Formation of mono- and dual-labelled antibody fragment conjugates reversible site-selective disulfide modification and proximity induced lysine reactivity.

Many protein bioconjugation strategies focus on the modification of lysine residues owing to the nucleophilicity of their amine side-chain, the generally high abundance of lysine residues on a protein's surface and the ability to form robustly stable amide-based bioconjugates. However, the plethora of solvent accessible lysine residues, which often have similar reactivity, is a key inherent issue when searching for regioselectivity and/or controlled loading of an entity. A relevant example is the modification of antibodies and/or antibody fragments, whose conjugates offer potential for a wide variety of applications. Thus, research in this area for the controlled loading of an entity reaction with lysine residues is of high importance. In this article, we present an approach to achieve this by exploiting the quantitative and reversible site-selective modification of disulfides using pyridazinediones, which facilitates near-quantitative proximity-induced reactions with lysines to enable controlled loading of an entity. The strategy was appraised on several clinically relevant antibody fragments and enabled the formation of mono-labelled lysine-modified antibody fragment conjugates the formation of stable amide bonds and the use of click chemistry for modular modification. Furthermore, through the use of multiple cycles of this novel strategy, an orthogonally bis-labelled lysine-modified antibody fragment conjugate was also furnished.