Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: Antibody internalization and EGFR degradation.

e13022 Background: Targeting the epidermal growth factor receptor (EGFR) with monoclonal antibodies has proven to be an effective therapy for the treatment of cancer. In addition to blocking activation, antibodies directed to the ligand-binding domain also induce EGFR degradation. A number of EGFR antibodies are approved for use in patients and/or in clinical development, including cetuximab (chimeric IgG1), necitumumab (human IgG1), and panitumumab (human IgG2). It is important to understand the similarities and differences between these antibodies to guide future drug development strategies. Methods: We used confocal live cell microscopy to measure EGFR antibody internalization and EGFR degradation. To obtain kinetic data on antibody induced receptor degradation, a HELA reporter cell line expressing EGFR fused to the green fluorescent protein (GFP) was utilized to quantify the decrease in sum pixel intensity of EGFR-GFP over a 24-hour time course in live cells. EGFR antibody trafficking to the lysosome was also monitored with a pH-activated dye (pHrodo) conjugated to an anti-human IgG FAB antibody. The fluorescence intensity signal from pHrodo increases upon decrease in pH, allowing the use of this dye as a surrogate for delivery of EGFR to a low-pH degradative compartment. We used these novel assays to directly compare cetuximab, necitumumab, and panitumumab. Results: Necitumumab and cetuximab were internalized at a faster rate, and resulted in more rapid degradation of EGFR, compared to panitumumab. Experiments performed in reporter cells revealed a 2-3 fold increase in the rate of antibody-mediated degradation of EGFR-GFP induced by necitumumab and cetuximab as compared to panitumumab. Similar studies using three NSCLC cell lines (HCC827, NCI-H1650, and EKVX) uncovered a 1.5-2 fold increase in the rate of EGFR-antibody delivery to lysosomes with necitumumab and cetuximab, as compared to panitumumab. Conclusions: Necitumumab and cetuximab are internalized to a low-pH compartment more quickly than panitumumab in NSCLC cells. This difference correlated with a more rapid degradation of EGFR induced by necitumumab and cetuximab, compared to that induced by panitumumab.