Abstract 231: Optimizing lysosomal activation of antibody-drug conjugates (ADCs) by incorporation of novel cleavable dipeptide linkers

Abstract

Abstract Peptides constitute a major linker class in antibody-drug conjugates (ADCs), designed to connect antibodies to cytotoxic drug molecules. An optimal linker for an ADC should be stable in circulation and be cleaved efficiently in lysosomes upon binding and internalization of the ADC in target cells, releasing drug molecules that exit lysosomes and inhibit key cellular functions. Previous studies have focused on a limited selection of peptide linkers and specific biochemical tools, such as a cathepsin B cleavage assay, leading to the selection of a valine-citrulline dipeptide linker with a p-aminobenzyloxycarbonyl (PABC) spacer, which is now often used in candidate ADCs. In this study, we screened a panel of dipeptide linkers for efficient lysosomal proteolysis. Dipeptide linkers bearing different amino acids (typically both L, with D used as a control) were synthesized with a fluorogenic leaving group, 7-amino-4-methylcoumarin, which provided a convenient model system for screening of peptide cleavage using lysosomal extracts from cancer cells as well as several individual cathepsins. The linkers were also tested for stability in mouse, rat, cynomolgus, and human plasma. Based on these screens, we identified several novel, previously unreported peptide linker designs and incorporated them into ADCs bearing a DNA-alkylating indolinobenzodiazepine (IGN) payload. These ADCs with different peptide linkers were assayed for in vitro cytotoxicity in multiple cancer cell lines, in vivo efficacy in human tumor xenograft models in mice, and ex vivo plasma stability. In addition, we measured the impact of peptide linkers on the kinetics of proteolytic processing of ADCs in cancer cell lines. We observed that several dipeptide linker designs were superior in rates of lysosomal processing compared to a reference standard L-Ala-L-Ala dipeptide linker. Citation Format: Paulin Salomon, Luke Harris, Emily E. Reid, Erin K. Maloney, Alan J. Wilhelm, Michael L. Miller, Ravi V. Chari, Thomas A. Keating, Rajeeva Singh. Optimizing lysosomal activation of antibody-drug conjugates (ADCs) by incorporation of novel cleavable dipeptide linkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 231.