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 Fce 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 FceRI
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.