Optimization of butyrylcholinesterase for the targeted activation of CPT-11.

Proc Amer Assoc Cancer Res, Volume 45, 2004 2198 The camptothecin derivative CPT-11 (Irinotecan/Camptosar) has shown clinical efficacy against a wide range of refractory neoplasms including carcinomas and hematologic malignancies. CPT-11 is a prodrug that is converted in vivo to the potent topoisomerase inhibitor SN38 by a variety of endogenous esterases. Despite its impressive efficacy, interpatient variability of prodrug activation and unpredictable toxicity are barriers to the effective use of this promising drug. Although CPT-11 is a logical candidate for antibody directed enzyme prodrug therapy (ADEPT), studies using cloned tissue carboxylesterases have underscored the need for an enzyme with higher specific activity for CPT-11 activation. Butyrylcholinesterase (BChE) is a human serum esterase that converts CPT-11 to SN38 with low efficiency. Using a system of directed evolution technologies in mammalian cells, we have optimized BChE for the activation of CPT-11. Libraries of BChE variants based on amino acids lining the active site gorge of the enzyme were expressed and screened for cytotoxicity mediated by enzyme-activated prodrug against SW48 colon carcinoma cells. Combinatorial screening of selected mutations led to the identification of variants exhibiting >3000-fold improvement in their ability to activate CPT-11 and a 5-fold improvement in Km for CPT-11 when compared with the wild-type enzyme. The optimized enzyme was used to used to construct an anti-CD20 antibody-BChE fusion protein which maintained potent binding specificity and enzymatic activity. Antigen-specific targeted killing of B-cell lines by bound fusion protein was demonstrated in vitro at pharmacologically relevant con\. centrations of CPT-11 (0.1-10uM). These studies support the further evaluation of antibody-BChE fusion proteins in animal tumor models for the targeted activation of CPT-11 in vivo .