72. Enhancement of Immune Responses to Measles Virotherapy with aPD-1 Therapy for Improved Efficacy in Glioblastoma Therapy

Glioblastoma (GBM) is one of the most lethal malignancies with a median survival of 12-16 months. There is urgent need for improved therapies. Use of the measles virus (MV) Edmonston vaccine strain lineage for GBM virotherapy is a promising direction due to the excellent safety record and demonstrated pre-clinical efficacy in multiple patient derived orthotopic models. GBM have been shown to be immune suppressive, subverting both the innate (da Fonseca et.al. 2013) and adaptive immune systems (Wei et.al. 2010). GBM have been shown to express PD-L1, MHC-1, and down regulate MHC-II compared to non-neoplastic tissue, thereby aiding in immune evasion and suppression. Furthermore GBM can induce PD-L1 expression on tumor associated macrophages and increase PD-1 expression on peripheral blood T cells, further bolstering an immune suppressive environment. Anti PD-1 therapy with mAB (aPD-1) blocks the binding of PD-1 to its ligands, thereby preventing T cell inactivation, and was previously reported to have synergistic effects in combination with radiation therapy in GBM (Zeng et.al. 2013). We therefore sought to combine aPD-1 therapy and MV therapy of GBM to enhance immune responses and improve therapeutic outcome. Herein we present our preliminary findings. We first sought to characterize PD-L1 expression on our primary patient derived GBM and murine GBM cells and found a significant up regulation of PD-L1 and HLA-ABC upon IFN-γ stimulation, indicating functionality of these immune evading mechanisms. Next we determined if T cells trafficked to GBM following MV therapy and whether aPD-1 therapy could affect T cell influx as assessed by MRI. Briefly, C57/Blk6 mice bearing GL261 syngeneic orthotopic GBM were treated with MV-EGFR, UV-MV-EGFR, or MV-EGFR+aPD-1 on days 5 and 9 post tumor implantation. Day 11 post tumor implantation mice received tail-vein injections of monocrystalline iron oxide particles conjugated to CD4 and CD8 mAB. MRI 24 hrs later demonstrated a significant influx of T cells into the brains of mice treated with MV-EGFR+aPD-1. To examine the effect of aPD-1 on the immune system in vitro, we established co-cultures of patient derived primary GBM39 or murine GL261 GBM cells, overlaid with BV2 microglia. GBM39 cells were infected with MV-GFP and murine GL261 cells were infected with MV-EGFR (MOI=3). Cultures were treated with either 1 or 20mg/ml of aPD-1 and overlaid with BV2. Twenty four hours later the co-cultures were harvested and rtQPCR performed: results showed a significant increase in mRNA for IFN-α, IFN-β in both GBM39 and GL261 co-culture models when aPD-1 was present at 1μg/mL. aPD-1 also increased BV2 migration towards MV-GFP infected GBM39 and MV-EGFR infected GL261 cells (MOI=3) in a transwell system. Lastly, survival analysis of mice bearing orthotopic syngeneic GL261 GBM treated with MV-EGFR (2×105 TCID50 × 4 doses), aPD-1 (10mg/kg × 3 doses), MV-EGFR+ aPD-1 or untreated showed a significant prolongation of survival for mice treated with MV-EGFR+aPD-1 compared to the other treatment groups (P=0.009, 0.038, and 0.003 respectively). Additional in vitro and in vivo experiments are ongoing. Funding by: Brain SPORE (P50 CA108961), R01 CA154348, T32 NS 07494.