in a substantial percentage of the population of individuals sensitive to the allergen (e.g., at least about 75%).
  • the therapeutic composition comprises the modified form of the protein or portion thereof, the recombinantly produced protein allergen, or the recombitope peptide, each as used in the Delayed Type Hypersensitivity test, and a pharmaceutically acceptable carrier or diluent.
  • the present invention also provides a method of producing Cryj II or fragment thereof comprising culturing a host cell containing an expression vector which contains DNA encoding all or at least one fragment of Cryj II under conditions appropriate for expression of Cryj II or at least one fragment.
  • the expressed product is then recovered, using known techniques.
  • Cryj II 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 oligonucleotide sequence is one which is 1) a sequence capable of hybridizing to a complementary oligonucleotide to which the sequence (or corresponding sequence portions) of Cryj II or fragments thereof hybridizes, or 2) the sequence (or corresponding sequence portion) complementary to Cryj II, 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 Cryj II.
  • 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 II allergen, it need only meet the third criterion).
  • the defatted pollen was extracted at 4°C overnight in 2L extraction buffer l ⁇
  • soybean trypsin inhibitor (2 ⁇ g/mL), leupeptin (1 ⁇ g/mL), pepstatin A (1 ⁇ g/mL) and phenyl methyl sulfonyl fluoride (0.17 mg/mL).
  • the insoluble material was re-extrated with 1.2L 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 resulting pellet was resuspended in 0.4 L of 50 mM Na- acetate, pH 5.0 containing protease inhibitors and was dialyzed extensively against the same buffer.
  • the sample was further subjected to purification by either one of the two methods described below.
  • the sample was applied to a 100 mL DEAE cellulose column (Whatman DE- 52) equilibrated at 4°C with 50 mM Na-acetate, pH 5.0 with protease inhibitors.
  • the unbound material (basic proteins) from the DEAE cellulose column was then applied to a 50 ml cation exchange column (Whatman CM-52) which was equilibrated with 10 mM Na-acetate, pH 5.0 at 4°C with protease inhibitors.
  • a linear gradient of 0-0.3 M NaCl was used to elute the proteins.
  • the early fractions were enriched in Cryj I whereas the later fractions were enriched in Cryj II.
  • the sample was applied to FPLC Superdex 75 16/60 column (Pharmacia, Piscataway, NJ) in 10 mM acetate buffer, pH 5.0 and 0.15 M NaCl at a flow rate of
  • the dialyzed sample from the ammonium sulfate precipitation was applied at 1 ml/min to an 5.0 ml Q-Sepharose Econapac anion exchange cartridge (BioRad,
  • FPLC gel filtration was performed using a 320 mL Superdex 75 26/60 (Pharmacia, Piscataway, NJ) column at 0.5 ml/min in 20 mM sodium acetate, pH
  • Cryj II The physiochemical properties of Cryj II were studied and summarized as below. Under non-reducing SDS-PAGE conditions Cryj II consists of two bands with molecular weights ranged 34000-32000. The molecular weights of both bands are shifted higher to about 38-36 kD under reducing conditions (Fig. lb). This shift in SDS-polyacrylamide gel has also been observed by others (Sakaguchi et al, -4//ergy45:309-312 (1990)). These results suggest that intra-disulfide bonds are probably present in the protein, and it is supported by the present findings that cloned Cryj II contains 20 cysteines deduced from the nucleotide sequence (Example 3). The pi of Cryj II estimated from IEF gel is about 10. The purified Cryj II binds human IgE of some allergic patients.
  • the two molecular weight bands of Cryj II were separated on a 12% SDS- polyacrylamide gel and was then electroblotted onto PNDF membrane (Applied Biosystems, Foster City, CA). The blot was stained with coomassie brilliant blue and was cut and subjected to ⁇ -terminal amino acid sequencing. (Example 2). The results showed that the upper and lower molecular weight bands had identical ⁇ - terminal sequences except the lower molecular weight band missed the first five amino acids.
  • the estimated molecular weight of the upper band based on the cD ⁇ A sequence is about 52,000, which is significantly higher than the molecular weight estimated from SDS-polyacrylamide gel either in the presence or absence of reducing reagent.
  • the two N-terminal sequences obtained from the purified Cryj II also contained the N-terminal sequence (10 amino acid) published by Sakaguchi et al ⁇ Allergy, 45:309-312(1990)) suggesting that the N-terminal of Cryj II is probably hydrolyzed. Since Sakaguchi et al. (supra), did not use any protease inhibitors in their purification, a higher degree of hydrolysis might have occurred.
  • Another approach which may be used to purify native Cryj II or recombinant Cryj II is immunoaffinity chromatography. This technique provides a very selective protein purification due to the specificity of the interaction between monoclonal antibodies and antigen. Murine polyclonal and monoclonal antibodies are generated against purified Cryj II. These antibodies are used for purification, characterization, analysis and diagnosis of the allergen Cry j II.
  • Cryj II protein was isolated as in Example 1.
  • the doublet band shown on SDS-PAGE (Fig. la) was electroblotted onto ProBlott (Applied Biosystems, Foster City, CA).
  • Sequencing was performed with the Beckman/Porton Microsequencer (model LF3000, Beckman Instruments, Carlsbad, CA), a Programmable Solvent Module (Beckman System Gold Model 126, Beckman Instuments, Carlsbad, CA) and a Diode Array Detector Module for PTH-amino acid detection (Beckman System
  • both the long and short forms of Cry j II contained the ten amino acids, NH 2 -AINIFNVEKY-COOH (SEQ ID NO: 6), previously described for Cryj II (Sakaguchi et al. 1990, supra).
  • the previously published ten amino acids correspond to amino acids ten through 19 of the long form described above.
  • DEPC diethyl pyrocarbonate
  • RNA was precipitated from the aqueous phase with 0.1 volume 3M sodium acetate and 2 volumes ethanol. The pellets were recovered by centrifugation, resuspended in 2 ml dH2 ⁇ and heated to 65 °C for 5 minutes. Two ml 4M lithium chloride was added to the preparation and the RNA was precipitated overnight at 0°C. The RNA pellets were recovered by centrifugation, resuspended in 1 ml dH2 ⁇ , and again precipitated with 3M sodium acetate and ethanol on dry ice for one hour.
  • Double stranded cDNA was synthesized from 4 ⁇ g pollen RNA or 8 ⁇ g flowerhead RNA using a commercially available kit (cDNA Synthesis System kit, BRL, Gaithersburg, MD).
  • the double-stranded cDNA was phenol extracted, ethanol precipitated, blunted with T4 DNA polymerase (Promega, Madison, WI), and then ligated to ethanol precipitated, self annealed, AT and AL oligonucleotides for use in a modified Anchored PCR reaction, according to the method of Rafnar et al.
  • Oligonucleotide AT has the sequence (SEQ ID NO: 10) 5 ' -GGGTCTAGAGGTACCG-TCCGTCCGATCGATC ATT-3 ' (Rafnar et al. supra).
  • Oligonucleotide AL has the sequence (SEQ ID NO: 11)
  • CP-11 has the sequence (SEQ ID NO: 12) 5'-ATACTTCTCIACGTTGAA-3' , wherein A at positon 1 can be G, C at position 4 can be T, C at position 7 can be T, I at position 10 is inosine to reduce degeneracy (Knoth et al. (1988) Nucleic Acids Res. 16: 10932), G at position 13 can be A, and G at position 16 can be A).
  • AP which has the sequence (SEQ ID NO: 13) 5'-GGGTCTAGAGGTA-CCGTCCG-3' , corresponds to nucleotides 1 through 20 of the oligonucleotide AT.
  • CP-11 is the degenerate oligonucleotide sequence that is complementary to the coding strand sequence substantially encoding amino acids PheAsnValGluLysTyr (SEQ ID NO: 14) (amino acids 59 to 64 of Fig. 4), which correspond to the carboxy terminus of the previously published Cry j II sequence (Sakaguchi et al. , supra) shown in Fig. 4. All oligonucleotides were synthesized by Research Genetics Inc., Huntsville, AL.
  • PCR Polymerase chain reactions
  • a commercially available kit GeneAmp DNA Amplification kit, Perkin Elmer Cetus, Norwalk, CT
  • 10 ⁇ l lOx buffer containing dNTPs was mixed with 100 pmoles of each oligonucleotide, 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.
  • 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 min, annealing of primers to the template at 45 °C for 1 min, and chain elongation at 72 °C for 1 min.
  • the final 20 rounds of amplification consisted of denaturation as above, annealing at 55 °C for 1 min, and elongation as above.
  • the primary PCR reaction was carried out with 100 pmol each of the oligonucleotides AP and CP-11. Five percent (5 ⁇ l) of this initial amplification was then used in a secondary amplification with 100 pmoles each of AP and CP-12.
  • CP- 12 has the sequence (SEQ ID NO: 15) 5'-CCTGCAGTACTTCT- CIACGTTGAAIAT-3' , wherein C at position 10 can be T, C at position 13 can be T, I at positions 16 and 25 are inosines to reduce degeneracy as above, G at position 19 can be A, and G at position 22 can be A.
  • CCTGCAG-3' (bases 1 through 7 of CP-12) represents a Pst I site added for cloning purposes; the remaining degenerate oligonucleotide sequence is complementary to the coding strand sequence that substantially encodes the amino acids IlePheAsnValGluLysTyr (SEQ ID NO: 17) (amino acids 58-64 of Fig. 4). Amplified DNA was recovered by sequential chloroform, phenol, and chloroform extractions, followed by precipitation on dry ice with 0.5 volumes of 7.5M ammonium acetate and 1.5 volumes of isopropanol.
  • the DNA was simultaneously digested with Xba I and Pst I in a 50 ⁇ l reaction, precipitated to reduce the volume to 10 ⁇ l, and electrophoresed through a preparative 2% GTG NuSeive low melt gel (FMC, Rockport, ME).
