8th International Conference on Oncolytic Virus ...

HUMAN GENE THERAPY 25:1–23 (Month 2014) ª Mary Ann Liebert, Inc. DOI: 10.1089/hum.2014.118

Meeting Report

8th International Conference on Oncolytic Virus Therapeutics Barbara-ann Guinn,1 Lynne Braidwood,2 Alan Parker,3 Kah-Whye Peng,4 and Leonard Seymour 5

Abstract

The 8th International Conference on Oncolytic Virus Therapeutics meeting was held from April 10–13, 2014, in Oxford, United Kingdom. It brought together experts in the field of oncolytics from Europe, Asia, Australasia, and the Americas and provided a unique opportunity to hear the latest research findings in oncolytic virotherapy. Presentations of recent work were delivered in an informal and intimate setting afforded by a small group of attendees and an exquisitely focused conference topic. Here we describe the oral presentations and enable the reader to share in the benefits of bringing together experts to share their findings.

dead and dying cells and presents antigens therein to stimulate the immune system (the ‘‘immune phase’’) to cause further tumor cell death. Vesicular stomatitis viruses (VSV) has low virulence in the population but causes blistering disease in ungulates (cattles, horse, pigs), while human exposure most commonly causes flulike symptoms. VSV has a broad tropism, and so this virus has been modified to express interferon-beta (IFNb and denoted VSV-IFNb). A second virus was developed to also express the sodium iodide symporter gene, NIS (VSV-IFNb-NIS). While both viruses express IFNb at higher levels, there remains a lack of VSV antibodies in the population. This low sero-prevalence makes VSV a suitable vector for systemic therapy. The Viral Vector Production Laboratory at the Mayo Clinic (VVPL, led by Mark Federspiel) makes VSV in a WAVE bioreactor > 99.5% purification with 30–50% yields and the Toxicology/Pharmacology Laboratory (Peng) is involved in the toxicology pharmacological assessment of VSV vectors prior to clinical trial. Mitesh Borad is the principal investigator for the clinical trials and is now overseeing a phase I trial of intratumoral injections of patients with advanced hepatocellular carcinoma (HCC), who are refractory or intolerant to sorafenib and not eligible for transplant. Six patients have been treated to date, three at 105 TCID50 and three at 106 TCID50 dose. The clinical trial is also open for patients with liver metastases. VSV-hIFNb has no NIS reporter so you cannot see the extent of virus replication. Next generation VSV-IFNb-NIS allows visualization of the virus in multiple myeloma (MM) mouse models, including human

Introduction

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any of the presentations showed a ground swell of excitement in the field of oncolytic viruses (OVs). Several clinical trials are now in progress and showing considerable promise. OVs have shown safety and efficacy in phase I/II clinical trials and in terms of responses there are a growing number of examples of patients achieving complete remission (CR) and extended survival. Now that oncolytic proof of principle has been established, we are entering a new era of OV development, with greater focus on enhanced targeting to tumor tissue, improved replication in situ, spread of virus through tumor tissue, and coexpression of transgenes, which enhance anti-cancer activity. There has also been an acceptance that the immune system can often play an important role in effective OV killing of tumor cells, providing several new strategies for design of ‘‘oncolytic vaccines.’’ Finally, most of these advances are founded on basic science, and several presentations showed new insights into the mechanisms of selectivity and action of oncolytic viruses, and how tumor-associated changes often provide a molecular environment that encourages efficient virus replication and spread.

Immuno-virotherapy

Kah Whye Peng, Mayo Clinic, Rochester, Minnesota, described how during the first phase of OV infection the virus kills infected cells (the ‘‘oncolytic phase’’), while often in a second phase the immune system comes to mop up 1

Department of Life Sciences, University of Bedfordshire, Park Square, Luton, LU1 3JU, United Kingdom. Virttu Biologics, Glasgow, G514WF, Scotland. 3 Institute of Cancer & Genetics, University of Cardiff, Wales CF14 4XN, United Kingdom. 4 Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905. 5 Department of Oncology, University of Oxford, Oxford OX2 6HE, United Kingdom. 2

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xenografts in severe combined immunodeficiency (SCID) mice. The study has shown potency (VSV), specificity (IFNb), and tracking (NIS imaging). They can see the virus is fast, following a single intravenous (IV) dose and then daily imaging, within 1 day the virus has homed to the tumor and you can see reduction in the NIS signal as the tumor is destroyed. Perivascular distribution of the virus at early time points, and every 6 hr a rapid expansion is observed, and by 48 hr, most of the infectious foci have coalesced. The speed of VSV spread in this model outpaces the immune system; 72 hr after administration the 5TGM1 tumor has been rapidly killed, and the group showed that IFNb is enhancing the antitumor immunity in mice. Neurotoxicity has not been seen in irradiated SCID mice given 108 TCID50 VSV-mIFN-NIS, and when contrasted to immune competent mice, the effect is not as stark but still good. VSV-IFNb-NIS translation in companion dogs, in this case beagles, received an IV push every 3–5 mins of VSV-hIFNb-NIS diluted in saline. Delivered doses were 108, 109, 1010, and 1011 and TCID50 was measured at each dose. Assessments were made of weight, pyruvate kinase (PK) levels, complete blood count, blood chemistry, coagulation, shedding of virus as detected by RTPCR and inhibitor of virus replication (IVR) assays were performed. Dose limiting toxicity was seen at 1011—including serious vomiting and diarrhea. A transient increase in alanine aminotransferase (ALT) liver enzyme levels were observed in both of the dogs, which had T cell lymphoma and correlated with a more sustained shrinkage of tumor. Currently 2 · 1010 viral particles (vp) dose has been shown to be safe, and minor increments in doses are now being tested. Richard Vile, Mayo Clinic, chose VSV as it is very sensitive to the type I interferon (IFN) response, which is shut down in normal cells but not in tumor cells. This is only the case if tumor cells are truly and completely defective in all aspects of the IFN response. VSV will be an excellent oncolytic. In reality many tumor cells still have the ability to produce and/or respond to IFN. By encoding multiple tumor associated antigens (TAAs) in VSV, the Vile group found that they need to identify relevant TAAs, release TAA for presentation to antigen presenting cells (APC), recruit and activate APC for presentation to TAA-specific T cells, and then increase the frequency of fully activated T cells. IV administration into B16 tumors has an oncolytic effect while depletion of CD4 cells led to removal of the effect, and the depletion of CD8 had a much smaller effect on tumor responses. VSV-TAA need all three self-antigens to generate a response. Systemic treatment of mice with intracranial (IC) tumors led to a significant improvement in survival, but the immunogenic targets differ between the locations. IC studies demonstrated the important combinations of VSV were hypoxia inducible factor-2a (HIF-2a), SOX10, TYRP-1, and c-MYC—giving an interleukin (IL)-17 recall response. Subcutaneously (SC), it was N-RAS, TYRP-1, and CYT-c that generated the largest IL-17 response when compared to the IC combination. When the group looked at tumors from the different sites and examined their antigen expression they could see significant differences in the profiles of antigen expression, even though B16 was used in the same strain of BL6 mice. The group demonstrated that it was the tumor microenvironment that imposed the phenotype on the tumor cells. Best survival was found with a combination of VSV-N-RAS + VSV-TYRP-1 + VSV-CYT-C in IC tissues

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delivered as 12 IC injections. If they added in T cell costimulation, the group showed you can move from extended survival to £ 80% cures. If the same tumor is then placed in different mice the group saw a difference in the combination of antigens that generated the best antitumor response. Avogadri and Wolchok (2012) showed that different antigens are needed to treat recurrent tumors compared to primary tumors. Recurrent B16 tumors can be treated with VSV-cDNA libraries that cross-protect against a range of tumors. TYRP1 is an antigen from ASMEL that is expressed in B16 and is expressed in tumors in both locations. Akseli Hemminki, University of Helsinki, Finland, showed data from the 290 patients who have been treated in an Advanced Therapy Access Program, which is not a trial but a process of individualized therapy, using 10 different viruses. Patients showed mild side effects (grade 1–2) while some serious adverse effects were observed in about 10% of patients (such as emboli). Overall patients required much less chemotherapy, and there were no treatment-related deaths. There was also evidence of immune responses including vitiligo, a classic indicator of antitumor immunity. Lymphocyte numbers decreased in the blood 1 day after treatment, when they increased in numbers in the tumor but recovered within 1 week. There was an induction of antitumor T cells followed by trafficking to tumors, and there was a high concordance between induction of antiviral and antitumoral immunity. In ovarian cancer patients the group could see benefits from oncolytic virotherapy including major molecular responses, molecular CR, and very long overall survival (OS). Cancer treatment with oncolytic Ad armed with different transgenes (GM-CSF, CD40L) with or without capsid modification were shown to be safe. Some patients benefitted from the therapy, and Oncos Therapeutics, Ltd, has completed phase I and is now planning randomized phase II. Adoptively transferred T cells act as a catalyst for preexisting T cells but fails as a therapy when there is lack of recruitment of transferred T cells to the tumor. Causes of this include anergy of transferred cells at the tumor, lack of human leucocyte antigen (HLA) in tumor cells, and a lack of propagation of transferred cells within the tumor and lymph node. Solutions may include cytokines coded by virus to provide trafficking and activation signals. Jean Rommelaere, DKZ-Heidelberg, Germany, described how H-1 parvovirus (H-1PV) is not associated with diseases in humans; it has been shown to be oncotropic and have oncolytic properties in cell and animal models. In addition, it exerts immunomodulating effects that contribute to its capacity for tumor suppression. Cell transformation results in both quantitative and qualitative up-modulations of the cytotoxic activity of the parvoviral NS1 protein. This protein will kill tumor cells in a way that circumvents resistance, unlike conventional anticancer treatments. The PDK1/PKC/PKB pathway represents a contributing factor for H-1PV oncotropism by enhancing the permissiveness of human cells for both nonstructural (NS1) production and functional activation through phosphorylation. The immunostimulating effects of H-1PV are mostly indirect and mediated by infected tumor cells. Compared to cells that are freeze-thawed or irradiated, H-1PV-infected tumor cell lysates are much more efficient at inducing dendritic cell (DC) maturation, tumor-associated antigen cross-presentation, and tumor-specific cytotoxic T lymphocyte (CTL) activation,

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as measured by IFNc production. Similarly, natural killer (NK) cell activation and antitumor responses are much enhanced when the target tumor cells are H-1PV-infected. The dual effect of H-1PV infection of tumor cells, namely direct oncolysis and immunostimulation, could also be demonstrated using patient pancreatic ductal adenocarcinoma cells (PDAC)-xenografted immunodeficient mice, which had been reconstituted with ex vivo PDAC-primed DC/T cells. Furthermore, immunodepletion experiments showed that in an immunocompetent rat model, H-1PV-mediated glioma suppression is in part CD8 + cell-mediated. Similarly, in a mouse glioma model, MVMp-mediated cure leads to a tumor-specific T cell memory response. An ideal OV should be able to not only kill tumor cells but also to be produced in large amounts, resulting in virus spread and infection of tumor cells that were not hit by the primary infection. Forced passaging of H-1PV in human glioma cell cultures led to the isolation of virus mutants endowed with an enhanced capacity for production and propagation in these cultures. Molecular changes associated with H-1PV adaption to enable their efficient production in human glioma cells consisted of single amino acid (aa) substitution(s) displayed on the capsid surface that affected early interactions of the virus with putative intracellular receptor(s), and a deletion in the NS proteins affecting viral DNA replication and progeny virus production. H-1PV is the subject of an ongoing phase I/IIa clinical trial, ParvOryx01, in patients with recurrent gliomablastoma multiforme (GBM) progressing in spite of surgery and radio/chemotherapy. ParvOryx01 is the first oncolytic virotherapy clinical trial approved in Germany and is composed of two arms differing in the first step of the treatment. In study group 1, the virus is applied intratumorally (IT) (single injection), while in study group 2, the virus is injected by IV (five daily infusions). After 9 days the tumor is resected and the same dose of virus is reapplied around the cavity. In the high dose group of IT treated patients, large necrotic areas were seen in two of the four resected tumors. Through fluorescent in-situ hybridization (FISH) and immunofluorescence/ immunohistochemistry (IHC) analyses, there was dosedependent detection of widespread IT virus replication and expression, and also glioma-infiltrating immune cells (in particular T-lymphocytes). Blood analyses gave evidence of transient viremia followed by virus-neutralizing seroconversion and induction of T cell responses mostly directed against viral NS proteins. Remarkably, H-1PV proved able to cross the blood-brain barrier and efficiently penetrate gliomas after IV administration. While no toxicity was observed at the first three dose levels tested, more time is needed to draw a clinical lesson from the trial that is still in progress. Carolien Koks, KU Leuven, Belgium, described her work on Newcastle disease virus (NDV) therapy for high grade glioma. In this work, she uses the murine GL261 glioma model, which is a fully immunocompetent, orthotopic tumor model. The group wanted to investigate the cell death pathways used by NDV in this model, as not many previous studies have investigated the pathways responsible for cancer cell demise following NDV treatment. They found an absence of Annexin V positivity in propidium iodide (PI) negative cells, but abundant Annexin V positivity in the PI positive fraction, both at early and at late time points after NDV infection, indicating that killing by NDV was of a

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necrotic and not an apoptotic nature. This was also clear from the morphology of infected cell cultures as viewed by microscopy. To further demonstrate this, they showed that indeed, inhibition of caspases did not rescue the cells from NDV-induced cell death. In fact, this action further diminished the viability of the cells, indicating the presence of necroptosis, a programmed form of necrosis. Accordingly, a specific inhibitor of necroptosis did restore cell viability. Together, this data demonstrated that NDV kills glioma cells in an apoptosis-independent fashion, with features of necroptosis. In their in vivo model, animals were challenged with tumors and treated IT with NDV or placebo after 7 days, when a tumor mass has been established in all mice. NDV therapy prolonged median OS of treated animals and cured 50% of established gliomas. There were no signs of virus-induced toxicity and no tumor recurrence for up to 100 days. Looking into the mechanism behind NDV therapy, they showed that NDV induces an immunogenic type of cell death in the GL261 cells, as seen by surface exposure of Calreticulin and release of the danger signal HMGB1. Simultaneously, infected cells upregulated their surface expression of tumor-associated antigens, thereby highlighting the immunogenicity of the cells. Secretion of adenosine triphosphate (ATP), another classical hallmark of an immunogenic type of cell death, was absent in this model. The group suggests that this is due to the actively ongoing viral replication within the cells, which requires additional ATP to be used up. NDV therapy increases the brain infiltration of IFNc + secreting T cells, and treated animals have less immunosuppressive myeloid-derived suppresser cells (monocytic and granulocytic) present in their brains. This confirms the first hallmark of immunogenic cell death in vivo, namely a shift toward more immune activation. To investigate the importance of these activated T cells, RAG2 - / - animals specifically lacking B and T cells were challenged with tumor cells and treated with NDV after 7 days. Though NDV could prolong median survival lightly but significantly, cure was only induced in fully immunocompetent animals. Untreated, immunocompetent animals also survived longer than their immunodeficient counterparts. These findings demonstrate that NDV therapy and the immune system seem to act together in a situation where either alone is not sufficient to induce a cure. Finally, long-term surviving immunocompetent animals rechallenged with GL261 cells resisted tumor outgrowth, showing the induction of a long-term antitumor memory response. In conclusion, though NDV was shown to exert a (rather limited) cytotoxic effect on the murine glioma cells in vitro, the in vivo curative effect of NDV therapy depends mainly on the elicited cellular antitumor effects. The group are now looking at combination therapies. Martine Lamfers, Erasmus MC, Rotterdam, The Netherlands, works with Delta24-RGD, which has a 24 bp deletion in the retinoblastoma (Rb) binding site of E1A, which makes it tumor selective, and has an RGD peptide motif modification leading to extended tropism to integrins. Delta24RGD is currently being tested in phase I/II clinical trials for glioblastoma. Oncolytic adenoviruses (Ad) lag behind other OVs with regards to their use as immunotherapeutic agents. To gain insight into this aspect, the group used an immune-competent orthotopic syngeneic mouse model C57BL/6 with the GL261 glioma cell line. Compared to human glioma cells, higher doses of virus are required to

