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WO1996013589A1 - Epitopes des lymphocytes t des allergenes majeurs provenant d'ambrosia artemisiifolia - Google Patents

Epitopes des lymphocytes t des allergenes majeurs provenant d'ambrosia artemisiifolia Download PDF

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Publication number
WO1996013589A1
WO1996013589A1 PCT/US1995/014362 US9514362W WO9613589A1 WO 1996013589 A1 WO1996013589 A1 WO 1996013589A1 US 9514362 W US9514362 W US 9514362W WO 9613589 A1 WO9613589 A1 WO 9613589A1
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seq
amb
rae
peptide
gly
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PCT/US1995/014362
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English (en)
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Mei-Chang Kuo
Richard Garman
Julia Greenstein
Sean Evans
Kent Amsberry
Zéev SHAKED
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Immulogic Pharmaceutical Corporation
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Priority to AU41467/96A priority Critical patent/AU4146796A/en
Publication of WO1996013589A1 publication Critical patent/WO1996013589A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Ambrosia artemisiifolia or short ragweed pollen is the major cause of late summer hayfever in North America and Canada and considerable effort has been expended in trying to identify the major allergens produced by this species.
  • Amb a I or Antigen E (AgE) has been reported to be the predominant allergen. (King, T.P., et al. Biochemistry, 2:458 (1964)). AgE has been characterized and reported to be a nonglycosylated protein of 38kD molecular mass (King, T.P., Adv. Immunol. 21-77 (1976); King, T.P., et al., Arch. Biochem. Biophys., 212: 127 (1981)).
  • Amb a ll (AgK) An immunochemically related protein, Amb a ll (AgK), has been reported to have similar properties (King, T.P., Adv. Immunol. 21-77 (1976); King. T.P., Biochemistry, ⁇ :367 (1972)).
  • Amb a I or AgE can be purified using conventional chromatographic or biochemical techniques. However, it has been reported that due to cleavage of the 38 kD single-chain precursors by the action of a trypsin-like pollen protease, purification often results in the isolation of two noncovalently associated chains of 26 and 12 kD molecular mass, designated a and ⁇ , respectively (King, T.P., et al., Arch. Biochem.
  • the present invention provides isolated peptides of the major protein allergens of Ambrosia artemisiifolia including peptides derived from the family of related proteins, previously designated Amb a IA, Amb a IB, Amb a IC, and Amb a ID. These allergens have been renamed according to the IUIS approved nomenclature as Amb a 1.1 ⁇ Amb a IA), Amb a 1.2 ⁇ Amb a IB), Amb a 1.3 ⁇ Amb a IC) and Amb a 1.4 ⁇ Amb a ID).
  • Peptides within the scope of the invention comprise at least one T cell epitope, preferably at least two T cell epitopes, of a protein allergen selected from the family of Amb a I allergens and Amb a II.
  • the invention further provides peptides comprising at least two regions, each region comprising at least one T cell epitope of a ragweed pollen allergen. The regions are derived from the same or from different ragweed pollen allergens.
  • 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.
  • Peptides of the invention are capable of modifying, in a ragweed pollen-sensitive individual to whom they are administered, the allergic response of the individual to a ragweed pollen allergen. Methods of treatment or of diagnosis of sensitivity to a ragweed pollen allergen in an individual and therapeutic compositions comprising one or more peptides of the invention are also provided and human clinical testing is described.
  • Fig. 1 shows Western blot analysis of IgE binding to recombinant Amb a ⁇ proteins.
  • Fig. 2 is a graphic representation of a direct binding assay of IgE from a single ragweed allergic patient to recombinant Amb a I and Amb a ⁇ proteins.
  • Fig. 3 is a graphic representation of the results of a direct binding assay of IgE from pooled human sera to native Amb a I, Amb a II, recombinant Amb a 1.1 . recombinant Amb a II and pollen extract.
  • Fig. 4A and 4B are graphic representations depicting the responses of lymph node cells isolated from mice tolerized in vivo with either Amb a I.l or PBS and CFA and challenged in vitro with various antigens.
  • Fig. 5 A-5F are graphic representations depicting the responses of lymph node cells isolated from mice tolerized in vivo with Amb a I.l or pollen extract, challenged with Amb a I.l, and tested with various antigens.
  • Fig. 6A-6F are graphic representations depicting the responses of lymph node cells isolated from mice tolerized with pollen extract challenged with pollen extract, and tested with various antigens.
  • Fig. 7 shows various peptides of desired length derived from the Amb a I.l, Amb a 1.2 and Amb a 1.3 protein allergens.
  • Fig. 8 is a graphic representation depicting the responses of T cell lines from 39 patients primed in vitro to recombinant Amb a 1.1 protein and analyzed for response to various overlapping Amb a 1.1 peptides and selected Amb a 1.2 and Amb a 1.3 peptides by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 9 shows selected peptides of desired lengths derived from the Amb a I. l protein allergen.
  • Fig. 10 is a graphic representation depicting the responses of T cell lines from 48 patients primed in vitro to recombinant Amb a I.l protein and analyzed for response to selected peptides derived from Region 1 of the Amb a I.l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 1 1 is a graphic representation depicting the responses of T cell lines from 48 patients primed in vitro to recombinant Amb a ⁇ . ⁇ protein and analyzed for response to selected peptides derived from Region 2 of the Amb a 1.1 protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 12 is a graphic representation depicting the responses of T cell lines from 48 patients primed in vitro to recombinant Amb a I.l protein and analyzed for response to selected peptides derived from Region 3 of the Amb a I. l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 13 is a graphic representation depicting the responses of T cell lines from 48 patients primed in vitro to recombinant Amb a I.l protein and analyzed for response to selected peptides derived from Region 4 of the Amb a 1.1 protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 14 shows selected peptides of desired lengths derived from the Amb a 1.1 protein allergen and the Amb a 1.3 protein allergen.
  • Fig. 15 is a graphic representation depicting the responses of T cell lines from 23 patients primed in vitro to recombinant Amb a l. l protein and analyzed for response to selected peptides derived from Region 1 of the Amb a I.l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 14 shows selected peptides of desired lengths derived from the Amb a 1.1 protein allergen and the Amb a 1.3 protein allergen.
  • Fig. 15 is a graphic representation depicting the responses of T cell lines from 23 patients primed in vitro to recombinant Amb a l. l protein and analyzed for response to selected peptides derived from Region 1 of the Amb a I.l protein, by
  • 16 is a graphic representation depicting the responses of T cell lines from 23 patients primed in vitro to recombinant Amb a 1.1 protein and analyzed for response to selected peptides derived from Region 2 of the Amb a I. l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 17 is a graphic representation depicting the responses of T cell lines from 23 patients primed in vitro to recombinant Amb a l.l protein and analyzed, for response to selected peptides derived from Region 3 of the Amb a I.l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 18 is a graphic representation depicting the responses of T cell lines from 23 patients primed in vitro to recombinant Amb a l ⁇ protein and analyzed for response to selected peptides derived from Region 4 of the Amb a I.l protein, by percent of positive responses within the individuals tested, the mean stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
  • Fig. 19 is a graphic representation depicting the responses of T cell lines of 9 patients primed in vitro to recombinant Amb a 1.1 protein or recombinant Amb a 1.3 protein and analyzed for response to selected peptides derived from Amb a I.l, by percent of positive responses within the individuals tested and the mean stimulation index of positive responses for the peptide.
  • Fig. 20 is a graphic representation depicting the responses of T cell lines of 9 patients primed in vitro to recombinant Amb a I.l protein or recombinant Amb a 1.3 protein and analyzed for response to selected peptides derived from Amb a 1.3, by percent of positive responses within the individuals tested and the mean stimulation index of positive responses for the peptide.
  • Fig. 21 is a graphic representation of a direct binding assay of IgE from a single ragweed allergic patient to peptides derived from Amb a I.
  • Fig. 22 is a graphic representation depicting the responses of T cell lines of 28 patients primed in vitro to recombinant Amb a 1.1 protein and analyzed for response to selected peptides derived from Amb a I.l by percent of positive responses within the individuals tested, the mean stimulation index of positive responses to the peptide and the ranked sum of peptide responses.
  • Fig. 23 is a graphic representation depicting the responses of T cell lines of 28 patients primed in vitro to recombinant Amb a l ⁇ protein and analyzed for response to selected peptides derived from Region 4 of Amb a I.l by percent of positive responses within the individuals tested, the mean stimulation index of positive responses to the peptide and the ranked sum of peptide responses.
  • Fig. 24 is a graphic representation depicting the responses of T cell lines of 32 patients primed in vitro to recombinant Amb a I.l protein and analyzed for response to six selected peptides derived from Amb a I.l by percent of positive responses within the individuals tested, the mean stimulation index of positive responses to the peptide and the ranked sum of peptide responses.
  • Fig. 25 shows various peptides derived from peptide RAE 70.1 which include modifications designed to increase the solubility of the peptide.
  • Fig. 26 is a graphic representation depicting the response of a T cell line from patient 956.2 primed in vitro to Fel d I and analyzed for response to various peptides derived from the Amb a 1.1 protein.
  • Fig. 27 is a graphic representation depicting the response of a T cell line from patient 1 19 primed in vitro with recombinant Amb a l ⁇ and analyzed for response to various modified peptides derived from Region 2 of the Amb a I.l protein by tritiated thymidine incorporation.
  • Fig. 28 is a graphic representation depicting the response of a T cell line from patient 1199 primed in vitro with recombinant Amb a l ⁇ and analyzed for response to various modified peptides derived from Region 2 of the Amb a 1.1 protein by tritiated thymidine incorporation.
  • Fig. 29 is a graphic representation depicting the response of a T cell clone generated by limiting dilution from an Amb a 1.1 specific T cell line stimulated with the AMB 2-10.1 peptide, primed in vitro w/recombinant Amb a I.l and analyzed for response to various modified peptides derived from Region 2 of the Amb a 1.1 protein by tritiated thymidine incorporation.
  • Fig. 30 is a graphic representation depicting the percent of total histamine release in blood samples from 8 ragweed-allergic patients in response to selected peptides derived from ihfm Amb a I. ⁇ protein.
  • Fig. 31 is a graphic representation depicting the responses of T cell lines from 39 patients primed in vitro to purified native Amb a l ⁇ protein and analyzed for response to various overlapping Amb a 1.1 peptides by percent of positive responses within the individuals tested (above the bar), the mean stimulation index of positive responses for the peptide (in parenthesis above the bar) and the ranked sum of peptide responses (X axis).
  • the present invention provides isolated peptides derived from the major protein allergens of Ambrosia artemisiifolia.
  • a peptide refers to an amino acid sequence having fewer amino acids than the entire amino acid sequence of a protein from which the peptide is derived.
  • Peptides of the invention include peptides derived from Amb a I ⁇ . Amb a 1.2, Amb a 1.3, Amb a 1.4 and Amb a II which comprise at least one T cell epitope of the allergen.
  • Peptides comprising at least two regions, each region comprising at least one T cell epitope of a protein allergen of Ambrosia artemisiifolia are also within the scope of the invention. Each region of such peptides is derived from the same or from different ragweed pollen allergens. Isolated peptides or regions of isolated peptides, each comprising at least two T cell epitopes of a ragweed pollen allergen are particularly desirable for increased therapeutic effectiveness. Peptides which are immunologically related (e.g., by antibody or T cell cross-reactivity) to peptides of the present invention are also within the scope of the invention.
  • Peptides immunologically related by antibody cross-reactivity are bound by antibodies specific for a peptide of a protein allergen of Ambrosia artemisiifolia.
  • Peptides immunologically related by T cell cross-reactivity are capable of reacting with the same T cells as a peptide of the invention.
  • the present invention also pertains to a ragweed pollen allergen encoded by a nucleic acid sequence of clone IPC 1/5.
  • the full-length nucleic acid sequence of clone IPC 1/5 has been determined and the encoded protein has been produced recombinantly in both the pSEM vector (as a fusion protein with ⁇ -galactosidase) and the pETl Id vector.
  • the recombinant protein was determined to bind approximately 10-20% of allergic serum IgE on a Western blot.
  • the protein encoded by clone IPC 1/5 was found to have a high degree of amino acid sequence homology with cysteine proteinase inhibitors in man and rice.
  • the protein has 66.6% homology with the rice protein oryzacystatin-I.
  • the nucleic acid sequence and deduced amino acid sequence of the allergen encoded by clone IPC 1/5 is represented in SEQ ID NO. 11 and 12.
  • Isolated proteins and isolated peptides of the invention can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid having a sequence encoding such protein or peptide.
