+

WO1995006728A2 - Epitopes de lymphocyte t pour l'allergene du ray-grass - Google Patents

Epitopes de lymphocyte t pour l'allergene du ray-grass Download PDF

Info

Publication number
WO1995006728A2
WO1995006728A2 PCT/US1994/009024 US9409024W WO9506728A2 WO 1995006728 A2 WO1995006728 A2 WO 1995006728A2 US 9409024 W US9409024 W US 9409024W WO 9506728 A2 WO9506728 A2 WO 9506728A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
lpk
lpi
lpix
peptide
Prior art date
Application number
PCT/US1994/009024
Other languages
English (en)
Other versions
WO1995006728A3 (fr
Inventor
Irwin J. Griffith
Mei-Chang Kuo
Mohammad Luqman
Original Assignee
Immulogic Pharmaceutical Corporation
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 Corporation filed Critical Immulogic Pharmaceutical Corporation
Priority to JP7508127A priority Critical patent/JPH09504167A/ja
Priority to AU75597/94A priority patent/AU7559794A/en
Priority to US08/737,904 priority patent/US7112333B1/en
Priority to NZ271818A priority patent/NZ271818A/en
Priority to EP94925803A priority patent/EP0714440A1/fr
Publication of WO1995006728A2 publication Critical patent/WO1995006728A2/fr
Publication of WO1995006728A3 publication Critical patent/WO1995006728A3/fr
Priority to FI960629A priority patent/FI960629L/fi
Priority to NO960553A priority patent/NO960553L/no

Links

Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof 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