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kill the murine GL261 cells. However, cytopathic effect– like morphology is observed, and up to a 10-fold increase in E1A copy number is detected at 96 hr post-treatment. In vivo, staining for hexon was detected up to day 14 post IT virus injection, and neutralizing antibodies were detected from day 3 post-injection. In this setting, Delta24-RGD cured 50% of mice using a dose of 108 vp. The group then used dexamethasone to knock out the immune system, resulting in complete loss of therapeutic efficacy of Delta24RGD. Analysis of tumor lysates showed that the biggest player was IFNc, which was completely inhibited by dexamethasone. Delta24-RGD infection attracted NK cells, macrophages, CD4 + , and CD8 + T cells to the tumor. Longterm survivors were reinjected with a tumor and were shown to reject it. Splenocytes from Delta24-RGD-treated mice recognize the virus and tumor. In an OVA modeling system, the group investigated whether it was the IFNc or the virus that caused the increased presentation of TAA to CD8 + T cells. It was found that the IFNc was enhancing the response, which correlated with increased major histocompatability complex (MHC) class I expression on the tumor cells. Delta24-RGD has also been shown to interfere with DNA repair systems, making cells more sensitive to standard of care agent temozolomide (TMZ). However, TMZ has pronounced effects on the immune system, which could hamper the viral-induced antitumor immune response. Therefore, the group tested different treatment regimens of combined Delta24-RGD + TMZ therapy. Administering TMZ prior to virus injection hampered the IT influx of DC, NK, CD4 +, and CD8 + cells, which was not the case when TMZ was administered post-virus injection. However, both combination treatment regimens significantly improved survival compared to TMZ alone. The TMZ post-virus regimen also prolonged survival significantly compared to the virus only. In summary, Delta24-RGD injection into IC glioma triggers an innate Th1 immune response followed by an adaptive antitumor response, which is abrogated by high-dose dexamethasone treatment. The addition of TMZ to Delta24-RGD treatment increases the therapeutic efficacy of the virus, especially when administered post-virus injection. Virus and the Immune System Oncolytic cell killing strategies

Paola Grandi, University of Pittsburgh, Pittsburgh, described his work with GBM, which has a median survival from diagnosis of 15 months. Overall strategies by the group include 1) Tumor selective virus human simplex virus-1 (HSV-1) infectivity based on tumor-related plasma membrane targets (retargeted strategies, K-NT-scEGFR: infection occurs only in cells expressing EGFR or EGFRvIII). 2) Control of virus growth based on tumor-specific changes in metabolism, particularly those essential to the tumor phenotype (e.g., miRNAs). ICP4-mir124: the virus replicate ONLY in the absence of mir-124 (cancer cells). 3) Vector expression of genes that modify the extracellular matrix (vector armed with MMP9). 4) Vector expression of genes that induce antitumor immunity.

Matteo Samuele Pizzuto, Imperial College London, United Kingdom, described recent work in which their group had demonstrated the ability of different avian influenza virus (AIV) to efficiently infect and induce high levels of apoptosis in pancreatic ductal adenocarcinoma (PDA) cells in vitro (Kasloff et al., 2014). These results had led to an investigation of the oncolytic potential of an engineered avian-origin influenza virus against PDA. They had focused on BxPC-3, a PDA cell line that was particularly sensitive to AIV infection, but which was previously shown resistant to other OVs (Murphy et al., 2012). Like many other PDA cell lines, BxPC-3 are IFN-deficient due to the loss of genetic material within the chromosome arm 9P, where the IFN genes are located. As such, a conditionally replicating H7N3 low pathogeneticity AIV (LPAIV) was generated through the truncation of the viral NS1 protein, whose major role is to antagonize the host IFN-mediated antiviral response. Production of a protein of only 77 aa’s (NS1-77) resulted in an almost complete loss of the ‘‘effector domain’’ (ED) predominantly involved in IFN evasion and apoptosis modulation. The resulting H7N3 NS1-77 virus lost the ability to counteract the IFN-mediated antiviral response in IFN-competent cells when compared to the wild type (WT) mainly because it was unable to limit posttranscriptional IFN-b induction. The effect of the NS1-77 truncation was shown to be strain specific as the difference in the IFN expression induced by the well characterized human H1N1 PR8 viruses with full length or truncated NS1 proteins was less pronounced. The H7N3 NS1-77 virus did not replicate efficiently in IFN-competent embryonated chicken eggs (ECE), which represent a standard laboratory medium for propagation of influenza viruses, while it reached the same titer as the WT in 7-day-old ECE, where the IFN system is not completely established. The truncated virus triggered higher levels of apoptosis compared to chemotherapeutic agents (Gemcitabine and Cisplatin) in BxPC3 cells, but not in HPDE6 cells derived from normal human pancreatic ducts. Even more interesting was the fact that H7N3 NS1-77 virus induced significantly higher levels of cell death in BxPC-3 than the corresponding WT virus. Nevertheless, whilst the NS1-77 truncation conferred to the H7N3 virus higher selectivity and further enhanced its apoptotic skills in BxPC-3 cells, it displayed the reduced replication observed in the parental virus. The idea then was that the virus was so efficient at promoting apoptosis during infection that it did not give enough time for its viral RNA to reach high copy number. Since the H7N3 virus triggered the intrinsic apoptotic pathway as shown by immunocytochemistry (ICC) targeting active caspases, to rebalance apoptosis induction in favor of virus replication their group investigated the viral PB1-F2 pro-apoptotic protein, which targets the mitochondrial membrane during infection, causing depolarization of membrane potential (DCm), release of cytochrome c (cyt c) and activation of caspase-9-mediated apoptotic pathway. A region near the Cterminus of PB1-F2 called mitochondrial targeting sequence (MTS) is necessary and sufficient for its inner MM localization. Specific leucine (Leu) residues within the MTS are considered to be responsible for mitochondrial targeting (Chen et al., 2010). Depending on the strength of the MTS region, PB1-F2 can also be found in the nucleus of infected cells, where it seems it is important for virus replication, as

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PB1-F2 has been previously suggested as an interaction partner of PB1, affecting viral polymerase activity. In addition to its role in inducing apoptosis, PB1-F2 has been shown to exert an IFN antagonist function by binding to the mitochondria antiviral signaling protein (MAVS), which, as a downstream adaptor for RIG-I, plays a central role in virus-triggered IFNb induction. Therefore, any decrease of PB1-F2 mitochondrial targeting could reduce MAVS inhibition, increasing IFN expression by healthy cells during infection, hence contributing to selectivity and containment of the virus. Based on these considerations to rebalance apoptosis induction in favor of virus replication, an H7N3 NS1-77 PB1-F2 L75H virus with a decreased number of specific Leu within the PB1-F2 MTS was generated. PB1-F2 L75H protein resulted in decreased mitochondrial targeting and a corresponding increased virus replication and polymerase activity in permissive IFN-deficient cells. H7N3 NS1-77 PB1-F2 L75H virus was still able to trigger higher levels of apoptosis in BxPC-3 cells compared to both chemotherapeutic agents and the corresponding full-length NS1 virus. Moreover, the decrease in mitochondrial targeting reduced the virus’s ability to antagonize IFN at the level of MAVS and further enhanced its preference to IFN-deficient systems. In SCID mice bearing BxPC-3-derived solid tumors, H7N3 NS1-77 PB1-F2 L75H virus produced a significant reduction in tumor growth compared to the control within 14 days from the first administration. In conclusion, the group generated an engineered avian-origin H7N3 virus with enhanced selectivity, apoptotic potential, and replication efficiency in IFN-deficient PDA cells, which also displayed a beneficial effect in a xenograft model. The results obtained also underline the importance of strain-specific characteristics like IFN-antagonism and/or apoptosis, emphasizing the possibility to modulate these features by manipulating virus genome to obtain optimal attenuation/replication and apoptosis/ replication balance. Moreover, the data clearly show that PDA cells resistant to other OVs (Murphy et al., 2012) are effectively eliminated in vitro and in vivo using influenza viruses, stressing the idea that research in the area of oncolytic virology should be focused on a broad range of viruses characterized by specific cancer cell permissiveness, and that influenza virus could play a role in further studies on treatment of PDA. Hiroshi Nakashima, Brigham and Women’s Hospital, Boston, described the novel GADD34-armed oncolytichuman simplex virus 1 (oHSV1) vector NG34, which has been developed to improved its safety profile and efficacy. HSV therapy mostly uses oncolytic mutants that originally lacked ICP34.5, as it’s a specific determinant of neurovirulence by suppressing autophagic function in neurons. The protein GADD34 is a homologous protein to viral ICP34.5 to function PP1-binding for enhancing translation via dephosphorylation of eIF2 alpha. However, it lacks the Beclin1 binding domain, therefore, it doesn’t restore the ICP34.5 associated neurovirulence. Hiroshi’s group had engineered c34.5 and UL39 double-null mutant HSV1 vector to express human GADD34 gene under the control of a glioma-active nestin promoter, called NG34. The group showed that NG34 infection led to GADD34 expression in cells and reverses the phosphorylation of eIF2 in glioma cells. Thus, GADD34 can functionally replace the viral ICP34.5 gene for enhanced oHSV1 replication. And NG34

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virus shows efficient cytotoxicity against primary GBM cells and didn’t show lethality at the dose wild-type HSV1 showed lethality after inoculating in mice brains. NG34 significantly prolongs the life of mice bearing an IC primary GBM. If TMZ is combined with NG34 then a synergy was shown using Chou Talaley plots at confidence interval (CI) < 1. Thus, NG34 is a potentially suitable therapeutic bioagent against brain tumors to achieve higher glioma killing effect but fewer side effects when it compares to ICP34.5harbored oHSV1 or ICP34.5 null mutant oHSV1. Cell Virus Interactions

Geoffrey L Smith, University of Cambridge, United Kingdom, described the use of vaccinia virus (VV) to eradicate smallpox and how it remains an enigma in the field of virology. It is the only vaccine to have been used to eradicate a human disease, and yet its origin and natural host are unknown. Scarcely had the ink dried on the WHO document proclaiming the eradication of smallpox in 1980, than two groups working in the United States developed techniques to construct live recombinant VVs that expressed foreign genes. Such engineered VV strains have been useful laboratory tools for cell biologists and immunologists, are being used as vectors for the development of new live vaccines, and can also be used as oncolytic agents, the subject of this meeting. These applications have ensured that interest in VV has endured long after the eradication of smallpox. In this presentation, Geoffrey described how the study of VV is providing new insights into virus–host interactions and will focus on one out of the 200 proteins encoded by this virus. Protein C16 is a 37 kDa intracellular protein that is made early during infection, is nonessential for virus replication, and contributes to virus virulence (Fahy et al., 2008). To understand how C16 functions, the protein was tandem affinity purification-tagged and binding partners were sought by proteomics. This identified the Ku proteins (Ku70 and Ku86) as the major binding partners for C16. In parallel, our group showed that DNA protein kinase (DNA-PK), a trimeric complex composed of the Ku proteins and DNA-PKcs, functions as a pattern recognition receptor for foreign DNA and activates IRF-3-dependent innate immunity (Ferguson et al., 2012). One function of C16 is to prevent DNA-PK binding to DNA and thereby block the innate immune response to DNA (Peters et al., 2013). However, the Ku proteins were not the only proteins that C16 bound, and prolyl hydroxylase domain containing protein 2 (PHD2) was identified as another binding partner (Mazzon et al., 2013). PHD2 is an oxygen sensor and under normoxic conditions will hydroxylate HIF-1a, thereby causing its ubiquitination and degradation by the proteasome. However, during low oxygen (hypoxia) PHD2 cannot hydoxylate its substrates and so HIF-1a remains stable, translocates into the nucleus, and activates the transcription of scores of genes bearing the HIF responsive element. These genes activate a wide range of changes including angiogenesis and a switch from oxidative phosphorylation to glycolysis (the Warburg effect seen in solid tumors). Protein C16 inhibits PHD2-mediated hydroxylation of HIF1a and thereby induces a hypoxic response during infection in normoxic conditions (Mazzon et al., 2013). By expressing C16, VV induces a reprogramming of cellular energy

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metabolism with increased utilization of glutamine and greater synthesis of nucleotides, aa’s, and fatty acid precursors for virus synthesis of virus macromolecules. Len Seymour, University of Oxford, United Kingdom, discussed what is now known as the ten hallmarks of cancer, with a particular focus on metabolism. In nonproliferating differentiated tissue most ATP is produced by the citric acid cycle (TCA) or Krebs cycle and oxidative phosphorylation. The original deduction of the TCA cycle by Hans Krebs used predominantly minced pigeon breast muscle, which have a high demand for energy but undergo only limited cellular replication (Krebs, 1940). In contrast, proliferating cells require anabolic precursors for synthesis of new proteins and nucleotides, and many of these can be garnered from products of glycolysis. This means that proliferating cells use predominantly more glycolysis than oxidative phosphorylation, and there is a five-fold decrease in ATP production for each glucose molecule consumed. This excessive use of glycolysis, even in the presence of oxygen, is also seen in many forms of cancer and termed the ‘‘Warburg Effect.’’ Virus infection of cells often also promotes glycolysis, to provide biosynthetic precursors for manufacture of daughter viruses. This could mean there is a potential cancer-selectivity of mutated viruses that fail to promote glycolysis, providing several options for design of selective virotherapy based on metabolic complementation (Table 1) (Thai et al., 2014). Aladar Szalay, University of Wuerzburg, UC San Diego, and Genelux Corp., San Diego, used vaccinia for his studies; it was chosen because of its attributes, which include the fact that it is not a human pathogen and replicates in the host cell cytoplasm (Chakrabarti et al., 1997). Different promoters with different strengths have been developed so the user can influence the dose delivered and the time at which the genes introduced. VV (Lister) with three insertional mutations constructed in 2003 (Zhang et al., 2007), the group had inserted three genes, these were Renilla luciferase, b-glactosidase, and b-glucuronidase. Visualization of VV colonized C6 gliomas injected with Renilla luciferase showed that the virus went to the tumor and replicated there (Yu et al., 2004). The group could see that the mouse blood vessels were not infected, only the tumor cells. Then the group looked to see whether they could track in real-time the occurrence of therapy (Zhang et al., 2007) using the human breast cancer cell line, GLV-1h68. They found that WT virus in the kidneys, lung, and spleen were cleared by

