A resource of high-quality and versatile nanobodies for drug delivery

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb_HSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nb_HSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected Nb_HSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of Nb_HSAs were drastically prolonged by 771-fold. Nb_HSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive Nb_HSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.