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Supplementary Figure 2. The EGFR-GC1118 interaction is not disrupted by excess amounts of high- or low-affinity EGFR ligands. The indicated colorectal cancer cell lines were pre-incubated with 0.1 ug/ml of cetuximab or GC1118 for 2 h prior to the addition of EGFR ligands for 10 min (EGF, HB-EGF, BTC, and TGF-�, 20~500 ng/ml; AREG and EREG, 40~1000 ng/ml). The blue and red lines indicate the relative % of cetuximab and GC1118, respectively, bound to EGFR in the presence of competing ligands compared with bound antibodies in the absence of blocking ligands. Antibody binding to EGFR was assessed by measuring the fluorescence intensity using flow cytometry. The results are expressed as the average{plus minus}SE of % maximal binding of the antibody of triplicate experiments.

Supplementary Figure 1. Kinetic binding interactions between GC1118 and EGFR as analyzed by SPR. SPR sensorgrams were obtained from injections of (A) GC1118 or (B) cetuximab over an EGFR-immobilized surface at a flow rate of 30 µl/min. The results of a quantitative evaluation of the experimental data are shown in Supplementary Table 2.

Supplementary Table 1. X-ray data collection and refinement statistics.

Surface plasmon resonance (SPR)

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR–GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251–63. ©2015 AACR.

Background: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. Findings: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. Conclusion: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy.