Structural Analysis of Human IgE Monoclonal Antibody Epitopes on Dust Mite Allergen Der p 2

Abstract

BackgroundHuman IgE monoclonal antibodies (hIgE mAb) against major mite allergen Der p 2 developed using human hybridoma technology, were used for IgE epitope mapping and analysis of epitopes associated with the human IgE repertoire. ObjectiveTo elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. MethodsX-ray crystallography was utilized to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human FcεRIα–transgenic mouse model of passive systemic anaphylaxis. ResultsThe structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by three overlapping hIgE mAb (1B8, 5D10, and 2G1). Compared to wildtype Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα–transgenic mice sensitized by hIgE mAb, which were susceptible to anaphylaxis when challenged with wildtype Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants. ConclusionThese data establish the structural basis of allergenicity of two hIgE mAb non-overlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.