HYPOALLERGENIC MOLECULES

The present invention relates to hypoallergenic molecules.

Worldwide 10-20 million people suffer from pollen allergies and among these patients around one third exhibits allergic reactions towards tree pollen. In the temperate climate zone Bet v 1, the major birch pollen allergen, accounts for most cases of tree pollinosis. Additionally between 50 and 93% of birch pollen allergic individuals develop hypersensitivity reactions towards pollen-related food mediated by cross-reactive IgE antibodies primarily directed against Bet v 1. This type of hypersensitivity is described as pollen-food syndrome (PFS). Thereby allergen contact with the oral mucosa causes immediate adverse reactions usually restricted to the oral cavity and the pharynx. In case of birch pollen allergy one of the allergens most frequently implicated with PFS is Mal d 1, the major apple allergen. Mal d 1 shares 64% amino acid sequence identity with Bet v 1 and cross-inhibition experiments demonstrated the presence of common IgE epitopes on both molecules.

In Ferreira F et al. (FASEB (1998) 12:231-242) the modulation of the IgE reactivity of Bet v is by introducing point mutations in the wild-type Bet v 1a is described. The authors introduced mutations at position 10, 30, 57, 112, 113 and/or 125 of Bet v 1a. Bet v 1a molecules having mutations at these positions do not lead to a complete loss of the three-dimensional structure and thus to an elimination of the IgE reactivity. Mutations at these positions led to decreased IgE reactivity for some patients but not for all, reflecting the polyclonal nature of IgE responses in predisposed individuals. Therefore, such mutations could not be used as an approach to generate therapeutic molecules for the population at large.

In Wallner et al. (Allergy Clin. Immunol., 120(2) (2007):374-380) chimeric proteins displaying low IgE reactivity and high T-cell reactivity are disclosed which have been selected from a library comprising shuffled allergens of Bet v 1 and Bet v 1 homologous allergens.

In Larsen et al. (Allergy Clin. Immunol., 109(1) (2002):164) the cross-reactivity between Bet v 1 and Mal d 1 is discussed.

Wallner et al. (Allergy Clin. Immunol., 109(1) (2002):164) refers to in vitro evolution of the Bet v 1 family using gene shuffling methods.

In Bolhaar S T H P et al. (Clin. Exp. Allergy. 35(12) (2005):1638-1644) a hypoallergenic variant of Mal d 1 comprising 5 point mutations is disclosed.

It is an object of the present invention to provide a hypoallergenic molecule which exhibits no or a significantly reduced IgE reactivity compared to the wild-type allergen which may be used to treat pollen-food syndrome caused by Bet v 1a cross reactive allergens.

Therefore, the present invention relates to a hypoallergenic molecule consisting of Bet v 1a (SEQ ID No. 1) or an allergen having at least 40% identity to Bet v 1a comprising mutations of at least four amino acid residues in the region of amino acids 100 to 125 of Bet v 1a or its corresponding region of the allergen having at least 40% identity to Bet v 1a.

It turned out that the mutation of at least four amino residues in the region of amino acids 100 to 125 of Bet v 1a or an allergen having at least 40% identity to Bet v 1a (preferably having an identity of at least 55%, 60%, 65%, 70%, 75% or 80%) results in a hypoallergenic molecule exhibiting a substantially reduced or even complete removal of IgE reactivity due to loss of its three-dimensional structure. The IgE reactivity in the mutated allergen is—compared to the corresponding wild-type allergen reduced. In antibody (IgE)-based biologic assays (e.g. inhibition ELISA or RAST, basophil inflammatory mediator release), at least 50, preferably at least 100, more preferably at least 500, even more preferred at least 1000 fold higher concentrations of the mutated allergen is required to reach half-maximal values obtained with wild type allergen. This is in the light of Ferreira F et al. (FASEB (1998) 12:231-242) surprising because therein it was shown that the mutation of three amino acid residues in this region did not result in a hypoallergenic molecule with the properties of the molecule of the present invention.

The hypoallergenic properties of the hypoallergenic molecules of the present invention are a consequence of the destruction of the three-dimensional structure compared to the wild-type allergen. It has been demonstrated by Akdis et al. (Akdis C A et al. Eur J Immunol. 28(3)(1998):914-25) that a native folded allergen, here the bee venom allergen phospholipase A (PLA), induced a different immune response as the non-native folded version of the very same protein. Folded PLA induced IgE antibodies in B cells and stimulated T helper (TH) 2 cells, both markers of an allergic immune response, whereas non-refolded or reduced-and-alkylated PLA both induced a TH1 dominated cytokine profile leading to an IgG4 response in B cells. A TH1 driven immune response is clearly favoured for molecules, which should be used as therapeutic agents for allergic diseases. Therefore, the unfolded nature of the molecules of the present invention is crucial.

It is in particular preferred to mutate, next to three other amino acid residues, at least amino acid residue 113 or 114 of Bet v 1a or an allergen having at least 40% identity to Bet v 1a.

Another aspect of the present invention relates to a hypoallergenic molecule consisting of Bet v 1a or an allergen having at least 40% identity to Bet v 1a comprising mutations of at least one amino acid residue in the region of amino acids 100 to 125 of Bet v 1a of its corresponding region of the allergen having at least 40% identity to Bet v 1a, wherein the mutation of at least one amino acid residue comprises the mutation of amino acid residue at position 114 of Bet v 1a or the allergen having at least 40% identity to Bet v 1a.

