Site-selective acylation of human insulin to engineer a long-acting monovalent insulin-Fc conjugate

Basal insulin therapy requires sustained and low-variability of insulin exposure; however, natural human insulin is rapidly cleared, thus necessitating frequent subcutaneous injections. Here we report a modular chemical platform for constructing monovalent insulin-Fc conjugates with structural homogeneity and long-term action. Site-selective acylation on three accessible amines of insulin (Gly^A1, Phe^B1, or Lys^B29) yields a panel of alkynyl insulin analogs (3a-3c), which could couple with an azido Fc protein by CuAAC to produce monovalent insulin-Fc conjugates. Among these conjugates, Fc conjugation at Lys^B29 of insulin, namely 3c-Fc, retains the bioactivity, while insulin at Gly^A1 (3a-Fc) and Phe^B1 (3b-Fc) were markedly deactivated, as assessed by the downstream Akt phosphorylation test. In vivo, the 3c-Fc conjugate exhibits a long pharmacokinetic and pharmacodynamic profile, which may result from the increase in the molecular size, the recycling by the neonatal Fc receptor, and the monovalence for slow insulin receptor-mediated clearance. Overall, we describe here the method of site-selective acylation and Fc conjugation to tune the basal-leaning pharmacology of insulin.