Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden
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Erik W. Martin1, Marguerite S. Buzza1, Kathryn H. Driesbaugh1, Shihui Liu2, Yolanda M. Fortenberry3, Stephen H. Leppla2, Toni M. Antalis1
1Center for Vascular and Inflammatory Diseases and the Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
2National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3Division of Pediatric Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Toni M. Antalis, e-mail: [email protected]
Keywords: anthrax toxin, membrane-anchored serine protease, testisin, hepsin, prodrug
Received: July 07, 2015 Accepted: September 03, 2015 Published: September 15, 2015
The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.
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