A FAMILY OF BICISTRONIC VECTORS TO ENHANCE BOTH LOCAL AND SYSTEMIC ANTITUMOR EFFECTS OF HSVTK OR CYTOKINE EXPRESSION IN A MURINE MELANOMA MODEL

The herpes simplex virus-thymidine kinase/ganciclovir (HSVtk/GCV) syst em produces both direct and immune-mediated tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with cytokines, alone and in comb ination, on the tumorigenicity and immunogenicity of B16 cells. With r espect to single gene modifications, only HSVtk/GCV, or high-level int erleukin-2 (IL-2) secretion, completely prevented tumor growth, wherea s granulocyte-macrophage colony-stimulating factor (GM-GSF) generated the best levels of long-term systemic protection. To augment both loca l killing and immune activation, we constructed bicistronic constructs that express HSVtk and a cytokine within the same cell. Go-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 env ironment preprimed with GM-CSF, generated the best long-term immune pr otection. However, in a short-term therapy model, continued IL-2 expre ssion was most effective against 3-day established tumors. This probab ly reflects differences in the activities of IL-2 and GM-GSF in genera ting short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery e xperiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled p opulations) and adenoviral vectors (5 x 10(9) virus/ml) and function a s 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 vect ors, and should allow easier preparation of vectors for the delivery o f multiple genes to patients' tumor cells.