Classifications

• • 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
• • 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
• • 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
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/24—Immunology or allergic disorders

Definitions

• the present invention relates to the field of allergy. More specifically, the invention relates to the identification of a novel allergen from pollen of species belonging to the Cupressaceae family and to diagnosis and treatment of allergy to such pollen and to specifically related food allergies.
• allergens that induce immediate and/or delayed types of hypersensitivity are primarily proteins or glycoproteins and referred to as allergens [1] which can be found in a variety of sources, such as pollens, dust mites, animal dander, insect venoms and foods of plant or animal origin.
• allergens proteins or glycoproteins
• the fundamental immunological mechanism of allergic disease is the formation of allergen-specific immunoglobulin E (IgE) antibodies against such allergens, commonly referred to as sensitization.
• IgE antibodies bind to basophils, mast cells and dendritic cells via the specific high affinity IgE receptor, FcsRI.
• allergen-specific IgE antibodies on the cell surface become crosslinked through the recognition of at least two different epitopes of each allergen molecule, leading to the release of inflammatory mediators such as histamine and leukotrienes from these cells.
• inflammatory mediators such as histamine and leukotrienes
• a diagnostic test procedure for allergen sensitization can either utilize an in vitro immunoassay for the detection of allergen-specific IgE antibodies in a patient’s blood sample, or a skin prick test (SPT) performed by topical application of an allergen extract on the patient’s skin [3]
• SPT skin prick test
• an allergen reagent comprising a protein extract from an allergen source is traditionally used. While such a test may have a high sensitivity and thereby a high negative predictive value, sensitization to constituents in a natural allergen extract does not necessarily imply that clinical manifestations of allergy will occur.
• Such a dissociation between detectable sensitization and clinical allergy is in part due to the unequal significance of different allergenic proteins present in a natural extract.
• sensitization to certain proteins in an allergen source is more closely associated with clinical disease than others has opened an avenue towards the development of diagnostic tests with improved clinical utility by employing such specific proteins in a pure form for diagnostic testing.
• diagnostic testing for IgE antibodies to individual allergenic proteins is often referred to as component-resolved diagnostics (CRD) [4]
• CRD has several distinct advantages as compared to conventional IgE analysis using allergen extracts.
• One important feature of CRD is its ability to distinguish primary sensitisation to an allergen source from sensitisation due to cross reactivity, where the former is characteristically associated with more severe symptoms and the latter with milder symptoms or clinical tolerance.
• primary sensitization is typically directed to abundant and often stable proteins. As a consequence, accidental intake of even a small amount of the food in question will bring about exposure to a significant dose of such a food protein and a high risk of a severe reaction in a sensitized individual.
• Another application of purified natural or recombinant allergen components is their use as spiking reagents to counteract an imbalance or shortage of the corresponding protein in a natural allergen extract. This may be particularly important in miniaturized or non-laboratory immunoassay, such as an allergen microarray or a doctor’s office test where the combination of less favourable assay conditions, lower capacity for antibody-binding allergen reagent and natural allergen extract of limited potency, may cause insufficient diagnostic sensitivity.
• EAACI European Academy of Allergy and Clinical Immunology
• AIT allergen immunotherapy
• allergen-specific IgG response that mainly consists of the lgG4 subclass.
• IgG antibodies may modulate the effect of IgE antibodies, either directly by blocking the allergen or indirectly by acting via Fc receptors [19-21] Since the IgG antibody response is considered to be part of the mechanism of successful immunotherapy [20, 21], the analysis of allergen specific IgG may be a way to monitor relevant immunological effects of the treatment.
• the measurement of allergen-specific IgG levels may reflect natural or induced tolerance to the allergen through environmental exposure or
• immunotherapy treatment may, in combination with IgE measurements, increase the clinical relevance of the diagnostic workup in allergy.
• Pollen allergens are a major cause of respiratory allergy in industrialized countries and pollinosis has steadily been increasing during the past decades, in part possibly as a result of climate changes [22] Pollinosis presents with a variety of symptoms such as seasonal rhinitis, conjunctivitis and asthma. Pollen grains are released from flowers of grasses, weeds or trees and are dispersed either by the wind or by insects. Most allergenic pollen from trees are wind-borne and produced by species belonging to the orders Fagales, Lamiales, Proteales and Pinales. Pinales are gymnosperms and characterised by having separate male and female flowers.
• the allergens are highly cross-reactive and share 70%-95% sequence identity [26, 27]
• the group 2 allergens (Cup s 2, Cup a 2, Jun a 2, Cha o 2 and Cry j 2) belong to the polygalacturonase family and exhibit 71 %-97% sequence identity.
• the rate of sensitisation may be as high as 80% among Cupressaceae pollen allergic patients and they can thus also be considered major allergens [24]
• Group 3 and group 4 Cupressaceae pollen allergens belong to the thaumatin-like protein family and polcalcin protein family, respectively, and have been described as minor allergens [27, 28] Beyond these four groups, around 15 other allergenic proteins in Cupressaceae pollen have been reported.
• Sensitisation to pollen is often associated with allergy to different plant foods due to cross reactivity between homologous pollen and food proteins.
• Such cross-reactivity occurs as a consequence of structural similarity between homologous proteins present in pollen and plant-derived foods.
• Extensive cross-reactivity can be expected between proteins having a sequence identity to one another of 60% or higher but may occur between less closely related proteins [29]
• Pollen-related food allergy is believed to be driven predominantly or entirely by pollen sensitization. It typically causes oral symptoms and is thus referred to as the oral allergy syndrome (OAS).
• OAS oral allergy syndrome
• the most well-known and widespread example of pollen-related food allergy is caused by birch pollen and involves the so-called PR-10 protein Bet v 1 and homologous proteins in a variety of fruits and vegetables [30]
• profilin- mediated food allergy which can be driven by any pollen sensitization, it is less frequent and may cause allergy to food such as melon, banana and other fruits [31 , 32]
• Pru p 7 is a 7 kDa, cystein-rich protein belonging to the gibberellin regulated protein (GRP) family which has also been reported as an important cause of fruit allergy in Japan
• GFP gibberellin regulated protein
• BP14 A 14 kDa protein from cypress pollen, referred to as BP14, has been reported to contain a peptide of 13 amino acid residues with sequence homology to potato protein snakin-1 [37], another member of the GRP family.
• This sequence stretch comprises a highly conserved GRP segment that is identical in GRPs from more than 40 plant species as evidenced by Blast searching using the 13-residue BP14 sequence, however not including any other available sequence from a Cupressaceae species.
• the reported BP14 peptide includes no sequence information characteristic and distinguishing of a Cupressaceae pollen GRP and therefore provides no guidance towards specific features of such proteins. So far no
• Cupressaceae pollen protein corresponding to Pru p 7 has been identified and characterized.
• Cupressaceae pollen allergens which can be used in the diagnosis, prognosis, treatment and/or prevention of Type 1 allergies, in particular allergens that display cross-reactivity with proteins in foods and may elicit sensitization causing food allergic reactions.
• the above mentioned needs have now been met, or at least mitigated by the identification and provision herein of a novel isolated 7 kDa allergen of Cupressaceae pollen (herein also referred to as Cup s GRP).
• the allergen is homologous with Pru p 7 in peach, a 7 kDa basic cysteine-rich protein belonging to the family Gibberellin regulating proteins (GRPs), and was shown herein to be a likely primary sensitizer in severe peach allergy mediated by Pru p 7.
• GRPs Gibberellin regulating proteins
• the herein identified allergen, Cup s GRP shares high sequence homology with the corresponding pollen proteins in other species from the Cupressaceae family. Therefore, it is also described herein the identification of allergens, including Cup s GRP, in three species from the Cupressaceae family.
