Screening, expression, and functional validation of camelid-derived nanobodies targeting RSPO2

RSPO2 (R-spondin 2) is a key regulator of the Wnt/β-catenin signaling pathway, involved in embryogenesis, tissue homeostasis, and cancer progression. Despite its therapeutic potential, effective agents targeting RSPO2 remain elusive. To address the unmet need for RSPO2-targeted therapies, we aimed to develop high-affinity nanobodies via phage display and prokaryotic expression, characterizing their binding specificity and functional blockade of RSPO2-LGR4 interactions. This study provides foundational insights into nanobody-mediated inhibition of Wnt signaling, supporting future therapeutic strategies against RSPO2-driven pathologies.

Recombinant RSPO2 proteins were constructed and purified using PCR-based recombination. Camels (Camelus bactrianus) were immunized with RSPO2, and phage display was employed to screen nanobody libraries. High-affinity nanobodies were cloned, expressed, purified, and assessed for specificity and binding affinity using biolayer interferometry and protein blotting. Functional validation was performed using TOPFLASH assays to evaluate their impact on Wnt/β-catenin signaling.

Nanobodies with high specificity and nanomolar-range affinity constants (KDs) for RSPO2 were identified. The nanobody effectively inhibited RSPO2-induced Wnt/β-catenin signaling in human renal epithelial cells.

The development of RSPO2-targeting nanobodies offers new prospects for treating RSPO2-related diseases. The nanobody serve as valuable tools for functional research and hold potential as diagnostic and therapeutic agents for RSPO2-driven conditions.