572. Augmentation of Immuno-Gene Therapy for Lung Cancer Using Chemotherapy

Abstract

RATIONALE: We reported that adenovirus-mediated murine IFN-beta (Ad.muIFN-beta) transfer eradicates murine malignant mesothelioma via a CD8+ T cell-dependent mechanism. However, Ad.muIFN-beta was much less effective in a minimally immunogenic model of lung cancer (LC), Lewis Lung Carcinoma (LLC). We hypothesized that combining chemotherapy (to induce cancer cell death and increase antigen availability) with Ad.muIFN-beta would be more effective. We used the drug gemcitabine (gem) since it is one of the most effective cytotoxic agents for human LC and has recently been reported to spare T-cell function. METHODS: C57BL/6 mice bearing flank LLC tumors (tumor volumes: 80–100 mm3) were injected intra-tumorally (IT) with 1×109 pfu of Ad.muIFN-beta with or without intra-peritoneal injection with 120 μg/g gem. Chemotherapy was given either 5 days before or after gene therapy. Effects of depletion of CD4+, CD8+, and NK cells were examined after antibody depletion. To determine if cancer cell death was important, we generated a gem-resistant LLC line by slowly increasing gem concentrations in media. NK cell activity was measured using a Cr51 release assay (Target cell: Cr51 labeled YAC-1, Effector cell: spleen cells from tumor bearing mice treated with gem, Ad.muIFN-beta or the combination) and cytolytic T-lymphocyte (CTL) activity from splenocytes was assessed using in vivo Winn assays. To explore mechanisms of the gem effect, we performed adhesion assays. A mouse endothelial cell line (H5V) was treated with gem for 24 hrs and isolated spleen cells labeled with Cr51 were then allowed to adhere on the test cells for 3 hrs. RESULTS: IT injection of either Ad.muIFN-beta or gem alone had minimal anti-tumor activity in this model. In contrast, combination treatment had significant anti-tumor activity, but only if gem was given after Ad.muIFN-beta. The combination worked equivalently in gem-resistant LLC suggesting the effect was not dependent on gem-induced tumor cell death, but was due to immunological effects. This was supported by experiments showing: 1) the anti-tumor effects were abolished by NK cell depletion; 2) elimination of CD8+ T cells did not effect initial anti-tumor effects, but blocked growth inhibition during later time points. CD4+ T cell depletion had no effect. Further studies showed that NK cells were activated by Ad.muIFN-beta. Winn assay showed that CD8+ T cells from mice 8 days after the combination treatment had LLC cell lytic activity, while CD8+ T cells from mice 4 days after the combination treatment were not lytic. Adhesion assays showed that significantly higher numbers of splenocytes adhered to H5V cells treated with gem. CONCLUSIONS: Our data suggests that although IT injection of Ad.muIFN-beta activates NK cell activity, the activated NK cells could only effectively enter the tumor site and subsequently induce a CD8+ T cell response if the tumor endothelium was “activated” by gem. This study shows an example of synergy between chemotherapy and immuno-gene therapy and suggests a novel mechanism by which this occurs. A better understanding of this process will lead to improved design of future clinical trials.