Chemoenzymatic Site-Specific Lysine Modification of Nanobodies and Subsequent Bioconjugation via Potassium Acyltrifluoroborate (KAT) Ligations
Jinling Wang, Kateryna A. Tolmachova, Jeffrey W. Bode
Abstract
Single chain camelid antigen binding domains, often called nanobodies, have emerged as powerful tools for diagnostics and therapy. Methods for their site-specific modification offer immense potential for enhancing their therapeutic applications, but established approaches, such as fusion proteins, have well-known limitations in the nanobody format. Here, we report a convenient and broadly applicable method for site-specifically functionalizing a single residue near the C-terminus of VHH nanobodies by employing lysine acylation using conjugating enzymes (LACE) to transfer short peptides bearing functional group handles for potassium acyltrifluoroborate (KAT) ligations onto a single lysine residue of the expressed nanobodies. This approach requires a LACE tag (4 residues or 11 residues) in the recombinant nanobodies and enables direct elaboration of the products via a rapid amide forming reaction. In this study, VHH nanobodies expressed in Escherichia coli could be efficiently modified through the transfer of specific chemical handles, enabling their conjugation to small molecules, nanobodies, and antibodies by chemoselective, amide-forming KAT ligations that operate at micromolar concentrations.