VV GLV-1h68 treatment. All treatments were delivered as a single injection in mice. GLV-1h68 effectively clears glioma tumors, and a number of pro-inflammatory cytokines were shown to be upregulated. GL-1h68 colonized and eradicated metastases (Donat et al., 2012). Within 21 days, hematogenous micrometastases in the lungs were removed. Single injection of GLV-1h68 virus resulted in dramatic reduction of circulating tumor cells (CTC) in the blood of mice bearing metastatic PC-3 prostate tumors. The group then wanted to investigate whether GLV-1h68 can work in combination with chemotherapeutic agents such as cisplatin (Yu et al., 2009) and Avastin (Frentzen et al., 2009) and with radiotherapy (Advani et al., 2012). Clinical trials were performed with GL-ONC1 to establish which route as well as key considerations—is it safe, shedding, systemic, or regional delivery (trials to date summarized in Table 2). The clinical trials demonstrated infection of tumor tissue, an anti-vaccinia antibody response, and that GL-ONC1 is safe. GL-ONC1-002/MA is a dose escalation trial; in nude mouse the group had used 1 · 105, so they went with a ten times lower dose in humans. Now in phase I/b using 1.667 – 5 · 109 plaque forming units (pfu) and 7–14 days cycle, the next 3 patients are planned with multiple injection times a week. Reported adverse effects were mild and moderate. GL-ONC1-004/TUE had mild side effects with IP administration. Phase I at UCSD Moores Cancer Center consisted of attenuated vaccinia IV and concurrent cisplatin and radiation, currently at 3 · 109 pfu—2 doses in dose escalation. In phase I at Memorial Sloan Kettering Cancer Center (MSKCC), intra-pleural breast cancer patients in cohort four are receiving 3 · 109 pfu. No viral shedding has been detected. Sustained viral production was confirmed in patients in the GL-ONC1-004/TUE clinical trial. In this trial patients to be were treated with an intraperitoneal injection, virus levels increased in the ascites, and this paralleled the production of viral-encoded proteins and fever. Inoculation dose re-isolated from treated patients was 1 · 107 pfu. In cases where tumor biopsies were taken, viral colonization was then confirmed by IHC. CTCs were green (GL-ONC1002/MA), showing the efficacy of the treatment in terms of targeting CTCs. Patients with colorectal cancer and liver metastases were treated and efficacy was confirmed by VPA and qPCR while anti-vaccinia antibody levels plateaued early. With a single dose per cycle there was a peak of antivaccinia antibody levels after 8 days. This cause and effect was even tighter. The peak occurred when multiple doses of

Table 1. Options for DNA Virus Complementation by Tumor Metabolism References Vaccinia C16 inhibits prolylhydroxylase and induces glycolysis via stabilized HIF (Mazzon et al., 2013) Herpes Virus EBV LMP1 inhibits prolylhydroxylase and induces glycolysis via stabilized HIF (Kondo et al., 2006) HSV1 activates glycolysis via 6-phophofructose-1-kinase (Abrantes et al., 2012) Human CMV (HHVS) activates Glut4 expression and activates PFK1 (McArdle et al., 2011; Yu et al., 2011) HHV8 LANA protein increases HIF mRNA (Cai et al., 2006) Adenovirus E4ORF1 induces glycolysis by stimulation of AKT/Myc (Thai et al., 2014) E4ORF4 deregulates AMPK energy sensor AMPK, AMP-activated protein kinase; CMV, cytomegalovirus; HIF, hypoxia inducible factor; HHV8, human herpesvirus 8; HHVS, human herpesvirus; HSV1, herpes simplex virus 1; mRNA, messenger RNA.

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Table 2. Clinical Trials Performed to Date with GL-ONC1 to Establish Which Route and Key Considerations Regarding Safety, Shedding, and Optimal Delivery Mode Clinical site location IV IV Tubingen UCSD MSKCC

Phase I Ib I/II I I

Disease indication Solid tumors Solid tumors Peritoneal carcinomatosis H & N carcinoma Mesothelioma; malignant pleural infusion

Route of delivery

Combination therapy

No. of patients treated

No. of total infusions given

IP IV Intra-pleural

——— ——— ——— Cisplatin + radiation ———

27 16 9 14 11

129 104 23 19 11

77

286

Totals

H & N, head and neck; IP, intraperitoneal; IV, intravenous; MSKCC, Memorial Sloan Kettering Cancer Center; UCSD, University of California, San Diego.

treatment were given per cycle but antibody levels did not maximize within the system. Phase I showed safety but also some clinical benefit and some stabilization of disease. OS of patients with stable (SD) versus progressive disease (PD) have longer survival but this is still a small group of patients. After 4 days of injections the group could see that 5– 10% of tumor cells were infected in the ascites, while this rose to 90% after 8 days. In a syngeneic mouse tumor model study the fluorescent intensity of green fluorescent protein (GFP) was found to be poor compared with TurboFP635 (courtesy N.H. Chen). Studies from the laboratory of Urs Greber, University of Zurich, Switzerland, focus on enhancing oncolytic efficacy of Ad comprising more than 60 different types, and they use at least 10 different receptors (Greber et al., 2013; Wolfrum and Greber, 2013). Virus binding to a receptor can trigger forward signaling, for example, releasing cytokines from immune cells or activating kinases during virus entry (Lutschg et al., 2011; Suomalainen et al., 2001). In addition, the cell can also signal in reverse to the virus, and thereby trigger virus uncoating (Burckhardt et al., 2011; Strunze et al., 2011). The group uses eukaryotic green fluorescent protein (eGFP) expressing replicating HAdV, such as an E3B-deleted HAdV-C2 and a GFP insertion mutant HAdVC2-GFP-V. The former encodes a soluble eGFP under the human cytomegalovirus virus promoter (Yakimovich et al., 2012), and the latter an eGFP-protein V fusion protein, which is incorporated into virions and allows the tracking of virions (Puntener et al., 2011). The group recently developed a method to track the adenoviral genome in the infected cells by using click chemistry. In this method the viral genome is tagged with ethynyl modified nucleosides added to infected cells, incorporated into virions, and then tracked in incoming viruses by covalent labeling using azide-modified fluorophores (Wang et al., 2013). A second interest in the Greber lab is the egress of adenovirus from infected cells, and the spread to neighboring cells. Studies in the human lung epithelia 549 cells show that HAdV-C2dE3B_GFP spreads by lytic infection (Yakimovich et al., 2012). Cell lysis always precedes viral spreading by a few hours. Whether this occurs stochastically or deterministically will have to be shown by mechanistic studies. Regardless of the mechanism, the nucleus breaks apart at later stages of infection, and viruses are released into the media (Puntener et al., 2011). Interestingly, the knock-down of the Golgi specific Brefeldin A resistant factor 1 (GBF-1) boosts

HAdV-triggered cancer cell killing (Prasad et al., Targeting the Secretory Pathway Enhances Human Adenovirus Infection and Cancer Cell Killing. In preparation). GBF1 knock-down is known to induce the unfolded protein response (UPR) pathway (Citterio et al., 2008). UPR downregulates cytoplasmic proteins and increases the protein folding capacity in the endoplastic reticulum by enhanced synthesis of chaperones. Further studies are ongoing to determine the mechanism of how UPR enhances cancer cell killing by adenovirus. Roberto Cattaneo, Mayo Clinic, described how measles virus (MV) spreads in the airway’s epithelium. These findings are relevant for oncolysis because the MV epithelial receptor nectin-4 is highly expressed in lung, breast, and ovarian cancer. Structure-guided studies of how the viral attachment protein interacts with nectin-4 indicates that it takes over the recently described nectin canonical adhesive interface (Harrison et al., 2012). Strikingly, MV infection spreads without cytopathic effects in well-differentiated primary human airways epithelia, and transepithelial resistance remains constant. Thus in this tissue there is ‘‘de´tente, not war,’’ as in other long-established virus-host interactions. Remarkably, viral spread occurs through ‘‘canals’’: cytoplasmic contents flow out from infected epithelial columnar cells to adjacent cells through fenestrations that open proximal to their apical side. It is thought that the viral membrane fusion apparatus exploits nectin-4 to open pores at the adherens junction. Since these pores are constrained by the actin belt cytoskeleton, nearby tight junctions, and desmosomes, they become stable canals. Other OVs that use junctional proteins as receptors (Miest and Cattaneo, 2014) may spread through the ‘‘make de´tente, not war’’ strategy; high levels of viral replication without much cell death have been documented in current human clinical trials. In these cases, it may be necessary to push the virus into the war mode after cancer cell entry. Tran Nguyen, Harvard Medical School, Boston, described the insights on an obstacle of OV spreading during the course of the host innate immune response, and focused particularly on the antiviral role of ubiquitin-like modifier, IFN-stimulated gene 15 (ISG15). ISG15 conjugates to newly synthesized proteins on polysomes in response to viral infection and immune responses, and therefore, has an emerging role in antiviral responses. Although the detailed antiviral mechanisms of ISG15 conjugation are largely unknown, it was found that ISG15 is involved in nutrient starvationindependent selective autophagy, and it was demonstrated

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that selective autophagy factors, histone deacetylase 6 (HDAC6), and p62 can directly bind ISG15. The ISG15 and HDAC6 binding domains were also identified by coimmunoprecipitation and pulldown assays. It was also found that during IFN stimulation, ISG15 localizes with HDAC6 and p62 in the cytoplasmic puncta areas in human gliomas, indicating ISG15 (and its conjugates) as selective autophagic degradation targets. In addition to the molecular mechanism, a novel inducible ISG15 conjugation system was developed in human glioma cells without IFN stimulation to evaluate pure ISG15 functional effects. Under ISG15 induction, replication kinetics of oncolytic HSV1 (o-HSV1) was impaired in response to ISG15 conjugation. The suggestion is if we can interfere with this mechanism, we could enhance OV therapy in the future. Targeting Delivery and Imaging

Gabriella Campadelli-Fiume, University of Bologna, Italy, in collaboration with G. Gatta, L. Menotti, V. Leoni, P-L Lollini, P. Nanni, A. Palladini, and P. Malatesta, highlighted that the success of T-Vec is an encouragement to pursue improvements of o-HSVs. The o-HSVs now in clinical trials are highly attenuated; their cancer specificity rests on defects of cancer cells to mount an innate response to the virus. Their strength is their safety. Their weakness is due to the fact that tumors differ with respect to genotype; hence, attenuated viruses are effective against some tumor genotypes but not against others. More robust viruses are needed. This aim has been partly achieved by arming the oHSVs with cytokines. The aim of Campadelli-Fiume’s lab was to leave the virus unattenuated and engineer an o-HSV that infects tumor cells only by retargeting the HSV tropism to cancer-specific receptors. The retargeted viruses carry modifications only in gD, the receptor-binding glycoprotein of HSV, and are otherwise wild-type. Why retarget HSV to HER2? HER2 is overexpressed in approximately 25% of breast and ovary tumors, it is found in gastric tumors and in a fraction of glioblastomas among others. It is associated with poor prognosis and high relapse rates. Anti-HER2 antibodies are the standard of care in some cancers, however, only 25% of patients respond to them, and of those who respond, many develop resistance within 1 year. To overcome or prevent resistance, antibodies to different sites of the HER2 receptor were developed. The overall retargeting strategy developed by the group consists of the insertion in gD of the sequence encoding a single chain antibody (ScFv) to HER2, coupled with appropriate deletions in gD, aimed at abolishing the virus tropism for the natural receptors. The group identified two privileged positions in gD and made two viruses: R-LM249, in which the core of gD is deleted and replaced with ScFv to HER2, and the other virus, named R-LM113, carries a deletion in the N-terminus of gD, and the insertion of ScFv to HER2. Both viruses carry GFP to monitor virus growth. Both viruses are fully retargeted, that is, they only infect cells through the HER2 receptor. The group looked at the efficacy of the retargeted o-HSVs against xenotransplanted tumors in nude mice. They found that 60% of mice were tumor free following multiple administrations with RLM249, compared with no survivors in the absence of treatment. The HER-2-retargetted RLM249 is effective against trastuzumab resistant human cells. R-

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LM113 was employed in a syngeneic glioma model, by stereotactic administration, with significant increase in the number of surviving mice. The group asked whether oHSV can be administered by IP and IV. Model SK-OV-3 cells were injected IP into Rag2-/-;IL-2rg-/- mice to mirror the peritoneal carcinogenesis observed in women. Subsequent treatment with RLM-249 led to significantly extended survival. GFP showed that GFP-HER2-HSV exclusively populates the tumor. Antitumor efficacy was observed also in model 2, consisting of Rag2 - / - ;IL-2rg - / - mice that develop multiorgan metastases upon IV injection of MDA-MB453 or BT-474 breast cancer cells. The ongoing studies were designed to evaluate the delivery of RLM249 to metastatic cancers by means of carrier cells. Infection was aided by carrier cells. Preliminary results show that the RLM249infected with carrier cells home to the tumor and enable the spread of progeny RLM249 to cancer cells in vivo. The group also found very low metastatic tumor burden in mice injected with virus-infected carrier cells. In summary they showed administration of HER2-retargeted HSV by three routes, following loco-regional administration against glioblastoma, IP administration against IP metastatic ovary and breast cancer, and IV administration via carrier cells. Kerry Fisher, University of Oxford, United Kingdom, explained how the systemic delivery of OVs has remained a substantial challenge for the field. Sequestration of virus particles by the reticulo-endothelial system together with neutralizing antibodies and complement take a heavy toll on the administered dose. A number of different strategies have been proposed or have been attempted in the clinics or preclinically, including cell-based delivery, dose scheduling, and vector modification. The Fisher group had focused on using a group B OV, Enadenotucirev (formerly called ColoAd1), that retains activity in human blood and can be produced in sufficient quantities to allow repeated administration of a ‘‘breakthrough dose.’’ A breakthrough dose is the amount of virus required to saturate neutralization or unwanted interactions with blood components. Dose scheduling and optimization of infusion rates can then be used to manipulate clearance by the innate immune system and maximize the number of viable virus particles in the blood stream in order to achieve tumor delivery. In clinical trials the group was able to show that a data-driven optimization of virus kinetics could achieve convincing delivery to tumors and expression of virus late proteins. This data validated the utility of Enadenotucirev as an oncolytic product candidate and provides a strong platform for next generation agents expressing therapeutic biologicals. Tony Reid, University of California, San Diego, discussed the endpoints used in clinical trials, stating that the size of a tumor is not always an indication of response. Sometimes tumor size is unchanged because of immune infiltration, so it is important that we choose appropriate end points. One of the best end points is probably SD over a prolonged time but that may impact on a desire to try other treatments if the one being used is not an improvement. However it may require 2 years to see the effect of a new treatment. This was seen with the ONYX-015 clinical trial, for example. If we consider the RECIST response evaluation criteria in solid tumors (Eisenhauer et al., 2009), this may not capture critical responses. It can take 2 years to see CR. Immunological criteria that should be considered may