  • isolated proteins and isolated peptides of the invention can also be produced by chemical synthesis. In certain limited situations, isolated peptides can be produced by chemical cleavage of a protein allergen.
  • host cells transformed with a nucleic acid having a sequence encoding the protein or peptide or the functional equivalent of the nucleic acid sequence are cultured in a medium suitable for the cells and protein or peptides can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying proteins and peptides including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or immunopurification with antibodies specific for the protein or peptide, the protein allergen of Ambrosia artemisiifolia from which the peptide is derived, or a portion thereof.
  • protein and peptides of the present invention are substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically.
  • Recombinant ragweed pollen proteins including recombinant Amb a 1.1 , Amb a 1.2, Amb a 1.3, Amb a 1.4, and Amb a II have been produced.
  • Suitable expression vectors for producing recombinant protein and recombinant peptides of the invention include pTRC, pGEX, pMAL, pRIT5, pETl Id and pCA.
  • the use of pTRC, pETl 1 d and pGEX as expression vectors will result in expression of ragweed pollen protein as an unfused protein.
  • the use of pMAL, pRIT5, pCA and pSEM as expression vectors will result in expression of ragweed pollen protein fused to maltose E binding protein (pMAL), protein A (pRIT5), truncated protein A (pCA), or ⁇ -galactosidase (pSEM).
  • Suitable expression vectors are commercially available. When produced as a fusion protein, recombinant ragweed pollen protein can be recovered from the fusion protein through enzymatic or chemical (e.g., cyanogen bromide or dilute acid) cleavage and biochemical purification. For example, enzymatic cleavage sites for Factor Xa or thrombin can be introduced at the fusion junction between the carrier protein (e.g., Protein A) and the ragweed pollen protein.
  • Suitable host cells for expression of recombinant ragweed pollen protein include bacteria, yeast and insect or mammalian cells. Appropriate vectors for expression in yeast include YepSec, pMFa and JRY88. These vectors are also commercially available.
  • a ragweed pollen allergen is divided into non-overlapping peptides of desired lengths or overlapping peptides of desired lengths as discussed in Example V which may be produced recombinantly, synthetically or in certain limited situations by chemical cleavage of the allergen.
  • Peptides comprising at least one T cell epitope are capable of reducing T cell responsiveness or inducing T cell nonresponsiveness.
  • 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 nonresponsiveness are defined as having T cell stimulating activity.
  • human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to a ragweed pollen allergen, (i.e., an individual who has an IgE mediated immune response to a ragweed pollen allergen) with a peptide derived from the allergen and determining whether proliferation of T cells occurs in response to the peptide as measured, e.g., by cellular uptake of tritiated thymidine.
  • stimulation indices for responses by T cells to peptides can be calculated as the maximum CPM in response to a peptide divided by the medium control CPM.
  • a peptide comprising at least one T cell epitope when determined by T cell stimulation requires a stimulation index of at least 2.0.
  • a peptide having a T cell stimulation index of 2.0 is considered useful as a therapeutic agent.
  • Preferred peptides have a stimulation index of at least 2.5, more preferably at least 3.5, and most preferably at least 5.0.
  • 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. If two or more peptides which share an area of overlap in the native protein sequence are found to have human T cell stimulating activity, as determined by T cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be tested by a similar procedure. Following this technique, 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 ragweed pollen, and the potential cross-reactivity of the peptide with Amb a I family members and Amb a ll.
  • the physical and chemical properties of these selected peptides e.g., solubility, stability
  • the ability of the selected peptides or selected modified peptides to stimulate human T cells e.g., induce proliferation, lymphokine secretion is determined.
  • preferred peptides of the invention do not bind immunoglobulin E (IgE) or bind IgE to a substantially lesser extent (i.e. preferably at least 100 fold and more preferably at least 1000 fold less) than the protein allergen from which the peptide is derived binds IgE.
  • Recombinant ragweed pollen allergens including recombinant Amb a I A, Amb a 1.2, Amb a 1.3, Amb a 1.4, and Amb a II have been produced and shown to have reduced IgE binding activity as compared to the corresponding native protein allergen (See Fig. 3).
  • the major complications of standard immunotherapy are 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 cells or basophils and the release of mediators (e.g., histamine, serotonin, eosinophil chemotacic factors).
  • mediators e.g., histamine, serotonin, eosinophil chemotacic factors.
  • 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 protein allergen (e.g., Amb a I.l).
  • a peptide or recombinant protein of the invention when administered to a ragweed pollen-sensitive individual, is capable of modifying the allergic response of the individual to the allergen.
  • peptides of the invention comprising at least one T cell epitope of a ragweed pollen allergen or at least two regions derived from a ragweed pollen allergen each comprising at least one T cell epitope, when administered to a ragweed pollen-sensitive individual are capable of modifying the T cell response of the individual to the allergen.
  • modification of the allergic response of a ragweed pollen-sensitive individual to a ragweed pollen allergen can be defined as non-responsiveness or diminution in symptoms to a ragweed pollen allergen, as determined by standard clinical procedures (see e.g., Vamey et al.. British Medical Journal 302: 265-269 (1990)).
  • peptides derived from ragweed pollen allergens comprising at least one T cell epitope have been produced. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to ragweed pollen allergen(s) which are responsible for the clinical symptoms of ragweed pollen 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, the recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies.
  • IgE is fundamentally important in the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted.
  • a T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acid residues essential to receptor recognition which may be contiguous and/or non-contiguous in the amino acid sequence of the protein.
  • Amino acid sequences which mimic those of T cell epitopes and which modify the allergic response to protein allergens of Ambrosia artemisiifolia are within the scope of this invention.
  • Exposure of ragweed pollen allergic patients to peptides of the present invention, in a non-immunogenic form, may induce T cell non-responsiveness of appropriate T cell subpopulations such that they become non-responsive to ragweed pollen allergen(s) and do not participate in mounting an immune response upon such exposure.
  • T cell non ⁇ responsiveness (which includes 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))
  • a peptide of the invention may be administered in nonimmunogenic form as discussed above, in conjunction with a reagent capable of blocking costimulatory signals such that the level of T cell nonresponsiveness is enhanced.
  • administration of a peptide of the present invention may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring ragweed pollen allergen or portion thereof (e.g., result in a decrease of IL-4 and or an increase in IL- 2).
  • exposure to a peptide of the invention may influence T cell subpopulations which normally participate in the response to ragweed pollen allergen(s) 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 peptide.
  • This redistribution of T cell subpopulations may ameliorate or reduce the ability of an individual's immune system to stimulate the immune response at the site of normal exposure to the ragweed pollen allergen(s), resulting in a diminution in allergic symptoms.
  • Isolated peptides of the invention comprise at least one T cell epitope of a protein allergen of Ambrosia artemisiifolia (i.e., the peptide comprises at least approximately seven amino acid residues of the protein allergen).
  • therapeutic compositions of the invention preferably comprise at least two T cell epitopes of a ragweed pollen allergen.
  • isolated peptides of the invention preferably comprise at least two T cell epitopes (i.e., the peptide comprises at least approximately eight amino acid residues, and preferably fifteen amino acid residues).
  • isolated peptides of the invention preferably comprise a sufficient percentage of the T cell epitopes of the entire protein allergen such that upon administration of the peptide to an individual sensitive to ragweed pollen, T cells of the individual are rendered non-responsive to the protein allergen.
  • Isolated peptides of the invention comprising up to approximately 45 amino acid residues in length, and most preferably up to approximately 30 amino acid residues in length are particularly desirable as increases in length may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g., immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived. All of the peptides shown in Fig.
  • a peptide (candidate peptide) or a combination of candidate peptides are likely contain a sufficient percentage of T cell epitopes of ragweed protein antigen to induce non-responsiveness in a substantial percentage of a population of individuals sensitive to the protein antigen.
  • an algorithm can be used. In accordance with one such algorithm, a human T cell stimulation index (discussed above) for the peptide(s) in an in vitro T cell proliferation assay is calculated for each individual tested in a population of individuals sensitive to ragweed protein allergen.
  • the remaining peptides in the in vitro T cell proliferation assay are overlapping peptides (overlapping by between about 5 - 10 amino acid residues) which cover the remainder of the protein not covered by the candidate peptide(s), which peptides are at least 12 amino acids long and which are preferably no longer than 30 and more preferably no longer than 25 amino acid residues in length.
  • a human T cell stimulation index for each such remaining peptide in the set of peptides produced in the in vitro T-cell proliferation assay with T-cells obtained from each individual in the population of individuals tested is calculated and added together.
  • the human T cell stimulation index for the candidate peptide(s) is divided by the sum of the human T cell stimulation indices of the remaining peptides in the set of peptides tested to determine a percent. This percent is obtained for at least twenty (20) and preferably at least thirty (30) individuals sensitive to the protein antigen of interest and a mean percent is determined (the percentage of positive T cell responses (S.I. greater than or equal to 2.0) in response to the candidate peptide or combination of candidate peptides).
  • Preferred peptides comprise all or a portion of the areas of major T cell reactivity within the Amb a I.l protein allergen, i.e., Region 1, Region 2, Region 3 and Region 4. Each area is broadly defined as follows: Region 1 comprises amino acid residues 48-107; Region 2 comprises amino acid residues 171-216; Region 3 comprises amino acid residues 278-322; and Region 4 comprises amino acid residues 331-377. Preferred areas of major T cell reactivity within each Region comprise: amino acid residues 57-101 ; amino acid residues 182-216; amino acid residues 280-322; and amino acid residues 342-377. Similar areas of major T cell reactivity can be found within the other Amb a I family members (i.e., Amb a 1.2.
  • Amb a 1.3 and Amb a 1.4), and Amb a II As shown in Example VIII, the Amb a I protein allergens and Amb a II demonstrate a high degree of T cell cross-reactivity. Given this cross- reactivity, shared areas of major T cell reactivity and shared T cell epitopes are likely to be found in conserved regions between Amb a I and the remaining Amb a I family members and Amb a ll. For example. Amb a 1.1 stimulated T cells have been shown to recognize both Amb a I.l derived peptides and homologous Amb a 1.3 derived peptides (See Example IX). Similarly, Amb a 1.3 stimulated T cell recognize both Amb a I.l and Amb a 1.3 derived peptides.
  • Preferred ragweed pollen peptides comprise all or a portion of the following peptides: RAE 67.1 (SEQ ID NO: 13); RAE 57.1 (SEQ ID NO: 14); RAE 24.E (SEQ ID NO: 15): RAE 24.1 (SEQ ID NO: 16); RAE 22.E (SEQ ID NO: 17); RAE 22.E-1 (SEQ ID NO: 18); RAE 3.D (SEQ ID NO: 19); RAE 3.1 (SEQ ID NO:20); RAE 22.E-2 (SEQ ID NO:21); RAE 5.D (SEQ ID NO:22); RAE 6.D (SEQ ID NO:23); RAE 6.1 (SEQ ID NO:24); RAE 7.D (SEQ ID NO:25); RAE 7.D-1 (SEQ ID NO:26); RAE 40.1-6 (SEQ ID NO:27); RAE 40.1-5 (SEQ ID NO:28); RAE 40.1-4 (SEQ ID NO:29); RAE 40.D (SEQ ID NO:30); R
  • AMB 4-9.1QP (SEQ ID NO: 146); AMB 4-9. IDA (SEQ ID NO: 147); amb 4-9. IDS (SEQ ID NO.148); AMB 4-9.1DG (SEQ ID NO:149); and RA-02.1 (SEQ ID NO:150), the amino acid sequences of such peptides being shown in Figs. 7, 14, 23, 24 and 25.
  • Particularly preferred peptides comprise all or a portion of the following peptides: AMB 1-2.1 (SEQ ID NO:86); AMB 2-6.1 (SEQ ID NO:93); AMB 2-4.1 (SEQ ID NO:90); Amb 2-36.1 (SEQ ID NO.T39); Amb 2-38.1 (SEQ ID NO: 141); RA-02.1 (SEQ ID NO:150); AMB 2-9.1 (SEQ ID NO:98); AMB 3-5.1 (SEQ ID NO:102); and AMB 4-9.1 (SEQ ID NO:110).
  • Another embodiment of the present invention provides peptides comprising at least two regions, each region comprising at least one T cell epitope of a protein allergen of
  • Ambrosia artemisiifolia (e.g., each region comprises at least approximately seven amino acid residues).
  • These peptides comprising at least two regions can comprise as many amino acid residues as desired and preferably comprise at least about 7, more preferably at least about 15, even more preferably about 30 and most preferably at least about 40 amino acid residues of a ragweed pollen allergen.
