Oncolytic Virotherapy and the Tumor Microenvironment.

Oncolytic viral therapy is a promising approach to treat many malignancies, including breast, colorectal, hepatocellular, and melanoma. The best results are seen when using "targeted and armed" viruses. These are viruses that have been genetically modified to selectively replicate within cancer cells and express specific transgenes that alter the tumor microenvironment to inhibit tumor progression. The products of these transgenes induce cell death, make the virus less virulent, compromise tumor vascularity, and are capable of modulating or enhancing the immune system-such as cytokines and chemokines. In addition, oncolytic viruses can induce anti-vascular effects and disrupt the extracellular matrix to improve viral spread within the tumor. Oncolytic viruses also improve crosstalk between fibroblasts, cytokine-induced killer cells, and cancer cells within the microenvironment, leading to enhanced tumor cell death.

Oncolytic Virus-Mediated Targeting of PGE2 in the Tumor Alters the Immune Status and Sensitizes Established and Resistant Tumors to Immunotherapy.

Immunotherapies are highly promising cancer treatments, but understanding the factors mediating their resistance remains critical. Successes in randomized clinical testing have supported the growing appreciation that oncolytic virotherapies primarily act as immunotherapies. Here we identified prostaglandin E2 (PGE2) in the tumor as a key mediator of resistance to immunotherapies, including oncolytic vaccinia virotherapy. Elevated levels of PGE2 coupled to suppressive chemokine profiles and high levels of granulocytic myeloid-derived suppressor cells resulted in loss of immunotherapeutic potential. Viral vectors engineered to target PGE2 were capable of overcoming localized immunosuppression leading to profound changes in the tumor's immune status. This allowed the viral vectors to raise robust anti-tumor adaptive immune responses and sensitized established and previously resistant tumors to immunotherapies.

Noninvasive Imaging of Fluorescent Reporters in Small Rodent Models Using Fluorescence Molecular Tomography.

The capacity to combine noninvasive whole animal imaging of genetic reporters and exogenously added probes in a single animal makes fluorescence imaging a powerful tool for investigating molecular events in live animals in preclinical research. However, the adsorption and diffraction properties of light passing through tissues mean that the choice of reporters, models, and imaging systems needs to be carefully considered. Here, we describe approaches to design and run experiments incorporating noninvasive whole animal fluorescence imaging into small animal imaging studies.

Manipulating TLR Signaling Increases the Anti-tumor T Cell Response Induced by Viral Cancer Therapies.

The immune response plays a key role in enhancing the therapeutic activity of oncolytic virotherapies. However, to date, investigators have relied on inherent interactions between the virus and the immune system, often coupled to the expression of a single cytokine transgene. Recently, the importance of TLR activation in mediating adaptive immunity has been demonstrated. We therefore sought to influence the type and level of immune response raised after oncolytic vaccinia therapy through manipulation of TLR signaling. Vaccinia naturally activates TLR2, associated with an antibody response, whereas a CTL response is associated with TLR3-TRIF-signaling pathways. We manipulated TLR signaling by vaccinia through deglycosylation of the viral particle to block TLR2 activation and expression of a TRIF transgene. The resulting vector displayed greatly reduced production of anti-viral neutralizing antibody as well as an increased anti-tumor CTL response. Delivery in both naive and pre-treated mice was enhanced and immunotherapeutic activity dramatically improved.

Adding STING to the Tale of Oncolytic Virotherapy.

The identification of STING as a key cytoplasmic innate recognition molecule for DNA viruses whose function is lost in a variety of cancers has coincided with the approval of IMLYGIC for metastatic melanoma. This represents the first replication competent viral therapy approved for the treatment of any cancer in the US. The role of STING pathway in the selectivity of HSV has been addressed for the first time in Xia et al (1).

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging.

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy.

PURPOSE: Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in preclinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective antitumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. EXPERIMENTAL DESIGN: Here, we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. RESULTS: Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody, and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8(+) and NK, but not CD4(+) cells mediating the effects. CONCLUSIONS: These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. Clin Cancer Res; 21(24); 5543-51. ©2015 AACR.See related commentary by Slaney and Darcy, p. 5417.

In vivo inflammation imaging using a CB2R-targeted near infrared fluorescent probe.

Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund's Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases.

Reciprocal cellular cross-talk within the tumor microenvironment promotes oncolytic virus activity.

Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-ß (TGF-ß) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.

Novel therapeutic strategies in human malignancy: combining immunotherapy and oncolytic virotherapy.