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include immune infiltration, absence of symptomatic deterioration, and the occurrence of new lesions. In the TRIBE study design, ROLFIRI + bevacizumab was given to patients for up to 12 cycles compared with FOLFOXFIRI + bevacizumab. The group looked at endpoints but noted that if you look at them too early then the novel treatment arm looks ineffective; however, when you look at later stages you can see the true impact of the new treatment on patient survival. You do need a quick read of how the clinical trial is progressing, but we may need to identify different criteria to do this. There are currently 50 small molecule inhibitors, and a number look like they don’t work until you pick a target population in whom you can see significant improvements in response rates and OS. You can see the impact of drugs, but this will require the utilization of personalized therapy. There are 138 patients nationwide with pancreatic cancer in whom to evaluate your drug, and then there are geographical, institutional, and financial considerations. We have now moved into an age of precision medicine, and we need to pick the right patients and the right endpoint. There is a growing list of response criteria (Table 3), and these should be considered to help us develop treatments for the right patients. Mark Ferguson, Queen Mary University of London, United Kingdom, used VV in his studies citing cytoplasmic replication and its quick life cycle (12–24 hr), natural oncotropism, availability for delivery IT or IV, and a good safety profile as reasons why. Systemic delivery is attractive for the treatment of metastatic disease as it treats both known and unknown disease deposits simultaneously. However, viruses delivered systemically must overcome the various immunological barriers that include complement, antibodies, and cellular components including resident macrophages in the spleen and lung. A total of 108 pfu of VV was delivered systemically in nude and immune competent mice bearing CT26 flank tumors, but only in the athymic mice was an effective infection established at the tumor sites. They immediately assumed T cells were central to this issue, but it soon became obvious they were not. It was macrophages that were playing a crucial role in early clearance of VV in the WT mice. The group used clondronate liposomes to deplete macrophages in these mice, after which they observed similar infections to those seen in nude mice. The problem with this approach is that clodronate liposomes irreversibly clears the macrophages, but this is not desirable, as these cells may play a beneficial role after the infection in the immune-mediated clearance of the tumors. So the group looked for something else to perturb the interaction between macrophages and virus early in the time-course of systemic delivery. They found that class I

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PI3 kinase inhibitors are potentially useful to transiently inhibit macrophages. IC87114, p110 delta inhibitor, is almost as good at reducing viral attachment to macrophages as the pan inhibitor Wortmannin. In further in vivo studies luciferase assays were used to show that pretreatment with IC87114 results in significantly more virus in the tumor than in its absence. Encouragingly, there was similar minimal off-target viral recovery in the mice treated with this optimized regime as compared to virus alone. The group then showed that this strategy enhances the efficacy of systemically delivered VVL15 in vivo. There was a modest survival advantage using this approach in both BALB/c mice bearing CT26 flank tumors and in an orthotopic 4T1 tumor model. So their data suggests that macrophages appear to be the key early cellular player in systemic clearance of VV, and p110 delta blockade is an effective strategy for enhancing systemic delivery of VV in preclinical models and could be a useful adjuvant in VV clinical trials. Virus Genetic Engineering

John Bell, Ottawa Hospital Research Institute, Canada, has been investigating strategies to enhance OV growth in tumors using either high throughput screens or rational combinations of cytokines. In collaboration with Jean Simon Diallo they have found that histone deacetylase (HDAC) inhibitors can in some cell lines conditionally regulate the growth of OVs. This provides a pharmaco-viral approach to potentially dynamically regulate OV replication in the clinic. Pancreatic tumors cultured in nonobese diabetic (NOD)SCID mice maintain the same histology and show the same metastastic profile as they do in patients. In one patient used to exemplify this point, you can see that both the tumor and the tumor stroma influences how the tumor behaves. Does there exist mutual interactions between cancer associated fibroblasts (CAFs) and tumor cells important for OV therapeutic outcome? Paracrine factors from CAFs promote OV infection using either rhabdovirus or vaccinia-based vectors. This activity stems from the secretion of fibroblast growth factor-2 (FGF-2) from CAFs that leads to sensitization of tumor cells. For instance, the pancreatic cancer cell line MIAPACA can form pancreatic tumors very quickly without stroma and is relatively resistant to OV infection; however, if grown with CAFs or FGF2 is added directly by injection then they become sensitized. FGF downregulates reteinoic acid inducible gene 1 (RIGI), indicating cross-talk with the IFN pathway. Tumor explants from PCa patients showed varying FGF2 levels, and the group found higher virus titers in patients with higher FGF levels. Add FGF2 into an oncolytoc virus (rhabdovirus virus) and use it to treat

Table 3. Response Criteria Judged Based on Serum Prostate-Specific Antigen Levels—Modification of Immune-Related Response Rate Described by Wolchok et al. (2009) Response Complete Response (CR) Partial Response (PR) Stable disease (SD) Progressive disease (PD) PSA, prostate-specific antigen.

Serum PSA levels PSA £ 4 ng/mL and cancer lesion not detected from biopsy specimens of the prostate ‡ 50% decrease in PSA < 50% decrease or < 25% increase in PSA ‡ 25% increase in PSA in two consecutive tests at least 4 weeks apart. The increase in PSA must be ‡ 2 ng/ml.

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tumors derived from the 786-0 kidney cancer cell line. Rhabdovirus virus (MG1) alone is not very good, but when you treat the tumors with G1-FGF then you observe extensive tumor killing and robust virus replication. This treatment also works with Mia-Paca and OvCar too. When MG1 or MG1-FGF2 is added to GM38 normal fibroblasts there is little effect, so the virus activity is tumor selective and unaffected by the addition of FGF2, and this is also the case with more potent OV. David Stojdl, Children’s Hospital of Eastern Ontario, Canada, has been engineering a new rhabdoviruses for brain cancer therapy. Using a bioprospecting approach to identify novel viruses in this family, his group have identified a candidate with improved safety margins in the brain for rhabdovirus oncolytic vaccine trials. The Farmington (FMT) virus, an oncolytic rhabdovirus, is nontoxic, has one of the highest therapeutic indices in the rhabdovirus family, lacks pathogenicity, has no preexisting immune responses in the human population even though it is endemic to the United States, is easily manufactured, and has been shown by these investigators to be well tolerated by IV and IC routes. High doses can be directly injected into the brains of susceptible mice with minimal inflammation, little neuronal damage, and no impact on normal motor function compared to saline controls. The receptor for FMT is LDLR, and anti-LDLRtreated tissue shows no virus infection. IFN is most likely the pathway responsible for maintaining natural FMT tumor selectivity, since FMT does not block type I IFN production, and infection is IFN-dependent. In addition, FMT appears to have a second, IFN-independent tumor selectivity mechanism targeting virus infection only to replicating cells, which may explain its unique lack of neurotoxicity. FMT shows in vivo tumor-selective growth and cytotoxicity with an EC50 that is low for many GBM-derived cell lines (MOI < 0.1). While all tumors tested to date are robustly infected by FMT, approximately 50% are partially or entirely resistant to cytotoxicity after virus infection. This group has shown that FMT kills primarily through the extrinsic apoptosis via the death receptor pathway. Infection leads to cytokine production and subsequent FAS, tumor necrosis factor (TNF), or death receptor expression; this likely explains why FMT doesn’t kill all GBM cell lines, as these proteins can be lost with disease progression. FMT induces acquired antitumor immune responses, which are CD8 + T cell–dependent. Thus, modifications to FMT that improve the immuno-stimulatory release of cancer cell antigens (in terms of the quality of death and the release of damage associated molecular patterns (DAMPs)) will likely consolidate antitumor immune activation and therapeutic efficacy. This group has generated recombinant FMT viruses expressing cell death transgenes to enhance the immunogenicity of tumor cell death by activating apoptosis, necrosis/ necroptosis or autophagy. Assays on four GBM cell lines (three human and one mouse) identified several ‘‘armed’’ FMT viruses with improved expression of immunological cell death markers, such as ATP. These transgene-expressing FMT viruses caused rapid lytic death in CT2A murine glioma cells but, importantly, maintained their safety profile in normal primary human fibroblasts. Nori Kasahara, University of California, Los Angeles (currently at the University of Miami), has developed retroviral replicating vectors (RRV), which replicate in a tumor-

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selective manner because of their requirement for actively dividing host cells to establish a successful infection, and because tumors generally exhibit local immune-suppression of innate and adaptive immunity. RRVs permanently integrate into the cancer cell genome, and spread efficiently through the tumor mass without immediate cell lysis. The group has inserted a prodrug activator gene, yeast cytosine deaminase (CD), which enzymatically converts the prodrug 5-fluorocytosine (5-FC), an antifungal agent, into the active anticancer drug 5-fluorouracil (5-FU), within the infected cancer cells. As the retrovirus stably integrates into the cancer cell genome, any residual infected tumor cells that are not killed by prodrug activation acts as a reservoir that continues to produce virus and thereby enables infectious spread to resume during tumor recurrence. Hence, multiple cycles of produg can be administered, and this strategy continues to show therapeutic efficacy in mice and rats after a single RRV injection. In immunodeficient xenograft models of human glioblastoma, it was previously shown that this approach significantly prolongs survival and effectively converts this rapidly lethal malignancy into a chronic disease (Tai et al., 2005), and in syngeneic glioma models in immunocompetent hosts, the immune system can subsequently eradicate minimal residual disease and effect a ‘‘cure’’ (Ostertag et al., 2012). RRV can infect and replicate in most human cancers, and Toca 511, an improved RRV with enhanced stability over serial passage and an optimized CD gene, has been shown to spread through animal tumors, is selective for the tumor, and kills tumor cells through CD expression combined with 5-FC administration (Perez et al., 2012). This RRV is currently being evaluated in three multicenter phase I dose-escalation clinical trials employing different routes of administration (www.clinicaltrials.gov, NCT01156584, NCT01470794, NCT01985256). In a TMZresistant Tu-2449 glioblastoma model, TMZ is ineffective alone, and in combination therapy Toca 511 + 5-FC does not further augment efficacy; if the dosing and scheduling regimen is not optimized, it can even detract from Toca 511 + 5-FC treatment efficacy. However, in a TMZ-sensitive U87 model, TMZ in combination with Toca 511 + 5-FC can vastly improve survival (Huang et al., 2013) over either agent alone. It was also shown that the use of cellular delivery vehicles such as human mesenchymal stem cells can serve as motile producer cells, which help to improve IT vector dissemination by local RRV production as the cells migrate through the tumor mass and thereby enhance tumor transduction results. Extracellular stroma or necrotic tissue may act as a barrier to virus replication, so human mesenchymal stem cells that show tumor-homing capabilities and convert into cancer-associated fibroblasts within the tumor microenvironment may help disseminate the virus more efficiently by seeking out nests of cancer cells that are not infected. Douglas Jolly, Tocagen Inc., San Diego, described their clinical data from first-in-human phase I dose-escalation trials with Toca 511 to treat patients with recurrent highgrade gliomas (stage III gliomas and stage IV GBM), currently being conducted at multiple clinical sites in the United States (UCLA, UC San Francisco, UC San Diego, Cleveland Clinic, Ohio State, Swedish Medical Center, Henry Ford Health System, and JFK Memorial). For GBM, current treatment options are limited and median OS from

OVC ANNUAL MEETING, OXFORD 2014

diagnosis in the United States is 10 months (Yabroff et al., 2012) and around 16 months with maximal therapy. In recurrent GBM patients, historical data indicate a median OS from recurrence of 30–42 weeks with the higher end representing patients at first recurrence. In the first clinical trial (www.clinicaltrials.gov, NCT01156584), Toca 511 is delivered transcranially by magnetic resonance imaging (MRI)–guided IT injection through a stereotaxic needle or catheter, with vector dose escalation performed according to standard 3 + 3 design, and followed by courses of Toca FC (an improved extended-release formulation of 5-FC) beginning 1 month after Toca 511 injection. Even in the lowest dose cohort, tumor samples recovered from a patient showed evidence of RRV infection in the tumor, and virus replication by quantitative PCR and IHC. The second clinical trial (www.clinicaltrials.gov, NCT01470794) is also a phase I dose-escalation study starting at a threefold higher dose, but the virus is injected into multiple sites around the tumor bed after surgical resection, followed by an interval of 7 weeks to allow virus spread, and then followed by Toca FC treatment. Evidence of antitumor activity was observed by MRI in some subjects after Toca FC. Of note, local inflammation after prodrug activator gene therapy can be mistaken for tumor progression, frequently referred to as ‘‘pseudo-progression.’’ For example, a patient in the lowest dose cohort appeared to show tumor progression after Toca 511 and Toca FC treatment, but when the tumor was subsequently resected, histology showed mostly dead tissue, dying cells, and lymphocytic infiltration indicating immune involvement, as well as residual tumor cells staining positive for virus and CD proteins, indicating viral persistence, and continued replication. In a recently initiated third clinical trial (www.clinicaltrials.gov, NCT01985256), Toca 511 is being administered by IV infusion with a dose escalation plan to administer higher doses than in the other trials. Professor Kasahara’s group has previously shown that IV delivery can enable RRV to reach multiple tumor sites in a CT26-Luc multifocal liver metastasis model of colorectal cancer (Hiraoka et al., 2007). Tocagen researchers have now shown that IV-administered RRV can spread to brain tumors in preclinical orthotopic models. In the current human trial, single patient dose cohorts are being employed to establish safety and feasibility at lower doses. In the first two trials spikes in viral RNA in serum and whole blood DNA were observed in about 10% of patients, which spontaneously cleared with emergence of antiviral antibodies. In summary, clinical evidence of benefit associated with alleviation of symptoms has been observed, consisting of disease regression and stabilization by MRI, and tumor necrosis was observed in resected tumor specimens after Toca 511 and Toca FC prodrug activator gene therapy. Median OS and landmark survival at 6 and 12 months (OS-6, OS-12) is higher than seen in historical patient data as reported in the postTMZ literature. Prodrug activator gene therapy with Toca 511 appears to be well-tolerated, infection has been observed to persist in residual or recurrent tumor but without infection of healthy cells, and evidence of clinical activity has been obtained. Jan Hanauer, Paul-Ehrlich-Institut, Langen, Germany, described the MV from vaccine strains, retargeted with DARPins (designed ankyrin repeat protein)—very stable and quite small (15 KDa) targeting domain. The issue of

