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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 Domain¹

Sabine Flicker^*, Susanne Vrtala^*, Peter Steinberger^*, Luca Vangelista, Albrecht Bufe^§, Arnd Petersen^§, Minoo Ghannadan, Wolfgang R. Sperr, Peter Valent, Lars Norderhaug^¶, Barbara Bohle^*, Hannes Stockinger^||, Cenk Suphioglu^#, Eng Kok Ong^#, Dietrich Kraft^* and Rudolf Valenta^2,*

^* Department of Pathophysiology, Division of Hematology, Department of Internal Medicine I, University of Vienna, Austria; Structural Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany; ^§ Research Center Borstel, Germany; ^¶ Department of Molecular Cell Biology, Institute of Biology, University of Oslo, Norway; ^|| Institute of Immunology, University of Vienna, Vienna, Austria; and ^# Department of Allergy and Clinical Immunology, Monash Medical School, Prahran, Australia

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 combinatorial library prepared from lymphocytes of a grass pollen-allergic patient 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 Phl 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 conformation consisting predominantly of an 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 allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

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