  • Each region of such peptide preferably comprises up to 45 amino acid residues in length, more preferably up to 40 residues in length and most preferably up to 30 amino acid residues in length as increases in length of a region may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g., immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived.
  • the amino acid sequences of the regions can be produced and joined by a linker to increase sensitivity to processing by antigen-presenting cells.
  • linker can be any non-epitope amino acid sequence or other appropriate linking or joining agent.
  • the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in the allergen.
  • the regions containing T cell epitope(s) can be arranged in a noncontiguous configuration and can preferably be derived from the same protein allergen.
  • Noncontiguous is defined as an arrangement of regions containing T cell epitope(s) which is different than that of an amino acid sequence present in the protein allergen from which the regions are derived.
  • the noncontiguous regions containing T cell epitopes can be arranged in a nonsequential order (e.g..
  • a peptide can comprise at least 15%, at least 30%, at least 50% or up to 100% of the T cell epitopes of a ragweed pollen allergen.
  • the individual peptide regions can be produced and tested to determine which regions bind immunoglobulin E specific for a ragweed pollen allergen and which of such regions would cause the release of mediators (e.g., histamine) from mast cells or basophils.
  • mediators e.g., histamine
  • Those peptide regions found to bind immunoglobulin E and cause the release of mediators from mast cells or basophils in greater than approximately 10-15% of the allergic sera tested are preferably not included in the peptide regions arranged to form peptides of the invention.
  • Preferred peptides of the invention comprise two or more regions derived from the same or from different ragweed pollen allergens (e.g., Amb a l. ⁇ , Amb a 1.2, Amb a 1.3, Amb a I A and Amb a II).
  • one region can be derived from Amb a l.l and one region can be derived from Amb a 1.2; one region can be derived from Amb a l ⁇ and one region can be derived from Amb a 1.3; one region can be derived from Amb a l ⁇ and one region can be derived from Amb a 1.4; one region can be derived from Amb a 1.2 and one region can be derived from Amb a 1.3; one region can be derived from Amb a 1.2 and one region can be derived from Amb a 1.4; one region can be derived from Amb a 1.3 and one region can be derived from Amb a 1.4; one region can be derived from Amb a I.l and one region can be derived from Amb a II; one region can be derived from Amb a 1.2 and one region can be derived from Amb a ll; one region can be derived from Amb a 1.3 and one region can be derived from Amb a
  • Regions of a peptide of the invention preferably comprise all or a portion of Region 1 , Region 2, Region 3 and Region 4 of Amb a I.l, and the above discussed preferred areas of major T cell reactivity within each Region. If Region 1, 2, 3 or 4 is found to bind IgE and cause the release of mediators from mast cells or basophils, then it is preferred that more than one region of the peptide comprise such Region and that the various regions of the peptide do not bind IgE or cause release of mediators from mast cells or basophils.
  • Examples of preferred regions include: AMB 1-1.1 (SEQ ID NO:85); AMB 1-2.1 (SEQ ID NO:86); AMB 1-3.1 (SEQ ID NO:87); AMB 1-4.1 (SEQ ID NO:84); AMB 1-5.1 (SEQ ID NO:83); AMB 1- 6.1 (SEQ ID NO:82); AMB 1 -4.15 (SEQ ID NO:88); AMB 1-2.15 (SEQ ID NO:89); AMB 2-4.1 (SEQ ID NO:90); AMB 2-3.1 (SEQ ID NO.91); AMB 2-5.1 (SEQ ID NO.92); AMB 2- 6.1 (SEQ ID NO:93); AMB 2-2.1 (SEQ ID NO.94); AMB 2-1.1 (SEQ ID NO:95); AMB 2- 7.1 (SEQ ID NO:96); AMB 2-8.1 (SEQ ID NO:97); AMB 2-9.1 (SEQ ID NO:98); AMB 2- 10.1 (SEQ ID NO.99); AMB 2-1 1.1 (SEQ ID NO.100); AMB 2-1.15 (S
  • Preferred peptides comprise various combinations of two or more regions, each region comprising all or a portion of Region 1, Region 2, Region 3 or Region 4 of Amb a I.l.
  • Preferred peptides comprise various combinations of two or more regions, each region having an amino acid sequence as shown in Fig. 14, such combination of regions including the following: AMB 4-6.1 and RAE 70.1 (SEQ ID NO:l 11 and SEQ ID NO:44); AMB 4-6.1 and AMB 2-5.1 (SEQ ID NO:l 11 and SEQ ID NO:92); AMB 4-9.1 and AMB 2-5.1 (SEQ ID NO: 110 and SEQ ID NO:92); AMB 4-9.1 and RAE 70.1 (SEQ.
  • AMB 4-6.1, AMB 2-5.1 and AMB 1-2.1 (SEQ ID NO.T 11, SEQ ID NO:92 and SEQ ID NO:86); AMB 4-9.1, AMB 2-5.1 and AMB 1-2.1 (SEQ ID NO: 1 10, SEQ ID NO.92 and SEQ ID NO:86); AMB 4-6.1, RAE 70.1 and AMB 1-2.1 (SEQ ID NO.T 1 1, SEQ ID NO:44 and SEQ ID NO:86); AMB 4-9.1, RAE 70.1 and AMB 1-2.1 (SEQ ID NO: l 10, SEQ ID NO:44 and SEQ ID NO:86); AMB 4-6.1, RAE 70.1, AMB 1-2.1 and AMB 3-5.1 (SEQ ID NOT 11, SEQ ID NO:44, SEQ ID NO:86 and SEQ ID NO: 102); AMB 4-9.1, RAE 70.1, AMB 1-2.1 and AMB 3-5.1 (SEQ ID NOT 11, SEQ ID NO:44, SEQ ID NO:86 and SEQ ID NO: 102); AMB 4-9.1, RAE 70.1, AMB 1-2.1
  • AMB 2-1.15 and AMB 4-3.15 (SEQ ID NOT01 , and SEQ ID NO.l 16); AMB 1-2.15, AMB 2-1.15 and AMB 4-3.15 (SEQ ID NO:89, SEQ ID NOT01, and SEQ ID NOT 16); and AMB 1-2.15, AMB 2-1.15, AMB 4- 3.15 and AMB 3-4.15 (SEQ ID NO:89, SEQ ID NO: 101, SEQ ID NOT 16 and SEQ ID NO: 107).
  • Peptides of protein allergens of Ambrosia artemisiifolia within the scope of the invention can be used in methods of treating and preventing allergic reactions to ragweed pollen allergens.
  • one aspect of the present invention provides therapeutic compositions comprising a peptide of Amb a l ⁇ .
  • Amb a 1.2, Amb a 1.3, Amb a 1.4 or Amb a II including at least one T cell epitope. or preferably at least two T cell epitopes. and a pharmaceutically acceptable carrier or diluent.
  • the therapeutic composition comprises a pharmaceutically acceptable carrier or diluent and a peptide comprising at least two regions, each region comprising at least one T cell epitope of a ragweed pollen allergen and is derived from the same or from different ragweed pollen allergens.
  • Administration of the therapeutic compositions of the present invention to desensitize an individual can be carried out using known techniques.
  • a peptide derived from a ragweed pollen allergen comprising at least one T cell epitope can be administered in combination with an appropriate diluent, or carrier.
  • peptides are administered in soluble form.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al.,
  • the therapeutic composition is preferably administered in non-immunogenic form, e.g., one that does not include adjuvant.
  • non-immunogenic form e.g., one that does not include adjuvant.
  • Such compositions will generally be administered by injection (e.g., intravenous, subcutaneous, intramuscular), oral administration, (e.g., as in the form of a capsule), inhalation, transdermal application or rectal administration.
  • therapeutic compositions are administed subcutaneously.
  • isolated and purified native ragweed pollen protien allergens see,
  • Example 1 for isolation and purification of ragweed pollen protein antigens) or portions thereof, can be administered orally.
  • the therapeutic compositions of the invention are administered to ragweed pollen- sensitive individuals at dosages and for lengths of time effective to reduce sensitivity (i.e., reduce the allergic response) of an individual to a ragweed pollen allergen.
  • a therapeutically effective amount of one or more of the same or of different therapeutic compositions can be administered simultaneously or sequentially to a ragweed pollen-sensitive individual. Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to ragweed pollen allergens, the age. sex, and weight of the individual, and the ability of the peptide to stimulate a T cell response in the individual.
  • Unit dosage form refers to physically discrete units suited as unitary dosages for human subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier.
  • the specification for the novel unit dosage 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 human subjects.
  • Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered over the course of days, weeks, months or years, or the dose may be proportionally increased or reduced with each subsequent injection as indicated by the exigencies of the therapeutic situation.
  • subcutaneous injections of therapeutic compositions are given once a week for 3-6 weeks. The dosage may remain constant for each injection or may increase or decrease with each subsequent injection.
  • a booster injection may be administered at intervals of about three months to about one year after initial treatment and may involve only a single injection or may involve another series of injections similar to that of the initial treatment.
  • a composition of the invention by other than parenteral administration, (i.e. oral administration) it may be necessary to coat the composition with, or co-administer the composition with, a material to prevent its inactivation or enhance its absorption and bioavailability.
  • a peptide formulation 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) J. Neuroimmunol. x 7:27).
  • the peptide When a peptide is suitably protected, 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, solutions, gels, 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.
  • the active compound may be incorporated into sustained-release or controlled release (steady state or pulsatile release) preparations and formulations.
  • compositions comprising at least two peptides (e.g., a physical mixture of at least two peptides). each comprising at least one T cell epitope of a protein allergen of Ambrosia artemisiifolia.
  • Compositions comprising several peptides or combinations of separate peptides can include as many peptides as desired (e.g., 5, 6, 7...) for therapeutic efficacy.
  • the peptides are derived from the same or from different ragweed pollen allergens.
  • Such compositions can be administered in the form of a therapeutic composition with a pharmaceutically acceptable carrier or diluent.
  • peptides are administered in soluble form.
  • compositions and preferred combinations of peptides which can be administered simultaneously or sequentially include the following combinations: AMB 4-6.1 and RAE 70.1 (SEQ ID NO:l 1 1 and SEQ ID NO:44); AMB 4-6.1 and AMB 2-5.1 (SEQ ID NOT 1 1 and SEQ ID NO:92); AMB 4-9.1 and AMB 2-5.1 (SEQ ID NOT 10 and SEQ ID NO:92); AMB 4-9.1 and RAE 70.1 (SEQ.
  • compositions and preferred combinations of peptides for therapeutic administration include the following combinations: AMB 1-2.1 and AMB 4-9.1 (SEQ ID NO:86 and SEQ ID NOT 10); AMB 1-2.1, Amb 2-38.1 and AMB 4-9.1 (SEQ ID NO:86, SEQ ID NO:141 and SEQ ID NOT 10); AMB 1-2.1, Amb 2-38.1, AMB 4-9.1 and AMB 2-4.1 (SEQ ID NO:86, SEQ ID NO: 141, SEQ ID NOT 10 and SEQ ID NO:90); AMB 1-2.1. Amb 2-38.1, AMB 4-9.1, AMB 2-4.1 and AMB 3-5.1 (SEQ ID NO:86, SEQ ID NO: 141.
  • SEQ ID NO: 110, SEQ ID NO:90, and SEQ ID NO: 102 AMB 1-2.1, Amb 2-36.1 and AMB 4-9.1 (SEQ ID NO:86, SEQ ID NO: 139 and SEQ ID NO:l 10); AMB 1-2.1 , Amb 2-36.1.
  • AMB 4-9.1 and AMB 2-4.1 SEQ ID NO:86, SEQ ID NO: 139, SEQ ID NO: 1 10 and SEQ ID NO:90
  • the multipeptide formulation includes at least two or more peptides of ragweed pollen protein allergen having human T cell stimulating activity in an in vitro T cell proliferation assay (i.e., comprising at least one T cell epitope).
  • Special considerations when preparing a multipeptide formulation include maintaining the solubility and stability of all peptides in the formulation at a physiologically acceptable pH. This requires choosing one or more pharmaceutically acceptable carriers such as excipients which are compatible with all the peptides in the multipeptide formulation.
  • suitable excipients include sterile water, sodium phosphate, mannitol or both sodium phosphate and mannitol or any combination thereof.
  • an agent such as EDTA to prevent dimerization.
  • a preferred multipeptide formulation includes at least one first peptide and at least one second peptide of ragweed pollen protein each having human T cell stimulating activity and soluble at a physiologically acceptable pH and selected from the group of peptides.
