Clostridium perfringens enterotoxin-targeted pancreatic cancer therapy using recombinant spores of Clostridium sporogenes

Abstract

A new “clostridial-directed toxin therapy” (CDTT) system was constructed in this study. The inherent sporulation-dependent intracellular CPE from C. perfringens could be successfully cloned into, constutively expressed and secreted from C. sporogenes with the aid of Tet system and three signal peptide sequences (colA´, pec´ and amyP´). The cpe gene was fused to the different N-terminal signal peptide sequences. The whole fusions were cloned downstream of the tetO1 promoter and the constitutive expression and secretion of the cytotoxic protein from C. sporogenes have been proven by Western Blots. This C. sporogenes CPE CDTT can be used for targeting overexpressed claudin-3/-4 on the surface of pancreatic cancer cells. For therapy, the immunologically inert spores of the recombinant strains C. sporogenes [pMTL-PtetO1-colA´-cpe(his)x6], C. sporogenes [pMTL-PtetO1-pec´-cpe(his)x6] and C. sporogenes [pMTL-PtetO1-amyP´- cpe(his)x6] could be intravenously injected into patients. Due to the obligatory anaerobic feature of C. sporogenes, spores will localize, germinate, and secrete CPE only in the anaerobic necrotic areas inside tumors. CPE expression and release are no longer sporulation dependent, rather a constitutive expression and secretion of CPE is now provided by this study. Secreted CPE will bind specifically to claudin-3/-4 on the surrounding tumor cells, which mediates calcium influx and stimulate cancer cell death. This can be considered as a highly promising therapy for refractory pancreatic cancer.