  • FMC FastG NuSeive low melt gel
  • the appropriate sized DNA area was visualized by ethidium bromide (EtBr) staining, excised, and ligated into appropriately digested pUC19 for sequencing by the dideoxy chain termination method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5476) using a commercially available sequencing kit (Sequenase kit, U.S.
  • CP-21 has the sequence (SEQ ID NO: 18) 5'-CCTGCAGTACTTCTCIACGTTGAAGAT-3 * wherein C at position 10 can be T, C at position 13 can be T, I at position 16 is inosine to reduce degeneracy as above, G at position 19 can be A, G at position 22 can be A, and G at position 25 can be A or T.
  • sequence (SEQ ID NO: 16) 5'-CCTGCAG-3 * (bases 1 through 7 of CP-21) represent a Pst I site added for cloning purposes; the remaining degenerate oligonucleotide sequence is the non-coding strand sequence corresponding to coding strand sequence substantially encoding amino acids IlePheAsnNalGluLysTyr (SEQ ID NO: 17) (amino acids 58 to 64 of Fig. 4).
  • a primary PCR was also performed on double-stranded, linkered cDNA using CP-23D and AP, as above, to attempt to amplify the 3' end of the Cryj II cDNA.
  • a secondary PCR was performed using 5 % of the primary reaction, using CP-24D and AP.
  • CP-23D sequence (SEQ ID NO: 19) 5'- GCIATTAATATTTTTAA-3' , wherein the T at position 6 can be C or A, T at position 9 can be C, T at position 12 can be C or A, and T at position 15 can be C
  • CP-23D sequence (SEQ ID NO: 19) 5'- GCIATTAATATTTTTAA-3' , wherein the T at position 6 can be C or A, T at position 9 can be C, T at position 12 can be C or A, and T at position 15 can be C
  • CP-23D sequence (SEQ ID NO: 19) 5'- GCIATTAATATTTTTAA-3' , where
  • oligonucleotide sequence of CP-24D substantially encodes amino acids AlalleAsnllePheAsnVal (SEQ ID NO: 23) (amino acids 55 to 61 of Fig. 4). Again, multiple clones were sequenced, none of which could be identified as Cryj II, and this approach was not pursued further.
  • CP-35 has the sequence (SEQ ID NO: 24) 5'-GCTTCGGTACAATCATGTTT-3' , wherein T at position 3 can also be C; G at position 6 can also be A, T or C; A at position 9 can also be G; A at position 12 can also be G; A at position 15 can be G; and T at position 18 can also be C; this degenerate oligonucleotide sequence is the non-coding strand sequence corresponding to coding strand sequence substantially encoding amino acids LysHisAspCysThrGluAla of Cry j II (SEQ ID NO: 25) (amino acids 71 to 77 of Fig. 4).
  • JC136 Five percent (5 ⁇ l) of this initial amplification, designated JC136, was then used in a secondary amplification with 100 pmoles each of AP and degenerate Cryj II primer CP-36, an internally nested Cryj II oligonucleotide primer with the sequence (SEQ ID NO: 26) 5'- GGCTGCAGGTACAATCATGTTTGCCATC-3' wherein A at position 11 can also be G; A at position 14 can also be G; A at position 17 can also be G; T at position 20 can also be C; G at position 23 can also be A, T, or C; and A at position 26 can also be G.
  • SEQ ID NO: 26 an internally nested Cryj II oligonucleotide primer with the sequence (SEQ ID NO: 26) 5'- GGCTGCAGGTACAATCATGTTTGCCATC-3' wherein A at position 11 can also be G; A at position 14 can also be G; A at position 17 can also be G; T at position
  • the nucleotides 5'-GGCTGCAG-3' represent a Pst I restriction site added for cloning purposes.
  • the remaining degenerate oligonucleotide sequence of CP-36 is the non-coding strand sequence corresponding to coding strand sequence substantially encoding amino acids AspGlyLysHisAspCysThr of Cryj II (SEQ ID NO: 28) (amino acids 69 to 75 of Fig. 4).
  • the dominant amplified product, designated JC137 was a DNA band of approximately 265 base pairs, as visualized on an EtBr-stained 2% GTG agarose gel.
  • 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 2% GTG SeaPlaque low melt gel (FMC, Rockport, ME). The appropriate sized DNA band was visualized by EtBr staining, excised, and ligated into appropriately digested pUC19 for sequencing by the dideoxy chain termination method (Sanger et al. (1977) Proc. Natl Acad Sci. USA 74: 5463-5476) using a commercially available sequencing kit (Sequenase kit, U.S. Biochemicals, Cleveland, OH).
  • the clones designated pUC19JC137a, pUC19JC137b, and pUC19JC137e were found to contain sequences encoding the amino terminus of Cryj II. All three clones had identical sequence in their regions of overlap, although all three clones had different lengths in the 5' untranslated region. Clone pUC19JC137b was the longest clone. The translated sequence of these clones had complete identity to the disclosed 10 amino acid sequence of Cryj II (Sakaguchi et al., supra.), as well as to the Cryj II amino acid sequence described in Example 2. Amino acid numbering is based on the sequence of the full length protein; amino acid 1 corresponds to the initiating memionine (Met) of Cryj II.
