Nanobodies targeting the follicle-stimulating hormone receptor achieve specific accumulation in Atlantic salmon gonads

Atlantic salmon (Salmo salar) aquaculture requires reliable sterility technologies to prevent genetic introgression from escapees and address welfare and production losses associated with precocious male maturation. Follicle-stimulating hormone receptor (Fshr) is a promising target for nanobody-based biologics because it is expressed in germ cell-supporting cells that are indispensable for gametogenesis. Here, we report the selection, characterization and in vivo validation of nanobodies targeting Atlantic salmon Fshr as delivery modules for future sterility-inducing biologics. Nanobodies are small (~15 kDa) binders with potential to make targeted therapies feasible in aquaculture. We selected nanobodies from a synthetic phage-display library against recombinant Atlantic salmon Fshr ectodomain and extracellular loop regions. Four Fshr-binding nanobodies were identified, showing apparent binding affinities of ~60-180 nM in monomeric format. To improve functional affinity and extend systemic exposure for in vivo evaluation, biparatopic constructs were generated and fused to salmonid half-life extension modules. The best-performing construct showed an avidity-driven improvement in apparent affinity to 6.6 nM in a mixed-antigen format. In vivo evaluation in Atlantic salmon parr demonstrated preferential accumulation of both monomeric and biparatopic Fshr-targeting constructs in gonads relative to blood and non-targeting controls. Gonadal enrichment was generally stronger in testes than ovaries, consistent with gonadal tissue architecture. Half-life-extended constructs remained detectable in circulation for up to 48 h, while intraperitoneal delivery appeared less optimal than intramuscular administration. These findings support further development of Fshr-targeting nanobodies as delivery modules for sterility-inducing biologics in salmon aquaculture.