Identification of Urokinase Plasminogen Activator Receptor (uPAR)-Binding Peptides Using Phage Display: Strategy and Downstream Validation

Background: Peptides that selectively bind to cancer-associated proteins are attractive candidates for molecular imaging and targeted therapeutic applications. The urokinase plasminogen activator receptor (uPAR) represents a promising molecular target as it is highly expressed in several cancers and has been linked to cancer aggressiveness, invasion, and metastasis. This study applied an NGS-informed biopanning strategy against uPAR, combined with multi-assay downstream validation, to identify and prioritize candidate peptides. Methods: An optimized biopanning protocol incorporating additional selection controls was combined with next-generation sequencing to identify candidate uPAR-binding peptides from a phage display library. Selected peptides were evaluated using complementary in vitro assays, including flow cytometry and a quantitative plate assay. Results: Three peptide candidates were identified and selected for further evaluation with complementary in vitro assays. The optimized workflow incorporates measures to reduce, but not eliminate, enrichment of target-unrelated peptides, and includes complementary validation of the peptides in in vitro assays. Differences observed between the validation methods highlighted the importance of evaluating peptide candidates using complementary assays. Conclusions: This study demonstrates the value of combining an optimized phage display selection with next-generation sequencing and complementary validation assays for identifying candidate peptides. The identified peptides represent promising candidates for further optimization toward molecular imaging and targeted therapeutic applications.