title: Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha.
abstract: Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. 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 Fc epsilon RI alpha. beta-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 Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 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 C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. 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 C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hunter
authors.forename: Athanassios P
authors.initials: AP
authors.lastname: Vratimos
authors.forename: Jonathan E M
authors.initials: JE
authors.lastname: Housden
authors.forename: Birgit A
authors.initials: BA
authors.lastname: Helm
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D005434:Flow Cytometry; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000276:immunology*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E; D001619:beta-N-Acetylhexosaminidases