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. 2011 Apr 1;286(13):11337-45.
doi: 10.1074/jbc.M111.223503. Epub 2011 Jan 31.

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

Nadège Gaborit  1 , Christel Larbouret, Julie Vallaghe, Frédéric Peyrusson, Caroline Bascoul-Mollevi, Evelyne Crapez, David Azria, Thierry Chardès, Marie-Alix Poul, Gérard Mathis, Hervé Bazin, André Pèlegrin
Affiliations

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

Nadège Gaborit et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of the NIH/3T3-HERs cell lines. A, flow cytometry analysis of cell surface EGFR (black) and HER2 (gray) expression using the anti-EGFR antibody m225 and the anti-HER2 antibody FSP77 coupled with a FITC-labeled secondary antibody in parental NIH/3T3 (wt), -R1 (EGFR overexpression), -R2 (HER2 overexpression) and -R1R2 (EGFR and HER2) cells. Controls (gray area) were cells incubated only with the FITC-labeled secondary antibody. B–D, EGF binding in A431 cells (positive control) (B), NIH/3T3-R1 (C), and NIH/3T3-R1R2 (D) cells. Cells were incubated with 4 nm EGF labeled with Lumi4 Tb cryptate and increasing concentrations of unlabeled EGF to compete for binding. Unlabeled EGF induced a dose-dependent inhibition of fluorescence emitted at 620 nm. Data are expressed as arbitrary units of fluorescence at 620 nm (AUF620) and represent the mean ± S.E. of triplicates. E and F, Western blot analysis in parental NIH/3T3 (wt), NIH/3T3-R1, NIH/3T3-R2 and NIH/3T3-R1R2 cells. After stimulation with 100 ng/ml EGF, cells were lysed and protein extracts used to determine the expression and the phosphorylation level of EGFR and HER2 with antibodies against total EGFR (E) or HER2 (F) and against phosphorylated EGFR (E) or HER2 (F). G, presence of EGFR/HER2 dimers in NIH/3T3-R1R2 and SKOV-3 cells. Whole cell lysates from EGF-stimulated cells were immunoprecipitated (IP) using the FSP77 anti-HER2 monoclonal antibody and probed with anti-EGFR and -HER2 polyclonal antibodies to detect HER2 and the amount of co-precipitated EGFR (as a dimerization partner).
FIGURE 2.
FIGURE 2.
Detection of HER homo- and heterodimers in NIH/3T3-R1R2 and SKOV-3 cells. A, schematic of the antibody-based TR-FRET assays to detect EGFR/EGFR or HER2/HER2 homodimers and EGFR/HER2 heterodimers using m425 (anti-EGFR) and/or FRP5 (anti-HER2) mAbs conjugated with terbium cryptate (Lumi4 Tb) or the d2 dye. B, detection of HER dimers in parental NIH/3T3 (wt: no detectable EGFR or HER2 expression), NIH/3T3-R1 (only EGFR), -R2 (only HER2) and -R1R2 and SKOV-3 cells (both EGFR and HER2) using the antibody-based TR-FRET assay. Cells were incubated with labeled antibodies overnight and the fluorescence signal was directly measured, without washes. Data are representative of three independent experiments.
FIGURE 3.
FIGURE 3.
Quantification of EGFR/HER2 dimers in different cancer cell lines using the antibody-based TR-FRET assay. A, 3.2 × 105 cells were incubated at 37 °C with d2-labeled m425 (anti-EGFR) Lumi4 Tb-labeled FRP5 (anti-HER2) antibodies overnight. Cells were then stained with 10 μg/ml of Hoechst 33342 and washed three times with KREBS buffer before detection of the fluorescence signals (see “Experimental Procedures” for a complete description of the protocol used). The ΔF665 value represents the EGFR/HER2 dimer concentration normalized to the DNA quantity (Hoechst fluorescence). Data are the mean ± S.E. of three independent experiments performed in triplicate. B, correlation curve between EGFR/HER2 dimer quantity and EGFR expression (determined previously using the quantitative immunofluorescence indirect assay, see Table 1) in cancer cell lines with higher HER2 than EGFR expression level. C, correlation curve between EGFR/HER2 dimer quantity and HER2 expression in cancer cell lines with higher EGFR than HER2 expression level.
FIGURE 4.
FIGURE 4.
Effect of therapeutic anti-EGFR or -HER2 mAbs and TKIs on EGFR/HER2 heterodimer concentration in SKOV-3 cells. 105 cells/well were treated with increasing concentrations (from 0.1 to 100 μg/ml) of Px (irrelevant antibody), Cetuximab, Trastuzumab, Cetuximab + Trastuzumab (ratio 1:1), and Pertuzumab (A and B); or with increasing concentrations (from 0.01 to 10 μm) of the TKIs Lapatinib and Erlotinib (C). Cells were then fixed with formalin for 2 min, and EGFR/HER2 heterodimers were quantified using the antibody-based TR-FRET assay with anti-EGFR (d2-labeled m425) and anti-HER2 (Lumi4 Tb-labeled FRP5) antibodies. After extensive washes, fluorescence was measured and the TR-FRET signal was expressed as ΔF665 (%) = ΔF665/F665Terbium (see “Experimental Procedures”). ΔF665 (%) represents the concentration of EGFR/HER2 dimers normalized to HER2 expression. Data are the mean ± S.E. of three independent experiments performed in triplicate. p, ***<0.001; **<0.01, n.s. non-significant, by one- and two-way analysis of variance (B and A, respectively), with Bonferroni's multiple comparison post-tests. D, Western blot analysis of SKOV-3 cells treated with increasing concentrations of TKIs without prior induction by EGF. Antibodies against total EGFR and HER2, phosphorylated EGFR and HER2, AKT and phosphorylated AKT and GAPDH were used to detect variations in EGFR and HER2 expression/phosphorylation and AKT phosphorylation.
FIGURE 5.
FIGURE 5.
Effect of therapeutic anti-EGFR and HER2 mAbs and of the TKI Lapatinib on the median survival and tumor progression in mice xenografted with SKOV-3 cells. A, median survival and tumor-free percentage of mice xenografted with 5 × 106 SKOV-3 cells and treated, when the tumor reached a minimum volume of 50 mm3, with Pertuzumab (2 or 10 mg/kg), Trastuzumab (2 or 10 mg/kg), Cetuximab + Trastuzumab (2 or 10 mg/kg of each mAb) or Lapatinib (100 or 300 mg/kg) for 4 weeks. Tumor dimensions were measured twice weekly. B, tumor progression curves in the mice treated with the highest doses of mAbs (10 mg/kg) and Lapatinib (300 mg/kg). Tumor dimensions were measured twice weekly.
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