Polyethylene glycol-based linkers as hydrophilicity reservoir for antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are an established therapeutic entity in which potent cytotoxic drugs are conjugated to a monoclonal antibody. In parallel with the great emphasis put on novel site-specific bioconjugation technologies, future advancements in this field also rely on exploring novel linker-drug architectures that improve the efficacy and stability of ADCs. In this context, the use of hydrophilic linkers represents a valid strategy to mask or reduce the inherent hydrophobicity of the most used cytotoxic drugs and positively impact the physical stability and in vivo performance of ADCs. Here, we describe the use of linkers containing monodisperse poly(ethylene glycol) (PEG) moieties for the construction of highly-loaded lysine-conjugated ADCs. The studied ADCs differ in the positioning of PEG (linear or pendant), the bonding type with the antibody (amide or carbamate), and the drug-to-antibody ratio (DAR). These ADCs were first evaluated for their stability in solution under thermal stress, showing that both the drug-linker-polymer design and the nature of the antibody-linker bonding are of great importance for their physical and chemical stability. Amide-coupled ADCs bearing two pendant 12-unit poly(ethylene glycol) chains within the drug-linker structure were the best performing conjugates, distancing themselves from the ADCs obtained with a conventional linear 24-unit PEG oligomer or the linker of Kadcyla®. The pharmacokinetic profiles of amide-linked ADCs, with a linear or pendant configuration of the PEG, were tested in mice in comparison to Kadcyla®. Total antibody pharmacokinetics paralleled the trends in aggregation tendency, with slower clearance rates for the ADCs based on the pendant drug-linker format. The above-mentioned findings have provided important clues on the drug-linker design and revealed that the positioning and configuration of a PEG unit have to be carefully tuned to achieve ADCs with improved stability and pharmacokinetics.