  • the position of the initiating Met was supported by the presence of an upstream in-frame-stop codon and by 78% homology of the surrounding nucleotide sequence with the plant consensus sequence that encompasses the initiating Met, as reported by Lutcke et al. (1987) EMBO J. 6:43-48.
  • cDNA encoding the remainder of Cryj II gene was cloned from the linkered cDNA by using oligonucleotides CP-37 (SEQ ID NO: 29) (which has the sequence 5'-ATGTTGGACAGTGTTGTCGAA-3') and AP in a primary PCR, designated JC138U.
  • Oligonucleotide CP-37 corresponds to nucleotides 129 to 149 of *
  • Fig. 4 is based on the nucleotide sequence determined for the partial Cryj II clone pUC19JC137b.
  • a secondary PCR reaction was performed on 5% of the initial amplification mixture, with 100 pmoles each of AP and CP-38 (SEQ ID NO: 30) (which has the sequence 5'-GGGAATTCAGAAAAGTTGAGCATTCTCGT-3'), the nested primer.
  • the nucleotide sequence (SEQ ID NO: 31) 5'-GGGAATTC-3' (bases 1 through 8 of CP-38) represents an Eco RI restriction site added for cloning purposes.
  • the remaining oligonucleotide sequence corresponds to nucleotides 177 to 197 of Fig. 4, and is based on the nucleotide sequence determined for the partial Cryj II clone pUC19JC137b.
  • the amplified DNA product, designated JC140iii was purified and precipitated as above, followed by digestion with Eco RI and Asp 718 and electrophoresis through a preparative 1 % low melt gel.
  • DNA was sequenced by the dideoxy chain termination method (Sanger et al. supra) using a commercially available kit (sequenase kit (U.S. Biochemicals,
  • CP-41 (SEQ ID NO: 33) has the sequence 5'- GTGTTAGGACT- GTCTCTCGG-3', which is the non-coding strand sequence that corresponds to nucleotides 720 to 739 of Fig. 4.
  • CP-42 (SEQ ID NO: 35) has the sequence 5'-TGTCCAGGCCAT-GGAATAAG-3', which corresponds to nucleotides 864 to 883 of Fig. 4 except that the first nucleotide was synthesized as a T rather than the correct G.
  • CP-43 has the sequence (SEQ ID NO: 35) 5'-
  • CGCTTACATGGACTGCAACC-3' which is the non-coding strand sequence that corresponds to nucleotides 1476 to 1495 of Fig. 4.
  • CP-44 has the sequence (SEQ ID NO: 36) 5'-TCCACGGGTCTGATAATCCA-3 ⁇ which corresponds to nucleotides 612 to 631 of Fig. 4.
  • CP-45 has the sequence (SEQ ID NO: 37)
  • CP-46 has the sequence (SEQ ID NO: 38) 5'-TACTGCACTTCAGCT-TCTGC-3', which corresponds to nucleotides 1077 to 1096 of Fig. 4.
  • CP-47 has the sequence (SEQ ID NO: 39) 5 ' -GGGGGTCTCCG AATTTATC A-3 ' , which is the non-coding strand sequence that substantially corresponds to nucleotides 1039 to 1058 of Fig. 4, except that the fifth nucleotide of CP-47 was synthesized as a G rather than the correct nucleotide, T.
  • CP-48 (SEQ ID NO: 40), which has the sequence 5'-
  • GGATATTTCAGTGGACACGT-3' corresponds to nucleotides 1290 to 1309 of Fig. 4.
  • CP-49 (SEQ ID NO: 41) has the sequence 5'-TATTAGAAGACC- CTGTGCCT-3', which is the non-coding strand sequence that corresponds to nucleotides 821 to 840 of Fig. 4.
  • CP-50 (SEQ ID NO: 42) has the sequence
  • CP-51 (SEQ ID NO: 43) has the sequence
  • 5'-ACACCTTTACCCATTAGAGT-3' which is the non-coding strand sequence that corresponds to nucleotides 486 to 505 of Fig. 4.
  • the sequence of clone pUC19JC140iiid was chosen as the consensus sequence since it had the longest 3' untranslated region.
  • the sequences of pUC19JC140iiid and pUC19JC137b were used to construct the composite Cryj II sequence shown in Fig. 4. In this composite, nucleotide 230 is reported as the A found in pUC19JC137b
  • Clone pUC19JC140-2a has C for nucloeotide 297 instead of T (changes amino acid 86 from Cys to Arg) and clone pUC19JC140-2b has G for nucleotide 753 instead of A (changes amino acid 238 from He to Val). Both clone pUC19JC140-2a and clone pUC19JC140-2b have a T at nucleotide 357 in place of C (no predicted change in amino acid 106). Two different PCR amplifications were also sequenced directly to verify the clonal Cryj II sequence using the Amplitaq Cycle Sequencing kit (Perkin Elmer Cetus, Norwalk, CT).
  • This procedure involves the [32p] -end-labelling of oligonucleotide sequencing primers which are then annealled (1.6 pmoles in 1 ⁇ l) to template DNA and elongated with dideoxy NTPs (methodology of Sanger et al.