Definitions

  • Allergens constitute the most abundant proteins of grass pollen, which is the major cause of allergic disease in temperate climates (Marsh (1975) Allergens and the genetics of allergy; in M. Sela (ed.), The Antigens, Vol. 3, pp 271-359, Academic Press Inc., London, New York)., Hill et al. (1979) Medical Journal of Australia, 1:426-429).
  • the first descriptions of the allergenic proteins in ryegrass showed that they are immunochemically distinct, and are known as groups I, II, HI and IV (Johnson and Marsh (1965) Nature, 206:935-942; and Johnson and Marsh (1966) Immunochemistry, 3:91-100).
  • Lolp I which is also known as Lolp N or Lolp lb (Singh et al. (1991) Proc. Natl. Acad. Sci, USA, 88:1384-1388).
  • Lolp N is defined as an allergen because of its ability to bind to specific IgE in sera of ryegrass-sensitive patients, to act as an antigen in IgG responses and to trigger T- cell responses.
  • the allergenic properties have been demonstrated by immunoblotting studies showing 80% of ryegrass pollen sensitive patients possessed specific IgE antibody that bound to Lolp N isoforms (PCT application publication number WO 93/04174, page 65). These results indicate that Lolp W is a major ryegrass allergen.
  • the present invention provides isolated peptides of Lolp N.
  • Peptides within the scope of the invention comprise at least one T cell epitope, preferably at least two T cell epitopes of Lolp N.
  • the invention further provides peptides comprising at least two regions, each region comprising at least one T cell epitope of Lolp N.
  • the invention also provides modified peptides having similar or enhanced therapeutic properties as the corresponding, naturally-occurring allergen or portion thereof, but having reduced side effects, as well as modified peptides having improved properties such as increased solubility and stability.
  • Therapeutic peptides of the invention are capable of modifying, in a.
  • an allergen immunologically cross-reactive Lolp V e.g. allergens derived from pollen belonging to the Poacea (Graminae) family such as D ⁇ ctylis glomer ⁇ t ⁇ , D ⁇ c g N.
  • Methods of treatment or of diagnosis of sensitivity to ryegrass pollen protein, Lolp V in an individual or to pollen proteins that are immunologically related to Lolp N such as D ⁇ c g N, and therapeutic compositions comprising one or more peptides of the invention are also provided.
  • the present invention also provides nucleic and amino acid sequences of D ⁇ c g N protein allergen which is immunologically cross
  • Fig. 1 shows the nucleotide sequence of cDNA clone 12R (SEQ ED NO:l) and its predicted amino acid sequence (SEQ ID NO:2).
  • Clone 12R is a full-length clone of Lolp
  • FIG. 2 shows peptides of the invention of various lengths derived from Lol p V
  • Fig. 3 shows peptides of various lengths derived from Lolp I (SEQ ID NO:30-53).
  • Fig. 4 is a graphic representation depicting the response of T cell lines from 19 patients primed in vitro with affinity purified Lolp V and analyzed for response to Lolp V peptides (derived from the Lol p V protein allergen) by percent of responses with a mean
  • Fig. 5 is a graphic representation derived from the same data shown in Fig. 4 showing the ranked sum for each peptide, the bar represents the cumulative rank of the peptide response in the group of 19 patients tested, above each bar in parenthesis is the percent of patients positively responding to each peptide, the S.I. is also indicated above each bar.
  • Fig. 6 is a graphic representation of the results of a direct ELIS A, the source of IgE was a sample of pooled human plasma (PHP) designated PHP- A, and wherein the antigen is either soluble pollen extract (SPE) of ryegrass pollen, or bacterially expressed recombinant Lol p V (rLolpV).
  • SPE soluble pollen extract
  • rLolpV bacterially expressed recombinant Lol p V
  • Fig. 7 is a graphic representation of the results of a direct ELISA, the source of IgE was a sample of pooled human plasma (PHP) designated PHP-B and wherein the antigen is either soluble pollen extract (SPE) of ryegrass pollen, rLolpV.
  • PPP pooled human plasma
  • SPE soluble pollen extract
  • Fig. 8 is a graphic representation of the results of a direct ELISA
  • the source of IgE was plasma from 4 individual patients, #1118, #1120, #1125, #1141, and wherein the antigen is ryegrass pollen SPE.
  • Fig. 9 is a graphic representation of the results of a direct ELISA the source of IgE was plasma from 4 individual patients, #1118, #1120, #1125, #1141 , and wherein the antigen is xLolp V.
  • Fig. 10 is a graphic representation of the results of a competition ELISA, the source of IgE was a sample of pooled human plasma designated PHP- A, IgE binding was measured in the presence of ryegrass pollen SPE, affinity purified native Lol p V or ⁇ l ⁇ ol p
  • Fig. 11 is a graphic representation of the results of a competition ELISA, the source of IgE was plasma from individual patient #706 as a source of IgE, IgE binding was measured in the presence of ryegrass pollen SPE, affinity purified Lol p N or rLol p V.
  • Fig. 12 is a graphic representation of a histamine release assay to ryegrass pollen
  • Fig. 13a and Fig. 13b each show a graphic representation of a direct ELISA using a sample of pooled human plasma designated PHP-B as a source of IgE, and wherein the antigen was either a selected peptide derived from Lol p N or x ⁇ Lolp N.
  • Fig. 14 is a graphic representation of a competition ELISA using a sample of pooled human plasma designated PHP-B as a source of IgE, and wherein the antigens were a mixture of affinity purified Lol p I and Lol p V or a mixture of recombinant Lolp I ⁇ xLolp I) or ⁇ Lolp V to compete for IgE binding to ryegrass pollen SPE.
  • Fig. 15 is a photograph of a Coomassie blue stained SDS-PAGE (12.5%) analysis of an AblB9-affinity purified native Lolp N, the sample was run under reducing conditions, the molecular weight standards are shown on the left.
  • Fig. 16 shows the nucleotide sequence of clone 259 of Dae g N, and its predicted amino acid sequence, the nucleotide sequence of nucleotides 1 to 699 has been confirmed, and the nucleotide sequence of nucleotides 700 to 1181 are unconfirmed.
  • the present invention provides isolated peptides derived from Lolp N.
  • the present invention also provides Dae g N protein allergen which is immunologically cross- reactive with Lolp N.
  • a "peptide” refers to any protein fragment of Lolp N that induces an immune response.
  • fragment and antigenic fragment refer to an amino acid sequence having fewer amino acid residues than the entire amino acid sequence of the protein from which the fragment is derived, and that induces an immune response.
  • isolated and purified refer to peptides of the invention which are substantially free of cellular material or culture medium when produced by recombinant D ⁇ A techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically.
  • peptide of the invention include peptides derived from I ⁇ olp N which comprise at least one T cell epitope of the allergen or a portion of such peptide which comprises at least one T cell epitope. Peptides comprising at least two regions, each region comprising at least one T cell epitope of Lolp V are also within the scope of the invention. Isolated peptides or regions of isolated peptides, each comprising at least two T cell epitopes of Lolp N protein allergen are particularly desirable for increased therapeutic effectiveness.
  • Peptides which are immunologically related e.g., by antibody or T cell cross-reactivity
  • peptides from Dae g N are also within the scope of the invention.
  • Peptides immunologically related by antibody cross-reactivity are bound by antibodies specific for a peptide of Lolp N.
  • Peptides immunologically related by T cell cross-reactivity are capable of reacting with the same T cells as a peptide of the invention.
  • Isolated peptides of the invention can be produced by recombinant D ⁇ A techniques in a host cell transformed with a nucleic acid having a sequence encoding such peptide.
  • the isolated peptides of the invention can also be produced by chemical synthesis.
  • host cells transformed with a nucleic acid having a sequence encoding a peptide of the invention or the functional equivalent of the nucleic acid sequence are cultured in a medium suitable for the cells 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 or immunopurification with antibodies specific for the peptide, the protein allergen from which the peptide is derived, or a portion thereof.
  • the present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the invention.
  • Nucleic acid coding for a Lolp V peptide of the invention or at least one fragment thereof may be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
  • bacterial cells such as E. coli, insect cells, 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.
  • 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. For example, a baculovirus system is commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commercially available (Pharmacia, Piscataway, NJ) for expression in mammalian cells.
  • suitable expression vectors include, among others, pTRC (Amann et al. (1988) Gene 69: 301-315); pG ⁇ X (Amrad Corp., Melbourne, Australia); pMAL (N. ⁇ . Biolabs, Beverly, MA); pRIT5 (Pharmacia, Piscataway, NJ); pET- 1 Id
  • Lol p V peptide of the invention When a Lol p V peptide of the invention is expressed as a fusion protein, it is particularly advantageous to introduce an enzymatic cleavage site at the fusion junction between the carrier protein and Lolp V peptide.
  • the Lolp V peptide 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 Company, St. Louis, MO and N.E. Biolabs, Beverly, MA.
  • 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 Lolp V peptides in different 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, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming the host cells may be found in Sambrook et al. supra, and other laboratory textbooks.
  • nucleic acid sequences of the invention may also be chemically synthesized using standard techniques (i.e. solid phase synthesis). Details of the isolation and cloning of clone 12R encoding Lolp V (described as Lolp lb.1) are given in PCT application Publication Number WO 93/04174 incorporated herein by reference in its entirety.
  • Inducible non-fusion expression vectors include pTrc (Amann et al, (1988) Gene, 69:301-315) and pETlld (Studier etal, Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990), 185:60-89).
  • target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc
  • expression of target genes inserted into pETl Id relies on transcription from the T7 gnlO-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
  • coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990), 185:119-128).
  • Another strategy would be to alter the nucleic acid sequence of the desired gene to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al. (1992) Nuc. Acids Res, 20:2111-2118).
  • Such alteration of nucleic acid sequences of the invention could be carried out by standard DNA synthesis techniques.
  • the nucleic acids of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent 4,598,049; Caruthers et al. U.S. Patent 4,458,066; and Itakura U.S. Patents 4,401,796 and 4,373,071, incorporated by reference herein).
  • the present invention also provides nucleic acid sequences encoding peptides of the invention.
  • Nucleic acid sequences used in any embodiment of this invention can be cDNAs encoding corresponding peptide sequences as shown in Fig. 2 (SEQ ID NO:3-29). Such oligodeoxynucleotide sequences can be produced chemically or mechanically, 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 Lolp V as shown in Fig. 1 or fragments thereof hybridizes, or 2) the sequence (the corresponding sequence portions complementary to the nucleic acid sequences encoding the peptide sequence derived from Lolp V as shown in Fig.
  • nucleic acid sequences of the invention also include RNA which can be transcribed from the DNA prepared as described above.
  • Preferred nucleic acids encode a peptide having at least about 50% homology to a Lolp V peptide of the invention, more preferably at least about 60% homology and most preferably at least about 70% homology with a Lol p V peptide of the invention.
  • Nucleic acids that encode peptides having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with Lolp V peptides of the invention are also within the scope of the invention.
  • Homology refers to sequence similarity between two peptides of Lolp V, or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide or amino acid, then molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • nucleic acid fragments encode peptides of at least 7 amino acid residues in length, and preferably 13-40 amino acid residues in length, and more preferably at least 16-30 amino acids residues in length
  • Nucleic acid fragments encoding peptides of at least 30 amino acid residues in length, at least 40 amino acid residues in length, at least about 80 amino acid residues in length, at least about 100 amino acid residues in length or more are also contemplated.
  • nucleic acid sequences encoding allergens immunologically cross-reactive with I ⁇ olp V such as full length Dae g V protein or peptides (Fig. 16).
  • Proteins and peptides of Dae g V may be produced recombinantly as discussed above, or synthetically.
  • Expression vectors and host cells transformed to express Dae g V protein or peptides thereof are also within the scope of the invention. Details of the cloning of Dae g V are given in the examples.
  • the present invention also provides a method of producing isolated Lol p V peptides of the invention or a portion thereof comprising the steps of culturing a host cell transformed with a nucleic acid sequence encoding a Lolp V peptide of the invention in an appropriate medium to produce a mixture of cells and medium containing said Lolp V peptide; and purifying the mixture to produce substantially pure I ⁇ olp Y peptide.
  • Host cells transformed with an expression vector containing DNA coding for a Lolp V peptide of the invention or a portion thereof are cultured in a suitable medium for the host cell.
  • Lolp V peptides of the invention 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 the Lolp V peptides or portions thereof of the invention.
  • Lolp V peptides of the invention can be used as "purified" allergens to standardize allergen extracts.
  • an animal such as a mouse or rabbit can be immunized with an immunogenic form of an isolated I ⁇ ol p V peptide of the invention capable of eliciting an antibody response.
  • Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well-known in the art.
  • the Lolp V peptide also can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
  • an ⁇ -I ⁇ olp V peptide antisera can be obtained and, if desired, polyclonal an ⁇ -I ⁇ olp V peptide antibodies from the serum.
  • antibody producing cells lymphocytes
  • immortalizing cells such as myeloma cells
  • Hybridoma cells can be screened immunochemically for production of antibodies reactive with the Lolp V peptides of the invention.
  • compositions and biological activity can be made and administered for therapeutic purposes (e.g. to modify the allergic response of a ryegrass pollen sensitive individual to pollen of such grasses or pollen of an immunologically related grass such as D ⁇ c g V).
  • Administration of such peptides may, for example, modify B-cell response to I ⁇ olp V allergen, T-cell response to Lolp V allergen or both responses.
  • Isolated peptides can also be used to study the mechanism of immunotherapy of ryegrass pollen allergy and to design modified derivatives or analogues useful in immunotherapy.
  • the present invention also pertains to T cell clones which specifically recognize Lolp V peptides of the invention. These T cell clones may be suitable for isolation and molecular cloning of the gene for the T cell receptor which is specifically reactive with a peptide of the present invention.
  • the T cell clones may be produced as described in Cellular and Molecular Immunology, Abdul K. Abbas et al., W.B. Saunders Co. (1991) pg. 139.