  • the multipeptide formulation includes Peptides Amb 1-2.1, Amb 4- 9.1, Amb 2-36.1, and modifications thereof, and sodium phosphate and mannitol.
  • Peptides Amb 1-2.1, Amb 4-9.1, and Amb 2-36.1 are in the form of a lyophilized powder which is reconstituted in a physiologically acceptable carrier, such as sterile water, prior to use.
  • a multipeptide formulation comprising the three peptides were produced and used in Phase I human clinical trials (see Example XV).
  • the peptides were combined during manufacturing to produce a vial containing a sterile, pyrogen free, lyophilized powder having the following composition: Active: Peptide Amb 1-2.1 , Peptide Amb 4-9.1. and Peptide Amb 2-36.1
  • Diluent Sterile Water for Injection, U.S. P. (initial reconstitution)
  • the multipeptide formulation of the invention can be provided in the form of a kit, including instructions for use.
  • the present invention also provides methods of detecting sensitivity in individuals to ragweed pollen allergens comprising combining a blood sample obtained from the individual with a peptide of the present invention, under conditions appropriate for binding of blood components with the peptide and determining the extent to which such binding occurs.
  • the extent to which binding occurs is determined by assessing T cell function. T cell proliferation or a combination thereof.
  • Radio-allergergosorbent test (RAST), paper radioimmunosorbent test (PRIST), enzyme linked immunosorbent assay (ELISA), radioimmunoassays (RIA), immuno-radiometric assays (IRMA), luminescence immunoassays (LIA), histamine release assays and IgE immunoblots.
  • RAST radio-allergergosorbent test
  • PRIST paper radioimmunosorbent test
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassays
  • IRMA immuno-radiometric assays
  • LIA luminescence immunoassays
  • histamine release assays and IgE immunoblots.
  • the presence in individuals of IgE specific for ragweed protein allergen and the ability of T cells of the individual to respond to T cell epitope(s) of the protein allergen can be determined by administering to the individuals an Immediate Type Hypersensitivity test and a Delayed Type Hypersensivity test.
  • the individuals are administered an Immediate Type Hypersensitivity test (see e.g. Immunology (1985) Roitt, I.M., Brostoff, J., Male, D.K. (eds), C.V. Mosby Co. , Gower Medical Publishing, London, NY, pp. 19.2-19.18; pp.
  • the Delayed Type Hypersensitivity test utilizes a modified form of ragweed pollen protein or a portion thereof, ragweed protein allergen produced by recombinant DNA techniques, or peptide derived from ragweed pollen protein, each of which has the ability to stimulate human T cells and each of which does not bind IgE specific for the allergen in a substantial percentage of the population of individuals sensitive to the allergen (e.g., at least about 75%).
  • the extent to which a specific Delayed Type Hypersensitivity reaction occurs in the individual to the protein allergen or ragweed pollen protein peptide indicating presence in the individual of T cells specific to T cell epitope(s) of the protein allergen or ragweed pollen protein peptide is determined.
  • Those individuals found to have both a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity reaction are diagnosed as having sensitivity to a ragweed allergen and may, if need be, administered a therapeutically effective amount of a therapeutic composition comprising the modified form of ragweed protein allergen or portion thereof, the ragweed protein allergen produced by recombinant DNA techniques, or peptide, each as used in the Delayed Type Hypersensitivity test, and a pharmaceutically acceptable carrier or diluent.
  • 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 non-responsiveness 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 such as, for example, with alanine and determination of the presence or absence of T cell reactivity).
  • residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance, diminish, but not eliminate, or not affect T cell reactivity.
  • 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 not eliminate binding to relevant MHC.
  • Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with alanine, gluatamic acid or a methyl amino acid.
  • peptide AMB 4-9.1 which contains an acid-sensitive aspartic acid-proline bond at amino acid residues 360-361, was modified to increase the stability of this peptide. For example, in peptide AMB 4-
  • Stability may also be enhanced in those peptides shown to be susceptible to degradation by deamidation (e.g. the peptide contains a labile Asn-Gly sequence susceptile to deamidation under various conditions. In such situations it has been found that lyophilization stabilized such peptides against deamidation.
  • Peptide Amb 4-9.1 contained a labile Asn-Gly sequence and was found to be susceptible to deamindation under accelerated conditions (e.g. increased buffer concentration, increased ionic strength and increased temperature). Lyophilization stabilized the peptide against deamidation with no significant increase in degradants following 6 months of storage at 5°C.
  • peptides can also be modified to incorporate one or more polymorphisms in the amino acid sequence of a 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 can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al., supra, to produce a 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, sj ⁇ ia); esterification (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 4J.: 199-215 (1971)).
  • peptides within an allergen group can be modified to enhance T cell reactivity.
  • an allergen group e.g., Amb a I or Amb a II
  • a peptide of one group allergen which may be less reactive than a peptide of another group allergen corresponding in amino acid position can have one or more amino acids substituted with one or more amino acids from the corresponding peptide.
  • peptides can be modified to incorporate a polymorphism in the amino acid sequence of a protein allergen resulting from natural allelic variation. Modification of peptides to include one or more of these polymorphisms may result in enhanced stability and/or reactivity.
  • 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, £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.
  • a selected peptide AMB 3- 4.1 was modified to increase its solubility by the addition of three naturally occurring sequential residues found in the Amb a I.l protein, "RHG", to the 5' end of the peptide. These residues are not necessary for T cell recognition and are also found in peptide AMB 3- 5.1.
  • peptide RAE 70.1 was divided into two fragments and modified to increase solubility.
  • a fragment of RAE 70.1 (“ORIGINAL" in Figure 25. amino acid residues 194-216, SEQ ID NO: 125) was modified by substitution or addition of charged amino acids to increase the hydrophilicity and decrease the pi of the peptide to thereby increase the solubility.
  • isoleucine was substituted with glutamic acid to decrease the pi and avoid precipitation of the peptide from solution at a physiological pH. Similar substitutions and additions are shown in Figure 25.
  • charged amino acids or charged amino acid pairs or triplets when added to the carboxy or amino terminus of the peptide may be particularly useful to increase the solubility of the peptide.
  • charged amino acids include, but are not limited to arginine (R), lysine (K), histidine (H), glutamic acid (E), and aspartic acid (D). Examples of such modifications are shown in Fig. 14. AMB 2-8.1, AMB 2-9.1. AMB 2-10.1. AMB 2-7.1 and AMB 2-11.1.
  • canonical protease sensitive sites can be recombinantly or synthetically engineered between regions, each comprising at least one T cell epitope.
  • the resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate portions of the peptide containing one or more T cell epitopes.
  • such charged amino acid residues can result in an increase in solubility of a peptide.
  • Site-directed mutagenesis of DNA encoding a peptide of the invention can be used to modify the structure of the peptide. Such methods may involve PCR with degenerate oligonucleotides (Ho et al.. Gene, 22:51-59 (1989)) or total synthesis of mutated genes (Hostomsky, Z., et al., Biochem. Biophys. Res. Comm., 161 :1056-1063 (1989)). To enhance bacterial expression, the aforementioned methods can be used in conjunction with other procedures to change the eucaryotic codons in DNA constructs encoding peptides of the invention to ones preferentially used in E. coli, yeast, mammalian cells or other eucaryotic cells.
  • Another aspect of the invention pertains to an antibody specifically reactive with Amb a I, or a fragment thereof.
  • the antibodies of this invention can be used to standardize allergen extracts or to isolate the naturally-occurring or native form of Amb ⁇ I. For example, by using proteins or fragments thereof based on the cDNA sequence of Amb ⁇ I, anti- protein/anti-peptide antisera or monoclonal antibodies can be made using standard methods.
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of such protein or an antigenic fragment which is capable of eliciting an antibody response.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • Amb ⁇ I or fragment thereof 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 ⁇ LISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • an -Amb ⁇ I antisera can be obtained and, if desired, polyclonal an ⁇ -Amb ⁇ I antibodies isolated from the serum.
  • antibody-producing cells lymphocytes
  • myeloma cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • immortalizing cells such as myeloma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with Amb a I and the monoclonal antibodies isolated.
  • the term antibody as used herein is intended to include fragments thereof which are also specifically reactive with Amb a I.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • the antibody of the present invention is further intended to include bispecific and chimeric molecules having an ⁇ m ⁇ -Amb a I portion.
  • T cell clones and soluble T cell receptors specifically reactive with Amb a I or a fragment thereof are also provided.
  • Monoclonal T cell populations i.e., T cells genetically identical to one another and expressing identical T cell receptors
  • Single Amb al MHC responsive cells can then be cloned by limiting dilution and permanent lines expanded and maintained by periodic in vitro restimulation.
  • Amb a I specific T-T hybridomas can be produced by a technique similar to B cell hybridoma production.
  • a mammal such as a mouse can be immunized with Amb a I or fragment thereof, T cells from the mammal can be purified and fused with an autonomously growing T cell tumor line. From the resulting hybridomas, cells responding to Amb a I or fragment thereof are selected and cloned. Procedures for propagating monoclonal T cell populations are described in Cellular and Molecular Immunology (Abul K. Abbas et al. ed.), W.B. Saunders Company, Philadelphia, PA (1991) page 139.
  • Soluble T cell receptors specifically reactive wi Amb a I or fragment thereof can be obtained by immunoprecipitation using an antibody against the T cell receptor as described in Immunology: A Synthesis (Second Edition), Edward S. Golub et al., ed., Sinauer Associates. Inc., Sunderland, MA (1991) pages 366-269.
  • T cell clones specifically reactive with Amb a I or fragment thereof can be used to isolate and molecularly clone the gene encoding the relevant T cell receptor.
  • a soluble T cell receptor specifically reactive with Amb a I or fragment thereof can be used to interfere with or inhibit antigen-dependent activation of the relevant T cell subpopulation, for example, by administration to an individual sensitive to a cat allergen.
  • Antibodies specifically reactive with such a T cell receptor can be produced according to the techniques described herein. Such antibodies can be used to block or interfere with the T cell interaction with peptides presented by MHC.
  • the present invention also provides nucleic acids having sequences encoding proteins and peptides of the invention.
  • Nucleic acid sequences used in any embodiment of this invention can be cDNA as described herein, or alternatively, can be any oligodeoxynucleotide sequence having all or a portion of a sequence represented herein, or their functional equivalents. Such oligodeoxynucleotide sequences can be produced chemically or 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 SEQ ID NO: 1, 3, 5, 7, 9 and 1 1 or fragments thereof hybridizes, or 2) the sequence (or corresponding sequence portion) complementary SEQ ID NO: 1, 3, 5, 7, 9 and 11 and/or 3) a sequence which encodes a product (e.g., a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) of SEQ ID NO: 1 , 3, 5, 7, 9 and 11.
  • a product e.g., a polypeptide or peptide
  • a functional equivalent must meet one or more criteria will depend on its use (e.g., if it is to be used only as an oligoprobe, it need meet only the first or second criteria and if it is to be used to produce a peptide of the present invention, it need only meet the third criterion).
  • Amb a I and Amb a II proteins have been recombinantly expressed in E. coli, purified and shown to have reduced binding to human allergic ragweed pollen IgE on Western blots.
  • Overlapping peptides derived from the Amb a 1.1 protein and various peptides derived from Amb a 1.3 and Amb a 1.2 were synthesized and used to identify regions of T cell reactivity within the protein. These regions of T cell reactivity were further defined by modifying selected Amb a I.l peptides and determining T cell reactivity to these peptides.
  • defatted short ragweed pollen (Greer Labs) was extracted in 500 ml .05 M Tris pH 7.95 containing protease inhibitors.
  • the extract was then depigmented by batch absorption with Whatman DE-52 DEAE cellulose (150 g dry weight) in the presence of 0.2 M NaCl at 4°C. Unbound material was dialysed against .025 M Tris pH 7.95 with protease inhibitors.
  • the depigmented sample was next applied to an 80 ml DEAE cellulose column (Whatman DE-52) equilibrated in .025 M Tris pH 7.95 containing protease inhibitors.
  • Acidic proteins were eluted with .025 M Tris, 0.2 M NaCl pH 7.95 at 4°C with inhibitors.
  • the acidic DEAE elution sample was fractionated by ammonium sulfate precipitation into 0-45% and 45-59% saturation samples (4°C).
  • the Amb a I-enriched 45-59% pellet was applied at 0.5 ml/min (4°C) to a 500 ml Sephacry] S200 (Pharmacia) column in .05 M ammonium bicarbonate containing inhibitors.
