Structure of intact IgE and the mechanism of ligelizumab revealed by electron microscopy

Abstract

Background: IgE is the central antibody isotype in TH2-biased immunity and allergic diseases. The structure of intact IgE and the impact of IgE-targeting molecules on IgE however remain elusive. In order to obtain insights into IgE biology and the clinical impact, we aimed for structure determination of IgE and the complex of IgE with the anti-IgE antibody ligelizumab. Methods: Structures of two distinct intact IgE with specificity for cross-reactive carbohydrate determinants and Der p 2 as well as complexes of ligelizumab-Fab with IgE and IgE Fc were assessed by negative stain electron microscopy and solution scattering. Inhibition of IgE binding and displacement of receptor-bound IgE were assessed using cellular assays, basophil activation testing and ELIFAB assays. Results: Our data reveal that the investigated IgE molecules share an overall rigid conformation. In contrast to the IgE Fc fragment, the IgE Fc in intact IgE is significantly less asymmetrically bent. The proximal and the distal Fabs are rigidly tethered to the Fc. Binding of ligelizumab to IgE in a 2:1 stoichiometry induces an extended and twofold symmetrical conformation of IgE, which retains a rigid Fab-Fc architecture. Analyses of effector cell activation revealed that ligelizumab inhibits IgE binding without displacing receptor-bound IgE. Together with an interference of CD23 binding, the data underline a functional activity similar to omalizumab. Conclusions: Our data reveal the first structures of intact IgE suggesting that the IgE Fab is fixed relative to the Fc. Furthermore, we provide a structural rationale for the inhibitory mechanism of ligelizumab.

Capturing transient antibody conformations with DNA origami epitopes

“…Cryogenic electron microscopy (cryo-EM) serves as a tool for imaging individual Abs at atomic resolution, offering unprecedented capability to determine complex structures. But important information on conformational flexibility and intermediate Ab-Ag complexes are likely lost due to the lack of temporal resolution ability of cryo-EM 13,14 .…”

Tuning IgE: IgE-Associating Molecules and Their Effects on IgE-Dependent Mast Cell Reactions

“…In IgE, things are more complicated. The hinge region is shorter than that of IgG and seems to have less flexibility [ 64 ]. Instead, the Fc portion of IgE can be acutely and asymmetrically bent ( Figure 4 A,B) [ 65 , 66 , 67 ], and an appropriate bend is required for the binding to FcεRI [ 64 , 68 , 69 ].…”

“…The hinge region is shorter than that of IgG and seems to have less flexibility [ 64 ]. Instead, the Fc portion of IgE can be acutely and asymmetrically bent ( Figure 4 A,B) [ 65 , 66 , 67 ], and an appropriate bend is required for the binding to FcεRI [ 64 , 68 , 69 ]. Interestingly, an antibody Fab fragment called aεFab can straighten this bend by binding from two sides at high concentrations and block IgE’s binding to FcεRI allosterically [ 67 ].…”

“…It was shown that the bent form of free IgE-Fc is the most stable in terms of the free energy level [ 67 ]. However, another recent negative stain electron microscopy study suggested a major free IgE conformation with less bent Cε2 and asymmetrically oriented two Fabs [ 64 ]. Since this conformation is incompatible with FcεRI binding, transient bending may be required for FcεRI engagement.…”

“…The contact mapping suggests that ligelizumab sterically blocks FcεRI engagement [ 99 ]. In addition, a negative electron microscopy revealed that the IgE in solution is bound by ligelizumab from two sides, constraining it in an extended conformation [ 64 ]. In contrast to omalizumab, ligelizumab did not show any dissociation activity on FcεRI-bound IgE [ 99 ].…”