Results from randomized clinical trials over the last several years have finally begun to demonstrate the potential of oncolytic viral therapies to treat a variety of cancers. One reason for these successes has been the realization that this platform is most effective when considered primarily as an immunotherapy. Cancer immunotherapy has also made dramatic strides recently with antibodies capable of blocking immune checkpoint inhibitors and adoptive T-cell therapies, notably CAR T-cells, leading a panel of novel and highly clinically effective therapies. It is clear therefore that an understanding of how and when these complementary approaches can most effectively be combined offers the real hope of moving beyond simply treating the disease and toward starting to talk about curative therapies. In this review we discuss approaches to combining these therapeutic platforms, both through engineering the viral vectors to more beneficially interact with the host immune response during therapy, as well as through the direct combinations of different therapeutics. This primarily, but not exclusively focuses on strains of oncolytic vaccinia virus. Some of the results reported to date, primarily in pre-clinical models but also in early clinical trials, are dramatic and hold great promise for the future development of similar therapies and their translation into cancer therapies.

Immunotherapeutic potential of oncolytic vaccinia virus.

The concept of oncolytic viral therapy was based on the hypothesis that engineering tumor-selectivity into the replication potential of viruses would permit direct destruction of tumor cells as a result of viral-mediated lysis, resulting in amplification of the therapy exclusively within the tumor environment. The immune response raised by the virus was not only considered to be necessary for the safety of the approach, but also something of a hindrance to optimal therapeutic activity and repeat dosing. However, the pre-clinical and subsequent clinical success of several oncolytic viruses expressing selected cytokines has demonstrated the potential for harnessing the immune response as an additional and beneficial mechanism of therapeutic activity within the platform. Over the last few years, a variety of novel approaches have been incorporated to try to enhance this immunotherapeutic activity. Several innovative and subtle approaches have moved far beyond the expression of a single cytokine transgene, with the hope of optimizing anti-tumor immunity while having minimal detrimental impact on viral oncolytic activity.

Arming viruses in multi-mechanistic oncolytic viral therapy: current research and future developments, with emphasis on poxviruses.

The field of oncolytic virology has made great strides in recent years. However, one key finding has been that the use of viral agents that replicate selectively in tumors is usually insufficient to achieve anything beyond small and transient responses. Instead, like most cancer therapies, oncolytic viruses are most effective in combination with other therapies, which is where they have proven therapeutic effects in clinical and preclinical studies. In cases of some of the smaller RNA viruses, effects can only be achieved through combination regimens with chemotherapy, radiotherapy, or targeted conventional therapies. However, larger DNA viruses are able to express one or more transgenes; thus, therapeutic mechanisms can be built into the viral vector itself. The incorporated approaches into arming oncolytic viruses through transgene expression will be the main focus of this review, including use of immune activators, prodrug converting enzymes, anti-angiogenic factors, and targeting of the stroma. This will focus on poxviruses as model systems with large cloning capacities, which have routinely been used as transgene expression vectors in different settings, including vaccine and oncolytic viral therapy.

The role of GM-CSF in enhancing immunotherapy of cancer.

Evaluation of: Kanerva A, Nokisalmi P, Diaconu I et al. Antiviral and anti-tumor T-cell immunity in patients treated with GM-CSF coding oncolytic adenovirus. Clin. Cancer Res. 19(10), 2734-2744 (2013). The field of oncolytic viral therapy has been reinvigorated recently with publication of clinical data from two leading vectors, one based on HSV and one on Vaccinia, both of which express GM-CSF. Part of the reason for the improved clinical results with these vectors appears to be the enhanced immunotherapeutic mechanism of tumor destruction mediated by GM-CSF expression itself. The article by Kanerva et al. extends this work to describe early clinical use of an oncolytic adenovirus expressing GM-CSF, although the data are too preliminary to describe significant therapeutic benefits of GM-CSF expression in this backbone. However, the description of enhanced antitumor immunity in those patients that developed greater antiviral immunity after treatment provides a potent demonstration of the immunotherapeutic potential of epitope spreading after treatment with oncolytic viral therapies.

Oncolytic vaccinia virus disrupts tumor-associated vasculature in humans.

Efforts to selectively target and disrupt established tumor vasculature have largely failed to date. We hypothesized that a vaccinia virus engineered to target cells with activation of the ras/MAPK signaling pathway (JX-594) could specifically infect and express transgenes (hGM-CSF, ß-galactosidase) in tumor-associated vascular endothelial cells in humans. Efficient replication and transgene expression in normal human endothelial cells in vitro required either VEGF or FGF-2 stimulation. Intravenous infusion in mice resulted in virus replication in tumor-associated endothelial cells, disruption of tumor blood flow, and hypoxia within 48 hours; massive tumor necrosis ensued within 5 days. Normal vessels were not affected. In patients treated with intravenous JX-594 in a phase I clinical trial, we showed dose-dependent endothelial cell infection and transgene expression in tumor biopsies of diverse histologies. Finally, patients with advanced hepatocellular carcinoma, a hypervascular and VEGF-rich tumor type, were treated with JX-594 on phase II clinical trials. JX-594 treatment caused disruption of tumor perfusion as early as 5 days in both VEGF receptor inhibitor-naïve and -refractory patients. Toxicities to normal blood vessels or to wound healing were not evident clinically or on MRI scans. This platform technology opens up the possibility of multifunctional engineered vaccinia products that selectively target and infect tumor-associated endothelial cells, as well as cancer cells, resulting in transgene expression, vasculature disruption, and tumor destruction in humans systemically.

