Combined p19Arf and Interferon-beta Gene Therapy: Evidence of Immune Response in Murine Models of Melanoma and Lung Carcinoma

Abstract

Background: Our previous work has included the development of viral vectors where transgene expression is controlled by the transcriptional functions of the p53 tumor suppressor protein. Since wild-type p53 is frequently maintained in melanoma, we propose that such vectors may provide an opportunity for interplay between endogenous and exogenous factors. Transfer of p19Arf, a functional partner of p53, should help activate endogenous p53, thus supporting both vector expression and killing of tumor cells. Interferon-beta (IFNβ) is known to activate the immune system, induce apoptosis and inhibit angiogenesis. Moreover, interactions of the p53/Arf and IFN pathways have been reported. Previously we have shown that combined, but not individual, transfer of p19Arf and IFNβ mediated by our p53-responsive Ad5 vector induced massive cell death of B16 (mouse melanoma) both in vitro and in vivo. Objective: Our current aims include revealing involvement of the immune system in response to gene transfer protocols utilizing p53-responsive Ad5 or AdRGD vectors. Methods/Results: B16 cells with forced expression of CAR were transduced ex vivo with the Ad5 vectors (called AdPG) and implanted subcutaneously in C57BL/6 mice. Seven days later, these same mice received a challenge with fresh B16 cells implanted s.c. in the contralateral flank. Transfer of IFNβ alone or in combination with p19Arf reduced tumor formation at the sites of the vaccination and challenge. However, the combined treatment resulted in smaller tumors with delayed progression and prolonged survival. In parallel, RGD-modified adenoviral vectors, AdRGDPG, were constructed and shown to increase viral tropism as well as provide the expected synergy between p19Arf and IFNβ in CAR-negative B16 cells. This new set of AdRGDPG vectors was used in a model of in situ gene therapy of Lewis Lung Carcinoma (LLC) where tumors were first established s.c. in C57BL/6 mice then treated in vivo with six rounds of viral transduction. Treatment with IFNβ alone or in combination with p19Arf was effective in retarding tumor progression. Strikingly, s.c. challenge tumors implanted in the contralateral flank were inhibited especially well only in the animals previously treated with the combination of p19Arf and IFNβ. Alternatively, LLC cells were implanted s.c. in Balb/c nude mice and treated in situ. In this case, we did not observe a reduction in tumor progression in any of the conditions, indicating the importance of the adaptive immune system for tumor inhibition in response to our gene transfer strategy. Conclusion: In mouse tumor cell lines that retain wild-type p53, treatment with the combination of p19 Arf and IFNβ appears to involve the immune system, induce immunological memory and may provide an advantage over mono-gene therapy.