Functional Dissection of the Epidermal Growth Factor Receptor Epitopes Targeted by Panitumumab and Cetuximab

Abstract

Cetuximab and panitumumab, two antibodies targeting the extracellular domain of the epidermal growth factor receptor (EGFR), are of major clinical importance particularly in the treatment of metastatic colorectal cancer. As patients may acquire resistance-mediating mutations within the extracellular EGFR domain, functional dissection of the exact binding sites of EGFR targeting antibodies may help predict treatment responses. We therefore assessed the epitope recognition of panitumumab by screening phage-displayed random cyclic 7mer and linear 12mer peptide libraries on this antibody. Phage screenings revealed two strong, potentially epitope-mimicking consensus motifs targeted by panitumumab. A computational approach was used to map the sequences back to the potential epitope region on domain III of EGFR. The presumed epitope regions (386)WPEXRT(391) and a biochemically similar though discontinuous region P349-F352-D355 on a neighboring loop of domain III could be confirmed as part of the functionally relevant binding site of panitumumab by site-directed mutational analysis. To more accurately differentiate the panitumumab epitope from the previously characterized cetuximab epitope, binding studies were performed on a broad range of additional mutants. Taken together, this analysis revealed two large, partially overlapping functional epitopes consisting of 17 critical amino acid positions. Four of these positions were selectively targeted by cetuximab (I467, S468, Q408, and H409), whereas another four were selectively recognized by panitumumab (W386, E388, R390, and T391). In view of the clinical significance of extracellular domain mutations, our data may help guide treatment decisions in selected patients receiving EGFR-targeted therapies.

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

“…15,16 More importantly, cetuximab-resistant patients who harbor the S492R mutation responded to treatment with panitumumab. 17 In keeping with the results, Voigt et al 18 revealed that the epitope of panitumumab differs from that of cetuximab, providing a possible explanation for the benefits from panitumumab treatment in some cetuximab-resistant patients. However, as a fully human IgG2 mAb, panitumumab is devoid of natural killer (NK) cellmediated classical ADCC.…”

“…17 This mutation may have a role in providing the clinical benefit of panitumumab in a subset of subjects with mCRC who do not respond to treatment with cetuximab. 38 Additionally, Voigit et al 18 described a distinct binding site for panitumumab that is different from that of cetuximab. Therefore, panitumumab has unique functional properties in EGFR-targeted therapy, especially for the patients who are refractory to cetuximab.…”

“…18 Based on the method described above, we successfully constructed Pan-P with target selectivity based on Pan. Upon incubation with normal tissues adjacent to tumor, Pan exhibited moderate binding activity while Pan-P showed no significant binding signals.…”

“…The sequence consists of blocking peptide (IYPPLLRTSQAM), substrate peptide (LSGRSDNH) and serine-glycine linker peptide (GSSGGSGGSGGSG). The selected blocking peptide, which binds specifically to panitumumab but not to cetuximab, was identified by Vogit et al 18 Protease uPA is known to be up-regulated in a variety of human carcinomas. 31 In recent years, it has been widely selected for developing prodrugs, which are inactive until they are converted to active drugs in tumor tissues.…”

Resistance to Anti-EGFR Therapy in Colorectal Cancer: From Heterogeneity to Convergent Evolution

“…Indeed, they reported that a patient harboring the S492R allele as a mechanism of secondary resistance to cetuximab was subsequently treated with panitumumab and responded transiently to this therapy. Notably, the crystal structure of cetuximab bound to the extracellular domain of the EGFR indicates that S492R likely interferes with ligand binding ( 72 ). Because other residues in the extracellular region could equally affect the binding of cetuximab to the EGFR, we postulated that molecular profi ling of these regions in tumors that developed resistance to EGFR antibodies may reveal additional mutations capable of conferring acquired resistance to cetuximab or panitumumab.…”