Abstract 5632: Immunoprotected, microencapsulated bacterial cytosine deaminase mediated conversion of 5-fluorocytosine to 5-fluorouracil

Abstract

Abstract Introduction Enzymes of non-human origin possess tremendous potential as anticancer agents, especially in enzyme-prodrug therapy. Unfortunately, these enzymes are recognized as foreign agents by the human immune system and are targeted by an immune response. This immune reaction limits enzymes’ efficacy, particularly in treatments requiring repeated dosing. Current strategies for deimmunizing these therapeutic enzymes are labor/time intensive and yield limited success. Encapsulating enzymes in a hydrogel, such as sodium alginate, can confer immunoprotection and enhance in vivo stability. Alginate serves as a barrier between enzyme and host and its porosity can be controlled to prevent antibody infiltration while allowing the diffusion of the prodrug and the drug. The bacterial enzyme cytosine deaminase (bCD) mediates the conversion of 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). We encapsulated the bCD in sodium alginate microbeads and tested enzyme efficacy post encapsulation as determined by conversion of 5-FC to 5-FU, with concomitant cell kill assays. Methods bCD was encapsulated in sodium alginate microbeads, ∼200 microns, using a NISCO microencapsulation system (www.nisco.ch). The beads were incubated with 5-FC (25, 50, 100 and 200 microM), and conversion to 5-FU was monitored over time using spectrophotometry. Unencapsulated bCD was used as controls. Then, microbeads were incubated with 9L rat glioma cells in the presence of 5-FC. Cytotoxicity of the enzyme-prodrug system to 9L cells was evaluated using an MTT assay. 5-FC alone in the absence of bCD and 5-FU were used as controls. Experiments were repeated using beads stored for 72 h at 4°C and 37°C and temperature effects on the stability of encapsulated bCD were noted. Results summary We observed the complete conversion of 5-FC to 5-FU for all concentrations of encapsulated enzyme, albeit at a slower rate than unencapsulated controls. Cytotoxicity of the encapsulated enzyme-prodrug system toward 9L cells was similar to that of 5-FU alone, and of unencapsulated controls, indicating that encapsulation had no deleterious effect on enzyme efficacy. Though the enzyme kinetics were slower for the stored beads (at 4°C and 37°C), these beads resulted in similar cell kill. Our results suggest that sodium alginate microencapsulation of bCD maintained the enzyme's functionality and may therefore be a suitable platform for immunoisolative enzyme-prodrug therapy. We are extending our work to other cancer cell lines and to in vivo study of the anti-tumor effects of these encapsulated enzymes. This system has the advantage of localized 5-FC to 5-FU conversion, thereby potentially reducing systemic toxicity and increasing the locally available dose of the toxic drug. The strategy can be extended to the encapsulation of enzyme-producing cells that serve as de novo drug factories. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5632. doi:1538-7445.AM2012-5632