WO2015054217A2 - Méthodes et utilisations pour réduire une réaction allergique chez un sujet - Google Patents

Méthodes et utilisations pour réduire une réaction allergique chez un sujet Download PDF

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WO2015054217A2
WO2015054217A2 PCT/US2014/059449 US2014059449W WO2015054217A2 WO 2015054217 A2 WO2015054217 A2 WO 2015054217A2 US 2014059449 W US2014059449 W US 2014059449W WO 2015054217 A2 WO2015054217 A2 WO 2015054217A2
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amino acid
seq
acid sequence
peptide
identity
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WO2015054217A3 (fr
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Alessandro Sette
Bjoern Peters
Véronique SCHULTEN
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La Jolla Institute For Allergy And Immunology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen

Definitions

  • the invention relates generally to the field of medical science and immunology, more specifically to the field of allergy immunotherapy with peptides and proteins.
  • Allergen-specific immunotherapy is a hyposensitizing immunotherapy introduced in clinical medicine almost a century ago for the treatment of a type 1 hypersensitivity immune response.
  • Type I allergy is mediated by activated allergen- specific Th2 cells, which produce cytokines such as IL-4, IL-5, and IL-13.
  • Th2 cells which produce cytokines such as IL-4, IL-5, and IL-13.
  • the allergen-specific T-cell response is mediated predominantly by Th1 cells.
  • SIT may reduce the ratio of Th2:Th1 cells and may alter the cytokine profile, reducing the production of IL-4, IL-5, and IL-13 and increasing the production of IFN- g in response to major allergens or allergen extracts.
  • full length wild type protein or variants thereof in which the peptides are present may have the same immunodiagnostic and/or therapeutic potential.
  • a first aspect of the invention relates to a therapeutic method for treating an allergic immune response against a grass pollen allergen.
  • the method comprises administering to a subject in need thereof a therapeutically sufficient amount of a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1 -125 set out in Tables 3a and 3b).
  • the method comprises administering a source protein comprising as part of its sequence one or more peptide sequences able to down regulate a T cell response during SIT.
  • the method comprises administering to a subject in need thereof a therapeutically sufficient amount of a protein comprising an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS:1-125 set out in Tables 3a and 3b.
  • the invention also relates to a peptide or a protein for use in treating an allergic immune response against a grass pollen allergen, wherein i) the peptide comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-125 set out in Tables 3a and 3b; or ii) the protein comprises an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 1 -125 set out in Tables 3a and 3b,
  • the invention relates to the use of a peptide or a protein for manufacturing a medicament for treating an allergic immune response against a grass pollen allergen, wherein i) the peptide comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-125 set out in Tables 3a and 3b; or ii) the protein comprises an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 1 -125 set out in Tables 3a and 3b.
  • peptides in diagnostic or immunotherapy is limited by their ability to bind a plurality of HLA Class II molecules present in a target population, the unique set of peptides have, either singly or in combination, diagnostic and therapeutic value with substantial population coverage in diverse ethnicities.
  • a therapeutic method comprising administering to a subject in need thereof a therapeutically sufficient amount of i) a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4, or ii) a protein comprising an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 126- 145 set out in Table 4, e.g. a protein comprising an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 146-149 set out in Table 6.
  • the invention also relates in one aspect to a peptide combination or a protein for use in treating an allergic immune response against a grass pollen allergen, wherein i) the peptide combination comprises 2-20 peptides, wherein a peptide in the combination comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4; and ii) the protein comprising an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4.
  • the invention also relates to the use of a peptide combination or a protein for manufacturing a medicament for treating an allergic immune response against a grass pollen allergen, wherein i) the peptide combination comprises 2-20 peptides, wherein a peptide in the combination comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4; and ii) the protein comprising an amino acid sequence with at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4.
  • a composition such as a
  • compositions comprising one or more of the peptides of Table 4.
  • the composition comprises a combination of 2-20 peptides, wherein a single peptide in the combination comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145 set out in Table 4.
  • Such compositions may have value as immunotherapeutic product and/or diagnostic reagent.
  • a still further aspect of the invention relates to source proteins (wild type proteins) found in grass pollen, which comprise one or more of the IL-5 modulating peptide amino acid sequences as part of its amino acid sequence.
  • This is for example proteins numbered herein 2, 49/54, 53, 62, 6, 7, 52 and 89, which been detected and identified as wild type proteins in aqueous extracts of grass pollen (Table 6).
  • a further aspect of the invention relates to a protein comprising an amino acid sequence with at least 65% identity over the length of a reference sequence selected from any of SEQ ID NOS: 146-154 set out in Table 6.
  • a still further aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more peptides defined herein or proteins defined herein as well as and one or more pharmaceutically acceptable ingredients or carrier.
  • a pharmaceutical composition is formulated for administration as an immunotherapeutic product, such as formulation for injection (e.g. subcutaneous or intradermal)
  • a pharmaceutical composition may comprise lyophilized peptides or proteins. Lyophilized peptides or proteins may either be re- dissolved in a suitable solvent before administration or be administered lyophilized as part of a solid dosage form or a device that delivers the lyophilized powders to an oral mucosa.
  • Figures 1A to 1 F Cytokine production by PBMCs in response to the TG extract and peptide pools. Peptides that induce the production of IL-5 (A and B), IFN-Y (C and D), and IL-10 (E and F) by PBMCs from 20 patients receiving SIT and 20 controls. Panels A, C, and E show the percentage of PBMC samples from patients who responded to each antigen. Panels B, D, and F show average responses of PBMCs from 20 patients to each antigen.
  • Figures 2A and 2B Cytokine production by PBMCs from control subjects and patients who received SIT in response to TG antigens. Circles indicate the number of SFCs in PBMCs from individual donors (black circles, from 20 controls; white circles, from 20 patients who received SIT), indicating the production of IL-5 (A) and IFN- ⁇ (B) in response to the TG extract, known allergens, and additional TG antigens. * P ⁇ .05, ** P ⁇ .005, based on the Student t test.
  • Figures 3A and B IL-5 production by PBMCs in response to modulated
  • ATGAs show the number of responding patients who received SIT (white bar) and controls (black bar) whose PBMCs produced IL-5 in response to a subset of ATGAs that show modulated IL-5 production after SIT treatment.
  • B shows the magnitude of the IL-5 response to the subset of antigens in PBMCs from patients who received SIT (white bar) or control (black bar) subjects.
  • Figures 4A to 4C Two-dimensional (2D) gel electrophoresis and immunoblot analysis of the TG extract, using pooled serum samples from controls and patients who received SIT.
  • Figure 4A shows the Coomassie-stained 2D gel of the TG extract.
  • Figures 4B and 4C show immunoblot analysis of proteins that react with IgG (green spots), IgE (red spots), or both (yellow spots) in sera from controls (B) and patients who received SIT (C). Yellow circles indicate locations of the 13 ATGAs that induce IL-5 production in PBMCs from controls.
  • Figures 5A to 5C Modulation of cytokine production by PBMCs from patients who received SIT in response to the MNA pool.