• the identified allergens from Cupressus sempervirens, Cryptomeria japonica and Juniperus ashei are herein named as Cup s GRP, Cry j GRP and Jun a GRP, respectively.
• the three pollen GRP allergens are also alternatively named Cup s 7, Jun a 7 and Cry j 7.
• an isolated allergenic protein i.e. the herein identified allergen(s)
• said protein comprising an amino acid sequence according to SEQ ID NO:4 (i.e. Cup s GRPa), or a functionally equivalent protein fragment or variant thereof having a sequence identity to SEQ ID NO:4 of at least 85%.
• an isolated allergenic protein or a functionally equivalent protein fragment or variant thereof having a sequence identity to SEQ ID NO:4 of at least 90% such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
• an isolated allergenic protein comprising or consisting of an amino acid sequence according to any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8 (i.e. Cup s GRPa, Cup s GRPb, Jun a GRP, and Cry j GRP, respectively).
• an isolated allergenic protein or a functionally equivalent protein fragment or variant thereof having a sequence identity to any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8, respectively, of at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
• isolated nucleic acid molecules encoding the respective isolated proteins presented herein.
• Such isolated nucleic acid molecules are represented by the sequence according to SEQ ID NO: 10 presenting a degenerated DNA sequence for pollen GRP comprising synonym codons and variants according to lUPAC ambiguity codes.
• an expression vector (also simply referred to herein as a vector) comprising an isolated nucleic acid molecule comprising an isolated nucleic acid sequence as disclosed elsewhere herein.
• an isolated host cell comprising an expression vector described herein. Said host cell is used for expressing the protein of interest encoded by the expression vector.
• a method for producing an allergen composition comprising the step of adding an isolated protein, or fragment or variant thereof, as described herein, to a composition comprising an allergen extract and/or at least one purified allergen component.
• an allergen composition obtainable by a method for producing an allergen composition as described herein, said allergen composition comprising an isolated protein, or fragment or variant thereof, as further defined elsewhere herein.
• an allergen composition comprising an isolated protein, or fragment or variant thereof, as described herein, and an allergen extract and/or at least one purified allergen component.
• kit of parts comprising an isolated protein, or a fragment or variant thereof, as disclosed herein, immobilized to a soluble or a solid support, said kit optionally further comprising a detection reagent and/or instructions for use.
• composition comprising an isolated protein, or fragment or variant thereof, as disclosed herein, and a pharmaceutically acceptable carrier and/or excipient.
• a method for the treatment or prevention of a Type 1 allergy comprising administering a pharmaceutically effective amount of an isolated protein, or fragment or variant thereof, as provided herein, or an allergen
• composition or a pharmaceutical composition, to a subject in need thereof.
• Figure 1 shows the first purification step of native Pru p 7 by cation exchange
• Figure 2 shows the second purification step of native Pru p 7 by size exclusion
• FIG. 1 shows the third purification step of native Pru p 7, comprising affinity immunoadsorption chromatography using an anti-Pru p 3 monoclonal antibody. Absorbance at 280 nm (A280) and conductivity are indicated by solid and hatched lines, respectively. Bracket indicates unbound material collected for further purification.
• Figure 4 shows the fourth purification step of native Pru p 7 by reversed phase
• Figure 5 shows the first purification step of recombinant Pru p 7 by cation exchange chromatography. Absorbance at 280 nm (A280) and conductivity are indicated by solid and hatched lines, respectively. Bracket indicates fractions pooled for further purification.
• Figure 6 shows the second purification step of recombinant Pru p 7 by size exclusion chromatography a) Chromatogram with absorbance at 280 nm (A280) and conductivity indicated by solid and hatched lines, respectively. Bracket indicates fractions pooled for further analysis b) SDS-PAGE analysis of reduced (lane 1) and non-reduced (lane 2) samples of the pool from the size exclusion chromatography step. Molecular weights of marker proteins (lane M) are indicated to the right.
• Figure 7 shows binding of rabbit anti-Pru p 7 IgG to a) recombinant Pru p 7 and b)
• Cupressus sempervirens pollen extract at different antiserum dilutions. Preimmune serum from the same rabbit was used as a negative control.
• Figure 8 shows the first step of purification of nCup s GRP from Cupressus sempervirens pollen extract by size exclusion chromatography. Absorbance at 214 nm (A214) and conductivity are indicated by solid and hatched lines, respectively. Bracket indicates fractions pooled for further purification.
• Figure 9 shows the second step of purification of nCup s GRP by ion exchange
• Figure 10 shows the third step of purification of nCup s GRP by size exclusion
• Figure 11 illustrates the amino acid sequence determination of nCup s GRP by MS/MS.
• Nucleotide sequence of EST record BY878079 (a cDNA sequence from male strobilus of Cryptomeria japonica) representing the best database match with the MS/MS data obtained from the purified 7 kDa Cupressus sempervirens protein.
• the start codon (ATG) and stop codons (TGA and TAG) of an interrupted, hypothetical open reading frame are underlined
• Figure 12 shows the third and final purification step of nJun a GRP by size exclusion chromatography a) Chromatogram with absorbance at 280 nm (A280) and conductivity indicated by solid and hatched lines, respectively. Brackets indicate pooled fractions of the three absorbance peaks (pool 1-3) shown in the chromatogram b) SDS-PAGE analysis of selected fractions from the size exclusion chromatography shown in Figure 12a. Fractions comprising pools 1-3 are indicated by brackets. Molecular weights of marker proteins (lane M) are indicated to the right c) Bar diagram showing levels of rabbit anti-Pru p 7 IgG binding by the three pools indicated in Figure 12a.
• Figure 13 shows the third and final purification step of nCry j GRP by size exclusion chromatography a) Chromatogram with absorbance at 280 nm (A280) and conductivity indicated by solid and hatched lines, respectively. Arrows indicate fractions selected for analysis by SDS-PAGE and binding of rabbit anti-Pru p 7 IgG. Bracket indicates fractions pooled for further purification b) SDS-PAGE analysis of the fractions indicated in Figure 13a. Molecular weights of marker proteins (lane M) are indicated to the right c) Bar diagram showing levels of rabbit anti-Pru p 7 IgG binding by the fractions indicated in Figure 13a.
• Figure 14 illustrates the amino acid sequence determination of nJun a GRP by MS/MS.
• the start codon (ATG), corrected previous stop codon (TGY) and terminal stop codon (TAG) of a hypothetical open reading frame are underlined
• a predicted signal peptide is underlined and a stop codon is represented by an asterisk c) Amino acid sequence of Jun a GRP determined by MS/MS, with amino acids deviating from the sequence hypothetically encoded by the amended BY878079 indicated by underlining d) Alignment of the amino acid sequences of Jun a GRP and Pru p 7. Vertical lines, colons and periods indicate identical, conserved and
• Figure 15 illustrates the amino acid sequence determination of nCry j GRP by MS/MS.
• a predicted signal peptide is underlined and a stop codon is represented by an asterisk c) Amino acid sequence of Cry j GRP determined by MS/MS. d) Alignment of the amino acid sequences of Cry j GRP and Pru p 7. Vertical lines, colons and periods indicate identical, conserved and semiconserved positions, respectively.
• Figure 16 shows sequence and eletrophoretic comparisons of the Cupressaceae pollen- derived GRPs.
• Molecular weights of marker proteins (lane M) are indicated to the right.
• Figure 17 shows an SDS-PAGE analysis of rCup s GRPa (lane 1), rCup s GRPb (lane 2), purified native Cup s GRP (lane 3) and rPru p 7 (lane 4). Molecular weights of marker proteins (lane M) are indicated to the left.
• Figure 18 shows a comparison of IgE binding to rPru p 7 and nCup s GRP among 44 peach allergic subjects. Values below the assay’s 0.1 kll A /L limit of quantitation (LoQ) were set to 0.1 kll A /L. Hatched diagonal line indicates 1 :1 slope.
