Oncolytic Herpesvirus Promising in Colon Cancer Liver Metastases

NEW ORLEANS—A mutant her-pesvirus has been shown, in vitro and in vivo, to be highly oncolytic against colon carcinoma cells in liver metastases. On-colysis induced by the replication of this virus, combined with cyclophosphamide prodrug activation, appears to hold therapeutic promise in this setting, according to research presented at the Society of Surgical Oncology Cancer Symposium.

Timothy Pawlik, MD, MPH, a resident at the University of Michigan, Ann Arbor, presented research he is conducting with Kenneth Tanabe, MD, of Massachusetts General Hospital, Boston.

A Replicating Vector

Most gene therapy approaches have used viral vectors modified so that they cannot replicate in humans. But this study used a virus that can replicate in humans, specifically, herpes simplex virus type 1 (HSV-1) The investigators chose HSV-1 because it has several properties that render it useful as a vector for cancer gene therapy, Dr. Pawlik explained.

First, HSV replication itself is cytotoxic to cells, including tumor cells. Progeny virion released from the cells can then infect adjacent tumor cells. The cytotoxic effect of the HSV vector is present even at an extremely low multiplicity of infection (MOI).

Further, he said, the HSV vectors can carry a great number of transgenes in sequence, making it possible to deliver multiple therapeutic transgenes to enhance therapeutic efficacy.

The HSV mutant, known as rRp450, expresses the cytochrome rRp450 trans-gene. The p450 component bioactivates the cancer prodrug cyclophosphamide. As an HSV-1 vector, rRp450 also ex-presses viral thymidine kinase, which bioactivates the cancer prodrug ganci-clovir (Cytovene).

In Vitro Studies

The virus’ cytotoxic efficacy was assessed in vitro by measuring survival of a panel of colon carcinoma cell lines after exposure to an increasing viral MOI. Even at a low MOI, rRp450 replication effectively destroyed all colon carcinoma cell lines.

“Cell lines were almost completely destroyed when as little as 1 viral unit per 100 tumor cells was added,” Dr. Pawlik reported.

To examine the effects of the prodrugs on rRp450-mediated cytotoxicity, the investigators infected each carcinoma cell line with rRp450 using an MOI that would cause 50% cell destruction in that cell line. Infected cells were then incubated in the presence of the prodrugs, cyclophosphamide or ganciclovir, or a control.

Cyclophosphamide requires p450 to be activated to its cytotoxic metabolite; therefore, it had no effect on cancer cell lines by itself, but when it was added to the rRp450-infected cells, it produced a 20% to 30% increase in cell destruction over the rRp450 infection alone.

“When rRp450-infected colon carcinoma cells were in the presence of cyclophosphamide, cell destruction was nearly complete,” Dr. Pawlik observed.

Effects of Ganciclovir

In contrast, when ganciclovir was added to rRp450-infected carcinoma cells, it actually diminished cell destruction.

These results indicate that these two prodrugs differ substantially in their ability to potentiate rRp450-mediated oncolysis. Cyclophosphamide enhances cell destruction, while ganciclovir reduces the cytotoxic effects of this viral vector.

Further experiments showed that this difference in oncolysis of colon carcinoma was probably due to a difference in the drugs’ effects on viral replication. Ganciclovir significantly decreases viral yield, and since viral replication produces tumor cell destruction, ganciclovir reduces rRp450’s oncolytic effects.

Experiments also showed that rRp450 has preferential replication in colon carcinoma vs normal hepatocytes.

In a murine model of experimental colon cancer metastases, a single injection of rRp450 into the portal vein effectively treated diffuse liver tumors. Mock-infected animals had abdominal distension, bloody ascites, innumerable tumor nodules, and livers that weighed twice that of the treated animals. All rRp450-treated animals, in contrast, appeared healthy, showing less than 10 tumor nodules per specimen.

The addition of cyclophosphamide to the rRp450 treatment further reduced the number of tumors per liver specimen, Dr. Pawlik reported.

He concluded that the HSV-1 rRp450 mutant is highly oncolytic, with viral replication able to destroy colon carcinoma cells. It selectively replicates in colon

cancer cells vs normal hepatocytes; it bioactivates cyclophosphamide, which augments its antineoplastic effects; and it significantly reduces liver metastases via a portal venous dose.