  • Purified Amb a I was recovered in the 38 kD region and dialysed against .04 M Tris pH 8.0.
  • Purified Amb a II was recovered in the 38 kD region and dialysed against .04 M Tris pH 8.0 at 25°C to separate contaminating Amb a I from Amb a II. Elution was performed with .04 M Tris pH 8.0 containing .08M NaCl.
  • the pollen (50g) was extracted overnight at 4°C with extraction buffer (lOml/g pollen) containing 50 mM Tris-HCl, pH8 and protease inhibitors in final concentrations: phenyl methyl sulfonyl fluoride (170 ⁇ g/ml); soybean trypsin inhibitor (l ⁇ g/ml); leupeptin (1 ⁇ g ml) and peptstatin A (1 ⁇ g/ml).
  • the soluble extract was clarified by sequential filtration through Whatman #1 paper (Whatman, Maidstone England) followed by an 0.8 micron filter.
  • the soluble pollen extract (SPE) was either used for IgE binding studies or subjected to further purification as described below.
  • the sample was then loaded onto an an ⁇ -Amb a 1 murine monoclonal antibody affinity column (mAb-4B5B7, Dr. D. Klapper, Univ. North Carolina, Chapel Hill, NC) which was determined to bind members of the Amb a l/Amb a II family.
  • Amb a I was eluted with 0.1 M glycine, pHl 1 and was immediately neutralized with 1 M sodium phosphage, pH 3.9
  • This preparation contained both Amb a 1.1 and Amb a 1.2 as detected by SDS-PAGE analysis and by Western blotting.
  • the affinity purified proteins were pooled, concentrated and dialyzed against 20 mM Tris, pH 8.5, extensively.
  • Amb a I.l and Amb a 1.2 were further separated by a Source Q ionic exchange column (Pharmacia, Piscataway, NJ), which was equilibrated with 20 mM Tris. pH 8.5.
  • the flow-through material contained Amb a 1.2 whereas Amb a ⁇ . ⁇ was eluted with step gradients of 24 M and 80 mM NaCl.
  • the latter peak also contained some ⁇ , ⁇ cleaved fragments of Amb a I.l.
  • Amb a I.l and Amb a 1.2 purity was verified by NH2- terminal amino acid sequencing as being greater than 95%.
  • nucleotide sequences and deduced amino acid sequences of the Amb a I family members are shown in the sequence listing as SEQ ID NOT and 2 ⁇ Amb a I.l), SEQ ID NO:3 and 4 ⁇ Amb a 1.2), SEQ ID NO:5 and 6 ⁇ Amb a 1.3), SEQ ID NO:7 and 8 ⁇ Amb a 1.4), and SEQ ID NO:9 and 10 ⁇ Amb a II).
  • the cDNAs encoding each allergen were cloned in frame with a polylinker encoding six sequential histidines, (CAC)g, that had been inserted into the 5' end of the pTrc99A vector as a NcoI/EcoRI synthetic adapter (Maniatis T., et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). These cDNA inserts were then cloned in-frame into the appropriate pTrc99. To further enhance expression, a retroregular stem-loop sequence was placed at the 3' end in the untranslated region (Skoglund, C. M., et al. Gene 88:1 (1990)).
  • the H6 leader sequence allowed purification using QIAGEN NTA-Agarose (Diagen GmbH, Dusseldorf, FRG), a Ni 2+ chelating support (Hochuli, E. et al., Biotechnology, 6: 1321 (1988)).
  • the cells were resuspended in lysozyme containing phosphate buffer (0.4 mg/ml) and incubated for 30 minutes on ice.
  • the cell suspension was frozen and quick thawed followed by sonication (Bond, J. G. et al., J. Immunol, 146:3380 (1991)).
  • Insoluble recombinant protein was recovered by a low speed centrifugation and solubulized in 10 ml (per 1 liter growth) of buffer containing 8 M urea, 50 mM Tris, pH 8.0, 2 mg/ml leupeptin, 2 mg/ml pepstatin and 1 mg/ml soybean trypsin inhibitor.
  • the urea solubilized preparation was subjected to a low speed centrifugation and the recombinant proteins in the supernatant isolated by metal ion chromatography (Hochui supra..
  • the pelleted bacteria were resuspended in 6 M guanidine HC 1.
  • This suspension was subjected to centrifugation at 15,000 X g, and the supernatant removed, adjusted to pH 8.0 with 10 N NaOH, and applied to an NTA agarose column that had been equilibrated in 6 M guanidine HCl, 100 mM NaPO4, 10 mM Tris pH 8.0.
  • the column was washed in 6 M guanidine HCl , 100 mM NaPO4, 10 mM Tris pH 8.0 until the OD28O of tne effluent reached background.
  • the column buffer was then switched to 8 M urea, 100 mM NaPO4, 10 mM Tris pH 8.0. After equilibration, a more stringent wash was performed in 8 M urea, 100 mM NaOAc, 10 mM Tris pH 6.3 until the OD28O of the effluent reached background.
  • Recombinant protein (as an Hg fusion) was then eluted in 8 M urea, 100 mM NaOAc, 10 mM Tris pH 4.5 and collected in aliquots whose OD28O profile was monitored. The protein peak was dialyzed 3 times into 500 volumes of PBS for human T-cells analysis. Yield ranged from 1-3 mg of recombinant protein per litre with purity of approximately 55% (as determined by densitometric scanning).
  • high yields e.g. 50-100 mg purified ⁇ Amb a I.l protein/L of fermentation broth
  • This embodiment involves the use of a construct which is different from the one described above in 3 ways: l)deletion of sequence coding for 36 non-Amb a I related amino acids, 2) attachment of a 6 histidine linker to the NH2-terminus, and 3) replacement of 2 existing arginine codons with E. c ⁇ /t-preferred arginine codon.
  • the resulting construct showed significantly higher expression levels than did the original construct. This phenomenon was true in both shaker flask and 10L fermentation cultures.
  • the purification also differed significantly from the procedure described above.
  • Cell paste from fermentors was homogenized and inclusion bodies were purified prior to solubilization in guanidine-HCL.
  • the solubilized inclusion bodies were then purified using metal chelate chromatography and buffer exchanged into acetate buffer.
  • the resulting protein is greater than 90% full length r Amb a l. l.
  • the antigens were loaded on the gels as follows: lane 1 SPE (Soluble Pollen Extract), 15 mg/lane; lane 2 xAmb a I.l (recombinant Amb a I.l), 3 mg/lane; lane 3 xAmb a 1.2, 4 mg/lane; lane 4 xAmb a 1.3, 3 mg/lane; lane 5 xAmb a 1.4, 3 mg/lane; and lane 6 xAmb a II.1. 4 mg/lane.
  • the blots were rinsed in blot solution (25 mM Tris-HCl, pH 7.5, 0.17 M NaCl, and 0.05% Tween 20) and stained for 1 hour with 0.1% India ink. All subsequent incubations with antibodies and washes were performed in blot solution at room temperature. The first antibody incubations were performed overnight then rinsed and incubated with the appropriate biotinylated second antibody (Kirkegaard Perry Laboratories. Gaithersburg. MD). The final incubation was performed using 125 -I streptavidin (1 mCi/25 ml blot solution) for 1 hour followed by removal of unbound labeled material and autoradiography at -80°C with an intensifying screen. Fig.
  • Fig. 2 shows the results of this analysis performed according to the following method.
  • Corning assay plates (#25882-96) were coated with each antigen listed in Fig. 2 at the following concentrations: Soluble Pollen Extract (SPE) 15 mg/ml; xAmb a I.l, 5 mg/ml; xAmb a 1.2, 20 mg/ml; xAmb a 1.3, 5 mg/ml; xAmb a 1.4, 15 mg/ml; and xAmb a II, 20 mg/ml. 50 mis/well of the above antigens were added and coating was carried out overnight at 4° C. The coating antigens were removed and the wells were blocked with 0.5% gelatin in PBS, 200ml/well for 2 hours at room temperature.
  • SPE Soluble Pollen Extract
  • Patient #143 plasma was serially diluted with PBS-Tween 20 (PBS with 0.05% nonionic detergent Tween-20 Sigma, St. Louis MO) and lOOml/well was added and incubated overnight at 4°C (plasma dilutions are tested in duplicate).
  • the second antibody biotinylated goat anti-Human IgE, 1 : 1000, Kirkegaard & Perry Laboratories Inc. Gaithersburg, MD
  • TMB Membrane Peroxidates Substrate system (Kirkegaard & Perry Laboratories) was freshly mixed, and added at l OOml/well. The color was allowed to develop for 2-5 minutes. The reaction was stopped by the addition of lOOml/well of 1 M phosphoric acid. Plates were read on a Microplate EL 310 Autoreader (Biotek Instruments, Winooski. VT) with a 450nm filter. The absorbance levels of duplicate wells was averaged.
  • an ELISA based assay system for serum IgE binding to peptides was developed which had greater sensitivity than direct ELISA, and creating tandem Amb a I peptide copies in the form of a recombinant trimer.
  • One strategy for constructing a recombinant trimer based on Amb a I peptide Amb 4-9.1 is as follows.
  • DNA sequences encoding Amb 4-9.1 are amplified by PCR from a plasmid containing Amb a I. l sequence. Three Amb 4-9.1 copies are made each containing unique restriction enzyme cleavage sites. The restriction sites are designed (inserted during the amplification reaction) to ensure peptide ligation to each other or ligation to plasmid. Amb 4-9.1 copies are ligated together and then the fused sequences are ligated into a sequencing vector, pUC19. After DNA sequence confirmation, the trimer sequences are excised from pUC19 and inserted into an expression vector pEt-l ld containing a 6 histidine tag.
  • the expression plasmid containing Amb 4-9.1 trimer sequence was expressed in E.coli BL21. Expressed protein was purified over a Ni2+ column, protein was analyzed by SDS-Page, Western, ELISA and protein sequencing. The Amb 4-9.1 trimer was greater than 95% pure and was soluble in PBS. The sequence of the Amb 4-9.1 trimer was confirmed as: MGHHHHHHEFELGTKDVLENGAIFVASGVDPVLTPEQSAGSRGTK DVLENG AIFV ASGVDPVLTPEQSAGLQGTKD VLENGAIFV ASGVDPVLTPEQS AG .
  • Balb/c mice H-2 d were immunized in the hind foot pads and at the base of the tail with an emulsion of Complete Freunds Adjuvant (CFA) and 50mg/mouse Amb a l.l . Seven days later, the popliteal lymph nodes, superficial inguinal lymph nodes, and the periaortic lymph nodes were harvested and cultured (1 x 10-- * lymph node cells + 2 x 10*5 irradiated spleen feeders) with various challenge antigens in vitro.
  • CFA Complete Freunds Adjuvant
  • the in vitro antigens consisted of various doses of Amb a l.l, Amb a 1.2, Amb a 1.3, Amb a I A, Amb a II, and a media control. Cultures were incubated for 3 days at 37°C in a CO2 incubator. On day 3, each culture was pulsed with lmCi -1H thymidine and on day 4 the cultures were harvested and proliferation was monitored by incorporation of -1H into DNA.
  • mice that were immunized with 50mg Amb a 1.1 + CFA have a good response to Amb a l.l, Amb a 1.3, and Amb a I A.
  • the response to Amb a 1.2 and Amb a ll is also good but less than other antigens. It appears as though Amb a l.l is very immunogenic in Balb/c mice and results in a response to each Amb a family member.
  • mice were immunized with PBS + CFA there was no significant response to any of the Amb a family members (Fig. 4B).
  • mice were tolerized with Amb a l.l .
  • the mice were tolerized with Amb a 1.1 and pollen extract and then challenged with Amb a l.l or pollen extract.
  • the outline of this experiment #5 is shown below:
  • Group (2) 6 Balb/c mice tolerized with 300mg Pollen Extract + IFA i.p.
  • Group (3) 6 Balb/c mice exposed to PBS + IFA i.p.
  • the animals were sacrificed by cervical dislocation on day 23 and the popliteal lymph nodes, superficial inguinal lymph nodes, and the periaortic lymph nodes were removed and placed in rinsing buffer (cold RPMI 1640 containing 1% FCS).
  • the nodes were rinsed with rinsing buffer and forced through a fine stainless steel mesh, using a glass pestal to suspend them in rinsing buffer.
  • the suspended cells were rinsed two times by centrifugation at 1000 rpm for 10 minutes and resuspended in rinsing buffer. An aliquot from each sample was taken in order to do a cell count.