  • Figure 5A shows cytokine production (measured as SFCs) from patients receiving SIT (white dots) or controls (black dots). Line indicates median values. * P ⁇ .05, *** P ⁇ .001 , determined by
  • Figure 5B shows IL-5 production by PBMCs from controls (black dots, left), patients who did not respond to SIT or were uncertain of their response (white dots, middle), or patients who reported improved symptoms after receiving SIT (dots with X, right) in response to the MNA pool.
  • the dashed line indicates a strong response.
  • Figure 5C shows a bar chart showing the percentage of patients in each group who had PBMC responses of more than 100 SFCs for IL-5. * P ⁇ .05, determined by Fisher exact test.
  • Figures 6A and 6B Intracellular cytokine staining to measure TH2 cytokines IL-4 and IL-13.
  • allergen is meant to designate a proteinaceous substance capable of eliciting a type 1 hypersensitivity immune response in an animal, such as a human. Typically, an allergen is able to induce specific IgE antibodies in an animal and bind to IgE antibodies.
  • the allergen may be a sensitizing allergen or an allergen capable of cross-reacting with an antibody able to bind the sensitizing allergen.
  • the term "sensitized to” is generally meant to encompass that the subject has been exposed to a protein in a manner that the individual's adaptive immune system displays memory to the protein, for example that the protein has induced detectable IgE antibodies against the protein and/or that T-cells stimulated in vitro are able to proliferate under the presence of the protein or fragments of the protein (e.g. linear peptides).
  • allergic immune response is meant to encompass a hypersensitivity immune response, e.g. type 1 immune response, such as typically an immune response that is associated with the production of IgE antibodies (i.e. IgE-mediated immune response).
  • An allergic immune response may be associated with an allergic disease, for example atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, anaphylaxis, food allergy and hay fever.
  • the term "immunotherapy” is meant to encompass treatment of a disease by inducing, enhancing, or suppressing an immune response.
  • the therapeutically active agent is an antigen, more particularly an allergen.
  • An antigen may be a protein or a fragment thereof (e.g. immunogenic peptide).
  • Immunotherapy usually encompasses repeated administration of a sufficient dose of the
  • antigen/allergen/protein/peptide usually in microgram quantities, over a prolonged period of time, usually for more than 1 year, such as 2 or 3 years, during which period the antigen may be administered daily or less frequent, such as several times a week, weekly, bi-weekly, or monthly, every second month or quarterly.
  • specific immunotherapy is meant to designate that immunotherapy is conducted with the administration of an antigen to which the subject is sensitized to, e.g. an antigen to which the patient has raised specific IgE antibodies to or has a T cell response to.
  • grass pollen is meant to designate pollen of the plant family Poaceae, for example pollen of the plant genus Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus, Hordeum, Lolium, Oryza, Paspalum, Phalaris, Phleum, Poa, Secale, Sorghum, Triticum and Zea.
  • subject is meant to designate a mammal having an adaptive immune system, such as a human, a domestic animal such as a dog, a cat, a horse or cattle.
  • treatment refers to any type of treatment that conveys a benefit to a subject afflicted with allergy, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the onset of symptoms, slowing the progression of symptoms, or induce disease modification etc.
  • Typical symptoms of an allergic reaction is nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function.
  • the treatment may also give the benefit that the patient needs less concomitant treatment with corticosteroids or H1 antihistamines to suppress the clinical symptoms.
  • treatment is not necessarily meant to imply cure or complete abolition of symptoms, but refers to any type of treatment that imparts a benefit to a patient.
  • Treatment may be initiated before the subject becomes sensitized to a protein. This may be realized by initiating immunotherapy before the subject has raised detectable serum IgE antibodies capable of binding specifically to the sensitizing protein or before any other biochemical marker indicative of an allergic immune response can be detected in biological samples isolated from the individual.
  • treatment may be initiated before the subject has evolved clinical symptoms of the allergic disease, such as symptoms of allergic rhinitis, allergic asthma or atopic dermatitis.
  • a therapeutically sufficient amount is meant to designate an amount effective to reduce, suppress, relieve or eliminate an allergic immune response, e.g. an amount sufficient to achieve the desirable reduction in clinical relevant symptoms or manifestations of the allergic immune response.
  • a therapeutically sufficient amount may be the accumulated dose of a peptide, a set of peptides, a protein and/or a set of proteins administered during a course of immunotherapy in order to achieve the intended effect or it may be the maximal dose tolerated within a given period.
  • the total dose or accumulated dose may be divided into single doses administered daily, twice a week or more, weekly, every second or fourth week or monthly depending on the route of administration and the pharmaceutical formulation used.
  • the total dose or accumulated dose may vary. It is expected that a single dose is in the microgram range, such as in the range of 5 to 500 microgram dependent on the peptide and protein.
  • patient responding to therapy such as “immunotherapy” is meant to designate that the patient has improvement in the symptoms of the allergic immune response caused by grass or grass pollen, including Timothy grass pollen.
  • Symptoms may be the clinically symptoms of allergic rhinitis, allergic asthma allergic conjunctivitis, atopic dermatitis, food allergy and/or hay fever.
  • the symptoms are the same as experienced with a flu/cold, sneezing, itching, congestion, coughing, feeling of fatigue, sleepiness and body aches.
  • nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes
  • respiratory symptoms in the form of decreased lung function.
  • a responder may also be evaluated by monitoring the patient's reduced need for concomitant treatment with corticosteroids or H1 antihistamines to suppress the clinical symptoms.
  • Symptoms may be subjectively scored or in accordance with official guidelines used in clinical trials of SIT.
  • adjuvant refers to a substance that enhances the immune response to an antigen. Depending on the nature of the adjuvant, it can promote either a cell- mediated immune response, humoral immune response or a mixture of the two.
  • An "analyte” as described herein refers to an entity that binds to a target such as a nucleic acid or polynucleotide sequence, or an amino acid sequence, protein, polypeptide, or peptide. Analytes that may be used in methods and uses of the present invention therefore include any molecular entity capable of binding to a target, typically that which is able to specifically bind to a target, for example, a cytokine such as IL-5, IL-4 or IL-13.
  • a peptide for use in any aspects of the invention consists of, consists essentially of or comprises a reference amino acid sequence set out in Tables 3a or 3b and Table 4 or a functional equivalent thereof.
  • Peptides shown in Tables 3a, 3b and 4 have been shown to be recognized by IL-5 producing T-cells obtained from patients allergic to a grass pollen allergen and thus contain at least one T-cell epitope, such as a Th2-cell epitope. Epitopes are considered to contain at least 9 amino acids.
  • a peptide for use in any aspects of the invention comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1 -125 set out in Tables 3a and 3b and 126-145 set out in Table 4.
  • the amino acid sequence has at least 70% identity or homology, such as at least 75%, 80%, 85%, 90% or 95% identity or homology over at least 9, such as at least 10, 11 , 12, 13, 14 or 15 or all contiguous amino acids of the reference sequence set out in Tables 3a, 3b or 4.