  • the template DNA was a PCR product that was recovered by sequential chloroform, phenol, and chloroform extractions, precipitated at -20°C with 0.5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol, then electrophoresed through a preparative 1 or 2% SeaPlaque low melt gel (FMC). Appropriate sized DNA bands were visualized by EtBr staining, excised, and treated with Gelase (Epicentre Technologies, Madison,
  • JC137U the 5' end PCR, (amplified from the 1° PCR JC136 above) was reamplified with oligonucleotides AP and CP-36
  • JC140U the 3' end PCR, (amplified from the 1 ° PCR JC138U above) was reamplified with oligonucleotides AP and CP-38.
  • CTGTCCAACATAATTTGGGC-3' is the non-coding strand sequence corresponding to nucleotides 120 to 139 of Fig. 4.
  • the oligonucleotide primers used for sequencing JC140U were CP-38, CP-40, CP-41, CP-42, CP-43, CP-44, CP-45, CP-46, CP-47, CP-49, CP-50, CP-54 (SEQ ID NO: 45), which has the sequence 5'- CATGGCAGGGTGGTTCAGGC-3 ' , corresponds to nucleotides 985 to 1004 of Fig.
  • Figs. 4 and 5 The nucleotide and predicted amino acid sequences of Cryj II are shown in Figs. 4 and 5. This is a composite nucleotide sequence from the two overlapping clones pUC19JC137b and pUC19JC140iiid. Sequencing of multiple independent clones and cycle sequencing of PCR product confirmed the nucleotide sequence of Figure 4. There were several nucleotide changes resulting in predicted amino acid changes, as cited above. However, all nucleotide polymorphisms, with the exception of the T for C substitition at nucleotide 357, were only observed in single clones or sequencing reactions. Although T was seen at nucleotide 357 in all clones except pUC19JC140iiid, both C and T encode Leu at amino acid 106.
  • the complete cDNA sequence for Cryj H is composed of 1726 nucleotides, including 41 nucleotides of 5' untranslated sequence, an open reading frame of 1542 nucleotides starting with the codon for an initiating Met (nucleotides 42-44 of Fig.
  • Sakaguchi et al., supra, as shown in Fig. 6) may suggest that the amino terminus of the mature Cry j II protein is blocked and that the sequences obtained by sequence analysis of purified protein represent proteolytic cleavage products.
  • the amino acid sequence of the long form of Cryj II begins at amino acid 46 and the amino acid sequence of the short form of Cryj II begins at amino acid 51 ; and the NH2-terminal sequence determed by Sakaguchi et al. begins at amino acid 54.
  • amino acids 1 to 45 represent the leader/pre-pro position of Cryj II that is enzymatically cleaved to give a functionally active protein beginning at amino acid 46 of Fig. 4.
  • sequences beginning at amino acids 51 and 54 represent breakdown products of the protein beginning at amino acid 46. There is a predicted cleavage site between amino acids 22 and 23 of Fig. 4 using the method of von Heijne (Nucleic Acids Res. (1986) 14:4683-4690). If the mature Cry j II protein started at amino acid 23 in Fig. 4, the protein would be 492 amino acids long with a predicted molecular weight of 54.2 kDa and a predicted pi of 9.0.
  • Cryj II is 43.3% homologous (33.3% identical to polygalacturonase of tomato (Lycopersicon esculentum) and 48.4% homologous (32.6% identical) to polygalacturonase of corn, Zea mays. All nucleotide and amino acid sequence analyses were performed using PCGENE (Intelligenetics, Mountain View, CA.).
  • 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 2 ml dH2 ⁇ 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 0°C.
  • the RNA pellets were recovered by centrifugation, resuspended in 1 ml dH2 ⁇ , 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.
  • 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.
  • PCRs were carried out using the Gene Amp DNA Amplification kit (Perkin Elmer Cetus) whereby 10 ⁇ l lOx buffer containing dNTPs was mixed with 100 pmol each of a sense oligonucleotide and an anti-sense oligonucleotide, cDNA (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 min, annealing of primers to the template at 45°C for 1 min, and chain elongation at 72°C for 1 min.
  • the final 20 rounds of amplification consisted of denaturation as above, annealing at 55°C for 1 min, and elongation as above.
  • CP-52 (SEQ ID NO: 50) has the sequence 5'- GCCGAATTCATGGCCATGAAATTAATT-3 ' where the nucleotide sequence 5'-GCCGAATTC-3' (SEQ ID NO: 51) (bases 1 through 9 of CP-52 represents an Eco RI restriction site added for cloning purposes, and the remaining sequence corresponds to nucleotides 42 to 59 of Fig. 4.
  • CP-53 (SEQ ID NO: 52) has the sequence 5'-CGGGGATCCTCATTATGGATG-GTAGAT-3' where the nucleotide sequence 5'-CGGGGATCC-3' (SEQ ID NO: 53) (bases 1 through 9 of CP-53 represents a Bam HI restriction site added for cloning purposes, and the remaining oligonucleotide sequence of CP-53 is complementary to coding strand sequence corresponding to nucleotides 1572 to 1589 of Fig. 4.