• Figure 19 shows a comparison of IgE binding to nCup s GRP and the two variants of recombinant Cup s GRP among 18 peach allergic subjects a) rCup s GRPa vs. nCup s GRP and b) rCup s GRPb vs. nCup s GRP. c) rCup s GRPb vs. rCup s GRPa. Hatched diagonal lines indicate 1 :1 slope.
• Figure 20 shows inhibition of IgE binding to rPru p 7 by native and recombinant Cup s GRPb in sera of four different subjects. Results are expressed as percent of a dilution buffer control.
• Figure 21 shows pairwise comparison of IgE binding to a) nJun a GRP and nCup s GRP, b) nCry j GRP and nCup s GRP and c) nCry j GRP and nJun a GRP among 18 peach allergic subjects. Hatched diagonal lines indicate 1 :1 slope.
• Figure 22 shows the prevalence and level of IgE antibodies to nCup s GRP in sera of 88 subjects sensitized to cypress pollen. Values below the assay’s 0.1 kll A /L limit of quantitation (LoQ) (marked by hatched vertical and horizontal lines) were set to 0.05 kll A /L. Hatched diagonal line indicates 1 :1 slope.
• Figure 23 shows immunological and sequence comparisons between the profilins Bet v 2, Cor a 2, Mai d 4, Pru av 4, Pyr c 4, Api g 4, Dau c 4 and Phi p 12.
• Figure 24 shows an alignment of Cup s GRPa, Cup s GRPb, Jun a GRP and Cry j GRP sequences and a Cupressaceae consensus sequence based on those four sequences. Only amino acids deviating from those of Cup s GRPa are shown in the sequences of Cup s GRPb, Jun a GRP and Cry j GRP. Amino acid positions that show variability are indicated by an X in the Cupressaceae pollen GRP consensus sequence (SEQ ID NO: 9) aligned on top of the other sequences.
• Figure 25 shows a multiple sequence alignment of 37 known GRP sequences. Asterisk indicates a phylogenetically conserved amino acid among all the compared sequences.
• Figure 26 shows a sequence alignment of the Cupressaceae pollen GRP consensus sequence, SEQ ID NO: 9, and Pru p 7.
• row A amino acid positions indicated with Z differ between Cupressaceae pollen GRP and Pru p 7.
• row B those amino acids are indicated that are conserved between Cupressaceae pollen and Pru p 7 but differ among the 37 sequences that were aligned as shown in Figure 25.
• row C those amino acids are indicated that are conserved among all the 37 aligned sequences.
• SEQ ID NO: 52 is a consensus sequence among Cupressaceae pollen GRP sequences and Pru p 7 where X represents amino acids that are non-conserved in this comparison.
• An“isolated” protein refers to a protein that has been isolated or removed from its natural environment. It also indicates that it has been produced through human intervention and has been substantially separated from the materials co-existing in a protein production environment.
• an“isolated protein” or“protein” is referred to herein, this may also refer to a fragment or a variant of said isolated protein, having a sequence identity to the isolated protein as further explained herein, and being functionally equivalent to the original full length protein.“Functionally equivalent” is further defined elsewhere herein.
• the terms“protein”,“isolated protein”,“allergenic protein”,“fragment or variant thereof”,“fragment or variant of an isolated allergenic protein”, and“allergen” may be used interchangeably and are envisaged in all contexts and aspects of the present disclosure.
• Sequence identity relates to the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, expressed as a percentage. “Alignment” in this regard relates to the process or result of matching up the nucleotide or amino acid residues of two or more biological sequences to achieve maximal levels of identity and, in the case of amino acid sequences, conservation, for the purpose of assessing the degree of similarity and the possibility of homology. The amino acid sequences of the respective isolated proteins as compared to the fragments or variants thereof, or the nucleic acid sequences encoding them may be used in an alignment in order to determine an “overall” sequence identity [38, 39]
• The“length” of a protein is the number of amino acid residues in the protein.
• a“fragment” of a protein should be construed as meaning a fragment having at least 85% sequence identity to the amino acid sequence of the original protein, as calculated over the entire length of the original protein. In other words, at least 85% of the amino acid residues of the full length original protein are present in a fragment according to the present disclosure.
• the original protein may have an amino acid sequence according to SEQ ID NO:4. Consequently, a“fragment” as disclosed herein may have at least 85% identity to the amino acid sequence of SEQ ID NO: 4, as calculated over the entire length of SEQ ID NO:4. Protein fragments may further comprise additional amino acids as a result of their production, such as a hexahistidine tag, linker sequences, or vector derived amino acids.
• A“variant” of a protein relates to a variant having a sequence identity of at least 85% to the amino acid sequence of said original protein, as calculated over the entire length of the variant protein.
• the original protein may have an amino acid sequence according to SEQ ID NO: 4. Consequently, a“variant” as disclosed herein may have at least 85% identity to the amino acid sequence of SEQ ID NO: 4.
• a number of software tools for aligning an original and a variant protein and calculating sequence identity are commercially available, such as Clustal Omega provided by the European Bioinformatics Institute (Cambridge, United Kingdom).
• Protein variants may further comprise additional amino acids as a result of their production, such as a hexahistidine tag, linker sequences, or vector derived amino acids.
• a“functionally equivalent protein fragment or variant” of a protein or a“functionally equivalent fragment or variant” of a protein, this is intended to mean that the protein and a fragment or variant thereof have comparable IgE binding properties. More particularly, in order to be a functionally equivalent variant or fragment of an original, isolated allergenic protein:
• the variant or fragment inhibits the binding, by the original allergenic protein, of IgE antibodies from a serum sample of a representative patient sensitized to the original allergenic protein, by at least 50% as compared to a mock inhibition with buffer alone (IgE diluent, Thermo Fisher Scientific).
• This property of a variant or fragment can be assayed by using any suitable inhibition assay as known in the art, e.g. as described in Example 9.
• the variant or fragment binds IgE antibodies at substantially the same level as the original allergenic protein. Binding levels can be measured by immobilising the variant or fragment on a solid phase and measuring the IgE reactivity of individual sera, as is described for example in Example 8, and comparing the measured IgE reactivity to the IgE reactivity of the original isolated allergenic protein.
• substantially the same level should be construed as meaning that the binding level of the variant differs from the binding level of the original protein by at the most 25%, 20%, 15%, 10%, or 5%.
• the variant or fragment contains all conserved amino acids of the Cupressaceae GRP consensus sequence (i.e. SEQ ID NO:9; see Figure 26 for conserved amino acids); and
• the variant has a maximum of 9 amino acid residues exchanged as compared to SEQ ID NO:4, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) exchanged compared to the SEQ ID NO:4, or
• the fragment has a maximum of 9 amino acid residues deleted compared to SEQ ID NO:4, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) deleted compared to SEQ ID NO:4, or
• the combined variant and fragment has a maximum of 9 amino acid residues exchanged or deleted compared to SEQ ID NO:4.
• Examples 10 and 12 demonstrate that IgE binding of allergenic proteins, due to cross reactivity, is very similar among closely related allergenic proteins within the same protein family. Homologous proteins which have a sequence identity to each other of at least 80%, such as 90% or higher, show remarkably similar IgE reactivity.
• amino acid residue in a certain position of an amino acid sequence is’’phylogenetically conserved”, sometimes simply worded“conserved”, among different proteins of the same protein family if the amino acid residue in said position is identical among the aligned protein sequences compared. It follows that an amino acid residue which is“non-conserved” among different proteins is not identical among the protein sequences compared.