  • a peptide comprises an amino acid sequence having at least 75% identity or homology over at least 9 contiguous amino acids of the reference sequence; or a peptide comprises an amino acid sequence having at least 85% identity or homology over at least 9 contiguous amino acids of the reference sequence; or a peptide comprises an amino acid sequence having at least 95% identity or homology over at least 9 contiguous amino acids of the reference sequence; a peptide comprises an amino acid sequence having at least 75% identity or homology over at least 12 contiguous amino acids of the reference sequence; or a peptide comprises an amino acid sequence having at least 85% identity or homology over at least 12 contiguous amino acids of the reference sequence; or a peptide comprises an amino acid sequence having at least 95% identity or homology over at least 12 contiguous amino acids of the reference sequence; a peptide comprises an amino acid sequence having at least 80% identity or homology over the length of the reference sequence; or a peptide comprises an amino acid sequence having at least 85% identity or homology over the length
  • peptides with high IL-5 modulating activity for example peptides comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-51 set out in Table 3a; or selected from any of SEQ ID NOS: 126-145 set out in Table 4; or selected from any of SEQ ID NOS: 136-138, 141-145 (peptides (reference sequences derived from proteins 49/54, 62 and 91 showing the highest IL-5 modulating activity). It might also be considered to at least treat with a peptide having both high IL-5 modulating activity (Table 4) and high population coverage (Table 11 ). Therefore, in some embodiments, a peptide comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of reference sequence with SEQ ID NOS: 141 , 126 or 128.
  • the administration may be with a single peptide or single protein or a combination of peptides or a combination of proteins. Due to the MHC restriction of peptides, various subjects do not respond to the same peptide. Therefore, in certain circumstances, it might be advantageous to combine several peptides to treat a greater population of subjects with the same immunotherapeutic product. Thus, in some embodiments there is administered at least two peptides, e.g.
  • a combination of 2-25 different peptides wherein a single peptide in the combination comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-125 set out in Tables 3a, 3b and 126-145 set out in
  • Table 4 One may consider to administer a combination of 3-25, 4-25, 5-25, 6-25, 7- 25 peptides defined herein, such as a combination of 2-20, 3-20, 4-20, 5-20, 6-20, 7- 20 peptides defined herein, such as a combination of 2-15, 3-15, 4-15, 5-15, 6-15, 7- 15 peptides defined herein, such as a combination of 2-12, 3-12, 4-12, 5-12, 6-12, 7- 12 peptides defined herein, such as a combination of 2-10, 3-10, 4-10, 5-10, 6-10, 7- 10 peptides defined herein.
  • one or more proteins may be administered, such as a combination of 2 to 5 proteins, such as a combination of 2, 3, 4, or 5 proteins defined herein.
  • the administration of one peptide or one protein may be simultaneously,
  • the combination of peptides includes only the peptides of Table 4.
  • the combination may at least comprise a peptide showing both high IL-5 modulating activity (Table 4) and high population coverage (Table 11 ).
  • a peptide combination comprises a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of reference sequence with SEQ ID NOS: 141 , 126 or 128.
  • the combinations may contain additional peptides, for example peptide combinations comprising 3 to 20, 4 to 20, 5 to 20, 6 to 20 or 7 to 20 peptides.
  • the peptide may contain additional peptides, for example peptide combinations comprising 3 to 20, 4 to 20, 5 to 20, 6 to 20 or 7 to 20 peptides.
  • combination comprises 20 peptides, wherein a single peptide comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence with SEQ ID NOS: 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 and/or 145.
  • any peptide administered in combination with another peptide may not derive from the same reference sequence or comprise the same reference sequence.
  • a single peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence with SEQ ID NO: 126 may not be combined with another peptide with an amino acid sequence of SEQ ID NO:126 (e.g. a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 12 contiguous amino acids of a reference sequence with SEQ ID NO: 126), but may be combined with a peptide with an amino acid sequence of another reference sequence selected from any of Tables 3a, 3b and 4 (e.g. a single peptide
  • the peptide combination comprises 20 peptides, wherein a single peptide comprises an amino acid sequence with SEQ ID NOS: 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 or 145, respectively, or a functional equivalent thereof.
  • a peptide utilized in the methods and uses of the invention can be soluble in aqueous solutions, such as buffered aqueous solutions or un-buffered aqueous solutions having pH in the range of 3 to 9, e.g. pH in the range of 4 to 8. Therefore, in some embodiments of the invention, the peptide contains less than 30 amino acids, but peptides containing more peptides are still applicable if sufficiently water soluble.
  • a peptide has a length of 9 to 30 amino acids, such as a length of 9 to 25; 9 to 20; 9 to 19; 9 to 18; 9 to 17; 9 to 16; 9 to 15 amino acids, such as a length of 10 to 30; 10 to 25; 10 to 20; 10 to 19; 10 to 18; 10 to 17; 10 to 16; 10 to 15 amino acids, such as a length of 11 to 30; 11 to 25; 11 to 20; 1 1 to 19; 11 to 18; 11 to 17; 1 1 to 16; 1 1 to 15 amino acids, such as a length of 12 to 30; 12 to 25; 12 to 20; 12 to 19; 12 to 18; 12 to 17; 12 to 16; 12 to 15 amino acids, such as a length of
  • 13 to 30 13 to 25; 13 to 20; 13 to 19; 13 to 18; 13 to 17; 13 to 16; 13 to 15 amino acids, such as a length of 14 to 30; 14 to 25; 14 to 20; 14 to 19; 14 to 18; 14 to 17;
  • the peptide comprises one or more amino acid residues in addition to the reference sequences of Tables 3a, 3b, and 4, wherein the additional amino acid residue(s) is/are selected from an amino acid residue or an amino acid sequence within the wild type protein of which the reference sequence is a part of.
  • the wild type amino acid residue or wild type amino acid sequence to be added to extend the length of the peptide may be adjacent to, subtended, comprised within, overlapping with or is a part of the reference sequence when present in its natural biological context within the wild type protein.
  • a variant sequence e.g. a functional equivalent, may be a longer peptide, which includes a peptide disclosed herein or a partial sequence thereof.
  • peptides herein may contain additional amino acid residues at the N- and/or C- terminal end of a reference sequence disclosed herein, such as additional amino acids selected from amino acids flanking the N- and/or C- terminal ends when the peptide is aligned with the source protein it is present in, based upon or derived from.
  • additional amino acids may be the amino acids flanking the N- and/or C-terminal ends of the peptide when aligned to protein 2.
  • a protein comprising one or more of the peptide sequences with IL-5 modulating activity may be administered to a subject in need thereof.
  • a protein comprising one or more reference sequence(s) set out in Tables 3a, 3b, and 4 may be employed in therapeutic methods and uses of the present invention.
  • a protein to be administered comprises an amino acid sequence with at least 65% identity or homology, such as at least 70%, 75%, 80%, 85%, 90% or 95% over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 1 -51 set out in Table 3a, SEQ ID NOS: 42-125 set out in Table 3b and/or SEQ ID NOS: 126-145 set out in Table 4, e.g. SEQ ID NOS: 136-138, 141 -145 that are reference sequences deriving from proteins 49/54, 53, 62 and 91 and have shown high IL-5 modulating activity.
  • the protein comprises at least 2, e.g. 2-25 or 2-20 different amino acid sequences, such as at least 3, 4, or 5 different amino acid sequences each having at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 1 -51 set out in Table 3a, SEQ ID NOS: 52-125 set out in Table 3b, SEQ ID NOS: 126-145 set out in Table 4, e.g. SEQ ID NOS: 136-138, 141 -145.
