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

  • Gaborit N
  • Larbouret C
  • Vallaghe J
 et al. 
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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

Author-supplied keywords

  • $$Medline
  • Animals
  • Antibodies
  • Antineoplastic Agents/pd [Pharmacology]
  • Antitumor/mt [Methods]
  • Carcinoma
  • Cell Line
  • Cell Proliferation
  • Cell Proliferation/de [Drug Effects]
  • Cellular
  • Combination
  • Cytotoxicity
  • Drug Screening Assays
  • Fibroblast Growth Factor/ai [Antagonists & Inhibit
  • Fibroblast Growth Factor/ge [Genetics]
  • Fibroblast Growth Factor/me [Metabolism]
  • Fluorescence
  • Fluorescence Resonance Energy Transfer
  • France
  • Humans
  • Induced
  • Light
  • Mice
  • Monoclonal
  • Murine-Derived/pd [Pharmacology]
  • NIH 3T3 Cells
  • Neoplasm/pd [Pharmacology]
  • Neoplasms/dt [Drug Therapy]
  • Neoplasms/ge [Genetics]
  • Neoplasms/me [Metabolism]
  • Phosphorylation
  • Phosphorylation/de [Drug Effects]
  • Protein Kinase Inhibitors/pd [Pharmacology]
  • Protein Multimerization/de [Drug Effects]
  • Quinazolines/pd [Pharmacology]
  • Receptor
  • Receptors
  • Research
  • Tumor
  • erbB-2/ai [Antagonists & Inhibitors]
  • erbB-2/ge [Genetics]
  • erbB-2/me [Metabolism]

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  • N Gaborit

  • C Larbouret

  • J Vallaghe

  • F Peyrusson

  • C Bascoul-Mollevi

  • E Crapez

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