Selective delivery of drugs to tumors continues to be a major goal in cancer chemotherapy. Pro-drugs activated by enzymes positioned at tumor sites either through ADEPT or GDEPT approaches offer great promise for enhancing the selectivity of cancer therapy. One such promising candidate is CPT-11 (irinotecan), a clinically approved cancer pro-drug, which is converted to the active form SN-38 by the major human liver carboxylesterase hCE-2. However, the inefficient conversion of CPT-11 by hCE-2 limits its development as the enzymatic component of an ADEPT-GDEPT strategy. Therefore, the primary objective of this study will be to evolve, through a variety of molecular techniques, a human CE-2 enzyme with significantly improved catalytic efficiency towards the pro-drug CPT-11.
The aims of this proposal are to generate diverse hCE-2 gene libraries, identify improved variants by employing high-throughput screening and perform biochemical/kinetic analyses on the improved enzymes. It is anticipated that the engineering of a highly evolved, human carboxylesterase will be of immense benefit clinically by maximizing the exposure of tumor cells to the cytotoxic agent, potentially limiting the systemic concentrations and reducing the toxicity associated with conventional CPT-11 therapy as well as circumventing potential problems associated with immunogenicity, particularly when used in an ADEPT therapeutic strategy.
Site-selective CPT-11 activation using an evolved human carboxylesterase conjugated to a monoclonal antibody directed against tumor-specific antigens or delivered by gene therapy formulations will lead to higher SN-38 levels than could be achieved through conventional CPT-11 therapy. This will result in a better therapeutic efficacy at well tolerated doses.