  • the present invention also pertains to soluble T cell receptors. These receptors may inhibit antigen-dependent activation of the relevant T cell subpopulation within an individual sensitive to Lol p V. Antibodies specifically reactive with such a T cell receptor can also be produced according to the techniques described herein.
  • Such antibodies may also be useful to block T-cell -MHC interaction in an individual.
  • Methods for producing soluble T cell receptors are described in Immunology; A Synthesis, 2nd Ed., Edward S. Golub et al., Sinaur Assoc, Sunderland Massachusetts, (1991) pp. 366-369.
  • Lolp Y is divided into non- overlapping peptides of desired length or overlapping peptides of desired lengths as discussed in Example 2 which can be produced recombinantly, synthetically, or in certain situations, by chemical cleavage of the allergen.
  • Peptides comprising at least one T cell epitope are capable of eliciting a T cell response, such as stimulation (i.e.
  • T cell non-responsiveness peptides comprising at least one T cell epitope
  • isolated peptides are tested by, for example, T cell biology techniques, to determine whether the peptides elicit a T cell response or induce T cell non-responsiveness.
  • Those peptides found to elicit a T cell response or induce T cell non-responsiveness are defined as having T cell stimulating activity.
  • T cell stimulatory activity is assayed by contacting a peptide of the invention with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture.
  • Presentation of a peptide of the invention in association with appropriate MHC molecules to T cells, in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4.
  • the culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines.
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86:1333 (1989) the pertinent portions of which are inco orated herein by reference.
  • a kit for an assay for the production of interferon is also available from Genzyme Corporation (Cambridge, MA).
  • a common assay for T cell proliferation entails measuring tritiated thymidine incorporation.
  • the proliferation of T cells can be measured in vitro by determining the amount of ⁇ H-labeled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
  • a peptide may also be screened for the ability to reduce T cell responsiveness.
  • the ability of a peptide known to stimulate T cells, to inhibit or completely block the activity of a purified native Lol p V protein allergen or portion thereof and induce a state of T cell nonresponsiveness or reduced T cell responsiveness can be deteraiined using subsequent attempts at stimulation of the T cells with antigen presenting cells that present a native Lol p V allergen following exposure to a peptide of the invention. If the T cells are unresponsive to the subsequent activation attempts, as determined by interleukin-2 synthesis and T cell proliferation, a state of nonresponsiveness has been induced. See, e.g., Gimmi, et al. (1993) Proc. Natl. Acad. Sci USA, 90:6586-6590; and Schwartz (1990) Science, 248:1349-1356, for assay systems that can be used as the basis for an assay in accordance with the present invention.
  • peptides comprising "cryptic epitopes” may be determined and are also within the scope of this invention.
  • Cryptic epitopes are those determinants in a protein antigen which, due to processing and presentation of the native protein antigen to the appropriate MHC molecule, are not normally revealed to the immune system.
  • a peptide comprising a cryptic epitope is capable of causing T cells to become non-responsive, and when a subject is primed with the peptide, T cells obtained from the subject will proliferate in vitro in response to the peptide or the protein antigen from which the peptide is derived.
  • Peptides which comprise at least one cryptic epitope derived from a protein antigen are referred to herein as "cryptic peptides".
  • cryptic peptides Peptides which comprise at least one cryptic epitope derived from a protein antigen are referred to herein as "cryptic peptides".
  • antigen- primed T cells are cultured in vitro in the presence of each peptide separately to establish peptide-reactive T cell lines.
  • a peptide is considered to comprise at least one cryptic epitope if a T cell line can be established with a given peptide and T cells are capable of proliferation upon challenge with the peptide and the protein antigen from which the peptide is derived.
  • a modified peptide 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.
  • 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).
  • Those 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, or not affect T cell reactivity.
  • those amino acid residues which are not essential for T cell receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance, diminish or not affect T cell reactivity but does 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 affect 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 affect, 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.
  • 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 peptide within the scope of this invention.
  • 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 polyethylene glycol
  • Modifications of peptides or portions thereof can also include reduction/ alkylation (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 4,939,239; or mild formalin treatment (Marsh, International Archives of Allergy and Applied Immunology, 41:199-215 (1971)).
  • 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
  • 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 be added to the amino or carboxy terminus of the peptide and can result in an increase in solubility of a peptide.
  • Site-directed mutagenesis of DNA encoding a peptide of the invention can be used to modify the structure of the peptide by methods known in the art. Such methods may, among others, include PCR with ohgonucleotides containing the sequences encoding the desired amino acids (Ho et al., Gene, 77:51-59 (1989)) or total synthesis of mutated genes (Hostomsky, Z. et al., Biochem. Biophys, Res. Comm., 161:1056-1063 (1989)).
  • the aforementioned methods can be used in conjunction with other procedures to change the eukaryotic codons in DNA constructs encoding protein or peptides of the invention to ones preferentially used in E. coli, yeast, mammalian cells, or other eukaryotic cells.
  • Peptides or antibodies of the present invention can also be used for detecting and diagnosing ryegrass pollinosis. For example, this could be done in vitro by combining blood or blood products obtained from an individual to be assessed for sensitivity to ryegrass pollen or another cross reactive pollen such as Dae g V, with isolated peptides of Lolp V, under conditions appropriate for binding of components in the blood (e.g., antibodies, Tcells, B cells) with the peptide(s) and determining the extent to which such binding occurs.
  • components in the blood e.g., antibodies, Tcells, B cells
  • Radio- allergergosorbent test (RAST), paper radioimmunosorbent test (PRIST), enzyme linked immunosorbent assay ( ⁇ LISA), radioimmunoassays (RIA), immuno-radiometric assays (IRMA), luminescence immunoassays (LIA), histamine release assays and Ig ⁇ immunoblots.
  • RAST radio- allergergosorbent test
  • PRIST paper radioimmunosorbent test
  • ⁇ LISA enzyme linked immunosorbent assay
  • RIA radioimmunoassays
  • IRMA immuno-radiometric assays
  • LIA luminescence immunoassays
  • histamine release assays and Ig ⁇ immunoblots.
  • 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. 22.1-22.10) utilizing the protein allergen or a portion thereof, or a modified form of the protein allergen or a portion thereof, each of which binds Ig ⁇ specific for the allergen.
  • the same individuals are administered a Delayed Type Hypersensitivity test prior to, simultaneously with, or subsequent to administration of the Immediate Type Hypersensitivity test.
  • the Delayed Type Hypersensitivity test utilizes a modified form of the protein allergen or a portion thereof, the protein allergen produced recombinantly, or a peptide derived from the protein allergen, each of which has human T cell stimulating activity and each of which does not bind Ig ⁇ specific for the allergen in a substantial percentage of the population of individuals sensitive to the allergen (e.g., at least about 75%).
  • Those individuals found to have both a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity reaction may be treated with a therapeutic composition comprising the same modified form of the protein or portion thereof, the recombinantly produced protein allergen, or the peptide, each as used in the Delayed Type Hypersensitivity test.
  • Isolated peptides of the invention when administered in a therapeutic regimen to a Lolp V-sensitive individual, or an individual allergic to an allergen cross-reactive with Lol p Y such as Dae g V, are capable of modifying the allergic response of the individual to Lolp V ryegrass pollen allergen or such cross-reactive allergen, 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 ryegrass pollen allergen or cross-reactive 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 the 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.
  • Lolp V peptides of the present invention which have T cell stimulating activity, and thus comprise at least one T cell epitope are particularly desirable for therapeutic purposes.
  • 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 or reducing allergic response to Lolp V can also be used.
  • 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.
  • 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.
  • 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.
  • Exposure of ryegrass pollen patients to isolated Lolp V peptides of the present invention which comprise at least one T cell epitope and are derived from Lolp V protein allergen may cause appropriate T cell subpopulations to become nonresponsive or have a reduced response to the protein allergen and thus do not participate in stimulating an immune response upon such exposure.
  • administration of a peptide of the invention or portion thereof which comprises at least one T cell epitope may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring Lolp V protein allergen or portion thereof (e.g. result in a decrease of IL-4 and/or an increase in B -2).
  • T cell subpopulations which normally participate in the response to the naturally occurring 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.
  • allergen e.g., nasal mucosa, skin, and lung
  • 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 diminution in allergic symptoms.
  • the isolated Lolp V peptides of the invention can be used in methods of diagnosing, treating and preventing allergic reactions to Lolp V allergen or a cross reactive protein allergen.
  • the present invention provides compositions useful in allergy diagnosis and/or useful in allergy therapy comprising isolated Lolp V peptides or portions thereof. Such compositions will typically also comprise a pharmaceutically acceptable carrier or diluent when intended for in vivo administration.
  • Therapeutic compositions of the invention may also comprise synthetically prepared Lolp V peptides 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.
  • Lolp V peptides or portions 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. 64:84-99) and liposomes (Strejan et al. (1984) J. Neuroimmunol, 7: 27).
  • compositions of the invention are administered to ryegrass allergen sensitive individuals or individuals sensitive to an allergen which is immunologically cross-reactive with house ryegrass allergen (i.e. Dactylis glomerata, or Sorghum halepensis, etc.).
  • allergen i.e. Dactylis glomerata, or Sorghum halepensis, etc.
  • therapeutic compositions of the invention are preferably administered in non-immunogenic form, e.g. which does not contain adjuvant.
  • T cell non responsiveness or reduced T cell responsiveness is induced as a result of not providing an appropriate costimulatory signal sometimes referred to as a "second signal”
  • second signal an appropriate costimulatory signal
  • stimulation of T cells requires two types of signals, the first is the recognition by the T cell via the T cell receptor of appropriate MHC-associated processed antigens on antigen presenting cells (APCs) and the second type of signal is referred to as a costimulatory signal(s) or "second signal" which may be provided by certain competent APCs.
  • a composition of the invention When a composition of the invention is administered without adjuvant, it is believed that competent APCs which are capable of producing the second signal or costimulatory signal are not engaged in the stimulation of appropriate T cells therefore resulting in T cell nonresponsiveness or reduced T cell responsiveness.
  • antibodies or other reagents capable of blocking the delivery of costimulatory signals such as the "second signal" which include, but are not limited to B7 (including B7-1, B7-2, and BB-1), CD28, CTLA4, CD40 CD40L CD54 and CD1 la/18 (Jenkins and Johnson, Current Opinion in Immunology, 5:361-367 (1993), and Clark and Ledbetter, Nature, 367:425-428 (1994))
  • B7 including B7-1, B7-2, and BB-1
  • CD28 including B7-1, B7-2, and BB-1
  • CTLA4 CD40L CD54 CTLA4, CD40 CD40L CD54 and CD1 la/18
  • a peptide of the invention may be administered in
  • Administration of the therapeutic compositions of the present invention to an individual to be desensitized can be carried out using known procedures at dosages and for periods of time effective to reduce sensitivity (i.e., reduce the allergic response) of the individual to the allergen.
  • Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to ryegrass pollen, the age, sex, and weight of the individual, and the ability of the protein or fragment thereof to elicit an antigenic response in the individual.
  • the active compound i.e., protein or fragment thereof
  • the active compound may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
  • the active compound may be coated within a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • preferably about 1 ⁇ g- 3 mg and more preferably from about 20-750 ⁇ g of active compound (i.e., protein or fragment thereof) per dosage unit may be administered by injection.
  • Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • To administer a peptide by other than parenteral administration it may be necessary to coat the protein with, or co-administer the protein with, a material to prevent its inactivation.
  • peptide or portion thereof may be co-administered with enzyme inhibitors or in liposomes.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) /. Neuroimmunol., 7:27).
  • the active compound may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethyline glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions of dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glyceral, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, parabens, chlorobutanol, phenol, ascorbic acid, thirmerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol and sorbitol or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about, including in the composition, an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating active compound (i.e., protein or peptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • active compound i.e., protein or peptide
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., protein or peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the peptide may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the peptide and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between from about 10 ⁇ g to about 200 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservative, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit from as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • Various isolated peptides of the invention derived from ryegrass pollen protein Lol p Y are shown in Fig. 2 (SEQ ID NO:3-29).
  • Peptides comprising at least two regions, each region comprising at least one T cell epitope of Lolp V are also within the scope of the invention.
  • a region may include the amino acid sequence of a peptide of the invention as shown in Fig. 2 or the amino acid sequence of a portion of such peptide.