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tumor heterogeneity (which may mean that some subpopulations of tumor cells are unaffected by monospecific targeted treatments that can lead to relapse) can be counteracted by coupling two different DARPins to functional, bispecific targeting domains. Pascal Buijs, Department of Surgery, Erasmus MC, Holland, explained his work on recombinant Newcastle disease virus (rNDV), an avian paramyxovirus (APMV-1: (-)ssRNA 15186nt), for the treatment of pancreatic adenocarcinoma. The virulence of NDV is dictated by the protease cleavage site of the fusion protein. NDV induces (mitochondrial) apoptosis, necro(pto)sis and the formation of syncytia. Although potentially fatal in birds, NDV only causes mild symptoms in infected humans. It also exhibits naturally tumor specific replication, however, there is a lot of variability between efficacy of oncolysis within human pancreatic cancer cell lines (HPACs), and most cells can be protected by pretreating them with IFN. Defects in innate immunity pathways in HPACs do not always correlate with susceptibility for NDV infection, replication, or cytotoxicity. Blocking innate immunity could therefore improve oncolytic activity, so the goal of this group was to improve oncolytic efficacy of rNDV without increasing virulence. The group cloned pNDVs and inserted either hIFNb between P/V and M to derive rNDV-hIFNb F0 or NS1 to derive rNDV-NS1 F0. These immunomodulating rNDVs were compared with a virulent rNDV that possesses a multibasic cleavage site, rNDV F3aa. Only this replicating rNDV F3aa was capable of overcoming inherent resistance in some of the HPACs, while IFN inducing or blocking rNDVs were capable of immunomodulation, but this did not lead to relevantly increased oncolytic efficacy. Similarly, treatment of tumors in an immune-deficient SC mouse model showed that rNDV F3aa was the only virus with a clear treatment efficacy, with two of three HPACs achieving CR (n = 5/6), rNDV F0 + / - hIFNb or NS1 were not effective. Alexander Muik, Georg-Speyer-Haus, Frankfurt am Main, Germany, described VSV as one of the most potent OV with impressive antitumor activity in preclinical models. In terms of pros there is no preexisting immunity, infections are asymptomatic, and VSV replication is fast and highly cytopathic. In terms of cons there is strong inherent neurotoxicity seen in rodents and nonhuman primates, which has hampered clinical development. To detarget VSV from neurons the group changed the neurotropic glycoprotein VSV-G in the genome for the non-neurotropic glycoprotein, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) of the LCMV WE54-strain. Although the titer is one log lower it mediates a broad tropism. The VSV/ LCMV chimera, rVSV(GP), lacks neurotoxicity, even when directly injected into mice brains (CD1 and Balb/c mice) at doses in excess of 108 pfu, whereas already 10 pfu VSV-WT is lethal. VSV-WT infection of the central nervous system (CNS) leads to CD3 + and CD11b + immune cell recruitment while rVSV(GP) does not. rVSV(GP) is also a safe systemic therapeutic as shown in mice even at 109 pfu. However, ALT levels rise transiently for both VSV-WT and rVSV(GP) but then drop down again. Moreover, rVSV(GP) is a potent oncolytic in vitro. rVSV(GP) is superior to parental virus in some cell lines including breast, brain, lung, and skin cancer. rVSV(GP) is a potent oncolytic in vivo. In an SC glioma tumor model a double dose of 25 pfu of VSVWT or rVSV(GP) showed that rVSV(GP) led to tumor

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regression within 50 days. In terms of survival rVSV(GP) is curative while VSV-WT is not. In ovarian and skin cancer SC tumor models the group saw delayed tumor development and/or enhanced survival with rVSV(GP) compared to rVSV as well. Most importantly, a triple dose of 108 pfu rVSV(GP) IV was able to cure > 80% of orthotopic U87 human glioma-bearing immunodeficient NOD/SCID mice, and a single IT dose of 108 pfu led to a cure rate of 50% and 81% in orthotopic syngeneic primary and secondary brain tumor models, respectively. In terms of humoral immunity, rVSV induces neutralizing antibodies while rVSV(GP) failed to induce neutralizing antibody responses even on multiple repeat injections. Moreover, rVSV(GP) is not inactivated by human serum complement, unlike rVSV, and so there is now an intended move to clinical trials by an academic spin-off called ViraTherapeutics. Alice Brown, PsiOxus Therapeutics, Oxfordshire, United Kingdom, described the next generation of ColoAd1 (Enadenotucirev, EnAd) viruses that are ‘‘armed’’ to deliver therapeutic antibodies to tumors. Their work focuses on targeting the tumor microenvironment by in situ delivery of mAbs, which could complement EnAd’s mechanism of action. EnAd is a chimeric subtype B adenovirus, developed by bioselection from a library of adenoviruses, which shows good selectivity and oncolytic activity for tumors (Kuhn et al., 2008). Clinically, EnAd virus delivery and production of viral protein has been demonstrated in patients’ tumors following IV administration of virus, indicating that EnAd may also be a good platform for local delivery of encoded therapeutic transgenes to tumors. To facilitate ‘‘arming’’ EnAd with a transgene of interest, the group developed a flexible cloning platform that allows straightforward production of a panel of viruses for evaluation. Initially a set of reporter viruses with different transgene cassette designs were used to optimize ‘‘armed’’ virus design for both transgene expression and the selectivity and oncolytic activity of the viruses. The group have now armed the virus genome with mAbs, including a full length humanized antivascular endothelial growth factor (VEGF) antibody, and have shown good mAb expression and virus activity despite the encoding of a complex protein. VEGF binding enzymelinked immunosorbent assay (ELISA) shows anti-VEGF antibody was expressed prior to infected tumor cell lysis, and that antibody production increased over at least 72 hr. Quantification of anti-VEGF antibody yield from virus infected cells indicates the potential for high local concentrations in tumors (*10–20 lg/ml). The group also demonstrated that this anti-VEGF antibody armed virus (termed NG-135) had comparable infection kinetics and oncolytic potency to EnAd in a panel of cancer cell lines, as well as good production of antibody in all carcinoma cell lines tested. In in vivo models bearing human xenograft tumors the replication of the NG-135 and EnAd viruses were also similar and anti-VEGF antibody was detected in tumors at all time-points post NG-135 treatment (days 3, 7, and 14) and at lower levels in the blood stream by day 14. Demonstration of good antibody delivery in situ has now led the group to also demonstrate EnAd activity and high levels of transgene expression when armed with antibody scFv’s. The potential to arm with several of these scFv antibody domains to target multiple pathways that influence the tumor microenvironment is now under investigation.

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Rob Hoeben, Leiden University Medical Center, The Netherlands, works on oncolytic reovirus, which is nonpathogenic in humans. There are ten different dsRNA segments that constitute the genome making it difficult to manipulate. The group’s mission was to expand the reovirus’s tropism, as it is currently limited by expression of its receptor. The group modified the spike protein by inserting a His-tag in the reovirus spike protein so that the modified spike can bind an scFv-His expressed on cells. The scFv-His can act as a surrogate receptor on cells lacking the reovirus receptor protein JAM-A (van den Wollenberg et al., 2008). Viruses were produced to express the His-tagged spike, but one had lost the His-tag. This mutant virus (jin-1) showed a much wider tropism to many cell lines which lack both JAM-A and the scFv-His. This and two more jin mutants have mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the tail of the spike protein (Reiter et al., 2011; van den Wollenberg et al., 2012) and depend on sialic acids for infection. There is no nice location to insert a transgene in the reovirus genome. The group considered decapitating the spike protein. As the head domain of the spike is dispensable for spike trimerization, the group made tail-domains mutant based on jin-2 and fused this with the codons for the iLOV protein. Replicating virus could be generated and passaged and the iLOV transgene is well expressed. This strategy allows arming replication-competent oncolytic reoviruses by insertion of therapeutic transgenes in the reovirus genome. Progress in Virotherapy Clinical Trials

Chantal Lemay, Ottawa Hospital Research Institute, Canada, described their work translating the oncolytic prime/ boost vaccination from a lab discovery into a first-in-man clinical trial. The work centers around an oncolytic rhabdovirus (Maraba virus, MG1 mutant) expressing MAGE A3 (MG1MA3) given IV as a boost. The priming vector is a nonreplicating adenovirus given intramuscularly 2 weeks previously, which also expresses MAGE A3 (AdMA3). This heterologous prime/boost vaccination ensures the immune response is focused on the tumor antigen, instead of the virus. Bench-to-bedside development has taken place over the last 3 years, leading to a multicenter phase I/IIa trial in Canada. The translational work was facilitated with a project management workflow and in-grained quality management. Assay development and process development were started early and are ongoing to ensure reliable results and quality products. A 28 cynomolgous macaques toxicity study was undertaken using the same treatment regimen as will be used in the trial. Tumorfree animals weighing approximately 5 kg were treated with up to 2 · 1011 pfu of MG1MA3 and demonstrated no grade 3–4 toxicities. Tissue and shedding analysis using RT-qPCR demonstrated MG1 viral genomes mostly in spleens, lymph nodes, and a few in urine and saliva. No replicating virus was detected. Immune analysis performed on peripheral blood demonstrated that animals treated with the prime/boost (but not the boost alone) has tremendous immune responses generated against MAGE A3. GMP-grade MG1MA3 and AdMA3 have been manufactured and released and regulatory submission is imminent. Mark J. Federspiel, Mayo Clinic, directs the Mayo Clinic VVPL that has specialized in manufacturing OVs for human

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phase 1/2 clinical trials. The VVPL is one part of the infrastructure developed at the Mayo Clinic for translating virus-based therapies to human clinical trials. This infrastructure has translated four different viruses into phase 1 clinical trials for 10 different cancers. Manufacturing and the manufacturing process for clinical products is a critical component of translation in that the process is the product and should manufacturing fail so will the product. A huge variety of viruses are potential oncolytic agents for cancer treatment. All virus groups have their own specific features, which affect manufacturing for human clinical use. Largescale propagation and purification of different viruses have many variables: the cell lines for propagation, the differences for enveloped and non-enveloped viruses, the vast variability of virus sizes that may preclude sterile filtration, cytotoxicity to cells they infect thereby precluding the use of producer lines, and because of the cytolytic nature of many of the viruses cellular genomic DNA contamination is a major problem. For the translation to human therapy, the dose level needs to be increased by 1,000–3,000 times the dose for efficacy in mice. The Food and Drug Administration (FDA) had purification concerns including the need for a guarantee that no intact cells pass through the manufacturing process and the goal of keeping the cellular genomic DNA contamination to < 10 ng per dose or digest products of < 500 bp. However, since oncolytic virotherapy is not a vaccine, the level of some impurities including residual cellular DNA can be higher as long as the manufacturer has demonstrated their due diligence in the purification process. The subsequent toxicology studies and the risk versus benefits for the patient are weighed by the FDA for approval of the product for use in humans. The VVPL has specialized in the production and purification of large, enveloped viruses that require aseptic processing throughout manufacturing. Two families of viruses have been the VVPL’s main products to date: VSV and MV. The VVPL has developed a general production and purification process that can be applied to a wide range of OVs but is especially efficient for large enveloped viruses like MVs and VSVs. We have used suspension cell lines and WAVE bioreactors to effectively scale-up cell biomass for virus production. A well-established virus infection time course is followed by harvest of the MV or VSV infected cell culture supernatants. The purification process begins with depth filters to remove intact cells and large debris followed by benzonase treatment, a product approved for clinical trial use, to digest residual nucleic acid. Hollow fiber tangential flow filtration is then used to purify, concentrate, and diafilter the virus into the final storage buffer. A final filtration step polishes the preparation before vialing and frozen storage. Translating VSV-hIFNb and VSV-hIFNb-NIS into clinical trials. The expression of the IFNb transgene by VSV limits VSV replication to the many cancer cell types that have lost the ability to respond to INFb. The IFNb expression also complicates the choice of production cells as well as the addition of the transgene, or two transgenes when combined with the human NIS, significantly slows replication and reduces maximum titers. VSV has a bullet-shaped structure that enlarges with the addition of 1–2 genes: VSV is 165 nm, VSV-hIFNb is 190 nm, while VSV-hIFNb-NIS is 236 nm according to E-M photographs. The size of the VSV and the production scale offered additional challenges for optimiz-

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ing virus yields and maximizing viral titers. The extensive specification and safety testing required for translating VSVs into the first in human clinical trial were reviewed. For OVs, the specification testing for unknown adventitious viruses that may be contaminating the therapeutic virus are a special challenge since the virus itself causes phenotypes that may indicate contamination. For VSVs, the VVPL developed a neutralization procedure combining a mouse monoclonal antibody with goal anti-VSV sera for this testing. The VVPL has translated two different MVs to human clinical trial: MV with a carcinoembryonic antigen gene (CEA) and an MV with the NIS gene. MV-NIS has recently demonstrated clinical efficacy in two patients with MM that were refractory to all standard care. One patient had a complete clinical response out to 15 months. The dose level that produced these clinical responses was one dose of 1011 TCID50 units delivered via IV. Three generations of MVNIS manufacturing and purification improvements enabled a > 100-fold increase in yields and titers to reach this dose level. In addition, the dose infusion rate was critical to achieve maximum systemic levels of MV-NIS. Matt Coffey, Oncolytics Biotech, Canada, presented data on progress with REOLYSIN, a reovirus-based oncolytic. His presentation began with an overview into their attempts to monitor reovirus biodistribution and tropism following intravascular delivery, since the only available data dates back to the 1960s. By monitoring S1 transcripts they evaluated biodistribution in mice across a number of time points, demonstrating fairly stable presence in the blood (up to 72 hr) with levels in the heart and liver peaking up to 72 hr post injection. Levels in the spleen peaked a little earlier (24 hr), with lower levels of transcripts also being detected in the lung, bone marrow, and ovaries. However, it was noted that detection of transcripts did not necessarily equate to a bona fide infection. Thus the team sought to use PCR and in situ hybridization to demonstrate replicative infection, and found good coverage in colorectal tissues and more varied coverage in melanoma, with clearance of virus most likely occurring in the lung and lymph nodes. Coffey then gave some updates on his team’s clinical experience using REOLYSIN in clinical trials in combination with gencitabine. In a phase I dose escalation trial, increasing doses were given five times, ranging from 3 · 108 to 3 · 1010 TCID50 per day, demonstrated that REOLYSIN is safe and well tolerated, with excellent transcript coverage in CD138 + cells. Although transcript levels were high in the relevant target cell population, levels of protein were low, perhaps indicating that a post transcriptional block may limit efficacy. In an effort to overcome this block, the team have noted that cell stress induces enhanced reovirus replication, and thus radiotherapy may augment efficacy, as noted previously in solid tumors. Thus, they are announcing a new phase I combination study of REOLYSIN in combination with dexamethasone and carfilzomib that will again be a dose escalation trial with an objective to see increased protein expression in the majority of patients, that is, to lift the translational block to deliver a productive viral infection in the target cells. Finally, the team sought to establish new predictive biomarkers that might stratify positive/negative outcomes in patients with MM treated with REOLYSIN, earmarking a potential role for cancer upregulated gene 2 (CUG2) with positive outcomes, and hyperammonia as a

