title: Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies.
abstract: In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U896, Université Montpellier1, CRLC Val d'Aurelle Paul Lamarque, Montpellier, France.
authors.forename: Nadège
authors.initials: N
authors.lastname: Gaborit
authors.forename: Christel
authors.initials: C
authors.lastname: Larbouret
authors.forename: Julie
authors.initials: J
authors.lastname: Vallaghe
authors.forename: Frédéric
authors.initials: F
authors.lastname: Peyrusson
authors.forename: Caroline
authors.initials: C
authors.lastname: Bascoul-Mollevi
authors.forename: Evelyne
authors.initials: E
authors.lastname: Crapez
authors.forename: David
authors.initials: D
authors.lastname: Azria
authors.forename: Thierry
authors.initials: T
authors.lastname: Chardès
authors.forename: Marie-Alix
authors.initials: MA
authors.lastname: Poul
authors.forename: Gérard
authors.initials: G
authors.lastname: Mathis
authors.forename: Hervé
authors.initials: H
authors.lastname: Bazin
authors.forename: André
authors.initials: A
authors.lastname: Pèlegrin
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000494:pharmacology*; D000912:Antibodies, Neoplasm / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D000069347:Erlotinib Hydrochloride; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D000077341:Lapatinib; D051379:Mice; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects*; D011799:Quinazolines / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000912:Antibodies, Neoplasm; D000970:Antineoplastic Agents; D000069347:Erlotinib Hydrochloride; D000077341:Lapatinib; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D017468:Receptors, Fibroblast Growth Factor; C508053:ERBB2 protein, human