  • human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to Lolp V allergen, (i.e., an individual who has an IgE mediated immune response to Lolp V 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". Positive results are used to calculate the mean stimulation index for each peptide for the group of patients tested.
  • the mean T cell stimulation index is indicated above the bar.
  • 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 in a significant number of ryegrass pollen sensitive patients tested is considered useful as a therapeutic agent.
  • Preferred peptides have a mean T cell stimulation index of at least 2.5, more preferably at least 3.0, more preferably at least 3.5, more preferably at least 4.0, more preferably at least 5.0 and most preferably at least about 6.
  • peptides of the invention having a mean T cell stimulation index of at least 5, as indicated by data shown in Figs 4 and 5, include peptides LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-8 (SEQ ID NO: 10), LPIX-17 (SEQ ID NO: 19) and LPIX-19 (SEQ ID NO:21).
  • preferred peptides have a positivity index (PJ.) of at least about 60, more preferably about 100, more preferably at least about 200 and most preferably at least about 300.
  • the positivity index for a peptide is determined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to ryegrass pollen (e.g., preferably a population of at least 15 individuals, more preferably a population of at least 30 individuals or more), who have a T cell stimulation index to such peptide of at least 2.0.
  • the positivity index represents both the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell response to a peptide in a population of individuals sensitive to ryegrass pollen.
  • the bar represents the positivity index and the percent of individuals tested who have a T cell stimulation index of at least 2.0 to that peptide are indicated in parenthesis above each bar (the mean T cell stimulation index is also indicated above each bar).
  • Lolp V peptide LPIX-5 (SEQ ID NO:7) has a mean S.I. of 5.8 and 26.3% of positive responses in the group of individuals tested resulting in a positivity index of 152.54.
  • Lolp V peptides having a positivity index of at least about 100 and a mean T cell stimulation index of at least about 4 include: LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), and LPIX-17 (SEQ ID NO:19).
  • the bar represents the cumulative rank of the peptide response in the group of patients tested as described in Example 2.
  • the 5 peptides with the highest S.I. in each individual were determined and assigned a numerical rank in descending order, with 5 representing the strongest response.
  • the ranks for each peptide were then summed for the entire group of patients tested to determine the cumulative rank for the peptide.
  • each bar is the mean S.I. for each peptide and the percent of positive responses (in parenthesis) with an S.I. of at least 2 to the peptide in the group of patients tested.
  • a peptide having T cell stimulating activity and thus comprising at least one T cell epitope as determined by T cell biology techniques is modified by addition or deletion of amino acid residues at either the amino or carboxy terminus of the peptide and tested to determine a change in T cell reactivity to the modified peptide.
  • peptides are selected and produced recombinantly or synthetically.
  • Peptides are selected based on various factors, including the strength of the T cell response to the peptide (e.g., stimulation index), the frequency of the T cell response to the peptide in a population of individuals sensitive to ryegrass pollen, and the potential cross-reactivity of the peptide with other allergens from other species of grasses as discussed earlier i.e. Dactylis glomerata.
  • the physical and chemical properties of these selected peptides e.g., solubility, stabiUty
  • the abiUty of the selected peptides or selected modified peptides to stimulate human T cells e.g., induce proliferation, lymphokine secretion
  • appropriate T cell populations to become non-responsive or have a reduced response to the protein allergen is determined.
  • peptides such as LPIX-4 (SEQ ID NO:6),-5 (SEQ ID NO:7),-6 (SEQ ID NO:8),-ll (SEQ ID NO:13),-12 (SEQ ID NO:14),- 16 (SEQ ID NO:18),-17 (SEQ ID NO:19)and -20 (SEQ ID NO:22) for purposes of increasing solubiUty or stabiUty.
  • Modifications to improve solubiUty include truncation from either the amino or carboxyl terminus of the peptide or both termini to remove hydrophiUc amino acids such as Val, He, Leu, Phe, Tyr and Trp.
  • Residues removed by truncation may also be replaced with charged hydrophiUc amino acids such as Asp, Glu, Lys and Arg or neutral hydrophilic amino acids such as Ser, Pro, Gly or Ala.
  • Such amino acids may be of either the R or S optical configuration.
  • hydrophiUc polymers to either the amino- or carboxy te-rminus of the peptides or to both.
  • Such polymers may be polyanionic, polycationic or neutral (such as polyoxyethylene).
  • Modifications to improve stabiUty include deletion or replacement of Asn and Gin residues and eUmination ot Asn-Gly, Asp-Gly and Asp-Pro sequences.
  • IgE- mediated responses such as anaphylaxis.
  • Immunoglobulin E is a mediator of anaphylactic reactions which result from the binding and cross-linking of antigen to IgE on mast ceUs or basophils and the release of mediators (e.g., histamine, serotonin, eosinophil chemotacic factors).
  • anaphylaxis in a substantial percentage of a population of individuals sensitive to Lolp V could be avoided by the use in immunotherapy of a peptide or peptides which do not bind IgE in a substantial percentage (e.g., at least about 75%) of a population of individuals sensitive to Lol p V allergen, or if the peptide binds IgE, such binding does not result in the release of mediators from mast cells or basophils.
  • the risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides which have reduced IgE binding.
  • peptides which have minimal IgE stimulating activity are desirable for therapeutic effectiveness.
  • Minimal IgE stimulating activity refers to IgE production that is less than the amount of IgE production and or IL-4 production stimulated by the native Lolp V protein aUergen. Similarly, IL-4 production can be compared, with reduced IL-4 production indicating lessened IgE stimulating activity.
  • a peptide of the invention is to be used as a diagnostic reagent, it is not necessary that the peptide or protein have reduced IgE binding activity compared to the native Lol p V aUergen.
  • IgE binding activity of peptides can be determined by, for example, using various types of enzyme linked immunosorbent assays (ELISA).
  • Preferred T cell epitope containing peptide of the invention when administered to a ryegrass poUen-sensitive individual or an individual sensitive to an aUergen which is immunologicaUy related to ryegrass poUen allergen such as Dae g I, in a therapeutic treatment regimen, is capable of modifying the aUergic response of the individual to the aUergen.
  • Such preferred Lolp V peptides of the invention comprising at least one T ceU epitope of Lol p V or at least two regions derived from Lol p Y, each comprising at least one T cell epitope, when administered to an individual sensitive to ryegrass poUen are capable of modifying T cell response of the individual to the allergen and are useful as therapeutics in addressing sensitivity to grasses.
  • a preferred isolated Lolp V peptide of the invention comprises at least one T ceU epitope of the Lolp V and accordingly the peptide comprises at least approximately seven amino acid residues.
  • preferred therapeutic compositions of the invention preferably comprise at least two T cell epitopes of Lolp V, and accordingly, a preferred peptide comprises at least approximately eight amino acid residues and preferably at least fifteen amino acid residues.
  • therapeutic compositions comprising preferred isolated peptides of the invention preferably comprise a sufficient percentage of the T ceU epitopes of the entire protein allergen (i.e.
  • SyntheticaUy produced peptides of the invention comprising up to approximately forty-five amino acid residues in length, and most preferably up to approximately thirty amino acid residues in length are particularly desirable as increases in length may result in difficulty in peptide synthesis.
  • Peptides of the invention may also be produced recombinantly as described earlier, and it is preferable that peptides of 45 amino acids or longer be produced recombinantly.
  • Peptides derived from the Lolp V protein allergen which can be used for therapeutic purposes comprise at least one T cell epitope of I ⁇ olp V and comprise all or a portion of the foUowing peptides: LPIX-1 (SEQ ID NO:3), LPIX-1.1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4), LPD -2.1 (SEQ ID NO: 4), LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6) LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPD -7 (SEQ ID NO:9), LPIX-8 (SEQ ID NO: 10), LPIX-9 (SEQ ID NO: 11), LPIX-10 (SEQ ID NO: 12), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LP-K-13 (SEQ ID NO:15), LPIX- 14 (SEQ ID NO:16), LPIX-15 (
  • the portion of the peptide preferably has a mean T ceU stimulation index (S.I.) equivalent to, or greater than the mean T cell stimulation index of the peptide from which it is derived (e.g. as shown in Fig. 5, the S.I. for LPIX-16 (SEQ ID NO:18) is shown above the bar to be 3.7, therefore any portion of LPD -16 preferably has a mean S.I. of 3.7).
  • S.I. mean T ceU stimulation index
  • peptides derived from the l ⁇ olp V protein aUergen which can be used for therapeutic purposes comprise all or a portion of the following peptides: LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPLX-8 (SEQ ID NO:10), LPDC-9 (SEQ ID NO:l 1), LPIX-11 (SEQ ID NO:13), LPD -12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX- 17 (SEQ ID NO:19), LPDC-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25), and LPIX-26 (SEQ ID NO:28) as shown in Fig.
  • peptides derived from Lol p V protein allergen which can be used for therapeutic purposes comprise all or a portion of the following peptides: LPIX-1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4), LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6), LPIX- 5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPDC-7 (SEQ ID NO:9), LPIX-8 (SEQ ID NO:10), LPDC-9 (SEQ ID NO:ll), LPIX-10 (SEQ ID NO:12).
  • LPK-11 SEQ ID NO:13
  • LPK-12 SEQ ID NO:14
  • LPK-13 SEQ ID NO:15
  • LPK-14 SEQ ID NO:16
  • LPD - 15 SEQ ID NO:17
  • LPIX-16 SEQ ID NO:18
  • LPIX-17 SEQ ID NO:19
  • LPIX-18 SEQ ID NO:20
  • LPIX-19 SEQ ID NO:21
  • LPIX-20 SEQ ID NO:22
  • LPIX-21 SEQ ID NO:23
  • LPIX-22 SEQ ID NO:24
  • LPD -23 SEQ ID NO:25
  • LPIX-24 SEQ ID NO:26
  • LPIX-26 SEQ ID NO:28
  • LPIX-27 SEQ ID NO:29
  • Y is an amino acid sequence selected from the group consisting of: LPIX-1 (SEQ ID NO:3), LPD -1.1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4), LPIX-2.1 (SEQ ID NO: 4), LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6) LPK-5 (SEQ ID NO:7), LPD -6 (SEQ ID NO:8), LPD -7 (SEQ ID NO:9), LPD -8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:ll), LPIX-10 (SEQ ID NO: 12), LPIX-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO: 14), LPIX-13 (SEQ ID NO: 15), LPIX-1 (SEQ ID NO:3), LPD -1.1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4), LPIX-2.1 (SEQ ID NO: 4), LPIX-3 (SEQ ID NO
  • X n are amino acid residues contiguous to the amino terminus of Y in the amino acid sequence of the protein aUergen and Zm are amino acid residues contiguous to the carboxy terminus of Y in the amino acid sequence of the protein allergen.
  • n is 0-30 and m is 0-30.
  • the peptide or portion thereof has a mean T ceU stimulation index equivalent to greater than the mean T ceU stimulation index of Y as shown in Fig. 4.
  • amino acids comprising the amino terminus of X and the carboxy terminus of Z are selected from charged amino acids, i.e., arginine (R), lysine (K), histidine (H), glutamic acid (E) or aspartic acid (D); amino acids with reactive side chains, e.g., cysteine (C), asparagine (N) or glutamine (Q); or amino acids with sterically small side chains, e.g., alanine (A) or glycine (G).
  • n and m are 0-5; most preferably n + m is less than 10.
  • Another embodiment of the present invention provides peptides comprising at least two regions, each region comprising at least one T cell epitope of Lolp V and accordingly each region comprises at least approximately seven amino acid residues.
  • These peptides comprising at least two regions can comprise up to 100 or more amino acid residues but preferably comprise at least about 14, even more preferably at least about 20, and most preferably at least about 30 amino acid residues of the Lol p V aUergen.
  • the amino acid sequences of the regions can be produced and joined by a Uriker to increase sensitivity to processing by antigen-presenting ceUs.
  • Such linker can be any non-epitope amino acid sequence or other appropriate Unking or joining agent.
  • the regions are arranged in the same or a different configuration from a naturaUy-occurring configuration of the regions in the aUergen.
  • the regions containing T cell epitope(s) can be arranged in a noncontiguous configuration and can preferably be derived from the same protein aUergen.
  • Noncontiguous is defined as an arrangement of regions containing T ceU epitope(s) which is different than that of the native amino acid sequence of the protein aUergen from which the regions are derived.
  • noncontiguous regions containing T cell epitopes can be arranged in a nonsequential order (e.g., in an order different from the order of the amino acids of the native protein aUergen from which the region containing T cell epitope(s) are derived in which amino acids are arranged from an amino terminus to a carboxy terminus).
  • a peptide of the invention can comprise at least 15%, at least 30%, at least 50% or up to 100% of the T cell epitopes of Lolp V but does not comprise the entire amino acid sequence of Lolp Y.
  • the individual peptide regions can be produced and tested to determine which regions bind immunoglobulin E specific for Lolp V and which of such regions would cause the release of mediators (e.g., histamine) from mast ceUs or basophils.
  • mediators e.g., histamine
  • Those peptide regions found to bind immunoglobulin E and to cause the release of mediators from mast ceUs or basophils in greater than approximately 10-15% of the allergic sera tested are preferably not included in the peptide regions arranged to form preferred peptides of the invention.
  • Examples of preferred peptide regions which do not appear to bind to IgE in preliminary IgE binding data studies include the amino acid sequences of such regions being shown in Fig. 2 (SEQ ID NO:3-29), or portions of said regions comprising at least one T ceU epitope.
  • Preferred peptides comprise various combinations of two or more of the above- discussed preferred regions, or a portion thereof.
  • Preferred peptides comprising a combination of two or more regions include the foUowing:
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ TD NO:7), LPD -6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO:14), LPD -16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ TD NO:7), LPIX-6 (SEQ ID NO:8), LPIX-17 (SEQ ID NO:6)
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8) and LPIX-20 (SEQ ro NO:22);
  • LPD -4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPK-6 (SEQ ID NO:8), LPIX-11 (SEQ ID NO:6)
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10), LPD -9 (SEQ ID NO:l 1), LPDC-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO:
  • LPIX-16 SEQ TD NO:18
  • LPIX-17 SEQ ro NO:19
  • LPIX-19 SEQ ID NO:14
  • LPK-4 (SEQ ID NO:6), LPIX-11 (SEQ TD NO: 13), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ ID NO:22);
  • LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and
  • LPIX-4 (SEQ fl) NO:6), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and
  • LPIX-20 (SEQ ID NO:22); LPIX-5 (SEQ ID NO:7), LPDC-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO: 18), and
  • LPK-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPDC-17 (SEQ TD NO: 19), and
