Development and characterization of nanobodies specific for Atlantic salmon serum albumin and apolipoprotein A-I as future half-life extenders of biologics in salmonids

Atlantic salmon ( Salmo salar ) is a high-value aquaculture species, yet challenges in control of pests and pathogens, and reliable induction of sterility persist. Targeted therapies could offer new solutions, however, conventional antibodies are too costly for aquaculture-scale use. Nanobodies (Nbs) are small single-domain binders that can be produced cheaply in bacteria, but their utility is limited by rapid systemic clearance. In mammals, half-life extension (HLE) of Nbs is commonly achieved via albumin binding; in salmonids, baseline Nb pharmacokinetics and optimal HLE targets have not been described. Using a fluorescence-based assay, we quantified the concentration of a non-targeting Nb in tissues and blood of juvenile Atlantic salmon. After intravenous administration, this control Nb displayed a short plasma half-life of ~43 min, with renal clearance and low hepatic load. To develop salmonid-specific HLE modules, we purified transport proteins from salmon serum and used them to generate immune llama phage-display libraries. We selected Nbs to Atlantic salmon serum albumin (asSA-1) and apolipoprotein A-I (ApoA-I). Target specificity was validated by co-elution of Nbs with serum proteins and by label-free LC–MS proteomics. In vivo, these HLE candidate Nbs showed prolonged circulation versus the control: the asSA-binding Nb exhibited a plasma half-life of ~12 h, while ApoA-I Nbs showed half-lives of ~17 h and ~ 48 h. Estimated absolute bioavailability after intramuscular dosing neared 100%. Together, these data establish a first Nb pharmacokinetic baseline in Atlantic salmon and provide validated asSA– and ApoA-I–binding HLE molecules to enable development and use of Nb-based biologics in salmonids.