PROJECT SUMMARY At the center of the pathogenesis of allergic diseases is the IgE molecule. In sensitized individuals, re- exposure to the offending allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. To date, studies of the human IgE molecule, and its targeted epitopes on allergens, have been very limited. Most of our knowledge of this process has come from studies using allergic patient serum, which contains a mixture of many antibodies, with many specificities, directed toward many different epitopes, and having many different affinities; thus the studies of the molecular interactions of IgE with target allergens are greatly flawed. The ideal way to study this process is to use naturally-occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many impassible intrinsic technical hurdles no such antibodies have previously ever been made. We have now established a method to grow, identify and immortalize IgE encoding B cells by making human hybridomas from the peripheral blood of allergic individuals. In this proposal, we develop the first comprehensive panels of naturally-occurring peanut allergen-specific human IgE mAbs to define the molecular interactions of the most potent inducers of anaphylaxis. We have already begun comprehensive mapping studies to identify key immunodominant antigenic sites on the major peanut allergen proteins Ara h 1, 2, 3, and 6. In Aim 1, functional antigenic site mapping via peanut induced anaphylaxis will be accomplished by passive sensitization of human FcεRI transgenic mice using IgE mAbs. In Aim 2, prototype mAbs that bind unique antigenic sites will be selected and expressed as recombinant IgG mAbs to use as tools for advanced mapping studies using human serum. IgG mAbs, which bind identically as the IgE mAbs from which they were made, will be employed in blocking studies using a panel of peanut allergic research subjects’ frozen serum and ImmunoCAP diagnostics. This will define the role that each antigenic site-specific population of IgE antibodies play within and between peanut allergic individuals. This information will be used to draw clinical correlates of disease and to select research subjects which possess IgE antibodies not blocked by our panels, allowing for the generation of new site-specific IgE mAbs in Aim 1. Finally, in Aim 3, we will obtain atomic resolution structures to precisely define the first ever naturally-occurring human IgE epitopes on Ara h 2 and 6 by X- ray crystallography. The goal of this work is to create a definitive, complete and comprehensive molecular map of the human IgE antibody response to the major allergen proteins of peanut. The results will serve as a much-needed road map to allow for the design of new immunotherapies and allergy vaccines.

PROJECT NARRATIVE In developed countries allergic diseases effect up to 1/5th of the population and in developing countries 2 billion people worldwide are infected with parasitic worms; these two diseases seem very much unrelated, however, both involve the human IgE antibody response. As industrialized countries eliminate parasitic worms the incidence of allergic diseases, particularly those to foods, has steadily risen – resulting in an “allergy epidemic”. Studying human IgE at the molecular level as monoclonal antibodies will allow for the most basic and critical interactions to be assessed between the human IgE antibody and the allergen proteins of peanut and will provide new insights into the often pathological but potentially protective aspect of the human adaptive immune system.