Abstract
BACKGROUND: Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with food extracts, may be effective but dangerous due to anaphylactic side-effects. The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting persistent and severe allergy to fish (cod) and fruit (peach). Both are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. FAST will apply hypo-allergenic recombinant major allergens for SIT. METHODS: Two approaches were evaluated for achieving hypo-allergenicity, i.e. site-directed mutagenesis and chemical modification. Wildtype (wt) natural and recombinant allergens and the hypo-allergens were extensively purified and characterized physico-chemically. Their stability was tested and allergenicity was compared by CAP-inhibition and histamine release experiments while immunogenicity was tested in T-cell proliferation experiments and rabbit and mice immunizations. RESULTS: For Cyp c 1, the mutant without calcium-binding site showed up to a 1000 times reduced allergenicity, while secondary fold and immunogenicity (tested in human PBMC stimulations and by immunization of laboratory animals) were retained. Chemically modified Cyp c 1 demonstrated a reduced capacity to stimulate T-cells and showed less immunogenicity in rabbits. The calcium-binding mutant has been produced under GMP conditions. For Pru p 3, 5 potential hypoallergens were compared. The allergenicity was reduced to a similar extent (∼1000-fold) for both variants in which disulfide bridges were disrupted, i.e. either by mutagenesis or by reduction/alkylation. The modification resulted in loss of alpha-helical secondary structure. However, unexpectedly, the immunogenicity was also significantly lowered/absent. CONCLUSIONS: For the Cyp c 1 calcium-binding mutant we are preparing to enter Phase I clinical trials. For Pru p 3, we need to evaluate new molecules to generate a hypoallergenic mutant that retains immunogenicity.