The herpes simplex virus-
thymidine kinase/
ganciclovir (HSVtk/GCV) system produces both direct and immune-mediated
tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with
cytokines, alone and in combination, on the tumorigenicity and immunogenicity of B16 cells. With respect to single gene modifications, only HSVtk/GCV, or high-level
interleukin-2 (IL-2) secretion, completely prevented
tumor growth, whereas
granulocyte-macrophage colony-stimulating factor (
GM-CSF) generated the best levels of long-term systemic protection. To augment both local killing and immune activation, we constructed bicistronic constructs that express HSVtk and a
cytokine within the same cell. Co-expression of HSVtk with
IL-2 or
GM-CSF enhanced the local antitumor activity of any gene alone. In a
tumor-prevention model, HSVtk killing, in an environment preprimed with
GM-CSF, generated the best long-term immune protection. However, in a short-term
therapy model, continued
IL-2 expression was most effective against 3-day established
tumors. This probably reflects differences in the activities of
IL-2 and
GM-CSF in generating short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery experiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled populations) and adenoviral vectors (5 x 10(9) virus/ml) and function as predicted within virally infected cells. This family of bicistronic vectors can be used to stimulate synergy between suicide and
cytokine genes, overcomes the problems of delivering two genes on separate vectors, and should allow easier preparation of vectors for the delivery of multiple genes to patients'
tumor cells.