  • a protein comprises all the amino acid sequences of all of the peptides in Table 4, i.e. a protein comprising the amino acid sequences with SEQ ID NOS: 126, 127, 128,
  • the protein is a protein found in grass pollen, for example in Timothy grass pollen.
  • the present invention relates in some aspects to a protein consisting of, consisting essentially of or comprising an amino acid sequence having at least 65% identity or homology over the length of a reference amino acid sequence selected from any of SEQ ID NOS: 146-154 set out in Table 6 (full length proteins numbered 2, 49/54, 53, 62, 6, 7, 52, 89 and a fragment of 45/54.).
  • the percent identity or homology is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% over the length of a reference amino acid sequence selected from any one of SEQ ID NOS: 146-154.
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 1-18 such as selected from any of SEQ ID NOS: 126-135.
  • An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 146 (NTGA 2).
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 25-28, 38- 40, such as selected from any of SEQ ID NOS: 136, 137, 138 and 141.
  • An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 148 or 154 (NTGA 49/54).
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 29-37, such as selected from any of SEQ ID NOS: 139 and 140.
  • An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 149 (NTGA 53).
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 41 -48, such as selected from any of SEQ ID NOS: 142 and 143.
  • An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 147 (NTGA 62).
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 144 and 145 (NTGA 91 ). In some embodiments, a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 52 to 75. An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 150 (NTGA 6). In some embodiments, a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 76-84. An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 151 (NTGA 7).
  • a protein comprises as part of its amino acid sequence one or more of the reference sequences selected from any of SEQ ID NOS: 112-125.
  • An example thereof is a protein comprising an amino acid sequence with at least 65% identity to SEQ ID NO: 153 (NTGA 89).
  • the invention provides the proteins in isolated and/or purified form, such as in compositions consisting essentially of the protein in isolation, such as in purified form.
  • isolated means that the proteins/compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment, such as separated from grass pollen or a plant cell.
  • isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more nucleic acids or cell membranes.
  • isolated does not exclude alternative physical forms of the composition, such as fusions/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host-cells produced by the hand of man.
  • An “isolated” protein/composition can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates in nature.
  • An isolated protein or peptide may be lyophilized, so as to extend physical or chemical stability.
  • Proteins and peptides can be prepared recombinantly, chemically synthesized, isolated from a biological material or source, and optionally modified, or any combination thereof.
  • the invention also relates to a nucleic acid encoding a protein of SEQ ID NOS: 146 - 154 set out in Table 6, e.g. a nucleic acid that is isolated or purified.
  • identity and “identical”, and grammatical variations thereof, mean that two or more amino acid sequences are the same. Thus, where a protein or a peptide is compared to a reference sequence and are identical, they have the same amino acid sequence.
  • the identity can be over a defined area of the reference sequence, such as over at least 9 contiguous amino acids or over at least 65% of the reference sequence when optimally aligned (with gaps permitted). Identity can be determined by comparing each position in aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as is known in the art, including the
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www. n cbi . n I m . n i h . g ov/). Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area.
  • the degree of homology may be determined between a peptide or protein and the reference sequence, optionally over a certain length of the reference amino acid sequence, such as over at least 9 contiguous amino acids up to the entire length of the reference sequence.
  • a peptide or protein as defined herein is a "homologue" of, or is “homologous” to, the reference sequence if the two sequences have substantial identity over a specified region when optimally aligned (with gaps permitted), such as at least 65% identity over at least 9 contiguous amino acids and a functional activity of the sequences is preserved or conserved, at least in part (as used herein, the term "homologous" does not infer nor exclude evolutionary relatedness).
  • homologous peptides and proteins include proteins or peptides of non-Timothy grass proteins, including for example homologous proteins found in other grass pollen in the family Poaceae, e.g. grass pollen of a genus selected from Anthoxanthum, Cynodon, Dactylis, Lolium, Phleum and Poa.
  • a protein having less than 100% identity to a reference sequence e.g. a reference sequence of Table 6) or a peptide having less than 100% identity to a reference sequence (e.g. a reference sequence of Tables 3a, 3b or 4) is considered to be a variant or derivative of the protein and peptide, respectively.
  • a variant refers to an amino acid sequence with amino acid deletions, additions and substitutions in relation to the reference amino acid sequences (e.g. reference amino acid
  • a variant therefore includes deletions, including truncations and fragments; insertions and additions, including tagged polypeptides and fusion proteins; substitutions, for example conservative
  • substitutions site-directed mutants and allelic variants; and modifications, including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications.
  • modifications including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications.
  • Non-limiting examples of variants include one or more amino acid substitutions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30- 50, 50-100, or more residues), additions and insertions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues) and deletions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100) of a reference amino acid sequence of Tables 2, 3a, 3b, 4, 5, 6.
  • amino acid substitutions e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30- 50, 50-100, or more residues
  • additions and insertions e.g., 1
  • Variants include naturally-occurring polymorphisms or allelic variants, strain variants, as well as synthetic proteins and peptides that contain a limited number of conservative amino acid substitutions of the amino acid sequence (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 conservative substitutions).
  • conservative amino acid substitutions e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 conservative substitutions.
  • a variety of criteria can be used to indicate whether amino acids at a particular position in a protein or peptide are similar.
  • substitutions of "like" amino acid residues can be basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the same function as the peptide or protein by use of routine testing, e.g. by testing the ability to secrete IL-5, IL-4 and/or IL-13 in a sample of PBMCs as described herein and further to show reduction in the specific IL-5, IL-4 and/or IL-13 response in allergic donors versus SIT donors.
  • a variant may include a functional equivalent, e.g. one that binds to same HLA Class II molecules, such as sharing the HLA DR, DQ and/or DP allelic variants, with the peptide or protein from which the variant derives. This may be assessed via MHC Class II binding prediction tools or assessed by in vitro HLA binding assays.
  • the functional equivalent sequence binds to the same, substantially the same or at least 80%, such as at least 85%, 90% or 95% or more of the Class HLA II alleles that binds to the reference sequence from which the functional equivalent sequence is modified from.
  • Class HLA II alleles consists of the group of HLA-DPA10201 -DPB10101 , HLA-DPA10103-DPB10201 , HLA-DPA10103-DPB10301 , HLA-DPA10103-DPB10401 , HLA-DPA10103- DPB10402, HLA-DPA10202-DPB10501 , HLA-DPA10201 -DPB11401 , HLA- DQA10501 -DQB10201 , HLA-DQA10501 -DQB10301 , HLA-DQA10301 -DQB10302, HLA-DQA10401 -DQB10402, HLA-DQA10101 -DQB10501 , HLA-DQA10102-
  • a functional equivalent is identified according to its binding to DRB1 alleles only,
  • a functional equivalent typically comprises a longer amino acid sequence of up to 30-60 amino acids long (e.g. up to 30 amino acids, e.g. up to 28 or 25 amino acids long) compared to the reference sequence that the functional equivalent derives from.
  • a variant sequence including a functional equivalent thereof, may share the ability to recognize a T cell epitope with the peptide it is modified from.
