Abstract 2575: Activation of antibody drug conjugate MDX-1203 by human carboxylesterase
Dan Derwin, David Passmore, Janette Sung, Daniel Tengco, Brian Lee, Barbara A. Aguilar, Tim Chen, Allen Zhang, Bilal A. Sufi, Charlie Cong, Adam Salles, Mary Huber, Alice Stevens, Chetana M. Rao, Sanjeev Gangwar, Shrikant Vishnu Deshpande, Vangipuram S. Rangan · Cancer Research · 2010
Abstract MDX-1203 is a human anti-CD70 antibody conjugated to a DNA alkylating cytotoxic Drug A. Drug A is an enzyme-activated prodrug that requires cleavage of its peptide linker and removal of an ester-linked protecting group for cytotoxic activity. Upon internalization of MDX-1203 in targeted cancer cells, peptide linker in drug A is cleaved by lysosomal enzymes to produce Drug B; which is further processed by intracellular enzymes to produce active drug. The purpose of this study was to determine if Drug B and n-acetyl-cysteine-conjugated Drug A (NAC-Drug A) are substrates of recombinant human carboxylesterase, and to determine kinetic parameters for the enzyme iso-forms rhCE1 and rhCE2. Furthermore, a comparison of rhCE1 and rhCE2 kinetics was made with CPT-11, a commercially available prodrug used to treat colon cancer that is preferentially activated by human CE2. Human carboxylesterase 1 and 2 with a C-terminal histidine tag were cloned and expressed in CHO cells. The enzymes were purified using immobilized metal ion affinity chromatography (IMAC), and their identities were confirmed by N-terminal protein sequencing and Western blot using an anti-his antibody. Enzyme reactions were carried out at 37°C by incubation with Drug A, NAC-Drug B, or CPT-11. The amount of enzymatically released drug product was determined by RP-HPLC. Drug B was determined to be a substrate for both rhCE1 and rhCE2; however, its Km was much lower with rhCE2 (1.5 uM) as compared to rhCE1 (53 uM), and the overall enzyme efficiency (Vmax/Km) was approximately 50 fold higher for rhCE2 as compared to rhCE1. Similarly, Drug B and NAC-Drug A were both found to be better substrates for rhCE2 as compared to rhCE1. Furthermore, recombinant human CE2 cleaves ester group in NAC-Drug A, 20 fold more efficiently as compared to CPT-11. These results indicate that expression of carboxylesterase 2 in targeted cancer cells is probably important for intracellular activation of MDX-1203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2575.