  • FMC preparative 1 % SeaPlaque low melt gel
  • Clones pUC19JC145a and pUC19JC145b were completely sequenced using M13 forward and reverse primers (N.E. Biolabs, Beverly, MA) and internal sequencing primers CP-41, CP-42, CP-44, CP-46, and CP-51.
  • the nucleotide and deduced amino acid sequences of clones pUC19JC145a and pUC19JC145b were identical to the Cryj II sequence of Fig. 4, with the following exceptions.
  • Clone pUC19JC145a was found to contain a single nucleotide difference from the previously known Cryj II sequence: it has a C at nucleotide position 1234 of Fig. 4 rather than the previously described T.
  • This nucleotide change results in a predicted amino acid change from He to Thr at amino acid 398 of the Cryj II protein.
  • Clone pUC19JC145b has a G at nucleotide position 1088 of Fig. 4 rather than the previously described A, and an A for a G at nucleotide 1339.
  • the nucleotide change at 1088 is silent and does not result in a predicted amino acid change.
  • the nucleotide change at position 1339 results in a predicted amino acid change from Ser to Asn at amino acid 433 of the Cryj II protein.
  • Cryj II Expression of Cryj II was performed as follows. Ten ⁇ g of pUC19JC145b was digested simultaneously with Eco RI and Bam HI. The nucleotide insert encoding Cryj II (extending from nucleotide 42 through 1589 of Fig. 4) 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-lld (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
  • a recombinant clone was used to transform Escherichia coli strain BL21-DE3, which harbors a plasmid that has an isopropyl- ⁇ - 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-1 Id.
  • IPTG isopropyl- ⁇ - D-thiogalactopyranoside
  • media Brain Heart Infusion Media, Difco
  • a negative control consisted of crude lysate from uninduced bacteria containing the plasmid with Cryj II. There was no notable increase in production of any recombinant E. coli protein in the range of 58 Kd, the size predicted for the recombinant Cryj II with the His6 leader.
  • the pET-1 ld ⁇ HRhis6JC145b.a clone was then grown on a larger scale to examine if there was any recombinant protein being expressed.
  • a 2 ml culture of bacteria containing the recombinant plasmid was grown for 8 hr, then 3 ⁇ l was spread onto each of 6 (100 x 15 mm) petri plates with 1.5% agarose in LB medium (Gibco-BRL, Gaithersburg, MD) containing 200 ⁇ g/ml ampicillin, grown to confluence overnight, then scraped into 6 L of liquid media (Brain Heart Infusion media, Difco) containing ampicillin (200 ⁇ g/ml).
  • the culture was grown until the absorbance at A600 was 1.0, IPTG added (1 mM final concentration), and the culture grown for an additional 2 hours.
  • Bacteria were recovered by centrifugation (7,930 xg, 10 min) and lysed in 50 ml of 6M Guanidine-HCl, 0.1M Na2HPO4, pH 8.0, for 1 hour with vigorous shaking. Insoluble material was removed by centrifugation (11,000 xg, 10 min, 4° C). The pH of the lysate was adjusted to pH 8.0, and the lysate applied to a 50 ml
  • Nickel NTA agarose column (Qiagen) that had been equilibrated with 6 M Guanidine HC1, 100 mM Na2HPO4, pH 8.0.
  • the column was sequentially washed with 6 M Guanidine HC1, 100 mM Na2HPO4, 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 had an A280_ ⁇ - 0-05.
  • the recombinant Cryj II protein 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-
  • the 24 kDa band accounts for about 90% of the total protein and may represent a degradation product of the recombinant Cryj II or an E. coli contaminant.
  • Another Cryy II expression construct was made by the ligation of the pUC19JC140iiid Cryj II insert into appropriately digested pETl ld ⁇ HR (with the 6 histidine leader).
  • the vector was derived from another pETlld ⁇ HR construct whose insert supplied an EcoR I site (at the 5' pETlld ⁇ HR-insert junction) and an Asp 718 site (at the 3' end of the insert); the construct was digested with these two enzymes, run on a low melt minigel as above, and the vector recovered as a band in low melt agarose.
  • the pUC19JC140iiid construct was digested with Eco R I and
  • RNA samples (first precipitated with 1/10 volume sodium acetate, 2 volumes ethanol to reduce volume and resuspended in 5.5 ⁇ l dH2O) were run with 10 ⁇ l formaldehyde/formamide buffer containing loading dyes with 15.5% formaldehyde, 42% formamide, and 1.3X MOPS solution, final concentration.
  • the samples were transferred to Genescreen Plus (NEN Research Products, Boston, MA) by capillary transfer in 10X
  • 2 ⁇ l dNTP mix (0.167mM dATP, 0.167mM dTTP, 0.167mM dGTP, and 0.033mM dCTP), 2 ⁇ l 10X PCR buffer, 10 ⁇ l 32 P-dCTP (100 ⁇ Ci; Amersham, Arlington Heights, II), 1 ⁇ l (100 pmoles) antisense primer CP-53, 0.5 ⁇ l Taq polymerase, and dH2O to 20 ⁇ l; the 10X PCR buffer, dNTPs and Taq polymerase were from Perkin Elmer Cetus (Norwalk, CT).
