Abstract 5675: Optimization of a Seneca Valley Virus (SVV) 3C protease substrate for virus-directed enzyme prodrug therapy.

Abstract

Abstract Lung cancer is the leading cause of death among cancer patients, killing more than prostate, breast, and colon cancers combined. Extensive stage small cell lung cancer (ES-SCLC) has a median survival time of less than 12 months with current cytotoxic therapies. Therefore, new and more effective therapies are desperately needed. Recently, a novel cancer therapeutic has emerged using oncolytic viruses to selectively kill cancer cells. Seneca Valley Virus (SVV), a newly discovered picornavirus, was found to selectively infect cancers with neuroendocrine features, such as SCLC and pediatric brain tumors. Although its method of tropism is still unknown, SVV has been shown to be highly effective in the eradication of both solid tumors and pre-formed metastases in multiple in vivo models. SVV is being tested as a therapy in SCLC and pediatric brain tumors in clinical trials currently. Neutralizing antibodies are generated against the virus one to three weeks after initial treatment, yet the virus has been shown to persist intratumorally for at least a month after administration. To further increase the eradication of tumor cells by SVV infection, we are designing and studying a prodrug which will be activated selectively by the virus’ own protease. Once SVV infects a tumor cell, the viral genome is replicated, transcribed, and translated into a single polyprotein. The SVV 3C protease, one of the mature viral proteins which cleaves the viral polyprotein, is generated in large amounts within and around the infected tumor during the virus life cycle. Using fluorogenic FRET proteins separated by both endogenous and exogenous protease substrates, we have identified the optimal peptide substrate specific for the SVV 3C protease. We determined that two of the ten proposed endogenous 3C substrates were efficiently cleaved in vitro by a recombinant SVV 3C protease. Surprisingly, the fastest native substrate, L/VP4, did not contain the picornavirus consensus Q↓GP motif. We substituted proline for the P2’ asparagine in L/VP4, based on the hypothesis that more closely mimicking the consensus sequence could further augment efficiency. In fact, this new substrate, L/VP4.1, demonstrated a ∼9 fold increase of kcat/KM over the endogenous substrate, L/VP4. Truncation analysis of L/VP4.1 determined that although the P6 amino acid is not essential for efficient cleavage, additional truncation significantly decreases or even abolishes proteolysis. This peptide substrate will be tested in cellular assays to characterize the kinetics of proteolysis during an SVV infection. The L/VP4.1 optimized substrate will be used as the basis for generating candidate peptide prodrugs for use in combination with SVV. Citation Format: Linde A. Miles, John T. Poirier, Charles M. Rudin. Optimization of a Seneca Valley Virus (SVV) 3C protease substrate for virus-directed enzyme prodrug therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5675. doi:10.1158/1538-7445.AM2013-5675