Abstract
Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR–528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.
Highlights
Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens
Epidermal growth factor receptor (EGFR) overexpression is widely observed in a variety of cancer cells, and in some cases, its signal is critical for cell survival and tumorigenesis
Heat treatment of sEGFR complex (sEGFR) will disrupt its tertiary structure. These experiments allow the preliminary investigation of sEGFR recognition by the 528 antibody with respect to establishing whether the full tertiary structure is required for binding or whether 528 binds a short segment of an amino acid sequence
Summary
Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Crystal structure analysis have revealed that these antibodies bind to domain III of EGFR and thereby inhibit binding of EGF, which in turn blocks the signaling and proliferation of cancer c ells[12,13,14].
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