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negative predictor, which appears to block production of reovirus in KMS-18 resistant melanoma cell lines. These findings could have important implications for reovirus oncolytic therapies in myeloma. Christine Blanc, PsiOxus Therapeutics, described the PsiOxus teams’ work with enadenotucirev (EnAd), an Ad11p/Ad3 chimeric Ad, developed through the use of directed evolution. Preclinically, EnAd has a broad spectrum of activity covering most major epithelial tumors and is selective for tumor cells. PsiOxus is currently conducting three clinical trials with EnAd: EVOLVE, a phase I study using IV dosing in subjects with epithelial tumors, which is near to completion; MOA, an ongoing mechanism of action study using IV and intratumoral (IT) delivery of EnAd; and OCTAVE, a phase I/II study of weekly intraperitoneal administration of EnAd alone or in combination with IV paclitaxel in platinum-resistant epithelial ovarian cancer, which is awaiting activation. The objectives for the EVOLVE phase I are to determine the IV dose and schedule of EnAd to be recommended for phase II studies and evaluate its safety and tolerability. Secondary objectives include antitumor activity, kinetics, and clearance, together with shedding and a variety of exploratory endpoints. Thirty-seven patients were included across eight separate doses and a schedule of administration cohorts. The dose escalation proceeded initially with 10-fold increases in EnAd dose, with EnAd administered on days 1, 3, and 5 as a single cycle. Doses ranged from 1 · 1010 to 1 · 1013 vp per infusion. The initial duration of infusion was 5 min but was increased after dose limiting toxicities (acute lung injury in one patient, and dyspnoea with hypoxia in another patient) were observed at the dose of 1 · 1013 over 5 min. Increases in various serum cytokines including IL2, IL6, IFNc, TNFa, and IL10 were observed at the highest doses administered, but these were decreased by lowering the dose and extending the duration of infusion. Blood pharmacokinetic data show a peak of EnAd after the third infusion, at a time when the cytokine levels are lowest. The last cohort of the study is evaluating repeat cycle dosing (every 21 days), using the dose and schedule planned for phase II studies. Side effects are as expected with OVs. The MOA study is a ‘‘window of opportunity’’ study evaluating IV and IT delivery of EnAd to tumor, lymph nodes, and normal tissue margins in cancer patients undergoing resection of their primary tumor. EnAd administration occurs before planned surgical resection. Distribution and expression of EnAd as well as inflammatory infiltrates were analyzed. Preliminary results using IHC staining for virus hexon protein in tumor sections demonstrate consistent replication and expression of EnAd protein in tumors following both IT and IV delivery. Robert Coffin described his work with HSV-based oncolytic immunotherapy. In particular, he described the results of a pivotal phase 3 study in melanoma (the OPTiM study), which met its primary endpoint (durable response rate), the first FDA approved phase 3 study of a gene therapy or oncolytic therapy to do so. Talimogene laherparepvec (T-VEC; formerly known as OncoVEXGM-CSF) is an HSV based OV. T-VEC is based on a new strain of HSV derived from a healthy volunteer. Deletion of ICP34.5 confers tumor selectivity and deletion of ICP47 improves antigen presentation. A transgene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) is inserted in place of ICP34.5. The new strain of HSV is more effective at killing

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a range of tumor cell types as compared to previous strains, including melanoma cells, and increased and earlier expression of US11 via ICP47 deletion improves tumor shrinkage in vivo. GM-CSF expression was shown to increase the antitumor effects in uninjected tumors and aid vaccination of animals against tumor cell re-challenge (Liu et al., 2003). The aims of treating patients with T-VEC are to (1) directly kill injected and local regional tumors through lytic virus replication, (2) provide an in situ patient-specific antitumor vaccine, and (3) vaccinate and protect against future progression and recurrence. Phase I and II studies in 114 patients demonstrated a good tolerability profile—mild influenza-like symptoms were observed 24–48 hr after injections. Local, locoregional, and distant effects were seen in melanoma, head and neck cancer, pancreatic cancer, and breast cancer. Virus spread and extensive necrosis was found in injected tumors and in tumors locoregional to the injection site. True systemic effects included immune-related side effects (vitiligo), objective responses in distant metastases, and induction of MART-1 reactive T cells. Responses were also durable. In a phase II study in 50 patients with stage IIIc-IVMc melanoma, an overall response rate of 28% was observed. Responses were seen at cutaneous, SC, nodal, and visceral disease sites, and 93% of responses lasted > 6 months. The response rate was highest in stage IIIc and IVM1a patients, that is, prior to patients having visceral disease (Senzer et al., 2009). A phase III study in melanoma enrolled 438 patients with Stage IIIbIVM1c disease, randomized 2:1 to T-VEC or SC GM-CSF. The primary endpoint was durable response rate (DRR), CR, or PR of all lesions within 12 months, which then lasts a further 6 months. Progression was allowed before response to allow for immune effects. A key secondary end point was survival. The objective response rate was 26.4% for T-VEC vs. 5.7% for GM-CSF. Durable response rates, the primary endpoint of the study, was 16.3% for T-VEC vs. 2.1% for GM-CSF ( p < 0.0001), resulting in study success. As for response rate in phase 2, DRR was also highest in first line patients and patients prior to visceral disease. Interim OS showed a strong trend toward statistical significance for the entire enrolled group (hazard ratio [HR] 0.79, p = 0.07). As for response rate and durable response rate, effects were most marked in first line patients and patients prior to visceral disease (stage IIIb/c/IVM1a HR 0.56, first line HR 0.49 vs. stage IVM1b HR 0.97, stage IVM1c HR 1.14, previously treated HR 1.13). OS was highly statistically significant in this exploratory subgroup analysis for the first line and previsceral disease groups. It was concluded that TVEC provides a potential new treatment for patients with advanced melanoma, particularly for first line use or prior to the appearance of visceral disease. James Burke, Jennerex, San Francisco, CA, provided an update on clinical development for bladder cancer using CG0070 being developed by Cold Genesys. Three GM-CSF expressing OVs are in clinical trials, at the moment, including CG0070. CG0070 is being developed by Cold Genesys (originally in development at Cell Genesys) in a phase II/III study for superficial bladder cancer. The proposed dual mechanism of action included direct lysis and stimulation of q systemic antitumor immune response. CG0070 is based on WT Ad5, with the E2F promoter controlling E1A gene expression and driving virus replication.

OVC ANNUAL MEETING, OXFORD 2014

The phase I first in human clinical trial (Burke et al., 2012) was based on a rationale for targeting nonmuscle invasive bladder cancer after Bacille de Calmette et Gue´rin (BCG) vaccination failure using the GM-CSF armed oncolytic Ad, CG0070. Immune-mediated mechanisms have been established in early bladder cancer but there remain limited options for patients after first line BCG therapy, with little opportunity for bladder preservation. At initial presentation, 70–80% bladder cancers are superficial, 25% invasive, and 5% metastatic. Carcinoma in situ (CIS) of the bladder has an approximately 5,000/yr incidence, and a high recurrence rate. First line treatment of superficial TCC with BCG results in a 70% response rate (RR). The phase I was an open label, dose escalation study of 35 patients with non-muscle invasive (Ta, T1, or CIS) transitional cell cancer of the bladder who had failed BCG. CG0070 treatment weekly · 6 and monthly · 3 was followed by quarterly disease assessment with cystoscopy and biopsy or cytology as indicated starting at the third month. Notably, there is a protective glycosaminoglycan (GAG) layer coating the urothelium that prevents virus access to target tumor cells. Prior to treatment with JX-594, a mild detergent is used to remove the GAG layer. Preclinical mouse studies demonstrated that GAG layer removal is required for efficient transduction of the urothelium. The group showed the detergent was compatable with the virus, and allowed CG0070 to dwell within the bladder for 45 min following intravesical instillation. Treatment was well-tolerated, mainly limited to local bladder toxicity (i.e., bladder spasms and dysuria) and flulike symptoms (fever, chills). Minimal systemic toxicity and detectable virus in the blood was demonstrated. GM-CSF expression peaks at day 2 across cohorts indifferent of whether patients had received single or multiple doses. Largest peak was after first administration, and the longer between doses the lower the peak. So the question remains, has the virus already infected most of the cells or is the immune response clearing the virus? The interval between virus input, decrease in detectable viral genomes in the urine, and resurgence of the PCR signal for viral genomes between days 3–5 suggests replication. Urine GM-CSF was detected in all 35 patients, with peak urine GM-CSF found on day 2. Most patients had baseline neutralizing antibodies; 50% of patients had a CR while the remainder had no response at all (in superficial bladder cancer, responses are either complete or no response; PR is not assessable). Following multiple doses (weekly · 6 and monthly · 3 pooled data) 63.6% had a CR compared with approximately 23% in single dose cohort. The weekly · 6 schedule resulted in a 78% CR rate and was selected for further clinical development. A phase II/III study targeting patients with CIS who have failed BCG and refused cystectomy has been initiated, which allocates treatment:control at a 2:1 ratio. In the experimental arm CG0007 1012 vp is administered weekly · 6 in conjunction with the preparative agent n-dodecy-b-Dmaltosidase (DDM). The control arm is one of several conventional chemotherapeutics chosen at the discretion of the treating physician. Phase II endpoints durable CR if there is no relapse for 6 months or longer, and phase III durable CR for 12 months. Secondary endpoints include safety, cystectomy free survival durability of response, OS, PR to muscle invasive disease, and subgroup analysis on patients who undergo cystectomy.

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Tomoki Todo, University of Tokyo, Japan, summarized the G47delta clinical trial in patients with recurrent glioblastoma that started in 2009. The group felt that conventional methods for efficacy evaluation such as WHO criteria and RECIST may not be adequate for OV therapy, and immune responses that immediately follow the OV injection should be taken into consideration. The group noted that a conventional dose escalation scheme for anticancer drugs may not be suited to the design of phase I trials for OV therapy because of the extent of viral replication, and the strength of immune responses vary considerably among patients. A preferred means of treatment using oHSV-1 may involve repeating local injections until the cancer is cured. Tomoki then explained about the G47delta clinical trial at the University of Tokyo for prostate cancer. This is a phase I clinical trial for recurrent prostate cancer without prostatectomy, which is refractory to hormonal therapy and is treated by direct injection into the prostate using ultrasound guidance. The group found that the response criteria and dose escalation scheme needed to be modified specifically for OVT following the first clinical trials. Olfactory neuroblastoma phase I clinical trial has just started, in which each patient is treated with a direct injection into the tumor with repeat injections every 4 weeks until tumor shrinkage or a PD. The clinical trials with G47delta in patients with recurrent glioblastoma, prostate cancer, and olfactory neuroblastoma are ongoing. The group concluded that a preferred treatment using oHSV-1 may be repeated local injections, and in light of this, a phase II clinical trial for patients with recurrent glioblastoma is planned that will involve up to six injections. Caroline Breitbach, Jennerex Biotherapeutics, provided an update on the clinical development of Pexa-Vec, an oncolytic and immunotherapeutic VV. More than 300 patients have been treated to date in active programs in first line liver cancer, renal cell cancer, and colorectal cancer. Data from a phase 1 IV dose escalation trial demonstrated Pexa-Vec administration was well-tolerated (no dose limiting toxicities were reported). Reproducible delivery of Pexa-Vec to metastatic tumors was demonstrated when treating at or above 1 · 109 pfu dose threshold (Breitbach et al., 2011). Furthermore, in a phase 2 randomized dose-finding study (n = 30) evaluating high-dose Pexa-Vec (1 · 109 pfu) versus low-dose Pexa-Vec (1 · 108 pfu) in patients with primary liver cancer, an improvement in OS was demonstrated with high-dose treatment (Heo et al., 2013). A phase 2b trial in liver cancer patients having failed sorafenib therapy was enrolled globally (n = 129). Patients were randomized 2:1 to receive Pexa-Vec plus best supportive care (BSC) versus BSC alone. The primary endpoint of improved OS was not met on this study in end stage cancer patients. A phase 3 clinical trial is planned in sorafenib naive liver cancer patients. Finally, data was presented on JX-929, a next-generation vaccinia designed for enhanced potency. Seventeen patients were enrolled on the phase 1 IT dose escalation trial led by Dave Bartlett, University of Pittsburgh Medical Center, JX-929 treatment was well-tolerated and no doselimiting toxicities were reported. There was early evidence of antitumor activity, which warrants future study. Darren Shafren, Viralytics Ltd, Australia, described his work with coxsackievirus A21, CAVATAKTM a common cold virus, a bioselected oncolytic strain of A21, positive-

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sense single-stranded RNA picornavirus that binds to the Nterminal domain of ICAM-1, which is highly expressed on many solid tumors including melanoma. The group showed that rapid replication with the biproduct of rapid lysis of tumor cells was observed with an associated release tumor antigen leading to a host systemic antitumor response. Phase I dose escalation was performed in Australia. There was a phase II clinical trial performed in the United States— ‘‘CALM study’’ for 54 late-stage melanoma patients; for inclusion, patients had to have at least one injectable lesion of at least 1 cm. Neutralizing antibody levels above the 1/16 cutoff were observed in 12.9% of screened patients. The patients had a series of 10 injections. The primary end-point was immune-related progression free survival (PFS), which was observed in approximately 35% of patients at 6 months post-treatment. If patients had an SD or a better response, then they were eligible for an extension study including additional injections to make a total nine cycles of multi-IT injections. CAVATAKTM is a non–genetically altered virus. One patient had multiple injectable lesions with CR in some of the injected and noninjected lesions. The group also saw a vitiligo halo around the noninjected lesions, indicating an antitumor immune response as well as regressions in noninjected common iliac lymph node (LN) and distant lung lesions. Some patients show pseudo progression at 18 weeks and then subsequent complete responses, and some patients showed regression of noninjected distant metastatic lesions. All patients were sero-negative at the start of the clinical trial and all converted to sero-positivity between days 8–20. This window of time is important, as all tumor responses were observed in the presence of neutralizing antibodies without circulating infectious virus. The majority of patients had high levels of virus in the serum about 30 mins after injection. A number of patients with significant levels of virus in the serum also had significant responses. Following treatment with CAVATAKTM, a lot of serum inflammatory cytokines were left unchanged except IFNc and IL-8, which were suggestive of a response. Combination Approaches

Ira Mellman, Genentech, San Francisco, focused on the desire of scientists and clinicians to mobilize the immune system to kill tumor cells. The immune response can be activated by virus, marshalling natural aspects of the immune system to react to virus and provide cunning approaches to making antitumor vaccines. A number of challenges remain so we can understand how they work, why they work, and how they can be optimized. The aim is to utilize the antigens already expressed by tumors and turn on the immune system to recognize them (Mellman et al., 2011). ‘‘Coley’s toxins’’ used bacteria emulsates to generate localized inflammation that led to some tumor responses. Three subsequent decades of work concentrating on various vaccination approaches, however, was with little impact and exhibited very few positive results. The oncogene phase focused on drug development, whereby the recombination of B-Raf and Mek inhibitors extended response time. About 50% of BRAF V600 mutated melanoma patients respond to vemurafenib, a class of inhibitors, with an average duration of response of 6.7 months. The problem is patients relapse after treatment with disease at least as serious as it was