  • LPIX-5 (SEQ ID NO:7), LPD -16 (SEQ ID NO:18), LPIX-17 (SEQ TD NO:19), and LPIX-20 (SEQ TD NO:22); LPIX-11 (SEQ ID NO: 13), LPD -16 (SEQ TD NO: 18), LPDM7 (SEQ ED NO:19), and
  • LPIX-4 (SEQ E) NO:6), LPD -11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22);
  • LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ TD NO:18), and LPIX-20 (SEQ TD NO:22); LPIX-4 (SEQ ro NO:6), LPIX- 17 (SEQ ro NO: 19), and LPIX-20 (SEQ ID NO:22);
  • LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22);
  • LPD -5 (SEQ ID NO:7), LPD -16 (SEQ ID NO: 18), and LPIX-20 (SEQ ro NO:22);
  • LPIX-11 SEQ ID NO: 13
  • LPIX-16 SEQ ID NO:18
  • LPIX-20 SEQ TD NO:22
  • LPIX-11 (SEQ ID NO: 13), LPD -17 (SEQ TD NO:19), and LPK-20 (SEQ TD NO:22); LPD -16 (SEQ ID NO: 18), LPIX-17 (SEQ ID NO: 19), and LPIX-20 (SEQ ID NO:22)
  • LPIX-5 (SEQ ID NO:7), LPIX-17 (SEQ TD NO: 19), and LPIX-20 (SEQ ID NO:22);
  • LPIX-4 (SEQ ID NO:6), LPIX-20 (SEQ ED NO:22);
  • LPIX-5 (SEQ ID NO:7), and LPIX-20 (SEQ ID NO:22);
  • LPIX-6 (SEQ ID NO:8), and LPIX-20 (SEQ ID NO:22); LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22);
  • LPIX-12 (SEQ ID NO: 14), and LPIX-20 (SEQ ID NO:22);
  • LPIX-16 SEQ ID NO:18
  • LPIX-20 SEQ ID NO:22
  • LPDC-17 (SEQ ID NO: 19), and LPD -20 (SEQ ID NO:22).
  • compositions comprising at least two peptides (e.g., a physical mixture of at least two peptides), each comprising at least one T ceU epitope of I ⁇ olp V.
  • compositions can be in the form of a composition additionally with a pharmaceutically acceptable carrier of diluent for therapeutic uses, or with conventional non-pharmaceutical excipients for reagent use.
  • an effective amount of one or more of such compositions can be administered simultaneously or sequentially to an individual sensitive to ryegrass pollen.
  • combinations of Lolp V peptides are provided which can be administered simultaneously or sequentiaUy.
  • Such combinations may comprise therapeutic compositions comprising only one peptide, or more peptides if desired.
  • Such compositions may be used simultaneously or sequentially in preferred combinations.
  • Lolp V peptides which can be administered or otherwise used simultaneously or sequentially (comprising peptides having amino acid sequences shown in Fig. 2) include the following combinations: LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ro NO:8), LPIX-16 (SEQ ID NO:6), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ro NO:8), LPIX-16 (SEQ
  • LPIX-4 (SEQ K> NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPD -12 (SEQ ID NO:6), LPIX-4 (SEQ K> NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPD -12 (SEQ ID NO:6), LPIX-4 (SEQ K> NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPD -12 (SEQ
  • LPIX-16 SEQ ID NO:18
  • LPD -17 SEQ ID NO:19
  • LPD -20 SEQ ID NO:22
  • LPD -4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPDC-6 (SEQ TD NO:8), LPIX-17 (SEQ ID NO:6)
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8) and LPDC-20
  • LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPD -6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:6)
  • LPIX-9 SEQ TD NO:ll
  • LPIX-11 SEQ ID NO.13
  • LPIX-12 SEQ ID NO:14
  • LPIX-16 SEQ ID NO:18
  • LPD -17 SEQ ID NO:19
  • LPIX-19 SEQ ID NO:10
  • LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ TD NO: 13), LPIX-16 (SEQ ID NO:18), and
  • LPIX-20 (SEQ ID NO:22); LPD -4 (SEQ ID NO:6), LPDC-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and
  • LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ TD NO:18), LPIX-17 (SEQ ID NO:19), and
  • LPIX-5 (SEQ fl) NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ro NO:18), and LPIX-20 (SEQ ID NO:22);
  • LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and
  • LPK-5 SEQ ro NO:7
  • LPDC-16 SEQ ID NO:18
  • LPDC-17 SEQ ID NO:19
  • LPIX-20 (SEQ ED NO:22); LPIX-11 (SEQ ro NO: 13), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and
  • LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ TD NO:22);
  • LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ ID NO:22);
  • LPIX-4 (SEQ ID NO:6), LPIX-17 (SEQ ID NO: 19), and LPIX-20 (SEQ ID NO:22); LPIX-5 (SEQ fl) NO:7), LPD -11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22); LPIX-5 (SEQ ID NO:7), LPIX-16 (SEQ TD NO:18), and LPD -20 (SEQ ID NO:22); LPD -11 (SEQ ID NO: 13), LPDC-16 (SEQ TD NO: 18), and LPIX-20 (SEQ TD NO:22); LPIX-11 (SEQ K> NO: 13), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); LPD -16 (SEQ TD NO:18), LPD -17 (SEQ TD NO:19), and LPD -20 (SEQ TD NO:22); LPIX-5 (SEQ ID NO:7), LPIX-17 (SEQ ID NO:
  • a therapeutic composition comprising at least two peptides (e.g. a physical mixture of at least two peptides, each peptide comprising at least one epitope) wherein at least one peptide, comprises an amino acid sequence or portion thereof derived from I ⁇ olp Y selected from the following group: LPIX-1 (SEQ ID NO:3), LPIX-2 (SEQ TD NO:4), LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ TD NO:8), LPIX-7 (SEQ ID NO:9), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:ll), LPIX-10 (SEQ ID NO:12), LPDC-11 (SEQ ID NO: 13), LPD -12 (SEQ ID NO: 14), LPD -13 (SEQ ED NO: 15), LPIX-14 (SEQ r
  • LPI-1 (SEQ ID NO:30), LPI-1.1 (SEQ TD NO:31), LPI-2 (SEQ ID NO:32), LPI-3 (SEQ ro NO:55), LPI-4 (SEQ TD NO:33), LPI-4.1 (SEQ ED NO:34), LPI-5 (SEQ ID NO:35), LPI-6 (SEQ ID NO:36), LPI-7 (SEQ TD NO:37), LPI-8 (SEQ ED NO:38), LPI-9 (SEQ ID NO:39), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ TD NO:41), LPI-12 (SEQ TD NO:42), LPI-13 (SEQ ID NO:43), LPI-14 (SEQ ID NO:44), LPI-15 (SEQ ID NO:45), LPI-16 (SEQ ro NO:46), LPI-16.1 (SEQ ID NO:47),
  • a therapeutic composition comprises at least four, five, six, seven, or eight peptides wherein at least two, three or four peptides are derived from Lol p V and are selected from the following group: LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),
  • LPI-6 (SEQ ID NO:36), LPK-11 (SEQ TD NO: 13), LPI-12 (SEQ ED NO:42), LPK-16
  • a preferred therapeutic composition comprises at least two peptides of Lolp I and two peptides of Lolp V, or two peptides of Lolp I and three peptides of Lolp V, or three peptides from Lolp I and three peptides from Lolp V, or three peptides from Lolp I and four peptides from Lolp Y, or four peptides from Lolp I and four peptides from Lol p V, or four peptides from Lol p I and three peptides from Lol p I and three peptid
  • a method comprising administering a combination of peptides or portions thereof derived from Lol p Y and Lol p I which can be administered simultaneously or sequentiaUy; each of such peptides can be in the form of a therapeutic composition with a pharmaceuticaUy acceptable carrier or dUuent.
  • preferred compositions and preferred combinations comprising Lolp Y and Lolp I peptides or portions thereof, which can be administered simultaneously or sequentiaUy comprise the following combinations:
  • LPI-16.1 (SEQ ro NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ TD NO:55), LPI-4.1 (SEQ TD NO:34), LPI-10 (SEQ TD NO:40), LPI-11 (SEQ TD NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ TD NO:52), LPK-4 (SEQ ID NO:6), LPIX-5 (SEQ TD NO:7), LP-K-6 (SEQ TD NO:8), LPIX-8 (SEQ ID NO:10), LPD -9 (SEQ ID NO:ll), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPLX-17 (SEQ ID NO:19), LPLX-19 (SEQ ID NO:21), LPLX-20 (SEQ ID NO:22),
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ TD NO:49), LPI-20, LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ TD NO:40), LPI-11 (SEQ K) NO:41), LPI-15 (SEQ TD NO:45), LPI-22 (SEQ TD NO:52), LPD -4 (SEQ ED NO:6), LPLX-5 (SEQ ED NO:7), LPDC-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO: 10), LPD -9 (SEQ ID NO:ll), LPIX-11 (SEQ ID NO:13), LPD -12 (SEQ ED NO:14), LPIX-16 (SEQ ED NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (S
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ TD NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPD -6 (SEQ ED NO:8), LPIX-9 (SEQ ID NO:ll), LPIX-12 (SEQ ID NO:14), LPD -16 (SEQ ID NO:18), LPIX-17 (SEQ TD NO: 19), LPIX-19 (SEQ TD NO:21), LPIX-20 (SEQ TD NO:22), LPIX-23 (SEQ ID NO:25);-
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ro NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPD -17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ TD NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ K ) NO:6), LPIX-5 (SEQ TD NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ TD NO:34), LPI-10 (SEQ TD NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ ID NO:52);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ K) NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ED NO:41), LPI-15 (SEQ ID NO:45), LPDC-4 (SEQ ED NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ED NO:8), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ fl) NO:l 1), LPIX-11 (SEQ TD NO: 13), LPIX-12 (SEQ ID NO: 14), LPDC-16 (SEQ TD NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ TD NO:21), LPDC-20 (SEQ ID NO:22), LPIX-23 (SEQ ID
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ TD NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ TD NO:40), LPI-11 (SEQ TD NO:41), LPI-15 (SEQ ID NO:45), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO: 14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPD -20 (SEQ E ) NO:22);
  • LPI-16.1 (SEQ K) NO:47), LPI-18 (SEQ TD NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ K) NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ ED NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45), LPIX-4 (SEQ ID NO:6), LPDC-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPK-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO: 19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ K ) NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ED NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ED NO:34), LPI-10 (SEQ ED NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO: 19), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ro NO:47), LPI-18 (SEQ TD NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ TD NO:34), LPD -4 (SEQ TD NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10),
  • LPK-9 (SEQ fl) NO:ll), LPIX-11 (SEQ ID NO:13), LPD -12 (SEQ ID NO:14), LPDC-16 (SEQ ro NO:18), LPK-17 (SEQ ED NO:19), LPIX-19 (SEQ ED NO:21), LPLX-20 (SEQ ID NO:22); LPD -23 (SEQ TD NO:25), LPD -26 (SEQ ID NO:28);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPDC-4 (SEQ TD NO:6), LPDC-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:ll), LPIX-11 (SEQ fl) NO:13), LPIX-12 (SEQ ID NO:14), LPDC-16 (SEQ TD NO:18), LPIX- 17 (SEQ fl) NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ED NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ K) NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ TD NO:34), LPIX-4 (SEQ ID NO:6), LPK-5 (SEQ TD NO:7), LPD -6 (SEQ ID NO:8), LPDC-9 (SEQ ID NO:ll), LP.lX-12 (SEQ K> NO: 14), LPD -16 (SEQ ID NO: 18), LPD -17 (SEQ ID NO: 19), LPK- 19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25); LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ED NO:55
  • LPI-16.1 (SEQ ro NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO: 19), LPIX-19 (SEQ TD NO:21), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ED NO:55), LPI-4.1 (SEQ TD NO:34), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ED NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO: 19), LPIX-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ DD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-22 (SEQ. ID NO:52), LPDC-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ED NO:8),
  • LPIX-8 (SEQ. ED NO:10), LPIX-9 (SEQ. ID NO:ll), LPIX-11 (SEQ. ID NO:13), LPK- 12 (SEQ. ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ED NO:19), LPIX-19 (SEQ. ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ. ID NO:25), LPD -26 (SEQ. ID NO:28);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ED NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ED NO:55), LPI-4.1 (SEQ DD NO:34), LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ED NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ TD NO:8), LPD -8 (SEQ ED NO: 10), LPIX-9 (SEQ ID NO: 11), LPIX-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO:14), LPD -16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPDC-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ED NO:53), LPI-3 (SEQ TD NO:55), LPI-4.1 (SEQ ID NO:34), LPI-22 (SEQ TD NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),
  • LPK-9 (SEQ ⁇ ) NO:ll
  • LPK-12 (SEQ TD NO:14)
  • LPIX-16 (SEQ TD NO:18)
  • LPK-17 (SEQ ID NO: 19)
  • LPDC-19 SEQ ID NO:21
  • LPIX-20 SEQ ID NO:22
  • LPK-23 SEQ ID NO:25
  • LPI-16.1 (SEQ DD NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ED NO:53), LPI-3 (SEQ TD NO:55), LPI-4.1 (SEQ TD NO:34), LPI-22 (SEQ ED NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ DD NO:8), LPIX-12 (SEQ ED NO:14), LPE -16 (SEQ ID NO:18), LPIX-17 (SEQ DD NO:19), LPIX- 19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22); LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ TD NO:56), LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ TD NO:34), LPI-22 (SEQ ED NO:52), LPD -4 (SEQ ID NO:6), LPD -5 (SEQ ID NO:7), LPD -6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO: 18), LPD -17 (SEQ TD NO: 19), LPK-20 (SEQ ID NO:22);
  • LPI-16.1 (SEQ fl) NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ A NO:53), LPI-3 (SEQ TD NO:55), LPIX-4 (SEQ ID NO:6), LPDC-5 (SEQ ED NO:7), LPIX-6 (SEQ ID NO:8), LPD -8 (SEQ ID NO: 10), LPIX-9 (SEQ ID NO: 11), LPK-11 (SEQ ID NO: 13), LPD -12 (SEQ TD NO: 14), LPIX-16 (SEQ ID NO: 18), LPD - 17 (SEQ ID NO: 19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ A NO:25), LPIX-26 (SEQ ED NO:28);
  • LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ro NO:53), LPD -4 (SEQ TD NO:6), LPD -5 (SEQ ED NO:7), LPD -6 (SEQ TD NO:8), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ TD NO: 19), LPIX-20 (SEQ ID NO:22).
  • a composition comprising at least two Lolp I peptides (e.g.
  • compositions can be administered in the form of a therapeutic composition with with a pharmaceutically acceptable carrier or diluent to treat ryegrass sensitivity and particularly, sensitivity to Lolp I protein allergen.
  • a pharmaceutically acceptable carrier or diluent to treat ryegrass sensitivity and particularly, sensitivity to Lolp I protein allergen.
  • Preferred compositions and preferred combinations of Lolp I peptides which can be administered simultaneously or sequentiaUy (comprising peptides having the amino acid sequences shown in Fig. 3 include the following combinations:
  • LPI-16 SEQ A NO:46
  • LPI-20 SEQ ID NO:56
  • LPI-18 (SEQ ID NO:49), and LPI-20 (SEQ ID NO:56);
  • LPI-20 (SEQ ro NO:56), and LPI-23 (SEQ ID NO:53);
  • LPI-16 (SEQ ID NO:46), LPI-18 (SEQ ID NO:49), and LPI-20 (SEQ ED NO:56);
  • LPI-16 (SEQ ID NO:46), LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53); LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53);
  • LPI-16 (SEQ ID NO:46), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), and LPI-23
  • compositions described herein are useful in the manufacture of a medicament for treating sensitivity to ryegrass poUen allergen or an immunologicaUy cross reactive aUergen in an individual.
  • Balb/c mice were immunized with crude Dactylis glomerata (orchard grass/cocksfoot grass) poUen extract and antibody secreting clones were generated as described (Walsh et al, Int. Arch. Allergy Appl. Immunol, 1990, 91: 419-425).
  • MAb 1B9 hybridoma clone which cross- reacts to Lolp Y was obtained from Dr. Walker (Univ. Birmingham, Wolfson Research Lab, Birmingham,UK).
  • Ascitic fluid generated from Balb/c mice was produced by contract (Babco, Richmond, CA). The antibodies were purified from ascites fluid by (NH4)2 SO4 precipitation (50% saturation).
  • the pellet was resuspended in lOmM phosphate buffer, pH 7.5 and dialyzed against the same buffer at 4°C overnight and then fractionated by ion-exchange chromatography on FPLC Q-Sepharose (Pharmacia, Piscataway, NJ) using linear gradient 0-0.5 M NaCl. IgG was eluted between 0.15-0.2 M NaCl concentration.
  • Purified 1B9 was coupled to Affigel-10 (Biorad, Richmond, CA) using 3-4 mg protein/mL of gel according to manufacturer's instructions.
  • PFLC Q-Sepharose purified mAb 1B9 was dialyzed against 0.1M MOPS buffer, pH 7.5 with two to three changes overnight at 4°C.
  • the Affigel-10 resin was washed with deionized cold H2O in a scintered glass funnel.
