Yeast Surface Display-Based Screening and Validation of Nectin-4-Targeting Nanobodies for Bladder Cancer

Background Advanced bladder cancer (BCa) is associated with a poor prognosis, highlighting the urgent need for novel therapeutic strategies with improved efficacy and reduced toxicity. Nectin-4 (PVRL4, here after referred to as N4) is a clinically validated therapeutic target; however, existing antibody-based agents are limited by inadequate tumor penetration and off-target toxicities. Nanobodies (Nbs) possess favorable characteristics, including small molecular size, enhanced tissue penetration, and ease of engineering. This study aimed to identify and validate N4-specific Nbs for BCa using a yeast surface display-based screening strategy. Results N4 was highly expressed in BCa and significantly associated with an unfavorable prognosis. The YSD platform supported efficient library expansion, surface display, and iterative enrichment of N4-binding clones. Among the identified Nbs, Nb-80 exhibited favorable binding activity, with an EC₅₀ of 0.150 µg/mL. BLI analysis demonstrated rapid association–dissociation kinetics. Conclusions An N4-targeting Nb was successfully identified using a YSD-based screening strategy. These findings support the utility of the YSD platform for Nb discovery and provide a foundation for developing Nb-based N4-targeted strategies in BCa.