It surprisingly turned out that the mutation of at least amino acid residue 114 may already lead to a hypoallergenic molecule having a three-dimensional structure being different to the corresponding wild-type allergen. The mutation of amino acid 114 of Bet v 1a or an allergen being at least 40% identity to Bet v 1a will lead to a hypoallergenic molecule. According to a preferred embodiment of the present invention this hypoallergenic molecule will comprise at least one (preferably at least two, three, four, five) further mutations within amino acids 100 to 125 of Bet v 1a or its corresponding region of allergen having at least 40% identity to Bet v 1a. Particularly preferred the hypoallergenic molecule comprises a mutation at amino acid residue 102 and/or 120 of Bet v 1a. In a particular preferred embodiment of the present invention amino acid residue 114 of Bet v 1a or an allergen having at least 40% identity to Bet v 1a is substituted by lysine (K), aspartic acid (D) or glutamic acid (E).

According to a preferred embodiment of the present invention the allergen having at least 40% identity to Bet v 1a is immunologically cross-reactive with Bet v 1a. Here immunologic cross-reactivity is defined by antibody binding to a certain molecule and can be determined by ELISA using affinity purified polyclonal rabbit anti Bet v 1a antibodies. Molecules having at least 40% identity to Bet v 1a and being recognized by these antibodies will be considered as immunological cross-reactive with Bet v 1a.

It is advantageous that the allergen having at least 40% identity to Bet v 1a is immunologically cross-reactive with Bet v 1a because in this case the hypoallergenic molecule of the present invention can be used to prevent or treat pollen-food syndrome (PFS), because the immune system is able to produce antibodies which are able to bind to Bet v 1a as well as to food allergens, which in many cases shows at least 40% identity to Bet v 1a.

It is even more advantageous when the allergen has at least 85%, preferably at least 90%, more preferably at least 95%, identity to Bet v 1a.

The term “identity”, as used herein, indicates whether any two (or more) peptide, polypeptide or protein sequences have amino acid sequences that are “identical” to a certain degree (“% identity”) to each other. This degree can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) PNAS USA 85: 2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) Nucleic Acids Res., 12, 387-395), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215: 403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al, (1988) SIAM J Applied Math 48: 1073). For instance, the BLAST tool of the NCBI database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison, Wis.)). Percent identity of protein molecules can further be determined, for example, by comparing sequence information using a GAP computer program (e.g. Needleman et al., (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter one of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and for non-identities) and the weighted comparison matrix of Gribskov et al. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

The allergen having at least 40% identity to Bet v 1a (Z80098) is preferably composed of Bet v 1 and an allergen selected from the group consisting of Dau c 1, in particular Dau c 1.0101 (U47087), Dau c 1.0102 (D88388), Dau c 1.0104 (Z81362), Dau c 1.0105 (Z84376), Dau c 1.0201 (AF456481) or Dau c 1.0103 (Z81361), Api g 1, in particular Api g 1.0101 (Z48967) or Api g 1.0201 (Z75662), Pet c 1 (X12573), Cas s 1 (AJ417550)), Clue a 1 (P85126), Mal d 1, in particular Mal d 1.0101 (X83672), Mal d 1.0102 (Z48969), Mal d 1.0109 (AY026910), Mal d 1.0105 (AF124830), Mal d 1.0106 (AF124831), Mal d 1.0108 (AF126402), Mal d 1.0103 (AF124823), Mal d 1.0107 (AF124832), Mal d 1.0104 (AF124829), Mal d 1.0201 (L42952), Mal d 1.0202 (AF124822), Mal d 1.0203 (AF124824), Mal d 1.0207 (AY026911), Mal d 1.0205 (AF124835), Mal d 1.0204 (AF124825), Mal d 1.0206 (AF020542), Mal d 1.0208 (AJ488060), Mal d 1.0302 (AY026908), Mal d 1.0304 (AY186248), Mal d 1.0303 (AY026909), Mal d 1.0301 (Z72425), Mald1.0402 (Z72427), Mald1.0403 (Z72428) or Mald1.0401 (Z72426), Pyr c 1, in particular Pyr c 1.0101 (065200), Pru av 1, in particular Pru av 1.0101 (U66076), Pru av 1.0202 (AY540508), Pru av 1.0203 (AY540509) or Pru av 1.0201 (AY540507), Pru p 1 (DQ251187), Rub i 1.0101 (DQ660361), Pru ar 1, in particular Pru ar 1.0101 (U93165), Cor a 1, in particular Cor a 1.0401 (AF136945), Cor a 1.0404 (AF323975), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974), Cor a 1.0301 (Z72440), Cor a 1.0201 (Z72439), Cor a 1/5 (X70999), Cor a 1/11 (X70997), Cor a 1/6 (X71000) or Cor a 1/16 (X70998), Bet v 1d (X77266), Bet v 1l (X77273), Bet v 1al-6 (Ferreira F et al. FASEB (1998) 12:231-242), Bet v 1 g (X77269), Bet v 1f (X77268), Bet v 1j (X77271), Bet v 1e (X77267), Bet v 1b (X77200), Bet v 1c (X77265), Bet v 1.0101 (X15877), Bet v 1.0901 (X77272), Bet v 1.1101 (X77599), Bet v 1.1201 (X77600), Bet v 1.1301 (X77601), Bet v 1.1401 (X81972), Bet v 1.1501 (Z72429), Bet v 1.1601 (Z72437), Bet v 1.1701 (Z72430), Bet v 1.1801 (Z72431), Bet v 1.1901 (Z72433), Bet v 1.2001 (Z72434), Bet v 1.2101 (Z72435), Bet v 1.2201 (Z72438), Bet v 1.2301 (Z72436), Bet v 1.2401 (Z80100), Bet v 1.2501 (Z80101), Bet v 1.2601 (Z80102), Bet v 1.2701 (Z80103), Bet v 1.2901 (Z80105), Bet v 1.3001 (Z80106), Aln g 1 (S50892), Car b 1, in particular Car b 1.0301 (Z80169), Car b 1.0302 (Z80170), Car b 1.0103 (Z80159), Car b 1.0105 (Z80161), Car b 1.0104 (Z80160), Car b 1/18, Car b 1.0101 (X66932), Car b 1.0102 (X66918), Car b 1/1b, Car b 1/2, Car b 1.0106a (Z80162), Car b 1.0106b (Z80163), Car b 1.0106c (Z80164), Car b 1.0106d (Z80165), Car b 1.0107a (Z80166), Car b 1.0107b (Z80167), Car b 1.0201 (X66933) or Car b 1.0108 (Z80168), Gly m 4.0101 (X60043), Vig r 1.0101 (AY792956), Ara h 8.0101 (AY328088), Asp ao PR10 (X62103), Bet p 1a (AB046540), Bet p 1b (AB046541), Bet p 1c (AB046542), Fag s 1 (AJ130889), Cap ch 17 kD a (AJ879115), Cap ch 17 kD b (AJ878871), Fra a 1.0101 (AY679601), Tar o 18 kD (AF036931).

Particularly suited is Mal d 1 (SEQ ID No. 2).

According to a preferred embodiment of the present invention the at least one mutation of at least four amino acid residues is an amino acid substitution, deletion or addition.

The mutation of the at least four amino acid residues within the allergen may be of any kind. However, it is preferred to substitute single amino acid residues or stretches of amino acid residues within the molecule, because in such a case the length of the hypoallergenic molecule is not affected at all compared to Bet v 1a.

According to a preferred embodiment of the present invention the mutated region of Bet v 1a or its corresponding region of the allergen having at least 40% identity to Bet v 1a comprises amino acids 105 to 120, preferably amino acids 108 to 118, more preferably amino acids 109 to 116, of Bet v 1a or its corresponding region of the allergen having at least 40% identity to Bet v 1a.

According to a preferred embodiment of the present invention the at least one mutation comprises the substitution of said region with the corresponding region of another allergen selected from the group consisting of Bet v 1a and an allergen having at least 40% identity to Bet v 1a.

Due to the substitutions of said regions it is possible not only to create a hypoallergenic molecule which comprises T-cell epitopes of Bet v 1a or an allergen having at least 40% identity to Bet v 1a but also molecules comprising potentially specific Bet v 1a epitopes or epitopes of the allergen having at least 40% identity to Bet v 1a. This allows administering to an individual suffering or being at risk to suffer a pollen-food allergy a vaccine comprising said hypoallergenic molecule, which is able to provoke the formation of an immune response not only against a first allergen but also against a second allergen.

According to another preferred embodiment of the present invention the molecule consists of Bet v 1 and the region of amino acids 109 to 116 of Bet v 1 is substituted with the corresponding region of Mal d 1 (resulting in SEQ ID No. 3).

According to a particular preferred embodiment of the present invention the molecule consists of Mal d 1 and the region of amino acids 109 to 116 of Mal d 1 is substituted with the corresponding region of Bet v 1.

Bet v 1a or an allergen sharing at least 40% identity with Bet v 1a may be modified by substituting at least a part of said defined region with an amino acid fragment which can be derived from another allergen, whereby said fragment is of the same region as of the region to be substituted.

Another aspect of the present invention relates to a nucleic acid molecule encoding a hypoallergenic molecule according to the present invention.

A further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.

The vector of the present invention comprises a nucleic acid fragment encoding for the hypoallergenic molecule of the present invention and vector elements which allow its reproduction in prokaryotic (e.g. bacteria) organisms. These vectors comprise functional polynucleotide sequences such as promoters, transcriptional binding sites etc. The vector may be a plasmid or viral vector.

In order to facilitate the purification of the hypoallergenic molecule, said molecule may be fused to a Tag, in particular to a histidine or gluthathione-S-transferase. Hence, suited expression vectors known in the art may be used.

Another aspect of the present invention relates to a vaccine preparation comprising a hypoallergenic molecule according to the present invention.

According to a preferred embodiment of the present invention said preparation comprises further at least one pharmaceutically acceptable excipient, diluent, adjuvant and/or carrier.

In order to provide a vaccine formulation which can be administrated subcutaneously, intramuscularly, intravenously, mucosally etc. to an individual the formulation has to comprise further excipients, diluents, adjuvants and/or carriers. Preferred adjuvants used comprise KLH (keyhole-limpet hemocyanin) and alum. Suitable protocols for the production of vaccine formulations are known to the person skilled in the art and can be found e.g. in “Vaccine Protocols” (A. Robinson, M. P. Cranage, M. Hudson; Humana Press Inc., U.S.; 2^ndedition 2003).

Another aspect of the present invention relates to the use of hypoallergenic molecule according to the present invention for the manufacture of a vaccine for the prevention and/or treatment of allergies.

The hypoallergenic molecule of the present invention can be used for treating and preventing allergies, in particular allergies associated with pollen-food syndrome, where patients suffering from pollinosis also experience food allergies (these may provoke e.g. itching and pruritus around the oral cavity to generalized urticaria and even anaphylaxis). In the pollen-food syndrome, an individual is first sensitized by inhaling allergens present in pollen. Thereafter, immediate-type symptoms begin to be provoked when the individual gets in contact with any plant-derived foods containing proteins cross-reactive to the sensitizing allergen. After the completion of per-oral sensitization, allergic symptoms are provoked whenever the patient gets in contact with the same protein. Bet v 1a is a highly cross-reactive allergen which is able to cross-react with a large number of food derived allergens, in particular derived from vegetables and fruits. Therefore, an individual suffering from a pollen allergy caused by Bet v 1a may also be allergic to food derived allergies.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

FIG. 1 shows (A) a schematic representation of the 160 amino acids long (including initiation methionine) mutant allergen BM1,2,3,5. The protein was constructed by grafting epitopes of Mal d 1.0108 consisting of 7 consecutive amino acids on the template allergen Bet v 1a. Stretches matching amino acids of Bet v 1a are shown in white, epitopes inserted from Mal d 1.0108 in grey. Amino acids composing the exchanged epitopes are listed in one-letter code. Residues identical in both proteins are presented in black, different amino acids in grey. (B) A model of BM1,2,3,5 was calculated and the structure was compared to the 3-dimensional fold of Bet v 1. Residues identical in BM1,2,3,5 and Bet v 1a are shown in grey, epitopes derived from Mal d 1.0108 are presented in black. The lower panel shows the proteins rotated by 180° C. (C) Secondary structure of BM1,2,3,5 was compared to Bet v 1 by circular dichroism (CD). Data is presented as mean residue molar ellipticity [θ] MRW at a given wavelength and baseline-corrected.