• a non-conserved amino acid residue may be“phylogenetically restricted”, or have“phylogenetically restricted variability”, across the protein sequences compared, which is intended to mean that the amino acid in a particular position is selected from a group consisting of a restricted number of amino acids which are found in a phylogenetic comparison of a group of similar proteins, e.g. from the GRP protein family.
• vector or“expression vector” relates to a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.
• A“host cell” relates to a bacterial, yeast, insect or mammalian cell which has been transformed by a vector as disclosed herein to express the protein, fragment or variant of interest.
• a protein“isoform” is a member of a group of proteins with a high similarity and that originate from allelic variants of a single gene or non-identical members of a gene family and is a result of genetic differences. Many isoforms perform the same or similar biological functions.
• the rabbit anti-Pru p 7 IgG antibodies only detected a 7 kDa protein (i.e. a protein half the size of BP-14) in C. sempervirens pollen extract, as well as in pollen extracts of Juniperus ashei and Cryptomeria japonica. Once identified, these proteins could be purified to homogeneity for analysis by mass spectrometry and for biochemical and immunological characterization.
• Cup s GRP Based on the sequence of Cup s GRP established and disclosed herein, recombinant Cup s GRP was produced in Pichia pastoris and shown to have similar biochemical properties and IgE reactivity as the natural purified protein. The recombinant protein inhibited the IgE binding to Pru p 7, demonstrating that there is cross-reactivity between the peach protein Pru p 7 and Cup s GRP of which the latter may act as the primary sensitizer.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:4, or a functionally equivalent protein fragment or variant thereof having a sequence identity to SEQ ID NO:4 of at least 85%.
• said sequence identity to SEQ ID NO:4 may be at least 90%, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
• the isolated protein is represented and exemplified herein by the highly homologous proteins Cup s GRP (protein isoform a (SEQ ID NO:4) and protein isoform b (SEQ ID NO:5)), Jun a GRP (SEQ ID NO:6) and Cry j GRP (SEQ ID NO:8).
• the amino acid sequence of Pru p 7 (Pru p7) comprises the following sequence:
• the amino acid sequence of Cup s GRPa comprises the following sequence:
• the amino acid sequence of Cup s GRPb comprises the following sequence:
• the amino acid sequence of Jun a GRP comprises the following sequence:
• the amino acid sequence of Cry j GRP comprises the following sequence:
• a Cupressaceae - Pru p 7 GRP consensus sequence (SEQ ID NO:52) has been designed and is disclosed herein.
• said consensus sequence according to SEQ ID NO:52 the positions in which amino acids vary among the different Cupressaceae pollen GRP disclosed herein (i.e. Cup s GRPa, Cup s GRPb, Jun a GRP, and Cry j GRP) as well as the positions in which amino acids vary between the Cupressaceae pollen GRP and the peach GRP disclosed herein (i.e. Pru p 7) are indicated by X ( Figure 26). In row A of Figure 26, the positions in which amino acids vary between the Cupressaceae pollen GRP and the peach GRP disclosed herein (i.e. Pru p 7) are indicated by Z.
• the present disclosure provides an isolated allergenic protein, wherein said protein comprises the following amino acid sequence according to SEQ ID NO:52:
• amino acids are identical to the amino acids in the corresponding positions of SEQ ID NO:4.
• the present disclosure provides an isolated allergenic protein, wherein said protein comprises the following amino acid sequence according to SEQ ID NO:52:
• amino acid residues listed in Table 3 are amino acid residues having phylogenetically restricted variability in positions X of SEQ ID NO:52.
• sequence identity to SEQ ID NO:5, 6 or 8, respectively may be at least 90%, such as 90%, 91%, 92%, 93%, 94% , 95% , 96% , 97% , 98% or 99% .
• amino acid sequences of these exemplified isolated proteins from the Cupressaceae family differ from each other only in a maximum of seven amino acid positions as illustrated in SEQ ID NO:9.
• a sequence alignment illustrating the differences and similarities between the above-mentioned family members is shown in Figure 24. It was proven in Example 10 herein that varying the amino acids residues at least in these positions did not affect the functionality of the proteins for their intended purpose. Accordingly, it is thereby illustrated a certain degree of structural flexibility in the amino acid sequence, meaning that functionally equivalent variants or fragments of an isolated protein illustrated by SEQ ID NO:4 are envisaged and proven useful in the context of the present disclosure. A definition of a functionally equivalent variant or fragment of an isolated protein according to SEQ ID NO:4 presented herein is described elsewhere herein.
• an isolated allergenic protein wherein said protein comprises the following amino acid sequence according to SEQ ID NO:9:
• SEQ ID NO:9 may be considered an example of a Cupressaceae pollen GRP consensus sequence representing the isolated proteins identified herein, wherein in said maximum of seven amino acid positions (positions 2, 18, 31 , 34, 41 , 46 and 52), the amino acid residues may be varied, i.e. exchanged, in one or more positions, for any other amino acid, with the proviso that X is not C, and provided that such a protein maintains an activity that is functionally equivalent to the original protein with regard to its intended purposes as described elsewhere herein. It is also envisaged herein other variants of SEQ ID NO:4 with the same sequence similarity but wherein additional or alternative amino acid residues are varied and wherein said variants maintain the same functional activity as the original protein, i.e. said variants being functionally equivalent to the original protein, or a protein where the amino acids at the above mentioned seven positions have been varied as further described herein. There is also provided herein a functionally equivalent fragment of an isolated protein according to SEQ ID NO:9.
• the amino acid is selected from a group consisting of phylogenetically restricted amino acids, as demonstrated in Example 13.
• X in position 2 is Q or H
• X in position 18 is A or L
• X in position 31 is K or E
• X in position 34 is H or N
• X in position 41 is A or Y;
• X in position 46 is V or S;
• X in position 52 is H or N.
• Said above-disclosed amino acid residues have phylogenetically restricted variability and are in accordance with those found in Cupressaceae pollen GRP, i.e. Cup s GRPa, Cup s GRPb, Jun a GRP and Cry j GRP as shown in the alignment in Figure 24.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 6, such as 5, 4, 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain any amino acid, with the proviso that X is not C; and wherein in the remaining position(s) X, the amino acids are selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q, H, A, E, S, D, T, L, or Y;
• X in position 18 is selected from any one of A, L, Y, I, V, F, M, or R;
• X in position 31 is selected from any one of K, E, D, Q, A, G, or S;
• X in position 34 is selected from any one of H, N, Q or K;
• X in position 41 is selected from any one of A, Y, F or S;
• X in position 46 is selected from any one of V, S, E, V, A, or Q;
• X in position 52 is selected from any one of H, N, D, or E.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 4, such as 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain any amino acid, with the proviso that X is not C;
• amino acids are selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q, H, A, E, S, D, T, L, or Y;
• X in position 18 is selected from any one of A, L, Y, I, V, F, M, or R;
• X in position 31 is selected from any one of K, E, D, Q, A, G, or S;
• X in position 34 is selected from any one of H, N, Q or K;
• X in position 41 is selected from any one of A, Y, F or S; X in position 46 is selected from any one of V, S, E, V, A, or Q; and/or
• X in position 52 is selected from any one of H, N, D, or E.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 6, such as 5, 4, 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain any amino acid, with the proviso that X is not C;
• amino acids are selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q or H;
• X in position 18 is selected from any one of A or L;
• X in position 31 is selected from any one of K or E;
• X in position 34 is selected from any one of H or N;
• X in position 41 is selected from any one of A or Y;
• X in position 46 is selected from any one of V or S;
• X in position 52 is selected from any one of H or N.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 4, such as 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain any amino acid, with the proviso that X is not C;
• amino acids are selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q or H;
• X in position 18 is selected from any one of A or L;
• X in position 31 is selected from any one of K or E;
• X in position 34 is selected from any one of H or N;
• X in position 41 is selected from any one of A or Y;
• X in position 46 is selected from any one of V or S;
• X in position 52 is selected from any one of H or N.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein in positions X, said amino acids are selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q, H, A, E, S, D, T, L, or Y;
• X in position 18 is selected from any one of A, L, Y, I, V, F, M, or R;
• X in position 31 is selected from any one of K, E, D, Q, A, G, or S;
• X in position 34 is selected from any one of H, N, Q or K;
• X in position 41 is selected from any one of A, Y, F or S;
• X in position 46 is selected from any one of V, S, E, V, A, Q; and/or
• X in position 52 is selected from any one of H, N, D, or E.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein:
• X in position 2 is Q or H
• X in position 18 is A or L
• X in position 31 is K or E
• X in position 34 is H or N
• X in position 41 is A or Y;
• X in position 46 is V or S;
• X in position 52 is H or N.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 4, such as 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain an amino acid selected from the following groups of amino acids:
• X in position 2 is selected from any one of Q, H, A, E, S, D, T, L, or Y;
• X in position 18 is selected from any one of A, L, Y, I, V, F, M, or R;
• X in position 31 is selected from any one of K, E, D, Q, A, G, or S;
• X in position 34 is selected from any one of H, N, Q or K;
• X in position 41 is selected from any one of A, Y, F or S;
• X in position 46 is selected from any one of V, S, E, V, A, Q; and/or
• X in position 52 is selected from any one of H, N, D, or E;
• amino acids are identical to the amino acids in the corresponding positions of SEQ ID NO:4.