  • the cells (4 x 10-5/ml) were incubated in culture media (RPMI 1640 media containing 10% FCS, 2mM L-glutamine, 50U/ml penicillin, 50 mg/ml streptomycin and 5 x 10" ⁇ M 2-mercaptoethanol) and test antigens at various concentrations.
  • culture media RPMI 1640 media containing 10% FCS, 2mM L-glutamine, 50U/ml penicillin, 50 mg/ml streptomycin and 5 x 10" ⁇ M 2-mercaptoethanol
  • the triplicate 0.1 ml cultures (U -bottom 96 well plates (Costar)) were incubated at 37°C and at 5% CO2- After 24 hours, 50ml of media from each well was placed in separate flat bottom 96 well plates (Costar) and frozen overnight at -20°C to eliminate carryover of live cells.
  • CTLL3 in log phase growth were rinsed 3 times by centrifugation at 1000 rpm for 10 minutes.
  • CTLL3's were added to the warmed culture supernatants (5 x 10-3 cells/well) and the IL-2 assay was incubated at 37°C and 5% CO2. After 24 hours, 1 mCi/well -1H thymidine was added in 50 ml/well and the CTLL3 cells were incubated for an additional 4-6 hours. Following the pulse with ---H, the plates were harvested on a Tom Tek 96 well cell harvester. The - ⁇ H incorporation in each well was counted by a Betaplate Model 1205 scintillation counter. Background counts were not subtracted.
  • Fig. 5 shows the lymph node responses to Amb a l.l, Amb a 1.2, Amb a 1.3, Amb a I A. and Amb a II in the mice that were tolerized with Amb a l.l or Phosphate Buffered Saline (PBS) and challenged with Amb a l.l.
  • Fig. 5A shows that the mice that were tolerized with Amb a I.l have a lower response to Amb a I.l than the mice that were tolerized with PBS.
  • Amb a I.l overlapping peptides as shown in Fig. 7 were synthesized using standard Fmoc/tBoc synthetic chemistry and purified by dialysis or Reverse Phase HPLC.
  • various peptides derived from Amb a 1.2 and Amb a 1.3 were synthesized.
  • the amino acid residues of the synthesized peptides are in brackets by the peptide name and the amino acid sequence (in single letter code) is next to the peptide name.
  • the peptide names are consistent throughout the Figures.
  • the relationship of the Amb a I family members at the level of T cell cross-reactivity as determined in Example VIII was considered.
  • the Amb a I protein allergens share a high degree of cross-reactivity.
  • Amb a 1.1 and Amb a II were found to have 55.2% cross- reactivity.
  • the amino acid sequences of the Amb a I family members and Amb a II were examined to identify conserved and variable regions. It was expected that conserved regions within the Amb a I family members and Amb a II would contain "shared" T cell epitopes.
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • Viable cells were purified by LSM centrifugation and cultured in complete medium supplemented with 5 units recombinant human IL-2/ml and 5 units recombinant human IL-4/ml for up to three weeks until the cells no longer responded to lymphokines and were considered "rested”. The ability of the T cells to proliferate to Amb a I ⁇ . Amb a 1.2 and Amb a 1.3 sequence-derived synthetic peptides was then assessed.
  • 2 x 10 4 rested cells were restimulated in the presence of 2 x 10 4 autologous Epstein-Barr virus (EBV)-transformed B cells (gamma-irradiated with 25,000 RADS) or 5 x 10 4 autologous PBMC (3,500 RADS) with various concentrations of Amb a 1.1 synthetic peptides in a volume of 200 ml complete medium in duplicate or triplicate wells in 96-well round bottom plates for 3 days. Each well then received 1 mCi tritiated thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. Table II shows the results of a representative assay.
  • EBV Epstein-Barr virus
  • the maximum response in a titration of each peptide is expressed as die S.I. or stimulation index.
  • the S.I. is the CPM incorporated by cells in response to peptide divided by the CPM incorporated by cells in medium only.
  • An S.I. value greater than the background level is considered "positive" and indicates that the peptide contains a T cell epitope.
  • only individual S.I. values above 2.0 were used in calculating mean stimulation indices for each peptide for the group of patients tested.
  • the results shown in Table II demonstrate that this patient (#466) responds very well to peptides RAE 7.D, RAE 69.1 , RAE 64.1 , and RAE 29.1. This indicates that these peptides contain Amb a I.l T cell epitopes recognized by T cells from this particular allergic patient.
  • the ranks for each peptide were then summed in the 39 patients to determine the cumulative rank for the peptide.
  • the number above each bar is the mean S.I. of the positive responses (S.I. of 2.0 or greater) from the group of patients to that peptide.
  • the positivity index (P.I) is the positivity index (P.I).
  • the P.I. for each peptide is determined by multiplying the mean S.I. by the percent of patients who responded to that peptide. The P.I. therefore represents both the strength of the response (S.I.) and the frequency of a response to a peptide in the group of patients tested.
  • peptide RAE 69.1 had the highest cumulative rank so it was the best peptide response in the overall population of 39 even through it did not have the highest mean S.I.
  • RAE 70.1 had the highest mean S.I but not the best cumulative rank or P.I.
  • the response to RAE 70.1 was strong when it occurred but it did not occur as frequently in the population as the response to other peptides.
  • the peptide with the highest P.I., RAE 29.1 also had a strong S.I. and the second highest cumulative rank. The response to this peptide was therefore generally strong and relatively frequent in this population.
  • T cell studies similar to those of Example VI were performed using these selected peptides to more precisely define the major areas of T cell reactivity within Regions 1-4 of the Amb a I.l protein.
  • PBMC from a single ragweed-allergic patient were isolated as described in Example VI and were stimulated with 20 mg/ml of recombinant Amb a l ⁇ as described above.
  • the results of proliferation assays with this one patient to selected peptides using irradiated (24,000 RADS) autologous EBV B cells as antigen presenting cells is shown in Table III. The data indicates that T cells from this patient respond well to RAE 7.D, RAE 70.1, RAE 40.1-4, and RAE 28.1-2. TABLE III
  • Fig. 10 indicates that the major area of T cell reactivity within Region 1 of the Amb a I.l protein is represented by peptides RAE 6.D, RAE 7.D, RAE 40.1 , RAE 40.1 -4, and RAE 40.1-5.
  • a preferred area of major T cell reactivity within Region 1 comprises amino acid residues 57-101.
  • Fig. 1 1 indicates that there is a broad area of weak T cell reactivity in Region 2 of the Amb a 1.1 protein relative to Region 1.
  • a preferred area of major T cell reactivity within Region 2 thus comprises amino acid residues 182-216.
  • Fig. 12 shows that the most frequent and dominant response within Region 3 of the Amb a 1.3 protein is to peptide RAE 64.1.
  • T cell reactivity is represented by peptide RAE 66.1.
  • a preferred area of major T cell reactivity within Region 3 comprises amino acid residues 280-322.
  • Fig. 13 indicates that the major area of T cell reactivity in Region 4 is represented by peptides RAE 28.1-2, RAE 28.1 -1 , and RAE 28.1. From this analysis, a preferred area of major T cell reactivity within Region 4 comprises amino acid residues 342- 377.
  • peptides derived from the Amb a 1.1 protein comprising T cell epitopes selected peptides from Regions 1-4 were further modified by addition or deletion of amino acid residues as described above. These selected modified peptides are shown in Fig. 14.
  • PBMC were stimulated with recombinant Amb a I.l and assayed with the selected peptides as described in Example VI, except in some cases autologous PBMC (irradiated 3.500 RADS) were used as antigen presenting cells.
  • the assay was followed with a number of patients and resulted in 23 positive experiments. In these assays an individual S.I.
  • Figs. 15-18 the bar represents the cumulative rank of each peptide. The best 3 peptide responses for each patient were ranked as described above. The mean S.I. for each peptide and percent positive are found above the bar.
  • 2 x 10 4 rested cells were restimulated in the presence of an equal number of autologous EBV-transformed B cells (irradiated with 25,000 RADS) with various concentrations (0-100 mg/ml) of recombinant Amb a l.l, 1.2, 1.3, 1.4, Amb a II, or ragweed pollen extract in a volume of 200 ml complete medium in duplicate or triplicate wells in 96-well round bottom plates for 3 days. Each well then received 1 mCi tritiated thymidine for 16-20 hours. The counts incorporated were assessed and analyzed as described in Example VI.
  • Table IV shows the S.I., P.I., percent of cultures positive for the assay antigen, and number of cultures analyzed. For these assays an individual S.I. greater or equal to 2.0 was used in calculating the mean S.I. Amb a I.l stimulated cells respond less well and less frequently to the least homologous family members 1.3 and 1.2. However, this level of cross-reactivity, 68.8% and 60% respectively, is still considered high. The lowest level of Amb a I.l stimulated T cell cross-reactivity is noted to the least homologous allergen, Amb a II. In addition, Amb a 1.1 reactive T cells are found at a high percentage from ragweed pollen extract stimulated cultures, again demonstrating the importance of the Amb a I.l allergen.
  • AMB 1-4.1 AENRKALADCAQGFGKGTVGGKDGD
  • AMB 2-1.1 GPAAPRAGSDGDAISISGSSQ
  • PBMC from ragweed allergic patients were stimulated with 20 mg/ml recombinant Amb a I.l protein as described in Example VI with the addition of cultures of PBMC which were stimulated with 20 mg/ml recombinant Amb a 1.3 protein.
  • Assays were performed as described in Example VI except the homologous Amb a 1.3 peptides were also tested at various doses. In these experiments an individual S.I of 2.0 or greater was used in calculating the mean S.I.
  • Fig. 19 shows the results from a set of 9 matched patients stimulated with either recombinant Amb a 1.1 protein or recombinant Amb a 1.3 protein and tested on the set of Amb a I.l peptides described above.
  • Each bar represents the P.I.
  • the dark bar represents cells stimulated with recombinant Amb a l.l protein and tested with Amb a I.l peptides
  • the stippled bar represents cells stimulated with recombinant Amb a 1.3 protein and tested with Amb a l peptides.
  • the results directly parallel each other, indicating that cells stimulated with recombinant Amb a 1.3 proteins recognize Amb a l ⁇ derived peptides comprising at least one T cell epitope.
  • One exception is that cells primed v ⁇ fhAmb a 1.3 protein recognize the RAE 7.D peptide poorly compared to recombinant Amb a I.l stimulated cells.
  • Example III To analyze IgE reactivity of peptides derived from the Amb a I.l protein, a direct binding ELISA was performed according to the procedure described in Example III.
  • the source of IgE for this analysis was a pool of ragweed allergic patient plasma from 38 ragweed skin test positive patients.
  • the ELISA protocol was the same as Example III except that the antigen coating with peptides and proteins was a concentration of 10 mg/ml at 100 mis/well.
  • Fig. 21 shows the graphed results of this assay demonstrating strong reactivity to both SPE and biochemically purified Amb l ⁇ . By this assay there is no detectable binding to any of the peptides.
  • a set of these assays was also run with rabbit and mouse antisera to demonstrate that the coating of the peptides onto these plates was successful.
  • Amb a I.l specific T cell reactivity selected peptides from Figure 14 were analyzed.
  • Amb a I.l specific T cells lines were derived from 28 ragweed allergic patients as described in Example VI. These lines were assessed for their ability to proliferate in response to the peptide in the presence of autologous EBV transformed antigen presenting cells by the uptake of tritiated thymidine as described previously.
  • Several lysine substituted peptides derived from the peptide RAE 70.1 sequence were designed to increase the solubility of the peptide from Region 2. These peptides shown in Figure 22 were tested against non-substituted controls to determine if the modification resulted in a change in T cell reactivity.
  • FIG. 22 shows the responses of the 28 T cell lines to these peptides as analyzed by the positivity index, the mean stimulation index and the percent of positive responses.
  • the positivity index as defined previously, is the mean stimulation index multiplied by the percent of patients responding to a peptide. Responses to peptides were considered positive if they were greater or equal to 2 fold over background.
  • Figure 22 demonstrates that certain lysine substituted peptides in the 182-216 sequence resulted in greater T cell responses, indicating that not all substitutions are recognized equally by a given T cell line.
  • the T cell responses to the lysine modified peptides having a substitution of cysteine at position 212 with serine reflect the responses of T cell lines tested with peptides without the lysine substitutions.
  • the decrease in response to AMB 2-10.1, with a substitution of leucine for cysteine at 212 reflects the decrease in the percentage of patients responding to this peptide relative to peptides AMB 2-9.1 and AMB 2-1 1.1 which contain serine and glutamic acid substitutions, respectively, at position 212.
