Structural analysis of epitopes recognized by two anti-CϵmX mAbs reveals intrinsic disorder of the CϵmX domain (P6002)

Abstract In human membrane-bound IgE (mIgE), a CϵmX domain containing 52 aa residues is located between the CH4 domain and the C-terminal membrane-anchoring segment. Antibodies specific for CϵmX, which can cause lysis of mIgE+ B cells, are being developed to treat allergic diseases and an anti-CϵmX mAb h47H4 is in phase IIb clinical trials. Hence, structural characterization of CϵmX free and complexed with anti-CϵmX mAb is of wide interest and importance. We have solved the crystal structures of (i) free CϵmX fused to IgG1-Fc and (ii) part of CϵmX complexed with an anti-CϵmX mAb, h4B12-Fab, at 1.94 Å resolution. The crystal structure of the IgG1-Fc•CϵmX fusion protein of homodimer exhibits a lack of well-defined electron density for residues 10 to 52 of A subunit and in absence of B subunit in dimeric CϵmX domain. The first 33 residues are also predicted to be intrinsically disordered. The 3D complex structure and epitope mapping by ELISA showed that the anti-CϵmX mAb, h4B12, binds to GLAGGSAQSQRAPDR, which has been reported to bind another anti-CϵmX mAb, h47H4, except for the first 6 residues (in italics). Comparison of the 3-D complex structure of h4B12 and h47H4 revealed different conformations of AQSQRAPDR, a feature characteristic of the intrinsically disordered regions in the CϵmX domain. This is the first report showing that the drug target, CϵmX, contains an intrinsically disordered region that can switch to different conformations upon binding to different anti-CϵmX mAbs.