657. Enhancing Therapeutic Efficacy of an Oncolytic Virus with a Beta-1 Integrin Blocking Antibody, OS2966

Cancer-Oncolytic Viruses II 655. CD133-Targeted Oncolytic Adenovirus Exhibits Anti-Tumor Effect by Attacking Colon Cancer Stem Cells

Mizuho Sato-Dahlman1, Yoshiaki Miura2, Masato Yamamoto1 1 Surgery, University of Minnesota, Minneapolis, MN, 2Internal Medicine (3), University of Toyama, Toyama, Japan Colorectal cancer is the third most common cancer in the world and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and and chemotherapy resistant of colorectal cancers. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers and is also generally accepted as a colorectal cancer stem cell marker. CD133 expression was correlated with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in colorectal cancer. It is therefore reasonable to develop CSCs-directed therapeutic strategies by employing CD133 as a target molecule. Recently, we have established a method for isolating transductionally-targeted adenovirus by high-throughput screening. In this study, the CD133-specific Oncolytic Adenovirus (OAd) was isolated by using this novel system, and the resultant virus was tested for the oncolytic activity both in vitro and in vivo. CD133-targeted OAd (AdML-TYML) showed strong binding to CD133-positive cells (293-CD133 and LoVo (CD133 (+) human colon carcinoma)), not to CD133-negative cells (parental 293 and LS174T (CD133 (-) human colon carcinoma)) and this virus also showed strong oncolysis selectively in LoVo cells, whereas there was no effect on LS174T cells. In the analyses of the effect of AdML-TYML on colorectal cancer stem cells, CD133-targeted OAd treatment leads to inhibition of tumor establishment of CD133-positive colorectal cancer cell lines in nude mice. 100% of the mice inoculated with non-infected LoVo cells had developed tumors, while 0% of the mice inoculated with AdML-TYML infected LoVo cells had done so. In addition, when the anti-tumor effect of the CD133-targeted OAd was analyzed in established tumors of CD133(+) colorectal cancer subcutaneous xenografts, intra-tumor (i.t.) administration of AdMLTYML exhibited significantly stronger antitumor effect compared to its counterpart with wild type fiber. We focused on the CD133 as a target molecule of CSCs in colon cancer and successfully identified potent CD133-targeting OAd using high-throughput screening system. The CD133-targeted OAd exhibited selective infectivity and oncolysis to CD133-positive cells and anti-tumorigenic activity against colon cancer cells. The ability of CSCs-targeted Ad such as CD133-targeted OAd to infect and kill CSCs has important implications for the prevention of metastases and relapses in a variety of cancers.

656. Effects of STAT3 Inhibition on the Innate Immune Response to OV Therapy

Alexander Weiss, George Koutras, Chelsea Bolyard, Balveen Kaur Neurological Surgery, The Ohio State Wexner Medical Center, Columbus, OH Objective. Our lab has established that an alteration in STAT3 signaling affects the replicative ability of oncolytic virus (OV) (Okemoto 2013). However, STAT3 inhibitors are currently in clinical trials for treating several cancers, including glioblastoma (NCT01904123). One STAT3 inhibitor, WP1066, has been shown to modulate the immune response to tumors (Hussein 2007). The innate immune response to OV is known to limit its therapeutic efficacy (Meisen 2015, Han 2015) This study examines the effects of STAT3 inhibition on the innate immune response to OV therapy. Methods. In vitro, we examined the effect of STAT3 inhibition on immune cell response to OV-infected glioma. We evaluated changes in migration of immune cells towards OV-infected tumor cells, using a modified Boyden chamber. Macrophages or microglia +/- STAT3 S260

inhibitor pretreatment were seeded on the upper chamber, and tumor cells +/- OV infection were plated on the lower chamber, acting as a migration stimulus. Migration was quantified after 6 hours. To evaluate the effect of STAT3 inhibition on the direct immune cell response to OV-infected GBM cells, we utilized a co-culture. Herein, we infected tumor cells with OV, and then overlaid them with immune cells +/- drug pretreatment. Virus replication was visualized through fluorescent imaging, and quantified via plaque assay. We utilized murine models of glioblastoma (GBM) to evaluate the effect of combining STAT3 inhibitors with OV therapy. Female nude mice were implanted intracranially or subcutaneously with human GBM cells, and then treated with PBS, pharmacologic STAT3 inhibitor, luciferase-expressing OV, or combination. Using bioluminescent in vivo live imaging, we monitored viral replication by measuring luciferase activity. We also monitored mice for survival. Results. Pharmacologic inhibition of STAT3 signaling in macrophage and microglial cell lines (RAW264.7 and BV2) reduced their migration towards OV-infected glioma cells. Furthermore, pre-treating immune cells with STAT3 inhibitor reduced their reactivity to OV-infected glioma cells, permitting increased replication. In vivo, combination therapy with STAT3 inhibitor and OV resulted in a significant increase in viral replication. Survival studies are ongoing. Conclusions. We hypothesize STAT3 inhibitors may alter the immune response to oncolytic virus, permitting increased viral replication and prolonged survival in vivo. Further research into this area may prove useful in developing new treatments to be used in combination with OV for treatment of GBM.

