Regulatable high-capacity gutless adenoviral vectors mediate expression of Pseudomonas exotoxin fused to IL-13

XIONG; CANDOLFI; KROEGER; PUNTEL; LOWENSTEIN; CASTRO

San Diego, California, USA

Congreso; American Association for Cancer Research; 2008

Abstract #4798Regulatable high-capacity gutless adenoviral vectors mediate expression of Pseudomonas exotoxin fused to IL-13Weidong Xiong, Marianela Candolfi, Kurt Kroeger, Mariana Puntel, Pedro Lowenstein and Maria CastroGene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CAGlioblastoma (GBM) expresses IL-132R, not present in normal brain. The fusion of IL-13 to Pseudomonas exotoxin (PE) generates a chimeric cytotoxin that proved effective in preclinical models, and is currently in clinical trials. However, the half life of the toxin in vivo is short. Further, GBMs usually recur. These conditions make a long term, stable and regulated delivery of IL13-PE within the tumor mass an attractive therapeutic approach. We developed a high capacity adenoviral (HC-Ad) plasmid expressing PE fused to a mutated IL-13 (mIL-13) with high affinity for the glioma-associated IL-132R and negligible binding to the physiological IL13/IL4R. We constructed HC-Ad plasmid; pSTK-mCMV-TetON-mIL4-TRE-mIL13-PE encoding domain II (cell entry and toxin activation) and domain III (cytotoxicity) of PE linked to mIL-13. Constructs encode a mutated IL-4 that binds to IL13/IL4R to reduce any putative binding of mIL-13-PE to normal cells. Expression is driven by the regulatable bidirectional TRE promoter, which is activated by a doxycycline(Dox)-dependent transactivator (TetON). We also included a trans-repressor that inhibits expression in the absence of Dox. During vector production, transgenes are expressed in the producing cells, we therefore rescued HC-Ads encoding IL-13-PE toxin in EF2 expressing 293 cells that are resistant to the toxin. Producer cells were stably transfected with pCI-neo-EF2, which encodes the gene for ADP ribozilation-resistant elongating factor-2 (EF2). HC-Ad vectors were generated using 5ug of HC-Ad plasmid DNA that was linearized and transfected into EF2-producer cells. Transfection of 293 and COS-7 cells with pSTK-mCMV-TetON-mIL4-TRE-mIL13-PE resulted in Dox-dependent transgene expression, as determined by immunocytochemistry of mIL-4, mIL-13 and PE. Secretion of IL-4 and IL-13 was assessed by ELISA. Transgene expression was negligible without Dox. 293 and COS-7 cell viability was unaffected by pSTK-mCMV-TetON-mIL4-TRE-mIL13-PE; however, their conditioned media induced cytotoxicity in IL-132R expressing U251 and IN859 human glioma cells. We conclude that mIL-13-PE released from HC-Ad infected cells will be taken up by neighboring GBM cells which express the IL-13R2, constituting a powerful regulated targeted approach. Since HC-Ad vectors are capable of sustaining long term gene expression even in the presence of a systemic anti-Ad immune response as would be encountered in patients undergoing clinical trials, they are excellent candidates for implementing this approach in humans.Funded by NINDS 1RO1 NS 42893.01, U54 NS045309-01, 1R21 NS047298-01 to P.R.L.; NINDS 1R01 NS44556.01, and NIDDK 1 RO3 TW006273-01 to MGC; the Linda Tallen & Paul Kane Foundation and the BOG-CSMS.