Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope

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.

Figure 1

(A) Dot blot of heat-treated sEGFR detected by antibodies 528 and cetuximab. Each blotting image was a consecutive membrane strip without cropping. (B,C) Competitive Biacore assay between 528 and EGF (B) and between 528 and cetuximab (C) for binding sEGFR. sEGFR (100 nM) was loaded onto the antibody-immobilized sensor chips with or without its competitor.

Figure 2

Negative-staining electron microscopy of the 528 Fab–sEGFR complex. (A) Two-dimensional (2D) class averages of the negatively stained 528 Fab–sEGFR complex. The purified complex was subjected to single particle image analysis via negative-staining electron microscopy. Upper panel: representative 2D class averages of the 528 Fab–sEGFR complex in their different orientation classes. Lower panel: dissociated 528 Fab. (B) Magnified representation of a 2D class average shown in (A) (orange square in (A), 90° rotated). Four domains of sEGFR are labeled. (C) An outline trace of the averaged image of (B) the sEGFR portion is shown in light purple and the 528 Fab portion is shown in green. (D) (1) A crystal structure of sEGFR (light purple, PDBID: 1NQL) overlapping with the outline trace (blue dashed line: sEGFR, green: 528 Fab). (2) A crystal structure of the cetuximab Fab–sEGFR complex (sEGFR: light purple, Cetuximab: orange, PDBID: 1YY9). (3) Cetuximab Fab onto the 1NQL sEGFR structure by superposing the domain III of both structures. The outline trace is overlapped onto the sEGFR structure (blue dashed line: sEGFR, green: 528 Fab). (E) Domain III surface from the view of the cetuximab side. Atoms within 3.5 Å from cetuximab and EGF are colored with orange (cetuximab), pink (EGF), and red (both). A possible binding area of 528 is indicated with a dashed green circle. Ser 492 is shown in blue.

Figure 3

(A) Superposed anti-sEGFR antibody structure. Crystal structures of cetuximab (PDBID 1YY9, orange), panitumumab (PDBID 5SX4, blue), and necitumumab (PDBID 6B3S, magenta) antibodies complexed with domain III of sEGFR (light purple). Possible positioning of 528 Fab deduced from the electron micrograph images is indicated with a green dashed line. A black arrow indicates the viewpoint direction for (B). A loop comprising residues 353–362 is shown in cyan. (B) Manual docking model between domain III (light purple) and 528 (green). CDR loops are colored in blue and the 353–362 loop of sEGFR is in cyan. (C) Mutational residues of domain III induced by the cetuximab treatment (labeled with black characters). The possible binding site of 528 is indicated with a green dashed circle. Panitumumab epitope is colored with purple. The panitumumab epitope overlapping with the cetuximab-induced mutation is colored with yellow. Ser 492 is shown in blue.