FIG. 2 shows (A) an SDS-PAGE and immunoblot analysis of affinity purified BM1,2,3,5 (each 5 μg per lane). Following SDS-PAGE proteins were visualized by Coomassie Brilliant Blue staining (left panel). For immunoblot analysis proteins were detected using a monoclonal mouse anti Bet v 1 antibody. (B) In ELISA IgE binding activity of Bet v 1a, Mal d 1.0108 and BM1,2,3,5 was assessed using sera of Bet v 1 allergic patients without clinical symptoms of PFS (n=12) or Bet v 1 allergic patients, suffering from PFS to apple and other Bet v 1-related foods (n=11). Allergens were titrated for coating and optimal antigen coating concentration was determined to be 0.5 mg/ml. No significant difference was observed for both groups of patients concerning IgE binding towards Bet v 1 (P>0.99). As expected, IgE binding towards Mal d 1 was stronger in the PFS group. Concerning the mutant BM1,2,3,5 patients with PFS showed significantly increased IgE binding (P<0.01). Symbols represent individual patients, bars means. P-values were calculated by t-test (**P<0.01).

FIG. 3 shows schematically the mutant allergen BM4 according to example 2.

FIG. 4 shows a clustal W alignment of Bet v 1a and BM4. The exchanged epitope of BM4 is underlined, the immunodominant T cell epitope of Bet v 1a is indicated in italic and bold. Amino acids in bold represent the “core” of the epitope exchange, which are amino acids found to be critical for patients' IgE binding of Bet v 1.

FIG. 5 shows a clustal W alignment of Mal d 1.0108 and BM4. The exchanged epitope of BM4 is underlined. Amino acids in bold represent the “core” of the epitope exchange, which are amino acids found to be critical for patients' IgE binding of Bet v 1.

FIG. 6 shows a SDS-Page of BM4.

FIG. 7 shows CD spectra of Bet v 1a in comparison to BM4 at 20° C. and at 95° C. All curves are baseline corrected; data are presented as mean residue molar ellipticity. Protein concentrations were determined by amino acid analysis. BM4 shows the typical CD spectrum of random-coiled proteins at 20° C. as well as at 95° C.

FIG. 8 shows an ELISA of Bet v 1a (Biomay), BM4 and ovalbumin. Ovalbumin was purchased from Sigma-Aldrich as irrelevant control antigen. Allergens (200 ng/well) were immobilized to the solid phase. All measurements were performed as duplicates; results are presented as mean OD values after background subtraction. Sera of 13 birch pollen allergic individuals were tested. As control NHS was uses which gave no signal to any of the proteins. BM4 shows virtually no patients' IgE binding in the ELISA.

FIG. 9 shows the allergenic activity of BM4 assessed by sensitizing huFcεRI-transfected RBL-2H3 cells with serum IgE from birch pollen-allergic patients. BM4 shows a 100-1000 fold reduced anaphylactic activity as compared to Bet v 1a.

FIG. 10 shows the determination of proliferative responses of peripheral blood mononuclear cells (PBMCs) from birch pollen allergic patients stimulated with 25, 12.5, 6.25 or 3 μg/ml of 6×his tagged BM4 or Bet v 1a. Average stimulation indices (SI) of BM4 were found to be higher than of Bet v 1a. Symbols represent individual patients, bars medians.

FIG. 11 shows the sequence of Bet v 1 (1bv1) obtained from the PDB protein data bank. The sequence area which has been replaced in the mutant protein BM4 is indicated by a box.

FIG. 12 shows the sequence area which has been replaced in the mutant protein BM4. A relevant T cell epitope of Bet v 1 (SILKISNKYHTK; SEQ ID No. 4) recognized by 41% of the patients is indicated in italic and bold. One peptide before (DGGSILKISNK; SEQ ID No. 5) is the recognized by 6%, the peptide after (KISNKYHTKGDH; SEQ ID No. 6) by none of the patients. The PBMC data so far did not show a reduced but rather a better T cell stimulation. Therefore no problems regarding T cell stimulation properties of BM4 can be expected.

FIG. 13 shows a Clustal W multiple sequence alignment of Bet v 1 and its homologous allergens found in other pollen sources, fruits, nuts or vegetables, including isoforms. The epitope replaced in Bet v 1a was replaced by the one of Mal d 1.0108 in BM9

FIG. 14 shows a calculation of Z-scores of different Bet v 1a mutants with grafted epitopes of Mal d 1.0108. Therefore overlapping non-sequence identical stretches (10aa; n=103) of Mal d 1.0108 were substituted on the Bet v 1a structure and Z-scores were calculated using the software tool ProSa II. This software uses knowledge-based potentials derived from known protein structures and captures the average properties of native globular proteins in terms of atom-pair and solvent interactions to generate scores reflecting the quality of protein structures (Sippl M. J, Proteins 17:355-362, 1993). The calculations revealed that the epitopes substitution of the amino acids 109-116 (SGSTIKSI; SEQ ID No. 7) of Mal d 1.0108 on Bet v 1a is one of the most destabilizing substitutions giving a high Z-score. With regard to protein structure this explains the unfolded nature of the mutant allergen BM4.

