Nathanaël Rakotoarinoro, Yan Dyck, Jeffrey L. Schloßhauer, Simon K. Krebs, Franziska Ramm, Dana Wenzel, Miriam-Kousso Assi, Anne Zemella, Maria K. Parr, Marlitt Stech

Design and generation of antibody-drug conjugates using non-canonical amino acids

The concept of antibody-drug conjugates (ADCs) is theoretically simple, but it is difficult to combine their components into an optimized and functional therapeutic agent. With most conjugation technologies, the choice of the conjugation site is limited, or pre-functionalization of the conjugation site is required. This leads to limited possibilities of optimization and extensive downstream processes, respectively. Against this background, we develop biochemical tools for the site-specific modification of antibodies using the amber suppression technology. We use two different approaches: Mammalian cell-based as well as novel mammalian cell-free systems. Here we demonstrate the synthesis of “ready-to-conjugate” antibodies containing site-specifically introduced 2-azidoethoxycarbonyl-L-lysine (AECK) by using an orthogonal tRNA/synthetase pair from the archaea Methanosarcina mazei in a mammalian cell-free system for the first time, as well as in a cell-based system. Using the cell-free system, we show the efficient one-step synthesis of radiolabeled and ready-to-click antibody. Using cell-based expression, we investigated the developability of an antibody-drug conjugate and we proof the position-specific coupling of DBCO-PEG3-MMAE to cell-based produced AECK labeled antibodies by hydrophobic interaction chromatography.