  • Amplification consisted of 30 rounds of denaturation at 94°C for 45 sec, annealing of primer to the template at 60°C for 45 sec, and chain elongation at 72°C for 1 min.
  • the reaction was stopped by addition of 100 ⁇ l TE, and the probe recovered over a 3cc G-50 spin column (2 ml G-50 Sephadex [Pharmacia, Uppsala, Sweden] in a 3cc syringe plugged with glass wool, equilibrated with TE) and counted on a 1500 TriCarb Liquid Scintillation Counter (Packard, Downers Grove, IL).
  • the probe was added to the prehybridizing buffer at 10 6 cpm/ml and hybridization was carried out at 60°C for 16 hrs.
  • the blot was washed in high stringency conditions: 3x15 min at 65°C with 0.2%SSC/1 % SDS, followed by wrapping in plastic wrap and exposure to film at -80°C.
  • a seven hour exposure of this Northern blot analysis revealed a single thick band at approximately 1.7 kb for both RNA collected from the Arboretum tree and the RNA collected from the pooled trees from Japan. This message is the expected size for Cryj II as predicted by PCR analysis of the cDNA.
  • the human plasma were serially diluted in PBS-Tween at a starting dilution of 1:2.
  • PBS-Tween For this set 23 plasma samples from patients symptomatic for Japanese cedar pollen allergy chosen for IgE binding analysis. The first antibody incubation proceeded overnight at 4°C. Following three washes with PBS-Tween the second antibodies were added (goat anti-mouse Ig or goat anti-human IgE both at 1:2000) and incubated for two hours at room temperature at 100 ⁇ L/well. This solution was removed and streptavidin-HRPO diluted to 1:10,000, was added at lOO ⁇ L/well. The color was allowed to develop for 2-5 minutes. The reaction was stopped by the addition of lOO ⁇ L/well of 1M phosphoric acid.
  • Fig. 7 the binding response of the monoclonal antibody, 4B11, and seven patients' (Batch 1) plasma IgE is shown to purified Cr y I as the coating antigen.
  • the monoclonal antibody, raised against purified Cr y I shows a saturating level of binding for the whole dilution series.
  • the individual patient samples show a variable response of IgE binding to the Cr y I preparation.
  • One patient, #1034, has no detectable binding to this protein preparation. All the patient samples were obtained from individuals claiming to be symptomatic for Japanese cedar pollen allergy and the results of their MAST scores are shown in Fig. 16.
  • Fig. 8 is a graph representing the binding of the same antibody set as in Fig. 7 to purified native Cry j II.
  • the anti- Cryj I monoclonal antibody, 4B11 is negative on this preparation demonstrating lack of cross-reactivity between the two allergen antigens. In general, there is a lower overall response to this allergenic component of cedar pollen with more patient samples showing decreased binding. However, patient #1034, that was negative on Cryy I shows very strong reactivity to Cryj II. In the last antigen set, Fig. 9, using recombinant Cryy II ( ⁇ Cryj II), monoclonal antibody 4B11 reactivity is negative and there is further reduction in binding of the human IgE samples compared to biochemically purified Cryy II.
  • Figs. 10-15 represent the application of the same antigen sets for the direct binding analysis of the next sixteen patients designated patient Batch 2 and patient Batch 3 in Figs. 10-15.
  • the table shown in Fig. 16 summarizes both the MAST scores, performed in Japan on the plasma samples before shipment using a commercially available kit, and the direct ELISA results outlined above.
  • Two patients were negative by the MAST assay, however, one of these patients, #1143, was positive on all the ELISA antigens.
  • the number of positive responses for each antigen is shown and this represents a measure relative allergenicity of the different allergen preparations.
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • T cell lines were established by stimulation of 2 X 10 ⁇ PBL/ml in bulk cultures of complete medium (RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5xlO" 5 M 2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heat inactivated human AB serum) with 10 ⁇ g/ml of partially purified native Cr y II for 7 days at 37°C in a humidified 5% CO2 incubator to select for Cry j II reactive T cells. This amount of priming antigen was determined to be optimal for the activation of T cells from most Japanese cedar pollen allergic patients.
  • Viable cells were purified by LSM centrifugation and cultured in complete medium supplemented with 5 units recombinant human IL-2/ml and 5 units recombinant human IL-4/ml for up to three weeks until the cells no longer responded to lymphokines and were considered "rested”.
  • the ability of the T cells to proliferate to peptides Cry j IIA and Cry j IIB, recombinant Cryy II ( ⁇ Cryj II), purified native Cr y * II, or purified native Cryj I was then assessed.