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before treatment. Ipilimumab (anti-CTLA-4) elicits low frequency but durable responses in metastastic melanoma (Hodi et al., 2010). Treatment with Ipilimumab led to patients dying more slowly and a quarter had long-term durable responses, a real breakthrough, for patients usually do not survive stage III–IV disease. Melanoma is unique as it is established as an ‘‘immunological’’ tumor and so other tumors are now being investigated. CTLA-4 puts a break on self-reacting T cells and stops regulatory T cells from expanding in an unrestrained fashion. Patients also develop adverse events following anti-CTLA-4 therapy at a high rate, as this product works in a nonspecific way (Chen and Mellman, 2013). Programmed death receptor-1 (PD-1) attenuates T cell function by binding PD-L1 during virus infection and acts as a negative regulator of T cell function. This reactivity can be regained if the source of PD-L1 is removed. This study showed how immunosuppression can be targeted by blocking the PD-L1/PD-1 pathway. A large percentage of tumors evade the immune system by circumventing the PD-L1/PD-1 pathway (Chen et al., 2012) as PD-L1 binds CD80 and facilitates T cell costimulation/ suppression. PD-L1 is broadly expressed in human cancer, including 50% of non-small cell lung cancer (NSCLC; SCC), 45% NSCLC (adeno), and 45% of colon cancer. This suggests a preexisting immunity in a large number of patients, and PD-L1 expression is found on many tumor cells and/or tumor-infiltrating immune cells. In phase Ia clinical trials using MPDL3280A, the team showed responses in lung cancer patients who received a 20 mg/kg dose (n = 39) (Herbst et al., 2013). In early cuts of the data they did not see all patients responding, but in those who do respond there is a quick response and a very low relapse rate. Diagnostic marker was PD-L1 expression in tumors and/or tumor infiltrating lymphocytes, and those with highest levels of PD-L1 were also found in the patients who had the highest response rates (Cho et al., 2013; Soria et al., 2013). When assessing all patients in this clinical trial, the response rate was 23% (12/53), but those patients with the highest PD-L1 staining (IHC) had an 83% response rate (n = 5/6), although numbers are recognized as being small overall. In contrast, PD-L2 status was not found to be associated with progression in this clinical trial. In all, 81% of the 53 patients were smokers and these patients showed the best response (23%) compared with the 10 nonsmokers who had a response rate of 10%. Lawrence et al. (2013) showed that the highest frequency of somatic mutations in cancers were seen in melanoma patients followed by lung (SCC), lung (ade), bladder, stomach, esophageal, and colorectal, and this may correlate with the good immune responses generated in these cancer types. Genetech monitor baseline gene expression using ImmunoChip (iChip) was used to determine baseline immunity. The group used mRNA from formaldehyde fixed-paraffin embedded (FFPE) tumors and examined the relative expression of PD-L1, Granzyme-A, Granzyme-B, Perforin, EOMES, IFNc, CXCL9, and CD8A in comparison to a housekeeping gene (Powderly et al., 2013). Only 25% of NSCLC patients respond to PD-L1 monotherapy, so to determine whether it can be combined with other therapies, proposed combinations have been examined in mouse models. One study examined the combination of chemotherapy oxaliplatin with PD-L1, which led to good antitumor growth responses and rejection of tumor

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challenge. V600E B-Raf inhibitors enhanced anti-CD8 + T cell responses but when combined did not improve responses. Combinations of a-PDL1 and MEK inhibitor were shown to improve PR and CR rates but did not lead to a cure. Also being examined is MEK inhibition, as this has been shown to increase MHC class I peptide presentation on tumors. Of note, Mellman stated that not all patients have preexisting tumor immunity (Chen and Mellman, 2013) and that this can impact on the efficacy of antitumor immune responses when patients are treated with immunotherapy. Kevin Harrington, The Institute of Cancer Research, United Kingdom, spoke of his research groups’ work from a single agent strategy to combining agents together (Harrington et al., 2010; Kyula et al., 2012). Reovirus has been shown to exert potent oncolytic effects in a head and neck cancer cell line independently of signaling in the EGFR pathway (Twigger et al., 2012). Combination reovirus with two chemotherapy drugs (cisplatin-paclitaxel), which are often used for the treatment of head and neck cancer, were shown to be highly synergistic in their action. There was no increase in the replication of the virus, and the virus did not overwhelm the cell by virtue of viral replication; the combination resetted the apoptotic rheostat in the cells as seen with caspase glo assays, and this translated into better survival in nude mice. In a phase I study of IV oncolytic reovirus (Adair et al., 2013; Vidal et al., 2008) the group isolated peripheral blood mononuclear cells then co-incubated the granulocytes and platelets with target cells and observed live virus coming off the target cells. Virus travelled through the blood, cloaked from the antibody response by hitchhiking on peripheral blood cells. Immunostaining showed virus factories in these tumor regions (Comins et al., 2010; Karapanagiotou et al., 2012). Karapanagiotou et al. also described the treatment of H&N tumor patients in a randomized phase I study, which is now completed. They have started a phase II clinical trial, and the initial response data shows no significant difference between controls and treated (Harrington et al., 2010). The group has also been investigating the effect of the N-Ras and K-Ras mutational background on combination therapy, including oncolytic vaccine virus and radiation. It has been noted that radiation alters sensitization of the cells to virus as if radiation was resetting the signaling pathways. The group hypothesized that they would see less virus replication in B-raf compared with WT and N-Ras carrying cells. In WT harboring cells they observed an upregulation of Jnk, while in A375 (a Braf mutant cells) there was a significant abrogration of apoptosis. Jnk inhibition phenocopies the effect of radiation only in the B-raf mutant melanoma, increasing the TNFa signal in B-raf mutants until radiation is administered. The group modeled vaccinia and RT relative to genetic background. Vaccinia in a Ras/B-raf was compared to WT/Ras mutant melanoma cells. They found increased p-p38, pJNK, p-ERK, and p-Akt levels leading to increases in TNFa, which delays apoptosis and facilitates viral replication. Alan Melcher, Leeds Institute of Cancer and Pathology, United Kingdom, described the clinical trials being performed in collaboration with Kevin Harrington and Hardev Pandha’s groups. Reovirus is a double-stranded RNA virus that most people are exposed to in childhood, so we all carry neutralizing antibodies against it. The REO13 trial showed that antibody levels increased after IV treatment. After

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systemic delivery, there was reovirus-RNA in the plasma of patients but in terms of ‘‘functionality,’’ there was replication competent virus only in blood cells, but not free in plasma. They found there was more reovirus protein in colorectal cancer tissue resected from the liver after IV reovirus than in the tumor stroma or the normal, surrounding liver. Replicating virus could be retrieved from tumor but not from normal liver, leading to the conclusion that virus can be selectively delivered to tumor even in the face of neutralizing antibodies. The question remains how can we improve delivery and, potentially, therapy in this system? If the number and/or activation state of virus carrier cells is boosted, we may be able to enhance both delivery to the tumor and immune-mediated reovirus therapy. The group therefore tested reovirus against melanoma in mice in combination with GM-CSF, G-CSF, and IL-2. They showed that reo/GM-CSF controlled tumor growth effectively, but counter-intuitively, only if the mouse was preimmunized against reovirus. This suggested a need for neutralizing antibodies to maximize both delivery and therapy with this combination. Reovirus could be taken up by GM-CSF-activated monocytes/macrophages, stimulating priming of the immune system against even reovirus-resistant tumor cells. If the group depleted NK cells or monocytes/macrophages in the mice, reo/GM-CSF therapy was lost; this did not happen when other immune cells were depleted, including T cells. Moving on to another tumor site, brain tumors—both high grade gliomas and metastases—remain a major unmet clinical need. Most oncolytic virotherapy to date for brain tumors has been given by direct IT injection, although the question remains as to whether IV therapeutic viruses can cross the blood-brain barrier into the tumor, which would make virotherapy far easier for widespread clinical application. Using a range of models, the group showed that IV reovirus can indeed access tumors within the brain after iv injection, and that Reo/GMCSF in preimmune mice is therapeutic, in the same way as it had been for flank tumors. Early data from the REO13 BRAIN study has shown that preoperative reovirus is delivered into primary and metastatic brain tumors in patients, as previously seen in colorectal liver metastases in REO13. Hence a systemic OV can access tumors in the brain, supporting the rationale for a trial of Reo/GMCSF in addition to chemoradiation as first line treatment for high grade glioma. Victoria Roulstone, The Institute of Cancer Research, United Kingdom, described her group’s interest in a combination of oncolytic reovirus with BRAF and/or MEK inhibitors in malignant melanoma. B-raf inhibitors switch off downstream MEK-ERK signaling in BRAF mutant melanoma lines, but paradoxically enhance MEK-ERK signaling if the cell line is RAS mutant (Heidorn et al., 2010). Since reovirus is selective for cells harboring activated/mutated RAS signaling, it was originally hypothesized that in the event that MEK-ERK signaling is further enhanced, such as this scenario in RAS mutant cell lines treated with a BRAF inhibitor, that we might see synthetic lethality. Contrary to this hypothesis, reovirus cell kill was not affected by inducing this environment of enhanced MEK-ERK signaling. Instead, switching off MEK-ERK signaling led to dramatic levels of cell kill compared to the single agent counterparts. This also holds true by inhibiting MEK-ERK using MEK inhibitors. The group discovered that the enhanced cell kill

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was due to increased apoptosis, mediated by the unfolded protein response. The combination of MEK-ERK inhibition and reovirus infection was causing unresolvable ER-stress, which led to apoptotic cell death induced by the chemotherapy combination of cyclophosphamide, doxorubicin (hydroxydaunomycin), vincristine (Oncovin), and prednisolone (a steroid), known as CHOP. Cells could be rescued from this cell kill through the use of salubrinal, an inhibitor of ER stress. The combination therapy of reovirus plus BRAF inhibitor PLX4720 is translatable in immunedeficient CD1 nude mice and immune-competent C57BL/6 mice bearing BRAF mutant murine 4434 tumor cells (Dhomen et al., 2009). Iain McNeish, University of Glasgow, United Kingdom, described their work investigating OV therapies and paclitaxel. They had previously identified that there was great variability in sensitivity to Ad in ovarian cancer cell lines in vitro (Connell et al., 2011). In Ad infection, expression of E1A allows cells to overcome multiple checkpoints and induces aberrant genomic DNA replication in sensitive cells. Paclitaxel (Wiernik et al., 1987) clinically is most effective when given weekly at low doses in ovarian cancer, rather than every 3 weeks (Katsumata et al., 2013). Rather than inducing mitotic arrest, low dose paclitaxel leads to multipolar mitosis, in which mitotic abnormalities and multinucleation are observed. Oncolytic Ad infection in ovarian cancer induces > 4n (tetraploidy) in sensitive cells, but this is not observed in resistant cell lines until you add low dose paclitaxel. The virus appears to be trying to drive the cells through mitosis, and by adding a low dose paclitaxel you enhance this and trigger cell death. In unpublished data using established cell lines, xenografts, and primary patient tumor samples, McNeish’s lab has also shown that paclitaxel resistance increases the efficacy of oncolytic Ad therapy—this partially results from increased infectivity and partially from dysregulated cell cycle progression. Ongoing work is elucidating the mechanisms behind these observations. Jean-Simon Diallo, Ottawa Hospital Research Institute, Canada, hypothesized that while OV therapy works by multiple mechanisms, including generation of an antitumor immune response, a prerequisite is that a certain number of tumor cells need to be infected to achieve meaningful therapeutic response. The group have noticed heterogeneity in the infectivity of tumors by OVs. Approximately 30 cancer patient tumor specimens showed a large range in viral titers output (several logs). Even within isolated subclones of cancer cell lines, there was significant variation in susceptibility to OV infection and this impacted therapeutic efficacy. Diallo describes their successes at leveling the playing field with drugs. High throughput screens have identified novel virus sensitizers or VSe drugs. They identified 15 compounds that enhance oncolytic VSV activity. VSe1, a previously uncharacterized compound, had among the most robust activity: It could enhance OV activity and spread in tumors in vitro, ex vivo, and in vivo. VSe1 dampens Type I IFN signaling and the expression of type I IFN response genes. VSe1 and novel derivatives can also enhance infectivity of HSV. In addition, they identified microtubule destabilizers (MDAs) such as colchicine and vinorelbine that can synergize with oncolytic rhabdoviruses. MDAs enhance viral spread by preventing efficient trans-

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lation and secretion of type I IFNs. In parallel, other unaffected virus-induced cytokines increase bystander killing of cancer cells treated with MDAs. A mathematical model (Le Boeuf et al., 2013) has described the interplay between these two effects and suggests that modulating bystander killing may be a more tumor selective approach to increase OV activity than altering IFN response, but shows that the combination of both is nonetheless safe, as observed experimentally. Yonghong Wan, McMaster University, Canada, demonstrated that vaccine-induced T cells are highly functional when they initially enter the tumor but become progressively dysfunctional as a result of an adaptive immunosuppressive response mounted by the tumor. They found this adaptive event in the tumor is surprisingly driven by infiltrating tumor antigen-specific T cells and their IFNc production. They provided evidence that this process can be overcome by rhabdoviral vaccines due to their robust boosting activity and direct oncolytic effect. Their studies indicated that IV delivery of oncolytic rhabdoviruses results in antigen presentation to memory T cells in splenic follicles and creates an inflammatory environment in the tumor, which enhances infiltration and tumor killing by T cells. The group showed that a unique synergy could be achieved by using oncolytic rhabdoviruses as a booster in the context of a prime boost regimen or in combination with adoptive cell therapy. Joe Conner, Virttu Biologics, Scotland, described their completed safety and proof of concept studies in 72 patients using SEPREHVIR (HSV1716). Phase I studies have been completed with 47 patients with high grade glioma, five patients with advanced melanoma, and 20 patients with squamous cell carcinoma. They described work on malignant pleural mesothelioma, which is a universally fatal and difficult to treat cancer associated with asbestos exposure. Survival with any treatment option is approximately 1 year. The incidence is increasing and it remains a major healthcare challenge, with limited therapeutic options available. Multifocal intrapleural disease can cause disabling symptoms of pain and breathlessness in the absence of distant metastases, so an intrapleural treatment approach is attractive. Patients with a histological diagnosis of malignant pleural mesothelioma and in-dwelling pleural catheters are being treated in an open label, dose escalation, phase I/IIa trial in a single clinical center at Sheffield in the United Kingdom. Patients will receive 1 · 107 i.u. of SEPREHVIR through their pleural catheter on one, two, or four occasions a week apart, in three separate cohorts. Primary objective is safety and tolerability when SEPREHVIR is given by single or repeat intrapleural administration. Prestudy compatibility testing involved monolayers of cells in tissue culture (TC), which were coated with pleural fluid and incubated with virus (HSV1716-GFP). HSV1716-GFP spread to all cells, showing virus replication was not prevented by pleural fluids. Samples from patients were analyzed for virus shedding (urine and mouth swabs), blood (chemistry; antiHSV-1 IgG/IgM status; PCR for HSV DNA; and biomarker assays for mesothelin, osteopontin, cell death markers, and VEGF) and pleural fluids were examined (fluid volume, evidence of HSV1716 replication, and biomarker assays). Three patients received one dose and exhibited no dose limiting toxicity (DLTs) and no safety issues. This was

