A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: Molecular, immunological, and structural characterization of the epitope-containing domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a co mbinatorial library prepared from lymphocytes of a grass pollen-allergic pa tient and studied its interaction with group 5 allergens. The IgE Fab cross -reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11,2-kDa N-terminal fragment of the major timothy grass pollen allergen Ph l p 5A, The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity, Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conf ormation consisting predominantly of an cu helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allerg enic activity in basophil histamine release as well as skin test experiment s. Thus, the rPhl p 5A domain represents an important allergen domain conta ining several IgE epitopes in a configuration optimal for efficient effecto r cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for hi ghly efficient effector cell activation and, perhaps later, for the develop ment of generally applicable allergen-specific therapy strategies.