Abstract 4511: Differential effects of GA201 and cetuximab on EGFR expression and endosomal recycling in non-small cell lung cancer cell lines
Authors: Kol Arjan , Jong Steven de , Pool Martin , Vries Elisabeth G.E. de , Gerdes Christian A. , Scheltinga Anton G.T. Terwisscha van 
Published: May 2015

Abstract
GA201 is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) that is optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and inhibition of EGFR signal transduction. Recently, combinations of EGFR mAbs were shown to be more potent in inducing EGFR downregulation and superior in controlling growth in several tumor types, including breast and lung cancer. Currently, it is unknown how internalization and membrane recycling of EGFR after treatment with GA201, and in combination with the anti-EGFR mAb cetuximab, will affect GA201-induced ADCC response and tumor growth inhibition. In the present study we have investigated the effects of the GA201 and cetuximab on EGFR expression and endosomal trafficking in non-small cell lung cancer cell lines (NSCLC).
Effects of GA201, cetuximab and the combination of these mAbs on EGFR were studied in a panel of NSCLC cell lines (A549, H441, H322 and H292). The influence of mAb binding on EGFR membrane expression, internalization and endocytic trafficking was determined using flow cytometry and immunofluorescence. For the internalization experiments, cells were pre-incubated with the mAbs for one hour at 4 °C. The effects on total EGFR protein levels and downstream signaling were studied using Western blotting.
Treatment of cells with GA201 or cetuximab at 37 °C triggered EGFR internalization as demonstrated with flow cytometry. During treatment with either antibody alone for 1, 2 and 4 hours, EGFR reappeared at the cell surface. Immunofluorescence demonstrated the presence of GA201 and cetuximab in early endosomes and recycling endosomes, which can explain the EGFR reappearance on the cell surface. Interestingly, after 24 and 72 hours incubation with GA201 membranous EGFR levels were diminished to 50%, whereas cetuximab had no effect on membranous EGFR expression levels. Treating cells with the combination of the two mAbs resulted in EGFR internalization, downregulation of membranous EGFR levels with 80% and a 2-fold reduction in cellular EGFR protein levels as shown by Western blotting. When combined, most of the internalized GA201 and cetuximab colocalized with the lysosomes, and were almost absent in early endosomes and recycling endosomes. Moreover, the combination efficiently inhibited EGF-induced phosphorylation of downstream signaling molecules.
In conclusion, GA201 downregulated membranous EGFR levels, whereas cetuximab had no effect. GA201 in combination with cetuximab leads to stronger downregulation of membranous and cellular EGFR levels by inhibiting endosomal recycling and increasing EGFR degradation in the endosmal/lysosomal compartment compared to both antibodies separately. We are currently investigating the effects of the single agents and the combination on EGFR signaling, ADCC response and tumor growth.
Funded by Hoffmann-La Roche AG and ERC grant OnQview
Citation Format: Arjan Kol, Steven de Jong, Martin Pool, Elisabeth G.E. de Vries, Christian A. Gerdes, Anton G.T. Terwisscha van Scheltinga. Differential effects of GA201 and cetuximab on EGFR expression and endosomal recycling in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2014-4511