FIG. 15 shows a schematic representation of the mutant allergens BM1, BM2, BM3, BM4, BM5, BM1,2,3,4,5 and BM1,2,3,5. The allergens were produced by epitope grafting from Mal d 1.0108 to the scaffold of Bet v 1a. The grafted epitope sequences are indicated in the Figure, amino acids identical to Bet v 1a are shown in black, amino acids inserted from Mal d 1.0108 are underlined. The mutant allergens were expressed in E. coli. Bet v 1-like folding of the different mutant allergens was either assessed by antibody binding in a dot blot or by circular dichroism spectroscopy of the purified allergens. Mutant allergens carrying the epitope 109-116 (SGSTIKSI; SEQ ID No. 7) of Mal d 1.0108 show no Bet v 1-like fold, however mutant allergen carrying other epitopes of Mal d 1.0108 were able to fold in a Bet v 1-like manner.

FIG. 16 demonstrates the reduced ability of BM4 to bind Bet v 1 specific patients' IgE, compared to wild-type Bet v 1a, as analyzed by ELISA inhibition. 200 ng/well of Bet v 1a were immobilized to an ELISA plate, patients' sera were incubated with serial dilutions of the respective inhibitor (e.g. Bet v 1a, BM4 or OVA as irrelevant control antigen). All measurements were performed as duplicates; results are presented as mean OD values after background subtraction. Sera of 5 birch pollen allergic individuals were tested. As control NHS was used which gave no signal to any of the proteins. Compared to Bet v 1a BM4 showed an approximately 100-1000 fold reduced patients' IgE binding capacity.

FIG. 17 shows an immunoblot of BM4, Bet v 1a and OVA used as irrelevant control antigen. Bound serum IgE was detected using I¹²⁵-labeled rabbit anti-human IgE (MedPro). BM4 shows virtually no IgE binding. As controls normal human serum or only the detection antibody were used.

FIG. 18 Homogeneity of BM4 was assessed by online-SEC light scattering using UV and triple detection array (SECTDA). BM4 appeared as single peak with a retention volume of 9.3 ml having an approximate MW of 20 kDa and a R_Hof >3.2 nm (a). Observed peak tailing might have been due to adsorptive effects and high R_Hdue to extended conformation of unfolded protein. Detectors were calibrated with bovine serum albumin (BSA) having a MW of 66 kDa and a R_Hof 3.1 nm. System suitability was checked by rBet v 1a having a MW of 17 kDa and a R_Hof 1.9 nm. More than 99% homogeneity of non-aggregated BM4 was observed as no further peak was detected between void (5.7 ml) and total retention volume (12 ml) of SEC (b). HPSEC runs were performed using a 7.8×300 mm TSKgel G2000_SWXL, column (Tosoh Bioscience, Stuttgart, Germany) on a HP1100 analytical chromatography system (Hewlett-Packard, San Jose, Calif., USA) at 0.5 ml/min in PBS. Using a combination of the built-in UV detector measuring at 280 nm and a sequential refractive index (RI, short-dashed), intrinsic viscosity (IV, dash-dotted), and light scattering (RALS, long-dashed) detection system (TDA 302, Viscotek Corp., Houston, Tex., USA) the approximate molecular weight (MW, solid) and hydrodynamic radius (R_H, dash-double dotted) were determined.

FIG. 19 shows the aggregation behavior of BM4 in solution by dynamic light scattering (DLS). More than 97% of BM4 appeared as monomeric molecule with a hydrodynamic radius (R_H) of 3.5 nm and a polydispersity of 18.4% (a). As reference, rBet v 1a displayed an R_Hof 2.1 nm with a polydispersity of 16.7% (b). Thus, BM4 adopted a higher R_H, which was probably due to its unfolded state. Besides, <3% of BM4 appeared as multimer of molecular weight>1MDa. Dynamic light scattering was performed on DLS 802 (Viscotek Corp., Houston, Tex., USA) upon centrifugation for 10 min at 14,000×g in 10 mM sodium phosphate. The solvent settings for water were used. Data were accumulated for 10−20×10 sec and the correlation function was fitted into the combined data curve, from which the mass distribution was calculated by the OmniSize™ software package (Viscotek).

FIG. 20 Purified recombinant Bet v 1, BM4, Mal d 1, Dau c 1, Api g 1, Cor a 1, Arabidopsis thaliana PR-10 protein, Thermus thermophilus PR-10 protein, and Methanosarcina mazei PR-10 protein (3 μg per lane) were subjected to SDS-PAGE and stained with coomassie blue (a). ELISA using polyclonal rabbit anti Bet v 1 antibodies (1:5000) for demonstration of cross-reactivity of BM4 and homologous proteins (b). Recombinant Bet v 1, BM4 and Cor a 1 were extensively recognized by the rabbit antibody, and structural cross-reactivity could also be demonstrated for Dau c 1, Mal d 1, and Api g 1. No positive signals were obtained with purified PR-10 proteins from Arabidopsis thaliana, Thermus thermophilus, and Methanosarcina mazei. Sequence identity of abovementioned PR-10 proteins in comparison to BM4 (c). Amino acid alignment and identity plot was done with AlignX (Vector NTI, Invitrogen).

FIG. 21 shows a schematic representation of the mutant allergens MB1, MB2, MB3, MB4, MB5 and MB1,2,3,4,5. The allergens were produced by epitope grafting from Bet v 1a to the scaffold of Mal d 1.0108. The grafted epitope sequences are indicated in the Figure, amino acids identical to Mal d 1.0108 are shown in black, amino acids inserted from Bet v 1a in grey. The mutant allergens were expressed in E. coli. The folding of Bet v 1 and its homologues found in other pollen as well as food sources was found very similar as demonstrated by X-ray crystallographic analysis (Gajhede M, Nat Struct Biol. 1996 December; 3(12):1040-5; Neudecker P, J Biol Chem. 2001 Jun. 22; 276(25):22756-63. Epub 2001 Apr. 3; Schirmer T, J Mol Biol. 2005 Sep. 2; 351(5):1101-9). This common fold results in good antibody cross-reactivity. Therefore the Bet v 1-like folding of the different Mal d 1.0108 mutant allergens was assessed by antibody binding in a dot blot using either serum from birch pollen allergic patients or affinity purified polyclonal rabbit anti Bet v 1 antibodies, which were shown to cross-react with Mal d 1. Based on these immunoblot results mutant allergens carrying the epitope 109-116 (DGGSILKI) of Bet v 1a do not show a Bet v 1-like fold; however mutant allergens carrying other epitopes of Bet v 1a were able to fold in a Bet v 1-like manner.

