Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: In vitro biological and binding activities.

Abstract

e13030 Background: The monoclonal antibodies cetuximab (chimeric IgG1), necitumumab (human IgG1), and panitumumab (human IgG2) competitively inhibit ligand binding to epidermal growth factor receptor (EGFR) in domain 3. These antibodies block ligand induced EGFR activation and downstream molecular signaling. Beyond protein backbone differences, further differentiation of these EGFR antibodies is an important area of investigation. Methods: The effect on the in vitro proliferation of DiFi human colorectal cancer cells was assessed by ATP luminescence. The binding of the antibodies to the soluble extracellular domain of EGFR was evaluated by ELISA and surface plasmon resonance (SPR). Inhibition of ligand induced EGFR activation and downstream signaling was evaluated utilizing electrochemiluminescence method. Finally, binding kinetics was analyzed by SPR using a capture method. Results: The necitumumab concentration producing a half-maximal anti proliferative response (IC50) in DIFI cells was comparable to that of cetuximab. In contrast, a 1.6 fold greater concentration of panitumumab was required to produce the similar level of anti- proliferative response. This observation is supported by in vitro binding assays, which indicated that cetuximab and necitumumab binds to EGFR more effectively than panitumumab. Furthermore, the IC50 of nectitumab for the inhibition of EGFR phosphorylation was at least two fold greater than that of panitumumab. When EGFR binding kinetics were analyzed by SPR, panitumumab showed a much higher affinity (Kd) to EGFR than necitumumab and cetuximab, with a lower binding rate (kon) and a much slower dissociation rate (koff). Given the results in functional assays and the SPR data indicates that the high binding affinity (Kd) of panitumumab does not correlate with improved ability to block EGFR activation, downstream pathway activation and tumor cell proliferation. Conclusions: Although, as published, panitumumab has a higher affinity (Kd) than cetuximab and necitumumab, in vitro functional assays indicate that necitumumab and cetuximab have equal or greater ability to impact EGFR signaling and effect downstream activation of EGFR.