Exatecan Payload-Based Antibody-Drug Conjugates with a Short Hydrophilic Cleavable Linker

Antibody-drug conjugates (ADCs) consist of a cytotoxin covalently linked to a target-specific antibody. ADCs are designed to release the cytotoxic drug upon internalization by cells expressing the select antigen. However, the true potential of an ADC becomes diminished by several factors, including high hydrophobicity of the biologic, which results in either aggregation in vitro or poor pharmacokinetics in vivo, thereby curtailing its efficacy. Hence, one of the goals in designing next generation ADCs is creating hydrophilic linker-payloads to overcome these challenges. Here, we describe generation and characterization of cathepsin and glucuronidase-cleavable linkers attached to a novel, short, stable, hydrophilic 1-amino-β-d-glucuronic acid spacer. These hydrophilic spacer-linkers, attached to the exatecan payload, were conjugated to the HER-2-targeting antibody trastuzumab to obtain ADCs with a drug-to-antibody ratio (DAR) of 8. The ADC with an amino-glucuronic acid spacer combined with a glucuronide linker (AV-L03) showed superior hydrophilicity compared to a cathepsin cleavable GGFG linker; both ADCs included the same hydrophilic short spacer and exatecan payload. The ADC with the hydrophilic AV-L03 linker-exatecan payload (AV-DL055) conjugated to trastuzumab displayed potent in vitro cytotoxicity, improved plasma stability, and remarkable in vivo efficacy in a mouse xenograft model compared to an ADC comprising trastuzumab conjugated to DXd payload via a tetrapeptide (hydrophobic GGFG) linker.