Oncolytic Viruses: The Power of Directed Evolution

Attempts at developing oncolytic viruses have been primarily based on rational design. However, this approach has been met with limited success. An alternative approach employs directed evolution as a means of producing highly selective and potent anticancer viruses. In this method, diverse viruses are grown under conditions that maximize diversity and then passaged under conditions meant to mimic those encountered in the human cancer microenvironment. Viruses which evolve to thrive under this selective pressure are isolated and tested to identify those with increased potency (i.e., ability to replicate and spread) and/or an increased therapeutic window (i.e., differentiated replication and spread on tumor versus normal cells), both of which have potential value but the latter of which defines an oncolytic virus. Using ColoAd1, an oncolytic virus derived by this approach as a prototype, we highlight the benefits of directed evolution, discuss methods to “arm” these novel viruses, and introduce techniques for their genetic modulation and control

Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer

Background Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses. Methodology/Principal Findings Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent. Conclusions/Significance Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies

Potency and therapeutic indices of ColoAd1.

<p>Potency values less than 1 indicates attenuation relative to Ad5.</p><p>The potencies of ColoAd1, Ad5, ONYX-015, Ad3 and Ad11p on colon cancer (HT-29, DLD-1) and normal cells (HUVEC, HMEC) were measured by MTS assay at day 4 post-infection and is represented in the table as an IC₅₀ value. The potency (as reflected in the IC₅₀) is the ratio of the IC₅₀ of a given virus on a given cell line relative to Ad5's IC₅₀ on that cell line. The Therapeutic Index was calculated using either HUVEC (primary endothelial) or HMEC (primary epithelial) cells and using the ratio of the IC₅₀ of a virus on these primary, normal cells and dividing it by its IC₅₀ on HT-29 tumor cells.</p

Anti-tumoral activity of ColoAd1 after systemic administration in a liver metastasis xenograft mouse model.

<p>HT-29 colon cancer cells were seeded to the liver of nude beige mice (n = 10 mice per treatment group). Plasma CEA level was used to monitor tumor establishment. <i>A</i> and <i>B</i>, Mice were treated by tail-vein (i.v.) injection with 1×10¹⁰, 5×10¹⁰, or 1×10¹¹ total viral particles of ColoAd1 per mouse. A fourth set of liver-tumor-bearing mice were i.v. injected with 1×10¹¹ total viral particles of a replication-defective [E1⁽⁻⁾] version of ColoAd1, ColoAd1CJ132. A fifth set of mice were injected with vehicle control (buffer). Tumor weight measurements demonstrate that ColoAd1 has anti-tumoral activity, which is dose-dependent (<i>A</i>). Blood CEA levels at the end of the study (day 12 post viral administration) corroborate the tumor weight data (<i>B</i>). <i>C and D,</i> Comparison of anti-tumoral activity of ColoAd1, Ad11p and ONYX-015 in the HT-29 liver metastasis xenograft mouse model. In a second study performed in the same model as in Panels A and B, ColoAd1 was compared to its parental virus, Ad11p, and to the clinically-approved oncolytic virus ONYX-015; each virus dosed i.v. to a total of 1×10¹¹ viral particles per mouse.</p

The Directed Evolution process and analysis of viruses and derivative viral pools by anion-exchange chromatography.

<p><i>A,</i> Representation of the Directed Evolution process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002409#s4" target="_blank">Materials and Methods</a> for detailed description). <i>B,</i> Chromatograms of each pure Ad serotype included in the mixed serotype starting pool from which ColoAd1 was selected. <i>C,</i> Chromatograms of the passage 20 viral pools derived on the HT-29, Panc-1, MDA-231, and PC-3 tumor cell lines, respectively. The differing retention times of these pools are consistent with the predominant serotype of the pool being Ad5 or Ad40 for the Panc-1 pool, Ad11p for the HT-29 pool, Ad3 or Ad4 for the PC-3 pool, and Ad5 or Ad40 for the MDA-231 pool.</p

Potency of ColoAd1 relative to Ad5 on a panel of colon cancer cell lines.

<p>Potency values less than 1 indicate attenuation relative to Ad5.</p><p>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 calculation IC₅₀ value Ad5 divided by the IC₅₀ value of ColoAd1 on the same colon tumor cell line.</p

Genomic sequence diagram of ColoAd1.

<p>The genomic differences between ColoAd1 and Ad11p are noted in the schematic. In the E2B region there are frequent substitutions of Ad3 sequences for Ad11p sequences between base pairs 6081 and 9322. In addition, ColoAd1 has a nearly complete (2,444 bp) E3 region deletion, and a smaller (25 bp), second deletion that maps to a putative E4orf4 region of the virus.</p