Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug

CPT-11 is a clinically approved anticancer drug used for the treatment of a dvanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that h as approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, ther e is a significant opportunity to improve its therapeutic efficacy and to d iminish its systemic toxicity by selectively activating the drug within tum or sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activa tion would be of interest, particularly, since the conjugate would likely b e minimally immunogenic, and the prodrug is clinically approved. Toward thi s end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes b ased on their abilities to effect the conversion and identified human carbo xylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 2 6-fold more active than human carboxylesterase 1 and was 65% as active as r abbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme kno wn. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that cou ld bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytot oxicity assays established that the conjugate led to the generation of acti ve drug, but the kinetics of prodrug activation (48 pmol min(-1) mg(-1)) wa s insufficient for immunologically specific prodrug activation. These resul ts confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.