• an isolated allergenic protein comprising an amino acid sequence according to SEQ ID NO:9, wherein a maximum of 4, such as 3, 2, or 1 , of said positions X of SEQ ID NO:9 contain an amino acid selected from the following groups of amino acids:
• X in position 2 is Q or H
• X in position 18 is A or L
• X in position 31 is K or E
• X in position 34 is H or N
• X in position 41 is A or Y;
• X in position 46 is V or S;
• X in position 52 is H or N.
• the present disclosure further provides an isolated allergenic protein comprising or consisting of an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8, respectively.
• a protein described herein can also have chemically modified amino acids added to the original sequence, which refers to an amino acid whose side chain has been chemically modified.
• a side chain may be modified to comprise a signalling moiety, such as a fluorophore or a radiolabel.
• a side chain may be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group.
• Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.
• An isolated protein, variant or fragment thereof as disclosed throughout herein may have been recombinantly produced.
• practical utilization of allergenic proteins in research, diagnostic or other applications is greatly facilitated by their availability in recombinant form.
• the expression system configuration, cultivation method and/or purification strategy may need to be extensively adapted or even newly developed in order to reach the goal of obtaining a functional protein in a useful quantity. How to achieve such an extensive adaption is not easily foreseeable even for the skilled person in the art.
• a gene encoding the protein can either be cloned in the form of a cDNA derived from mRNA prepared from the allergenic source material, or synthesized according to the desired DNA sequence. Due to the redundancy of the genetic code, with up to six different codons specifying the same amino acid, a given amino acid sequence can be encoded by a large number of synonymous DNA sequences. If required, functional additions or modifications to the protein can be introduced through the design of a synthetic gene, by site-specific mutagenesis or as part of the cloning strategy.
• a gene encoding the allergen of interest can be cloned in any of a variety of different expression vectors and introduced into any of a variety of prokaryotic or eukaryotic expression hosts [40]
• Common expression hosts include but are not limited to the gram negative bacterium Escherichia coii, the yeasts Saccharomyces cerevisiae and Pichia pastoris, insect cell lines derived from Spodoptera frugiperda or Drosophila melanogaster, and mammalian cell lines.
• the recombinant protein may be expressed intracellularly in soluble or insoluble form or secreted into the culture medium. Recovery and purification of the recombinant protein can be performed by a variety of well-known methods or combinations thereof. Common chromatographic techniques include immobilized metal ion affinity chromatography (IMAC), anion and cation exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography and size exclusion chromatography.
• IMAC immobilized metal ion affinity chromatography
• anion and cation exchange chromatography anion and cation exchange chromatography
• hydrophobic interaction chromatography hydrophobic interaction chromatography
• reversed phase chromatography reversed phase exclusion chromatography
• An isolated protein as presented herein, when recombinantly produced may be intentionally modified for a specific purpose, thereby resulting in a non-naturally occurring protein, without functionally affecting the protein in regard to e.g. antibody binding properties.
• a non-naturally occurring protein can be a recombinant protein which has been fused with another protein to enhance expression level or solubility.
• fusion partners include thioredoxin (TRX), maltose binding protein (MBP) and glutathione-S-transferase (GST).
• TRX thioredoxin
• MBP maltose binding protein
• GST glutathione-S-transferase
• TRX thioredoxin
• MBP maltose binding protein
• GST glutathione-S-transferase
• Another example is the addition of a signal peptide enabling the secretion of the protein into the culture medium from which it can be easily recovered in a soluble form.
• a non-naturally occurring protein may also be a recombinant protein where a short peptide tag has been genetically grafted onto said protein for the purpose of enabling affinity purification.
• peptide tags include hexahistidine for conferring
• a short peptide sequence for site-specific proteolytic cleavage may be inserted between the protein of interest and the fusion partner or peptide tag.
• target sites for proteolytic enzymes include DDDDK for enterokinase, IEGR for factor Xa, ENLYFQA for TEV protease and EKREAEAEF for Kex2/Ste13 for in vivo processing in P. pastoris.
• a few amino acid residues of such a target sequence may remain following cleavage, for example EAEFEF or a part thereof in the case of secreted expression in P. pastoris.
• an isolated nucleic acid molecule encoding an isolated protein, fragment or variant as disclosed herein.
• the isolated nucleic acid may be encoded by SEQ ID NO:10.
• SEQ ID NO:10 is a degenerated DNA sequence encoding the consensus
• Cupressaceae amino acid sequence (i.e. SEQ ID NO:9), which is based on the Cup s GRPa, Cup s GRPb, Jun a GRP and Cry j GRP sequences described herein, i.e. an isolated protein as disclosed herein, comprising synonymous codons and variants according to the ambiguity codes of lUPAC (International Union of Pure and Applied Chemistry; htps://iupac.org/).
• the consensus Cupressaceae nucleic acid sequence according to SEQ ID NO: 10 was constructed starting from the Cup s GRPa amino acid sequence (i.e.
• SEQ ID NO: 10 i.e. the Cupressaceae consensus nucleic acid sequence:
• GCN CAN ATH GAY TGY GAY AAR GAR TGY AAY MGN MGN TGY WSN AAR GCN WSN BYN CAY GAY 60 MGN TGY YTN AAR TAY TGY GGN ATH TGY TGY RAR AAR TGY MAY TGY GTN CCN CCN GGN ACN 12 0 KMN GGN AAY GAR GAY DBN TGY CCN TGY TAY GCN MAY YTN AAR AAY WSN AAR GGN GGN CAY 18 0 AAR TGY CCN 18 9 wherein:
• N T, C, A or G
• R A or G
• H T, C or A
• SEQ ID NO:50 i.e. Cup s GRPa backtranslated and taking into account synonymous codons:
• SEQ ID NO:51 i.e. Cup s GRPa backtranslated, in which the nucleotides encoding the seven variable amino acid positions (marked-up in bold text) have been changed to those nucleotides encoding the amino acids present in Cup s GRPb, Jun a GRP and/or Cry j GRP:
• variable nucleotides have the same meaning as in SEQ ID NO: 10, as defined above.
• nucleic acid molecule comprising a nucleic acid sequence according to any one of SEQ I D NO: 10, 50 or 51 , or a sequence having at least 85% sequence identity therewith, such as at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity therewith.
