nes used in this study, two additional tumor cell lines, and on a panel of colon tumor cell lines, comparing it to Ad5. ColoAd1 was over 2 logs more potent than Ad5 on the cognate cell line, HT-29. Additionally, ColoAd1 demonstrated potency equal to or greater than Ad5 on some human tumor cell lines, but was attenuated on the Panc1 cell line. ColoAd1 displayed significantly increased potency relative to Ad5 on all colon cancer tumor cell lines screened, with the exception of Colo320DM. This suggests that colon tumor cell lines have properties that make them significantly susceptible to infection and lysis by ColoAd1. To test whether ColoAd1 was selective for tumor cells over normal cells and thus, by definition, an Isoxazole 9 oncolytic virus, ColoAd1 was examined in two different colon tumor cell lines, and on primary endothelial and epithelial cells comparing its potency in each MTS assay to Ad5 and ONYX-015/H101. The results show that ONYX-015 and Ad5 are significantly less potent than ColoAd1 on the HT-29 and DLD-1 cell lines. In contrast, the potency of ColoAd1 
on HUVEC cells was the same as Ad5 and slightly more potent than ONYX-015. On HMEC cells, ColoAd1 was less potent than Ad5 and ONYX-015. To quantitate these differences between ColoAd1, ONYX-015, and Ad5, an in vitro therapeutic window was calculated, defined as the ratio of the IC50 of a given virus on normal cells, HUVEC or HMEC, divided by the IC50 on the colon tumor cell lines HT-29 or DLD-1. These calculations establish that ColoAd1 has a therapeutic window that is 3 to 4 logs greater than that of Ad5 or ONYX-015/ H101 in 17804601 these in vitro assays. ColoAd1 is a chimeric virus that displays enhanced potency over its parent virus, Ad11p ColoAd1 is a highly potent and selective oncolytic virus Since the viral pool passaged on HT-29 cells displayed the greatest increase in potency on its cognate cell line, viruses within A Novel Virus for Colon Cancer potency over both Ad11p and Ad3 on the colon tumor cell lines, demonstrating that this recombinant virus was superior in potency to both of its parent viruses. Interestingly, Ad11p, and not Ad3, displayed an inherent therapeutic window as defined here via MTS analysis. Thus, ColoAd1 is a derivative of Ad11p whose differences with Ad11p enhance the potency of the virus without altering the serotype’s natural ability to selectively replicate in tumor cells versus primary normal cells. A Novel Virus for Colon Cancer Cell Line HT-29 Virus Ad5 Wt0 HT29 IC50 20 20.03 40 8.0 9.5 30 80 25 9.5 15 3.5 Potency Cell Line HT-29 Virus Ad5 ColoAd1 IC50 73 0.06 35 0.35 8 0.21 0.72 0.02 13 0.57 1 0.06 9 1 3 Potency 1282 1 667 DLD-1 Ad5 ColoAd1 100 PC-3 Ad5 Wt0 PC-3 5 4.2 LS1034 Ad5 ColoAd1 38 MDA231 Ad5 Wt0 MDA231 HCT116.37 1.2 LS174T Ad5 ColoAd1 Ad5 ColoAd1 36 23 Panc-1 Ad5 Wt0 Panc-1 .07 2.7 SW48 Ad5 ColoAd1 17 Potency values less than 1 indicates attenuation relative to Ad5. The potency of each viral pool that underwent 20 passages of Directed Evolution was compared to the starting pool and to Ad5 on their respective cognate cell line. Viral potencies were measured by MTS assay. doi:10.1371/journal.pone.0002409.t001 SW403 Ad5 ColoAd1 9 Colo320DM Ad5 0.03 ColoAd1 18690793 r2 value 0.82 105 Potency values less than 1 indicate attenuation relative to Ad5. Potencies of ColoAd1 and Ad5 were measured by MTS on the mixed panel of tumor cell lines to derive an IC50 value for each virus. These IC50 values were used to derive the potency of ColoAd1 relative to Ad5 using the