657. Enhancing Therapeutic Efficacy of an Oncolytic Virus with a Beta-1 Integrin Blocking Antibody, OS2966

Tejaswini Reddy Nallanagulagari Neurological Surgery, The Ohio State University, Columbus, OH Glioblastoma Multiforme (GBM) is a destructive cancer of the central nervous system with poor outlook for its patients, which prompts for development of novel therapeutic approaches such as oncolytic viruses (OVs). OVs are genetically engineered viruses with tumor specific replication and lead to lysis and destruction of cancer cells. Our lab has developed a novel oncolytic Herpes Simplex Virus (oHSV), 34.5ENVE, which expresses the anti-angiogenic protein Vasculostatin. This virus has shown unparalleled antitumor efficacy against intracranial glioma in mice. OV therapy is limited by the activation and infiltration of monocytic cells that promote virus clearance and inhibit tumor destruction. β1 integrins are cell surface molecules involved in cellular proliferation, invasion and inflammation. We revealed that oHSV-induced cysteine-rich 61 protein (CCN1) binds with integrins on the surface of glioma cells and macrophages, triggering antiviral type-I interferon and chemokine responses that increase macrophage infiltration and activation, which causes increased virus clearance in glioblastoma cells and limits oncolytic virus efficacy. To improve OV therapeutic efficacy, we combined oHSV with OS2966, a β1 integrin neutralizing antibody, which has been humanized in anticipation of clinical trials. We hypothesized that OS2966 would mitigate CCN1- β1 integrin induced inflammation and increase oHSV replication. Combination treatment with OS2966 increased oHSV replication and glioma cell killing. Migration assays revealed that treatment with OS2966 strongly inhibited oHSV-induced raw246.7 macrophage cell migration toward infected glioblastoma cells. From our results, we demonstrate that β1 integrins limit oHSV replication. Furthermore, we concluded that OS2966 could rescue CCN1- β1 integrin mediated macrophage and microglia migration toward oHSV infected GBM cells. Our findings show how a β1 integrin neutralizing antibody (OS2966) inhibits the

Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

Cancer-Oncolytic Viruses II innate immune response triggered upon oHSV treatment. This work suggests that β1 integrin inhibition may be utilized in the development of a novel oHSV therapeutic strategy.

658. Bortezomib Treatment Sensitizes Oncolytic Virus Treated Tumors to NK Cell Immunotherapy Ji Young Yoo The Ohio State University, Columbus, OH

Background: Bortezomib, a proteasome inhibitor and oncolytic herpes simplex virus-1 (oHSV) are currently FDA-approved and continue to be evaluated in several human cancers. Various combinatorial treatment modalities are being investigated to enhance the efficacy of each treatment. In this study, bortezomib-mediated oHSV killing sensitization and anti-tumor immunity were evaluated. Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated using Chou-Talalay analysis. Western blot, flow cytometry, and caspase 3/7 activity assays were used to evaluate the induction of necroptotic cell death, JNK activation, and apoptosis. Production of reactive oxygen species (ROS) was measured. Inhibitors/shRNA targeting ROS, JNK and RIP1 kinase (RIPK1) were utilized to investigate the mechanism of cell killing. Natural killer (NK) cells isolated from normal human blood and co-cultured with tumor cells at an Effect/Target ratio of 2:1. Q-PCR, ELISA, and FACS analysis were used to evaluate NK cell activation. Intracranial tumor xenografts were utilized to evaluate anti-tumor efficacy. Results: Combination treatment with bortezomib and oHSV induced necroptotic cell death with increased production of mitochondrial ROS and phosphorylation of JNK. Inhibitors/shRNA of RIPK1 and JNK rescued synergistic cell killing. Combination treatment also increased HMGB1 and IL-1α secretion and significantly enhanced NK cell activation and tumor cell killing. Moreover, combinatorial therapy enhanced NK cell therapy. Conclusions: This study provides a significant rationale for triple combination therapy of bortezomib, oHSV, and NK cells to achieve synergistic efficacy, leading to future clinical testing of oHSV with bortezomib in patients.