EXAMPLES
Example 1

The pollen-food syndrome (PFS) is an association of food allergies to fruits, nuts, and vegetables in patients with pollen allergy. Mal d 1, the major apple allergen, is one of the most commonly associated food allergen for birch pollen-allergic patients suffering from PFS. Immunologically this is caused by cross-reactive IgE antibodies originally raised against the major birch pollen allergen, Bet v 1.

Although Bet v 1-specific IgE antibodies cross-react with Mal d 1, not every Bet v 1-allergic patient develops clinical reactions towards apple. This shows that distinct IgE epitopes present on both homologous allergens are responsible for the clinical manifestation of PFS. To test this four Mal d 1 stretches were grafted onto Bet v 1. The grafted regions were 7-amino acids long encompassing amino acids residues shown to be crucial for IgE recognition of Bet v 1.

The Bet v 1-Mal d 1 chimeric protein designated BM1,2,3,5 was expressed in E. coli and purified to homogeneity. BM1,2,3,5 was tested with patients' sera of i) Bet v 1-allergic patients displaying no clinical symptoms upon ingestion of apples and ii) Bet v 1-allergic patients displaying allergic symptoms upon ingestion of apples and other Bet v 1-related foods. Patients' IgE binding was assessed by ELISA.

Patients suffering from PFS reacted stronger with BM1,2,3,5 compared to solely birch allergic individuals. Thus B-cell epitopes of Bet v 1 implicated with apple allergy were successfully identified.

Methods

Patients' Sera:

Birch pollen allergic patients with and without PFS were selected based on typical case history, positive skin prick test and radioallergosorbent test (RAST) class 3. Correlation between clinical symptoms and relative IgE binding to Bet v 1 and Mal d 1 was confirmed for both groups of patients by ELISA using the respective antigens.

Cloning of BM1,2,3,5

BM1,2,3,5 was generated by PCR amplification of mutated fragments of Bet v 1a (X15877). Internal mismatch primers used for cloning were

Mut10F

(SEQ ID No. 8)

5′CGCCATTGTTTTCAATTACGAAAaTGAGttCACCTCTGagATC3′,

Mut30F

(SEQ ID No. 9)

5′GATGGCGATAATCTCaTTCCAAAGaTTGCACCCCAA3′,

Mut30R

(SEQ ID No. 10)

5′ATGGCTTGGGGTGCAAtCTTTGGAAtGAGATTATCG3′,

Mut57F

(SEQ ID No. 11)

5′ATTAAGAAGATCAcCTTTggCGAAGGCTTC3′,

Mut57R

(SEQ ID No. 12)

5′GAAGCCTTCGccAAAGgTGATCTTCTTAAT3′,

Mut125F

(SEQ ID No. 13)

5′CACACCAAAGGTaACatTGAGaTcAAGGCAGAGCAG3′,

Mut125R

(SEQ ID No. 14)

5′CTGCTCTGCCTTgAtCTCAatGTtACCTTTGGTGTG3′.

Bases exchanged are indicated in lower case. Mutated fragments were gel-purified, pooled and assembled in a primerless PCR. Full length genes were amplified with the primers BetF 5′GGCCCATATGGGTGTTTTCAATTACGAA3′ (SEQ ID No. 15) and BetR 5′TCGGCTCGAGGTTGTAGGCATCGGAGTG3′ (SEQ ID No. 16). Restriction sites are underlined. BM1,2,3,5 was cloned into a pHis-Parallel2 vector using Nde I and Xho I restriction sites (Wallner M et al., Methods 2004, 32(3):219-26).

Expression and Purification of BM4

Expression plasmids were transformed in E. coli BL21 (DE3) pLysS cells (Stratagene) and grown at 37° C. to and OD600 of 0.8 in LB medium supplemented with 100 mg/L ampicillin. Cultures were cooled to 16° C. and protein expression was induced by addition of 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After incubation for 18 h, cells were harvested by low speed centrifugation and resuspended in appropriate lysis buffer. BM1,2,3,5 was expressed 6×His tagged fusion protein and purified from soluble bacterial lysates by immobilized metal affinity chromatography (Wallner M et al., Methods 2004, 32(3):219-26). Recombinant proteins were dialyzed against 10 mM sodium phosphate buffer, pH 7.4 and stored at −20° C.

SDS-PAGE and Immunoblots

E. coli lysates and purified proteins were analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% gels. Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Biorad).

For immunoblot analysis proteins separated by SDS-PAGE were electroblotted on nitrocellulose membrane (Schleicher & Schuell). Proteins were detected using the monoclonal anti Bet v 1 antibody BIP-1 (1:10.000) (Ferreira F et al., FASEB 1998, 12:231-242). Bound BIP-1 was detected with an AP-conjugated rabbit anti mouse IgG+IgM (Immunoresearch Laboratories Inc.).

Circular Dichroism

Circular dichroism (CD) spectra of proteins were recorded in 5 mM sodium phosphate pH 7.4 with a JASCO J-810 spectropolarimeter (Jasco) fitted with a Neslab RTE-111M temperature control system (Thermo Neslab Inc.). Obtained curves were baseline-corrected; results are presented as mean residue molar ellipticity [θ]MRW at a given wavelength. Protein concentrations for normalizing CD signals were determined at OD280.

