Abstract
Antibody prodrugs provide a strategy to reduce systemic toxicity by masking therapeutic antibodies until activation in specific physiological environments. Nivolumab, an anti-PD-1 checkpoint inhibitor used in cancer immunotherapy, can cause immune-related adverse events. As a first step toward a prodrug version of nivolumab, we screened an Escherichia coli affibody library using MACS and FACS, identifying affibodies that effectively mask its PD-1-binding regions. Deep sequencing revealed an unexpected enrichment of proline-rich variants, possibly mimicking a PD-1 loop. AlphaFold modeling suggested that these affibodies form interactions with nivolumab despite low alpha-helical content. Biosensor assays confirmed effective masking in a nivolumab prodrug format, with PD-1 binding restored upon proteolytic cleavage. These findings support further exploration of PD-1-mimicking affibodies as masking domains, advancing the development of more selective nivolumab prodrugs with improved safety profiles.