659. Oncolytic Adenovirus Loaded with Bioactive Modified Peptide as a Novel Approach to Treat Cancer

Mariangela Garofalo1, Barbara Iovine2, Lukasz Kuryk1, Cristian Capasso1, Mari Hirvinen1, Andrea Vitale2, Marjo Yliperttula1, Maria Assunta Bevilacqua2, Vincenzo Cerullo1 1 University of Helsinki, Helsinki, Finland, 2University of Naples, Naples, Italy

Cancer is still a leading cause of death worldwide. Although many kinds of treatment have been developed during the past decades, there is still a lack of effective therapy for advanced cancer. Currently treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase patient life expectancy. Therefore new treatment strategies against cancer are in high demand. Efficient anticancer agent and its targeted delivery into the tumor mass is a key prerequisite for the successful cancer therapy. Oncolytic virotherapy is emerging as a potential approach to treat cancer, using viruses, which are specifically engineered to selectively infect, replicate in and kill cancer cells without causing damage to normal cells. Their combination with chemotherapeutic agents have shown promising results due to the synergistic effect of viruses and drugs; therefore the combinatorial therapy is considered a beneficial approach for cancer treatment. Taken into account these considerations we optimized a strategy to conjugate peptides on the viral capsid, based on electrostatic interaction and used this strategy to deliver an active anti-tumor dipeptide. We used L-carnosine, a naturally occurring histidine dipeptide with anti-proliferative activity. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

A modified L-carnosine, positively charged was absorbed onto the viral capsid of an oncolytic adenovirus to generate a virus-carnosine complex. The complex showed enhanced anti tumor efficacy in vitro and in vivo and higher infectious titer compared to a naked oncolytic adenovirus in colorectal and lung cancer cells. The in vivo efficacy of the complex was analyzed in lung and colon cancer xenograft models, displaying a significant reduction in tumor growth and synergistic effect between virus and dipeptide. Moreover, we studied the molecular mechanisms underlying the effects of complex on tumor growth reduction. Complex can induce apoptosis in both cells lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in further studies also for specific delivery of other active drugs.

660. Enhance Antitumor Effect by Combining Oncolytic Virus HF10 and Bevacizumab in the Treatment of Human Breast Cancer Xenograft

Gewen Tan1, Hideki Kasuya2, Zhiwen Wu2, Tsutomu Fujii2, Maki Tanaka3, Saori Fukuda2, Toyone Kikumori4, Nengquan Sheng1, Zhigang Wang1, Yasuhiro Kodera2 1 Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China, 2Department of Surgery II, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3TAKARA BIO INC, Shiga, Japan, 4Department of Breast and Endocrine Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan Background: The high prevalence and poor prognosis of breast cancer provides a strong rationale for developing new treatment strategies. Oncolytic herpes simplex viruses has a promising prospect because of its selectivity, and the replicating and tumor killing ability. In our study, the antitumor effect by combining oncolytic virus HF10 and Bevacizumab in the treatment of human breast cancer xenograft is evaluated. Methods: The VEGFA gene transcription and protein expression were measured in candidate cell lines (MCF-7,T47D and MDA-MB-231) by RT-PCR, Western blot and ELISA. The MTT analysis was applied to evaluate the efficiency of the combination therapy in vitro. Viral replication was assessed by PCR and plaque assay. Animal models were formed by implanting MDA-MB-231 tumor in the flank site of female BALB/c nude mice. The HF10 group of advanced tumor model received two injections of 106 pfu/ dose intratumorally on Day 1 and Day 14. The HF10 group of single tumor model received single injection of 106 pfu/dose intratumorally on Day 1. The Bevacizumab group received 5μg/g Bevacizumab intra-peritoneally twice a week for two weeks. The combination group received both intratumoral HF10 and intraperitoneal Bevacizumab at the same dose of single treatment groups. The tumor diameter was measured twice a week. On Day 3 and Day 36, the tumors were collected and observed respectively. Histopathological parameters were HIF1α, VEGFA, CD31 driven microvascular density, Caspase 3 and HSV-1 antigen. Results: MDA-MB-231 cells have the highest level of VEGFA expression, while T47D cells have the lowest level. The cytotoxic effect of HF10 is time- and dose- dependent in vitro. The combination therapy does not affect viral replication in vitro. The combination group has the smallest tumor volume comparing with other groups in both animal models (P<0.05). The combination therapy induces synergistic antitumor effect in both animal models. Viral distribution is significantly enhanced in the combination group compared to the HF10 group on both Day 3 and Day 36. Enhanced tumor hypoxia and the up-regulation of angiogenesis gene as well as enlarged population of apoptotic cells in the combination group are also demonstrated in the tumor sample on Day 3. Conclusions: Increased angiogenesis effect and limited viral distribution remain obstacles of oncolytic viral therapy. Anti-angiogenesis reagent is considered to be effective to achieve better antitumor effect of S261