• the isolated nucleic acid molecule may encode an isolated protein or a fragment or variant thereof as disclosed herein, and may hence comprise or consist of any nucleic acid sequence disclosed herein.
• an isolated host cell comprising a vector or an expression vector as described herein.
• said vector or expression vector comprises a nucleic acid molecule encoding an isolated protein or fragment or variant thereof as disclosed elsewhere herein.
• an allergen composition comprising a step of adding an isolated protein or a functionally equivalent fragment or variant thereof as described herein to a composition comprising an allergen extract and/or at least one purified allergen component.
• an allergen composition obtainable by such a method.
• Such an allergen composition can be“spiked” with an isolated protein, fragment or variant thereof as presented herein.
• Such an allergen composition may be an allergen extract or a mixture of purified or recombinant allergen components having no or a low content of the isolated proteins presented herein, wherein the isolated protein, fragment or variant thereof is added to said allergen composition (i.e.
• this aspect relates to a method for producing such a composition, which method comprises the step of adding said protein to an allergen composition, such as an allergen extract (as mentioned optionally spiked with other components) or a mixture of purified native or recombinant allergen components.
• an allergen composition comprising an isolated protein, or fragment or variant thereof, as described herein, and an allergen extract and/or at least one purified allergen component.
• Type 1 allergic symptoms are elicited by pollen of Cupressaceae species.
• said Type 1 allergy is a pollen-associated food allergy, with symptoms elicited by foods such as peach, apricot, plum, citrus fruits or pomegranate.
• Detection or measurement of allergen-specific IgE antibodies in a human or animal specimen can be performed in several different ways but normally includes an initial step of capture of antibodies binding to the allergen in question, followed by a washing step to remove unbound antibodies, application of an IgE detection reagent, washing to remove unbound such reagent and a final step of generating and recording a signal from the IgE detection reagent. Allergen may be immobilized on a solid or soluble support for capture of allergen-specific antibodies or complexed with the antibodies in solution for subsequent capture and quantitation of such complexes.
• the allergen detection reagent is typically a monoclonal antibody conjugated either with a reporter substance or with an enzyme that can catalyse the formation of a product quantifiable with fluorometric or colorimetric methods.
• An assay for measurement of allergen-specific antibodies also includes a calibration system allowing the conversion of primary response units to antibody concentration units. The same assay principles apply to the measurement of allergen-specific antibodies of other isotypes, the only difference being the specificity of detection regent used.
• a method for the in vitro diagnosis or assessment of Type 1 allergy comprising the steps of: contacting an immunoglobulin- containing body fluid sample from a subject suspected of having type 1 allergy with an isolated protein, or fragment or variant thereof as disclosed herein; and in said sample, determining the presence of antibodies specifically binding to said protein, fragment or variant thereof, such as IgE antibodies; wherein the presence of antibodies in said sample specifically binding to said protein is informative in relation to Type 1 allergy in said subject.
• IgE antibodies are present in said sample and specifically bind to said protein, this is indicative of a Type I allergy in said subject.
• a body fluid sample may be a blood or serum sample from the subject, wherein said body fluid sample is brought into contact with the isolated protein, or fragment or variant thereof, or a composition containing said protein, or fragment or variant thereof, to determine if said subject sample contains IgE antibodies that bind specifically to the isolated protein, variant or fragment thereof.
• fragments or variants of any isolated protein mentioned herein may be a natural or a man-made fragment or variant being functionally equivalent to the original protein.
• kits of parts comprising an isolated protein, a fragment or variant thereof, immobilized to a soluble or a solid support, wherein said kit optionally further comprises a detection reagent and/or instructions for use.
• a solid support may be selected from the group of nitrocellulose, glass, silicon, and plastic and/or is a microarray chip, or any other suitable solid supports available in the art.
• the kit may further also comprise a detection agent capable of binding to antibodies, such as IgE antibodies bound to the immobilised protein.
• detecting agents may e.g. be anti-lgE antibodies labelled with detectable labels, such as dyes, fluorophores or enzymes, as is known in the art of immunoassays.
• detectable labels such as dyes, fluorophores or enzymes, as is known in the art of immunoassays.
• Supports suitable for the immobilization of proteins and peptides are well-known in the art, and herein, in this aspect, it is encompassed any support which does not negatively impact the immunogenic properties of the protein or protein fragment to any substantial extent.
• the term“immobilized” may be any kind of attachment suitable for a specific support.
• the isolated protein or protein fragment may be immobilized to a solid support suitable for use in a diagnostic method, such as ImmunoCAP, EliA or VarelisA.
• a diagnostic method such as ImmunoCAP, EliA or VarelisA.
• the protein or protein fragment may be immobilized to a natural or synthetic polymeric structure in solution, such as one or more dendromeric structures in solution.
• an isolated protein, fragment or variant thereof as described herein which has been provided with a label or a labelling element.
• a protein or protein fragment or variant which has been provided with a luminescent label, such as a photoluminiscent label, a fluorescent label or phosphorescent label, a chemiluminescent label or a radioluminescent label.
• a luminescent label such as a photoluminiscent label, a fluorescent label or phosphorescent label, a chemiluminescent label or a radioluminescent label.
• an isolated protein, fragment or derivative thereof which has been derivatized with an element which may be identified, such as an affinity function. Affinity functions for the labelling of proteins and peptides are well-known in the art, and the skilled person will be able to choose any suitable function, such as biotin.
• an isolated protein or a functionally equivalent fragment or variant thereof for use in the treatment or prevention of Type 1 allergy.
• the Type 1 allergy may be caused by pollen of Cupressaceae species, and/or may be a Cupressaceae pollen- associated food allergy with symptoms elicited by ingestion of fruits such as peach, apricot, plum, citrus fruits or pomegratate.
• an isolated protein or a functionally equivalent fragment or variant thereof as disclosed herein in the manufacture of a medicament for the treatment or prevention of Type 1 allergy.
• the Type 1 allergy may be caused by pollen of Cupressaceae species, and/or may be a Cupressaceae pollen-associated food allergy with symptoms elicited by ingestion of fruits such as peach, apricot, plum, citrus fruits or pomegratate.
• composition comprising an isolated protein or a functionally equivalent fragment or variant thereof and a pharmaceutically acceptable carrier and/or excipient.
• a pharmaceutically acceptable carrier and/or excipient herein refers to a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.
• a pharmaceutically- acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.
• Each component must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
• a method for the treatment or prevention of a Type 1 allergy comprising administering a pharmaceutically effective amount of an isolated protein, fragment or variant thereof as described herein, an allergen composition, or a pharmaceutical composition comprising said isolated protein, fragment or variant thereof, to a subject in need thereof.
• a pharmaceutically effective amount of an isolated protein, fragment or variant thereof as described herein, an allergen composition, or a pharmaceutical composition comprising said isolated protein, fragment or variant thereof, to a subject in need thereof comprising administering a pharmaceutically effective amount of an isolated protein, fragment or variant thereof as described herein, an allergen composition, or a pharmaceutical composition comprising said isolated protein, fragment or variant thereof, to a subject in need thereof.
• the isolated protein may be used in its natural form or in a recombinant form displaying biochemical and immunological properties similar to those of the natural protein.
• the isolated protein may be used in a modified form, generated chemically or genetically. Examples of modifications to the isolated protein include, but are not limited to, fragmentation, truncation, tandemerization or aggregation of the protein, deletion of internal segment(s), substitution of amino acid residue(s), domain rearrangement, or disruption at least in part of the tertiary structure by disruption of disulfide bridges or its binding to another macromolecular structure, or other low molecular weight compounds.
• Any suitable methods of administration of a pharmaceutical composition as disclosed herein can be used depending on the purpose of administration of the isolated protein.
