Abstract
IgE is a minute class of immunoglobulin that mediates immediate-type hypersensitivity reactions responsible for various allergic symptoms. IgE is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on the surface. The e chain of human mIgE contains a membrane-anchoring peptide and an extra 52 a.a.-long domain (referred to as CemX) between the membrane anchor and the CH4 domain. CemX is uniquely present in the me chain of human membrane-bound IgE (mIgE) and provides an attractive site for immunological targeting of mIgE-expressing B cells in order to down-regulate IgE level. This study was designed to evaluate the effects of monoclonal antibodies (mAbs) specific for CemX to target IgE-expressing B cells and decrease IgE production. A CemX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting mAbs specific for CemX were obtained. Characterization of the mAbs using ELISA, immunoprecipitation, and immunoblotting methods showed that they could bind to both native and denatured forms of CemX. The mAbs exhibited mutual inhibition of binding to mIgE. Epitope mapping using synthetic peptides revealed that all five mAbs recognize the same epitope, RADWPGPP, located near the C-terminus of CemX. Binding of one of the mAbs to mIgE on SKO-007 cells induced the cross-linking of mIgE molecules on the cell surface, resulting in their patching and capping. One of the anti-CemX mAbs, designated a20, was employed for functional analysis. In vitro analysis revealed that the mAb can cause complement-mediated cytotoxicity on mIgE.Fc-expressing transfectants. In vivo effects were explored with “KM” transgenic mice, which harbor human immunoglobulin miniloci. These mice were first investigated for the profiles of antigen-specific IgE and total IgE production and their suitability for studying the effects of immuno-targeting of IgE-expressing B cells. Human IgE was found to be present over a large range in the blood of KM mice. The mice could produce human ovalbumin-specific IgE when they were immunized with the ovalbumin. The me chain of KM mice contains CemX and mAb a20 could bind to mIgE-expressing B cells. The treatment with a20 inhibited the production of ovalbumin-specific human IgE. The results indicate that KM mice can be employed for studying the immunological targeting of mIgE-expressing B cells. We conclude that anti-CemX mAbs that are specific to human mIgE on B cells have been prepared. The potential of the mAbs for targeting mIgE+ B cells and hence down-regulating IgE was demonstrated by showing that the anti-CemX mAb can inhibit the production of antigen-specific IgE in KM transgenic mice.