Engineering a conditionally active cetuximab prodrug via affibody-based paratope masking

The application of monoclonal antibodies as targeted drugs has revolutionized cancer therapy, yet their efficacy is sometimes limited by toxicities arising from target expression in healthy tissues. Various strategies have emerged to overcome these issues, including the design of antibody-based prodrugs. Here, we report the generation of affibody-based masking domains designed to specifically bind and mask the paratope of the anti-epidermal growth factor receptor (EGFR) antibody, cetuximab. Bacterial display of affibody library and cell sorting were employed to isolate suitable candidates. Both experimental and computational analyses confirmed that several of these selected affibodies effectively blocked cetuximab from binding EGFR. A cetuximab prodrug was created by fusing the most promising masking candidate to the antibody heavy chains. The prodrug demonstrated over 400-fold difference in growth inhibition effect between its masked and unmasked states in vitro. A biodistribution study comparing cetuximab and its prodrug demonstrated the feasibility of conditional activation of the cetuximab prodrug in a xenograft mouse model. This study is the first to describe the use of an affibody masking domain to create an antibody-based prodrug, demonstrating significant potential for further development of safer and more efficacious cancer therapies.