Project Summary IgE antibodies and receptors are important mediators of the allergic cascade, but may also provide distinct functions in anti-tumor immunotherapy. IgE engages its high affinity IgE Fc receptor (FceRI) on mast cells and basophils as well as a low affinity receptor (FceRII/CD23) expressed on B cells and epithelial cells. IgE binding to FceRI sensitizes allergic effector cells to allergens and is very stable, with the complexes persisting for months even in the presence of potent anti-IgE antibodies. We have developed approaches to rapidly destabilizing IgE:FceRI complexes and desensitizing allergic effector cells using hybrid, bispecific IgG/DARPin molecules that recognize two distinct epitopes on the IgE-Fc. Here in Aim 1 we will explore alternative approaches to target and remove IgE:FceRI complexes from cell surfaces using novel bispecific anti-IgE antibodies. In addition, we will use two anti-IgE antibodies that we have produced to gain greater insights into the structure and dynamics of intact IgE. The IgE interaction with CD23 also mediates important biological effects in B cells, together with CD21. Here, in Aim 2, we will use yeast display to evolve higher affinity variants of CD23 for its ligands to enable structural studies of these complexes and to probe CD23-dependent regulation of IgE production by B cells. Finally, in Aim 3, we seek to isolate and study new anti-FceRIa antibodies using yeast display, to develop novel reagents for the FceRI-directed modulation of allergic effector cell activities.