Abstract 2052: Efficient thermoregulation of heat-induced enzyme-prodrug therapy using recombinant E. coli containing a modified λpR promoter
Authors: Nemani Venkata K. , Gimi Barjor 
Published: July 2016

Abstract
Introduction: The λpR-cI857 transcriptional promoter cassette drives heat-induced protein expression in recombinant E. coli by inhibiting transcription below 37 °C (1). We have earlier reported on the development of magnetic nanoparticle hyperthermia (MNP) induced enzyme-prodrug therapy based on recombinant E. coli cells. The E. coli are designed to express cytosine deaminase (CD) under the transcriptional control of the λpR-cI857 promoter (2). CD converts non-toxic 5-fluorocytosine (5-FC) to the toxic 5-fluorouracil (5-FU). E. coli cells were co-encapsulated with MNP in immunoisolative alginate microcapsules and heated to 43 °C using an external alternating magnetic field (AMF). Using in vitro MTT assays, we demonstrated that the cytotoxicity of the heat activated E. coli in the presence of 5-FC, against 9L, MCF-7 and PC-3 tumor cells, was comparable to direct 5-FU treatment (2). However, we observed significant cytotoxicity at 37 °C resulting from basal CD expression. We now report on an improved strain of E. coli cells using a modified λpR promoter to minimize CD expression at 37 °C.
Methods: A single base mutation was introduced in the λpR promoter during PCR amplification (1) from the pLDR20 vector. The modified promoter was ligated to the linearized pNEB206A vector and transformed into NEB-α cells using the USER™ cloning protocol (www.neb.com). NM522 cells containing the modified λpR-cI857 cassette and the CD gene were constructed and characterized following procedures established in our laboratory (2). The E. coli were then encapsulated in alginate microcapsules and heated at 43 °C to trigger CD expression. Cytotoxicity against CT26 colon cancer cells was evaluated using a MTT assay following the incubation of the cancer cells with the heated microcapsules and 5-FC (0.1 mM, 72 h, 37 °C).
Results summary: We observed thermoselective CD expression and catalytic activity in E. coli heated at 43 °C. MTT cell viability assay shows that the cytotoxicity of the encapsulated, heat-activated E. coli cells against CT26 colon cancer cells was comparable to direct treatment with 5-FU. Importantly, the basal cytotoxicity at 37 °C was significantly lower than the E. coli cells activated at 43 ºC. We are extending our work to express CD periplasmically in endotoxin free hosts for improved catalytic efficiency and to eliminate cytotoxicity arising from bacterial lipopolysaccharides. We expect that the improved E. coli cells should facilitate de novo synthesis of 5-FU, on-demand and locally in the tumor, thereby reducing systemic toxicity and increasing therapeutic gain.
Acknowledgement: This work is supported by a pilot grant from the Hitchcock Foundation.
References:
(1) Jechlinger et al, FEMS Microbiol Lett. 1999;173(2):347-52
(2) Nemani et al, J Biotechnol. 2015, 203:32-40.
Citation Format: Venkata K. Nemani, Barjor Gimi. Efficient thermoregulation of heat-induced enzyme-prodrug therapy using recombinant E. coli containing a modified λpR promoter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2052.