  • the washed resin was mixed with the 1B9 antibody for four hours at 4°C, foUowed by an one-hour blocking step with 1 M ethanolamine, pH 8.0. Resin was packed into a column, washed with PBS and then stored in PBS + 0.05% sodium azide.
  • lOOg defatted ryegrass poUen (purchased from Greer Laboratories, Lenoir, NC) was extracted in 1 Uter extraction buffer containing 0.05 M phosphate buffer, pH 7.2, 0.15 M NaCl, phenyl methyl sulfonyl fluoride (170 ⁇ g/mL), leupeptin (1 ⁇ g/mL), pepstatin (l ⁇ g/mL) and soybean trypsin inhibitor (1 ⁇ g/mL).
  • the pollen was extracted by stirring the solution overnight at 4°C, followed by centrifugation at 12,000 x g for 100 minutes.
  • the insoluble materials were re-extracted in 0.5- 1.0L extraction buffer and then the supernatants were combined and depigmented by batch absorption onto 100 mL DE-52 cellulose (Whatman, Maidstone, England) equilibrated with 0.05 M phosphate buffer + 0.3 M NaCl, pH 7.2.
  • the unbound materials were loaded onto the 1B9-Affigel-10 column at a flow rate of
  • the 1B9 affinity-purified material was analyzed by SDS-PAGE. As shown in Fig. 15, Lol p Y exists as multiple bands with molecular weight ranged from 29,000 - 22,000. AU these components were reactive with 1B9 by Western blotting analysis (data not shown). These components were electroblotted onto ProBlott membrane (Applied Biosystems, Foster City, CA), stained by Coomassie blue and the three major bands were excised and sequenced on a Beckman LF-3000 sequencer (Beckman Instruments, Carlsbad, CA). N-terminal amino acid sequence of the three bands are shown in Table I. The sequencing data shows that the middle and lower molecular weight bands represent N-terminal cleavage products of the higher molecular weight component.
  • the N-terminus sequence was identical to the cloned Lolp Y (12R) (see PCT application pubUcation number WO93/04174).
  • the 5 proline residues at the N-terminus were found to be aU hydroxyprolines, which seemed to be common to Group V aUergens from Northern grasses (Matthiesen, F. et al., 1991, Clin. Exp. Allergy, 21:297-307).
  • the N-terminal sequence was determined from the three major bands electroblotted onto ProBlott membrane.
  • the upper band starts with amino acid 1 whereas the middle and the lower bands start at amino acid 9 and 18, respectively.
  • the arrows indicate the cleavage sites.
  • V peptides used in these studies The peptide names are consistent throughout.
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • T ceU lines were estabUshed by stimulation of 2xl0 6 PBIJml in bulk cultures of complete medium (IRPMI-164), 2 mM L-glutamine, 100 U/ml penicUUn/streptomycin, 5xlO" 5 M 2-mercaptoethanol, and 10 mM HEPES, supplemented with 5% heat-inactivated human AB serum, with 10 ⁇ g/ml of affinity purified native Lolp
  • T ceUs for 6 days at 37°C in a humidified 5% CO2 incubator to select for Lolp V reactive T Cells.
  • This amount of priming antigen was determined to be optimal for the activation of T ceUs from most grass-aUergic patients.
  • Viable cells were purified by LSM centrifiigation 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 3 weeks until the desired cell number were achieved. The ceUs were aUowed to rest for 4-6 days.
  • 2X10 4 rested ceUs were restimulated in the presence of 2X10 4 autologous Epstein-Barr virus (EBV)-transformed B cells (prepared as described below) or 5X10 4 irradiated PBL with 2-50 mg/ml of xLolp I, purified native Lol p Y, xFel d I (Chain I), or xLol p I, in a volume of 200 ml complete medium in duplicate weUs in 96-weU round-bottom plates for three days. Each weU then received 1 mCi tritiated thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scinitiUation counting.
  • EBV Epstein-Barr virus
  • the varying antigen dose in assays with xLolp V, purified native Lolp V, and recombinant Lol p I and antigenic peptides synthesized as described above were determined.
  • the titrations were used to optimize the dose of peptides in T ceU assays.
  • the maximum response in a titration of each peptide is expressed as the stimulation index (S.I.).
  • the S.I. is the counts per minute (CPM) incorporated by ceUs 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 ceU epitope.
  • Fig. 4 shows the mean S.I. for that peptide.
  • the numbers enclosed in the parentheses denote percentage of patients responding to that particular peptide.
  • the bar represents the positivity index for each peptide (% of patients responding multiplied by mean S.I.).
  • Fig. 5 shows the ranked sum for each peptide derived from the same data as described above.
  • the bar represents the cumulative rank of the peptide response in the group of the 19 patients tested. To determine the cumulative rank, the 5 peptides with the highest S.I.
  • TMB 3, 3', 5, 5'-tetramethylbenzidine (TMB)
  • KPL 3, 3', 5, 5'-tetramethylbenzidine
  • KPL 3, 3', 5, 5'-tetramethylbenzidine
  • the competition ELISA were carried out using the same protocol with the foUowing changes: a single dUution of patient plasma (or pooled human plasma (PHP)) was used as the source of IgE; seriaUy diluted antigen was mixed with the plasma and aUowed to incubate O/N at 4°C. This plasma was then incubated on dupticate weUs. The results are plotted as the absorbance vs. the log of the concentration of competing antigen.
  • soluble poUen extract SPE
  • xLolp V purified native Lolp V may have a smaU amount of Lolp I; use of recombinant material assures that the IgE binding is only to Lolp V ) and human IgE antibody binding to these antigens was analyzed.
  • PHP consisting of an equal volume of plasma from 20 patients with a ryegrass prick test score of 3+ or greater (PHP- A), or PHP consisting of equal aUquots of plasma from 40 grass skin test reactive patients with high IgE binding as measured by direct ELISA (PHP-B), or plasma from individual patients were compared in this assay.
  • ELISA weUs were coated with ryegrass pollen SPE and then allergic patient IgE binding was measured in the presence of competing ryegrass poUen SPE, purified native Lolp Y, or xt ⁇ olp V.
  • the source of aUergic IgE in this assay was PHP-A ( Figure 10) or individual patient plasma ( Figure 11).
  • the competition assays confirm that a significant portion of IgE against Lol p SPE is specific for Lol p V.
  • a histamine release assay was performed on one ryegrass aUergic individual, using
  • This assay is a measure of IgE reactivity through human basophU mediator release, and it is based on the detection of an acylated derivative of histamine using a specific monoclonal antibody (Morel, A.M. and Delaage, M.A.; 1988, J. AUergy CUn. Immunol. 82: 646-654).
  • the reagents for this radioimmunoassay are sold as a kit by Amac Inc. (Westbrook, ME).
  • Lol p I and Lol p V constitute the major aUergens of ryegrass pollen
  • Lolp I and Lolp V together constitute the major IgE binding proteins of ryegrass pollen SPE.
  • PHP-B was used to examine the abUity of a mixture of native purified Lol p I and Lol p V or a mixture of xLol p I and xl ⁇ ol p Y to compete for IgE binding to ryegrass pollen SPE.
  • the mixture of purified native proteins competes to background level the IgE binding to ryegrass pollen SPE.
  • the mixture of xLolp I and xLolp V is also able to substantiaUy reduce the amount of IgE ava able to bind to the SPE coating the plate.
  • the majority of human IgE directed against all of the ryegrass poUen proteins was bound up by the mix of just two proteins (Lol p I and Lol p V) found in the complex mix of ryegrass poUen SPE proteins. This data impUes that these two proteins are major allergens of ryegrass poUen.
  • Lolp V was performed as follows. The ⁇ gt ⁇ clone 12R was digested with EcoRI. The insert encoding L ⁇ //) V was tigated into pGEX. A pGEX vector containing Lol p V (clone 12R) was digested with EcoRI . The Lol p V insert (containing the nucleotide sequence shown in Fig. 1) was isolated by electrophoresis of this digest through a 1% SeaPlaque low melt agarose gel. The insert was then ligated into EcoRI digested expression vector pET-lld (Novagen, Madison, WI; Jameel et al. (1990) J. Virol.
  • 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-lld, which has a T7 promoter.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • the pET-lld containing the Lolp V (clone 12R) was confirmed by dideoxy sequencing (Sanger et al., (1977) Proc. Natl. Acad. Sci., (USA) 74:5460-5463) to be a Lol p Y clone in the correct reading frame for expression.
  • the pET-1 Id Lolp V clone was grown on a large scale for recombinant protein expression and purification.
  • a 2 ml culture of bacteria containing the recombinant plasmid was grown for 8 hr, then streaked onto soUd media (e.g. 6 petri plates (100 x 15 mm) with 1.5% agarose in LB medium (Gibco-BRL, Gaithersburg, MD) containing 200 ⁇ g ml ampiciUin), grown to confluence overnight, then scraped into 9 L of Uquid media (Brain Heart Infusion media, Difco) containing ampiciUin (200 ⁇ g/ml).
  • soUd media e.g. 6 petri plates (100 x 15 mm) with 1.5% agarose in LB medium (Gibco-BRL, Gaithersburg, MD) containing 200 ⁇ g ml ampiciUin
  • Uquid media Brain Heart Infusion media, Difco
  • Bacteria were recovered by centrifugation (7,930 x g, 10 min), and lysed in 90 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 x g, 10 min, 4° C).
  • the pH of the lysate was adjusted to pH 8.0, and the lysate appUed to an 80 ml Nickel NTA agarose column (Qiagen, Chatsworth, CA) that had been equUibrated with 6 M Guanidine HCl, 100 mM Na2HPO4, pH 8.0.
  • the column was sequentiaUy washed with 6 M Guanidine HCl, 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 protein, xLolp V was eluted with 8 M urea, 100 mM sodium acetate, 10 mM Tris-HCl, pH 4.5, and coUected in 10 ml aUquots.
  • the protein concentration of each fraction was determined by absorbance at A280 and the peak fractions pooled.
  • An aliquot of the collected recombinant protein was analyzed on SDS- PAGE (data not shown) according to the method in Sambrook et al., supra.
  • the first 9 Uter preparation yielded 12 mg of rLol p V with approximately 60-70% purity. Purity of the preparation was determined by densitometry (Shimadzu Hying Spot Scanner, Shimadzu Scientific Instruments, Inc., Braintree, MA) of the coomassie-blue stained SDS-PAGE gel.
  • Dactylis glomerata pollen was purchased from Greer Laboratories (Lenoir, NC). RNA was isolated as previously described in PCT/US92/05661 (WO93/01213) and polyA ⁇ RNA was isolated using MICRO-FAST TRACK® mRNA isolation kit from Invitrogen (San Diego,CA). Double stranded cDNA was made with the BRL cDNA SYNTHESIS PLUS® kit (Gaithersburg,MD). A cDNA Ubrary was made in ⁇ gtlO using the cDNA CLONING SYSTEM-1GT10® (Amersham, Arlington Heights, IL). The D.
  • glomerata double stranded cDNA was Ugated with adaptor arms, containing Eco RI, Bam HI, Kpn I and Nco I restriction sites and ligated into ⁇ gtlO vector arms using the manufacturer's suggested protocols.
  • the Ubrary was packaged and titred also using the manufacturer's suggested protocols.
  • the Ubrary was plated out and over 100,000 independent phage plaques were screened using random primed (RANDOM PRIMED DNA LABELING KIT®, Boehrninger Mannheim Corporation, IndianopoUs, IN) or nick- translated probe [Sambrook J et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1989].
  • the Ubrary was screened with the 1.2 kb Lolp V clone 12R cDNA (SEQ ID NO:l) [Singh MB et al, Proc Natl Acad Sci USA, 1991; 88: 1384-1388].
  • phage clones 228, 235, 236, 259, 267, and 285, were positive after this tertiary screening and high titred stocks were prepared as described [Sambrook J et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1989].
  • the cDNA inserts were isolated from the selected phage using standard techniques [Sambrook J et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1989].
  • the insert from clone 228 was approximately 500 base pairs (bp).
  • the insert from clone 235 was approximately 1,000 bp.
  • the insert from clone 236 was approximately 1200 bp.
  • the insert from clone 259 was approximately 1,200 bp.
  • the insert from clone 267 was approximately 1,000 bp.
  • the insert from clone 285 was approximately 800 bp.
  • the isolated inserts were cloned into appropriately digested pUC18 and/or pUC19 for subsequent analysis.
  • the cDNA inserts were sequenced using the SEQUENASE® kits (USB, Cleveland, OH) based on the standard dideoxy chain termination method of Sanger et al. [Sanger F et al. Proc Natl Acad Sci USA, 1977; 74: 5460-5463].
  • Partial sequences for aU of the clones were determined. All were found to contain Dae g V sequences by comparison with Lolp V clone 12R sequence (SEQ ID NO:l) [Ong EK et al. Gene, 1993; 134: 235-240].
  • the partial translated sequences of clones 235 and 236 were very similar to each other, although they started at different sites in the sequence (not shown), and appear to represent one isoform of Dae g V.
  • the partial translated sequence of clone 259 was different from that of clones 235 and 236 and appear to represent a second isoform of D ⁇ c g Y.
  • the partial translated sequence of clone 259 is most homologous to the sequence of Lolp V clone 12R (SEQ ID NO:2) [Ong EK et al. Gene 1993; 134: 235-240].
  • the partial translated sequences of clones 235 and 236 are most closely homologous to the sequence of Lolp V clone 19R [Ong EK et al. Gene 1993; 134: 235-240].
  • Clone 259 was sequenced in its entirety. It was sequenced from both ends using standard forward and reverse primers (New England Biolabs, Beverly, MA). Subconstructs were prepared by digestion of isolated insert with Eco RI and Pst I and the fragments were cloned into appropriately digested pUC18 for internal sequencing.
  • the Eco R ⁇ /Pst I insert that corresponded to the 5' portion of the Dae g V gene was isolated and further digested with Stu I or Sau 3A and Xho I and Ugated into appropriately digested pUC19 for further sequence analysis.
  • the nucleotide (SEQ ID NO:57) and deduced amino acid (SEQ ID NO:58) sequence of clone 259 is shown in Figure 16.
  • Nucleotides 1- 25 correspond to adaptor sequence. The sequence ends with the poly A tract; the adaptor sequence is not shown at the 3' end of the sequence.
  • the nucleotide sequence from 700 to 1181 is only preUminary and some bases may be misidentified.
  • nucleotide 712 has been tentatively identified as a "C". However, this is the third position of the codon encoding Glyl96 and the presence of another nucleotide at residue 712 would not change the predicted amino acid. It is difficult to sequence the Group V grass allergens due to their high GC content. Clone 236 and 259 have been deposited with the ATCC. As Group V aUergens tend to have very conserved regions, the major T cell epitope containing peptides of Lolp V as described herein, are likely to be the major T ceU epitopes of Dae g V, particularly where the regions are highly conserved between the related grasses.
  • Phe Lys lie Ala Ala Thr Ala Ala Asn Ala Ala Pro Thr Asn Asp Lys 160 165 170
  • CAG GCA CAG AAG GCC GGC AAA CCC GCT GCC GCC GCT GCC ACA GGC GCC 870
  • ATGTATGTGC ATGATCCGGG CGGCGAGTGG TTTTGTTGAT AATTAATCTT CGTTTTCGTT 1032 TCATGCAGCC GCGATCGAGA GGGCTTGCAT GCTTGTAATA ATTCAATATT TTTCATTTCT 1092
  • Val lie Ala Gly Ala Leu Glu Val His Ala Val Lys Pro Ala Thr Glu 120 125 130 135 Glu Val Pro Ala Ala Lys lie Pro Thr Gly Glu Leu Gin lie Val Asp
  • Lys lie Asp Ala Ala Phe Lys lie Ala Ala Thr Ala Ala Asn Ala Ala 155 160 165
  • Lys lie Pro Thr Gly Glu Leu Gin lie Val Asp Lys lie Asp Ala Ala 1 5 10 15
  • Ala lie Thr Ala Met Thr Gin Ala Gin Lys Ala Gly Lys Pro Ala Ala 1 . 5 10 15
  • Val Glu Lys Ala Ser Asn Pro Asn Tyr Leu Ala lie Leu Val Lys Tyr 1 5 10 15
  • Lys Gly Lys Asp Lys Trp lie Glu Leu Lys Glu Ser Trp Gly Ala Val 1 5 10 15 Trp Arg lie Asp
  • MOLECULE TYPE peptide
  • FRAGMENT TYPE N-terminal
  • GCA GTG CAG CAG TAC ACG GTG GCG CTG TTC CTG GCC GTG GCC TCG TGT 103
  • Leu Ser Glu Ala Leu Arg lie lie Ala Gly Thr Leu Glu Val His Ala 125 130 135
  • Lys Phe lie Pro Thr Leu Glu Ala Ala Val Lys Gin Ala Tyr Ala Ala 205 210 215
  • GCATGCATGC CGTGGCGCCG CGCAAGTTTG CTCATAATTA ATTCTTGGTT TTCGTTGCTT 1081
  • MOLECULE TYPE protein
  • Val Lys Pro lie Pro Ala Gly Glu Leu Gin lie Val Asp Lys lie Asp 145 150 155 160

