Fabs targeting a Cε2 epitope disassemble IgE-receptor complexes and suppress anaphylaxis.

Abstract

IgE is a key molecule that plays a role in allergic disorders. Compared to other subtypes of immunoglobulins, IgE has a unique immunoglobulin-like domain, Cε2, which is implicated in stabilizing the complex of IgE and its high-affinity receptor, FcεRI. However, whether human IgE Cε2 domain can be targeted as a functional domain by antibody therapeutics remains elusive. We aimed to investigate competitive and dissociating effects of fragment antigen-binding (Fab) fragments targeting Cε2 on IgE-receptor interactions. Fab libraries against human IgE Cε2-4 were generated by rabbit immunization and subsequent phage display screening. The sublibrary against Cε2 portion was subjected to competition and removal screening on IgE-FcεRI interactions. The binding sites of 3 potent Fab clones were determined by mutational analysis. The competition and removal activities against human FcεRI and CD23 on cell surface, and consequences on anaphylactic reactions were assessed in vitro and in vivo. Anti-Cε2 Fab clones exhibited various disruptive activities on preformed IgE-FcεRI complexes, which were correlated with the competing activities. Selected potent Fab clones exhibited removal activities on IgEs of IgE-presensitized mast cells, leading to suppression of antigen-induced immediate reactions in vitro and in vivo. However, the competing and disruptive activities on IgE-CD23 complex varied among these clones. The core responsible epitopes were found in close proximity to the fifth β-sheet and helix regions in Cε2. These results highlight the crucial role of human Cε2 in stabilizing IgE-receptor complexes and its potential as a target for anti-IgE therapeutics.