  • FIG. 23 shows the ranked sum of the strongest three peptide responses in the 28 T cell lines. The ranked sum was determined as described previously with the strongest response to a peptide given a value of 3, the second strongest a 2, and the third strongest a value of 1. The values from all 28 lines were then added to obtain the ranked sum.
  • Figure 23 indicates that the modified peptide AMB 4-9. IDA elicits T cell responses similar to those of the native sequence AMB 4-9.1. In contrast, other substitutions, while eliciting strong responses do not rank as highly in this analysis.
  • Toxicity defined as proliferation, as assessed by incorporation of tritiated thymidine, which is less than half the proliferation of media control of an Amb a I.l stimulated T cell line plus APC.
  • RAE 70.1 SEQ ID NO:44
  • peptide RAE 70.1 was divided into two fragments and modified to increase solubility and to further define the residues necessary for T cell reactivity.
  • a fragment of RAE 70.1 amino acid residues 194-216 was modified by substitution or addition of amino acids which would increase the hydrophilicity and decrease the pi of the peptide to thereby increase solubility.
  • Peptides Amb 2-18.1 (SEQ ID NO: 126), Amb2-19.1 (SEQ ID NO.127), Amb2-20.1 (SEQ ID NOT28) and Amb2-21.1 (SEQ ID NO: 129) correspond to different lengths of the original peptide which were synthesized (including the serine substitution at position 212) and analyzed to determine solubility. It was found that peptide Amb2-19.1 (residues 200- 217) was most soluble. Thus, substitutions and/or amino acid additions to this peptide were made in order to further increase the solubility while maintaining T cell reactivity.
  • isoleucine at position 201 was substituted with glutamic acid (Amb2-22.1) or lysine (Amb2- 23.1 ) to lower the pi of the resulting peptide and avoid precipitation of the peptide at physiological pH.
  • glutamic acid Amb2-22.1
  • lysine Amb2- 23.1
  • the following peptides were also synthesized with various substitutions or additions designed to decrease the pi and increase the hydrophilicity of the peptide.
  • Figures 27 and 28 show representative examples of proliferation of two individual Amb a 1.1 specific T cell lines to peptides selected from those shown in Figure 25. Assays were performed as described previously. Both Figure 27 and Figure 28 indicate a hierarchy of responses to these peptides. Patient 119 shown in Figure 27, has a hierarchy of response from strongest to weakest as follows: AMB2-23.1>AMB2-22.1>AMB2-30.1>AMB2-
  • a T cell clone was generated by limiting dilution from an Amb ⁇ I. l specific T cell line stimulated with AMB 2-10.1. Briefly, an A mb ⁇ I.l specific T cell line from patient 776 was shown to respond to AMB 2-10.1 in a proliferation assay as described previously.
  • Amb a l.l specific T cells were plated at 0.3 cells/well in a V-bottom 96 well plate with 20,000 irradiated autologous EBV transformed antigen presenting cells, AMB 2-10.1 at 40 mg/ml, leukoagglutin at 1 mg/ml, and recombinant human IL-2 and IL-4 at 10 units/ml in a total volume of lOOml/well.
  • Plating the T cells at 0.3 cells/well insures not more than 1 T cell/well can potentially proliferate to the peptide with the progeny of that T cell representing a clonal population.
  • IL-2 and IL-4 were recombinant human IL-2 and IL-4 to expand peptide-specific T cells.
  • This addition of IL-2 and IL-4 was repeated again every three days for the duration of the culture period. Twelve days after the initiation of culture, T cells in the wells were restimulated with 20,000 irradiated EBV antigen presenting cells and 40 mg/ml AMB 2-10.1. This was repeated again 20 days after the initial stimulation. T cells from wells which showed signs of growth were separated from cell debris by density centrifugation as described previously in the generation of T cell lines. The T cell clone was then expanded with additional IL-2 and IL-4 at 10 units/ml until there were significant numbers to assay for proliferation.
  • the objective of the histamine release analysis was to compare the effects of Amb a 1.1 or Amb a l.l -derived peptides in an in vitro allergic response system.
  • the release of histamine via IgE recognition and IgE receptor crosslinking on viable cells directly assays the allergic potential of a protein antigen.
  • the histamine release assay used for these studies 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. Allergy Clin. Immunol. 82:646-654).
  • the assay was performed in two steps: 1) the release of histamine from basophils present in heparinized whole blood in the presence of different concentrations of protein or peptide; and 2) the assay of histamine present in the supernatants of the release reactions following cell removal by centrifugation.
  • the reagents for this second step are available commercially as a competitive radioimmunoassay from AMAC Inc. (Westbrook, ME).
  • test protein Amb a 1.1 and peptides AMB 1-2.1, AMB 2-9.1 , AMB 4-9.1 , and AMB 3-5.1 were each diluted to 2X the final release concentration in PACM buffer (PIPES 25mM, NaCl 1 1 OmM, KC1 5.0 mM, human serum albumin 0.003% (w/v), CaCl2 5mM, MgCl2 2mM, pH7.3) and 0.2 ml of each dilution was added to a 1.5 ml polypropylene tube. A 0.2ml aliquot of blood was then added to each tube and the release reactions started by inversion of the tubes.
  • PACM buffer PPES 25mM, NaCl 1 1 OmM, KC1 5.0 mM, human serum albumin 0.003% (w/v), CaCl2 5mM, MgCl2 2mM, pH7.3
  • a negative control of 0.2 ml blood incubated with 0.2 ml PACM buffer was also included. The release reactions were performed for 30 minutes at 37°C. The tubes were then centrifuged in a microfuge at 1500 RPM for 3 minutes and the supernatants were carefully removed and analyzed or stored at -20°C for later analysis. To analyze the total histamine released, 0.1 ml blood was diluted with 0.9 ml PACM buffer and boiled 3 minutes. This tube was then centrifuged 2 minutes at 12,000 RPM and the supernatant was removed and save for analysis.
  • a 50 ml aliquot of each release supernatant was mixed with 150 ml of histamine release buffer (supplied with the kit from AMAC).
  • the diluted supernatant 100 ml was added to a kit tube containing an acylation reagent.
  • a 50 ml aliquot of acylation buffer was then immediately added and the tube mixed by vortexing.
  • the acylation reactions were incubated at least 30 minutes at room temperature.
  • a set of histamine standards supplied with the kit were acylated at the same time. 50 ml of each acylation reaction was then placed in a tube coated with a monoclonal antibody which specifically recognizes the acylated form of histamine.
  • a standard curve was generated from the histamine standard counts and graphed on a semi-log plot. Since this is a competitive assay (the *2->-labelled tracer competes with acylated histamine in the samples for binding to the antibody-coated tubes), the lower the number of radioactive counts measured, the greater the amount of histamine in the sample. The amount of histamine in each sample data point was extrapolated from the standard curve using a computer statistical program (StatView for the Macintosh). The assay is sensitive to 0.2 nM histamine.
  • AMB 3-5.1 Six or more 10-fold dilutions of Amb a 1.1 starting at 10 mg/ml (approximately 0.66 mM) were analyzed. Five five-fold dilutions of each peptide were also analyzed (ranging from 50 mg/ml to 0.08 mg/ml, this is approximately 15 mM to 24 nM in concentration for each peptide). The peptide concentrations were selected to encompass the higher end of the concentration curve.
  • Figure 30 shows the representative results from 1 of the patients (#1273). The graph shows the concentration of each antigen in mg ml versus th percent of total histamine released.
  • Plasma samples from 94 ragweed-allergic individuals, who tested positive for IgE binding to Amb a I. l were analyzed for IgE binding to peptides Amb 1-2.1, Amb 2-36.1 and Amb 4-9.1 by direct binding ELISA as described above. Plasma samples were studied at a 1 :3 and 1 :30 dilution. None of the 94 patients tested had measurable IgE binding to either Amb 1-2.1 or Amb 2-36.1 (data not shown). Six patients had a low level of IgE binding to Amb 4-9.1 (data not shown).
  • All peptide candidates were also examined for their ability to release histamine in vitro from basophils of ragweed-allergic patients.
  • the objective of the histamine release analysis was to measure the effects of Amb a I.l peptides Amb 1-2.1, Amb 2-36.1 and Amb 4-9.1 in vitro allergic response system. Heparinized whole blood from ragweed-allergic patients was incubated with different concentrations of each peptide (at 0.08-50 ⁇ g/mL) or Amb a I.l (at 10" ⁇ to 10 ⁇ g/mL). The levels of histamine release were measured using commercially available radioimmunoassay (Amac, Inc., Westbrook, ME).
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • Viable cells were purified by LSM centrifugation and cultured in complete medium supplemented with 5 units recombinant human IL-2/ml and 5 units recombinant human IL-4/ml for up to three weeks until the cells no longer responded to lymphokines and were considered "rested”. The ability of the T cells to proliferate to Amb a I.l sequence-derived synthetic peptides was then assessed.
  • 2 x 10 4 rested cells were restimulated in the presence of 2 x 10 4 autologous Epstein-Barr virus (EBV)-transformed B cells (gamma-irradiated with 25,000 RADS) or 5 x 10 4 autologous PBMC (3,500 RADS) with various concentrations of Amb a I.l synthetic peptides in a volume of 200 ml complete medium in duplicate or triplicate wells in 96-well round bottom plates for 3 days. Each well then received 1 mCi tritiated thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. Table III shows the results of a representative assay.
  • EBV Epstein-Barr virus
  • the maximum response in a titration of each peptide is expressed as the S.I. or stimulation index.
  • the S.I. is the CPM incorporated by cells in response to peptide divided by the CPM incorporated by cells in medium only.
  • An S.I. value greater than the background level is considered "positive" and indicates that the peptide contains a T cell epitope.
  • only individual S.I. values above 2.0 were used in calculating mean stimulation indices for each peptide for the group of patients tested.
  • the results shown in Table VI demonstrate that this patient (#1715) responds very well to peptides RAE 74.1 , Amb 4-9.1 , RAE 61.1, RAE 62.1. This indicates that these peptides contain Amb a 1.1 T cell epitopes recognized by T cells from this particular allergic patient.
  • the ranks for each peptide were then summed in the 57 patients to determine the cumulative rank for the peptide.
  • the number above each bar in paranthesis is the percentage of the positive responses (S.I. of 2.0 or greater) from the group of patients to that peptide. In parentheses above each bar is the mean S.I. of the positive responses.
  • the cumaltive rank sum represents both the strength of the response (S.I.) and the frequency of a response to a peptide in the group of patients tested. For example, peptide AMB 4-9.1 had the highest cumulative rank so it was the best peptide response in the overall population of 57 even through it did not have the highest mean S.I. Similarly, RAE 27.1 had the highest mean S.I but not the best cumulative rank.
  • Candidate peptides are selected based, at least in part on the mean human T cell stimulation index for each of the candidate peptides in the set of peptides tested and the positivity index (the mean S.I. multiplied by the percent of positive responses) of the candidate peptides in the set of peptides tested. For example, the results in Example XIII (Fig.
  • Nt flanking peptide refers to a peptide which comprises amino acid resides which overlap with amino acid residues located at the N-terminus of the candidate peptide in d e amino acid sequence of the protein antigen from which the peptide is derived;
  • C t flanking peptide refers to a peptide which comprises amino acid residues which overlap with amino acid residues located at the C-terminus of the candidate peptide in the amino acid sequence of the protein antigen from which the peptide is derived.
  • stimulation indices for the candidate peptide, the N-terminal flanking peptide and the C-terminal flanking peptide are added and divided by the sum total of the stimulation indices for the entire set of overlapping peptides to obtain a percent of T cell reactivity for the candidate peptides. If a combination of two or more candidate peptides is selected each of which contains amino acid residues which overlap, this calculation cannot be used to determine a percent of T cell reactivity for each candidate separately. However this is not the case for selected candidates, Amb 1-2.1 , Amb 2-36.1 and Amb 4-9.1.
  • the values obtained for the percentage of T cell reactivity for the candidate peptides in each individual tested can be expressed as a range of lower and higher values of the results of the above described calculations.
  • the mean percentage of T cell reactivity elicited by the candidate peptides can then be determined by averaging the values obtained for the individual responses to the candidate peptides.
  • candidate peptides Amb 1-2.1, Amb 2-36.1 and Amb 4-9.1, secondary A mb a I.l
  • T cell cultures derived from 57 ragweed-allergic subjects were analyzed for reactivity to an overlapping set of Amb a l.l peptides, and the highest stimulation index greater than or equal to 2.0 in response to each peptide was recorded for each individual tested.
