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Mestre Borras A, Mehari H, Ståhl S, Löfblom J
Protein Eng. Des. Sel. 39 (-) - [2026-01-09; online 2026-04-16]
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.
NGI Stockholm (Genomics Production) [Service]
National Genomics Infrastructure [Service]
PubMed 41989091
DOI 10.1093/protein/gzag010
Crossref 10.1093/protein/gzag010
pmc: PMC13122598
pii: 8655119