  • a T cell epitope may be determined by in-vitro T cell proliferation and cytokine assays as described herein.
  • substitutions include conservative and non- conservative amino acid substitutions.
  • a "conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy a biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge, or are hydrophilic and/or hydrophobic.
  • a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, which includes amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine,
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspart
  • phenylalanine, tryptophan particularly examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Val, lie, and Ala).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • Conservative changes can also include the substitution of a chemically derivatized moiety for a non-derivatized residue, for example, by reaction of a functional side group of an amino acid.
  • Variants include forms having a limited number of one or more substituted residues.
  • An addition can be a covalent or non-covalent attachment of any type of molecule.
  • Specific examples of additions include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitination, and derivatization by protecting/blocking groups and any of numerous chemical modifications.
  • Additional specific non-limiting examples of an addition are one or more additional amino acid residues, such as addition of one or more amino acid residues to a reference sequence of Tables 3a, 3b, 4 or 6. Accordingly, proteins and peptides can be a part of or contained within a larger molecule, such as another protein or peptide sequence, such as a fusion or chimera with a different (distinct) sequence.
  • an addition is a fusion (chimeric) sequence, an amino acid sequence having one or more molecules not normally present in a reference wild type sequence covalently attached to the sequence.
  • chimeric and grammatical variations thereof, when used in reference to a sequence, means that the sequence contains one or more portions that are derived from, obtained or isolated from, or based upon other physical or chemical entities.
  • a chimera of two or more different proteins may have one part of a protein or peptide and a second part of the chimera may be from a different sequence, or unrelated protein sequence.
  • Linkers such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity.
  • Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain.
  • Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence.
  • the length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g., U.S. Patent No. 6,087,329).
  • Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo- SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST).
  • sulfo-SMCC sulfo-succinimidyl derivatives
  • DSS disuccinimidyl suberate
  • DSG disuccinimidyl glutarate
  • DST disuccinimidyl tartrate
  • a variant of a peptide may include changes to avoid oxidation, improve solubility in aqueous solution, avoid aggregation, overcome synthesis problems etc.
  • a variant sequence may thus include the following:
  • a variant sequence comprising one or more modifications selected from the following: (a) any cysteine residues in the wild type sequence of the peptide are replaced with serine or 2-aminobutyric acid; (b) hydrophobic residues in the up to three amino acids at the N or C terminus of the wild type sequence of the peptide are deleted; (c) any two consecutive amino acids comprising the sequence Asp-Gly in the up to four amino acids at the N or C terminus of the wild type sequence of the peptide are deleted; and/or (d) one or more positively charged residues are added at the N- and/or C-terminus.
  • a variant sequence may comprise one, two or more lysine or arginine amino acid residue(s) added to the N- or C-terminus of the peptide to be modified, which may improve the aqueous solubility.
  • Peptides are typically provided in the form of a salt, for example as a pharmaceutically acceptable and/or a physiologically acceptable salt.
  • the salt may be an acid addition salt with an inorganic acid, an acid addition salt with an organic acid, a salt with a basic inorganic acid, a salt with a basic organic acid, a salt with an acidic or basic amino acid or a mixture thereof.
  • Typical examples on an acid addition salts with an inorganic acid is selected from any of salts with
  • An acid salt with an organic acid may be selected from any of salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, or the like.
  • Salts with an inorganic base may be selected from a salt of an alkali metal salts such as sodium salts and potassium salts; alkali earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts.
  • Salts with a basic organic base may be selected from any salt with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, ⁇ , ⁇ -dibenzylethylenediamine, caffeine, piperidine, and pyridine.
  • Salts with a basic amino acid may be selected from any salt with arginine, lysine, ornithine, or the like.
  • Salts with an acidic amino acid may be selected from any salt with aspartic acid, glutamic acid, or the like.
  • a salt, such as a pharmaceutically acceptable salt is an acetate salt.
  • the grass pollen allergen is a Timothy grass pollen (Phleum pratense). Timothy grass belongs to the grass family Poaceae and within this family there exists high homology between major allergens of the genus Anthoxanthum,
  • the allergic response is caused by an allergen from pollen of the genus Phleum and in still further embodiments, the allergic response is caused by an allergen from pollen of the genus Phleum, Anthoxanthum, Cynodon, Dactylis, Lolium and Poa.
  • the subject in need of treatment is sensitized to a Timothy grass pollen allergen, such as one or more of the allergens Phi p 1 , Phi p 5, and/or Phi p 6 or is sensitized to an homologous allergen thereof, e.g. a group 1 , 5 and/or 6 grass pollen allergen of the grass family Poaceae, e.g. from pollen of the genus Phleum, Anthoxanthum, Cynodon, Dactylis, Lolium and Poa.
  • a Timothy grass pollen allergen such as one or more of the allergens Phi p 1 , Phi p 5, and/or Phi p 6
  • an homologous allergen thereof e.g. a group 1 , 5 and/or 6 grass pollen allergen of the grass family Poaceae, e.g. from pollen of the genus Phleum, Anthoxanthum, Cynodon, Dactylis, Lolium and
  • the treatment is by immunotherapy.
  • the route of administration may be any route usually applied in the field of
  • immunotherapy for example the subcutaneous, intradermal, epicutaneous or sublingual route, but other routes of administration may also be suitable, such as the intranasal route, oral route, intralymphatic, topical or rectal route.
  • routes of administration such as the intranasal route, oral route, intralymphatic, topical or rectal route.
  • peptides and proteins may have different modes of action, for example that proteins may be processed by antigen presenting cells and peptides must be loaded on MHC molecules.
  • the protein/peptide may be repeatedly administered during a longer period before the desirable effect is achieved, such as administered daily, bi-weekly, weekly, every second week, monthly for a period of at least 2-6, for example 3 to 12 months or more.
  • the first dose may be administered before the grass pollen season and that an up-dosing phase with administration of increasing doses is implemented to reach the maintenance dose within days, weeks or months.
  • the protein/peptide may be the only therapeutically active ingredient to be administered.
  • the peptides or proteins are not administered concomitantly with another therapeutically active ingredient, for example a major allergen of grass pollen or an allergen extract of grass pollen (e.g. allergen extract containing Timothy grass pollen extract), so as to avoid competing immunological mechanisms, such as production of danger signals and/or inflammation derived from the administration of a major allergen.
  • the peptides and proteins may be administered in a therapeutically sufficient amount.
  • the sufficient amount may be determined by the amount sufficient to reduce a clinical relevant symptom of an allergic response or may be an amount sufficient to reduce the in vitro specific IL-5 cytokine secretion from PBMCs (peripheral blood monocytes) in response to at least the peptide(s) and/or proteins that the subject is administering.
  • PBMCs peripheral blood monocytes
  • the specific IL-5 secretion is reduced in comparison to before start of treatment.
  • the specific cytokine IL-5 response to peptides shown in Table 4 are significantly down-regulated under immunotherapy and have been shown to correlate well with reduction in allergic symptoms. Therefore, the treatment effect or dose-related response, dose-finding may be monitored by determining the reduced in-vitro specific cytokine IL-5 secretion in cultured PBMCs obtained from the patient in response to one or more of the peptides shown in Table 4.
