RATIONALE: Recombinant allergens can be safer alternatives to extract-based immunotherapies. Novozymes has recombinantly expressed the cysteine-protease Der p 1 from the house dust mite (HDM) D. pteronyssinus, and hypoallergenic variants thereof. METHODS: pro-Der p 1 was expressed in S. cerevisiae by fusion to a fungal signal-peptide. Mutation of the N-glycosylation site, resulted in production of un-glycosylated pro-Der p 1, which was purified by ultra-filtration followed by two chromatographic steps. The pro-peptide was removed and mature recombinant (r-) Der p 1 was obtained. r-Der p 1 was compared to natural (n-) Der p 1, from mites, in biochemical and immunological assays, using blood samples from 23 HDM allergic donors. Putative B-cell epitopes were predicted by in silico epitope mapping and in vitro by phage display. Predicted epitopes were changed by protein engineering and hypo-allergenic variants were expressed, purified and screened. RESULTS: r-Der p1 has cysteine-protease activity, correct N-terminal sequence and displays the same CD-specter as n-Der p 1. In addition, r-Der p 1 had similar IgE binding, T cell activation and histamine release profile as n-Der p 1. Several hypoallergenic variants were identified, with reduced ability to bind patient IgE, and release histamine (up to 6-fold) from blood basophils. The hypoallergenic variants maintained the ability to activate T cells. CONCLUSIONS: r-Der p 1 expressed in S. cerevisiae is biochemically and immunologically equivalent to n-Der p 1, and may provide a safer alternative to HDM-extract based immunotherapy. Additionally, several hypoallergenic Der p 1 variants were identified, with the potential for producing even safer therapies.

The cysteine protease Der p1 from dust mite of the genus Dermatophagoides pteronyssinus is a major type I allergen. About 80% of house dust mite (HDM) allergic individuals are reactive to this protease in standard assays for detection of IgE. A curative treatment for atopic allergy is immunotherapy (IT) with HDM extracts which are complex mixtures occasionally resulting in anaphylactic reactions. Novozymes focuses on developing a recombinant variant of Der p1 which exhibit lowered risk of IgE‐mediated allergic reactions, while maintaining its ability to trigger proper Th‐cell responses. This may provide a safer alternative for specific IT of HDM allergy. A secreted recombinant form of pro‐Der p 1 expressed by Saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. The N‐glycosylation site of Der p1 was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of 35 kDa. Protein purification procedure was developed to obtain nearly pure Der p1 protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor E64. The deglycosylated recombinant pro‐Der p 1 revealed immunologic similarity to the native Der p 1 molecule when compared in basophile histamine release, IgE‐binding assays and T‐cell proliferation assays. By in silico epitope mapping of a modelled 3‐dimensional structure of Der p1, five putative IgG and IgE epitopes were predicted. By protein engineering, the predicted epitopes were removed one by one in Der p1 and screening for hypoallergenic variants was performed.