  • 2 X 10 4 rested cells were restimulated in the presence of 2 X 10 4 autologous Epstein- Barr virus (EBN)-transformed B cells (prepared as described below) (gamma- irradiated with 25,000 RADS) with 2-50 ⁇ g/ml of rCryy II, purified native Cryy II, peptides Cry j IIA and Cry j HB, of purified native Cryj I, in a volume of 200 ⁇ l complete medium in duplicate or triplicate wells in 96-well round bottom plates for 2-4 days. The optimal incubation was found to be 3 days. Each well then received 1 ⁇ Ci tritiated thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. The maximum response in a titration of each peptide is expressed as the stimulation index
  • the S.I. is the counts per minute (CPM) incorporated by cells in response to peptide, divided by the CPM incorporated by cells in medium only.
  • An S.I. value equal to or greater than 2 times the background level is considered "positive" and indicates that the peptide contains a T cell epitope.
  • the results of this assay indicated that peptides Crj II, and Cr y HB did noit appear to contain a T cell epitope for this particular allergenic patient. However, additional Japanese cedar pollen allergic patients will be tested in this assay system and one or both of these peptides may contain T cell epitopes for other allergic individuals.
  • EBN-transformed cell lines were made by incubating 5 X 10 6 PBL with 1 ml of B-59/8 Marmoset cell line (ATCC CRL1612, American Type Culture Collection, Rockville, MD) conditioned medium in the presence of 1 ⁇ g/ml phorbol 12-myristate 13-acetate (PMA) at 37°C for 60 minutes in 12 X 75 mm polypropylene round-bottom Falcon snap cap tubes (Becton Dickinson Labware, Lincoln Park, ⁇ J).
  • B-59/8 Marmoset cell line ATCC CRL1612, American Type Culture Collection, Rockville, MD
  • PMA phorbol 12-myristate 13-acetate
  • CTGTTCTTCA ATGGGCCATG TCAACCTCAC TTTACTTTTA AGGTAGATGG GATAATAGCT 240 GCGTACCAAA ATCCAGCGAG CTGGAAGAAT AATAGAATAT GGTTGCAGTT TGCTAAACTT 300
  • CCATGTCAAC CTCACTTTAC TTTTAAGGTA GATGGGATAA TAGCTGCGTA CCAAAATCCA 240
  • AACAGTCCCG AATTTCATTT AGTTTTTGGG AATTGTGAGG GAGTAAAAAT CATCGGCATT 600
  • AGTATTACGG CACCGAGAGA CAGTCCTAAC ACTGATGGAA TTGATATCTT TGCATCTAAA 660
  • CAAGATGTGA CATACAAGAA CATACGTGGG ACATCAGCAA
  • AAACACCCAA AAACTGTAAT GGTTGAAAAT ATGCGAGCAT ATGACAAGGG TAACAGAACA 1320
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:47: TTGGGGTCGA GGCCTCCGAA 20

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EP0655500B1 (de) * 1993-11-05 1999-05-12 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Allergenisches Polypeptid von japanischen Zederpollen und diese kodierende DNA
JP4176820B2 (ja) * 1993-11-05 2008-11-05 明治乳業株式会社 スギ花粉アレルゲンＣｒｙｊＩＩエピトープ
JP3649460B2 (ja) * 1993-11-05 2005-05-18 明治乳業株式会社 スギ花粉アレルゲンＣｒｙ ｊ ＩＩエピトープ
AU748104B2 (en) * 1994-09-02 2002-05-30 Merck Patent Gmbh Peptide compositions capable of down regulating an antigen specific immune response
EP0783322A1 (de) * 1994-09-02 1997-07-16 Immunologic Pharmaceutical Corporation Peptidzusammensetzungen mit der fähigkeitantigenspezifische immunantworten herunterregulieren zu können
US6759234B1 (en) 1994-09-02 2004-07-06 Immulogic Pharmaceutical Corporation Compositions and methods for administering to humans, peptides capable of down regulating an antigen specific immune response
EP0700929A3 (de) * 1994-09-10 1999-09-01 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Von Zederpollen-Allergene abgeleitete Peptide, sowie deren Verwendungen
EP0923940B1 (de) * 1996-03-10 2008-09-03 Meiji Dairies Corporation Auf peptiden basierendes immunotherapeutisches mittel gegen allergien
ES2314515T3 (es) 1996-03-21 2009-03-16 Circassia Limited Peptidos cripticos y metodo para su identificacion.
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
ATE360641T1 (de) 1996-06-14 2007-05-15 Meiji Dairies Corp Peptide aus t-zellepitopen
DE19957904A1 (de) * 1999-12-01 2001-06-07 Merck Patent Gmbh Insektengift-Allergene mit verminderter IgE-Reaktivität und Verfahren zu ihrer Herstellung
JP6041490B2 (ja) 2009-10-30 2016-12-07 日本製紙株式会社 スギ花粉の免疫原性を有するタンパク質、当該タンパク質をコードするポリヌクレオチド及びこれらの用途
LT2861240T (lt) 2012-06-15 2021-01-25 Immunomic Therapeutics, Inc. Nukleorūgštys skirtos alergijoms gydyti

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WO1992003551A1 (en) * 1990-08-13 1992-03-05 Biomay Biotechnik Produktions- Und Handelsgesellschaft M.B.H. Birch pollen allergen p14 for diagnosis and therapy of allergic diseases

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