A Family of Bicistronic Vectors to Enhance Both Local and Systemic Antitumor Effects of HSVtk or Cytokine Expression in a Murine Melanoma Model

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Abstract
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 × 105 virus/ml from pooled populations) and adenoviral vectors (5 × 109 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. To enhance both the local killing and immune activating properties of herpes simplex virus-thymidine kinase (HSVtk) expression, we constructed bicistronic vectors to express HSVtk and a cytokine gene within the same cell. We compared the effects of HSVtk/GCV alone, cytokines alone (interleukin-2, granulocyte-macrophage colony-stimulating factor, or interferon-γ), or combinations of HSVtk and a cytokine, on the tumorigenicity and immunogenicity of B16 melanomas. Co-expression of HSVtk with IL2 or GM-CSF significantly decreased B16 tumorigenicity. HSVtk killing, in an environment preprimed with GM-CSF, was superior to other single, or double, gene modifications in generating long-term immune protection, but continued IL-2 expression was superior in a short-term therapy model. We also demonstrated that the HSVtk internal ribosome entry site (IRES) cytokine cassettes operate efficiently when incorporated into plasmid, retroviral, and adenoviral vectors, making them suitable for future in vivo delivery of combination gene therapies.