  • the data were then analyzed by the equation above for each individual tested, and the mean percentage of T cell reactivity was determined by averaging all the values obtained for the individual responses to the candidate peptides, Amb 1-2.1 , Amb 2-36.1 and Amb 4-9.1 , with the following results.
  • Candidate Peptides Range of mean percentage Frequency of response to at of T cell reactivity least one peptide
  • the drug product was a multipeptide formulation comprising three peptides, Amb 1-2.1 , Amb 2-36.1 and Amb 4-9.1, which together were determined to cover a sufficient percentage of T cell epitopes of a population of individuals as discussed in Example XIV.
  • Each peptide was purified to homogeneity (at least 97% pure) by known methods and a multipeptide formulation was prepared in accordance with procedures discussed earlier.
  • the multipeptide formulation used in this Phase I clinical Study was in the form of a freeze-dried powder cake comprising 1500 ⁇ g/L (when reconstituted) of each of the peptides, Amb 1-2.1, Amb 2-36.1 and Amb 4-9.1, in a single vial.
  • the excipients used were 5% mannitol and 0.05 M sodium phosphate buffer system in a single vial.
  • the formulation was reconstituted just prior to use with sterile water for injection resulting in a solution with a pH of ca. 7.5. Normal saline (0.9%) was used for any dilutions beyond initial reconstitution.
  • This study was a double blind placebo-controlled safety study of thirty-six patients conducted at Johns Hopkins Asthma and Allergy Center Baltimore. Twenty-four patients received active therapy while twelve received placebo.
  • Cohort B often patients (6 active, 4 placebo) initiated a five week course of weekly fixed dose therapy (250 ⁇ g per peptide or placebo).
  • a third Cohort (Cohort C) often patients (6 active, 4 placebo) began a five-week course of weekly fixed dose therapy (750 ⁇ g per peptide or placebo).
  • ADDRESSEE IMMULOGIC PHARAMCEUTICAL CORPORATION
  • B STREET: 610 Lincoln Street
  • Lys Val Glu lie lie Asn Ala Gly Phe Thr Leu Asn Gly Val Lys Asn
  • AAA AAT GTC CTA GGA AGG CAT GGT GAA GCC GCC GCA GAG TCG ATG AAG 1008
  • ATG ATT CCA GCC GAA CCA GGA GAG TCC GCT CTA AGC CTC ACT AGT AGT 1152 Met lie Pro Ala Glu Pro Gly Glu Ser Ala Leu Ser Leu Thr Ser Ser 370 375 380
  • GCT GGT GTA CTC TCA TGC CAA CCC GGA GCA CCT TGC TAAGCACCCG 1198
  • Lys Val Glu lie lie Asn Ala Gly Phe Thr Leu Asn Gly Val Lys Asn 145 150 155 160
  • ATC AAG AAA AAT GTC TTA GCG AGG ACT GGT ACT GGC AAC GCA GAG TCG 1008 He Lys Lys Asn Val Leu Ala Arg Thr Gly Thr Gly Asn Ala Glu Ser 325 330 335
  • AAG AAA AAT GTC CTA GCG AGG ACT GGT ACA GGC GCT GCT GAG TCG ATG 1008 Lys Lys Asn Val Leu Ala Arg Thr Gly Thr Gly Ala Ala Glu Ser Met 325 330 335
  • GCCAATTCTC CTAAGCTTTT GCAATGATCA AAAATACTTT TTTATTTTAT TTTTAATATT 1261 TTATATGTAC TGGAAATGAA CCATTACCTT CTAGTACTCT ATAACATGTT TTGCATTTA 1320
  • GCG AAA GTT GAA ATC ATT AAC GCT GGT TTC GCC ATC TAT AAT GTC AAG 480
  • GGC ATG ATG CAA GCT GAA CCG GGA GAT ATG GTT CCA CAA CTC ACC ATG 1152 Gly Met Met Gin Ala Glu Pro Gly Asp Met Val Pro Gin Leu Thr Met 370 375 380
  • MOLECULE TYPE protein

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Abstract

La présente invention se rapporte à des peptides isolés des allergènes protéiniques majeurs d'Ambrosia artemisiifolia ou du pollen d'ambrosiacées. Les peptides de l'invention comprennent au moins un épitope des lymphocytes T, ou de préférence au moins deux épitopes des lymphocytes T d'un allergène protéinique sélectionné parmi les allergènes Amb a I.1, Amb a I.2, Amb a I.3, Amb a I.4 et Amb a II. Des peptides modifiés présentent des propriétés thérapeutiques similaires ou de meilleures propriétés que l'allergène naturel correspondant ou d'une partie de celui-ci, mais ont moins d'effets secondaires. L'invention se rapporte également à des acides nucléiques ayant des séquences codant les peptides de l'invention. Des procédés de traitement ou de diagnostic de la sensibilité d'un individu aux allergènes du pollen d'ambrosiacées, ainsi que des compositions thérapeutiques comprenant au moins un peptide de l'invention sont également décrits, y compris des formulations multipeptidiques à usage thérapeutique s'appliquant à l'homme.
PCT/US1995/014362 1994-10-27 1995-10-24 Epitopes des lymphocytes t des allergenes majeurs provenant d'ambrosia artemisiifolia WO1996013589A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012213A3 (fr) * 1996-09-23 1998-04-30 Peptides contenant cysteine
EP1369483A1 (fr) * 2002-06-04 2003-12-10 BIOMAY Produktions- und Handels- Aktiengesellschaft Allergène du pollen de l'armoise
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
EP1958645A1 (fr) * 2007-02-13 2008-08-20 Biomay AG Peptides dérivés du allergène majeur de l'ambrosie (Ambrosia artemisiifolia) et leur utilisation
EP2153841A1 (fr) * 2008-08-15 2010-02-17 Circassia Limited Vaccin comprenant des peptides de Amb 1 a pour le traitement de l'allergie des ambroisies
JP2012500194A (ja) * 2008-08-15 2012-01-05 サーカッシア リミテッド Ｉｌ−１０産生の刺激のためのアレルゲン由来のｔ細胞抗原
WO2013001362A3 (fr) * 2011-06-27 2013-07-11 Anergis S.A. Peptides contigus se chevauchant destinés au traitement de l'allergie au pollen d'ambroisie
AU2013257437B2 (en) * 2007-02-13 2016-03-03 Biomay Ag Peptides derived from the major allergen of ragweed (ambrosia artemisiifolia) and uses thereof
EP3295956A1 (fr) 2016-09-20 2018-03-21 Biomay Ag Construction de polypeptide comprenant des fragments d'allergenes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021321A2 (fr) * 1992-04-09 1993-10-28 Immulogic Pharmaceutical Corporation EPITOPES DE LYMPHOCYTES T DES PRINCIPAUX ALLERGENES D'$i(AMBROSIA ARTEMISIIFOLIA)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021321A2 (fr) * 1992-04-09 1993-10-28 Immulogic Pharmaceutical Corporation EPITOPES DE LYMPHOCYTES T DES PRINCIPAUX ALLERGENES D'$i(AMBROSIA ARTEMISIIFOLIA)

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
WO1998012213A3 (fr) * 1996-09-23 1998-04-30 Peptides contenant cysteine
EP1369483A1 (fr) * 2002-06-04 2003-12-10 BIOMAY Produktions- und Handels- Aktiengesellschaft Allergène du pollen de l'armoise
WO2003102189A1 (fr) * 2002-06-04 2003-12-11 Biomay Produktions-Und Handels-Aktien Gesellschaft Allergene du pollen d'armoise
AU2003238199B2 (en) * 2002-06-04 2007-09-20 Biomay Produktions-Und Handels-Aktiengesellschaft Allergen from mugwort pollen
EP2491949A1 (fr) 2007-02-13 2012-08-29 Biomay Ag Peptides dérivés du allergène majeur de l'ambrosie (ambrosia artemisiifolia) et leur utilisation
AU2013257437B2 (en) * 2007-02-13 2016-03-03 Biomay Ag Peptides derived from the major allergen of ragweed (ambrosia artemisiifolia) and uses thereof
US9360486B2 (en) 2007-02-13 2016-06-07 Biomay Ag Allergen fragments
JP2010518813A (ja) * 2007-02-13 2010-06-03 ビオマイ アクチエンゲゼルシャフト ブタクサ（アンブロシアアルテミシーフォリア）の主要アレルゲンから得られるペプチドおよびこれらの使用
WO2008098749A3 (fr) * 2007-02-13 2008-11-27 Biomay Ag Fragments d'allergène
JP2015078218A (ja) * 2007-02-13 2015-04-23 ビオマイ アクチエンゲゼルシャフト ブタクサ（アンブロシアアルテミシーフォリア）の主要アレルゲンから得られるペプチドおよびこれらの使用
AU2008214852B2 (en) * 2007-02-13 2013-08-29 Biomay Ag Peptides derived from the major allergen of ragweed (Ambrosia artemisiifolia) and uses thereof
EP2491948A1 (fr) 2007-02-13 2012-08-29 Biomay Ag Peptides dérivés du allergène majeur de l'ambrosie (ambrosia artemisiifolia) et leur utilisation
EP1958645A1 (fr) * 2007-02-13 2008-08-20 Biomay AG Peptides dérivés du allergène majeur de l'ambrosie (Ambrosia artemisiifolia) et leur utilisation
EP2153841A1 (fr) * 2008-08-15 2010-02-17 Circassia Limited Vaccin comprenant des peptides de Amb 1 a pour le traitement de l'allergie des ambroisies
EP2444100A3 (fr) * 2008-08-15 2012-07-25 Circassia Limited Vaccin comprenant des peptides Amb a 1 pour une utilisation dans le traitement de l'allergie à l'herbe à poux
US20120108524A1 (en) * 2008-08-15 2012-05-03 Circassia Limited Vaccine comprising amb a 1 peptides for use in the treatment of ragweed allergy
JP2012500193A (ja) * 2008-08-15 2012-01-05 サーカッシア リミテッド ブタクサアレルギーの治療用のＡｍｂａ１ペプチド
EP2433639A3 (fr) * 2008-08-15 2012-06-27 Circassia Limited Vaccin comprenant des peptides Amb a 1 pour une utilisation dans le traitement de l'allergie à l'herbe à poux
US8491910B2 (en) 2008-08-15 2013-07-23 Circassia Limited Vaccine comprising AMB A 1 peptides for use in the treatment of ragweed allergy
JP2012500194A (ja) * 2008-08-15 2012-01-05 サーカッシア リミテッド Ｉｌ−１０産生の刺激のためのアレルゲン由来のｔ細胞抗原
US9347937B2 (en) 2008-08-15 2016-05-24 Circassia Limited Vaccine comprising Amb a 1 peptides for use in the treatment of ragweed allergy
AU2009281013B2 (en) * 2008-08-15 2015-01-29 Circassia Limited Vaccine comprising Amb a 1 peptides for use in the treatment of ragweed allergy
US8821887B2 (en) 2008-08-15 2014-09-02 Circassia Limited T-cell antigen peptide from allergen for stimulation of IL-10 production
CN104710512A (zh) * 2008-08-15 2015-06-17 切尔卡西亚有限公司 用于治疗豚草变态反应的包含Amb a 1肽的疫苗
CN102170897A (zh) * 2008-08-15 2011-08-31 切尔卡西亚有限公司 用于治疗豚草变态反应的包含Amb a 1肽的疫苗
WO2010018378A3 (fr) * 2008-08-15 2010-07-22 Circassia Limited Peptides d'ambroisie pour vaccin
US9005627B2 (en) 2011-06-27 2015-04-14 Anergis S.A. Contiguous overlapping peptides for treatment of ragweed pollen allergy
JP2014520791A (ja) * 2011-06-27 2014-08-25 アネルギ エスアー ブタクサ花粉アレルギーの治療のための連続する重複ペプチド
WO2013001362A3 (fr) * 2011-06-27 2013-07-11 Anergis S.A. Peptides contigus se chevauchant destinés au traitement de l'allergie au pollen d'ambroisie
AU2012277491B2 (en) * 2011-06-27 2017-03-23 Anergis S.A. Contiguous overlapping peptides for treatment of ragweed pollen allergy
EP3295956A1 (fr) 2016-09-20 2018-03-21 Biomay Ag Construction de polypeptide comprenant des fragments d'allergenes
WO2018054993A1 (fr) 2016-09-20 2018-03-29 Biomay Ag Construction polypeptidique comprenant des fragments d'allergènes
US11926651B2 (en) 2016-09-20 2024-03-12 Worg Pharmaceuticals (Zhejiang) Co., Ltd. Polypeptide construct comprising fragments of allergens

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