David L. Bartlett, MD

Development of tumor selective viral vectors and development of surgical procedures for the regional treatment of malignant neoplasms

Summary:

The laboratory is developing viral vectors which can be administered systemically to a tumor-bearing host, and selectively target tumor cells for the expression of genes for enzyme/prodrug therapy and local immune responses. We have shown that a vaccinia virus which has a deletion in the thymidine kinase gene specifically replicates in tumor tissue in vivo and expresses high levels of a marker gene selectively in tumor cells. This selective expression can be translated into improved survival and cures when the virus expresses either the cytosine deaminase gene (converts nontoxic 5-FC to toxic 5-FU) or purine nucleoside phosphorylase gene (converts nontoxic 6-MPDR to toxic 6-MP) in a murine model of hepatic metastases from colorectal cancer. In addition, treatment with the prodrug can rescue the mice from toxic effects of the virus. We have now isolated a vaccinia mutant which has a deletion in both the thymidine kinase and vaccinia growth factor (VGF) genes.

It has previously been shown that a deletion in VGF markedly attenuates the virus in normal tissues, but replication in transformed cells is unaffected. This double mutant may allow for improved tumor selectivity and improved therapeutic results. We have completed a non-human primate study demonstrating the safety of this deleted vector in an isolated limb perfusion. In parallel with the study of new virus is the development of more potent and specific enzyme/prodrug systems. We are constructing a virus which expresses a fusion gene combining a secreted B-lactamase and the scFv against a melanoma antigen. B-lactamase can convert nontoxic cephalosporin conjugated chemotherapeutic agents into toxic agents. This protein may expand the toxic effect beyond the cells expressing the gene, while maintaining specificity for melanoma.

Because neutralizing antibodies to vaccinia in patients immunized against small pox may prevent viral infection and gene expression, we are exploring other pox vectors which may have similar selectivity without cross reactivity. We have shown that the yaba like disease (YLD) virus can be grown in high titres in CV-1 cells, similar to vaccinia, and that it infects human cell lines. We have shown that it does not cross react with vaccinia antibodies. We have sequenced the YLD thymidine kinase gene and created a recombinant virus expressing a marker gene (green fluorescent protein). We have found that this recombinant YLD infects, replicates, and efficiently expresses genes in certain human tumors. We are in the process of testing this virus as a gene delivery vector in vivo with human tumors in nude mice.

We are also exploring other potential therapeutic transgenes that induce non-specific local inflammation which may lead to a bystander killing of un-infected tumor cells, including fas ligand, superantigen, and GmCSF.

As part of our clinical effort in regional therapy for metastatic cancer, we have developed hyperthermic peritoneal perfusion for peritoneal carcinomatosis. This has been developed in a phase I trial with cisplatin delivered intraoperatively in the context of surgical debulking and hyperthermia. Extensive cisplatin pharmacokinetics have been performed, describing this system. This has been extended to include post-operative dwell chemotherapy with paclitaxel and 5-FU, and a phase I trial escalating these agents in the early post-operative period has been completed. We are now testing it in a trial for peritoneal mesothelioma, mucinous appendiceal cancer, and colon cancer with peritoneal metastases. The most promising results have been seen with peritoneal mesothelioma, where many patients have had complete resolution of ascites, prolonged disease free intervals, and responses to re-treatment.

We are also developing a simplified technique for isolated liver perfusion for patients with unresectable metastatic tumors in the liver. We originally described a 75 to 80 percent response rate for various histologies after a single 60-minute perfusion with melphalan and TNF. We have continued to use melphalan alone with similar response rates, and in some cases have included a post-operative infusional regimen of FUDR/leucovorin via an implantable pump in order to improve the durability of the response. In colorectal cancer the median duration of response has been 14.5 months. This technique of isolated hepatic perfusion has taken a mean of 8 hours in the operating room and a hospital stay of 10 days. Our current goal is to simplify the procedure to allow for widespread applicability.

Finally, combining our expertise in regional delivery and gene therapy leads to the regional delivery of poxviruses for cancer gene therapy. We have completed primate studies, which demonstrate the ability for an isolated hyperthermic perfusion to deliver poxvirus to the tissues, improve the safety over intravenous injection, and avoid circulating antibody neutralization. We are developing clinical trials with this treatment strategy.

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