  • the specific in-vitro cytokine IL-5 secretion is determined subsequent to contacting cultured PBMCs isolated from the patient to a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145.
  • the specific IL-4 and/or IL-13 secretion may also be determined or replace the determination of the IL-5 secretion as IL-4 and IL-13 also are cytokines relevant for Th2 cells.
  • Some of the peptides have shown high reduction in IL-5 response and one may choose to determine the specific IL-5 secretion in response to at least a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 136-138, 141 -145.
  • secretion in response to at least a peptide(s) that is administered to the subject or a peptide derived from a protein administered to the subject.
  • secretion may be determined utilizing at least one of the peptides showing high reduction of specific IL-5 secretion (Table 4) and high HLA population coverage in Table 1 1 , e.g. a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of reference sequence with SEQ ID NOS: 141 , 126 or 128.
  • the specific cytokine IL-5 secretion may be determined in response to a peptide combination comprising 20 peptides, wherein a single peptide thereof comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence with SEQ ID NOS: : 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 or 145, respectively. It should be understood that the reference sequence is different for each of the single peptides of the combination.
  • the determination of the specific IL-5 secretion is not based upon the same reference sequence.
  • the peptide combination comprises peptides each consisting of, consisting essentially of or comprising an amino acid sequence with SEQ ID NOS: 26, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 and/or 145, or a functional equivalent thereof.
  • the specific cytokine IL-5 secretion may be compared before initiating treatment versus during and/or after completion of treatment.
  • any beneficial effect on the allergic immune response may be assessed by determining that the in-vitro cytokine IL-5 secretion is reduced by a factor of at least 2, when comparing the specific IL-5 secretion before initiating treatment versus during and/or after completion of treatment.
  • the factor is at least 3, 4, 5, 10, 15, 20, 50 or 100 or more.
  • the specific IL-5 secretion in samples obtained before start of treatment and during treatment be determined under the same experimental conditions, for example same in-vitro assay conditions.
  • the ability of a peptide or protein of the invention to produce a specific IL-5, or alternatively IL-4 and/or IL-13 response in a subject may be determined in a sample of PBMCs (peripheral blood monocytes) obtained from the subject, such as typically from a blood sample.
  • PBMCs peripheral blood monocytes
  • the blood sample may be further processed to obtain samples enriched with PBMCs, such as processed further by centrifugation.
  • the sample of PBMCs may be further processed in vitro to expand the number of cells or to produce clones of the desired cells.
  • the desired cells may be those responding specific to a grass pollen allergen, in particular to a Timothy grass pollen allergen, such as responding to an extract of grass pollen, in particular to an extract of Timothy grass pollen with the secretion of specific IL-5 response, IL-4 and/or IL-13 response.
  • the desired cells may be those responding to a peptide or protein for use in the present therapeutic methods and uses.
  • the expansion of cells is performed by culturing the sample in vitro.
  • IL-5 response as used herein is meant to designate either the secretion of IL-5 from a T cell in response to a peptide or protein defined herein, or the IL-5 mRNA gene expression in a T cell in response to a peptide or protein defined herein.
  • IL-4 response and “IL-13 response”, respectively is meant to encompass the secretion of IL-4 and IL-13, respectively from a T-cell or the IL-4 and IL-13 mRNA gene expression
  • cytokine secretion and cytokine mRNA gene expression are commonly well-known in the art.
  • sample of PBMCs e.g. expanded and cultured PBMCs
  • a peptide, a protein, combination of peptides or combination of proteins and the specific IL-5 response, IL-4 response and/or IL-13 response is determined by immuno assays well known in the art.
  • the secretion of IL-5, IL-4 or IL-13 may be determined by ELISA techniques, such as multiplex cytokine assay or by
  • intracellular cytokine staining typically, such assays include contacting the sample of cells stimulated with a peptide or protein with an anti-IL-5, anti-IL-4 or anti-IL-13 reagent/analyte, which is further detected with labeled reagents like antibodies suitable selected for the purpose.
  • labeled reagents like antibodies suitable selected for the purpose.
  • the IL-5 mRNA gene expression or IL-4/IL-13 gene expression is usually measured by PCR techniques including microarray gene expression analysis.
  • the invention also relates to a composition, such as a pharmaceutical composition
  • a composition such as a pharmaceutical composition
  • a pharmaceutical composition comprising i) a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-51 set out in Table 3a, SEQ ID NOS:51 -125 set out in Table 3b and/or SEQ ID NOS: 126-145 set out in Table 4, such as a reference sequence selected from any of SEQ ID NOS: 136-138, 141 -148, such as SEQ ID NO: 141 , 126 and/or 128.
  • a composition comprises one or more peptides and/or one or more proteins described herein. Therefore, in certain embodiments of the invention, the
  • composition comprises a combination of 2-25 different peptides defined herein, such as a combination of 3-25, 4-25, 5-25, 6-25, 7-25 peptides defined herein, such as a combination of 2-15, 3-15, 4-15, 5-15, 6-15, 7-15 peptides defined herein, such as a combination of 2-12, 3-12, 4-12, 5-12, 6-12, 7-12 peptides defined herein, such as a combination of 2-10, 3-10, 4-10, 5-10, 6-10, 7-10 peptides defined herein.
  • the combination of peptides is 2-20 peptides, such as 3 to 20, 4-20, 5 to 20, 6 to 20 or 7 to 20 peptides, wherein each peptide comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 1-51 sett out in Table 3a, SEQ ID NOS: 52-125 set out in Table 3b and/or SEQ ID NOS: 126-145 set out in Table 4.
  • a further aspect of the invention relates to a composition
  • a composition comprising a combination of 2-20 peptides, wherein a peptide in the combination comprises an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence selected from any of SEQ ID NOS: 126-145, for example selected from any of SEQ ID NOS: 136-138, 141-145, for example wherein the peptide
  • combination at least comprises a peptide comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of reference sequence with SEQ ID NOS: 141 , 126 and/or 128.
  • the peptide combination comprises the 20 different peptides (reference sequences) of Table 4 or peptide variants thereof, for example those comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a reference sequence with SEQ ID NOS: 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 and/or 145.
  • the reference sequence is different for any peptide in a peptide.
  • the peptide combination comprises peptides each comprising an amino acid sequence with at least 65% identity or homology over at least 9 contiguous amino acids of a different reference sequence selected from any of SEQ ID NOS: 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144 and/or 145.
  • composition comprises a combination of at least two peptides or at least two proteins
  • the combination may be contained in one composition or alternatively in separate compositions each containing different peptides or proteins including combinations thereof.
  • a composition is a vaccine product, i.e. immunotherapeutic product, including but not limited to a vaccine to treat against an allergic immune response, allergic disease, or one or more physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with an allergen.
  • a vaccine product i.e. immunotherapeutic product, including but not limited to a vaccine to treat against an allergic immune response, allergic disease, or one or more physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with an allergen.
  • the composition is a diagnostic reagent and may be supplied in accurate quantities (standardized) and supplied in a suitable container that allows the composition to be re-dissolved in a solvent, preferably for direct application in an assay that determines, for example, the specific IL-5 secretion in response to the composition.
