title: Differential and synergistic effects of epidermal growth factor receptor antibodies on unliganded ErbB dimers and oligomers.
abstract: Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e., ligand binding, a change in conformation, or the monomer-dimer transition. In this work, we demonstrate that wild-type EGFR and the truncated de2-7-EGFR (tumor-associated mutant) formed unliganded homo-oligomers and examined the effects of two clinically relevant antibodies on the conformation and quaternary state of these ligand-free EGFR oligomers on the surface of cells. The EGFR antibodies were mAb528, a ligand-blocking antibody that binds domain III, and mAb806, a conformationally sensitive antibody that binds near the dimer interface in domain II. We used a model cellular system, BaF/3 cells, with GFP-tagged receptors in the absence of interference from secreted ligands or other erbB receptor members. Different antibody-mediated effects (conformational transition, receptor cross-linking, or receptor dissociation) were distinguished by combining two complementary biophysical techniques: image correlation spectroscopy (submicrometer scale clustering) and homo-Forster resonance energy transfer (association and/or conformation on a 1-10 nm scale). mAb528 cross-linked EGFR into an inactive EGFR dimer of dimers but had no effect when added to de2-7-EGFR oligomers. mAb806 had a minor effect on EGFR dimers as expected from its poor binding to a conformationally shielded epitope on wtEGFR but bound de2-7-EGFR oligomers, causing a conformational change in the intracellular C-terminal GFP-tagged tail. The combination of the two antibodies had synergistic effects, increasing the level of cross-linking of de2-7-EGFR, but did not lead to enhanced cross-linking of EGFR. The results reveal new modes of receptor-antibody interactions for EGFR and de2-7-EGFR.
authors.affiliation: Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Royal Melbourne Hospital, Victoria 3050, Australia.
authors.forename: Noga
authors.initials: N
authors.lastname: Kozer
authors.forename: Marcus P
authors.initials: MP
authors.lastname: Kelly
authors.forename: Suzanne
authors.initials: S
authors.lastname: Orchard
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scott
authors.forename: Andrew H A
authors.initials: AH
authors.lastname: Clayton
mesh_terms: D000818:Animals; D016880:Anisotropy; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D066246:ErbB Receptors / Q000737:chemistry / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008024:Ligands; D051379:Mice; D009154:Mutation; D018739:Oncogene Proteins v-erbB / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D008024:Ligands; D018739:Oncogene Proteins v-erbB; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors