Expression of Humanized Fab Fragments That Recognize the IgE-Binding Domain of Human FcεRIα in COS and CHO Cells

Abstract

Interfering with the binding of IgE to high-affinity IgE receptor α chain (FcεRIα) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human FcεRIα inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human FcεRIα Fabs without papain -digestion of the whole antibody. Four Fabs with different C-termini of CHI were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-FcεRIα binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human FcεRIα will provide useful information for the design of therapeutic reagents for allergy and asthma.