Crosstalk between immune cell and oncolytic vaccinia therapy enhances tumor trafficking and antitumor effects.

The combination of an oncolytic virus, that directly destroys tumor cells and mediates an acute immune response, with an immune cell therapy, capable of further enlisting and enhancing the host immune response, has the potential to create a potent therapeutic effect. We have previously developed several strategies for optimizing the delivery of oncolytic vaccinia virus vectors to their tumor targets, including the use of immune cell-based carrier vehicles and the incorporation of mutations that increase production of the enveloped form of vaccinia (extracellular enveloped viral (EEV)) that is better adapted to spread within a host. Here, we initially combine these approaches to create a novel therapeutic, consisting of an immune cell (cytokine-induced killer, CIK) preloaded with an oncolytic virus that is EEV enhanced. This resulted in direct interaction between the viral and immune cell components with each assisting the other in directing the therapy to the tumor and so enhancing the antitumor effects. This effect could be further improved through CCL5 expression from the virus. The resulting multicomponent therapy displays the ability for synergistic crosstalk between components, so significantly enhancing tumor trafficking and antitumor effects.

Theranostic potential of oncolytic vaccinia virus.

Biological cancer therapies, such as oncolytic, or replication-selective viruses have advantages over traditional therapeutics as they can employ multiple different mechanisms to target and destroy cancers (including direct cell lysis, immune activation and vascular collapse). This has led to their rapid recent clinical development. However this also makes their pre-clinical and clinical study complex, as many parameters may affect their therapeutic potential and so defining reason for treatment failure or approaches that might enhance their therapeutic activity can be complicated. The ability to non-invasively image viral gene expression in vivo both in pre-clinical models and during clinical testing will considerably enhance the speed of oncolytic virus development as well as increasing the level and type of useful data produced from these studies. Further, subsequent to future clinical approval, imaging of reporter gene expression might be used to evaluate the likelihood of response to oncolytic viral therapy prior to changes in tumor burden. Here different reporter genes used in conjunction with oncolytic viral therapy are described, along with the imaging modalities used to measure their expression, while their applications both in pre-clinical and clinical testing are discussed. Possible future applications for reporter gene expression from oncolytic viruses in the phenotyping of tumors and the personalizing of treatment regimens are also discussed.

Next-generation oncolytic vaccinia vectors.

Oncolytic vaccinia viruses have made some impressive advances over the last 5 years, with a range of -different backbones displaying significant antitumor responses in preclinical models, and some exciting clinical results being reported against liver cancers. Because the virus is capable of rapid spread within the tumor, has evolved to spread relatively undetected within the blood stream, does not integrate into the host cell chromosome, and can infect almost any cell type, it is well-suited to the requirements for a successful oncolytic. In addition, the extensive clinical use of this virus means that contraindications to its use are known, and approved and experimental antivirals are available. Furthermore, because the virus has a large array of virulence genes whose deletion may target different properties of the cancer cell, and a large cloning capacity allowing for insertion of multiple transgenes, the possibilities for further development of novel and next-generation oncolytic vectors are multitude.

Targeted and armed oncolytic poxviruses for cancer: the lead example of JX-594.

Oncolytic viruses (OVs) are designed to replicate in, and subsequently lyse cancer cells. Numerous oncolytic virus platforms are currently in development. Here we review preclinical and clinical experience with JX-594, the lead candidate from the targeted and armed oncolytic poxvirus class. JX-594 is derived from a vaccinia vaccine strain that has been engineered for 1) enhanced cancer targeting and 2) has been "armed" with the therapeutic transgene granulocytemacrophage colony stimulating factor (GM-CSF) to stimulate anti-tumoral immunity. Poxviruses have many ideal features for use as oncolytic agents. The development of oncolytic vaccinia viruses is supported by a large safety database accumulated in the smallpox eradication program. In addition, poxviruses have evolved unique capabilities for systemic spread through the blood that can be harnessed for the treatment of metastatic disease. JX-594 demonstrates a high degree of cancer selectivity and systemic efficacy by multiple mechanisms-of-action (MOAs) in preclinical testing. Data from Phase 1 and 2 clinical trials has confirmed that these features result in potent and systemic efficacy in patients with treatment refractory metastatic cancers.