Recombinant CPE fused to tumor necrosis factor targets human ovarian cancer cells expressing the claudin-3 and claudin-4 receptors

Abstract

Using gene expression profiling, others and we have recently found that claudin-3 (CLDN3) and claudin-4 (CLDN4) are two of the most highly and consistently up-regulated genes in ovarian carcinomas. Because these tight junction proteins are the naturally occurring receptors for Clostridium perfringens enterotoxin (CPE), in this study, we used the COOH-terminal 30 amino acids of the CPE (CPE_290-319), a fragment that is known to retain full binding affinity but have no cytolytic effect, to target tumor necrosis factor (TNF) to ovarian cancers. We constructed a pET32-based vector that expressed the fusion protein, designated here as CPE_290-319-TNF, in which CPE_290-319 was fused to TNF at its NH₂-terminal end. Western blotting confirmed presence of both CPE_290-319 and TNF in the fusion protein. The TNF component in CPE_290-319-TNF was 5-fold less potent than free TNF as determined by a standard L-929 TNF bioassay. However, the CPE_290-319-TNF was >6.7-fold more cytotoxic than free TNF to 2008 human ovarian cancer cells, which express both CLDN3 and CLDN4 receptors. shRNAi-mediated knockdown of either CLDN3 or CLDN4 expression in 2008 markedly attenuated the cytotoxic effects of CPE_290-319-TNF. The fusion construct was efficiently delivered into target cells and located in both cytosol and vesicular compartments as assessed by immunofluorescent staining. We conclude that CPE_290-319 effectively targeted TNF to ovarian cancer cells and is an attractive targeting moiety for development of CPE-based toxins for therapy of ovarian carcinomas that overexpress CLDN3 and CLDN4. [Mol Cancer Ther 2009;8(7):1906–15]