Seneca Valley Virus 3C(pro) Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C(pro) is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C(pro), we measured cleavage kinetics of predicted SVV-001 3C(pro) substrates. An efficient substrate, L/VP4 (k(cat)/K(M) = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k(cat)/K(M) = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C(pro) in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C(pro), with direct implications for anti-cancer therapeutic development.