Molecular Modelling

Modelling was performed using the comparative modelling tool MODELLER and evaluated by ProSa2003. All models are based on the PDB structure file 1bv1 (Bet v 1) and presented using PyMOL 0.98.

ELISA Experiments

For IgE ELISA experiments, Maxisorp plates (NUNC) were coated with allergen titrations in 50 μl PBS/well overnight at 4° C. Plates were blocked with TBS, pH 7.4, 0.05% (v/v) Tween, 1% (w/v) BSA and incubated with patients' sera diluted 1:5 overnight at 4° C. Bound IgE was detected with alkaline phosphatase-conjugated monoclonal anti-human IgE antibodies (BD Biosciences Pharmingen), after incubation for 1 h at 37° C. and 1 h at 4° C. Alternatively, Bet v 1 and homologous proteins were coated at 4 μg/ml in 50 μl PBS/well overnight at 4° C. Blocking was done as described above and incubated with polyclonal rabbit anti rBet v 1 antibody using different dilutions (1:5.000-1:20.000). Detection was performed with an alkaline phosphatase-conjugated goat anti rabbit antibody. 10 mM 4-Nitrophenyl phosphate (Sigma-Aldrich) was used as substrate and OD was measured at 405/492 nm. Measurements were performed as triplicates (IgE ELISA) or duplicates (rabbit anti Bet v 1 antibody); results are presented as mean OD values.

Results

The aim of the present example was the identification of cross-reactive B cell epitopes responsible for the clinical manifestation of Bet v 1-related PFS towards apple. Therefore the mutant allergen BM1,2,3,5 was designed by implanting distinct epitopes of Mal d 1.0108 at corresponding positions of the Bet v 1a sequence (FIG. 1A). The grafted regions were defined as stretches of 7 consecutive amino acids encompassing residues already described to be crucial for IgE recognition of Bet v 1 and homologues. These particular residues (Thr10, Phe30, Ser57 and Asp125 in Bet v 1a) could not only alter IgE binding to Bet v 1, also IgE recognition of Mal d 1 is highly dependent of amino acids at the corresponding positions. The introduction of distinct mutations at the described sites does not change the overall structure of the allergens as demonstrated by circular dichroismus (CD), though this can drastically influence allergenicity of the molecules. By investigating patients IgE binding towards BM1,2,3,5 the grafted epitopes serve as indicator for cross-reactivity of Bet v 1 and Mal d 1. Since IgE epitopes of Bet v 1 are conformational an intact structure of the hybrid was essential. The 3-dimensional fold of BM1,2,3,5 was first evaluated by calculating a molecular model which was then compared to the 3-D structure of Bet v 1. The model showed the same conserved shape as the template allergen. All 4 mutated epitopes were exposed on the protein surface and therefore could influence antibody binding to BM1,2,3,5 (FIG. 1B). The calculated mutant allergen was cloned, expressed in E. coli as 6×His tagged fusion protein and purified to homogeneity. To verify in silico data on protein structure far UV CD spectra of BM1,2,3,5 and Bet v 1a were recorded and compared after normalizing the signals to [θ]MRW units. The overlay of both spectra indicated almost identical secondary structures (FIG. 1C). Further evidence of similar folding was provided by antibody binding of a monoclonal anti Bet v 1 antibody which was equivalent for both proteins a further indication of a Bet v 1-like fold of BM1,2,3,5 (FIG. 2A). To investigate IgE antibody binding of BM1,2,3,5, ELISA experiments were done with 2 groups of patients: i) Bet v 1 allergic patients without PFS and ii) Bet v 1 allergic patients showing PFS symptoms following apple ingestion. As reference the allergens Bet v 1a and Mal d 1.0108 were used. No significant difference of patients' IgE binding towards Bet v 1 (P>0.99) was observed in any of the two groups. IgE binding to Mal d 1.0108 was stronger for the PFS group compared to the non-PFS patients, though the latter also recognized the major apple allergen in ELISA. However IgE binding to BM1,2,3,5 was significantly reduced in the non-PFS group compared to patients with PFS (P<0.01) (FIG. 2B). The ELISA data demonstrate that the grafted epitopes are implicated in birch pollen PFS. IgE antibodies of Bet v 1 allergic individuals without PFS could not efficiently bind to BM1,2,3,5 however cross-reactive antibodies of individuals with PFS could still recognize the mutated allergen.

Example 2

The mutant allergen BM4 is based on the protein backbone of Bet v 1a where an epitope of 8 amino acids has been replaced by an epitope of Mal d 1.0108. The incorporation of the Mal d 1 epitope results in a protein which cannot fold in a Bet v 1-like manner and remains unfolded; however the unfolded protein stays in solution and is stable.

BM4 was cloned with 5′ Nco I, 3′ Eco R I in the vector pHis Parallel 2 and produced as 6×his tagged fusion protein with an N-terminal his tag in E. coli BL21 (DE3). The protein was purified from the insoluble fraction of E. coli using a 6M Urea containing Ni²⁺ buffer, loaded on a Ni²⁺ column, refolded on column and eluted with imidazole. The purified his tagged protein was subsequently cleaved with rTEV protease, non-tagged BM4 was purified again by IMAC and dialyzed against 10 mM sodium phosphate buffer pH 8. The yield of a 1 L LB Amp culture was approximately 200 mg of BM4 after purification.

Protein purity was monitored by SDS-PAGE indicating a purity of over 99%, the correct mass of the intact protein was verified by ESI-Q TOF mass spectrometry (measured mass 17690; calculated average protein mass 17689 Da). Aggregation status of BM4 was determined by size exclusion chromatography. The content of higher molecular weight aggregates was below 0.5%, 99.5% of the protein was found to be monomeric. Protein secondary structure was determined by circular dichroism spectroscopy. The protein was found to be unfolded.

