Inhibition of IgE-Dependent Histamine Release from Human Peripheral Blood Basophils by Humanized Fab Fragments That Recognize the Membrane Proximal Domain of the Human FcεRI α-Chain

Abstract

Background: Inhibition of the interaction between IgE and the α-chain of FcΕRI (FcΕRIα) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases. Objective: The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human FcΕRIα monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of FcΕRIα. Methods: The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind FcΕRIα and inhibit IgE binding to FcΕRIα were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured. Results: The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to FcΕRIα as the original mouse CRA2. Although the FcΕRI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the FcΕRI. Conclusion: In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of FcΕRIα expressing cells are involved.