Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with FcɛRIα

Identification of the domain(s) of canine IgE that interact with FcɛRIα may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage FcɛRIα. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine FcɛRIα. β-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine FcɛRIα for antigen induced mediator release. Replacing canine Cɛ2 with human Cɛ2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine FcɛRIα or human FcɛRIα. Substitution of canine Cɛ4 with human Cɛ4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine Cɛ2 and Cɛ4 were substituted with human Cɛ2 and Cɛ4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine Cɛ3 is the only domain essential for binding to canine or human FcɛRIα, species specific residues in canine Cɛ2 and Cɛ4 inhibit dissociation of the ligand from the receptor.