576. Antitumoral Effect Mediated by Two SFV-IL-12 Expression Vectors in a Murine Model of Colon Adenocarcinoma

Abstract

Two Semliki Forest Virus (SFV) based vectors expressing murine interleukin-12 (IL-12) have been developed and their antitumoral efficacy has been tested in the MC38 mouse model of colon carcinoma. In SFV-IL-12 vector, genes coding for IL-12 subunits were cloned under a single viral subgenomic promoter using an IRES between them. SFV-enhIL-12 vector carries each gene fused to the SFV capsid translation enhancer, under independent viral subgenomic promoters. Recombinant SFV viral particles were produced and IL-12 expression was tested in supernatants of BHK and MC38 infected cells by ELISA. Biologically active IL-12 was expressed at high levels from both vectors with a 9-fold increase in the case of SFV-enhIL-12. Single tumor nodules were implanted in the flank of C57BL/6 mice by subcutaneous injection of MC38 colon carcinoma cells and treated with a single intratumoral injection of SFV-IL-12, SFV-enhIL-12, and SFV-LacZ or saline as controls. With 108 viral particles of SFV-IL-12 or SFV-enhIL-12 more than 80% of treated mice experienced complete tumor regression with long-term tumor-free survival. However, when lower doses of vectors were used SFV-enhIL-12 was more efficient than SFV-IL-12 in inducing antitumoral responses. In order to compare the SFV vectors with other viral vectors more commonly used in gene therapy some groups of mice were treated in parallel with a first generation adenovirus vector coding IL-12 (Ad-IL-12). Both SFV vectors were more efficient inducing antitumoral responses than Ad-IL-12. Treatment with repeated intratumoral injection of suboptimal doses of SFV-enhIL-12 increased significatively the antitumoral response. This effect was also observed with Ad-IL-12 but only when 108 PFU were injected. In addition, all mice that rejected tumors showed specific protection against tumor rechallenge. CTL assays and depletion studies showed a CD8+ T cell dependent response. Intratumoral expression was measured in tumor homogenates after infection with SFV IL-12 vectors resulting in a maximum cytokine level at 24 hours postinjection. The length of expression was also determined in vivo by intratumoral injection of a SFV vector expressing luciferase (SFV-Luc) using the Xenogen IVIS system. Luciferase expression localized into the tumor mass for at least 6 days after infection. Systemic administration of SFV-Luc in tumor bearing mice by intraperitoneal or intravenous injection produced high luciferase expression in large body areas but it did not localized at the tumor nodule.