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Botany (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pulmonology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention a pour objet des peptides isolés de Lol pV, un allergène protéique essentiel de l'espèce Lolium perenne. Les peptides thérapeutiques entrant dans le champ de l'invention comprennent au moins un épitope de lymphocyte T, ou de préférence au moins deux épitopes de lymphocyte T et un allergène protéique de Lol pV. Les peptides de diagnostic entrant dans le champ de l'invention lient l'IgE. L'invention prévoit également des peptides modifiés possédant les mêmes propriétés thérapeutiques que l'allergène naturel correspondant ou ses fragments, ou améliorées par rapport à ceux-ci, ou d'autres propriétés souhaitables de ceux-ci. L'invention prévoit en outre un codage de séquences d'acide nucléique pour les peptides selon l'invention. L'utilisation des compositions thérapeutiques comporant un ou plusieurs peptides selon l'inveniton dans la fabrication de médicaments destinés à traiter la sensibilité à Lol pV ou un allergène immunologiquement apparenté à Lol pV, ou la sensibilité générale au ray-grass chez un individu, est également prévue. L'invention décrit également un codage d'acides nucléiques pour l'allergène protéique Dac gV ainsi que la séquence d'acides aminés de l'allergène Dac gV.
PCT/US1994/009024 1993-08-13 1994-08-05 Epitopes de lymphocyte t pour l'allergene du ray-grass WO1995006728A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP7508127A JPH09504167A (ja) 1993-08-13 1994-08-05 ドクムギ花粉アレルゲンのｔ細胞エピトープ
AU75597/94A AU7559794A (en) 1993-08-13 1994-08-05 T cell epitopes of ryegrass pollen allergen
US08/737,904 US7112333B1 (en) 1994-08-05 1994-08-05 T cell epitopes of ryegrass pollen allergen
NZ271818A NZ271818A (en) 1993-08-13 1994-08-05 T cell epitopes of ryegrass pollen antigen
EP94925803A EP0714440A1 (fr) 1993-08-13 1994-08-05 Epitopes de lymphocyte t pour l'allergene du ray-grass
FI960629A FI960629L (fi) 1993-08-13 1996-02-12 Raiheinäsiitepöly allergeenin T-soluepitoopit
NO960553A NO960553L (no) 1993-08-13 1996-02-12 T-celle-epitoper av raigresspollen-allergen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10601693A 1993-08-13 1993-08-13
US08/106,016 1993-08-13

