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.
Highlights
The human ErbB family of receptor tyrosine kinase comprises four members: epidermal growth factor receptor (EGFR),2 HER2
Characterization of the NIH/3T3-HERs Cell Lines—First, the ectopic expression of human EGFR (NIH/3T3-R1 cells) and HER2 (NIH/3T3-R2 cells) or both (NIH/3T3-R1R2 cells) in these cell lines was confirmed by fluorescence-activated cell sorting (FACS) using saturating concentrations of the monoclonal antibodies (mAbs) m225 and FSP77
The dose-dependent competition curve obtained by addition of increasing concentration of unlabeled EGF in the three cell lines suggests a specific recognition of EGFR by EGF in these cells
Summary
Antibodies and Reagents—The anti-EGFR mAbs Cetuximab and m425 were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively. Cells were plated at 5 ϫ 104 per well in 96-well sterile, black microplates (Greiner Bio-One, Frickenhausen, Germany) in DMEM medium (without phenol red) supplemented with 10% FCS and after 24 h they were washed with KREBS buffer (146 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 1 g/liter glucose, and 1 g/liter bovine serum albumin, Sigma-Aldrich) and incubated with 4 nM EGF labeled with Lumi4 Tb cryptate (Cisbio Bioassays, Bagnol sur Ceze, France) and increasing concentrations of unlabeled EGF (Sigma-Aldrich) in KREBS with 0.02% azide (Sigma-Aldrich) at room temperature in the dark for 90 min. Time-resolved Fluorescence Resonance Energy Transfer (TRFRET) Assay—This assay was performed using the anti-EGFR mAb m425 and the anti-HER2 mAb FRP5 labeled with Lumi Tb cryptate (donor) and d2 dye (acceptor) (Cisbio Bioassays) These mAbs were chosen because their targeting epitopes are different from those of the therapeutic mAbs used in this study (see supplemental Table S1). College Station, TX: StataCorp LP.) (xenograft experiments) and Prism GraphPad (TR-FRET experiments)
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