  • the composition may be supplied in an-hydrous formulation, e.g. lyophilized and eventually mixed with an inert carrier.
  • the composition may contain each of the peptides/ proteins in equimolar concentrations or in substantially equimolar concentrations.
  • each peptide may be present in a molar concentration of 1 to 1000 ⁇ , for example in the range of 10 to 800 ⁇ , for example in the range of 20 to 500 ⁇ , for example in the range of 20 to 300 ⁇ .
  • a pharmaceutical composition comprises in addition to a peptide or protein described herein pharmaceutically acceptable or physiologically acceptable excipients, which usually are therapeutically inactive ingredients.
  • Excipients for use in pharmaceutical compositions are well-known to the person skilled in the art and include solvents, emulsifiers, wetting agents, plasticizers, colouring substances, fillers, preservatives, anti-oxidants, anti-microbial agents, viscosity adjusting agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, mucoadhesive substances, and the like. Examples of formulation strategies are well-known to the person skilled in the art.
  • the peptide or protein of the invention may be any suitable amino acid sequence.
  • the peptide or protein of the invention may be any suitable amino acid sequence.
  • the adjuvant may be any conventional adjuvant, including oxygen-containing metal salts, e.g. aluminium hydroxide, chitosan, heat-labile enterotoxin (LT), cholera toxin (CT), cholera toxin B subunit (CTB), polymerised liposomes, mutant toxins, e.g. LTK63 and LTR72, microcapsules, interleukins (e.g.
  • IL-1 BETA, IL-2, IL-7, IL-12, INFGAMMA GM-CSF
  • MDF derivatives CpG oligonucleotides
  • LPS LPS
  • MPL MPL- derivatives
  • phosphophazenes Adju-Phos(R)
  • glucan antigen formulation
  • liposomes DDE, DHEA, DMPC, DMPG, DOC/Alum Complex
  • Freund's incomplete adjuvant ISCOMs(R), LT Oral Adjuvant, muramyl dipeptide, monophosphoryl lipid A, muramyl peptide, and phospatidylethanolamine.
  • the peptide or protein can be coupled with another protein, such as ovalbumin or keyhole limpet hemocyanin (KLH), thyroglobulin or a toxin such as tetanus or cholera toxin, or they can be mixed with adjuvants.
  • a pharmaceutical composition may be formulated as a solid dosage form, such as a tablet or capsule or the composition may be a liquid including a solution, a suspension, a dispersion, and a gelled liquid.
  • the composition may be an emulsion or a re-dissolvable powder, granulate or lyophilisate, which eventually can be dissolved to form a liquid before being administered.
  • the peptide/protein exhibits poor stability to a biological fluid, such as the gastric juice with which it may come in contact after delivery
  • the peptide/protein is administered in a form avoiding the contact with the biological fluid, such as the gastric juice.
  • a biological fluid such as the gastric juice
  • the peptide/protein is administered in a form avoiding the contact with the biological fluid, such as the gastric juice.
  • This may be accomplished by incorporating the peptide/protein in pharmaceutical compositions suitably formulated to resist the biological fluid, such as the gastric juice or by incorporating other pharmaceutical delivery techniques able to avoid the degradation of peptides/proteins in the biological fluid.
  • compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group,
  • compositions can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • Exemplary routes of administration for contact or in vivo delivery for which a composition can optionally be formulated include inhalation, intranasal, oral, buccal, sublingual, subcutaneous, intradermal, epicutaneous, rectal, transdermal, or intralymphatic.
  • the pharmaceutical composition is aqueous and, in other embodiments, the composition is non-aqueous solutions, suspensions or emulsions of the peptide/protein, which compositions are typically sterile and can be isotonic with the biological fluid or organ of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, vegetable or synthetic oils.
  • a composition can take the form of for example a solid dosage form, e.g. tablets or capsules, optionally formulated as fast- integrating tablets/capsules or slow-release tablets/capsules.
  • the tablet is a freeze-dried, optionally fast-disintegrating tablet suitable for being administered under the tongue.
  • a solid dosage form optionally is sterile, optionally anhydrous.
  • the pharmaceutical composition may also be formulated into a "unit dosage form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of a
  • Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
  • HLA human leukocyte antigen
  • Donor population Donors were recruited following Institutional Review Board approval (Federal Wide Assurance number 00000032), informed consent was obtained and a study identification number was assigned. Immunological reactivity was confirmed for all donors by skin prick tests to extracts from 32 common allergens. Timothy grass pollen allergic donors were identified as having a skin reaction with a wheal of > 5 mm in diameter to Timothy grass pollen and a clinical history consistent with seasonal grass pollen allergy. For SIT samples, donors who had received subcutaneous specific immunotherapy shots within the last 12 months and were still in maintenance treatment phase were recruited. Non-allergic donors were identified as having negative skin prick tests to all allergens and no clinical history of allergy or symptoms consistent with allergic disease.
  • PBMC isolation and in vitro expansion of Timothy grass pollen extract- specific T cells to screen novel peptide panel were isolated by density gradient centrifugation from one unit of blood (-450 ml) and cryo-preserved as described in Oseroff C et al, 2010.
  • PBMCs were thawed and cultured in RPMI 1640 ( ⁇ Scientific, Tarzana, CA) supplemented with 5% human AB serum (Cellgro, Herndon, VA) at a density of 2 x 10 6 cells/ml in 24-well plates (BD Biosciences, San Jose, CA) and stimulated with Timothy grass pollen extract (50 Mg/ml) (Greer, Lenoir, NC).
  • Cells were kept at 37°C, 5% C0 2 and additional IL-2 and IL-7 (10U/ml, eBioscience, San Diego, CA) was added every 3 days after initial antigenic stimulation. On day 14, cells were harvested and screened for reactivity against peptide pools consisting on average of 20 peptides. On day 17, peptides from positive pools were tested individually to identify the reactive epitopes.
  • ELISPOT assays The production of IL-5 from cultured PBMCs in response to stimulation with a peptide or a protein or allergen extracts was measured by
  • ELISPOT as described in Oseroff C et al, 2010. Briefly, flat-bottom 96-well nitrocellulose plates (Millipore, Bedford, MA) were prepared according to the manufacturer's instructions and coated with either 10 ⁇ g/ml anti-human IL-5 and anti-human IFN- ⁇ or 10 ⁇ g/ml anti-human IL-10 (clone 9D7; Mabtech). The next day, plates were washed, blocked and cells (1 x 10 5 cells/well) were plated and incubated with peptide, peptide pool or Timothy grass pollen extract (10 ⁇ g/ml, 5 ⁇ g/ml and 50 ⁇ g/ml, respectively).
  • Alkaline phosphatase-conjugated spots were developed with Vector Blue Substrate Kit (Vector Labs, Burlingame, CA). Horseradish Peroxidase-conjugated spots were developed with 3-amino-9-ethylcarvazole solution (Sigma-Aldrich, St. Louis, MO). Criteria for peptide pool positivity were 100 SFCs/10 6 PBMCs, p ⁇ 0.05, and a stimulation index (SI) > 2. Criteria for individual peptides were the same except a minimum of 20 SFCs was counted as positive.