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followed by dose escalation to two doses in which no safety issues were uncovered. Patients are now being treated with four doses. So far 6 patients—5 males (late onset, average age 69yrs)—have been treated with pleural fluid samples indicating evidence of virus replication and patients receiving two doses had strong serum anti-HSV IgG responses. David Mansfield, The Institute of Cancer Research, United Kingdom, showed vaccinia virus (VV-NIS) is effective against prostate cancer cell lines. All cell lines are susceptible but there is some variation in susceptibility. Irradiation can enhance gene expression. aH2AX staining of DNA doublestrand breaks and CellProfiler automated quantification shows increased 131I induced DNA damage in infected cells and also an increase in DNA damage in neighboring noninfected cells, indicating a bystander effect. The group took their work into in vivo models (PC3 and DU145) and showed that 131I and VV-NIS are an effective combination to cause tumor cell death. The group wanted to use a triple therapy and combine with irradiation. However, the xenograft models are not robust enough to determine any additional effect of the triple therapy. In the future, the group plans to use an immune competent model with a radiation resistant cell line to try to see if a third therapy will further improve outcome. Nick Lemoine, QMUL, United Kingdom, talked about their use of heterologous prime-boost vaccination utilizing two OVs. It is important to induce immunogenic responses that stimulate multiple arms of the immune system against the tumor. Strategies to enhance the potency of two OVs, including enhancing selectivity and potency, have been utilized but there is a double-edged sword between activating a host immune response against the OV and developing an adaptive immune response against tumor cells, which can induce cell death and release TAAs and peptides stimulating activated CTLs to clear the malignancy systemically. The group hypothesized that the use of two different viruses would help avoid host immunity to the second virus and to act as a prime and boost combination. The Syrian hamster is a model that can support the replication of Ad and VV, and has a host immune response similar to humans. A wide range of tumor cell lines and transplantable tumor models are also available, including pancreatic and kidney cancers. Tumors were established SC and allowed to grow to 100 mm3 when they were injected with Ad · 6 or VV · 6 or the combination (Ad · 3, VV · 3, or VV · 3 followed by Ad · 3). Injections were given six times, every other day. The most effective treatment strategy was Ad followed by VV, which led to tumor regression and longterm survival in almost all animals with both tumor types. More lymphocyte infiltration was observed in this treatment scenario, and the tumors displayed widespread apoptosis. This process is T cell–dependent as shown by anti-CD3 antibody administration the day before treatment started (Tysome et al., 2012). Sequential use of Ad and VV induces effective and long-lasting tumor-specific immunity. The animals were able to reject the same tumor as previously cleared but not a completely different one. The group is now arming the virus for a prime and boost. Ad-FLT3L/GMCSF/IL-12 provides a local release of cytokines to attract T cells and DCs. The boost is armed to amplify the T cells in situ (VV-IL15/R; VV-IL-21). Arming Ad-triple deleted (TD) with IL-12 enhances antitumor immunity and longterm survival, even with much larger tumors (treatment

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started at 300 mm3), in animals that would otherwise need to be sacrificed within 1 week. Understanding the Immune System: Toward the Development of Combination Therapies— Immunotherapy and Virotherapy Microenvironment

Timothy Cripe, Nationwide Children’s Hospital and Ohio State University, Columbus, explained that in the United States 1 in 285 children age 0–19 years are diagnosed with cancer; of these 42% are solid tumors, 18% are central nervous system (CNS) tumors, and 40% hematopoietic or lymphatic. Cripe is involved in a few clinical studies in young patients; JX594 has recruited six patients ( Jennerex) while HSV1716 has recruited six patients as part of an ongoing trial at Nationwide Children’s Hospital in Columbus and a hospital in Cincinnati. Cripe focused his talk on the preclinical data. His group has a number of tumor models including mouse models of rhabdomyosarcoma (76-9, HGF116, M-39M), xenografts of rhabdomyosarcoma (RD, Rh18, Rh30, Rh40), Ewing sarcoma (A673, 5838), and neuroblastoma [CHLA20, CHP134, SKNAS, SKNBE(2)]. In terms of definitions, susceptibility measures viral entry to cell by PCR, permissivity looks for infectious particles by plaque assay, and sensitivity examines cytotoxicity through assays such as MTT. The work presented focused on HSVrRp450 virus (from the Chiocca group). This virus has a cyp2b1 proenzyme in the icp6 region, but this study doesn’t look at the enzyme. There was an observed discordance between in vitro and in vivo results. Based on in vitro viral replication assays the team expected A673 to work better (in terms of tumor killing by oncolysis), but 5838 is better in vivo where 75% of mice survive ( p = 0.003), so the result is highly significant. The group believes this difference is explained by the tumor microenvironment, which likely differentiates immune cell recruitment. The group looked at various myeloid cell populations and saw an influx of leucocytes and NK cells and a fall in macrophages in the 5838 model. In 5838, NK cells are attracted by the increase in chemokine levels while chemokines that attract neutrophils show no change. They found an increase in TNFa and no change in TGFb levels. By FACs analysis of primary human NK cells added to cell line cultures, they saw an increase in TRAIL expression in 5838 cells, which is what was expected when NK cells are exposed to virally infected cells. No TRAIL was found in virus-infected A673 cells. In immunocompromised NOD/SCID mice they didn’t see the antitumor effect seen in the immunocompetent model. When they repeated the experiment with another line that is resistant to virus in vitro, there was no correlation between sensitivity in vitro and antitumor effect in vivo in syngeneic mouse models. M-3-9M is a sensitive line in vitro, and in nude mice the tumors don’t shrink in response to virus treatment, but 80% of the mice survive in the syngeneic model and the mice resist rechallenge, suggesting a vaccination effect. In some models (Currier et al., 2008) NK inhibition is good for the virus, but in Cripe’s model, increasing NK cells is good (in terms of tumor shrinkage). It appears that every model is different, thus making it difficult to generalize. Miriam Bazan-Peregrino, VCN Biosciences, Spain, talked about working with VCN-01, a modified oncolytic

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Ad that secretes hyaluronidase, which helps degrade the extracellular matrix. There was replication selectivity of the virus for pancreatic cancer cells. Following a high IV dose of VCN-01, the virus was present in many organs on day 2 in hamster models, but by day 8 the virus has restricted presence. On day 2, VCN-01 was in the corpus luteum of the ovary (2/6 hamsters), a structure that contains no genetic material to be transmitted to the germ line, and no virus was detected on day 8, so no risk of genetic transmission was determined with the help of a pathologist. VCN-01 plus gemcitabine significantly lowered the IC50, and there was no accumulative toxicity in mice or hamsters treated with the combination of VCN-01 and gemcitabine, as indicated by weight loss and biochemistry (transaminases and platelets). IV administration of VCN-01 with IP gemcitabine significantly inhibited NP-9 tumor growth compared to either therapy alone, and there were low levels of hyaluronic acid within treated tumors. The group hypothesis was that hyaluronidase is degrading the extracellular matrix, making the tumor more accessible to the chemotherapy. This treatment has been shown to be very effective even in Syrian hamster treatment-resistant pancreatic duotal tumour cell line, HP-1 cells when VCN-01 and gemcitabine were used in combination, with little or no effect when treated independently. Now VCN-01 is being used in two independent phase I clinical trials. Patients will receive VCN-01 oncolytic Ad either IT or IV. To calculate the initial dose the results from animal studies were considered. IV dose escalation from 1011 vp VCN-01 in one patient ramping up to 1 · 1013 vp VCN-01 in the final 3–6 patients recruited. Inclusion criteria considers neutralizing antibodies. So the titer needs to be < 1/320 so at least 50% of the virus is free and available to have some type of effect. Phase I starts with VCN-01 and gemcitabine, although there is little effect of gemcitabine, but it is given as the first line of treatment normally in these patients. Once established that it is safe, the clinicians will administer virus alone. Primary objectives include safety and viral shedding assessment. Secondary end points include efficacy by imaging such as positron emission tomography–computed tomography and elastography. Mark Tangney, University College Cork, Ireland, described how OVs and bacteria share the property of tumorselective replication following systemic administration. Bacterial tumor selectivity is based on the nature of the environment in tumors being suitable for bacteria due to its leaky and disorganized vasculature, permitting bacteria to lodge within tumor tissue where they can hide from the immune system, often concentrating in necrotic and anaerobic/hypoxic regions. The Tangney lab has also recently shown that a range of bacteria exists within tumors and adjacent tissue in cancer patients (Cummins and Tangney, 2013). Nonpathogenic (noninvasive) bacteria are particularly selective for tumors, grow extracellularly within tumor stroma, and are ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. The Bell lab has previously shown the ability of the type 1 IFN antagonist B18R to enhance the replication and spread of VSV by overcoming related cellular innate immunity (Le Boeuf et al., 2010). Together, the groups exploited nonpathogenic probiotic bacteria expressing B18R to facilitate tumorspecific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus ‘‘preconditioning’’

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the tumor to dramatically enhance subsequent tumor destruction by the OV. This strategy is the first example in which two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome. Christine Engeland, National Centre for Tumor Diseases and German Cancer Research Centre, Germany, described their investigations of MV-mediated checkpoint blockade to enhance immunovirotherapy. The group cloned oncolytic MV vectors that encode either anti-CTLA4, anti-PD-L1, IgGFc, or EGFP. There was no impairment of modified vectors compared with parental vectors. The group confirmed transgene expression and binding of the antibodies to their cognate antigens. Since MV tropism is restricted to primate cells, the group developed a syngeneic murine melanoma model, B16-CD20. B16-CD20 cells express hCD20, and therefore can be infected by MV retargeted to hCD20. In this immunocompetent model, treatment with MV-anti-CTLA4 significantly delayed tumor progression and treatment with MV-anti-PD-L1 significantly prolonged median OS compared to mock and Fc controls. MV-antiCTLA4 and MV-anti-PD-L1 were equally efficient compared to parental vectors in a NOD/SCID xenograft model of human melanoma, with high rates of CRs. Transduction of primary melanoma in vitro showed efficient spread and cell lysis. Progeny particles, transduction, and replication were also examined and the MV showed efficient lysis, although kinetics varied between individual patient samples. A phase I clinical trial is planned at the National Centre for Tumor Diseases with intralesional application of MV in combination with an anti-PD-L1 antibody. Matthew Mulvey, BeneVir Biopharm, Inc, Rockville, MD, explained how the cellular antigen presentation system allows the immune system to rid the body of viruses. Antigen presentation is accomplished in three steps: (1) cytosolic proteins are degraded by the proteasome, which are then (2) transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP), and then (3) loaded onto MHC-I for display on the cell surface. CD8 + CTL recognizes and kills virally infected cells when peptides derived from viral proteins are displayed by MHCI. CTL killing of infected cells blocks viral spread and leads to viral elimination. Some viruses encode functions that inhibit TAP in order to preclude presentation of viral antigens on MHC-I and evade clearance by CTL. To determine the role TAP inhibition may play in OV efficacy, BV-TAPi and BV-TAPi-FS were constructed. BV-TAPi is an attenuated, replication competent HSV-1 variant encoding a TAP inhibitor (TAPi). BV-TAPi-FS is genetically identical to BVTAPi except for a single frameshift (FS) mutation in the TAPi gene. Mice harboring bilateral SC MBT-2 bladder tumors received injections of BV-TAPi or BV-TAPi-FS in one of the two tumors. Both infected and contralateral tumors in mice treated with BV-TAPi were significantly smaller than in animals treated with BV-TAPi-FS and correlated with induction of CTL that secrete IFN-c in response to stimulation with tumor cells. BV-TAPi outperformed the FS control in an orthotopic 4T1 mouse breast cancer metastasis model. Depletion of CD8 + T cells in the 4T1 model erased the advantage conferred by TAP inhibition suggesting that BV-TAPi enhances induction of antitumor CTL. This conclusion is supported by the observation that

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significantly fewer lung metastases were observed in mice treated with BV-TAPi compared to the FS control. In a bilateral MB49 bladder cancer model, BV-TAPi outperformed an isogenic variant encoding mouse GM-CSF in place of TAPi. This indicates that TAP inhibitors may be superior to GM-CSF as OV armaments. Many tumor cells downregulate or mutate TAP in order to block display of tumor antigens. Like OV armed with TAPi, these cells evade clearance by antitumor CTL. However, these immuneevading cells display ‘‘neoantigens’’ bound to MHC-I on their cell surface. Neoantigens mark the cells as antigen presentation deficient. Furthermore, because neoantigens are not presented by APC during thymic selection, CTLs exist that are able to recognize and eliminate antigen presentation deficient tumor cells. However, APC are unable to activate CTL that recognize neoantigens because APC have normal antigen presentation capability. It is possible that APC in the tumor microenvironment become infected by BV-TAPi, which could lead to display of neoantigens on the APC surface and activation of CTL that recognize neoantigens. To determine if BV-TAPi is able to induce long-term protective immunity against immune-evading tumor cells, mice cured of MB49 tumors by treatment with BV-TAPi were challenged with the TAP-deficient metastatic lung carcinoma cell line CMT167. Compared to age-matched naive controls, BV-TAPi mice were protected against CMT167 cells to which they had no prior exposure. The data presented by Dr. Mulvey demonstrates that arming an OV with a TAP inhibitor improves therapeutic efficacy, presumably by improving viral spread in the tumor microenvironment, which enhances induction of CTL that recognize tumor antigens. Preliminary data in the CMT167 challenge experiment implicates a role for induction of a new type of CTL that is capable of recognizing and eliminating tumor cells that have evolved the ability to evade elimination by CTL that recognize tumor antigens. Immune-evading tumor cells are intrinsically resistant to conventional immunotherapies and associated with poor clinical outcomes. TAP inhibitors may confer multiple addition mechanisms of action to OV platforms and enhance patient outcomes. Summary

The 8th Oncolytic Virus Conference overviewed the state of the art in oncolytic viral therapy. There was a definite recognition of the benefit of combination therapies, complementation of standard therapy and virotherapy, the ideal delivery of virus IV, and working toward single doses where possible. Preference is to avoid patients with neutralizing antibodies or to overcome the limitations of treating patients with neutralizing antibodies. Modifications of viruses to enhance desirable characteristics include targeted infections, efficient spread, optimal mechanisms of death, and induction of humoral responses against tumor rather than against the virus. The importance of the immune system during delivery is also increasingly recognized, and strategies to circumvent the mechanisms that trigger antivirus responses have improved virus infection and spread. Finally, there is often a benefit from a local immune response to create inflammation and to mop up infected cells, presenting tumor antigens to the immune system for an extended effect, especially the killing of distant and micrometastatic disease.

21 Acknowledgment

Many thanks to Christina Woodward for all her assistance in the preparation of this manuscript. Author Disclosure Statement

Dr. Peng and the Mayo Clinic have a financial interest in the associated technologies described in this report. For all other authors, no competing financial interests exist. References

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Address correspondence to: Dr. Barbara Guinn Department of Life Sciences University of Bedfordshire Park Square, Luton, LU1 3JU United Kingdom E-mail: [email protected] Received for publication September 26, 2014; accepted September 29, 2014. Published online: October 1, 2014.