Publications (2)

Publication Number Publication Date
WO1995006728A2 true WO1995006728A2 (fr) 1995-03-09
WO1995006728A3 WO1995006728A3 (fr) 1995-05-04

Family

ID=22309029

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/009024 WO1995006728A2 (fr) 1993-08-13 1994-08-05 Epitopes de lymphocyte t pour l'allergene du ray-grass

Country Status (10)

Country Link
EP (1) EP0714440A1 (fr)
JP (1) JPH09504167A (fr)
AU (1) AU7559794A (fr)
CA (1) CA2168565A1 (fr)
FI (1) FI960629L (fr)
IL (1) IL110619A0 (fr)
NO (1) NO960553L (fr)
NZ (1) NZ271818A (fr)
WO (1) WO1995006728A2 (fr)
ZA (1) ZA946068B (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007428A1 (fr) * 1994-09-02 1996-03-14 Immulogic Pharmaceutical Corporation Compositions peptidiques pouvant reguler a la baisse une reponse immunitaire specifique a un antigene
WO1997005258A2 (fr) * 1995-08-02 1997-02-13 Biomay Produktions- Und Handelsgesellschaft Mbh Panallergene vegetal recombine de 60 kda (phosphoglycerate-mutase independante du cofacteur; e.c. 5.4.2.1.)
FR2809416A1 (fr) * 2000-05-29 2001-11-30 Tabacs & Allumettes Ind Clonage et sequencage de l'allergene dac g5 du pollen de dactilys glomerata, sa preparation et son utilisation
WO2003025009A1 (fr) 2001-09-20 2003-03-27 The University Of Melbourne Allergene de recombinaison presentant une fixation limitee a ige mais une antigenicite illimitee aux lymphocytes t
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
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
US7112333B1 (en) * 1994-08-05 2006-09-26 Heska Corporation T cell epitopes of ryegrass pollen allergen
WO2010125186A1 (fr) * 2009-04-30 2010-11-04 Stallergenes S.A. Procédé d'identification d'une espèce herbagère
US8551493B2 (en) 2007-08-15 2013-10-08 Circassia Limited Peptide with reduced dimer formation
US8551492B2 (en) 2007-06-01 2013-10-08 Circassia Limited Vaccine peptide combinations against cat allergy
US8753644B2 (en) 2009-02-05 2014-06-17 Circassia Limited Grass peptides for vaccine
WO2017004561A1 (fr) 2015-07-01 2017-01-05 Alk-Abelló A/S Associations de peptides et leurs utilisations dans le traitement de l'allergie aux graminées
US11524019B2 (en) 2017-08-21 2022-12-13 Glycom A/S Synthetic composition for reducing allergy symptoms

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004174A1 (fr) * 1991-08-16 1993-03-04 The University Of Melbourne Allergene du pollen de raygrass
WO1994004564A1 (fr) * 1992-08-14 1994-03-03 The University Of Melbourne EPITOPES DE LYMPHOCYTES T DE l'ALLERGENE DE POLLEN DE L'IVRAIE VIVACE

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004174A1 (fr) * 1991-08-16 1993-03-04 The University Of Melbourne Allergene du pollen de raygrass
WO1994004564A1 (fr) * 1992-08-14 1994-03-03 The University Of Melbourne EPITOPES DE LYMPHOCYTES T DE l'ALLERGENE DE POLLEN DE L'IVRAIE VIVACE

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLINICAL AND EXPERIMENTAL ALLERGY, vol.22, no.4, April 1992, OXFORD GB pages 491 - 497 KLYSNER ET AL. 'Group V allergens in grass pollens:IV.Similarities in amino acid compositions and NH2-terminal sequences of the Group V allergenes from Lolium perenne, Poa pratensis and Dactylis glomerata' cited in the application *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7112333B1 (en) * 1994-08-05 2006-09-26 Heska Corporation T cell epitopes of ryegrass pollen allergen
WO1996007428A1 (fr) * 1994-09-02 1996-03-14 Immulogic Pharmaceutical Corporation Compositions peptidiques pouvant reguler a la baisse une reponse immunitaire specifique a un antigene
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
WO1997005258A2 (fr) * 1995-08-02 1997-02-13 Biomay Produktions- Und Handelsgesellschaft Mbh Panallergene vegetal recombine de 60 kda (phosphoglycerate-mutase independante du cofacteur; e.c. 5.4.2.1.)
WO1997005258A3 (fr) * 1995-08-02 1997-03-27 Biomay Prod & Handel Panallergene vegetal recombine de 60 kda (phosphoglycerate-mutase independante du cofacteur; e.c. 5.4.2.1.)
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
FR2809416A1 (fr) * 2000-05-29 2001-11-30 Tabacs & Allumettes Ind Clonage et sequencage de l'allergene dac g5 du pollen de dactilys glomerata, sa preparation et son utilisation
WO2001092532A1 (fr) * 2000-05-29 2001-12-06 Seita Groupe Altadis Clonage et sequençage de l'allergene dac g5 du pollen de dactylis glomerata, sa preparation et son utilisation
US7285650B2 (en) 2000-05-29 2007-10-23 Seita Groupe Altadis Cloning and sequencing of the allergen Dac g5 of Dactylis glomerata pollen, its preparation and its use
US7666428B2 (en) 2001-09-20 2010-02-23 The University Of Melbourne Recombinant allergen with reduced IgE binding but undiminished T-cell antigenicity
EP1434793A1 (fr) * 2001-09-20 2004-07-07 The University Of Melbourne ALLERGENE DE RECOMBINAISON PRESENTANT UNE FIXATION LIMITEE A IgE MAIS UNE ANTIGENICITE ILLIMITEE AUX LYMPHOCYTES T
WO2003025009A1 (fr) 2001-09-20 2003-03-27 The University Of Melbourne Allergene de recombinaison presentant une fixation limitee a ige mais une antigenicite illimitee aux lymphocytes t
EP1434793A4 (fr) * 2001-09-20 2006-08-30 Univ Melbourne ALLERGENE DE RECOMBINAISON PRESENTANT UNE FIXATION LIMITEE A IgE MAIS UNE ANTIGENICITE ILLIMITEE AUX LYMPHOCYTES T
US8551492B2 (en) 2007-06-01 2013-10-08 Circassia Limited Vaccine peptide combinations against cat allergy
US9168295B2 (en) 2007-06-01 2015-10-27 Circassia Limited Vaccine peptide combinations
US9340580B2 (en) 2007-08-15 2016-05-17 Circassia Limited Peptide with multiple epitopes
US9744222B2 (en) 2007-08-15 2017-08-29 Circassia Limited Peptide for vaccine
US8551493B2 (en) 2007-08-15 2013-10-08 Circassia Limited Peptide with reduced dimer formation
US8652485B2 (en) 2007-08-15 2014-02-18 Circassia Limited Peptide for vaccine
US8753644B2 (en) 2009-02-05 2014-06-17 Circassia Limited Grass peptides for vaccine
EP2891662A1 (fr) * 2009-02-05 2015-07-08 Circassia Limited Peptides d'herbe pour vaccin
EP2249162A1 (fr) * 2009-04-30 2010-11-10 Stallergenes Sa Procédé pour l'identification d'espèces gazeuses
WO2010125186A1 (fr) * 2009-04-30 2010-11-04 Stallergenes S.A. Procédé d'identification d'une espèce herbagère
WO2017004561A1 (fr) 2015-07-01 2017-01-05 Alk-Abelló A/S Associations de peptides et leurs utilisations dans le traitement de l'allergie aux graminées
US11013781B2 (en) 2015-07-01 2021-05-25 Alk-Abelló As Peptide combinations and uses thereof for treating grass allergy
US11524019B2 (en) 2017-08-21 2022-12-13 Glycom A/S Synthetic composition for reducing allergy symptoms

Also Published As

Publication number Publication date
WO1995006728A3 (fr) 1995-05-04
AU7559794A (en) 1995-03-22
FI960629A0 (fi) 1996-02-12
NZ271818A (en) 1997-11-24
JPH09504167A (ja) 1997-04-28
FI960629L (fi) 1996-04-12
ZA946068B (en) 1995-03-28
IL110619A0 (en) 1994-11-11
EP0714440A1 (fr) 1996-06-05
CA2168565A1 (fr) 1995-03-09
NO960553L (no) 1996-04-12
NO960553D0 (no) 1996-02-12

Similar Documents

Publication Publication Date Title
US5710126A (en) T cell epitopes of ryegrass pollen allergen
AU684501B2 (en) T cell epitopes of ryegrass pollen allergen
WO1994001560A1 (fr) Proteines et peptides allergenes tires du pollen du cedre du japon
EP0714440A1 (fr) Epitopes de lymphocyte t pour l'allergene du ray-grass
AU2300492A (en) Allergenic proteins and peptides from japanese cedar pollen
NZ252013A (en) T cell receptor epitopes for short ragweed pollen
AU5668194A (en) Allergenic proteins and peptides from japanese cedar pollen
WO1994011512A9 (fr) Proteines et peptides allergenes provenant du pollen du cedre du japon
CA2142370C (fr) Epitopes de cellules t d'un allergene de pollen de ray-grass anglais
US7112333B1 (en) T cell epitopes of ryegrass pollen allergen
EP0659214A1 (fr) Proteines et peptides allergenes tires du pollen du cedre du japon
AU697491B2 (en) Nucleic acids encoding a house dust mite allergen, Der p III, and uses therefor
US6090386A (en) T cell peptides of the CRX JII allergen
WO1996013589A1 (fr) Epitopes des lymphocytes t des allergenes majeurs provenant d'ambrosia artemisiifolia
AU2003279352A1 (en) Dna sequence and recombinant production of the grass pollen allergen phl p4
JP4176820B2 (ja) スギ花粉アレルゲンＣｒｙｊＩＩエピトープ
AU3438493A (en) Allergenic proteins and peptides from japanese cedar pollen

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 1994925803

Country of ref document: EP

AK Designated states

Kind code of ref document: A2

Designated state(s): AT AU BB BG BR BY CA CH CN CZ DE DK ES FI GB HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2168565

Country of ref document: CA

Ref document number: 271818

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 960629

Country of ref document: FI

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1994925803

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 08737904

Country of ref document: US

WWW Wipo information: withdrawn in national office

Ref document number: 1994925803

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载