  • Intracellular cytokine staining Cultured PBMCs were re-stimulated with 5 ⁇ g/ml of a pool of the peptides set out in Table 4 in complete RPMI medium at 37°C in 5% CO 2 . Unstimulated PBMCs were used to assess nonspecific/background cytokine production. After the initial 2 hours of stimulation, 4 ⁇ /ml Golgiplug (BD Biosciences) was added. After 7 hours of total incubation time, cells were harvested and stained for cell surface antigens CD4, CD3, CD8, CD14 and CD19 and Fixable Viability Dye (all from eBioscience).
  • the potential population coverage afforded by the panel of epitopes identified herein was determined on the basis of predicted capacity to bind a panel of 47 different HLA DR, DQ and DP molecules (Table 8).
  • the panel of class II specificities utilized for the analysis was selected considering the availability of reagents allowing further characterization of responses, including MHC-peptide binding assays (Greenbaum J et al, 2011 ), tetramer reagents and single allele transfected cell lines (McKinney DM et al, 2013). Binding predictions were performed utilizing the prediction tools available at the internet site ⁇ URL: http://www.iedb.org.html> and described in Wang P et al, 2008 and 2010, respectively.
  • T cell cultures were then assayed for the production of IL-5, chosen as a prototype Th2 cytokine, IL-10 (Treg) and IFN- ⁇ (Th1 ) in response to pools consisting of peptides from either novel proteins (NTGA) or known allergens (KTGA) of Timothy grass pollen. 822 peptides from the NTGA were assembled into 42 pools and screened alongside 5 pools of 105 peptides from the 10 KTGA (-20 peptides per pool).
  • NTGA novel proteins
  • KTGA known allergens
  • IFN- ⁇ responses in both donor populations were in general of similar magnitude and frequency across the panel of pools tested
  • IL-10 was detected in >10% of donors for only 1 of 5 KTGA-derived pools for both allergic and SIT-treated donor cohorts (average magnitude 23 SFC and 44 SFC, respectively). Stimulation with NTGA -derived peptide pools yielded responses in >10% of donors for 3 of 42 pools in allergic and SIT-treated donors (average magnitude of 44 and 24 SFC, respectively) ( Figures 1 e and d).
  • IL-5 and IFN- ⁇ production observed in response to peptides from each antigen is also summarized in Table 8.
  • IFN- ⁇ 5.5 allergic donors responded on average, (3197 SFC), compared to 3.2 SIT donors responding (1795 SFC) (Table 8).
  • Th2/Th 1 reactivity to a defined epitope cocktail is specifically modulated in SIT
  • ELISPOT screen detected a significant increase in IFN- ⁇ , a decrease in IL-5 and no change in IL-10 production in response to the MNA pool in this second cohort of SIT, compared to allergic donors (Figure 5a).
  • Th2 cytokine staining To investigate whether the modulation of Th2 responses detected for IL-5 could be generalized to other Th2 cytokines intracellular cytokine staining (ICS) to assess the production of the Th2 cytokines IL-4 and IL-13 and the Th1 cytokine IFN- ⁇ as a control was performed.
  • the ICS shows a reduction in Th2 cytokine production (IL-4 and IL-13) in response to restimulation with the modulated peptide pool in SIT donors as compared to allergic donors ( Figure 6a and b).
  • the 20 peptides in the peptide pool of Table 4 were ranked by HLA binding prediction and HLA population coverage analysis.
  • Population coverage is calculated essentially as previously described (Bui et al. 2006; Sidney et al. 1996; Sidney et al. 2010a; Sidney et al. 2010b) based on data available at dbMHC (NCBI) (Meyer et al. 2007) and allele frequencies.net (Middleton et al. 2003).
  • NCBI dbMHC
  • NCBI Network et al. 2007
  • allele frequencies.net Middleton et al. 2003.
  • the redundancy (depth) of coverage by a panel of epitopes is defined as the total number of different HLA/peptide combinations potentially presented by a given individual and thus yielding a potentially immunogenic signal.
  • the percentages of individuals yielding any given number of HLA/peptide combinations can be calculated using a Monte Carlo analysis (Osborne and Rubinstein 1994). In this type of analysis a model population is developed for each ethnic group under
  • Model populations are constructed without considering linkage disequilibrium.
  • the number of epitopes presented by each individual in the model population is then determined by tabulating, for each model individual, the number of HLA/epitope combinations associated with, for example, a binding capacity (IC50) ⁇ 500 nM in the case of Class I, or ⁇ 1000 nM in the case of Class II.
  • IC50 binding capacity
  • a histogram can be generated to summarize the fraction of individuals in the population as a function of the number of HLA/epitope combinations presented.
  • a cumulative plot can also be generated to determine the minimal number of HLA/epitope combinations presented by a specified fraction of the individuals in a given population.
  • population coverage and promiscuity determined using a consensus prediction score ⁇ 10% as determinative of binding for each individual allele included in the prediction panel.
  • Population coverage factors only DRB1 , DQB1 and DPB1 alleles.
  • Average haplotype and phenotype frequencies for individual alleles are based on data available at dbMHC. dbMHC data considers prevalence in Europe, North Africa, North-East Asia, the South Pacific (Australia and Oceania), Hispanic North and South America, American Indian, South-East Asia, South-West Asia, and Sub-Saharan Africa populations.
  • DP, DRB1 and DRB3/4/5 frequencies consider only the beta chain frequency, given that the DRA chain is largely monomorphic, and that differences in DPA are not hypothesized to significantly influence binding. Frequency data are not available for DRB3/4/5 alleles. However, because of linkage with DRB1 alleles, coverage for these specificities may be assumed as follows: DRB3 with DR3, DR11 , DR12, DR13 and DR14; DRB4 with DR4, DR7 and DR9; DRB5 with DR15 and DR16. Specific allele frequencies at each B3/B4/B5 locus is based on published associations with various DRB1 alleles, and assumes only limited variation at the indicated locus. Example 8
  • the modulation of Th function can be generalized to known and novel antigens, the present data underlines that the impact of SIT at the T cell level is much broader than currently recognized.
  • the data herein identifies a set of epitopes that shows dramatic down-regulation of Th2 cytokines and upregulation of Th1 responses. To support the notion that this defined set of epitopes is useful for peptide immunizations, the pool of peptides afforded good population coverage, and that the coverage is estimated to span through all major population groups worldwide.
  • ID No refers to the ID number of the NTGA, e.g. 1 , as well as the peptide derived thereof, e.g. peptide number 12.
  • IFN-Y IL-5 IFN-Y IL-5
  • IFN-Y IL-5 IFN-Y IL-5
  • IFN-Y IL-5 IFN-Y IL-5
  • SEQ ID NO: 154 refers to a fragment of Protein 49/54.

Abstract

La présente invention concerne l'utilisation thérapeutique d'antigènes dérivés de pollen de plante herbacée qui ont montré leur capacité à réguler à la baisse la réaction à l'IL-5 spécifique chez des patients allergiques à des plantes herbacées pendant une immunothérapie spécifique. Des antigènes spécifiques, notamment des protéines, des peptides et des combinaisons peptidiques ont été identifiés comme présentant une grande activité modulatrice de l'IL-5.
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