Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, antitumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk 1 tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4 1 and CD8 1 T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-g (IFN-g), tumor necrosis factor-a (TNF-a) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk 1 cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo. Int.

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, antitumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk 1 tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4 1 and CD8 1 T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-g (IFN-g), tumor necrosis factor-a (TNF-a) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk 1 cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo. Int.

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.

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Targeting Cytotoxic I m m u not he rapy compared with 7 for the conventional conjugate.This eliminates the need for a clearing antibody together with its immunogenicity. The problem of immunogenicity is diminished to that of CPG2, the variable region of MFE-23 and any novel epitopes at the junction of the antibody and enzyme.These preliminary studies indicate that such fusion proteins may have useful advantages over conventional reagents for targeted cancer therapy.