• the dose and timing of administration will be determined by the physician as being suitable for the subject being treated.
• the protein may be purified from its natural source. It may also be produced by recombinant DNA technology or be chemically synthesized by methods known to a person skilled in the art or as described in the present application.
• Type 1 allergy is a Type 1 allergy caused by pollen of Cupressaceae species, and/or is a Cupressaceae pollen-associated food allergy with symptoms elicited by ingestion of fruits such as peach, apricot, plum, citrus fruits or pomegratate.
• Table 1 below identifies the SEQ ID NOs according to the sequence listing, which is part of the present disclosure, and the corresponding definitions/names of said sequences.
• Table 2 List of peptide sequences identified with MS/MS analysis of a) nCup s GRP, b) isoform variants of nCup s GRP, c) nJun a GRP and d) nCry j GRP.
• Table 3 identifies amino acids having phylogenetically restricted variability in positions X of SEQ ID NO:52, i.e. the Cupressaceae - Pru p 7 GRP consensus sequence having 21 variable positions.
• amino acid in positions 1 , 3, 4, 7, 8, 10, 11 , 17, 19, 20, 44, 51 , 59, and 60 is conserved among the four Cupressaceae GRP proteins disclosed herein but differ from the amino acid in the corresponding positions in Pru p 7. Said positions are indicated by a Z in row A of Figure 26 but are indicated by an X in SEQ ID NO: 52.
• amino acid in positions 2, 18, 31 , 34, 41 , 46, and 52 differ among the four Cupressaceae GRP proteins disclosed herein. Said positions are indicated by an X in both SEQ ID NO: 9 and SEQ ID NO: 52.
• Native Pru p 7 was purified from canned peaches using four chromatographic steps. Briefly, canned peaches were mixed with a kitchen blender in 130 mM NaAc pH 4.5 and incubated 2 hrs under agitation with grinding balls (Haldenwanger MTC, Berkshire, UK) at 4°C. The extract was clarified by centrifugation, filtered and loaded on a SP Sepharose FF column equilibrated with 130 mM NaAc pH 4.5. Washing and elution were performed by isocratic steps of 0.15 and 0.5 M NaCI, respectively, in 130 mM NaAc pH 4.5 ( Figure 1).
• Bound protein was eluted in a linear 0-55% acetonitrile gradient. Fractions were analysed by SDS-PAGE (Figure 4b) and those containing a pure 7 kDa band were pooled as indicated in Figure 4a and desalted to 20 mM MOPS, 0.15 M NaCI pH 7.6 on a Sephadex G25 column.
• the pool of native Pru p 7 was analysed by MS/MS analysis on an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific, CA, USA) after reduction, alkylation and enzymatic cleavage with either trypsin or chymotrypsin. Data analysis was made on a combination of MS spectra obtained from these digests. The data were analyzed against the Viridiplantae database Taxonomy ID 33090 which confirmed the identity of the purified protein as Pru p 7 (Sequence ID: NO 1). No trace of Pru p 3 or other peach proteins was detected in the preparation.
• Example 1 describes the purification of native Pru p 7 and confirmation of its identity by MS/MS. The preparation was subsequently used for the production of polyclonal rabbit antibodies against Pru p 7.
• a plasmid DNA construct containing a synthetic gene encoding Pru p 7 was prepared and transformed into the yeast Pichia pastoris strain X-33.
• the transformed strain was grown and induced to produce recombinant Pru p 7 in a 3-litre bioreactor (Belach Bioteknik, Skogas, Sweden).
• the culture medium was harvested by centrifugation and the supernatant was collected.
• the supernatant was applied to an SP Sepharose FF column equilibrated with 50 mM NaAc pH 4.5.
• the recombinant protein was eluted in a linear 0-1 M NaCI gradient in the same buffer ( Figure 5).
• rPru p 7 containing fractions were collected and further purified by SEC on a Superdex 75 pg column in 50 mM NaAc pH 4.5, 150 mM NaCI (Figure 6a). Fractions containing rPru p 7 were pooled and the concentration determined by absorbance at 280 nm, using a calculated extinction coefficient of 0.72 mg 1 ml_ cm -1 . Purity and identity of the allergen preparation were verified by SDS-PAGE ( Figure 6b) and mass spectrometry. Experimental ImmunoCAP tests (Thermo Fisher Scientific, Uppsala, Sweden) were prepared as previously described [42] and the immunological activity was evaluated using relevant patient serum samples.
• Example 2 describes the expression of rPru p 7 in Pichia pastoris and purification of the recombinant protein.
• Recombinant Pru p 7 could be used to characterize polyclonal rabbit antibodies raised against nPru p 7 and to study IgE reactivity to Pru p 7 in relevant patient sera.
• nPru p 7 Purified nPru p 7, prepared as described in Example 1 , was used to raise polyclonal rabbit antibodies against Pru p 7.
• a rabbit was immunized with nPru p 7 according to a protocol comprising four booster injections of antigen. Prior to immunization, a preimmune serum sample was taken from the rabbit, to serve as control in subsequent experiments. All procedures were performed at Agrisera AB (Vannas, Sweden) under a regional ethics approval.
• the obtained anti-Pru p 7 antiserum was tested in a series of dilutions against both rPru p 7 and Cupressus sempervirens pollen extract, immobilised on ImmunoCAP solid phase.
• the antiserum showed strong IgG binding to the immobilised rPru p 7 as compared to the pre serum, even at the highest dilution (1 :8000) ( Figure 7a), confirming its content of Pru p 7- reactive IgG.
• the serum displayed binding to C. sempervirens pollen extract ( Figure 7b), suggesting the presence of a hitherto unknown protein cross-reactive with Pru p 7. Consequently, the anti-Pru p 7 antiserum could be used as a probe to trace this potential Cupressaceae pollen homologue of Pru p 7 in efforts to purify it, as demonstrated in the following examples.
• EXAMPLE 4 Purification of a novel. Pru p 7 related C. sempervirens pollen allergen
• Example 3 polyclonal rabbit IgG antibodies raised against Pru p 7 bound to an immobilised protein extract of C. sempervirens pollen. Utilizing the same antibodies, a C. sempervirens pollen protein cross-reactive with Pru p 7 could be identified, purified and characterised. Briefly, C. sempervirens pollen (Allergon, Valinge, Sweden) was extracted in 50 mM NaAc, 1 M NaCI pH 4.5 under agitation for 72 hrs at 4°C, clarified by centrifugation, filtered through a Whatman GF/F glass microfiber filter and desalted on a Sephadex G25 column equilibrated with 50 mM NaAc pH 4.5 ( Figure 8).
• Protein-containing fractions eluting in the void volume were pooled and loaded on an SP Sepharose FF column equilibrated with 50 mM NaAc pH 4.5. Elution was performed with a linear gradient from 0 to 1 M NaCI in the same buffer ( Figure 9a).
• Figure 9b Nine selected fractions were immobilised on ImmunoCAP solid phase and assayed for anti-Pru p 7 IgG binding activity ( Figure 9b) and analysed by SDS-PAGE ( Figure 9c).
• Fractions 7-9 which showed the highest binding of anti-Pru p 7 IgG and contained a prominent 7 kDa protein band, were pooled as indicated in Figure 9a and concentrated on an SP Sepharose HP column.
• the concentrated pool was further purified by SEC on Superdex 30 pg column, equilibrated with 20 mM NaAc pH 4.5, 250 mM NaCI. Elution was performed with the same buffer ( Figure 10a). Eight selected fractions were immobilised on ImmunoCAP solid phase and tested for anti-Pru p 7 IgG binding activity as described above ( Figure 10b) and analysed by SDS-PAGE ( Figure 10c). The highest binding of anti-Pru p 7 IgG was found in fractions 3-5 which showed a single distinct band at 7 kDa. Hence, fractions 3-5 were pooled as indicated in Figure 10a and this extensively purified Pru p 7 related C.
