Jayson Hardcastle

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1−/− mice and a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

Phase IB Study of Gene-Mediated Cytotoxic Immunotherapy Adjuvant to Up-Front Surgery and Intensive Timing Radiation for Malignant Glioma

PURPOSE Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect. PATIENTS AND METHODS Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 10(10), 1 × 10(11), or 3 × 10(11) vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment. RESULTS Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3(+) T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months. CONCLUSION AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.

Oncolytic HSV-1 Infection of Tumors Induces Angiogenesis and Upregulates CYR61

Oncolytic viral therapy is under evaluation for toxicity and efficacy in clinical trials relating to several different tumors. We report a significant increase in the angiogenic index of oncolytic virus (OV)-treated glioma-matrigel implants (2.83-fold, P < 0.02). In a rat intracranial glioma model, large tumors from OV-treated animals were significantly more angiogenic than the phosphate-buffered saline (PBS)-treated control tumors (OV: 101 +/- 21.6; PBS: 19.8 +/- 10; P = 0.0037). Transcript profiling of OV-treated tumors revealed dysregulation of several transcripts involved in glioma angiogenesis. OV-mediated induction of CYR61 gene expression (8.94-fold, P = 0.001) correlated significantly with the presence of OV in tumor tissue in vivo (R = 0.7, P < 0.001). Further, induction of CYR61 mRNA and protein were confirmed in multiple human cancer cell lines and primary human tumor-derived cells in vitro, and in tumor lysate and cerebrospinal fluid (CSF) in vivo. Finally, we show that treatment of glioma cells with Cilengitide, known to counter CYR61-induced integrin activation, significantly suppressed the proangiogenic effect of OV treatment of gliomas (P < 0.05).

Enhanced antitumor efficacy of vasculostatin (Vstat120) expressing oncolytic HSV-1.

Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy.

Oncolytic viruses driven by tumor-specific promoters.

Oncolytic viruses can selectively replicate in and lead to tumor cell lysis with minimal infection/replication potential in adjoining non-neoplastic tissue. Because of paramount safety concerns, first-generation oncolytic viruses were designed to be significantly attenuated in their lytic potential. Results from recent clinical trials have revealed the safety of this approach, but have underscored the urgency for design and testing of more tumor-selective and -potent viruses to realize the full therapeutic potential of this revolutionary treatment modality. With the discovery of various molecular/genetic changes associated with neoplasia, tumor-specific transcriptional targeting of viral virulence is being tapped to generate tumor- and tissue-specific variants. This review will focus on the various strategies exploited to generate viruses whose virulence is governed by tumor-specific transcriptional events.

The Impact of Macrophage- and Microglia-Secreted TNFα on Oncolytic HSV-1 Therapy in the Glioblastoma Tumor Microenvironment

Purpose: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM. Experimental Design: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo. Results: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors. Conclusions: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy. Clin Cancer Res; 21(14); 3274–85. ©2015 AACR.

Immunovirotherapy with measles virus strains in combination with anti–PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment

Background Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. Methods In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. Results In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. Conclusions This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

BAI1 Orchestrates Macrophage Inflammatory Response to HSV Infection—Implications for Oncolytic Viral Therapy

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV). Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1−/− mice were used. Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1−/− or wild-type non–tumor-bearing mice revealed the safety of this approach. Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809–19. ©2016 AACR.

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.