Screening and functional studies of Norrin-related nanobodies

Norrin is a crucial regulator of the Wnt/β-catenin signaling pathway, playing a key role in retinal vascular development, blood-brain barrier maintenance, and neuroprotection. In this study, a Norrin fusion protein (Norrin-Fc) was successfully expressed and purified using an insect cell expression system. Camels were immunized with purified Norrin-Fc to generate nanobodies, which consist solely of the heavy chain variable domains of heavy chain antibodies (VHH) with a molecular weight of approximately 15 kDa. Two nanobody strains, Nb 1C4 and Nb 2B10, were identified for their specific binding to Norrin-Fc, and their binding affinities were further characterized. Flow cytometry analysis confirmed that Nb 1C4 and Nb 2B10 specifically bound to the Norrin-FZD4 fusion protein. Luciferase reporter assay results demonstrated that both nanobodies effectively disrupted LGR4-induced Wnt/β-catenin signaling upon Norrin stimulation. This study represents the first successful development of nanobodies targeting Norrin, providing a strong foundation for the advancement of Norrin-related diagnostic tools and antibody therapeutics.