• Example 4 describes how a novel C. sempervirens pollen protein, hereinafter referred to as Cup s GRP, was purified using a series of chromatographic steps and the anti- Pru p 7 IgG antibodies described in Example 3.
• Example 4 To establish the identity and primary structure of the 7 kDa C. sempervirens pollen protein purified in Example 4, it was analysed by MS/MS on an Orbitrap Fusion Tribrid instrument. Prior to the MS analysis, the protein was reduced by DTT, alkylated with acrylamide and enzymatically cleaved with either trypsin, chymotrypsin or Lys-C. The MS data analysis was made on a combination of the spectra obtained from the three digests of the 7 kDa protein.
• Cup s GRP Figure 11c, Seq ID: NO 4
• the Cup s GRP sequence differed from the amended BY878079-derived sequence in four further positions, as indicated in Figure 11c.
• the MS/MS analysis demonstrated a complete coverage of this amino acid sequence, corresponding to residues 55-117 of the sequence encoded by the amended BY878079 record. Examples of identified peptides being part of this sequence are listed in Table 2a.
• Cup s GRPa a sequence containing both the alternative amino acid, i.e. Leu at position 18 and His at position 52, is referred to as Cup s GRPb (Seq ID: NO 5).
• Cup s GRPb a sequence containing both the alternative amino acid, i.e. Leu at position 18 and His at position 52.
• the amino acid sequence encoded by EST record BY878079 contained a predicted 24- residue signal peptide (underlined sequence in Figure 11b). In addition, between that signal peptide and the sequence matching Cup s GRP peptide 1 (Pep 1 in Figure 11b) identified by MS/MS, a stretch of 30 amino acids was present to which no matching Cup s GRP peptide was identified.
• Cup s GRP In order to determine whether the purified Cup s GRP nevertheless contained such an N- terminal peptide, the protein was subjected to N-terminal sequencing by Edman degradation as described in [43] The first four amino acid residues of the protein were identified as Ala- Gln-lle-Asp which exactly matched the N-terminal part of Cup s GRP peptide 1 identified by MS/MS.
• a precursor of Cup s GRP might include a portion corresponding to residues 25-54 of the BY878079-derived sequence, it is cleaved off and no longer present in the mature Cup s GRP protein.
• Example 5 describes how the amino acid sequence of Cup s GRP was determined by MS/MS and N-terminal sequencing.
• MS analysis the mass of the intact protein was determined and found to be in perfect agreement with the calculated theoretical mass.
• the 63-residue sequence was shown to have alternative amino acids in two positions, resulting in four possible isoforms of the protein in its native state.
• Peak 2 was found to contain a dominant protein band at approximately 7 kDa and showed strong antibody binding activity.
• Figure 14a shows an amended version of BY878079 where the erroneous TGA stop codon at position 302-304, identified as such in Example 5, has been replaced by a cysteine codon TGY (Seq ID: NO 3), where Y represents C or T as defined by the lUPAC ambiguity code system [44]
• Figure 14b the amino acid sequence encoded by nucleotides 44-394 of the modified BY878079 (Seq ID: NO 6) is shown and Jun a GRP peptides identified by MS/MS are aligned below.
• the Cry j GRP preparation was analysed by MS/MS on an Orbitrap Fusion Tribrid instrument after sample preparation as described in Example 5.
• the best database match of the obtained MS/MS spectra was EST record BY900480, a cDNA sequence from male strobilus of C. japonica ( Figure 15a, Seq ID: NO 7).
• Figure 15b the translated amino acid sequence of the open reading frame spanning nucleotide positions 15-365 of BY900480 is shown and Cry j GRP peptides identified by MS/MS are aligned below.
• a listing of selected Cry j GRP peptides identified by MS/MS, representing the entire protein, is shown in Table 2d.
• the complete amino acid sequence of Cry j GRP is shown in Figure 15c, Seq ID: NO 8. No polymorphism was detected in the analysis and the sequence displayed 68% identity to Pru p 7 ( Figure 15d).
• Cry j GRP lacked the first 54 residues of the amino acid sequence encoded by the best matching database record. Again, the first 24 residues comprise a predicted signal peptide and the following 30 residues are concluded to represent a propeptide cleaved off during protein maturation.
• Example 6 describes the purification, amino acid sequence determination and mass determination of the Pru p 7-related pollen proteins Jun a GRP and Cry j GRP from J. ashei and C japonica, respectively.
• EXAMPLE 7 Cloning and purification of two recombinant Cup s GRP isoforms
• Synthetic genes designed to encode the amino acid sequence of Cup s GRPa and Cup s GRPb from Example 5 were cloned into a expression vector pPICZa A and transformed into Pichia pastoris strain X-33.
• the two recombinant proteins were expressed and purified using the same procedures as those described for rPru p 7 in Example 2.
• MS/MS analysis confirmed the identity and integrity of the purified recombinant proteins.
• a comparison of the two recombinant isoforms of Cup s GRP, nCup s GRP and rPru p 7 by SDS-PAGE demonstrated a nearly identical electrophoretic appearance of the four protein preparations, with an apparent molecular weight of 7 kDa ( Figure 17).
• Example 7 describes the cloning and purification of two recombinant isoforms of Cup s GRP, representing two of the amino acid sequence variants determined in Example 5.
• EXAMPLE 8 IgE binding activity of native and recombinant Cup s GRP in comparison with rPru p 7
• Example 8 confirms the immunological relationship between peach allergen Pru p 7 and C. sempervirens pollen protein Cup s GRP first established by rabbit IgG antibodies also in regard to recognition by human IgE antibodies. Secondly, the higher IgE level of IgE binding to Cup s GRP than to Pru p 7 suggests that Cup s GRP may act as a primary sensitizer, eliciting IgE antibodies cross-reacting with Pru p 7.
• EXAMPLE 10 Immunological similarity between Cup s GRP, Jun a GRP and Cry j GRP
• EXAMPLE 11 Prevalence of sensitization to Cup s GRP among subjects with cypress pollinosis
• EXAMPLE 12 Analysis of IgE reactivity of closely related proteins from the same protein family - profilins
• this example demonstrates that IgE reactivity is, due to cross reactivity, very similar among closely related proteins within the same protein family.
• the proteins were soluble and folded proteins of small size, with a pairwise sequence identity of around 80%.
• the studies described above were all performed with naturally occurring protein variants, it is highly likely that also artificial variants of these proteins with a high sequence identity to a specific profilin will demonstrate highly similar IgE reactivity, provided that the variant is a soluble folded protein.
• Artificial variants of profilins that are still soluble and folded may be designed by a limited number of amino acid substitutions in positions where the amino acid is not phylogenetically conserved. If such substitutions are made with amino acids that occur in other profilins at any such position, this will increase the likelihood of producing a soluble folded protein.
• EXAMPLE 13 Analysis of the experimentally determined sequences of GRP from pollen of different Cupressaceae species
• Cupressaceae pollen identified here are phylogenetically relatively distant from GRP proteins present in foods, such as Pru p 7.
• Cupressaceae pollen consensus sequence see row B of Figure 26. Amino acid exchanges in these positions will have a high probability of inducing structural changes that would affect the protein’s IgE binding capacity. Finally, there are 14 amino acid residues that differ between Cupressaceae pollen consensus sequence and Pru p 7, indicated by Z in row A of Figure 26. Amino acid substitution of any one or more of these 14 amino acid positions Z, to an amino acid residue that occurs in phylogenetically related proteins, as listed in table 3, would much likely not disturb the overall structure of the protein.
• Rashid, R.S., et al., Pollen-food syndrome is related to Bet v 1/PR-10 protein

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