Canarypox Virus Vectors for Gene Transfer in Cancer Immunotherapy
Authors: H. Levitsky
Journal: JNCI Journal of the National Cancer Institute (1997)

Thirty years ago, Lindenmann and Klein (1) demonstrated that mice immunized with a lysate of tumor cells that had been infected with influenza virus were rendered immune to a subsequent challenge with the original tumor. This pivotal observation represented the first report of using genetically altered tumor cells to prime systemic antitumor immunity. Since that time, considerable effort has gone into dissecting the mechanism responsible for this effect, as well as exploring related strategies that may enhance the magnitude of the response that is generated. One proposed explanation for the enhanced ‘‘immunogenicity’’ of virally infected tumor cells is that the strong antiviral immune response mounted by the host leads to the local production of inflammatory mediators at the site of tumor inoculation. These mediators then facilitate the processing and presentation of undefined tumor antigens for recognition by T cells. Subsequently, primed tumor-specific T cells disseminate to distant sites and mediate the rejection of the challenging parental tumor. This mechanistic hypothesis led to the prediction that immunization with tumor cells transfected to express genes encoding molecules that play a role in the regulation of T-cell responses should work at least as well as virally infected tumor cells in generating systemic antitumor immunity. During the past 7 years, this prediction has been borne out by numerous studies [reviewed in (2-4)] that have explored immunization with tumor cells that were genetically engineered to produce a wide range of immunologically relevant molecules, such as cytokines, chemokines, and T-cell co-stimulatory molecules. Although the general theme of these studies is quite clear (i.e., such vaccination can result in the generation of substantial antitumor immune responses), taken together, they have led to the development of a somewhat perplexing literature describing differing degrees of antitumor activity associated with various strategies of genetically modified tumor cell vaccination. In particular, the relative merits of any single strategy seem to vary considerably, depending on the model studied. Not surprisingly, so do the immunologic mechanisms that mediate these responses. While all of this continues to be worked out in the mouse, the acute need for promising therapeutic strategies to be tested in the clinic has provided an impetus for moving to phase I trials of genetically modified autologous tumor cell vaccines for several human cancers, including melanoma, renal cell carcinoma, lymphoma, and prostate cancer. One hurdle in the reduction of this approach to clinical practice is the need for high-efficiency gene transfer if autologous tumor cells are to be used as the source of antigen. Although plasmid transfection was often used in the initial mouse experiments, the problems inherent in selecting and expanding a rare transfectant in a primary tumor cell culture led to the use of virally mediated gene transfer for most of the current clinical trials. In one sense, this might be viewed as a return to the original approach of Lindenmann and Klein, although the ability to generate replication-defective recombinant viruses that encode a given gene of interest has enabled a somewhat more defined approach to activating the immune system. In this issue of the Journal, Kawakita et al. (5) report on the use of recombinant canarypox virus as a vector for gene transfer in the creation of tumor cell-based vaccines. This gene transfer strategy offers several appealing features, which are highlighted in the article by Kawakita et al. and include a high efficiency of infection, a broad range of tropism (enabling its use with most histologic types of tumor), and high levels of recombinant gene expression. Importantly, canarypox virus constructs are likely to be extremely safe, since productive viral replication is restricted to avian species, so that infection of mammalian cells fails to generate additional infectious virus (i.e., the virus can get in, but it can’t get out). In fact, this latter feature has made the canarypox virus vector a prime candidate for use in patients who may have impaired immune systems, a cohort previously demonstrated to be at substantial risk of serious toxic effects upon inoculation with live vaccinia (cowpox) virus, which can productively infect human cells. Infection with canarypox virus and expression of the desired gene product occur quickly, minimizing the period of tumor cell culture that is required before immunization. Furthermore, like the related vaccinia virus genome, the canarypox genome has abundant ‘‘room’’ for accommodating large genetic insertions. Finally, T-cell responses against vaccinia antigens (present in most of the adult population as a consequence of immunization against smallpox) do not significantly cross-react with canarypox antigens, obviating the concern that such pre-existing immunity would preclude immunization with cells infected with canarypox virus. In their study, Kawakita et al. (5) used a mouse prostate cancer model to explore the generation of immunity upon inoculation with live tumor cells infected with recombinant canarypox virus encoding interleukin 2 (IL-2), interferon gamma, tumor necrosis factor-a (TNF-a), or the T-cell co-stimulatory molecule B7-1. In vitro analysis demonstrated that infection resulted in a high percentage of cells expressing the gene of interest (shown directly in the case of B7-1) and that a high level of expression was maintained over a period of several days. It is interesting that in no case were mice able to reject live tumor cells infected with just one of these constructs, although a significant delay in the outgrowth of tumor cells producing TNF-a was observed. In contrast, most of the mice challenged with prostate cancer cells producing TNF-a plus IL-2 rejected the tumor. An analysis of the injection site revealed evidence of necrosis and a granulocytic infiltrate as well as a notable absence