This proposal tests the hypothesis that sustained, limited in vivo crosslinking of the high affinity IgE receptor, Fc?RI, on mast cells (MCs) and basophils (BAs) rapidly desensitizes these cells and safely and effectively prevents IgE-mediated diseases, such as food allergy and anaphylaxis. Rapid desensitization (RD) is traditionally a procedure in which MCs and BAs from people who have IgE-mediated allergy to a specific allergen are made temporarily unresponsive to that allergen by exposure to increasing allergen concentrations over several hours. We modified RD by inoculating mice with increasing concentrations of an anti Fc?RI ? chain mAb. These studies showed that: (1) RD with anti-Fc?RI? mAb is effective, safer and longer lasting than RD with allergen; (2) RD with anti-Fc?RI? mAb is antigen-nonspecific; (3) MC unresponsiveness can be safely sustained by maintaining serum levels of this mAb; (4) this process works with anti-human (hu) Fc?RI? mAbs in mice that express hu, rather than mouse Fc?RI? (huFc?RI? mice); and (5) RD depends both on induction of MC anergy and removal of MC Fc?RI and IgE. Recently, we have found that IgE-mediated anaphylaxis can be fully suppressed in huFc?RI? mice, without adverse effect, by repeatedly injecting <1 g of IE7, a mouse mAb that binds to huFc?RI? regardless of Fc?RI association with IgE. These mice can also be safely and dose- dependently desensitized by a single injection of a monovalent (mv) form of IE7 that has human IgG1-derived constant regions (mv huIgG1 IE7). mv huIgG1 IE7 also depletes blood BAs, and removes ~90% of MC IgE, but little Fc?RI?. The loss of MC IgE and particularly, its disproportionate loss compared to Fc?RI, was unexpected, because IE7 does not cause MCs to lose IgE in vitro and this mv Ab should only crosslink Fc?RI if the Ab becomes multivalent by aggregating or by binding to cellular Fc? or complement receptors. This proposal builds on these observations.
Aim 1 uses flow cytometry, structural studies, anti-Fc?R mAb, and hu IgG isotype switch variants of mv IE7 to determine whether mv huIgG1 IE7 crosslinks Fc?RI in vivo and, if so, through what mechanism(s).
Aim 1 also evaluates whether mv huIgG1 IE7 aggregation or internalization of IgE-loaded Fc?RI is required for the disproportionate in vivo loss of MC IgE.
Aim 2 applies Aim 1 results to use mv IE7 to safely desensitize actively sensitized, hyperallergic huFc?RI? mice that express a mutant IL-4 receptor as well as passively sensitized hu cord blood-reconstituted, hu cytokine-secreting, immunodeficient mice that develop large numbers of activated hu MCs and BAs; both models have been difficult to desensitize with increasing doses of divalent IE7.
Aim 3 applies aim 2 results to desensitize rhesus monkeys, which express Fc?RI? that reacts with IE7. Together, these aims should optimize the use of mv anti- Fc?RI? mAb for RD and suggest whether and how it could be used to suppress hu IgE-mediated disease.
Mast cell/Fc?RI/IgE-mediated allergy is common, causes considerable morbidity, and can be life-threatening. Allergen rapid desensitization only suppresses allergy to targeted allergens, has adverse effects and typically requires daily allergen exposure. We have developed the reagents and protocols for rapid desensitization with a monovalent anti-Fc?RI? antibody and have shown that this procedure is effective, safe, simple, and quick in mouse models. Determining how it works and testing its safety and efficacy in highly allergic humanized mice and rhesus monkeys will allow optimization of this approach and set the stage for clinical trials.