No IgE binding to BM4 was detected in immunoblots using a serum pool from Bet v 1 allergic patients and I¹²⁵-labeled rabbit anti-human IgE (MedPro) as detection antibody. In mediator release assays using rat basophilic leukaemia cells transfected with a human FcεRI receptor and sera of Bet v 1 allergic patients, a 100-1000 fold reduction of anaphylactic potential was observed for BM4 as compared to Bet v 1a.

T cell proliferation of BM4 was determined. Proliferative responses of human peripheral blood mononuclear cells (PBMCs) established from birch pollen-allergic patients were found to be higher for BM4 than for Bet v 1a or Mal d 1.0108.

Pre-clinical models are required to further characterize BM4. Mice were immunized with BM4 and immunologic parameters have been assessed: IgG as well as IgE titers towards Bet v 1a and BM4 were determined by ELISA. Further IgE binding was assessed by mediator release assays using rat basophilic leukaemia cells. The induction of blocking antibodies (IgG) against Bet v 1a was determined by indirect ELISA with end-point titrations of sera from immunized mice. T cell responses of mice were analyzed by ELISpot assays, T helper cells induced by immunization of BM4 were compared to those induced by immunization of mice with Bet v 1a.

An untagged BM4 construct was cloned as follows:

BM4 was inserted with 5′ Nco I and 3′ Eco R I in a pET 28b vector (Kan R) and transformed into E. coli BL21 Star™ (DE3) (Invitrogen) cells. The construct was sequenced and protein expression and purification tests were performed.

The products were produced as follows. Transformed cells were grown in a shaker flask in 1 L LB amp medium, protein expression was induced with 0.5 mM IPTG at OD600 of 0.8. The cells were harvested by low speed centrifugation, broken and the BM4 was recovered from the insoluble inclusion bodies with 6M UREA, 20 mM imidazole pH 7.4. The protein was refolded by dialysis against 20 mM sodium phosphate buffer. Secondary structure elements of BM4 were analysed by circular dichroism spectroscopy. BM4 produced as untagged recombinant protein in E. coli was found to be unfolded.

Example 3

In order to show that the mutation of amino acid residues 102, 114 and 120 of Bet v 1a results in a hypoallergenic molecule several Bet v 1a variants have been constructed. All of these molecules exhibit a reduced IgE reactivity compared to wild-type Bet v 1a.

TABLE 1

Z-Score of Bet v 1a and variants therof. The Z-score

can be used to determine the 3D structure of the protein

Description of mutant
Number

protein
of residues
Z-combined
Z-pair wise
Z-surface

wild type Bet v 1a
1bv1_I102I_L114L_Y120Y
159
−9.18
−6.45
−7.01

1bv1_I102K_L114D_Y120Q
159
−7.60
−5.74
−5.79

1bv1_I102K_L114K_Y120R
159
−7.60
−5.77
−5.76

1bv1_I102D_L114K_Y120K
159
−7.59
−5.87
−5.72

1bv1_I102E_L114D_Y120K
159
−7.59
−5.74
−5.80

1bv1_I102E_L114K_Y120D
159
−7.59
−5.92
−5.68

1bv1_I102K_L114E_Y120Q
159
−7.59
−5.81
−5.76

1bv1_I102K_L114N_Y120E
159
−7.59
−5.76
−5.77

1bv1_I102D_L114D_Y120E
159
−7.58
−5.66
−5.83

1bv1_I102E_L114E_Y120K
159
−7.58
−5.81
−5.77

1bv1_I102K_L114D_Y120D
159
−7.58
−5.82
−5.72

1bv1_I102D_L114E_Y120E
159
−7.57
−5.73
−5.79

1bv1_I102K_L114E_Y120D
159
−7.57
−5.90
−5.68

1bv1_I102K_L114K_Y120S
159
−7.57
−5.90
−5.65

1bv1_I102K_L114Q_Y120E
159
−7.57
−5.73
−5.78

1bv1_I102Q_L114K_Y120E
159
−7.57
−5.71
−5.78

1bv1_I102K_L114K_Y120N
159
−7.56
−5.87
−5.66

1bv1_I102K_I114K_Y120P
159
−7.55
−5.82
−5.64

1bv1_I102D_L114K_Y120E
159
−7.54
−5.79
−5.70

1bv1_I102E_L114D_Y120E
159
−7.54
−5.65
−5.79

1bv1_I102E_L114K_Y120K
159
−7.54
−5.86
−5.68

1bv1_I102K_L114K_Y120G
159
−7.54
−5.86
−5.65

1bv1_I102E_L114E_Y120E
159
−7.53
−5.72
−5.75

1bv1_I102K_L114D_Y120K
159
−7.53
−5.76
−5.72

1bv1_I102K_L114E_Y120K
159
−7.52
−5.83
−5.68

1bv1_I102K_L114K_Y120Q
159
−7.51
−5.77
−5.67

1bv1_I102E_L114K_Y120E
159
−7.49
−5.77
−5.67

1bv1_I102K_L114D_Y120E
159
−7.48
−5.67
−5.70

1bv1_I102K_L114K_Y120D
159
−7.48
−5.85
−5.60

1bv1_I102K_L114E_Y120E
159
−7.47
−5.74
−5.66

1bv1_I102K_L114K_Y120K
159
−7.44
−5.79
−5.59

1bv1_I102K_L114K_Y120E
159
−7.38
−5.70
−5.58

Sippl, M. J. et al. Stat.mechanics, protein struct & protein.substrate interactions; 0, pp. 297-315, (1994)

The description of mutant proteins indicates the pbd file of the template protein, which has been used to as scaffold for the mutations, in this context Bet v 1 (pdb entry 1bv1). Further the positions which have been mutated are listed (e.g I120K meaning I at position 102 has been replaced by K).

HYPOALLERGENIC MOLECULES

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