Paired-agent imaging for detection of head and neck cancers

Head and neck cancers overwhelmingly overexpress epidermal growth factor receptor (EGFR). This overexpression has been utilized for head and neck cancers using molecular targeted agents for therapy and cancer cell detection. Significant progress has been made in using EGFR-targeted fluorescent antibody and Affibody molecule agents for fluorescent guided surgery in head and neck cancers. Although success in achieving tumor-to-background ratio of 3-5 have been achieved, the field is limited by the non-specific fluorescence in normal tissues as well as EGFR specific fluorescence in the oral cavity. We propose that paired-agent imaging (PAI) could improve the contrast between tumor and normal tissue by removing the fluorescent signal arising from non-specific binding. Here, ABY-029 - an anti-EGFR Affibody molecule labeled with IRDye 800CW - and IRDye 680RD conjugated to Affibody Control Imaging Agent molecule (IR680-Aff_ctrl) are used as targeted and untargeted control agents, respectively, in a panel of head and neck squamous cell carcinomas (HNSCC) to test the ability of PAI to increase tumor detection. Initial results demonstrate that binding potential, a value proportional to receptor concentration, correlates well to EGFR expression but experimental limitations prevented pixel-by-pixel analysis that was desired. Although promising, a more rigorous and well-defined experimental protocol is required to align ex vivo EGFR immunohistochemistry with in vivo binding potential and fluorescence intensity. Additionally, a new set of paired-agents, ABY-029 and IRDye 700DX, are successfully tested in naïve mice and will be carried forward for clinical translation.